Modified nucleoside monomer and oligonucleotide prepared therefrom

Abstract

The present invention relates to the field of biomedicine, and specifically relates to a modified nucleoside monomer compound, and an oligonucleotide prepared therefrom. The modified oligonucleotide has an extrahepatic delivery targeting effect, has high stability and high inhibitory activity, and can provide a more excellent treatment solution for treating diseases at targeted positions such as the CNS and ophthalmologic targeted positions, or delaying the progress of said diseases.

Description

A modified nucleoside monomer and oligonucleotides prepared therefrom

[0001] This application claims priority to the following prior patent applications:

[0002] The applicant filed an earlier application with the China National Intellectual Property Administration on May 31, 2024, with patent application number 202410695135.8 and title "A modified nucleoside monomer and oligonucleotides prepared therefrom".

[0003] The applicant filed an earlier application with the China National Intellectual Property Administration on August 30, 2024, with patent application number 202411211681.6 and titled "A nucleic acid molecule that inhibits the expression of the vegfa gene and / or ang-2 gene".

[0004] The full text of the aforementioned prior patent applications is incorporated herein by reference. Technical Field

[0005] This invention relates to the field of biomedicine, and more specifically, to a modified nucleoside monomer compound, an oligonucleotide prepared therefrom having an extrahepatic delivery effect, and the use of the oligonucleotide in preparing a drug for regulating the expression of target genes in mammalian cells. Background Technology

[0006] Oligonucleotides can upregulate or downregulate the expression of target genes or induce alternative splicing of mRNA by generating stable and specific hybridization with target sequences, utilizing principles such as RNA interference (RNAi), RNA activation (RNAa), antisense nucleic acid technology, and exon skipping. In some respects, functional oligonucleotides can also be nucleic acid structures that generate stable and specific binding with target proteins.

[0007] Delivering oligonucleotides to target cells in vivo requires tissue-specific targeting and overcoming barriers from the extracellular environment. One of the factors limiting the experimental and therapeutic applications of oligonucleotides in vivo is the ability to efficiently deliver intact oligonucleotides.

[0008] Currently, delivery vectors for oligonucleotides are mainly divided into viral vectors and non-viral vectors. Non-viral vectors include: GalNac (N-acetylgalactosamine) conjugated vectors, lipid nanoparticles (LNPs), polymers, exocrine bodies, peptide conjugations, antibody conjugations, and other conjugated vectors. Among these, GalNac conjugated vectors and LNPs are the two most commonly used delivery vectors, both of which are liver-targeting. However, delivering oligonucleotides to extrahepatic tissues still faces challenges, thus limiting their application.

[0009] One challenge in treating ocular-related disorders with oligonucleotides is overcoming the internal limiting membrane barrier caused by retinal transfection. Additionally, it has been shown that negatively charged sugars in the vitreous interact with positive DNA-transfectant complexes, promoting their aggregation, which hinders diffusion and cellular uptake. A difficulty in treating CNS-related disorders with oligonucleotides lies in the inability of free oligonucleotides to cross the blood-brain barrier (BBB). Currently, one method for delivering oligonucleotides to the CNS is intrathecal delivery. However, oligonucleotides also need to be effectively internalized into target cells of the CNS to achieve the desired therapeutic effect.

[0010] WO2019217459A1 discloses a double-stranded iRNA with an internally conjugated lipophilic moiety, which is effective for extrahepatic delivery. The structure of the representative nucleotide monomer conjugated with the lipophilic moiety is as follows: However, the targeting efficiency, bioactivity, and persistence of double-stranded siRNAs containing this modified nucleotide monomer still need to be improved.

[0011] Therefore, there is a continued need for new and improved methods for delivering oligonucleotides in vivo to achieve stronger targeting and / or enhanced therapeutic effects.

[0012] Invention Overview

[0013] In view of the various deficiencies of the prior art, the present invention provides a modified nucleoside monomer compound, an oligonucleotide prepared therefrom having an extrahepatic delivery effect, and the use of the oligonucleotide in preparing a drug for regulating the expression of target genes in mammalian cells, thereby providing a solution to the problem of treating diseases that can be downregulated or upregulated by target genes.

[0014] As a first aspect of the present invention, the present invention provides a modified nucleoside monomer with the structure shown in formula (I):

[0015] Wherein, Base is a natural nucleobase or a modified nucleobase.

[0016] R1 and R2 are independently H, hydroxyl protecting groups, or phosphoramide groups, respectively;

[0017] R3 is C 14-22 Saturated or unsaturated hydrocarbon groups, or C 23-25 Saturated or unsaturated hydrocarbon groups;

[0018] Furthermore, when Base is uracil, R1 and R2 are not both H.

[0019] In some embodiments, the structure of the nucleoside monomer is shown in formula (I):

[0020] Wherein, Base is a natural nucleobase or a modified nucleobase.

[0021] R1 and R2 are independently H, hydroxyl protecting groups, or phosphoramide groups, respectively;

[0022] R3 is C 14-22 Saturated or unsaturated hydrocarbon groups;

[0023] Furthermore, when Base is uracil, R1 and R2 are not both H.

[0024] In some embodiments, the structure of the nucleoside monomer is shown in formula (I):

[0025] Wherein, Base is a natural nucleobase or a modified nucleobase.

[0026] R1 and R2 are independently H, hydroxyl protecting groups, or phosphoramide groups, respectively;

[0027] R3 is C 23-25 Saturated or unsaturated hydrocarbon groups;

[0028] Furthermore, when Base is uracil, R1 and R2 are not both H.

[0029] In some embodiments, the nucleobase is a purine base or a pyrimidine base.

[0030] In some embodiments, the nucleobase is selected from one, two or more of the following: adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U).

[0031] In some embodiments, the modified nucleobase is any one, two, or more nucleobases that are independently modified at each site, and the modification is selected from one, two, or more of the following: amino protecting group, C 1-3 Alkyl, C 3-5 cycloalkyl, halogenated C 1-3 Alkyl, C 1-3 alkyl carbonyl, C 1-3 Alkoxy C 1-3Alkyl, benzoyl, benzyl, dimethylaminomethylene, dimethylaminoC 1-3 Alkyl, oxo, thio, amino, C 2-6 alkenyl, C 2-6 alkynyl, mercapto, C 1-3 Alkyl thio, C 1-3 Alkylamino, di(C) 1-3 Alkyl)amino, hydroxyl, C 1-3 alkoxycarbonyl C 1-3 Alkyl, amino C 1-3 Alkoxy, amino C 1-3 Alkylaminocarbonyl, amino C 1-3 Alkylaminocarbonylvinyl and amino C 1-3 Alkyl hydroxyl group. In some embodiments, the modification is selected from one, two, or more of the following: amino protecting group, C... 1-3 Alkyl, C 3-5 cycloalkyl, halogenated C 1-3 Alkyl, C 1-3 alkyl carbonyl, C 1-3 Alkoxy C 1-3 Alkyl, benzoyl, benzyl, dimethylaminomethylene and dimethylaminoC 1-3 alkyl.

[0032] In some embodiments, R3 is a saturated or unsaturated hydrocarbon group. In some embodiments, R3 is a straight-chain or branched hydrocarbon group. In some embodiments, R3 is C 14-18 Saturated or unsaturated hydrocarbon group. In some embodiments, R3 is C 15-17 Saturated or unsaturated hydrocarbon group. In some embodiments, R3 is C 15 Alkyl, C 16 Alkyl or C 17 Alkyl group. In some embodiments, R3 is C6. 15-17 Branched or straight-chain hydrocarbon groups. In some embodiments, R3 is a straight-chain C. 15-17 Alkyl groups, such as straight-chain C 15 Alkyl, straight-chain C 16 Alkyl or straight-chain C 17 Alkyl group. In some embodiments, R3 is C6. 16 Alkyl group. In some embodiments, R3 is a straight-chain C. 16 alkyl.

[0033] In some implementations, R3 is C 22 Saturated or unsaturated hydrocarbon group. In some embodiments, R3 is C 22 Branched or straight-chain hydrocarbon groups. In some embodiments, R3 is a straight-chain C. 22 alkyl.

[0034] In some implementations, R3 is C 23-25 Saturated or unsaturated hydrocarbon group. In some embodiments, R3 is C 23 Alkyl, C 24 Alkyl or C 25 Alkyl group. In some embodiments, R3 is C6. 23-25 Branched or straight-chain hydrocarbon groups. In some embodiments, R3 is a straight-chain C. 23-25 Alkyl groups, such as straight-chain C 23 Alkyl, straight-chain C 24 Alkyl or straight-chain C 25 alkyl.

[0035] In some embodiments, the "amino protecting group" is selected from one, two, or more of the following: benzoyl (Bz), acetyl (Ac), benzyloxycarbonyl (CBz), tert-butoxycarbonyl (Boc), 9-phosphomethoxycarbonyl (Fmoc), p-methoxybenzyl (PMB), benzyl (Bn), triphenylmethyl (Tr), p-toluenesulfonyl (Tos), phthaloyl (Pht), allyloxycarbonyl (Alloc), and trifluoroacetyl (Tfa). Benzoyl (Bz), acetyl (Ac), benzyloxycarbonyl (CBz), tert-butoxycarbonyl (Boc), and 9-phosphomethoxycarbonyl (Fmoc) are preferred.

[0036] In some embodiments, the "phosphamide group" is selected from one, two, or more of the following structures: R4 and R5 are each independently C 1-3 Straight-chain or branched alkyl groups, R6 is C 1-3 Straight-chain or branched alkyl groups, or C groups substituted with cyano (-CN). 1-3 Straight-chain or branched alkyl groups.

[0037] In some implementation schemes, R1 and R2 are independently selected from H, Wherein, Cat is a cation, independently selected from sodium ion, potassium ion, triethylammonium ion, tripropylammonium ion, tributylammonium ion, and tetrabutylammonium ion; r is an integer from 0 to 5 (e.g., 0, 1, 2, 3, 4, 5); and Solid support is a solid-phase support.

[0038] In some embodiments, the "hydroxyl protecting group" is selected from one, two, or more of the following: Wherein, Cat is a cation, independently selected from sodium ion, potassium ion, triethylammonium ion, tripropylammonium ion, tributylammonium ion, and tetrabutylammonium ion; r is an integer from 0 to 5 (e.g., 0, 1, 2, 3, 4, 5); and Solid support is a solid-phase support.

[0039] In some embodiments, the “hydroxyl protecting group” is selected from one, two or more of the following: methoxymethyl ether (MOM), 2-tetrahydropyran (THP), tert-butyldimethylchlorosilyl (TBS), trimethylsilyl (TMS), triethylsilyl (TES), triphenylmethyl (Tr), 4,4'-bismethoxytriphenylmethyl (DMTr), 4,4',4'-trimethoxytriphenylmethyl (TMTr), benzyl (Bn), benzoyl (Bz), acetyl (Ac), p-methoxybenzyl (PMB), preferably triphenylmethyl (Tr), 4,4'-bismethoxytriphenylmethyl (DMTr), 4,4',4'-trimethoxytriphenylmethyl (TMTr), benzyl (Bn), benzoyl (Bz) and acetyl (Ac).

[0040] In some implementations, R1 is independently selected from the following structures: H, Preferably, R1 is independently selected from H,

[0041] In some implementation schemes, R2 is independently selected from: H, Preferably, R2 is independently selected from H,

[0042] In a preferred embodiment of the present invention, Cat is independently selected from sodium ions, triethylammonium ions, and tetrabutylammonium ions; r is an integer from 1 to 2, specifically 1 and 2; preferably, r is 1; Solid support is selected from controllable microporous glass CPG and polystyrene PS.

[0043] In some embodiments, R1 and R2 are independently a hydroxyl protecting group or a phosphoramide group. In some embodiments, R1 is DMTr and R2 is a phosphoramide group. In some embodiments, R1 is DMTr and R2 is...

[0044] In some implementations, R1 is selected from DMTr, and R2 is...

[0045] In some embodiments, the compound of formula (I) has the following structure:

[0046] In some embodiments, the compound of formula (I) has the following structure:

[0047] In some embodiments, the compound of formula (I) has the following structure:

[0048] In some embodiments, the compound of formula (I) has the following structure:

[0049] In some embodiments, the compound of formula (I) has the following structure:

[0050] In some embodiments, the compound of formula (I) has the following structure:

[0051] In some embodiments, the compound of formula (I) has the following structure:

[0052] In some embodiments, the compound of formula (I) has the following structure:

[0053] In some embodiments, the compound of formula (I) has the following structure:

[0054] In some embodiments, the compound of formula (I) has the following structure:

[0055] In some embodiments, the compound of formula (I) has the following structure:

[0056] In some embodiments, the compound of formula (I) has the following structure:

[0057] In some embodiments, the compound of formula (I) has the following structure:

[0058] In some embodiments, the compound of formula (I) has the following structure:

[0059] In some embodiments, the compound of formula (I) has the following structure:

[0060] In some embodiments, the compound of formula (I) has the following structure:

[0061] In some embodiments, the compound of formula (I) has the following structure:

[0062] In some embodiments, the compound of formula (I) has the following structure:

[0063] In some embodiments, the compound of formula (I) has the following structure:

[0064] A second aspect of the present invention is to provide an oligonucleotide comprising an antisense strand complementary to a target gene, the oligonucleotide containing one, two, or more nucleoside monomers as shown in formula (II):

[0065] The definitions of Base and R3 are the same as those in equation (Ⅰ) above, that is:

[0066] The Base is a natural or modified nucleobase. In some embodiments, the nucleobase is a purine or pyrimidine base. In some embodiments, the nucleobase is adenine (A), guanine (G), thymine (T), cytosine (C), or uracil (U). In some embodiments, the modified nucleobase is any one, two, or more sites that are independently modified, and the modification is selected from one or more of the following: amino protecting group, C... 1-3 Alkyl, C 3-5 cycloalkyl, halogenated C 1-3 Alkyl, C 1-3 alkyl carbonyl, C 1-3 Alkoxy C 1-3 Alkyl, benzoyl, benzyl, dimethylaminomethylene, dimethylaminoC 1-3 Alkyl, oxo, thio, amino, C 2-6 alkenyl, C 2-6 alkynyl, mercapto, C 1-3 Alkyl thio, C 1-3 Alkylamino, di(C) 1-3 Alkyl)amino, hydroxyl, C 1-3 alkoxycarbonyl C 1-3 Alkyl, amino C 1-3 Alkoxy, amino C 1-3 Alkylaminocarbonyl, amino C 1-3 Alkylaminocarbonylvinyl and amino C 1-3 Alkyl hydroxyl. In some embodiments, the modification is selected from one or more of the following: amino protecting group, C 1-3 Alkyl, C 3-5 cycloalkyl, halogenated C 1-3 Alkyl, C 1-3alkyl carbonyl, C 1-3 Alkoxy C 1-3 Alkyl, benzoyl, benzyl, dimethylaminomethylene and dimethylaminoC 1-3 alkyl.

[0067] R3 is C 14-22 Saturated or unsaturated hydrocarbon groups, or C 23-25 Saturated or unsaturated hydrocarbon group. In some embodiments, R3 is a saturated or unsaturated hydrocarbon group. In some embodiments, R3 is a straight-chain or branched hydrocarbon group. In some embodiments, R3 is C 14-18 Saturated or unsaturated hydrocarbon group. In some embodiments, R3 is C 15-17 Saturated or unsaturated hydrocarbon group. In some embodiments, R3 is C 15 Alkyl, C 16 Alkyl or C 17 Alkyl group. In some embodiments, R3 is C6. 15-17 Branched or straight-chain hydrocarbon groups. In some embodiments, R3 is C 15-17 Straight chain C 15 Alkyl, C 16 Alkyl or C 17 Alkyl group. In some embodiments, R3 is a straight-chain C. 15-17 Alkyl groups, such as straight-chain C 15 Alkyl, straight-chain C 16 Alkyl or straight-chain C 17 Alkyl group. In some embodiments, R3 is C6. 16 Alkyl group. In some embodiments, R3 is a straight-chain C. 16 Alkyl group. In some embodiments, R3 is C6. 22 Saturated or unsaturated hydrocarbon group. In some embodiments, R3 is C 22 Branched or straight-chain hydrocarbon groups. In some embodiments, R3 is a straight-chain C. 22 Alkyl group. In some embodiments, R3 is C6. 23-25 Saturated or unsaturated hydrocarbon group. In some embodiments, R3 is C 23 Alkyl, C 24 Alkyl or C 25 Alkyl group. In some embodiments, R3 is C6. 23-25 Branched or straight-chain hydrocarbon groups. In some embodiments, R3 is a straight-chain C. 23-25 Alkyl groups, such as straight-chain C 23 Alkyl, straight-chain C 24 Alkyl or straight-chain C 25 alkyl.

[0068] The "amino protecting group" is selected from one, two, or more of the following: benzoyl (Bz), acetyl (Ac), benzyloxycarbonyl (CBz), tert-butoxycarbonyl (Boc), 9-phosphomethoxycarbonyl (Fmoc), p-methoxybenzyl (PMB), benzyl (Bn), triphenylmethyl (Tr), p-toluenesulfonyl (Tos), phthaloyl (Pht), allyloxycarbonyl (Alloc), and trifluoroacetyl (Tfa). Benzoyl (Bz), acetyl (Ac), benzyloxycarbonyl (CBz), tert-butoxycarbonyl (Boc), and 9-phosphomethoxycarbonyl (Fmoc) are preferred.

[0069] In some embodiments, the oligonucleotide is RNA. In some embodiments, the oligonucleotide is DNA. In some embodiments, the oligonucleotide is a hybrid molecule of RNA and DNA. In some embodiments, the oligonucleotide has a double-stranded structure. In some embodiments, the oligonucleotide has a single-stranded structure. In some embodiments, the oligonucleotide includes, but is not limited to, one of: double-stranded RNA, microRNA, single-stranded RNA, antisense nucleic acid (antisense DNA, antisense RNA), nucleic acid aptamers, guide RNA, stem-loop RNA, or mRNA fragments. In some embodiments, the oligonucleotide is double-stranded RNA, single-stranded RNA, or nucleic acid having a secondary or three-dimensional structure.

[0070] In some implementations, the oligonucleotide is a small interfering RNA.

[0071] In some implementations, the oligonucleotide is a small activating RNA.

[0072] In some embodiments, the oligonucleotide is an antisense oligonucleotide (ASO).

[0073] In some embodiments, the antisense strand of the oligonucleotide is complementary to the coding region of the target gene. In some embodiments, the antisense strand of the oligonucleotide is complementary to the non-coding region of the target gene. In some embodiments, the antisense strand is complementary to the mRNA of the target gene. In some embodiments, the antisense strand is complementary to the pre-mRNA of the target gene. In some embodiments, the antisense strand is complementary to the mature mRNA of the target gene.

[0074] In some embodiments, at least one of the four to eight positions (e.g., positions 4, 5, 6, 7, or 8) of at least one chain of the oligonucleotide starting from the 5' end contains a nucleoside monomer represented by formula (II). At least one of the six, seven, or eight positions of the oligonucleotide oligonucleotide starting from the 5' end contains a nucleoside monomer represented by formula (II).

[0075] In some embodiments, the antisense strand of the oligonucleotide is 14 to 30 nucleotides (nt). In some embodiments, the antisense strand of the oligonucleotide is 19 to 25 nucleotides, and the antisense strand is complementary to at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides of the target gene. In some embodiments, the antisense strand of the oligonucleotide is 21-23 (21, 22, or 23) nucleotides. In some embodiments, the antisense strand of the oligonucleotide is 21-23 (21, 22, or 23) nt, and the antisense strand is complementary to at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides of the target gene.

[0076] In some embodiments, the oligonucleotide is a double-stranded RNA comprising an antisense strand complementary to the target gene and a sense strand complementary to the antisense strand, each strand being independently 14 to 30 nucleotides long. In some embodiments, the length of the sense and antisense strands of the double-stranded RNA is independently 19 to 25 nt. In some embodiments, the length of the sense and antisense strands of the double-stranded RNA is independently 21 to 23 nt.

[0077] In some embodiments, the double-stranded region of the double-stranded RNA is 15-25 bp in length (e.g., 15 bp, 16 bp, 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, or 25 bp). In some embodiments, the double-stranded region of the double-stranded RNA is 18-23 bp in length. In some embodiments, the double-stranded region of the double-stranded RNA is 19 bp, 20 bp, or 21 bp in length.

[0078] In some embodiments, the double-stranded RNA includes at least one single-stranded overhang at one end, such as a 3' and / or 5' overhang of 1-5 (e.g., 1, 2, 3, 4, 5) nucleotides in length. In one embodiment, the single-stranded overhang is 1, 2, or 3 nucleotides in length. In some embodiments, the double-stranded RNA may also have a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand), or vice versa. In some embodiments, the double-stranded RNA includes a 3' overhang at the 3' end of the antisense strand and optionally a blunt end at the 5' end of the antisense strand. In some embodiments, the double-stranded RNA has a 5' overhang at the 5' end of the sense strand and optionally a blunt end at the 5' end of the antisense strand. In some embodiments, the double-stranded RNA has two blunt ends at both ends of the double strand.

[0079] In some embodiments, the double-stranded RNA has a sense strand of 21 nucleotides and an antisense strand of 23 nucleotides, wherein the sense and antisense strands are complementary to form a double-stranded region of 21 consecutive base pairs, and the double-stranded RNA has a single-stranded overhang of 2 nucleotides at the 3' end of the antisense strand.

[0080] In some embodiments, the sense and / or antisense strands of the double-stranded RNA contain at least one of the 6th, 7th, and 8th positions starting from the 5' end, which is a nucleoside monomer of formula (II).

[0081] In some embodiments, at least one of the 6th, 7th, and 8th positions of the positive strand of the double-stranded RNA, starting from the 5' end, contains a nucleoside monomer represented by formula (II). In some embodiments, at least one nucleotide of the oligonucleotide is modified; the modification includes, but is not limited to, one, two, or more of the following modifications: locked nucleic acid modification, ring-opening or non-locked nucleic acid modification, 2′-methoxyethyl modification, 2′-O-methyl modification, 2′-O-allyl modification, 2′-C-allyl modification, 2′-fluoro modification, 2′-deoxy modification, 2′-hydroxyl modification, thiophosphate backbone modification (i.e., phosphothioester bond modification, methylphosphothioester bond modification), DNA modification (such as thymidine nucleotide), 5′-vinylphosphonate (5′-Vp) modification, fluorescent probe modification, and ligand modification. In some embodiments, the modification is selected from one, two or more of the following: 2′-O-methyl modification, 2′-fluoro modification, thiophosphate skeleton modification, 2′-deoxy modification and 5′-vinylphosphonate modification.

[0082] In some embodiments, all nucleotides in the oligonucleotide are modified. In some embodiments, at least 95%, 90%, 85%, or 80% of the nucleotides in the oligonucleotide are modified.

[0083] In some embodiments, the sense and / or antisense strands of the double-stranded RNA contain one nucleoside monomer of formula (II) located at the 6th, 7th, or 8th position starting from the 5' end, one, two, three, four, five, or six 2′-fluoromodified nucleotides, and the remaining nucleotides having a 2′-O-methyl modification or a 2′-deoxy modification (DNA) at the 2′ position.

[0084] In some embodiments, the nucleoside monomer of formula (II) has the following structure:

[0085] In some embodiments, the nucleoside monomer of formula (II) has the following structure:

[0086] In some embodiments, the nucleoside monomer of formula (II) has the following structure:

[0087] In some embodiments, the nucleoside monomer of formula (II) has the following structure:

[0088] In some embodiments, the nucleoside monomer of formula (II) has the following structure:

[0089] In some embodiments, the nucleoside monomer of formula (II) has the following structure:

[0090] In some embodiments, the nucleoside monomer of formula (II) has the following structure:

[0091] In some embodiments, the nucleoside monomer of formula (II) has the following structure:

[0092] In some embodiments, the nucleoside monomer of formula (II) has the following structure:

[0093] In some embodiments, the nucleoside monomer of formula (II) has the following structure:

[0094] In some embodiments, the oligonucleotides of the present invention may or may not contain a desialyl glycoprotein receptor (ASGPR) ligand.

[0095] In a third aspect, the present invention provides a pharmaceutical composition comprising an effective amount of the above-described oligonucleotide and pharmaceutically acceptable excipients.

[0096] "Effective amount" refers to the amount of oligonucleotides that can effectively produce the expected pharmacological therapeutic effect. Acceptable carriers include water, saline, glucose, buffers (such as PBS), excipients, diluents, disintegrants, binders, lubricants, sweeteners, flavorings, preservatives, or combinations thereof.

[0097] In some embodiments, the pharmaceutical composition comprises an effective amount of the aforementioned oligonucleotide, pharmaceutically acceptable excipients, and a delivery system.

[0098] In some embodiments, the delivery system for the pharmaceutical composition may be selected from one, two or more of the following: liposomes (e.g., liposome 2000), viral vectors, extracellular vesicles (EVs), and lipid nanoparticles (LNPs).

[0099] In some embodiments, the viral vector includes, but is not limited to, adenovirus-associated virus (AAV), lentivirus, adenovirus, and retrovirus.

[0100] In some embodiments, the pharmaceutical composition may be administered via the following routes or in the following forms: (1) orally (aqueous or non-aqueous solution or suspension), tablets (e.g., tablets targeted for buccal, sublingual and systemic absorption), pills, powders, granules, pastes for application to the tongue; (2) non-oral administration, such as by subcutaneous, intramuscular, intravenous, epidural, rectal, transdermal, intranasal, pulmonary, intraventricular (ICV), intrathecal (IT) or ocular administration (e.g., intravitreal administration).

[0101] A fourth aspect of the invention also relates to the use of the above-described nucleoside monomers, oligonucleotides, or pharmaceutical compositions in the preparation of medicaments for regulating (inhibiting, silencing, or enhancing) the expression of target genes in cells.

[0102] In some embodiments, the present invention relates to the use of the above-described nucleoside monomers, oligonucleotides, or pharmaceutical compositions in the preparation of a medicament for delivering oligonucleotides to a specific target site in a subject.

[0103] In some embodiments, if the above-mentioned nucleoside monomer, oligonucleotide, or pharmaceutical composition is additionally linked to a GalNAc ligand, the target gene is located in or expressed in the liver.

[0104] In some implementations, the target gene is located in an extrahepatic location or expressed in an extrahepatic location.

[0105] In some implementations, the target gene is located in the sheath or eye, CNS, muscle, lung, or fat.

[0106] In some implementations, the target gene is located in the brain, spinal cord, or eye tissue.

[0107] In some implementations, the drug is used to regulate (e.g., reduce or enhance) the expression of target genes in brain, spinal cord, or eye tissues.

[0108] In some embodiments, the target gene is located in one, two, or more of the following locations: hippocampus, cortex, substantia nigra, striatum, cerebellum, cervical vertebrae, lumbar vertebrae, thoracic vertebrae, retina, choroid plexus, heart, fat (e.g., subcutaneous fat), and muscle (e.g., quadriceps femoris and tibialis anterior).

[0109] In some embodiments, the drug is used to treat a subject with a central nervous system (CNS) or ocular disorder. In some embodiments, the CNS or ocular disorder is selected from one, two, or more of the following: Alzheimer's disease, amyotrophic lateral sclerosis, frontotemporal dementia, Huntington's disease, Parkinson's disease, spinocerebellar disease, prions, age-related macular degeneration, and diabetic macular edema.

[0110] In some embodiments, the target genes of the drug are selected from one, two, or more of the following: SOD1, APP, ATXN1, ATXN2, ATXN3, C9orf72, TARDBP, MAPT, HTT, LRRK2, SNCA, VEGFA, ANG-2, VEGFR1, VEGFR2, SCA1, SCA7, and SCA8. In some embodiments, the target genes of the drug are selected from one, two, or more of SOD1, VEGFA, ANG-2, and SNCA.

[0111] In some embodiments, the subject has a disease mediated by the SOD1, VEGFA, and ANG-2 genes. In some embodiments, the subject has a disease mediated by the SOD1, and / or VEGFA, and / or ANG-2, and / or SNCA genes. In some embodiments, the subject has amyotrophic lateral sclerosis (ALS) and neurodegenerative diseases, age-related macular degeneration, and / or diabetic macular edema, etc.

[0112] A fifth aspect of the invention also relates to a method for regulating (inhibiting or enhancing) the expression of a target gene in cells, the method comprising the steps of: introducing the above-described oligonucleotide into cells in vivo or in vitro, wherein the oligonucleotide, upon contact with a cell expressing a target gene, can at least regulate the expression of the target gene, for example, by increasing or inhibiting the expression of the gene by 95%, 90%, 85%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5%.

[0113] The detection of target gene, target RNA, or target protein levels can be used to predict or assess activity, efficacy, or treatment outcomes.

[0114] In some embodiments, the single-dose pharmaceutical composition of the present invention can have a long-lasting effect. For example, the oligonucleotides of the present invention that reduce SOD1 expression can cause a sustained decrease in SOD1 expression for at least 7, 28 days, or longer. The oligonucleotides of the present invention that reduce VEGFA and ANG-2 expression can cause a sustained decrease in VEGFA and ANG-2 expression for at least 14 days, respectively. The oligonucleotides of the present invention that reduce SNCA expression can cause a sustained decrease in SNCA expression for at least 28 days, respectively.

[0115] A sixth aspect of the invention also relates to a method for treating or preventing diseases related to abnormal upregulation or downregulation of target gene expression, the method comprising administering a therapeutically effective amount or a preventatively effective amount of one or more of the above-described oligonucleotides to a patient in need of such treatment, prevention, or control.

[0116] In some embodiments, the target gene is selected from one, two, or more of the following: SOD1, APP, ATXN1, ATXN2, ATXN3, C9orf72, TARDBP, MAPT, HTT, LRRK2, SNCA, SOD1, VEGFA, ANG-2, VEGFR1, VEGFR2, SCA1, SCA7, and SCA8. In some embodiments, the target gene is selected from one, two, or more of SOD1, VEGFA, ANG-2, and SNCA.

[0117] A seventh aspect of the invention also relates to a method for regulating (inhibiting or enhancing) the expression of a target gene in a cell, the method comprising the steps of: introducing the above-mentioned oligonucleotide into a cell, wherein the oligonucleotide, upon contact with a cell expressing a target gene, can at least enhance or inhibit the expression level of the target gene by 40%.

[0118] In some embodiments, the present invention also relates to a method for treating or preventing diseases associated with abnormal upregulation of target gene expression, the method comprising administering a therapeutically effective amount or a preventatively effective amount of one, two, or more of the oligonucleotides described herein to a patient requiring such treatment, prevention, or control.

[0119] In some embodiments, the oligonucleotides described above can be used to inhibit target gene expression. The antisense strand of the oligonucleotide contains a nucleotide sequence substantially complementary to at least a portion of the mRNA encoding the target gene, and the length of the complementary region is less than 30 nucleotides, typically 19-24 nucleotides. Upon contact with cells expressing the target gene, the oligonucleotide can inhibit at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the target gene expression.

[0120] In some embodiments, the target gene is selected from one, two, or more of the following: SOD1, APP, ATXN1, ATXN2, ATXN3, C9orf72, TARDBP, MAPT, HTT, LRRK2, SNCA, VEGFA, ANG-2, VEGFR1, VEGFR2, SCA1, SCA7, and SCA8. In some embodiments, the target gene is selected from one, two, or more of SOD1, VEGFA, ANG-2, and SNCA.

[0121] In one embodiment of the present invention, the oligonucleotide is selected from one, two, or more of the following:

[0122] (1) A positive strand consisting of a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the positive strand of nucleic acid molecule S1-(6-C16-S) (i.e., the naked sequence is SEQ ID NO: 5, and the modification motif is NmsNmsNmNmNmN(hdt)NfNmNfNfNmNmNmNmNmNmNmNmsNmsNmNm) and an antisense strand consisting of a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the negative strand of nucleic acid molecule S1-(6-C16-S) (i.e., the naked sequence is SEQ ID NO: 6, and the modification motif is NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmsNmsNmsNmNm);

[0123] Or (1') a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sense strand of nucleic acid molecule S1-(6-C22-S) (i.e., the naked sequence is SEQ ID NO: 5, and the modified motif is NmsNmsNmNmNmN(dat)NfNmNfNfNmNmNmNmNmNmNmsNmsNm) and an antisense strand of nucleic acid molecule S1-(6-C22-S) (i.e., the naked sequence is SEQ ID NO: 5, and the modified motif is NmsNmsNmNmNmNmNmNmNmNmNmsNmsNm) NO: 6, an antisense strand consisting of a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the modified motif Vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmsNmsNm)

[0124] (2) A positive strand consisting of a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the positive strand of nucleic acid molecule V14-(7-C16-S) (i.e., the naked sequence is SEQ ID NO: 1, and the modified motif is NmsNmsNmNmNmNmN(hdt)NmNfNfNmNmNmNmNmNmNmNmsNmsNm) and an antisense strand consisting of a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the negative strand of nucleic acid molecule V14-(7-C16-S) (i.e., the naked sequence is SEQ ID NO: 2, and the modified motif is NmsNfsNmNmNmNmNfNmNmNmNmNmNmNmNmNmsNmsNmsNm);

[0125] (3) A positive strand consisting of a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the positive strand of nucleic acid molecule A12-(7-C16-S) (i.e., the naked sequence is SEQ ID NO: 3, and the modification motif is NmsNmsNmNmNmNmN(hdt)NmNfNfNmNmNmNmNmNmNmNmsNmsNm) and an antisense strand consisting of a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the negative strand of nucleic acid molecule A12-(7-C16-S) (i.e., the naked sequence is SEQ ID NO: 4, and the modification motif is NmsNfsNmNmNmNmNfNmNmNmNmNmNmNmNmNmsNmsNmsNm);

[0126] (4) A positive strand consisting of a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the positive strand of nucleic acid molecule A12-(6-C16-S) (i.e., the naked sequence is SEQ ID NO: 3, and the modified motif is NmsNmsNmNmNmN(hdt)NfNmNfNfNmNmNmNmNmNmNmsNmsNm) and an antisense strand of nucleic acid molecule A12-(6-C16-S) (i.e., the naked sequence is SEQ ID NO: 3, and the modified motif is NmsNmsNmNmNmNmNmNmNmNmNmNmsNmsNm) and an antisense strand having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the negative strand of nucleic acid molecule A12-(6-C16-S) (i.e., the naked sequence is SEQ ID NO: 3, and the modified motif is NmsNmsNmNmNmNmNmNmNmNmNmNmNms ... NO: 4, an antisense strand consisting of a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the modified motif Vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmsNmsNm)

[0127] (5) A positive strand consisting of a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the positive strand of the nucleic acid molecule SN10-(6-C16-S) (i.e., the naked sequence is SEQ ID NO: 9, and the modification motif is NmsNmsNmNmNmN(hdt)NfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm) and an antisense strand consisting of a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the negative strand of the nucleic acid molecule A12-(7-C16-S) (i.e., the naked sequence is SEQ ID NO: 10, and the modification motif is NmsNfsNmNmNmNfNmNfNmNmNmNmNmNmNmNmsNmsNmsNm);

[0128] (6) A sense strand (i.e., sense strand with the modified sequence AmsUmsGmUmGmA(hdt)AfUmGfCfAfGmAmCmCmAmAmGmsAm) consisting of a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sense strand of nucleic acid molecule V22.3 (i.e., the naked sequence is SEQ ID NO: 25, and the modified motif is NmsNmsNmNmNmNmN(hdt)NfNmNfNfNfNmNmNmNmNmNmNmsNmsNm) and an antisense strand (i.e., the naked sequence is SEQ ID NO: 25) with ...7%, 98%, 99%, or 100% identity with the modified motif NmsUmsGmUmGmA(hdt)AfUmGfCfAfGmAmCmCmAmAmAmGmsAmsAm) and an antisense strand of nucleic acid molecule V22.3 (i.e., the naked sequence is SEQ ID NO: 25). NO: 26, an antisense strand consisting of a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the modified motif Vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmsNmsNm)

[0129] (7) A sense strand (i.e., sense strand with the modified sequence specifically CmsCmsAmUmGmA(hdt)UfGmAfUfCfCmGmAmCmCmAmGmCmsAmsGm) consisting of a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sense strand of nucleic acid molecule A12.2 (i.e., the naked sequence is SEQ ID NO: 3, and the modified motif is NmsNmsNmNmNmNmN(hdt)NfNmNfNfNmNmNmNmNmNmNmsNmsNm) and an antisense strand (i.e., the naked sequence is SEQ ID NO: 3) with ... modified motif NmsCmsAmUmGm) and the modified motif NmsCmsAmsGm of nucleic acid molecule V22.3 (i.e., the naked sequence is SEQ ID NO: 3). NO: 4, an antisense strand consisting of a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the modified motif Vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmsNmsNm)

[0130] In the above sequence and other parts of the present invention, Nm indicates that the nucleotide at the corresponding position is modified with a 2′-O-methyl group; Nf indicates that the nucleotide at the corresponding position is modified with a 2′-fluoro group; and (s) indicates that the nucleotides on both sides are connected by a thiophosphate backbone (thiophosphate diester bond). In the present invention, N in the modified motif corresponds one-to-one with nucleotides from 5′ to 3′ in the sequence having the modified motif. Optionally, other conventional chemical modifications can be made to the sugar ring, base, or phosphate ester bond of any nucleotide in the oligonucleotide to enhance stability.

[0131] Unexpected technical effects of the present invention

[0132] The modified oligonucleotides of this invention have a targeted effect for extrahepatic delivery. Compared with the prior art, the modified oligonucleotides of this invention have higher stability and higher and more durable inhibitory activity. Furthermore, the modified oligonucleotides can enter target cells and extrahepatic tissues without transfection reagents, reducing the negative effects of transfection reagents, such as cell or tissue toxicity.

[0133] The modified oligonucleotides of this invention exhibit high stability while maintaining high inhibitory activity. Combined with their extrahepatic delivery targeting effect, they can provide a superior treatment option for extrahepatic diseases (such as CNS diseases, eye diseases, muscle diseases, lung diseases, or fat diseases).

[0134] Invention Details

[0135] definition:

[0136] Unless otherwise defined herein, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. In the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions.

[0137] This is the covalent linkage site for the functional group.

[0138] The terms “optional,” “arbitrary,” “optionally,” or “arbitrarily” refer to events or conditions that are subsequently described but are not required to occur, and the description includes both cases where said events or conditions occur and cases where said events or conditions do not occur.

[0139] Unless otherwise specified, the term "alkyl" is synonymous with "saturated hydrocarbon group" and "saturated aliphatic hydrocarbon group," referring to a monovalent saturated aliphatic hydrocarbon group. An alkyl group with a specified number of carbon atoms refers to a straight-chain or branched alkyl group containing the specified number of carbon atoms, such as C... 16 -C 18 Saturated hydrocarbon groups refer to alkyl groups with 16-18 carbon atoms, i.e., C64-184 carbon atoms. 16 Alkyl, C 17 Alkyl, C 18 Alkyl groups, such as "C 1-6 "Alkyl" refers to an alkyl group with 1 to 6 carbon atoms in its carbon chain (specifically, 1, 2, 3, 4, 5, or 6). If the number of carbon atoms is not specified, it generally refers to a straight-chain or branched alkyl group with 1 to 20 carbon atoms, preferably 1 to 10 (i.e., C20-C20). 1-10 Alkyl groups, more preferably having 1-8 carbon atoms (C46-C56). 1-8 Alkyl groups, more preferably alkyl groups with 1-6 carbon atoms (i.e., C64-C ... 1-6Alkyl groups, including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, n-heptyl, n-octyl, etc.

[0140] Unless otherwise specified, the term "unsaturated hydrocarbon group" is synonymous with "unsaturated aliphatic hydrocarbon group," referring to hydrocarbon compounds containing carbon-carbon double bonds (alkene bonds) and / or triple bonds (alkynyl bonds) at any position in their structure. An unsaturated hydrocarbon group with a specified number of carbon atoms refers to a straight-chain or branched unsaturated hydrocarbon group containing that number of carbon atoms, such as C... 16 -C 18 Unsaturated hydrocarbon groups refer to unsaturated hydrocarbon groups with 16-18 carbon atoms, i.e., C64-C ... 16 Unsaturated hydrocarbon group, C 17 Unsaturated hydrocarbon group, C 18 Unsaturated hydrocarbon groups, such as "C 1-6 "Unsaturated hydrocarbon group" refers to an unsaturated hydrocarbon group with 1-6 carbon atoms in its carbon chain (specifically, 1, 2, 3, 4, 5, or 6). If the number of carbon atoms is not specified, it generally refers to a straight-chain or branched unsaturated hydrocarbon group with 1-20 carbon atoms, preferably 1-10 carbon atoms (i.e., C2C2). 1-10 Unsaturated hydrocarbon groups), further preferably 1-8 carbon atoms (C 1-8 Unsaturated hydrocarbon groups), more preferably 1-6 carbon atoms (i.e., C64-C ... 1-6 (unsaturated hydrocarbon group).

[0141] Unless otherwise specified, the term "alkenyl" refers to an unsaturated aliphatic hydrocarbon group consisting of a straight or branched chain of carbon and hydrogen atoms, having at least one double bond. The alkenyl group can have 2-20 carbon atoms, preferably 2-10 carbon atoms (i.e., C2-C2). 2-10 Alkenyl), further preferably 2-8 carbon atoms (C 2-8 Alkenyl), more preferably 2-6 carbon atoms (i.e., C14-C2 ... 2-6 alkenyl), 2-5 carbon atoms (i.e., C) 2-5 alkenyl), 2-4 carbon atoms (i.e., C) 2-4 alkenyl), 2-3 carbon atoms (i.e., C) 2-3 Alkenyl), or 2 carbon atoms (i.e., C2 alkenyl), for example "C 2-6 "Alkenyl" refers to a group that is alkenyl and has 2 to 6 carbon atoms in its carbon chain (specifically, 2, 3, 4, 5, or 6). Non-limiting examples of alkenyl groups include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, isobutenyl, and 1,3-butadienyl.

[0142] Unless otherwise specified, the term "alkynyl" refers to an unsaturated aliphatic hydrocarbon group consisting of a straight or branched chain of carbon and hydrogen atoms, having at least one triple bond. The alkynyl group can have 2-20 carbon atoms, preferably 2-10 carbon atoms (i.e., C2H2O). 2-10 Alkyne group), further preferably 2-8 carbon atoms (C 2-8 Alkyne group), more preferably 2-6 carbon atoms (i.e., C64-C ... 2-6 acetylsyl group), 2-5 carbon atoms (i.e., C64) 2-5 acetylsyl group), 2-4 carbon atoms (i.e., C64) 2-4 acetylsyl group), 2-3 carbon atoms (i.e., C64) 2-3 Alkynyl group), or 2 carbon atoms (i.e., C2 alkynyl group), for example "C 2-6 "Alynyl" refers to a group that is alkynyl and has 2 to 6 carbon atoms in its carbon chain (specifically 2, 3, 4, 5, or 6). Non-limiting examples of alkynyl include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and 1-butynyl.

[0143] Unless otherwise specified, the term "alkoxy" refers to -O-alkyl, where the alkyl group is defined as above, i.e., the number of carbon atoms is 1-20, preferably 1-10, more preferably 1-8, and even more preferably 1-6 (specifically 1, 2, 3, 4, 5, or 6). Examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 1-methylpropoxy, 2-methylpropoxy, tert-butoxy, pentoxy, 1-methylbutoxy, 2-methylbutoxy, 3-methylbutoxy, 1,1-dimethylpropoxy, 1,2-dimethylpropoxy, 2,2-dimethylpropoxy, 1-ethylpropoxy, etc.

[0144] Unless otherwise specified, the terms "halogen" or "halogenated" refer to F, Cl, Br, and I. The term "halogenated alkyl" refers to an alkyl group as defined above in which one, two, or more hydrogen atoms, or all hydrogen atoms, are replaced by a halogen. Representative examples of halogenated alkyl groups include CCl3, CF3, CHCl2, CH2Cl, CH2Br, CH2I, CH2CF3, and CF2CF3.

[0145] Unless otherwise specified, the term "cycloalkyl" refers to a monocyclic saturated aliphatic hydrocarbon group having a specific number of carbon atoms, preferably 3-12 carbon atoms (i.e., C12-C12). 3-12 cycloalkyl), more preferably 3-10 carbon atoms (C 3-10 cycloalkyl groups, more preferably 3-7 carbon atoms (C 3-7 cycloalkyl groups, 4-6 carbon atoms (C 4-6 cycloalkyl groups), or 5-6 carbon atoms (C 5-6 cycloalkyl groups), 3-5 carbon atoms (C 3-5(Cycloalkyl). Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methylcyclopropyl, 2-ethyl-cyclopentyl, dimethylcyclobutyl, etc.

[0146] Unless otherwise specified, the term "amino protecting agent (amino group)" refers to the portion that temporarily blocks the amine reactive site in a compound. Preferably, the amino protecting group is selectively removable by chemical reaction and, if necessary, can be removed using methods known in the art. Commonly used amino protecting groups include, but are not limited to: benzoyl (Bz), acetyl (Ac), benzyloxycarbonyl (CBz), tert-butoxycarbonyl (Boc), 9-phosphomethoxycarbonyl (Fmoc), p-methoxybenzyl (PMB), benzyl (Bn), triphenylmethyl (Tr), p-toluenesulfonyl (Tos), phthaloyl (Pht), allyloxycarbonyl (Alloc), and trifluoroacetyl (Tfa). Benzoyl (Bz), acetyl (Ac), benzyloxycarbonyl (CBz), tert-butoxycarbonyl (Boc), and 9-phosphomethoxycarbonyl (Fmoc) are further preferred amino protecting agents.

[0147] The term "solid support" refers to a matrix of inorganic particles, polymers, or other solid materials that can be linked to target compounds by surface modification with active groups (such as NH2), thereby enabling the synthesis of oligonucleotides. Examples of solid supports include, but are not limited to, controllable microporous glass (CPG) and polystyrene (PS).

[0148] Unless otherwise specified, the term "hydroxyl protecting group" refers to a portion that temporarily blocks the reactive hydroxyl site in a compound. Preferably, the hydroxyl protecting group is selectively removable by chemical reaction and, if necessary, by methods known in the art. Commonly used hydroxyl protecting groups include, but are not limited to, methoxymethyl ether (MOM), 2-tetrahydropyran (THP), tert-butyldimethylchlorosilyl (TBS), trimethylsilyl (TMS), triethylsilyl (TES), triphenylmethyl (Tr), 4,4'-dimethoxytriphenylmethyl (DMTr), 4,4',4'-trimethoxytriphenylmethyl (TMTr), benzyl (Bn), benzoyl (Bz), acetyl (Ac), and p-methoxybenzyl (PMB). More preferably, the hydroxyl protecting group is triphenylmethyl (Tr), 4,4'-dimethoxytriphenylmethyl (DMTr), 4,4',4'-trimethoxytriphenylmethyl (TMTr), benzyl (Bn), benzoyl (Bz), or acetyl (Ac).

[0149] "Unmodified" or "natural" nucleobases, also simply called bases, are often heterocyclic bases. The two most common classes of such heterocyclic bases are purines and pyrimidines, such as the purine nucleobases adenine (A) and guanine (G), and the pyrimidine nucleobases thymine (T), cytosine (C), and uracil (U).

[0150] "Modified" or "non-natural" nucleobases, also simply referred to as "modified" or "non-natural" bases, refer to nucleobases obtained by modifying or replacing "unmodified" or "natural" bases. Such modifications or substitutions generally do not affect the unwinding behavior, intracellular enzyme recognition, or biological activity of nucleotides, but may improve the physicochemical properties of nucleotides, such as stability and enzyme resistance. For example, certain "modified" or "non-natural" bases can enhance the nuclease resistance of nucleotides. Exemplary modified nucleobases include, but are not limited to: inosine, xanthine, hypoxanthine, muscarin, isoguanosine, tuberculin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(meththio)-N6-(isopentenyl)adenine, 6-( Alkyl)adenine, 6-(methyl)adenine, 7-(deazo)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxy)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, N6-(isopentyl)adenine, N6-(methyl)adenine, N 6 N 6 2-(dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(denitro)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxy)guanine, 8-(thioalkyl)guanine 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deazo)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N 4-(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-( (aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidinyl)uracil, 5-(1,3-diazol-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N 3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2-(thio)pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil ) pseudouracil, 1-(aminocarbonylvinyl)-pseudouracil, 1-(aminocarbonylvinyl)-2(thio)-pseudouracil, 1-(aminocarbonylvinyl)-4-(thio)pseudouracil, 1-(aminocarbonylvinyl)-2,4-(dithio)pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-pseudouracil, 1-(aminoalkylamino-carbonylvinyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3 ...2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2- (Aza)-Phenoxazine-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-(guanidinylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 7-(guanidinylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-(guanidinyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 7-(guanidinyl-hydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, 2-aza-inosine, 7-deaza-inosine, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindole, pyrrolopyrimidinyl, 3-(methyl)isoquinolone, 5-(methyl)isoquinolone, 3-(methyl)-7-(propynyl)isoquinolone, 7-(aza)indole, 6-(methyl)-7-(aza)indole, imidazopyridyl, 9-(methyl)imidazopyridyl, pyrrolopyrazinyl, isoquinolone, 7-(propynyl)isoquinolone alkyl, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthyl, anthraceneyl, phenanthrene, pyrene, stilbene, tetraphenyl, pentaphenyl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymidine, 2-pyridone, 5-nitroindol, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidine, N, 2 -substituted purines, N 6 -substituted purines, O 6 -Substituted purines, substituted 1,2,4-triazoles, pyrrolopyrimidin-2-one-3-yl, 6-phenylpyrrolopyrimidin-2-one-3-yl, para-substituted 6-phenylpyrrolopyrimidin-2-one-3-yl, ortho-substituted 6-phenylpyrrolopyrimidin-2-one-3-yl, di-ortho-substituted 6-phenylpyrrolopyrimidin-2-one-3-yl, p-(aminoalkylhydroxy)-6-benzyl 2-O-alkyl-pyrrolopyrimidin-2-one-3-yl, o-(aminoalkylhydroxy)-6-phenyl-pyrrolopyrimidin-2-one-3-yl, bis-o-(aminoalkylhydroxy)-6-phenyl-pyrrolopyrimidin-2-one-3-yl, pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl, 2-oxo-pyridopyrimidin-3-yl or any O-alkylated or N-alkylated derivative thereof.

[0151] The methods for preparing the modified nucleobases described above are well known to those skilled in the art.

[0152] The term "nucleotide" as used herein refers to one or more monomeric subunits of a DNA molecule (or sequence) or an RNA molecule (or sequence), namely, a compound or nucleotide residue comprising a nucleobase moiety, a sugar moiety, and a phosphate group, as conventionally defined in the art. It should be understood herein that the term "nucleotide" may be used in the context of modified DNA molecules, RNA molecules, or nucleotide substitutes, and may also refer to nucleotides modified at one or more positions.

[0153] The terms "oligonucleotide," "oligonucleotide," and "oligonucleic acid" are used interchangeably to refer to a single-stranded or double-stranded nucleic acid molecule (which may be RNA or DNA) with a length of less than 100, 90, 80, or 70 nucleotides (or the longer strand when double-stranded) that binds to the target gene by pairing according to the Watson-Crick base pairing rule to inhibit or enhance the expression of the target gene. In some embodiments, the oligonucleotide molecule has a length of 8-60, 8-50, 10-45, 15-40, 15-35, 15-30, or 15-25 nucleotides. In some embodiments, the oligonucleotide is RNA. In some embodiments, the oligonucleotide is DNA. In some embodiments, the oligonucleotide is a hybrid molecule of RNA and DNA. In some embodiments, examples of oligonucleotides include, but are not limited to, one of: double-stranded RNA (dsRNA), small interfering RNA (siRNA), microRNA, single-stranded RNA (ssRNA), guide RNA, antisense oligonucleotide (ASO), antisense DNA, antisense RNA, nucleic acid aptamer, stem-loop RNA, mRNA fragments, and small activating RNA (saRNA). In some embodiments, the oligonucleotide is selected from one of siRNA, shRNA, miRNA, gRNA, antisense oligonucleotide (ASO), and small activating RNA. The terms “target gene” and “target nucleic acid” as used herein are interchangeable to refer to any nucleic acid molecule (DNA or RNA) whose expression or activity can be regulated (inhibited or enhanced) by the aforementioned oligonucleotide (such as siRNA). Target genes include, but are not limited to, DNA encoding a target protein, RNA transcribed from DNA encoding a target protein (including, but not limited to, mRNA precursors and mRNA or portions thereof), non-coding RNA, and cDNA derived from such RNA, and miRNA. For example, the target gene can be a cellular gene (or mRNA transcribed from a gene) whose expression is associated with a specific disorder or disease state. In some embodiments, the target gene can be a nucleic acid molecule derived from an infectious agent. In some embodiments, the target genes of the present invention are selected from the group consisting of: SOD1, APP, ATXN1, ATXN2, ATXN3, C9orf72, TARDBP, MAPT, HTT, LRRK2, SNCA, VEGFA, ANG-2, VEGFR1, VEGFR2, SCA1, SCA7, and SCA8 genes, etc.

[0154] As used in this article, "sod1" or "SOD1" refers to the soluble SOD1 enzyme (also known as Cu / Zn superoxide dismutase), one of the superoxide dismutases that defend against oxidative damage to biomolecules by catalyzing the dismutation of superoxide into hydrogen peroxide (H2O2). Superoxide anion (O2) is a potentially harmful cellular byproduct, primarily produced by errors in oxidative phosphorylation in mitochondria. Mutations in the SOD1 gene are associated with the dominant inheritance pattern of ALS, a disease characterized by selective degeneration of upper and lower motor neurons.

[0155] The term "VEGFA" used in this article refers to vascular endothelial growth factor a. VEGFA is secreted by various cells, including endothelial cells and tumor cells, and is an endothelial growth factor and a regulator of vascular permeability.

[0156] The “ANG-2” used in this article refers to angiopoietin 2. ANG-2 is angiopoietin 2, and angiopoietin is a class of cell regulatory factors that act on vascular endothelial cells and play an important role in the process of new angiogenesis.

[0157] The “SNCA” used in this article refers to the α-synuclein gene, which is one of the important pathogenic genes of hereditary Parkinson’s disease (PD). The abnormal aggregation of the α-synuclein encoded by the gene is closely related to the pathological mechanism of PD.

[0158] Unless otherwise stated, when describing the relationship between the first and second nucleotide sequences, the term "complementarity" as used herein refers to the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing the second nucleotide sequence under certain conditions and form a double helix structure, as will be understood by those skilled in the art. Such conditions may, for example, be stringent conditions, which may include 400 mM NaCl, 40 mM PIPES pH 6.4, and 1 mM EDTA at 50°C or 70°C for 12–16 hours, followed by washing. Other conditions may also be applied, such as physiologically relevant conditions that may be encountered in vivo. Those skilled in the art can determine the most suitable series of conditions for the two-sequence complementarity assay based on the final application of the hybridized nucleotides.

[0159] This includes base pairing of the oligonucleotide or polynucleotide containing the first nucleotide sequence with the oligonucleotide or polynucleotide containing the second nucleotide sequence across the full length of the first and second nucleotide sequences. These sequences are referred to herein as “perfectly complementary” to each other. However, when referred to herein as “substantially complementary” to the first and second sequences, the two sequences may be perfectly complementary or form one or more mismatched base pairs upon hybridization, but typically no more than four, three, or two mismatched base pairs, while retaining hybridization capability under conditions most relevant to their final application. However, when two oligonucleotides are designed to hybridize to form one or more single-stranded overhangs, such overhangs should not be considered mismatches in light of the definition of complementarity. For example, in a dsRNA containing one 21-nucleotide oligonucleotide and another 23-nucleotide oligonucleotide, the longer oligonucleotide containing a 21-nucleotide sequence perfectly complementary to the shorter oligonucleotide is also considered “perfectly complementary” for the purposes of this invention.

[0160] As used herein, the terms "double-stranded ribonucleic acid," "double-stranded RNA," or "dsRNA" refer to a complex of ribonucleic acid molecules having a double-stranded structure and comprising two antiparallel, substantially complementary nucleic acid strands as described above. The two strands forming the double-stranded structure can be different portions of the same larger RNA molecule or separate RNA molecules. If the two strands are separate RNA molecules, such dsRNAs are often referred to in the literature as siRNAs ("small interfering RNAs"). If the two strands are portions of a larger molecule and are linked by an uninterrupted nucleotide chain between the 3'-end of one strand and the 5'-end of the other forming the double-stranded structure, then the linked RNA strands are referred to as "hairpin loops," "short hairpin RNAs," or "shRNAs." If the two strands are covalently linked in a manner other than an uninterrupted chain between the 3'-end of one strand and the 5'-end of the other forming the double-stranded structure, then the linking structure is referred to as a "linker." The RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the shortest strand in the dsRNA minus the number of nucleotides remaining after removing any dangling ends present in the double strand. In addition to a double-stranded structure, dsRNA may contain one or more nucleotide overhangs. Furthermore, as used herein, "dsRNA" may include chemical modifications of ribonucleotides, including modifications of multiple nucleotides, and includes all types of modifications disclosed herein or known in the art. For the purposes of this specification and the claims, "dsRNA" covers all such modifications used for siRNA-type molecules.

[0161] As used in this article, a "nucleotide overhang" or "nucleotide dangling end" refers to one or more unpaired nucleotides protruding from the double-stranded structure of a dsRNA when the 3' end of one strand extends beyond the 5' end of the other strand, or vice versa. A "blunt end" or "offset" refers to a dsRNA without any unpaired nucleotides at its ends, i.e., without a nucleotide dangling end. A "blunt-terminated" dsRNA is a dsRNA that is double-stranded throughout its entire length, meaning there is no nucleotide dangling end at any of its ends. For clarity, when determining whether siRNA has an overhang or a blunt end, chemical caps or non-nucleotide chemical portions bound to the 3' or 5' ends of the siRNA are not considered.

[0162] The term "antisense strand" refers to a nucleic acid strand containing a region substantially complementary to the target sequence, which may exist alone or hybridize with another strand to form a double strand. The term "complementary region" as used herein refers to a region on the antisense strand substantially complementary to a sequence (e.g., the target sequence) as defined herein. If the complementary region is not perfectly complementary to the target sequence, the most permissible mismatch occurs in the terminal region, and if a mismatch exists, it is typically located in the terminal region, or, for example, within 6, 5, 4, 3, or 2 nucleotides at the 5′ and / or 3′ ends.

[0163] As used in this article, the term "sense strand" or "sense chain" refers to a nucleic acid strand containing a region substantially complementary to the antisense strand region, which may be located in the target sequence or may form a functional molecule (e.g., siRNA, shRNA) through hybridization complementarity with the antisense strand.

[0164] The "conventional chemical modification" of nucleotides described in this invention mainly includes conventional chemical modification of sugar rings and phosphate ester bonds to enhance stability. These chemical modifications include, but are not limited to, locked nucleic acid modification, ring-opening or non-locked nucleic acid modification, DNA modification (such as thymidine monophosphate), 5'-vinylphosphonate (5'-Pv) modification, 2'-modification of sugar rings, fluorescent probe modification, ligand modification, etc. Examples of 2'-modification of sugar rings include, but are not limited to: 2'-methoxyethyl, 2'-O-methyl (2'-OMe), 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluorine, 2'-ON-methylacetamido (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), etc. Examples of conventional chemical modification of phosphate ester bonds include, but are not limited to, replacing the internucleotide phosphate ester bond with other linkages such as phosphothioester bonds. More than one such modification can be used, meaning that the nucleotide can contain both sugar ring and phosphate ester bond modifications at the same time.

[0165] The monomers of the oligonucleotides of the present invention (including the modified monomers of the present invention) are linked by phosphate esters or modified phosphate esters, which can be represented by the following formula:

[0166] Y1 and Y2 independently represent =O, =S, -O-, and -OH(-O) respectively. - -SH(-S) - -BH3, -OCH2CO2Rx, -OCH2C(S)ORx, and -ORx, where Rx represents a C1-C6 alkyl group and the wavy line indicates the connection to the remaining portion. One, each, or both unconnected oxygen atoms in the modified phosphate group can independently be any one of S, Se, B, C, H, N, or OR (R is an alkyl or aryl group). Preferably, the modified phosphate group is a thiophosphate group. Thiophosphate groups include dithiophosphates (i.e., where Y1 represents =S and Y2 represents -S). - ) and monothiophosphates (i.e., where Y1 represents -O) - And Y2 represents =S, or where Y1 represents =0 and Y2 represents -S. - More preferably, the modified phosphate group is a monothiophosphate (unless otherwise specified, thiophosphate or thiophosphate diester in this invention refers to monothiophosphate).

[0167] As those skilled in the art will understand, the term "introduced into cells (or introduced into cells)" in the context of dsRNA refers to facilitating uptake or absorption into cells. Absorption or uptake of dsRNA can occur through non-assisted diffusion or active cellular processes, or through the aid of reagents or devices. This term is not limited to in vitro cells; dsRNA can also be "introduced into cells (or introduced into cells)" where the cells are part of a living organism. In this case, introduction into cells will include delivery to the organism. For example, in in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. Introduction into in vitro cells includes methods known in the art, such as electroporation and lipofection.

[0168] Unless otherwise specified, the terms "pharmaceutically acceptable salt" or "medicinal salt" refer to a salt that, within the bounds of reasonable medical judgment, is suitable for contact with the tissues of mammals, particularly humans, without excessive toxicity, irritation, allergic reactions, etc., and is proportionate to a reasonable benefit / risk ratio. Medically acceptable salts of amines, carboxylic acids, and other types of compounds are well known in the art. The salts can be prepared in situ during the final separation and purification of the compounds of this invention, or solely by reacting a free base or free acid with a suitable reagent.

[0169] Unless otherwise specified, the term "isotope derivative" refers to compounds of the present invention that can exist in an isotopically traced or enriched form, containing one or more atoms whose atomic weights or mass numbers differ from the atomic weights or mass numbers of the most abundant atoms found in nature. Isotopes can be radioactive or non-radioactive. Commonly used isotopes for isotopic labeling are: hydrogen isotopes, 2 H and 3 H; Carbon isotopes: 13 C and 14 C; Chlorine isotopes: 35 Cl and 37 Cl; Fluorine isotopes: 18 F; Iodine isotopes: 123 I and 125 I; Nitrogen isotopes: 13 N and 15 N; oxygen isotopes: 15 O、 17 O and 18 O and sulfur isotopes 35 S. These isotope-labeled compounds can be used to study the distribution of pharmaceutical molecules in tissues. Especially 2 H and 13 C, because they are easy to label and convenient to detect, are more widely used. Some heavy isotopes, such as deuterium (… 2 Substitution with H can enhance metabolic stability and prolong the half-life, thereby achieving the goal of reducing dosage and providing therapeutic advantages. Isotope-labeled compounds are generally synthesized from labeled starting materials using known synthetic techniques, just like non-isotope-labeled compounds.

[0170] Unless otherwise specified, the terms "solvent" or "solvent compound" refer to the physical association of the compound of the present invention with one or more solvent molecules (organic or inorganic). This physical association includes hydrogen bonding. In some cases, such as when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate can be separated. The solvent molecules in the solvate may be present in a regular and / or disordered arrangement. The solvate may contain stoichiometric or non-stoichiometric solvent molecules. "Solvent compound" encompasses both solution phases and separable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanol compounds, methanol compounds, and isopropanol compounds. Solvation methods are well known in the art.

[0171] Unless otherwise specified, the term "stereoisomer" refers to compounds having the same chemical structure but with different spatial arrangements of atoms or groups. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometric isomers (cis / trans) isomers, and inhibited isomers. Any mixture of stereoisomers obtained can be separated into pure or substantially pure geometric isomers, enantiomers, and diastereomers based on differences in the physicochemical properties of the components, for example, by chromatography and / or fractional crystallization.

[0172] Unless otherwise specified, the term "tautomer" refers to structural isomers with different energies that can interconvert through a low energy barrier. If tautomerism is possible (e.g., in solution), chemical equilibrium can be achieved in the tautomer. For example, proton tautomers (also called proton transfer tautomers) involve interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers involve interconversions via the rearrangement of some bonding electrons.

[0173] Unless otherwise indicated, the structural formulas described in this invention include all isomers (e.g., enantiomers, diastereomers, and geometric isomers (or conformational isomers)): for example, R and S configurations containing an asymmetric center, (Z) and (E) isomers of double bonds, and (Z) and (E) conformational isomers. Therefore, any single stereochemical isomer of the compounds of this invention, or its enantiomers, diastereomers, or mixtures of geometric isomers (or conformational isomers), is within the scope of this invention.

[0174] Unless otherwise specified, the term "prodrug" refers to a drug that is converted into a parent drug in vivo. Prodrugs are generally useful because they can improve certain, undesirable physical or biological properties. Physical properties are generally related to solubility (excessive or insufficient lipid or water solubility) or stability, while problematic biological properties include metabolism that is too rapid or poor bioavailability, which may itself be related to physicochemical properties. For example, they can be bioavailable orally, whereas the parent drug cannot. Prodrugs also have improved solubility in pharmaceutical compositions compared to the parent drug. An example of a prodrug, but not limited thereto, can be any compound of the present invention administered as an ester ("prodrug") to facilitate transmembrane transport, where water solubility is detrimental to migration but beneficial once inside the cell, and which is subsequently metabolized and hydrolyzed into a carboxylic acid, the active entity. Another example of a prodrug can be a short peptide (polyamino acid) bound to an acid group, where the peptide is metabolized to exhibit the active moiety.

[0175] As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of oligonucleotides and a pharmaceutically acceptable excipient. The terms “pharmacologically effective amount,” “therapeuticly effective amount,” or “effective amount” as used herein refer to the amount of oligonucleotides that effectively produce the desired pharmacological, therapeutic, or preventative outcome. For example, if a given clinical treatment is considered effective if it reduces a measurable parameter associated with a disease or lesion by at least 25%, then the therapeutically effective amount of a drug used to treat said disease or lesion is the amount necessary to reduce said parameter by at least 25%. The term “pharmaceutically acceptable carrier” refers to a carrier used to administer the therapeutic agent. Such carriers include, but are not limited to, saline solutions, buffered saline solutions, glucose, water, glycerol, ethanol, and combinations thereof.

[0176] The term "silencing" refers to at least partially suppressing the expression of a target gene.

[0177] Unless otherwise specified, the "nucleoside monomer" of the present invention can be not only its specific compound, but also its various tautomers, stereoisomers, solvates, isotope derivatives or pharmaceutically acceptable salts thereof; the "oligonucleotide" of the present invention can be not only its specific compound, but also its various prodrugs, tautomers, stereoisomers, solvates, isotope derivatives or pharmaceutically acceptable salts thereof. Attached Figure Description

[0178] Figures 1a-1d: Efficacy assays of SOD1 candidate and control modified sequences at mRNA levels in different tissues of SD rats. Figure 1a shows the brain region.

[0179] Figure 1b shows the cervical spine region, Figure 1c shows the lumbar spine region, and Figure 1d shows the thoracic spine region.

[0180] Figure 2: Efficacy test of VEGFA candidate and control modified sequences at the level of mRNA in mouse retina.

[0181] Figure 3: Efficacy test of ANG-2 candidate and control modified sequences at the level of mRNA in mouse retina.

[0182] Figures 4a-4b: Leakage area statistics and leakage score statistics in the disease model using VEGFA / ANG-2 siRNA combination. Figure 4a shows the leakage area statistics.

[0183] Figure 4b shows the leakage score statistics.

[0184] Figure 5: Expression of the SNCA gene in cells after in vitro transfection of candidate SNCA siRNA molecular sequences.

[0185] Figures 6a-6b: Relative expression levels of SNCA gene mRNA after IT administration. Figure 6a shows the brain tissue region, and Figure 6b shows the spinal region.

[0186] Figure 7: Relative expression levels of ANG-2 gene mRNA after IVT administration.

[0187] Figure 8: mRNA expression levels of the SOD-1 gene in different tissues. Detailed implementation method:

[0188]

[0189] “G”, ​​“C”, “A”, “T” and “U” usually represent nucleotides with guanine, cytosine, adenine, thymine and uracil as bases, respectively.

[0190] REL (Relative expression level): relative expression level of mRNA.

[0191] Modifications: N = RNA nucleotide; dN = DNA nucleotide; Nm = 2'OMe-modified RNA nucleotide; Nf = 2'F-modified RNA nucleotide; (s) = PS backbone (i.e., thio-modified phosphate backbone); Vp = 5'-ethylenephosphonate. N(hd) = 2'-O-hexadecyl-modified RNA nucleotide. N(hdt) = 2'-S-hexadecyl-modified RNA nucleotide. N(dat) = 2'-S-dodecyl-modified RNA nucleotide.

[0192] siRNA and double-stranded RNAi agents have similar meanings and can be used interchangeably in the embodiments of this invention.

[0193] IT injection: Intrathecal administration. IVT injection: Intravitreal administration. ICV: Intraventricular injection.

[0194] aCSF: Artificial cerebrospinal fluid.

[0195] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of the present invention. The preferred embodiments and materials shown herein are for illustrative purposes only.

[0196] Example 1: Synthesis of Modified Nucleoside Monomer Compounds

[0197] 1.1 Preparation of compounds 2-4

[0198] Synthesis of compound 2:

[0199] 4.52 g of compound 1, 25.83 g of 1-hexadecylthiol, 11.51 g of tetramethylguanidine, and 100 mL of N,N-dimethylformamide were added to a 250 mL single-necked flask, and the mixture was stirred at 100 °C for 24 hours. After the reaction was complete, the reaction solution was extracted with ethyl acetate and water. The upper organic phase was washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 6.20 g of compound 2, in 62% yield. LCMS(ESI)C 25 H 44 N₂O₅S[M+H] + The theoretical value of m / z is 485.30, and the measured value is 485.3.

[0200] Synthesis of compound 3:

[0201] 5.90 g of compound 2 and 60 mL of pyridine were added to a 100 mL single-necked flask. After stirring at room temperature for 10 minutes, 4.50 g of 4,4'-dimethoxytriphenylmethyl chloride was added, and stirring was continued at room temperature for 2 hours. The reaction was quenched with 5 mL of ethanol. The reaction solution was concentrated under vacuum, extracted with dichloromethane and water, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 5.70 g of compound 3, in 59% yield. LCMS(ESI)C 46 H 62 N₂O₇S[M+H] + The theoretical value of m / z is 787.44, and the measured value is 787.4.

[0202] Synthesis of compound 4:

[0203] 5.70 g of compound 3, 2.62 g of bis(diisopropylamino)(2-cyanoethoxy)phosphine, and 60 mL of dichloromethane were added to a 100 mL three-necked flask. The mixture was stirred at room temperature for 10 minutes under nitrogen protection, followed by the addition of 0.40 g of tetrazolium. Stirring continued for 6 hours at room temperature. After the reaction was complete, the reaction solution was washed successively with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 4.0 g of compound 4, in 55% yield. LCMS(ESI)C 55 H 79 N4O8PS[M+H] + The theoretical calculated value of m / z is 987.54, and the measured value is 987.6.

[0204] 1.2 Preparation of compounds 6-9

[0205] Synthesis of compound 6:

[0206] 5.0 g of compound 5, 28.7 g of 1-hexadecylthiol, 12.8 g of tetramethylguanidine, and 100 mL of N,N-dimethylformamide were added to a 250 mL single-necked flask, and the mixture was stirred at 100 °C for 24 hours. After the reaction was complete, the reaction solution was extracted with ethyl acetate and water. The upper organic phase was washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 10.0 g of compound 6, in 86% yield. LCMS(ESI)C 25 H 45 N3O4S[M+H] + The theoretical value of m / z is 484.32, and the measured value is 484.3.

[0207] Synthesis of compound 7:

[0208] 9.0 g of compound 6, 18.8 g of triethylamine, 0.23 g of 4-dimethylaminopyridine, and 100 mL of dichloromethane were added to a 250 mL three-necked flask. The mixture was stirred at 0 °C for 10 minutes, followed by the addition of 8.09 g of trimethylchlorosilane. Stirring continued at room temperature for 2 hours. The reaction mixture was cooled to 0 °C, and 3.14 g of benzoyl chloride was added dropwise. Stirring continued overnight at room temperature. After the reaction was complete, the reaction mixture was washed and extracted successively with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 7.30 g of compound 7, in 67% yield. LCMS(ESI)C 32 H 49 N3O5S[M+H] + The theoretical value of m / z is 588.35, and the measured value is 588.4.

[0209] Synthesis of compound 8:

[0210] 6.0 g of compound 7 and 60 mL of pyridine were added to a 100 mL single-necked flask. After stirring at room temperature for 10 minutes, 3.8 g of 4,4'-bismethoxytriphenylmethyl chloride was added, and stirring was continued at room temperature for 2 hours. The reaction was quenched with 4 mL of ethanol. The reaction solution was concentrated under vacuum, extracted with dichloromethane and water, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 5.50 g of compound 8, in 61% yield. LCMS(ESI)C 53 H 67 N3O7S[M+H] + The theoretical calculated value of m / z is 890.48, and the measured value is 890.5.

[0211] Synthesis of compound 9:

[0212] 5.0 g of compound 8, 2.0 g of bis(diisopropylamino)(2-cyanoethoxy)phosphine, and 50 mL of dichloromethane were added to a 100 mL three-necked flask. The mixture was stirred at room temperature for 10 minutes under nitrogen protection, followed by the addition of 0.35 g of tetrazolium. Stirring continued for 8 hours at room temperature. After the reaction was complete, the reaction solution was washed successively with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 3.0 g of compound 9, in 49% yield. LCMS(ESI)C 62 H 84 N5O8PS[M+H] + The theoretical value of m / z is 1090.59, and the measured value is 1090.6.

[0213] 1.3 Preparation of compounds 13-17

[0214] Synthesis of compound 12:

[0215] According to the literature (J.Org.Chem.2021,86,4944-4956), 21.6 g of compound 12 was synthesized from 15.0 g of compound 10 through two steps, with a two-step yield of 60%.

[0216] Synthesis of compound 13:

[0217] 20.0 g of compound 12, 5.69 g of potassium thioacetate, and 100 mL of N,N-dimethylformamide were added to a 250 mL single-necked flask and stirred overnight at room temperature. After the reaction was complete, the reaction solution was concentrated under vacuum, and the residue was extracted with ethyl acetate and a 5% aqueous solution of sodium bicarbonate. The upper organic phase was washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum to obtain the crude product.

[0218] The crude product and 200 mL of ammonia-methanol solution (7.0 M) were added to a 500 mL single-necked flask and stirred at 0 °C for 4 hours. After the reaction was complete, the reaction solution was concentrated under vacuum, and 14.27 g of 1-bromohexadecane, 6.04 g of diisopropylethylamine, and 150 mL of acetonitrile were added. The mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction solution was concentrated under vacuum, and the residue was extracted with dichloromethane and sodium bicarbonate aqueous solution (5%). The lower organic phase was washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 9.35 g of compound 13, with a three-step yield of 40%. LCMS(ESI)C 38 H 71 N5O4SSi2[M+H] +The theoretical calculated value of m / z is 750.49, and the measured value is 750.5.

[0219] Synthesis of compound 14:

[0220] 9.2 g of compound 13, 12.4 g of triethylamine, 0.15 g of 4-dimethylaminopyridine, and 70 mL of dichloromethane were added to a 250 mL three-necked flask. The mixture was stirred at 0 °C for 10 minutes, then 5.33 g of trimethylchlorosilane was added, and stirring continued for 2 hours at room temperature. The reaction mixture was cooled to 0 °C, and 2.07 g of benzoyl chloride was added dropwise to the reaction mixture. The mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was washed and extracted successively with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 7.50 g of compound 14, in 72% yield. LCMS(ESI)C 45 H 75 N5O5SSi2[M+H] + The theoretical calculated value of m / z is 854.51, and the measured value is 854.5.

[0221] Synthesis of compound 15:

[0222] 7.2 g of compound 14 and 25 mL of tetrabutylammonium fluoride-tetrahydrofuran solution (1 M) were added to a 50 mL three-necked flask and stirred at room temperature for 2 hours. After the reaction was complete, ethyl acetate was added, and the organic phase was extracted by washing with water and saturated brine successively. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 4.80 g of compound 15, in 93% yield. LCMS(ESI)C 33 H 49 N5O4S[M+H] + The theoretical value of m / z is 612.36, and the measured value is 612.4.

[0223] Synthesis of compound 16:

[0224] 4.5 g of compound 15 and 50 mL of pyridine were added to a 100 mL single-necked flask. After stirring at room temperature for 10 minutes, 2.74 g of 4,4'-dimethoxytriphenylmethyl chloride was added, and stirring was continued at room temperature for 2 hours. The reaction was quenched with 3 mL of ethanol. The reaction solution was concentrated under vacuum, extracted with dichloromethane and water, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 4.6 g of compound 16, in 68% yield. LCMS(ESI)C 54 H 67 N5O6S[M+H] + The theoretical value of m / z is 914.49, and the measured value is 914.5.

[0225] Synthesis of compound 17:

[0226] 4.2 g of compound 16, 1.66 g of bis(diisopropylamino)(2-cyanoethoxy)phosphine, and 50 mL of dichloromethane were added to a 100 mL three-necked flask. The mixture was stirred at room temperature for 10 minutes under nitrogen protection, followed by the addition of 0.26 g of tetrazolium. Stirring continued for 8 hours at room temperature. After the reaction was complete, the reaction solution was washed successively with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 2.8 g of compound 17, in 55% yield. LCMS(ESI)C 63 H 84 N7O7PS[M+H] + The theoretical value of m / z is 1114.60, and the measured value is 1114.6.

[0227] 1.4 Preparation of compounds 21-25

[0228] Synthesis of compound 20:

[0229] According to the literature (J.Am.Chem.Soc.2014,136,10609-10614), 23.2 g of compound 20 was synthesized from 20.0 g of compound 18 through two steps, with a two-step yield of 50%.

[0230] Synthesis of compound 21:

[0231] 22.0 g of compound 20, 6.11 g of potassium thioacetate, and 110 mL of N,N-dimethylformamide were added to a 250 mL single-necked flask and stirred overnight at room temperature. After the reaction was complete, the reaction solution was concentrated under vacuum, and the residue was extracted with ethyl acetate and a 5% aqueous solution of sodium bicarbonate. The upper organic phase was washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum to obtain the crude product.

[0232] The crude product and 250 mL of ammonia-methanol solution (7.0 M) were added to a 500 mL single-necked flask and stirred at 0 °C for 4.5 h. After the reaction was complete, the reaction solution was concentrated under vacuum, and 15.32 g of 1-bromohexadecane, 6.48 g of diisopropylethylamine, and 175 mL of acetonitrile were added. The mixture was stirred at room temperature for 16 h. After the reaction was complete, the reaction solution was concentrated under vacuum, and the residue was extracted with dichloromethane and sodium bicarbonate aqueous solution (5%). The lower organic phase was washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 13.9 g of compound 21, with a three-step yield of 54%. LCMS(ESI)C 38 H 71 N5O5SSi2[M+H] + The theoretical calculated value of m / z is 766.48, and the measured value is 766.5.

[0233] Synthesis of compound 22:

[0234] 13.0 g of compound 21, 5.05 g of N,N-dimethylformamide methyl acetal, and 100 mL of N,N-dimethylformamide were added to a 250 mL single-necked flask. The mixture was stirred overnight at room temperature under nitrogen protection. After the reaction was complete, the reaction solution was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 11.3 g of compound 22, with a three-step yield of 81%. LCMS(ESI)C 41 H 76 N6O5SSi2[M+H] + The theoretical value of m / z is 821.52, and the measured value is 821.5.

[0235] Synthesis of compound 23:

[0236] 11.0 g of compound 22 and 50 mL of tetrabutylammonium fluoride (1 M) / acetic acid (0.5 M)-tetrahydrofuran solution were added to a 100 mL three-necked flask, and the mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was concentrated under vacuum. The residue was dissolved in dichloromethane, concentrated under vacuum, and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 7.0 g of compound 23, in 90% yield. LCMS(ESI)C 29 H 50 N6O4S[M+H] + The theoretical value of m / z is 579.37, and the measured value is 579.4.

[0237] Synthesis of compound 24:

[0238] 6.5 g of compound 23 and 70 mL of pyridine were added to a 100 mL single-necked flask. After stirring at room temperature for 10 minutes, 4.19 g of 4,4'-dimethoxytriphenylmethyl chloride was added, and stirring was continued at room temperature for 2 hours. The reaction was quenched with 5 mL of ethanol. The reaction solution was concentrated under vacuum, extracted with dichloromethane and water, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 7.03 g of compound 24, in 71% yield. LCMS(ESI)C 50 H 68 N6O6S[M+H] + The theoretical calculated value of m / z is 881.50, and the measured value is 881.5.

[0239] Synthesis of compound 25:

[0240] 6.0 g of compound 24, 2.46 g of bis(diisopropylamino)(2-cyanoethoxy)phosphine, and 60 mL of dichloromethane were added to a 100 mL three-necked flask. The mixture was stirred at room temperature for 10 minutes under nitrogen protection, followed by the addition of 0.38 g of tetrazolium. Stirring continued for 8 hours at room temperature. After the reaction was complete, the reaction solution was washed successively with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 4.3 g of compound 25, in 58% yield. LCMS(ESI)C 59 H 85 N8O7PS[M+H] + The theoretical value of m / z is 1081.61, and the measured value is 1081.6.

[0241] 1.5 Preparation of compounds 26-28

[0242] Synthesis of compound 26:

[0243] 7.5 g of compound 1, 13.62 g of 1-docoethanethiol, 9.54 g of tetramethylguanidine, and 150 mL of N,N-dimethylformamide were added to a 250 mL single-necked flask, and the mixture was stirred at 100 °C for 24 hours. After the reaction was complete, the reaction solution was extracted with ethyl acetate and water. The upper organic phase was washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 12.05 g of compound 26, in 64% yield. LCMS(ESI)C 31 H 56 N₂O₅S[M+H] + The theoretical calculated value of m / z is 568.39, and the measured value is 568.8.

[0244] Synthesis of compound 27:

[0245] 11.5 g of compound 26 and 115 mL of pyridine were added to a 250 mL single-necked flask. After stirring at room temperature for 10 minutes, 7.19 g of 4,4'-dimethoxytriphenylmethyl chloride was added, and stirring was continued at room temperature for 2 hours. The reaction was quenched with 5 mL of ethanol. The reaction solution was concentrated under vacuum, extracted with dichloromethane and water, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 11.0 g of compound 27, in 62% yield. LCMS(ESI)C 52 H 74 N₂O₇S[M+H] + The theoretical value of m / z is 870.52, and the measured value is 871.2.

[0246] Synthesis of compound 28:

[0247] 10.8 g of compound 27, 4.48 g of bis(diisopropylamino)(2-cyanoethoxy)phosphine, and 108 mL of dichloromethane were added to a 250 mL three-necked flask. The mixture was stirred at room temperature for 10 minutes under nitrogen protection, followed by the addition of 0.78 g of tetrazolium. Stirring was continued at room temperature for 4 hours. After the reaction was complete, the reaction solution was washed successively with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 8.0 g of compound 28, in 60% yield. LCMS(ESI)C 61 H 91 N4O8PS[M+H] + The theoretical value of m / z is 1070.63, and the measured value is 1070.9.

[0248] 1.6 Preparation of compounds 29-32

[0249] Synthesis of compound 29:

[0250] 6.0 g of compound 5, 10.90 g of 1-docoethanethiol, 7.93 g of tetramethylguanidine, and 120 mL of N,N-dimethylformamide were added to a 250 mL single-necked flask, and the mixture was stirred at 100 °C for 24 hours. After the reaction was complete, the reaction solution was extracted with ethyl acetate and water. The upper organic phase was washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 10.5 g of compound 29, in 86% yield. LCMS(ESI)C 31 H 57 N3O4S[M+H] + The theoretical value of m / z is 567.41, and the measured value is 567.8.

[0251] Synthesis of compound 30:

[0252] 10.0 g of compound 29, 17.82 g of triethylamine, 0.21 g of 4-dimethylaminopyridine, and 100 mL of dichloromethane were added to a 250 mL three-necked flask. The mixture was stirred at 0 °C for 10 minutes, then 7.65 g of trimethylchlorosilane was added, and stirring continued for 2 hours at room temperature. The reaction mixture was cooled to 0 °C, and 2.97 g of benzoyl chloride was added dropwise. The mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was washed and extracted successively with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 6.5 g of compound 30, in 55% yield. LCMS(ESI)C 38 H 61 N3O5S[M+H] + The theoretical value of m / z is 671.43, and the measured value is 972.0.

[0253] Synthesis of compound 31:

[0254] 6.0 g of compound 30 and 60 mL of pyridine were added to a 100 mL single-necked flask. After stirring at room temperature for 10 minutes, 3.33 g of 4,4'-dimethoxytriphenylmethyl chloride was added, and stirring was continued at room temperature for 2 hours. The reaction was quenched with 2 mL of ethanol. The reaction solution was concentrated under vacuum, extracted with dichloromethane and water, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 6.51 g of compound 31, in 75% yield. LCMS(ESI)C 59 H 79 N3O7S[M+H] + The theoretical value of m / z is 973.56, and the measured value is 974.4.

[0255] Synthesis of compound 32:

[0256] 6.0 g of compound 31, 2.23 g of bis(diisopropylamino)(2-cyanoethoxy)phosphine, and 50 mL of dichloromethane were added to a 100 mL three-necked flask. The mixture was stirred at room temperature for 10 minutes under nitrogen protection, followed by the addition of 0.35 g of tetrazolium. Stirring continued for 20 hours at room temperature. After the reaction was complete, the reaction solution was washed successively with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 4.2 g of compound 32, in 58% yield. LCMS(ESI)C 68 H 96 N5O8PS[M+H] + The theoretical value of m / z is 1173.67, and the measured value is 1174.5.

[0257] 1.7 Preparation of compounds 33-37

[0258] Synthesis of compound 12:

[0259] According to the literature (J.Org.Chem.2021,86,4944-4956), 21.6 g of compound 12 was synthesized from 15.0 g of compound 10 through two steps, with a two-step yield of 60%.

[0260] Synthesis of compound 33:

[0261] 20.0 g of compound 12, 5.69 g of potassium thioacetate, and 100 mL of N,N-dimethylformamide were added to a 250 mL single-necked flask and stirred overnight at room temperature. After the reaction was complete, the reaction solution was concentrated under vacuum, and the residue was extracted with ethyl acetate and a 5% aqueous solution of sodium bicarbonate. The upper organic phase was washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum to obtain the crude product.

[0262] The crude product and 200 mL of ammonia-methanol solution (7.0 M) were added to a 500 mL single-necked flask and stirred at 0 °C for 4 hours. After the reaction was complete, the reaction solution was concentrated under vacuum, and 18.21 g of 1-bromodocosahexadecane, 6.04 g of diisopropylethylamine, and 150 mL of acetonitrile were added. The mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction solution was concentrated under vacuum, and the residue was extracted with dichloromethane and sodium bicarbonate aqueous solution (5%). The lower organic phase was washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 10.43 g of compound 33, with a three-step yield of 40%. LCMS(ESI)C 44 H 83 N5O4SSi2[M+H] + The theoretical value of m / z is 833.57, and the measured value is 834.2.

[0263] Synthesis of compound 34:

[0264] 9.6 g of compound 33, 11.64 g of triethylamine, 0.14 g of 4-dimethylaminopyridine, and 96 mL of dichloromethane were added to a 250 mL three-necked flask. The mixture was stirred at 0 °C for 10 minutes, then 5.0 g of trimethylchlorosilane was added, and stirring continued for 2 hours at room temperature. The reaction mixture was cooled to 0 °C, and 1.94 g of benzoyl chloride was added dropwise to the reaction mixture. The mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was washed and extracted successively with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 6.82 g of compound 34, in 63% yield. LCMS(ESI)C 51 H 87 N5O5SSi2[M+H] + The theoretical value of m / z is 937.60, and the measured value is 938.2.

[0265] Synthesis of compound 35:

[0266] 6.0 g of compound 34 and 25 mL of tetrabutylammonium fluoride-tetrahydrofuran solution (1 M) were added to a 50 mL three-necked flask and stirred at room temperature for 2 hours. After the reaction was complete, ethyl acetate was added, and the organic phase was extracted by washing with water and saturated brine successively. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 4.0 g of compound 35, in 90% yield. LCMS(ESI)C 39 H 61 N5O4S[M+H] + The theoretical value of m / z is 695.44, and the measured value is 696.0.

[0267] Synthesis of compound 36:

[0268] 3.5 g of compound 35 and 35 mL of pyridine were added to a 100 mL single-necked flask. After stirring at room temperature for 10 minutes, 1.87 g of 4,4'-dimethoxytriphenylmethyl chloride was added, and stirring was continued at room temperature for 2 hours. The reaction was quenched with 2 mL of ethanol. The reaction solution was concentrated under vacuum, extracted with dichloromethane and water, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 4.3 g of compound 36, in 86% yield. LCMS(ESI)C 54 H 67 N5O6S[M+H] + The theoretical value of m / z is 997.58, and the measured value is 998.4.

[0269] Synthesis of compound 37:

[0270] 4.2 g of compound 36, 1.52 g of bis(diisopropylamino)(2-cyanoethoxy)phosphine, and 42 mL of dichloromethane were added to a 100 mL three-necked flask. The mixture was stirred at room temperature for 10 minutes under nitrogen protection, followed by the addition of 0.24 g of tetrazolium. Stirring continued for 8 hours at room temperature. After the reaction was complete, the reaction solution was washed successively with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 3.1 g of compound 37, in 62% yield. LCMS(ESI)C 69 H 96 N7O7PS[M+H] + The theoretical value of m / z is 1197.68, and the measured value is 1198.6.

[0271] 1.8 Preparation of compounds 38-42

[0272] Synthesis of compound 20:

[0273] According to the literature (J.Am.Chem.Soc.2014,136,10609-10614), 23.2 g of compound 20 was synthesized from 20.0 g of compound 18 through two steps, with a two-step yield of 50%.

[0274] Synthesis of compound 38:

[0275] 22.0 g of compound 20, 6.11 g of potassium thioacetate, and 110 mL of N,N-dimethylformamide were added to a 250 mL single-necked flask and stirred overnight at room temperature. After the reaction was complete, the reaction solution was concentrated under vacuum, and the residue was extracted with ethyl acetate and a 5% aqueous solution of sodium bicarbonate. The upper organic phase was washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum to obtain the crude product.

[0276] The crude product and 250 mL of ammonia-methanol solution (7.0 M) were added to a 500 mL single-necked flask and stirred at 0 °C for 4.5 h. After the reaction was complete, the reaction solution was concentrated under vacuum, and 19.54 g of 1-bromodocosahexadecane, 12.97 g of diisopropylethylamine, and 175 mL of acetonitrile were added. The mixture was stirred at room temperature for 16 h. After the reaction was complete, the reaction solution was concentrated under vacuum, and the residue was extracted with dichloromethane and sodium bicarbonate aqueous solution (5%). The lower organic phase was washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 15.2 g of compound 38, with a three-step yield of 53%. LCMS(ESI)C 44 H 83 N5O5SSi2[M+H] +The theoretical calculated value of m / z is 849.57, and the measured value is 850.4.

[0277] Synthesis of compound 39:

[0278] 14.0 g of compound 38, 5.89 g of N,N-dimethylformamide methyl acetal, and 100 mL of N,N-dimethylformamide were added to a 250 mL single-necked flask. The mixture was stirred overnight at room temperature under nitrogen protection. After the reaction was complete, the reaction solution was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 12.1 g of compound 39, with a three-step yield of 81%. LCMS(ESI)C 47 H 88 N6O5SSi2[M+H] + The theoretical value of m / z is 904.61, and the measured value is 905.5.

[0279] Synthesis of compound 40:

[0280] 12.0 g of compound 39 and 60 mL of tetrabutylammonium fluoride (1 M) / acetic acid (0.5 M)-tetrahydrofuran solution were added to a 100 mL three-necked flask, and the mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was concentrated under vacuum. The residue was dissolved in dichloromethane, concentrated under vacuum, and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 7.2 g of compound 40, in 82% yield. LCMS(ESI)C 35 H 62 N6O4S[M+H] + The theoretical value of m / z is 662.46, and the measured value is 663.0.

[0281] Synthesis of compound 41:

[0282] 7.0 g of compound 40 and 70 mL of pyridine were added to a 100 mL single-necked flask. After stirring at room temperature for 10 minutes, 3.94 g of 4,4'-bismethoxytriphenylmethyl chloride was added, and stirring was continued at room temperature for 2 hours. The reaction was quenched with 2 mL of ethanol. The reaction solution was concentrated under vacuum, extracted with dichloromethane and water, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 7.23 g of compound 41, in 71% yield. LCMS(ESI)C 56 H 80 N6O6S[M+H] + The theoretical value of m / z is 964.59, and the measured value is 965.4.

[0283] Synthesis of compound 42:

[0284] 7.0 g of compound 41, 2.62 g of bis(diisopropylamino)(2-cyanoethoxy)phosphine, and 70 mL of dichloromethane were added to a 100 mL three-necked flask. The mixture was stirred at room temperature for 10 minutes under nitrogen protection, followed by the addition of 0.41 g of tetrazolium. Stirring was continued at room temperature for 20 hours. After the reaction was complete, the reaction solution was washed successively with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum and purified by silica gel column chromatography (n-heptane / ethyl acetate) to give 4.5 g of compound 42, in 53% yield. LCMS(ESI)C 65 H 97 N8O7PS[M+H] + The theoretical value of m / z is 1064.69, and the measured value is 1165.5.

[0285] Following the above synthetic route and procedures, and using conventional raw materials and synthetic methods in this field, it is possible to prepare compounds with other bases and other hydrocarbon groups at the 2'-S position (such as C...). 14 -C 22 Nucleoside monomers with alkyl groups, 3' / 5' protecting groups on the sugar ring, etc. Table a shows representative modified nucleoside monomers prepared and used in this invention.

[0286] Table a: Representative modified nucleoside monomers prepared and used in this invention

[0287] Note: The monomers of this invention need to carry relevant protecting groups before synthesizing dsRNA. The protecting groups are removed during the synthesis of dsRNA, resulting in the specific structure in the oligonucleotide.

[0288] Example 2: In vivo drug efficacy detection - mRNA detection

[0289] I. siRNA Design

[0290] Based on the human SOD1, VEGFA, ANG-2, and SNCA mRNA sequences, SOD1, VEGFA, ANG-2, and SNCA siRNAs were designed at different sites. All designed individual siRNAs can target all transcripts of the target genes (as shown in Table 1). The above sequences (as shown in Table 2) showed the lowest homology with all other non-target gene sequences after sequence similarity comparison with sequence similarity software.

[0291] Table 1 Target Genes

[0292] Table 2 Design Sequence

[0293] II. siRNA Synthesis

[0294] The siRNA used in this embodiment of the invention comprises ribonucleotides and ribonucleotides modified with 2'-methoxy or 2'-fluoro, 2'-O-hexadecyl and / or 2'-S-hexadecyl, 2'-O-docodialkyl and / or 2'-S-docodialkyl. The siRNA was synthesized to a theoretical yield of 1 μmol using the commonly used 1 μmol Frit vector. All oligonucleotides were prepared using an LK-192X synthesizer. All phosphoramide monomers (including conventional phosphoramide monomers and those of this invention) were diluted 1:30 (g / mL) with anhydrous acetonitrile and coupled twice for 3 min. Deprotection was performed using 3% DCA, followed by activation with 0.6 M benzylthiotetrazole acetonitrile solution, and capping and oxidation were performed using CAPA / CAPB and 50 mM I2 solutions, respectively. After the trityl-off synthesis, the solid support was transferred to a 2 mL centrifuge tube, 1.2 mL of ammonia was added, and the tube was heated at 36 °C for 16 h to remove the protecting group. The solution was then cooled to room temperature and concentrated under vacuum for 30 min. It was filtered through a 0.22 μm filter into a sample vial and purified using a semi-preparative reversed-phase purification system for single-strand purification. The elution gradient was 7%–30% (ACN: 100 mM TEAA) for 12 min at a flow rate of 5 mL / min. After purification, the solution was concentrated under vacuum and evaporated to dryness at room temperature. The sample was then dissolved in water, and each solution was desalted on a GE Hi-Trap desalting column to elute the final oligonucleotide product. All characteristics and purities were confirmed using ESI-MS and IEX HPLC. The concentration was determined using a microplate reader under UV light. Equimolar amounts of the sense and antisense strands were mixed and transferred to a new delivery tube. The mixture was heated at 95°C for 5 min and then slowly annealed to room temperature. Finally, it was evaporated to dryness at room temperature using a vacuum concentrator to obtain the final product.

[0295] The basic modification patterns of the naked siRNA oligonucleotide sequences used in the embodiments of this invention are shown in Table 3. Several chemical modifications are employed, as listed in Table 4.

[0296] Table 3. siRNA motif modifications prepared according to the present invention

[0297] Table 4. Lipophilic modification methods of siRNA prepared in this invention

[0298] III. Real-time quantitative PCR analysis of target mRNA levels

[0299] 1. Extract total RNA from cells using a column extraction kit.

[0300] 2. One-step real-time PCR:

[0301] Using the GAPDH gene as an internal reference, real-time quantitative PCR was performed using the Taqman one-step Real-time PCR kit and a Bio-Rad CFX96 real-time PCR instrument. The primer sequences used are shown in Table 5.

[0302] Table 5 Primer sequence information

[0303] Example 3: In vivo drug efficacy detection - SOD1-mRNA

[0304] Male, 6-8 week old SPF-grade SD rats (Jiangsu Jinzhihe Co., Ltd.) were used in the experiment. The rats were administered intraperitoneal (IT) drugs. On day 28, brain tissue and spinal cord (cervical, lumbar, and thoracic vertebrae) were collected, and the rats were subsequently euthanized. The collected tissues were used to detect SOD1-mRNA levels.

[0305] Table 6. Information on siRNA molecules used in Example 3

[0306] Note: For details on the modification methods in the table, please refer to Table 3-4.

[0307] Table 7 Experimental protocol for IT-induced siRNA drug in SD rats

[0308] SOD1-mRNA inhibition effect detection

[0309] 1. Method

[0310] Different brain regions and spinal cord tissues from frozen rats were ground using an automated grinder. Total RNA was extracted from different brain and spinal cord tissues of rats using the Trizol method, and RNA purity and concentration were determined using K550. The mRNA expression level of the SOD1 gene was detected using a qRT-PCR Starter Kit (Novizan), and inter-group comparisons were performed.

[0311] 2. Results

[0312] The experimental results (Figures 1a-1d) showed that the relative expression level of SOD1 gene mRNA was lower than that of the control group to varying degrees after IT administration, and the difference was statistically significant (P<0.01). In addition, compared with the control group, after 28 days of drug administration: in the brain region, the SOD1 mRNA levels in groups G2, G3, and G4 were reduced by 23.03%, 31.28%, and 55.89%, respectively (Figure 1a); in the cervical spine region, the SOD1 mRNA levels in groups G2, G3, and G4 were reduced by 35.20%, 30.74%, and 53.46%, respectively (Figure 1b); in the lumbar spine region, the SOD1 mRNA levels in groups G2, G3, and G4 were reduced by 5.81%, 13.95%, and 73.92%, respectively (Figure 1c); and in the thoracic spine region, the SOD1 mRNA levels in groups G2, G3, and G4 were reduced by 11.85%, 24.74%, and 56.39%, respectively (Figure 1d). The above experimental results show that the S-C16 modification of the present invention has a better CNS delivery effect than the O-C16 modification and can significantly downregulate the mRNA expression level of the SOD1 gene in rat brain and spinal cord tissues.

[0313] Example 4: In vivo efficacy detection - VEGFA-mRNA

[0314] Male, 6-8 week old SPF-grade C57BL / 6J mice (Guangzhou Cyagen Biotech Co., Ltd.) were used for the experiment. IVT was administered to the mice. Retinal tissue was collected from the mice on day 14 post-administration, and the mice were subsequently euthanized. The collected tissue was used to detect VEGFA-mRNA levels.

[0315] Table 8. Information on siRNA molecules used in Example 4

[0316] Note: For details on the modification methods in the table, please refer to Table 3-4.

[0317] Table 9. Experimental protocol for IVT administration in C57BL / 6J mice.

[0318] VEGFA-mRNA detection

[0319] 1. Method

[0320] Frozen mouse retinal tissue was collected and ground using an automated homogenizer. Total RNA was extracted from the mouse retinal tissue using the Trizol method, and RNA purity and concentration were determined using a K550 assay. The mRNA expression level of the VEGFA gene was detected using a qRT-PCR Starter Kit (Novizan), and intergroup comparisons were performed.

[0321] 2. Results The experimental results (Figure 2) showed that after IVT administration, the relative expression levels of VEGFA gene mRNA were significantly lower than those in the control group (P<0.01). Furthermore, compared to the control group, after 14 days of administration, the mRNA levels in groups G2, G3, and G4 (Table 9) were downregulated by 12.18%, 28.66%, and 36.11%, respectively. The results indicate that the C16-S modified siRNA of this invention can significantly downregulate the mRNA expression level of the VEGFA gene in the mouse eye, and its efficacy is superior to that of the C16-O modified siRNA.

[0322] Example 5: In vivo efficacy detection - ANG-2-mRNA

[0323] Male, 6-8 week old SPF-grade C57BL / 6J mice (Guangzhou Cyagen Biotech Co., Ltd.) were used for the experiment. IVT administration was performed. Retinal tissue was collected from the mice on day 14 post-administration, and the mice were subsequently euthanized. The collected tissue was used to detect ANG-2-mRNA levels.

[0324] Table 10 Information on siRNA molecules used in Example 5

[0325] Table 11 Experimental protocol for IVT administration in C57BL / 6J mice

[0326] ANG-2-mRNA inhibition effect detection

[0327] 1. Method

[0328] Frozen mouse retinal tissue was collected and ground using an automated homogenizer. Total RNA was extracted from the mouse retinal tissue using the Trizol method, and RNA purity and concentration were determined using a K550 assay. The mRNA expression level of the ANG-2 gene was detected using a qRT-PCR Starter Kit (Novizan), and intergroup comparisons were performed.

[0329] 2. Results

[0330] The experimental results (Figure 3) showed that after IVT administration, the relative expression level of ANG-2 gene mRNA was lower than that in the control group to varying degrees, and the difference was statistically significant (P<0.01). In addition, compared with the control group, after 14 days of administration, the mRNA levels in groups G2, G3, and G4 in Table 11 were downregulated by 49.82%, 57.33%, and 59.14%, respectively.

[0331] Experimental results show that the C16-S modified siRNA of the present invention can significantly downregulate the mRNA expression level of the ANG-2 gene in the mouse eye, and its efficacy is better than that of the C16-O modified siRNA.

[0332] Example 6: Detection using VEGFA / ANG-2 siRNA combination

[0333] The C16-S modification of the present invention was applied to the combination of VEGFA / ANG-2 siRNA to further verify the efficacy of the compound in vivo. The siRNA sequences of the two targets are shown in Tables 12 and 13 below.

[0334] Table 12 VEGFA / ANG-2siRNA combination

[0335] Table 13 V22 naked sequence information

[0336] The validation experiment used male Dutch rabbits, weighing 1.3-2.5 kg. One rabbit was placed in a stainless steel cage (900mm x 600mm x 500mm). The room for the rabbits was well-ventilated with a filter ventilation rate of 10-20 air changes per hour. The temperature was maintained between 16-26℃, and the relative humidity was 40-70%. Lighting conditions included 12 hours of fluorescent lighting and 12 hours of no lighting per day. Two to four months after DL-α-aminoadipic acid (DL-AAA) intravitreal injection to induce retinal modeling, retinal angiogenesis and leakage were assessed using free radical scintillation (FFA) before administration. Sufficient successfully modeled eyes without severe inflammation or hemorrhage were selected for the study. Grouping and administration details are shown in Table 14.

[0337] Table 14 IVT-administered siRNA drug – Rabbit experimental protocol

[0338] Fundus fluorescein angiography (FFA) images were taken of animals in each group 2 days before drug administration and on days 7, 21, 42, 56, 70, 84, and 98 after drug administration. Early (within 1.5 minutes) and late (after 3 minutes) images were acquired after injection of sodium fluorescein solution. The acquired images were scored, and the leakage area was quantified. The condition 2 days before drug administration was used as 100% baseline, and the leakage score was graded from 0 to 4. Animals were anesthetized by inhaling 1%–4% isoflurane before testing. Animals were euthanized after all tests were completed.

[0339] Results: As shown in Figures 4a and 4b, the combined use of VEGFA / ANG-2 siRNA in the disease model showed superior results in both leakage area and leakage score statistics compared to the currently available yangshen drug Eylea. Four months after administration, the efficacy was excellent, highlighting the advantages of C16-S modification.

[0340] Example 7 In vitro and in vivo efficacy detection - SNCA-mRNA

[0341] 1. In vitro transfection of cells with SNCA siRNA

[0342] U87MG cells (human brain glioblastoma cells) were placed in DMEM medium containing 10% fetal bovine serum and cultured in a 5% CO2 incubator at 37°C. When the cells were in the logarithmic growth phase and in good condition (70% confluence), they were plated and transfected. The cell density was adjusted to 2 × 10⁶ cells / year. 5 1 mL of cell solution was added to each well of a 24-well plate and incubated overnight at 37°C with 5% CO2. To prepare the transfection complex: 250 μL of Opti-MEM and 5 μL of 1 nM siRNA naked sequence were mixed to form a mixture. 250 μL of Opti-MEM and 2.5 μL of Lipofectamine 2000 transfection reagent were mixed to form another mixture. The mixtures were allowed to stand for 5 min. The two mixtures were then combined and allowed to stand for 20 min to form the transfection complex. The transfection complex was added to each well of a 24-well plate and incubated at 37°C with 5% CO2 for 6 h. The supernatant was aspirated, and 1 mL of the corresponding complete culture medium was added to each well. The plates were then incubated at 37°C with 5% CO2 for 24 h.

[0343] In addition to the experimental group, a control group was set up for each cell transfection: the control group (NC) was given only culture medium and transfection reagent. Both the experimental group and the control group were independently replicated three times.

[0344] 2. Real-time quantitative PCR analysis of target mRNA levels

[0345] 1) After transfection for 24 hours in step 1, cells were lysed and total RNA was extracted from the cells using a column extraction kit (Novizan).

[0346] 2) One-step real-time PCR:

[0347] Using the GAPDH gene as an internal reference gene, real-time quantitative PCR was performed using the Taqman one-step Real-time PCR kit and a Bio-Rad CFX96 real-time PCR instrument.

[0348] 3) Data Analysis

[0349] After the PCR reaction, the Ct error of 9 replicates of a sample (3 transfection replicates and 3 qPCR replicates per sample) was within ±0.5, and relative quantification was performed using CFX 2.1. Table 15 shows the average expression levels of the screened siRNA sequences relative to the target gene expression levels of the NC group (with the relative mRNA expression level of the NC group as 1).

[0350] Cellular activity screening of SNCA siRNA sequences was performed in U87MG cells. After transfecting cells with candidate SNCA siRNA molecular sequences in vitro, the expression of SNCA genes in the cells is shown in Figure 5. The three independent replicate test values ​​of the relative expression level of the target gene relative to the NC group in Figure 5 are listed in Table 16.

[0351] Table 15 Information on siRNA molecules used in Example 7

[0352] Table 16 Results of Real-time Quantitative PCR Detection of SNCA Gene Expression

[0353] 3. In vivo efficacy testing of SCNA siRNA

[0354] The experiment used male, 6-8 week old SPF-grade SNCA (A53T) transgenic mice (Jinzhihe Biotechnology Co., Ltd.), administered the drug via intraperitoneal injection (IT). On day 28 post-administration, spinal cord and brain tissue were collected from the mice, which were then euthanized. The collected tissues were used to detect SNCA mRNA levels.

[0355] Table 17 Experimental protocol for IT-induced hSNCA (A53T) mouse therapy

[0356] 1) Method

[0357] Frozen mouse spinal cord and brain tissue were collected and ground using an automated grinder. Total RNA was extracted from the mouse tissues using the Trizol method, and RNA purity and concentration were determined using a K550 assay. The mRNA expression level of the vegfa gene was detected using a qRT-PCR Starter Kit (Novizan), and intergroup comparisons were performed.

[0358] 2) Results

[0359] The experimental results (Figures 6a and 6b) showed that after IT administration, the relative expression levels of SNCA gene mRNA were significantly lower than those in the control group (P < 0.01). Furthermore, compared to the control group, 28 days after administration, in brain tissue regions, the SNCA mRNA levels in the G2 (SN10-(6-C16-S)) and G3 (SN16-(6-C16-S)) groups were downregulated by 30.44% and 43.36%, respectively. In the spinal region, the SNCA mRNA levels in the G2 (SN10-(6-C16-S)) and G3 (SN16-(6-C16-S)) groups were downregulated by 48.60% and 27.46%, respectively.

[0360] The above experimental results show that the application of the S-C16 modification of the present invention at different target sites has a CNS delivery effect and can significantly downregulate the mRNA expression level of SNCA gene in mouse brain and spinal cord tissues.

[0361] Example 8: In vivo efficacy detection of ANG-2 mRNA

[0362] Male, 6-8 week old SPF-grade C57BL / 6J mice (Guangzhou Cyagen Biotech Co., Ltd.) were used for the experiment. IVT administration was performed. Retinal tissue was collected from the mice on day 14 post-administration, and the mice were subsequently euthanized. The collected tissue was used to detect ANG-2-mRNA levels.

[0363] Table 18 Information on siRNA molecules used in Example 8

[0364] Table 19 Experimental protocol for IVT administration in C57BL / 6J mice

[0365] ANG-2 mRNA detection was performed as follows: Frozen mouse retinal tissue was picked and ground using an automated homogenizer. Total RNA was extracted from the mouse retinal tissue using the Trizol method, and RNA purity and concentration were determined using a K550 assay. The mRNA expression level of the ANG-2 gene was detected using a qRT-PCR Starter Kit (Novizan), and intergroup comparisons were performed.

[0366] The experimental results, as shown in Figure 7, indicated that the relative expression levels of ANG-2 gene mRNA after IVT administration were significantly lower than those in the control group (P<0.01). Furthermore, compared to the control group, the mRNA levels in groups G3 and G4 (Table 19) were downregulated by 57.33% and 59.14% respectively after 14 days of administration. These results demonstrate that the C16-S modified siRNA of this invention can significantly downregulate the mRNA expression level of the ANG-2 gene in the mouse eye.

[0367] Example 9: In vivo efficacy detection of SOD1 mRNA

[0368] The experiment used 6-8 week old SPF-grade male C57BL / 6 mice (Shanghai Southern Model Biotechnology Co., Ltd.), with 5 mice in each group. Each mouse was given a single dose of the test drug shown in the table below (5 mg / kg, subcutaneous injection). Mice were sacrificed on day 28, and total RNA was extracted from the heart, liver, kidney, subcutaneous fat, quadriceps femoris muscle, and tibialis anterior muscle. Gene expression levels were detected using real-time PCR.

[0369] Table 20 Information on siRNA molecules used in Example 9

[0370] The results (Figure 8) show that the C22-S modified siRNA of the present invention can significantly downregulate the mRNA expression level of SOD-1 gene in the heart, subcutaneous fat, quadriceps femoris muscle and tibialis anterior muscle of mice, and its efficacy is better than that of C22-O modified siRNA.

[0371] Example 10: In vitro activity detection of SOD1 gene-dsRNA with different modifications

[0372] SOD1 dsRNA in vitro free uptake assay in B16 cells: B16 cells were cultured in DMEM medium containing 10% fetal bovine serum in a 5% CO2 incubator at 37°C. After cell digestion, the cell density was adjusted to 1×10⁶ cells / year. 5 Cells were seeded at a rate of 450 μL / well in 24-well plates. 100 μM siRNA (diluted with 1×PBS buffer) was added at 50 μL / well to each well containing cell suspension, resulting in a final siRNA concentration of 10 μM. The plates were then incubated at 37°C with 5% CO2 for 48 h.

[0373] Real-time quantitative PCR analysis: Cells were lysed 48 h after transfection, and total RNA was extracted using the Novizan FastPure Cell / Tissue Total RNA Isolation Kit V2 (refer to Novizan RC112-01 instruction manual). cDNA was reverse transcribed using Takara PrimeScript RT Master Mix RR036Q. Mouse GAPDH genes (m-GAPDH-F-2 and m-GAPDH-R-2; primer information see Table 5) were used as internal control genes, and mouse SOD-1 genes (m-SOD-1-F and m-SOD-1-R; primer information see Table 5) were used as target genes. PCR reactions were performed using a Bio-Rad CFX96 real-time PCR instrument. The Mock group (PBS group) was used as a control for normalization, ensuring that the SOD1 mRNA expression level in the Mock group was 1.

[0374] Data Analysis: After the PCR reaction, relative quantification was performed using the reference gene (GAPDH) mRNA as a standard and CFX96 software, followed by statistical analysis using GarphPad software. The results are shown in Table 21, indicating that the lipid-modified monomers of this invention exhibit excellent inhibitory activity against the target gene at most positions on the sense strand.

[0375] Table 21 Results of single-dose (10 μM) detection of dsRNA in B16 cells

[0376] Note: S1-base sequence 1 is the S1-unattached mentioned above, which is the combination of SEQ ID NO: 5 and SEQ ID NO: 6 of the base sequence 1 modification pattern.

Claims

A nucleoside monomer with the structure shown in formula (Ⅰ): in, Base is a natural nucleobase or a modified nucleobase. R1 and R2 are independently H, hydroxyl protecting groups, or phosphoramide groups, respectively; R3 is C 14-22 saturated or unsaturated hydrocarbyl, or is C 23-25 saturated or unsaturated hydrocarbyl; Furthermore, when Base is uracil, R1 and R2 are not both H. The nucleoside monomer as described in claim 1, characterized in that, The structure of the nucleoside monomer is shown in formula (Ⅰ): Wherein, Base is a natural nucleobase or a modified nucleobase. R1 and R2 are independently H, hydroxyl protecting groups, or phosphoramide groups, respectively; R3is C 14-22 saturated or unsaturated hydrocarbon group; Furthermore, when Base is uracil, R1 and R2 are not both H. The nucleoside monomer as described in claim 1, characterized in that, The structure of the nucleoside monomer is shown in formula (Ⅰ): Wherein, Base is a natural nucleobase or a modified nucleobase. R1 and R2 are independently H, hydroxyl protecting groups, or phosphoramide groups, respectively; R3is C 23-25 saturated or unsaturated hydrocarbon group; Furthermore, when Base is uracil, R1 and R2 are not both H. The nucleoside monomer according to any one of claims 1-3 is characterized in that: The nucleobase is a purine base or a pyrimidine base; preferably, the nucleobase is adenine (A), guanine (G), thymine (T), cytosine (C), or uracil (U). The nucleoside monomer according to any one of claims 1-3 is characterized in that: the modified nucleobase is independently modified at any one or more positions with one or more modifications selected from the group consisting of: an amino protecting group, C 1-3 alkyl, C 3-5 cycloalkyl, haloC 1-3 alkyl, C 1-3 alkylcarbonyl, C 1-3 alkoxyC 1-3 alkyl, benzoyl, benzyl, dimethylaminomethyl, dimethylaminoC 1-3 alkyl, oxo, thio, amino, C 2-6 alkenyl, C 2-6 alkynyl, mercapto, C 1-3 alkylthio, C 1-3 alkylamino, di(C 1-3 alkyl)amino, hydroxy, C 1-3 alkoxycarbonylC 1-3 alkyl, aminoC 1-3 alkoxy, aminoC 1-3 alkylaminocarbonyl, aminoC 1-3 alkylaminocarbonylvinyl and aminoC 1-3 alkylhydroxy; preferably, the modification is selected from the group consisting of: an amino protecting group, C 1-3 alkyl, C 3-5 cycloalkyl, haloC 1-3 alkyl, C 1-3 alkylcarbonyl, C 1-3 alkoxyC 1-3 alkyl, benzoyl, benzyl, dimethylaminomethyl, and dimethylaminoC 1-3 alkyl. The nucleoside monomer according to any one of claims 1-3 is characterized in that: said R3 is C 14-18 saturated or unsaturated hydrocarbyl; preferably, said R3 is C 15-17 saturated or unsaturated hydrocarbyl; preferably, said R3 is C 15 alkyl, C 16 alkyl or C 17 alkyl; preferably, said R3 is linear C 15 alkyl, linear C 16 alkyl or linear C 17 alkyl; or said R3 is C 22 saturated or unsaturated alkyl; preferably said R3 is C 22 branched or straight chain alkyl; preferably said R3 is straight chain C 22 alkyl; or said R3 is C 23-25 saturated or unsaturated alkyl; preferably said R3 is C 23 alkyl, C 24 alkyl or C 25 alkyl; preferably said R3 is C 23-25 branched or straight chain alkyl; preferably said R3 is straight chain C 23-25 alkyl, for example straight chain C 23 alkyl, straight chain C 24 alkyl or straight chain C 25 alkyl. The nucleoside monomer as described in claim 1, characterized in that, The hydroxyl protecting group is selected from one, two, or more of the following: Wherein, Cat is a cation, independently selected from sodium ion, potassium ion, triethylammonium ion, tripropylammonium ion, tributylammonium ion, and tetrabutylammonium ion; r is an integer from 0 to 5; Solid support is a solid phase support; Preferably, the hydroxyl protecting group is selected from any one or more of the following: methoxymethyl ether (MOM), 2-tetrahydropyran (THP), tert-butyldimethylchlorosilyl (TBS), trimethylsilyl (TMS), triethylsilyl (TES), triphenylmethyl (Tr), 4,4'-bismethoxytriphenylmethyl (DMTr), 4,4',4'-trimethoxytriphenylmethyl (TMTr), benzyl (Bn), benzoyl (Bz), acetyl (Ac), and p-methoxybenzyl (PMB); More preferably, the hydroxyl protecting group is selected from one or more of the following: triphenylmethyl (Tr), 4,4'-dimethoxytriphenylmethyl (DMTr), 4,4',4'-trimethoxytriphenylmethyl (TMTr), benzyl (Bn), benzoyl (Bz), and acetyl (Ac). The nucleoside monomer according to any one of claims 1-3 is characterized in that, The phosphoramide group is selected from one or more of the following structures: wherein R4, R5are each independently C 1-3 straight-chain or branched alkyl, R6is C 1-3 straight-chain or branched alkyl, or C 1-3 straight-chain or branched alkyl. The nucleoside monomer as described in claim 1, characterized in that, R1 and R2 are independently selected from H, Wherein, Cat is a cation, independently selected from sodium ion, potassium ion, triethylammonium ion, tripropylammonium ion, tributylammonium ion, and tetrabutylammonium ion; r is an integer from 0 to 5; Solid support is a solid phase support; Preferably, R1 and R2 are independently DMTr or phosphoramide groups; more preferably, R1 is DMTr and R2 is a phosphoramide group; more preferably, R1 is DMTr and R2 is... The nucleoside monomer as described in claim 1, characterized in that, The nucleoside monomer is selected from the following compounds: An oligonucleotide comprising an antisense strand complementary to a target gene, said antisense strand being 14 to 30 nucleotides in length, preferably 19 to 25 nucleotides in length, said oligonucleotide containing one or more nucleoside monomers as shown in formula (II): in, The definitions of Base and R3 are the same as those in any one of the terms (I) of claims 1-10. The oligonucleotide of claim 11, which is a double-stranded RNA, further comprises a sense strand complementary to the antisense strand, wherein the length of the sense strand and the antisense strand is independently 14 to 30 nucleotides; preferably, the length of the sense strand and the antisense strand is independently 19 to 25 nucleotides; more preferably, the length of the sense strand and the antisense strand is independently 21 to 23 nucleotides. The oligonucleotide as claimed in claim 11 or 12, wherein at least one of its strands contains at least one nucleoside monomer at positions 4 to 8 starting from the 5' end; preferably, at least one of its strands contains at least one nucleoside monomer at positions 6, 7 and 8 starting from the 5' end; preferably, at least one of its positive strands contains at least one nucleoside monomer at positions 6, 7 and 8 starting from the 5' end. The oligonucleotide according to any one of claims 11-13, wherein at least one nucleotide is modified; preferably, the modification is selected from one or more of the following: 2′-O-methyl modification, 2′-fluoro modification, thiophosphate backbone modification, 2′-deoxy modification and 5′-vinylphosphonate modification. A pharmaceutical composition comprising an oligonucleotide as described in any one of claims 11-14, and a pharmaceutically acceptable excipient; preferably, the pharmaceutical composition further comprises a delivery carrier. Use of the nucleoside monomer of any one of claims 1-10, the oligonucleotide of any one of claims 11-14, or the pharmaceutical composition of claim 15 in the preparation of a medicament for regulating the expression of target genes in cells. Use of the oligonucleotide of any one of claims 11-14 or the pharmaceutical composition of claim 15 in the preparation of a medicament for regulating the expression of a target gene in cells, wherein the oligonucleotide is conjugated with a GalNAc ligand, and the target gene is located in or expressed in the liver. The use as described in claim 16, wherein the target gene is located in an extrahepatic location or is expressed in an extrahepatic location; Preferably, the target gene is located in the intrathecal or ocular region, central nervous system (CNS), muscle, lungs, or fat. Preferably, the target gene is located in the brain, spinal cord tissue, or eye tissue; Preferably, the target gene is located in one or more of the following locations: hippocampus, cortex, substantia nigra, striatum, cerebellum, cervical vertebrae, lumbar vertebrae, thoracic vertebrae, retina, choroid plexus, heart, fat (e.g., subcutaneous fat), lungs, and muscles. Use of the nucleoside monomer of any one of claims 1-10, the oligonucleotide of any one of claims 11-14, or the pharmaceutical composition of claim 15 in the preparation of a medicament for treating a subject suffering from a central nervous system (CNS), eye, muscle, lung, or fat disorder. The use as described in claim 19, wherein the CNS, eye, muscle, lung, or fat disorder is selected from one or more of the following: Alzheimer's disease, amyotrophic lateral sclerosis, frontotemporal dementia, Huntington's disease, Parkinson's disease, spinocerebellar disease, prions, age-related macular degeneration, and diabetic macular edema. The use according to any one of claims 16-20, wherein the target gene is selected from one or more of the following: SOD1, APP, ATXN1, ATXN2, ATXN3, C9orf72, TARDBP, MAPT, HTT, LRRK2, SNCA, VEGFA, ANG-2, VEGFR1, VEGFR2, SCA1, SCA7, and SCA8.

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