Dual-target nucleic acid molecule for inhibiting expression of vegfa gene and ang-2 gene

Abstract

Provided is a dual-target nucleic acid molecule for inhibiting the expression of the vegfa gene and ang-2 gene. A linker contains 2-10 nucleotides or abasic nucleotides, and is used for linking two independent oligonucleotides. One or more nucleotides or abasic nucleotides in the linker contain a lipophilic moiety, and the lipophilic moiety is a C12-26 saturated or unsaturated hydrocarbon chain. Compared to a situation where a specific lipophilic modification is located inside the two linked moieties of a dual-nucleic acid molecule structure, the nucleic acid molecule exhibits better efficacy and a longer-lasting therapeutic effect when the specific lipophilic modification is located on the linker.

Description

A dual-target nucleic acid molecule that inhibits the expression of the vegfa and ang-2 genes.

[0001] Cross-reference to related applications

[0002] This invention claims priority to the following three prior applications: Patent Application No. 202410695135.8, filed with the China National Intellectual Property Administration on May 31, 2024, entitled "A Modified Nucleoside Monomer and Oligonucleotides Prepared Therefrom"; Patent Application No. 202411211681.6, filed with the China National Intellectual Property Administration on August 30, 2024, entitled "A Nucleic Acid Molecule Inhibiting the Expression of the VEGFA Gene and / or Ang-2 Gene"; and Patent Application No. 202510411738.5, filed with the China National Intellectual Property Administration on April 2, 2025, entitled "A Linker and a Dual-Target Nucleic Acid Molecule Containing the Linker Inhibiting the Expression of the VEGFA Gene and Ang-2 Gene". The full text of these three prior applications is incorporated herein by reference. Technical Field

[0003] This application relates to the field of biomedicine, specifically to a dual-target nucleic acid molecule that inhibits the expression of the vegfa gene and the ang-2 gene. Background Technology

[0004] RNA interference (RNAi) is a phenomenon widely observed in natural species. It refers to the highly efficient and specific degradation of targeted mRNA induced by double-stranded RNA (dsRNA). dsRNA, typically 18–30 bp in length, is a crucial tool in RNAi technology. In natural organisms, longer dsRNAs, upon entering cells, are specifically recognized and cleaved into shorter dsRNAs (siRNAs) by the Dicer enzyme. The resulting dsRNAs form a complex (RISC) with certain proteins, which then cleaves into single strands. RISCs bind to mRNAs complementary to the antisense strand of the dsRNA within the cell, cleaving and degrading the mRNA, preventing protein synthesis and resulting in gene silencing. In industrial production, the chemical synthesis and modification of siRNAs are preferred to further improve the stability and efficacy of siRNA drugs.

[0005] Angiogenesis in the eye typically plays a vital role, such as supplying oxygen and other essential nutrients and supporting normal tissue development. However, excessive and abnormal blood vessel growth can induce eye diseases such as wet age-related macular degeneration (AMD) and diabetic macular edema (DME), which can lead to blindness in some cases. AMD is a disease caused by degeneration of the retinal pigment epithelium lining in the macula of the eye, resulting in vision loss and is one of the leading causes of blindness worldwide. AMD occurs in "wet" and "dry" forms; wet AMD is the result of abnormal blood vessel growth in the retina. In the pathogenesis of wet AMD and DME, abnormally elevated vascular endothelial growth factor A (VEGF-A) acting on vascular endothelial cells promotes angiogenesis, while elevated angiopoietin-2 (Ang-2) acting on pericytes leads to vascular leakage.

[0006] Currently, there are several treatments that can slow down AMD / DME, including ranibizumab, aflibercept, bevacizumab, conbercept, and fagrizumab. However, all of these treatments require frequent injections, leading to poor patient adherence. Therefore, it is still necessary to develop other inhibitors or combination formulations targeting VEGFA or ANG-2 to improve efficacy, duration of action, specificity, stability, targeting, and tolerability, thereby reducing injection frequency and improving adherence.

[0007] Application Overview

[0008] This application provides a dual-target nucleic acid molecule containing a linker that inhibits the expression of the vegfa and ang-2 genes. The linker has a specific lipophilic modification; compared to when this specific lipophilic modification is located inside the two connected parts of the dual nucleic acid molecule structure, when the specific lipophilic modification is located on the linker, the nucleic acid molecule exhibits better efficacy and a longer-lasting therapeutic effect. Furthermore, the nucleic acid molecule screened by the inventors can more specifically silence the corresponding genes and inhibit the expression of the corresponding proteins, and the nucleic acid molecule of this application has the advantage of long-lasting therapeutic effect.

[0009] The first aspect of this application provides a linker comprising 2-10 nucleotides or abasic nucleotides for linking two independent oligonucleotides, wherein one or more nucleotides or abasic nucleotides in the linker contain a lipophilic moiety, the lipophilic moiety being C 12-26 Saturated or unsaturated hydrocarbon chains;

[0010] Wherein, one or more alkylene groups on the saturated or unsaturated hydrocarbon chain may be independently and optionally replaced with -O-, -S-, -C(O)-, -C(S)-, -S(O)-, -S(O)2-, -SS-, -N(G)C(O)-, -C(O)N(G)-, -N(G)-, -P(O)(OH)-, -P(O)(SH)-, -P(S)(SH)-, In this context, G is independently selected from hydrogen, deuterium, halogen, hydroxyl, amino, and C each time it appears. 1-6 Alkyl, C 1-6 Alkoxy, C 3-10 Cycloalkyl, or C substituted with one or more of deuterium, halogen, hydroxyl, or amino groups. 1-6 Alkyl, C 1-6 Alkoxy, C 3-10 cycloalkyl;

[0011] Wherein, one or more alkylene groups on the saturated or unsaturated hydrocarbon chain may also be independently and optionally substituted by one or more of the following: deuterium, halogen, hydroxyl, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-10 Cycloalkyl, or C substituted with one or more of deuterium, halogen, hydroxyl, or amino groups. 1-6 Alkyl, C 1-6 Alkoxy and C 3-10 Cycloalkyl.

[0012] In some embodiments, the linker described above may be independently and optionally replaced with -O-, -S-, -C(O)-, -N(G)C(O)-, or -C(O)N(G)-. Alternatively, the linker may be independently and optionally replaced with -O- or -S-. Alternatively, the linker may be independently and optionally replaced with -C(O)-, -N(G)C(O)-, or -C(O)N(G)-.

[0013] In some embodiments, one or more alkylene groups on the saturated or unsaturated hydrocarbon chain may also be independently, optionally, substituted with one or more of the following: halogen, hydroxyl, amino, C 1-6 alkyl.

[0014] In some embodiments, the linker G is independently selected from hydrogen and C each time it appears. 1-6 alkyl.

[0015] In some embodiments of the linker, at least one nucleotide or abasic nucleotide in the linker has a lipophilic moiety at the 1' and / or 2' position of its sugar ring; optionally, the 1' position of the one or more abasic nucleotides has a lipophilic moiety; optionally, the 2' position of the one or more nucleotides or abasic nucleotides has a lipophilic moiety.

[0016] In some embodiments of the linker described above, the lipophilic portion is C. 14-24 A saturated or unsaturated hydrocarbon chain; optionally, the lipophilic moiety is C. 16-22 Saturated or unsaturated hydrocarbon chains.

[0017] In some embodiments of the linker described above, the lipophilic portion is C. 12-26 A saturated hydrocarbon chain; optionally, the lipophilic moiety is C. 14-24 A saturated hydrocarbon chain; further optionally, the lipophilic moiety is C. 16-22 saturated hydrocarbon chains.

[0018] In some embodiments of the linker described above, the saturated or unsaturated hydrocarbon chain of the lipophilic portion is a straight chain or a branched chain; optionally, the saturated or unsaturated hydrocarbon chain of the lipophilic portion is a straight chain.

[0019] In some embodiments, the lipophilic portion of the linker is connected to a nucleotide or a non-base nucleotide via -O-, -S-, -C(O)-, -N(G)C(O)-, or -C(O)N(G)-; alternatively, the lipophilic portion is connected to a nucleotide or a non-base nucleotide via -O- or -S-; alternatively, the lipophilic portion is connected to a nucleotide or a non-base nucleotide via -C(O)-, -N(G)C(O)-, or -C(O)N(G)-. In this case, it can be seen that the alkylene group at the connection position between the lipophilic part and the nucleotide or abasic nucleotide is replaced by -O-, -S-, -C(O)-, -N(G)C(O)-, or -C(O)N(G)-, respectively. That is, the connected part is part of the lipophilic part. In addition, when the 2' hydroxyl group of the nucleotide or abasic nucleotide is connected to the 2' position of the sugar ring, it can be considered that the -O- at that position is not a lipophilic part, or it can be considered that the -O- at that position is a lipophilic part. That is, the alkylene group at the connection position between the lipophilic part and the nucleotide or abasic nucleotide is replaced by -O-, -S-, -C(O)-, -N(G)C(O)-, or -C(O)N(G)-, respectively.

[0020] In some embodiments of the linker described above, the lipophilic portion of one or more nucleotides or non-base nucleotides in the linker is independently selected from: 2'-SC 14-22 Alkyl (2'-SC) 14 Alkyl, 2'-SC15 Alkyl, 2'-SC 16 Alkyl, 2'-SC 17 Alkyl, 2'-SC 18 Alkyl, 2'-SC 22 alkyl), 2'-OC 14-22 Alkyl (2'-OC) 14 Alkyl, 2'-OC 15 Alkyl, 2'-OC 16 Alkyl, 2'-OC 17 Alkyl, 2'-OC 18 Alkyl, 2'-OC 19 Alkyl, 2'-OC 20 Alkyl, 2'-OC 21 Alkyl, 2'-OC 22 alkyl), 2'-C(O)NH-C 14-22 Alkyl, 2'-C(O)NH-C 2-6 Alkyl-C(O)NH-C 10-18 Alkyl, 2'-C(O)NH-C 2-6 Alkyl-NHC(O)-C 10-18 Alkyl, 2'-C(O)NH-C 10-18 Alkyl-C(O)NH-C 2-6 Alkyl, 2'-C(O)NH-C 10-18 Alkyl-NHC(O)-C 2-6 Alkyl, 2'-NHC(O)-C 14-22 Alkyl, 2'-NHC(O)-C 2-6 Alkyl-NHC(O)-C 10-18 Alkyl, 2'-NHC(O)-C 2-6 Alkyl-NHC(O)-C 10-18 Alkyl, 2'-NHC(O)-C 10-18 Alkyl-C(O)NH-C 2-6 Alkyl, 2'-NHC(O)-C 10-18 Alkyl-NHC(O)-C 2-6 Alkyl, 2'-OC 2-6 Alkyl-C(O)NH-C 10-18 Alkyl, 2'-OC 2-6 Alkyl-NHC(O)-C 10-18 Alkyl, 2'-OC 10-18 Alkyl-C(O)NH-C 2-6 Alkyl, 2'-OC 10-18 Alkyl-NHC(O)-C 2-6 Alkyl, 2'-SC 2-6 Alkyl-C(O)NH-C10-18 Alkyl, 2'-SC 2-6 Alkyl-NHC(O)-C 10-18 Alkyl, 2'-SC 10-18 Alkyl-C(O)NH-C 2-6 Alkyl and 2'-SC 10-18 Alkyl-NHC(O)-C 2-6 alkyl.

[0021] In some embodiments of the linker described above, the lipophilic portions of one or more nucleotides or non-base nucleotides in the linker are independently selected from: 2'-SC 14 Alkyl, 2'-SC 15 Alkyl, 2'-SC 16 Alkyl, 2'-SC 17 Alkyl, 2'-SC 18 Alkyl, 2'-OC 14 Alkyl, 2'-OC 15 Alkyl, 2'-OC 16 Alkyl, 2'-OC 17 Alkyl, 2'-OC 18 Alkyl; optionally, the lipophilic portion of one or more nucleotides or baseless nucleotides in the linker is 2'-SC. 16 Alkyl; further optionally, one, two, or three nucleotides or non-basic nucleotides in the linker have a lipophilic moiety of 2'-SC. 16 Alkyl; more preferably, one, two, or three nucleotides or abase-free nucleotides in the linker are independently 2'-SC. 16 Straight-chain alkyl-modified uridine [U(hdt)] or 2'-SC 16 Straight-chain alkyl-modified thymidine [dT(hdt)].

[0022] In some embodiments, each nucleotide in the linker is independently selected from deoxyribonucleotides or ribonucleotides; alternatively, the linker is a hybrid molecule of DNA and RNA.

[0023] In some embodiments, the length of the connector is 2-7; optionally, it is 3, 4, 5, 6 or 7.

[0024] In some embodiments, the connector described above has the structure shown in Formula I, where X = 0. - or S - :

[0025] Alternatively, X = O -, and / or, -C 16 H 33 The group is a straight-chain alkyl group; further optionally, X = O - And -C 16 H 33 The group is a straight-chain alkyl group. The linker is -dTUdT-, where U is 2'-SC- 16 H 33 Modification, dT and U are linked by a phosphate ester bond (i.e., X = O) - ), -C 16 H 33 When the group is a straight-chain alkyl group, it is -dTU(hdt)dT-. From a chemical structural perspective, in formula I, the linker... Linkage can be completed as long as it can form a covalent bond with the corresponding group from other nucleotides. For example, 5'-5', 3'-3', and 3'-5' links can be achieved by designing the left and right oligonucleotides. However, if the two ends are connected to undesigned nucleotides, the most common way for nucleotides to link is through a 5'-3' phosphate ester bond or a thiophosphate ester bond. In this case, in the linker of formula I... This indicates that the 3' end of the left oligonucleotide is connected via a linker. This represents the 5' end of the right oligonucleotide being linked by a linker.

[0026] A second aspect of this application provides the use of the above-described linker in the preparation of nucleic acid molecules or pharmaceutically acceptable salts thereof, the nucleic acid molecules comprising a dual nucleic acid molecular structure; optionally, the nucleic acid molecule is a dual-target nucleic acid molecule having a dual nucleic acid molecular structure.

[0027] A third aspect of this application provides the aforementioned nucleic acid molecule or a pharmaceutically acceptable salt thereof, wherein the nucleic acid molecule comprises a dual-nucleic acid molecular structure, the dual-nucleic acid molecular structure comprising a nucleic acid molecule targeting a first target, a nucleic acid molecule targeting a second target, and the aforementioned linker, wherein the nucleic acid molecule targeting the first target and the nucleic acid molecule targeting the second target are connected by the aforementioned linker; optionally, the nucleic acid molecule is a dual-target nucleic acid molecule, and its structure is a dual-nucleic acid molecular structure. In this application, the descriptions of the first target and the second target are merely designations; the first target and the second target may be the same or different.

[0028] In some embodiments, the nucleic acid molecules or their pharmaceutically acceptable salts are selected independently from the following molecular types: siRNA, ASO.

[0029] In some embodiments, when both the nucleic acid molecule targeting the first target and the nucleic acid molecule targeting the second target are siRNAs, the linker is connected via the positive-sense strand of the siRNA targeting the first target and the positive-sense strand of the siRNA targeting the second target; optionally, the connection direction is as follows: (5') positive-sense strand of the siRNA targeting the first target (3') - (5') linker (3') - (5') positive-sense strand of the siRNA targeting the second target (3'), and its schematic structure is shown in Formula II:

[0030] In some embodiments, the above-mentioned nucleic acid molecule or its pharmaceutically acceptable salt may be a first target of the vegfa gene and a second target of the ang-2 gene; or a first target of the ang-2 gene and a second target of the vegfa gene.

[0031] In some embodiments, the aforementioned nucleic acid molecules or their pharmaceutically acceptable salts are sodium, potassium, or ammonium salts.

[0032] A fourth aspect of this application provides a dual-target nucleic acid molecule or a pharmaceutically acceptable salt thereof for inhibiting the expression of the vegfa gene and the ang-2 gene, comprising a vegfa siRNA for inhibiting vegfa gene expression and an ang-2 siRNA for inhibiting ang-2 gene expression linked by a linker. The vegfa siRNA comprises complementary sense and antisense strands, or is composed of complementary sense and antisense strands, and the ang-2 siRNA comprises complementary sense and antisense strands, or is composed of complementary sense and antisense strands. The linker comprises 2-10 nucleotides or a base-free nucleotides for linking two independent oligonucleotides. One or more nucleotides or a base-free nucleotides in the linker contain a lipophilic moiety, the lipophilic moiety being C 12-26 Saturated or unsaturated hydrocarbon chains;

[0033] Wherein, one or more alkylene groups on the saturated or unsaturated hydrocarbon chain may be independently and optionally replaced with -O-, -S-, -C(O)-, -C(S)-, -S(O)-, -S(O)2-, -SS-, -N(G)C(O)-, -C(O)N(G)-, -N(G)-, -P(O)(OH)-, -P(O)(SH)-, -P(S)(SH)-, In this context, G is independently selected from hydrogen, deuterium, halogen, hydroxyl, amino, and C each time it appears. 1-6 Alkyl, C 1-6 Alkoxy, C 3-10Cycloalkyl, or C substituted with one or more of deuterium, halogen, hydroxyl, or amino groups. 1-6 Alkyl, C 1-6 Alkoxy, C 3-10 cycloalkyl;

[0034] Wherein, one or more alkylene groups on the saturated or unsaturated hydrocarbon chain may also be independently and optionally substituted by one or more of the following: deuterium, halogen, hydroxyl, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-10 Cycloalkyl, or C substituted with one or more of deuterium, halogen, hydroxyl, or amino groups. 1-6 Alkyl, C 1-6 Alkoxy and C 3-10 Cycloalkyl.

[0035] In some embodiments, one or more alkylene groups on the saturated or unsaturated hydrocarbon chain may be independently and optionally replaced with -O-, -S-, -C(O)-, -N(G)C(O)-, -C(O)N(G)-; alternatively, they may be independently and optionally replaced with -O-, -S-; alternatively, one or more alkylene groups on the saturated or unsaturated hydrocarbon chain may be independently and optionally replaced with -C(O)-, -N(G)C(O)-, -C(O)N(G)-;

[0036] In some embodiments, the lipophilic moiety of the aforementioned dual-target nucleic acid molecule or its pharmaceutically acceptable salt is linked to a nucleotide or a non-base nucleotide via -O-, -S-, -C(O)-, -N(G)C(O)-, or -C(O)N(G)-; alternatively, the lipophilic moiety is linked to a nucleotide or a non-base nucleotide via -O- or -S-; alternatively, the lipophilic moiety is linked to a nucleotide or a non-base nucleotide via -C(O)-, -N(G)C(O)-, or -C(O)N(G)-. In this case, it can be seen that the alkylene groups at the lipophilic moiety and the nucleotide or abase nucleotide linkage position are replaced by -O-, -S-, -C(O)-, -N(G)C(O)-, or -C(O)N(G)-, respectively. In addition, when the 2' hydroxyl group of the nucleotide or abase nucleotide is attached to the 2' position of the sugar ring, it can be considered that the -O- at that position is either not a lipophilic moiety or is a lipophilic moiety.

[0037] In some embodiments, one or more alkylene groups on the aforementioned dual-target nucleic acid molecule or its pharmaceutically acceptable salt may also be independently, optionally, substituted with one or more of the following: halogen, hydroxyl, amino, C 1-6 alkyl.

[0038] In some embodiments, the aforementioned dual-target nucleic acid molecules or their pharmaceutically acceptable salts, G is independently selected from hydrogen and C each time it appears. 1-6 alkyl.

[0039] In some embodiments, the aforementioned dual-target nucleic acid molecules or their pharmaceutically acceptable salts contain lipophilic moieties independently at the 1' and / or 2' positions of the sugar ring of at least one nucleotide or a baseless nucleotide in the linker.

[0040] In some embodiments, the lipophilic moiety of the aforementioned dual-target nucleic acid molecule or its pharmaceutically acceptable salt is C. 14-24 A saturated or unsaturated hydrocarbon chain; optionally, the lipophilic moiety is C. 16-22 Saturated or unsaturated hydrocarbon chains.

[0041] In some embodiments, the lipophilic moiety of the aforementioned dual-target nucleic acid molecule or its pharmaceutically acceptable salt is C. 12-26 A saturated hydrocarbon chain; optionally, the lipophilic moiety is C. 14-24 A saturated hydrocarbon chain; further optionally, the lipophilic moiety is C. 16-22 saturated hydrocarbon chains.

[0042] In some embodiments, the saturated or unsaturated hydrocarbon chain of the lipophilic moiety of the aforementioned dual-target nucleic acid molecule or its pharmaceutically acceptable salt is a straight chain or a branched chain; optionally, the saturated or unsaturated hydrocarbon chain of the lipophilic moiety is a straight chain.

[0043] In some embodiments, the lipophilic portion of one or more nucleotides or a baseless nucleotide in the linker is independently selected from: 2'-SC 14-22 Alkyl (2'-SC) 14 Alkyl, 2'-SC 15 Alkyl, 2'-SC 16 Alkyl, 2'-SC 17 Alkyl, 2'-SC 18 Alkyl, 2'-SC 22 alkyl), 2'-OC 14-22 Alkyl (2'-OC) 14 Alkyl, 2'-OC 15 Alkyl, 2'-OC 16 Alkyl, 2'-OC 17 Alkyl, 2'-OC 18 Alkyl, 2'-OC 19 Alkyl, 2'-OC 20 Alkyl, 2'-OC 21 Alkyl, 2'-OC 22alkyl), 2'-C(O)NH-C 14-22 Alkyl, 2'-C(O)NH-C 2-6 Alkyl-C(O)NH-C 10-18 Alkyl, 2'-C(O)NH-C 2-6 Alkyl-NHC(O)-C 10-18 Alkyl, 2'-C(O)NH-C 10-18 Alkyl-C(O)NH-C 2-6 Alkyl, 2'-C(O)NH-C 10-18 Alkyl-NHC(O)-C 2-6 Alkyl, 2'-NHC(O)-C 14-22 Alkyl, 2'-NHC(O)-C 2-6 Alkyl-NHC(O)-C 10-18 Alkyl, 2'-NHC(O)-C 2-6 Alkyl-NHC(O)-C 10-18 Alkyl, 2'-NHC(O)-C 10-18 Alkyl-C(O)NH-C 2-6 Alkyl, 2'-NHC(O)-C 10-18 Alkyl-NHC(O)-C 2-6 Alkyl, 2'-OC 2-6 Alkyl-C(O)NH-C 10-18 Alkyl, 2'-OC 2-6 Alkyl-NHC(O)-C 10-18 Alkyl, 2'-OC 10-18 Alkyl-C(O)NH-C 2-6 Alkyl, 2'-OC 10-18 Alkyl-NHC(O)-C 2-6 Alkyl, 2'-SC 2-6 Alkyl-C(O)NH-C 10-18 Alkyl, 2'-SC 2-6 Alkyl-NHC(O)-C 10-18 Alkyl, 2'-SC 10-18 Alkyl-C(O)NH-C 2-6 Alkyl and 2'-SC 10-18 Alkyl-NHC(O)-C 2-6 alkyl.

[0044] In some embodiments, the lipophilic portions of one or more nucleotides or non-basic nucleotides in the linker are independently selected from: 2'-SC 14 Alkyl modification, 2'-SC 15 Alkyl modification, 2'-SC 16 Alkyl modification, 2'-SC17 Alkyl modification, 2'-SC 18 Alkyl, 2'-OC 14 Alkyl modification, 2'-OC 15 Alkyl modification, 2'-OC 16 Alkyl modification, 2'-OC 17 Alkyl modification, 2'-OC 18 Alkyl; optionally, the sugar ring of one or more nucleotides or abase-free nucleotides in the linker has a 2'-SC position at the 2' position. 16 Alkyl modification; further optionally, one, two, or three nucleotides or baseless nucleotides in the linker have a lipophilic moiety of 2'-SC. 16 Alkyl modification; more preferably, one, two, or three nucleotides or abase-free nucleotides in the linker are independently 2'-SC. 16 Straight-chain alkyl-modified uridine (U) or 2'-SC 16 Straight-chain alkyl-modified thymidine (T).

[0045] In some embodiments of the aforementioned dual-target nucleic acid molecule or its pharmaceutically acceptable salt, each nucleotide in the linker is independently selected from deoxyribonucleotides or ribonucleotides; optionally, the linker is a hybrid molecule of DNA and RNA. In some embodiments of the aforementioned dual-target nucleic acid molecule or its pharmaceutically acceptable salt, the linker is 2-7 in length; optionally, it is 3, 4, 5, 6, or 7.

[0046] In some embodiments, the linker structure of the aforementioned dual-target nucleic acid molecule or its pharmaceutically acceptable salt is as shown in Formula I, where X = 0. - or S - :

[0047] Alternatively, X = O - , and / or, -C 16 H 33 The group is a straight-chain alkyl group; further optionally, X = O - And -C 16 H 33 The group is a straight-chain alkyl group. The linker is -dTUdT-, where U is 2'-SC- 16 H 33 Modification, dT and U are linked by a phosphate ester bond (i.e., X = O) - ), -C 16 H 33 When the group is a straight-chain alkyl group, it is -dTU(hdt)dT-. From a chemical structural perspective, in formula I, the linker... Linkage can be completed as long as it can form a covalent bond with the corresponding group from other nucleotides. For example, 5'-5', 3'-3', and 3'-5' links can be achieved by designing the left and right oligonucleotides. However, if the two ends are connected to undesigned nucleotides, the most common way for nucleotides to link is through a 5'-3' phosphate ester bond or a thiophosphate ester bond. In this case, in the linker of formula I... This represents the connection of the 3' end of the left-hand oligonucleotide molecule via a linker. This represents the 5' end of the right-hand oligonucleotide molecule being linked by a linker.

[0048] In some embodiments, the linker of the aforementioned dual-target nucleic acid molecule or its pharmaceutically acceptable salt links the vegfa siRNA and ang-2 siRNA by linking them to the positive strand of the vegfa siRNA and the positive strand of the ang-2 siRNA, respectively.

[0049] In some embodiments, the connection direction of the positive strand of vegfa siRNA and the positive strand of ang-2 siRNA is as follows: (5')vegfa siRNA positive strand (3')-(5')linker (3')-(5')ang-2 siRNA positive strand (3').

[0050] In some embodiments, the antisense strand of the above-mentioned dual-target nucleic acid molecule or its pharmaceutically acceptable salt has a nucleotide length of 18-23 nt, and the antisense strand comprises or is a sequence of 18-21 consecutive nucleotides, consisting of no more than 3 nucleotides distinct from the 1st to 21st nucleotides starting from the 5' end of any one of the sequences SEQ ID NO: 6, 10, 20, 28, 44, 46.

[0051] And / or, the antisense strand of the ang-2siRNA has a nucleotide length of 18-23 nt, and the antisense strand comprises or is a continuous nucleotide sequence of 18-21 nucleotides from the 5' end of any one of the sequences SEQ ID NO:48, 60, 62, 64, 70, 90, consisting of no more than 3 distinct nucleotides from the 1st to 21st nucleotides.

[0052] In some embodiments, the aforementioned dual-target nucleic acid molecule or its pharmaceutically acceptable salt may have a difference of no more than 3 nucleotides, which may be a difference of 3 nucleotides, a difference of 2 nucleotides, a difference of 1 nucleotide, or complete sameness.

[0053] In some embodiments, the 18-21 consecutive nucleotide sequences of the aforementioned dual-target nucleic acid molecules or their pharmaceutically acceptable salts are 18, 19, 20, or 21 consecutive nucleotide sequences.

[0054] In some embodiments, the aforementioned dual-target nucleic acid molecules or their pharmaceutically acceptable salts may further include 1-6 overhanging nucleotides at their 3' and / or 5' ends of the antisense strands of the vegfa siRNA and / or ang-2 siRNA; optionally, the overhangs may be 2-6 nucleotides, 1-5 nucleotides, 2-5 nucleotides, 1-4 nucleotides, 2-4 nucleotides, 1-3 nucleotides, 2-3 nucleotides, 1-2 nucleotides, or 2 nucleotides.

[0055] In some embodiments, the antisense strands of the aforementioned dual-target nucleic acid molecules or their pharmaceutically acceptable salts differ in length by more than two nucleotides.

[0056] Optionally, the complementary region formed by the sense and antisense strands of the vegfa siRNA is 21 bp, and the 3' end of the antisense strand of the vegfa siRNA has two protruding ends; wherein the complementary region formed by the sense and antisense strands of the ang-2 siRNA is 21 bp, and the antisense strand of the ang-2 siRNA does not have protruding ends.

[0057] In some embodiments, the antisense strand of the above-mentioned dual-target nucleic acid molecule or its pharmaceutically acceptable salt, in the vegfa siRNA, is a polynucleotide sequence shown in SEQ ID NO:6, 10, 20, 28, 44 or 46, or the antisense strand is a polynucleotide sequence shown in positions 1-21 of SEQ ID NO:6, 10, 20, 28, 44 or 46;

[0058] And / or, in ang-2 siRNA, the antisense strand is a polynucleotide sequence as shown in SEQ ID NO:48, 60, 62, 64, 70 or 90, or the antisense strand is a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO:48, 60, 62, 64, 70 or 90.

[0059] In some embodiments, the aforementioned dual-target nucleic acid molecules or their pharmaceutically acceptable salts, in vegfa siRNA:

[0060] The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 5, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 6;

[0061] The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 9, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 10;

[0062] The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 19, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 20;

[0063] The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 27, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 28;

[0064] The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 43, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 44;

[0065] Alternatively, the sense strand may be a polynucleotide sequence as shown in SEQ ID NO: 45, and the antisense strand may be a polynucleotide sequence as shown in SEQ ID NO: 46.

[0066] In some embodiments, the aforementioned dual-target nucleic acid molecules or their pharmaceutically acceptable salts, wherein: ang-2 siRNA contains:

[0067] The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 47, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 48 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 48;

[0068] The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 59, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 60 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 60;

[0069] The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 61, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 62 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 62;

[0070] The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 63, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 64 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 64;

[0071] The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 69, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 70 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 70;

[0072] Alternatively, the sense strand may be a polynucleotide sequence as shown in SEQ ID NO: 89, and the antisense strand may be a polynucleotide sequence as shown in SEQ ID NO: 90 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 90.

[0073] In some embodiments, the above-mentioned dual-target nucleic acid molecule or its pharmaceutically acceptable salt, in the vegfa siRNA: the sense strand is a polynucleotide sequence as shown in SEQ ID NO: 43, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 44;

[0074] In ang-2siRNA: the sense strand is a polynucleotide sequence as shown in SEQ ID NO: 69, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 70 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 70.

[0075] In some embodiments, the motifs of the above-mentioned dual-target nucleic acid molecules or their pharmaceutically acceptable salts, wherein the motifs of the positive strand of the vegfa siRNA and the positive strand of the ang-2 siRNA independently comprise or are one of the following motifs:

[0076] (1) The 2' position of the 7th and 9th-11th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methyl.

[0077] (2) The 2' position of the 7th, 9th and 11th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methyl.

[0078] (3) The 2' position of the 9th and 11th nucleotides starting from the 5' end of the positive strand is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the positive strand is 2'-O-methyl.

[0079] (4) The 2' position of the 7th and 9th-12th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methyl.

[0080] Among them, the deoxyribonucleotides corresponding to ribonucleotides A, C, and G are dA, dC, and dG, and the deoxyribonucleotide corresponding to ribonucleotide U is dT.

[0081] In some embodiments, the motifs of the above-mentioned dual-target nucleic acid molecules or their pharmaceutically acceptable salts, wherein the antisense strands of the vegfa siRNA and the ang-2 siRNA independently contain or are one of the following motifs:

[0082] (1) The 2' position of the nucleotides at positions 2, 14 and 16 of the antisense strand starting from the 5' end is 2'-fluorinated, and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methyl.

[0083] (2) The 2' position of the nucleotides at positions 2, 14 and 16 of the antisense strand starting from the 5' end is 2'-fluorinated, and the nucleotides at positions 5 and 7 are the corresponding deoxyribonucleotides (dN). The 2' position of the remaining nucleotides of the antisense strand is 2'-O-methylated, of which: the corresponding deoxyribonucleotides of ribonucleotides A, C and G are dA, dC and dG, and the corresponding deoxyribonucleotide of ribonucleotide U is dT;

[0084] Optionally, the antisense strands of vegfa siRNA and ang-2 siRNA also independently contain a 5'-VP modification at the 5' position of the first nucleotide starting from the 5' end.

[0085] In some embodiments, the motif combinations consisting of the sense and antisense strand motifs of the aforementioned dual-target nucleic acid molecules or their pharmaceutically acceptable salts, wherein the vegfa siRNA and ang-2 siRNA independently comprise or are one of the following motif combinations:

[0086] (1) Sensitive strand motif: The 2' position of the 7th and 9th-11th nucleotides starting from the 5' end is 2'-fluorinated, and the 2' position of the remaining nucleotides in the sense strand is 2'-O-methylated; Antisense strand motif: The 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end is 2'-fluorinated, and the 2' position of the remaining nucleotides in the antisense strand is 2'-O-methylated.

[0087] (2) Sensitive strand motif: The 2' position of the 7th, 9th and 11th nucleotides starting from the 5' end is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the positive strand is 2'-O-methylated; Antisense strand motif: The 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end is 2'-fluorinated, the 5th and 7th nucleotides are the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methylated.

[0088] (3) Sensitive strand motif: The 2' position of the 9th and 11th nucleotides starting from the 5' end is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the positive strand is 2'-O-methylated; Antisense strand motif: The 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end is 2'-fluorinated, the 5th and 7th nucleotides are the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methylated.

[0089] (4) Sensitive strand motif: The 2' position of the 7th and 9th-12th nucleotides starting from the 5' end is 2'-fluorinated, and the 2' position of the remaining nucleotides in the sense strand is 2'-O-methylated; Antisense strand motif: The 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end is 2'-fluorinated, and the 2' position of the remaining nucleotides in the antisense strand is 2'-O-methylated.

[0090] Among them: the corresponding deoxyribonucleotides of ribonucleotides A, C, and G are dA, dC, and dG, and the corresponding deoxyribonucleotide of ribonucleotide U is dT;

[0091] Optionally, the motif combination of the sense strand motif and the antisense strand motif of the vegfa siRNA and ang-2 siRNA independently includes, in addition, the 5' position of the first nucleotide of the antisense strand starting from the 5' end being modified with 5'-VP.

[0092] In some embodiments, the above-mentioned dual-target nucleic acid molecules or their pharmaceutically acceptable salts are linked by phosphate thioester bonds between the first 1-3 nucleotides of the vegfa siRNA antisense strand and the ang-2 siRNA antisense strand, both starting from their 5' ends and starting from their 3' ends.

[0093] In some embodiments, the above-mentioned dual-target nucleic acid molecules are connected by a linker to form the positive strand of vegfa siRNA and the positive strand of ang-2 siRNA in the following structure: (5')vegfa siRNA positive strand (3')-(5')linker (3')-(5')ang-2 siRNA positive strand (3'):

[0094] The positive strands of vegfa siRNA and ang-2 siRNA are linked by phosphate thioester bonds, starting from the 5' end and the 3' end, respectively.

[0095] Alternatively, the first three nucleotides of the positive strand of vegfa siRNA are linked by thiophosphate bonds starting from its 5' end, and the first three nucleotides of the positive strand of ang-2 siRNA are linked by thiophosphate bonds starting from its 3' end, and the first three nucleotides of the positive strand of vegfa siRNA are linked by phosphate bonds starting from its 3' end, and the first three nucleotides of the positive strand of ang-2 siRNA are linked by phosphate bonds starting from its 5' end.

[0096] In some embodiments, the aforementioned dual-target nucleic acid molecule or its pharmaceutically acceptable salt is selected from the following molecules: VA4, VA4-17, VA4-18, VA4-20, VA5, VA5-17, VA5-18, and VA5-20.

[0097] Wherein: Am, Um, Cm, and Gm represent ribonucleotides A, U, C, and G modified with 2′-O-methyl, respectively; Af, Uf, Cf, and Gf represent ribonucleotides A, U, C, and G modified with 2′-fluorine, respectively; s between adjacent nucleotides indicates that adjacent nucleotides are linked by a thiophosphate bond, and the absence of an s indicates that adjacent nucleotides are linked by a phosphate bond; dC and dG represent the deoxyribonucleotides corresponding to ribonucleotides C and G, respectively, and dT represents deoxyribonucleotide T; 5′VP represents 5′-vinylphosphonate modification; hdt represents 2′-S-linear C16 alkyl modification.

[0098] In some embodiments, the pharmaceutically acceptable salt of the aforementioned dual-target nucleic acid molecule or its pharmaceutically acceptable salt is a pharmaceutically acceptable salt selected from the following molecules: VA4, VA4-17, VA4-18, VA4-20, VA5, VA5-17, VA5-18, and VA5-20; optionally, the pharmaceutically acceptable salt is a sodium salt, potassium salt, or ammonium salt.

[0099] The fifth aspect of this application also provides a nucleic acid delivery system comprising the multi-target nucleic acid molecule of the aforementioned third aspect or a pharmaceutically acceptable salt thereof, or the dual-target nucleic acid molecule of the aforementioned fourth aspect that inhibits the expression of the vegfa gene and the ang-2 gene or a pharmaceutically acceptable salt thereof.

[0100] In some embodiments, the aforementioned nucleic acid delivery body is a liposome, lipid nanoparticle or other polymer, endosome, exosome or vesicle.

[0101] A sixth aspect of this application provides a cell comprising the multi-target nucleic acid molecule of the third aspect above or a pharmaceutically acceptable salt thereof, or the dual-target nucleic acid molecule of the fourth aspect above that inhibits the expression of the vegfa gene and the ang-2 gene or a pharmaceutically acceptable salt thereof.

[0102] The seventh aspect of this application provides a pharmaceutical composition comprising the multi-target nucleic acid molecule of the third aspect or a pharmaceutically acceptable salt thereof, or the dual-target nucleic acid molecule of the fourth aspect that inhibits the expression of the vegfa gene and the ang-2 gene or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients.

[0103] The eighth aspect of this application provides the use of the multi-target nucleic acid molecule of the aforementioned third aspect or a pharmaceutically acceptable salt thereof, the dual-target nucleic acid molecule of the aforementioned fourth aspect that inhibits the expression of the vegfa gene and the ang-2 gene or a pharmaceutically acceptable salt thereof, the nucleic acid delivery body of the aforementioned fifth aspect, the cell of the aforementioned sixth aspect, or the pharmaceutical composition of the aforementioned seventh aspect in the preparation of a medicament.

[0104] In some embodiments of the above-described uses, the drug is a drug that inhibits the expression of the vegfa gene and / or ang-2 gene in the subject; optionally, inhibiting the expression of the vegfa gene and / or ang-2 gene in the subject is for the prevention and / or treatment of ocular disorders; further optionally, the ocular disorder is age-related macular degeneration and / or diabetic macular edema; even further optionally, the ocular disorder is wet age-related macular degeneration and / or diabetic macular edema.

[0105] The ninth aspect of this application also provides a method for inhibiting the expression of the vegfa gene and / or ang-2 gene in a subject, comprising the following steps:

[0106] The administration of an effective dose of the aforementioned third aspect of a multi-target nucleic acid molecule or a pharmaceutically acceptable salt thereof, the aforementioned fourth aspect of a dual-target nucleic acid molecule or a pharmaceutically acceptable salt thereof that inhibits the expression of the vegfa gene and / or ang-2 gene, the aforementioned fifth aspect of a nucleic acid delivery body, the aforementioned sixth aspect of a cell, or the aforementioned seventh aspect of a pharmaceutical composition to a subject who requires inhibition of the expression of the vegfa gene and / or ang-2 gene.

[0107] In some embodiments of the above method, the expression of the vegfa gene and / or ang-2 gene in the subject is inhibited to prevent or treat the subject's ocular impairment; optionally, the ocular impairment is age-related macular degeneration and / or diabetic macular edema; further optionally, the ocular impairment is wet age-related macular degeneration and / or diabetic macular edema.

[0108] The tenth aspect of this application also provides a multi-target nucleic acid molecule of the aforementioned third aspect or a pharmaceutically acceptable salt thereof, a dual-target nucleic acid molecule of the aforementioned fourth aspect that inhibits the expression of the vegfa gene and ang-2 gene or a pharmaceutically acceptable salt thereof, a nucleic acid delivery body of the aforementioned fifth aspect, a cell of the aforementioned sixth aspect, or a pharmaceutical composition of the aforementioned seventh aspect, for preparing a drug; optionally, the drug is a drug that inhibits the expression of the vegfa gene and / or ang-2 gene in a subject; further optionally, inhibiting the expression of the vegfa gene and / or ang-2 gene in a subject is for the prevention and / or treatment of an ocular disorder; even further optionally, the ocular disorder is age-related macular degeneration and / or diabetic macular edema; even further optionally, the ocular disorder is wet age-related macular degeneration and / or diabetic macular edema.

[0109] The eleventh aspect of this application provides a vegfa siRNA or a pharmaceutically acceptable salt thereof for inhibiting vegfa gene expression, comprising or composed of complementary sense and antisense strands, wherein the nucleotide length of the antisense strand of the vegfa siRNA is 18-23 nt, and the antisense strand comprises or is a continuous nucleotide sequence of 18-21 nucleotides, consisting of no more than 3 nucleotides distinct from the 1st to 21st nucleotides starting from the 5' end of any one of the sequences SEQ ID NO: 6, 10, 20, 28, 44, 46.

[0110] In some embodiments, the difference of no more than 3 nucleotides in the above-mentioned vegfa siRNA or its pharmaceutically acceptable salt may be a difference of 3 nucleotides, a difference of 2 nucleotides, a difference of 1 nucleotide, or complete sameness.

[0111] In some embodiments, the vegfa siRNA or its pharmaceutically acceptable salt may have a 18-21 consecutive nucleotide sequence, which may be 18, 19, 20 or 21 consecutive nucleotide sequences.

[0112] In some embodiments, the above-mentioned vegfa siRNA or its pharmaceutically acceptable salt may further include 1-6 overhanging nucleotides at its 3' and / or 5' ends; optionally, the overhangs may be 2-6 nucleotides, 1-5 nucleotides, 2-5 nucleotides, 1-4 nucleotides, 2-4 nucleotides, 1-3 nucleotides, 2-3 nucleotides, 1-2 nucleotides, or 2 nucleotides.

[0113] And / or, wherein the vegfa siRNA antisense strand further comprises 1-6 overhanging nucleotides at its 3' end and / or 5' end; optionally, the overhangs may be 2-6 nucleotides, 1-5 nucleotides, 2-5 nucleotides, 1-4 nucleotides, 2-4 nucleotides, 1-3 nucleotides, 2-3 nucleotides, 1-2 nucleotides, or 2 nucleotides.

[0114] In some embodiments, the above-mentioned vegfa siRNA or its pharmaceutically acceptable salt may be a polynucleotide sequence as shown in SEQ ID NO:6, 10, 20, 28, 44, 46 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO:6, 10, 20, 28, 44, 46.

[0115] In some embodiments, the above-mentioned vegfa siRNA or its pharmaceutically acceptable salts, wherein:

[0116] The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 5, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 6 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 6;

[0117] The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 9, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 10 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 10;

[0118] The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 19, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 20 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 20;

[0119] The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 27, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 28 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 28;

[0120] The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 43, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 44 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 44;

[0121] Alternatively, the sense strand may be a polynucleotide sequence as shown in SEQ ID NO: 45, and the antisense strand may be a polynucleotide sequence as shown in SEQ ID NO: 46 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 46.

[0122] In some embodiments, the above-mentioned vegfa siRNA or its pharmaceutically acceptable salt, wherein the motif of the sense strand of said vegfa siRNA comprises or is one of the following motifs:

[0123] (1) The 2' position of the 7th and 9th-11th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methyl.

[0124] (2) The 2' position of the 7th, 9th and 11th nucleotides starting from the 5' end of the positive strand is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the positive strand is 2'-O-methyl.

[0125] (3) The 2' position of the 9th and 11th nucleotides starting from the 5' end of the positive strand is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the positive strand is 2'-O-methyl.

[0126] (4) The 2' position of the 7th and 9th-12th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methyl.

[0127] (5) The 6th position of the justice chain starting from the 5' end is 2'-S-straight chain C. 16 Alkyl modification or 2'-O-straight-chain C 16 Alkyl modification: the 2' position of the 7th and 9th-11th nucleotides starting from the 5' end of the sense chain is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense chain is 2'-O-methyl.

[0128] (6) The 7th position of the justice chain starting from the 5' end is 2'-S-straight chain C. 16 Alkyl modification or 2'-O-straight-chain C 16Alkyl modification: the 2' position of the nucleotides at positions 9-11 starting from the 5' end of the sense chain is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense chain is 2'-O-methyl.

[0129] Among them, the corresponding deoxyribonucleotides of ribonucleotides A, C, and G are dA, dC, and dG, and the corresponding deoxyribonucleotide of ribonucleotide U is dT.

[0130] In some embodiments, the above-mentioned vegfa siRNA or its pharmaceutically acceptable salt, wherein the motif of the antisense strand of the vegfa siRNA comprises or is one of the following motifs:

[0131] (1) The 2' position of the nucleotides at positions 2, 14 and 16 of the antisense strand starting from the 5' end is 2'-fluorinated, and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methyl.

[0132] (2) The 2' position of the nucleotides at positions 2, 14 and 16 of the antisense strand starting from the 5' end is 2'-fluorinated, and the nucleotides at positions 5 and 7 are the corresponding deoxyribonucleotides (dN). The 2' position of the remaining nucleotides of the antisense strand is 2'-O-methylated. Among them, the corresponding deoxyribonucleotides of ribonucleotides A, C and G are dA, dC and dG, and the corresponding deoxyribonucleotide of ribonucleotide U is dT.

[0133] Optionally, the antisense motif also includes a 5'-VP modification at the 5' position of the first nucleotide starting from the 5' end of the antisense strand.

[0134] In some embodiments, the above-mentioned vegfa siRNA or its pharmaceutically acceptable salts, wherein the motif combination consisting of the sense and antisense strand motifs of the vegfa siRNA comprises or is one of the following motif combinations:

[0135] (1) The 2' position of the 7th and 9th-11th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methylated; the 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end of the antisense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methylated.

[0136] (2) The 2' position of the 7th, 9th and 11th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methylated; The 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end of the antisense strand is 2'-fluorinated, the 5th and 7th nucleotides are the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methylated.

[0137] (3) The 2' position of the 9th and 11th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methylated; The 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end of the antisense strand is 2'-fluorinated, the 5th and 7th nucleotides are the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methylated.

[0138] (4) The 2' position of the 7th and 9th-12th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methylated; the 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end of the antisense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methylated.

[0139] Among them, the corresponding deoxyribonucleotides of ribonucleotides A, C, and G are dA, dC, and dG, and the corresponding deoxyribonucleotide of ribonucleotide U is dT;

[0140] Optionally, the motif assembly also includes a 5'-VP modification at the 5' position of the first nucleotide of the antisense strand starting from the 5' end.

[0141] In some embodiments, the above-mentioned vegfa siRNA or its pharmaceutically acceptable salt is linked by phosphate thioester bonds between the first 1-3 nucleotides of the antisense strand of the vegfa siRNA starting from its 5' end and / or between the first 1-3 nucleotides starting from its 3' end.

[0142] In some embodiments, the above-mentioned vegfa siRNA or its pharmaceutically acceptable salt is linked by phosphate thioester bonds between the first 1-3 nucleotides of the positive strand of the vegfa siRNA starting from its 5' end and / or between the first 1-3 nucleotides starting from its 3' end.

[0143] In some embodiments, the above-mentioned vegfa siRNA or its pharmaceutically acceptable salt is selected from the following molecules (see Table 1 for motif descriptions of the present invention):

[0144] The sense strand of the nucleic acid molecule is SEQ ID NO: 43 with modification motif 5, and the antisense strand is SEQ ID NO: 44 with modification motif 7.

[0145] The sense strand of the nucleic acid molecule is SEQ ID NO: 43 with modification motif 11, and the antisense strand is SEQ ID NO: 44 with modification motif 12.

[0146] The sense strand of the nucleic acid molecule is SEQ ID NO: 43 with modification motif 14, and the antisense strand is SEQ ID NO: 44 with modification motif 12.

[0147] The sense strand of the nucleic acid molecule is SEQ ID NO: 43 with modification motif 15, and the antisense strand is SEQ ID NO: 44 with modification motif 7.

[0148] The sense strand of the nucleic acid molecule is SEQ ID NO: 43 with modification motif 5, and the antisense strand is nucleotides 1-21 from 5' of SEQ ID NO: 44 with modification motif 10.

[0149] The sense strand of the nucleic acid molecule is SEQ ID NO: 43 modified with motif 11, and the antisense strand is nucleotides 1-21 from 5' of SEQ ID NO: 44 modified with motif 13.

[0150] The sense strand of the nucleic acid molecule is SEQ ID NO: 43 modified with motif 14, and the antisense strand is nucleotides 1-21 from 5' of SEQ ID NO: 44 modified with motif 13.

[0151] The sense strand of the nucleic acid molecule is SEQ ID NO: 43 with modification motif 15, and the antisense strand is nucleotides 1-21 from 5' of SEQ ID NO: 44 with modification motif 10.

[0152] Optionally, the three nucleotides at the 3' end and the three nucleotides at the 5' end of the sense strand of the nucleic acid molecule are linked by phosphate thioester bonds, and / or, the three nucleotides at the 3' end and the three nucleotides at the 5' end of the antisense strand of the nucleic acid molecule are linked by phosphate thioester bonds.

[0153] In some embodiments, the above-mentioned vegfa siRNA or its pharmaceutically acceptable salt may be a sodium salt, potassium salt, or ammonium salt.

[0154] The twelfth aspect of this application provides an ang-2 siRNA or a pharmaceutically acceptable salt thereof for inhibiting ang-2 gene expression, comprising or composed of complementary sense and antisense strands, wherein the nucleotide length of the antisense strand of the ang-2 siRNA is 18-23 nt, and the antisense strand comprises or is a sequence of 18-21 consecutive nucleotides, consisting of no more than 3 nucleotides distinct from the 1st to 21st nucleotides from the 5' end of any one of the sequences SEQ ID NO:48, 60, 62, 64, 70, 90.

[0155] In some embodiments, the difference of no more than 3 nucleotides in the above-mentioned ang-2siRNA or its pharmaceutically acceptable salt is defined as a difference of 3 nucleotides, a difference of 2 nucleotides, a difference of 1 nucleotide, or complete sameness.

[0156] In some embodiments, the 18-21 consecutive nucleotide sequence of the above-mentioned ang-2siRNA or its pharmaceutically acceptable salt is 18, 19, 20 or 21 consecutive nucleotide sequences.

[0157] In some embodiments, the above-mentioned ang-2siRNA or its pharmaceutically acceptable salt further comprises 1-6 overhanging nucleotides at its 3' end and / or 5' end; optionally, the overhangs may be 2-6 nucleotides, 1-5 nucleotides, 2-5 nucleotides, 1-4 nucleotides, 2-4 nucleotides, 1-3 nucleotides, 2-3 nucleotides, 1-2 nucleotides, or 2 nucleotides;

[0158] And / or, wherein the ang-2siRNA antisense strand further comprises 1-6 overhanging nucleotides at its 3' end and / or 5' end; optionally, the overhangs may be 2-6 nucleotides, 1-5 nucleotides, 2-5 nucleotides, 1-4 nucleotides, 2-4 nucleotides, 1-3 nucleotides, 2-3 nucleotides, 1-2 nucleotides, or 2 nucleotides.

[0159] In some embodiments, the above-mentioned ang-2siRNA or its pharmaceutically acceptable salt is wherein the antisense strand is a polynucleotide sequence as shown in SEQ ID NO:48, 60, 62, 64, 70, 90 or a polynucleotide sequence as shown in SEQ ID NO:48, 60, 62, 64, 70, 90, 1-21.

[0160] In some embodiments, the above-mentioned ang-2 siRNA or its pharmaceutically acceptable salts, wherein:

[0161] The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 47, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 48 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 48;

[0162] The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 59, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 60 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 60;

[0163] The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 61, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 62 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 62;

[0164] The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 63, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 64 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 64;

[0165] The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 69, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 70 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 70;

[0166] Alternatively, the sense strand may be a polynucleotide sequence as shown in SEQ ID NO: 89, and the antisense strand may be a polynucleotide sequence as shown in SEQ ID NO: 90 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 90.

[0167] In some embodiments, the motif of the above-mentioned ang-2siRNA or its pharmaceutically acceptable salt comprises or is one of the following motifs:

[0168] (1) The 2' position of the 7th and 9th-11th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methyl.

[0169] (2) The 2' position of the 7th, 9th and 11th nucleotides starting from the 5' end of the positive strand is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the positive strand is 2'-O-methyl.

[0170] (3) The 2' position of the 9th and 11th nucleotides starting from the 5' end of the positive strand is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the positive strand is 2'-O-methyl.

[0171] (4) The 2' position of the 7th and 9th-12th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methyl.

[0172] (5) The 6th position of the justice chain starting from the 5' end is 2'-S-straight chain C. 16 Alkyl modification or 2'-O-straight-chain C 16 Alkyl modification: the 2' position of the 7th and 9th-11th nucleotides starting from the 5' end of the sense chain is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense chain is 2'-O-methyl.

[0173] (6) The 7th position of the justice chain starting from the 5' end is 2'-S-straight chain C. 16 Alkyl modification or 2'-O-straight-chain C 16 Alkyl modification: the 2' position of the nucleotides at positions 9-11 starting from the 5' end of the sense chain is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense chain is 2'-O-methyl.

[0174] Among them, the corresponding deoxyribonucleotides of ribonucleotides A, C, and G are dA, dC, and dG, and the corresponding deoxyribonucleotide of ribonucleotide U is dT.

[0175] In some embodiments, the motif of the antisense strand of the ang-2siRNA or a pharmaceutically acceptable salt thereof comprises or is one of the following motifs:

[0176] (1) The 2' position of the nucleotides at positions 2, 14 and 16 of the antisense strand starting from the 5' end is 2'-fluorinated, and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methyl.

[0177] (2) The 2' position of the nucleotides at positions 2, 14 and 16 of the antisense strand starting from the 5' end is 2'-fluorinated, and the nucleotides at positions 5 and 7 are the corresponding deoxyribonucleotides (dN). The 2' position of the remaining nucleotides of the antisense strand is 2'-O-methyl.

[0178] Among them, the corresponding deoxyribonucleotides of ribonucleotides A, C, and G are dA, dC, and dG, and the corresponding deoxyribonucleotide of ribonucleotide U is dT;

[0179] Optionally, the antisense motif also includes a 5'-VP modification at the 5' position of the first nucleotide starting from the 5' end of the antisense strand.

[0180] In some embodiments, the motif combination of the above-mentioned ang-2siRNA or its pharmaceutically acceptable salt, wherein the motif combination of the sense strand motif and the antisense strand motif of the ang-2siRNA comprises or is one of the following motif combinations:

[0181] (1) The 2' position of the 7th and 9th-11th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methylated; the 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end of the antisense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methylated.

[0182] (2) The 2' position of the 7th, 9th and 11th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methylated; The 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end of the antisense strand is 2'-fluorinated, the 5th and 7th nucleotides are the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methylated.

[0183] (3) The 2' position of the 9th and 11th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methylated; The 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end of the antisense strand is 2'-fluorinated, the 5th and 7th nucleotides are the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methylated.

[0184] (4) The 2' position of the 7th and 9th-12th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methylated; the 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end of the antisense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methylated.

[0185] Among them, the corresponding deoxyribonucleotides of ribonucleotides A, C, and G are dA, dC, and dG, and the corresponding deoxyribonucleotide of ribonucleotide U is dT;

[0186] Optionally, the motif assembly also includes a 5'-VP modification at the 5' position of the first nucleotide of the antisense strand starting from the 5' end.

[0187] In some embodiments, the above-mentioned ang-2siRNA or its pharmaceutically acceptable salt is linked by phosphate thioester bonds between the first 1-3 nucleotides of the antisense strand of the ang-2siRNA starting from its 5' end and / or between the first 1-3 nucleotides starting from its 3' end.

[0188] In some embodiments, the above-mentioned ang-2siRNA or its pharmaceutically acceptable salt is linked by phosphate thioester bonds between the first 1-3 nucleotides of the positive strand of the ang-2siRNA starting from its 5' end and / or between the first 1-3 nucleotides starting from its 3' end.

[0189] In some embodiments, the nucleic acid molecule or its pharmaceutically acceptable salt is selected from the following molecules (motif descriptions are shown in Table 1):

[0190] The sense strand of the nucleic acid molecule is SEQ ID NO: 69 with modification motif 5, and the antisense strand is SEQ ID NO: 70 with modification motif 7.

[0191] The sense strand of the nucleic acid molecule is SEQ ID NO: 69 with modification motif 11, and the antisense strand is SEQ ID NO: 70 with modification motif 12.

[0192] The sense strand of the nucleic acid molecule is SEQ ID NO: 69 with motif 14 modified, and the antisense strand is SEQ ID NO: 70 with motif 12 modified.

[0193] The sense strand of the nucleic acid molecule is SEQ ID NO: 69 with modification motif 15, and the antisense strand is SEQ ID NO: 70 with modification motif 7.

[0194] The sense strand of the nucleic acid molecule is SEQ ID NO: 69 with modification motif 5, and the antisense strand is nucleotides 1-21 from 5' of SEQ ID NO: 70 with modification motif 10.

[0195] The sense strand of the nucleic acid molecule is SEQ ID NO: 69 with modification motif 11, and the antisense strand is nucleotides 1-21 from 5' of SEQ ID NO: 70 with modification motif 13.

[0196] The sense strand of the nucleic acid molecule is SEQ ID NO: 69 with motif 14 modified, and the antisense strand is nucleotides 1-21 from 5' of SEQ ID NO: 70 with motif 13 modified.

[0197] The sense strand of the nucleic acid molecule is SEQ ID NO: 69 with modification motif 15, and the antisense strand is nucleotides 1-21 from 5' of SEQ ID NO: 70 with modification motif 10.

[0198] Optionally, the three nucleotides at the 3' end and the three nucleotides at the 5' end of the sense strand of the nucleic acid molecule are linked by phosphate thioester bonds, and / or, the three nucleotides at the 3' end and the three nucleotides at the 5' end of the antisense strand of the nucleic acid molecule are linked by phosphate thioester bonds.

[0199] In some embodiments, the aforementioned ang-2siRNA or its pharmaceutically acceptable salt may be a sodium salt, potassium salt, or ammonium salt.

[0200] The preferred embodiments of this application have been described in detail above; however, this application is not limited thereto. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solutions of this application, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in this application and are all within the protection scope of this application. The aspects and embodiments of this application described herein include aspects and embodiments described as "comprising," "forming," and "substantially consisting of." Attached Figure Description

[0201] Figure 1a shows the effect of the vegfa siRNA candidate sequence on the expression of the vegfa gene in ARPE-19 cells in vitro at a transfection concentration of 10 nM; Figure 1b shows the effect of the vegfa siRNA candidate sequence on the expression of the vegfa gene in ARPE-19 cells in vitro at a transfection concentration of 1 nM.

[0202] Figure 2a shows the effect of the ang-2 siRNA candidate sequence on the expression of the ang-2 gene in ARPE-19 cells in vitro at a transfection concentration of 10 nM; Figure 2b shows the effect of the ang-2 siRNA candidate sequence on the expression of the ang-2 gene in U87MG cells in vitro at a transfection concentration of 10 nM.

[0203] Figure 3a shows the effect of administering the vegfa siRNA candidate modification sequence to mice on vegfa gene expression in mouse retinal tissue; Figure 3b shows the effect of administering the vegfa siRNA candidate modification sequence to rabbits on vegfa gene expression in rabbit retinal tissue.

[0204] Figure 4a shows the effect of administering the ang-2 siRNA candidate modification sequence to mice on the expression of the ang-2 gene in the mouse retina; Figure 4b shows the effect of administering the ang-2 siRNA candidate modification sequence to rabbits on the expression of the ang-2 gene in the rabbit retina.

[0205] Figure 5 shows the effect of administering V14, a candidate vegfa siRNA sequence with different modifications, to mice on the expression of the vegfa gene in the mouse retinal tissue.

[0206] Figure 6 shows the effect of administering mice with different modified ang-2 siRNA candidate sequences A12 on ang-2 gene expression in mouse retinal tissue.

[0207] Figure 7a shows the effect of each group on the expression of vegfa protein in the retinal tissue of mice after administration in Example 6; Figure 7b shows the effect of each group on the expression of ang-2 protein in the retinal tissue of mice after administration in Example 7.

[0208] Figure 8 shows the leakage area of ​​each group of molecules in the rabbit disease model after administration in Example 7.

[0209] Figure 9 shows the leakage area of ​​each group of molecules in the rabbit disease model after administration in Example 8.

[0210] Application details

[0211] This application provides a linker and a dual-target nucleic acid molecule containing it that inhibits the expression of the vegfa and ang-2 genes. The linker has a specific lipophilic modification; compared to when this specific lipophilic modification is located inside the two connected parts of the dual nucleic acid molecule structure, when the specific lipophilic modification is located on the linker, the nucleic acid molecule exhibits better efficacy and longer-lasting therapeutic effect. Furthermore, the nucleic acid molecules screened by the inventors can more specifically silence the corresponding genes and inhibit the expression of the corresponding proteins. The nucleic acid molecules of this application have the advantage of long-lasting therapeutic effect. In particular, the modified siRNA molecule, while maintaining high inhibitory activity and stability, also has a good ability to promote endocytosis, which can reduce the amount of siRNA molecule used, thereby reducing toxicity and lowering costs. The modified siRNA molecule can enter target cells and tissues without transfection reagents, reducing the negative effects of transfection reagents, such as cell or tissue toxicity.

[0212] the term

[0213] For the purposes of this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which the described technology pertains. All technical and patent disclosures referenced in this application are incorporated herein by reference in their entirety.

[0214] As used in this application, the term "linker" has a meaning well-known in the art. As the name suggests, it functions by linking together various parts, such as UUUUUUUU or dTdTdTdTdTdT, which are common nucleotide linkers. The linked parts can be two, three, or more; each part can function independently or synergistically; the parts can be identical or different.

[0215] As used in this application, the term "dual nucleic acid molecular structure" refers to a nucleic acid molecule having two parts connected by a linker, which can play their respective roles or work synergistically; these two parts can be identical or different; the targets they target can be the same or different; when these two parts target the same target, they can target the same sequence at the same position of the same target, or they can target different positions of the same target or sequences with partial overlap.

[0216] As used in this application, the term "nucleic acid molecule" can be used to refer to any molecule having a nucleotide sequence consisting of two or more nucleotides linked together, the linkage including: linkage via phosphate ester bonds, linkage via modified phosphate ester bonds (e.g., thiophosphate ester bonds), or linkage via other linkage methods.

[0217] As used in this application, the term "nucleotide sequence" refers to a polynucleotide chain composed of nucleotides arranged in a specific order. This polynucleotide chain can constitute a nucleic acid molecule or a segment of a chain within a nucleic acid molecule. Therefore, a "nucleotide sequence" can be represented as a precise polynucleotide sequence composed of various nucleotides (e.g., ATCG), or as nucleotides at specific positions within a sequence. Without limitation, a "nucleotide sequence" can be RNA or DNA, or a hybrid molecule of RNA and DNA.

[0218] As used in this application, the term "oligonucleotide" refers to a linear polynucleotide fragment linked together. However, there is no strict requirement regarding the number of nucleotide residues when using this term; a polynucleotide fragment with fewer than 10 nucleotides can be called an oligonucleotide, as can a polynucleotide containing 30 or more nucleotides. Compared to nucleotide sequences, oligonucleotides do not emphasize the sequence composition of nucleotides.

[0219] As used herein, the term "nucleotide" refers not only to naturally occurring ribonucleotide or deoxyribonucleotide monomers, but also, as used herein, to related structural variants, including derivatives and analogs, which are functionally equivalent in relation to the specific context in which the nucleotide is used, unless the context explicitly indicates otherwise. For example, "nucleotide" refers to deoxyribonucleotides or ribonucleotides. Nucleotides can be standard nucleotides (i.e., adenosine, guanosine, cytidine, thymidine, and uridine), nucleotide isomers, or nucleotide analogs. Nucleotide analogs refer to nucleotides having modified purine or pyrimidine bases or modified ribose moieties. Nucleotide analogs can be naturally occurring nucleotides (e.g., inosine) or non-naturally occurring nucleotides. Non-limiting examples of modifications to the sugar or base moieties of nucleotides include the addition (or removal) of acetyl, amino, carboxyl, carboxymethyl, hydroxy, methyl, phosphoryl, and thiol groups, as well as the substitution of the carbon and nitrogen atoms of the base by other atoms (e.g., 7-denitropurine). Nucleotide analogs also include dideoxynucleotides, 2'-O-methylnucleotides, and locked nucleic acids (LNAs). In some embodiments, the term "nucleotide" in this application does not include non-natural nucleotides with modified bases. In some embodiments, the term "nucleotide" in this application does not include nucleotides with modified bases. In this application, "G", "C", "A", "T", and "U" generally represent nucleotides with guanine, cytosine, adenine, thymine, and uracil as bases, respectively. However, in the context of RNA and in RNA sequences, unless otherwise specified, "T" refers to uridine or uracil. It should be understood that in the context of nucleotide sequences in this application, "nucleotide", "nucleotide residue", and "base" are used interchangeably. The number of nucleotide pairs and base pairs is measured in bp, where one bp represents one nucleotide pair or one base pair. The number of nucleotides is measured in nt, where one nt represents one nucleotide.

[0220] As used in this application, a dual nucleic acid molecular structure refers to a structure in which two nucleic acid molecules are linked together by a linker.

[0221] As used in this application, the formation of hydrogen bonds between bases or nucleotides according to the Watson-Crick base pairing principle is referred to as complementarity, pairing, or matching, such as A pairing with T or U, C pairing with G or I. All other base pairings are referred to as non-complementarity. When referring to "complementarity" between polynucleotide sequences, nucleic acid chains, or polynucleotide sequences and nucleic acid chains, it means complete complementarity or complementarity of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. In complementarity, one or more mismatches are permissible, as are one or more bumps (buffs) in the polynucleotide sequence or nucleic acid chain. Therefore, the formed complementary regions can be continuous or spaced. If one of the two complementary polynucleotide sequences is shorter than the other, the calculation is based on the number of nucleotides in the shorter sequence. For example, if the sense strand is 21 nt and the antisense strand is 23 nt, the length of the complementary region is 21 bp, then it is calculated as 21 / 21, which is 100% complementary, i.e., completely complementary.

[0222] As used in this application, antisense and sense strands have the common meaning in the art. Taking the target sequence vegfa gene mRNA sequence or ang-2 gene mRNA sequence mentioned in this application as an example, antisense strand refers to the sequence in the nucleic acid molecule that is substantially complementary to the vegfa gene mRNA sequence or ang-2 gene mRNA sequence, and sense strand refers to the sequence in the nucleic acid molecule that has at least 18 consecutive identical nucleotides to the vegfa gene mRNA sequence or ang-2 gene mRNA sequence.

[0223] As used in this application, "18-21 consecutive nucleotide sequences that differ by no more than 3 nucleotides" means that when compared with 18-21 consecutive nucleotide sequences (a specific reference range in the reference sequence) from the 5' end of any one of the sequences SEQ ID NO: 6, 10, 20, 28, 44, 46, or any one of the sequences SEQ ID NO: 48, 60, 62, 64, 70, 90), the corresponding 18-21 consecutive nucleotide sequences in the nucleotide sequence used for comparison (i.e., the nucleic acid molecule of this application) differ from the specific reference range in the reference sequence by no more than 3 nucleotides. The different nucleotides can be located either in the middle or at both ends of the nucleotide sequence to be compared in the nucleic acid molecule of this application.

[0224] As used in this application, "VEGFA" or "vegfa" refers to vascular endothelial growth factor α. VEGFA is secreted by various cells, including endothelial cells and tumor cells, and is an endothelial growth factor and a regulator of vascular permeability. Its sources include, but are not limited to, any vertebrate or mammalian origin, specifically primates (e.g., humans, monkeys, and further, cynomolgus monkeys), cattle, chickens, rodents (e.g., mice, rats, guinea pigs), pigs, sheep, etc. As used herein, "ANG-2" refers to angiopoietin-2. Angiopoietin is a class of cellular regulatory factors that act on vascular endothelial cells and play an important role in angiogenesis. Targeting and inhibiting two key pathways, Ang-2 and / or VEGF-A, that cause various retinal diseases can inhibit pathological angiogenesis, reduce vascular leakage and inflammation, enhance vascular stability, improve long-term visual benefits, and improve patients' quality of life, thereby achieving stable disease control while extending treatment intervals and reducing the number of injections.

[0225] As used in this application, “VEGFA gene” or “vegfa gene” refers to the vascular endothelial growth factor a gene; “ANG-2 gene” or “ang-2 gene” refers to the angiopoietin 2 gene.

[0226] As used in this application, "VEGFA gene mRNA" refers to the mRNA encoding vascular endothelial growth factor α, which can be transcribed from VEGFA gene DNA and can be mature mRNA or pre-mRNA, thus it may or may not contain introns. Because the VEGFA gene may have a few nucleotide mutations in different individuals, unless otherwise specified, the VEGFA gene mRNA sequence in this application is intended to include all mRNA sequences transcribed from VEGFA gene mutants. Human VEGFA gene mRNA sequences can be found, for example, in gene bank accession numbers. Other examples of VEGFA gene mRNA sequences are readily available using publicly available databases, such as GenBANK. In this application, VEGFA gene mRNA is one of the target sequences.

[0227] As used herein, "ANG-2 gene mRNA" refers to the mRNA encoding angiopoietin 2, which can be transcribed from ANG-2 gene DNA and can be mature mRNA or pre-mRNA, thus it may or may not contain introns. Because the ANG-2 gene may have a few nucleotide mutations in different individuals, unless otherwise specified, the ANG-2 gene mRNA sequence in this application is intended to include all mRNA sequences transcribed from ANG-2 gene mutants. Human ANG-2 gene mRNA sequences can be found, for example, in gene bank accession numbers. Other examples of ANG-2 gene mRNA sequences are readily available using publicly available databases, such as GenBANK. In this application, ANG-2 gene mRNA is one of the target sequences.

[0228] As used herein, the term "siRNA" has the meaning known in the art as small interfering RNA, which is a primary tool for RNAi. When "siRNA" is incorporated into the RNA-induced silencing complex (RISC), one or more helicases in the RISC unwind the siRNA double helix. When it binds to a target mRNA complementary to the antisense strand of the siRNA, one or more endonucleases in the RISC cleave the target, inducing gene silencing. Typically, most or all nucleotides of each strand of the siRNA molecule are ribonucleotides, but this does not preclude the inclusion of one or two non-ribonucleotides, such as deoxyribonucleotides and / or non-natural nucleotides, in any one or two strands. In some embodiments, the siRNA molecule does not contain non-natural nucleotides. In some embodiments, each nucleotide in the dsRNA is a ribonucleotide. As used herein, siRNA may contain one or more chemically modified nucleotides or may not contain chemically modified nucleotides.

[0229] As used in this application, "protruding nucleotide" is relative to "flat-ended" or "flat-ended," which means that there are no unpaired nucleotides at the 3' and 5' ends of a double-stranded nucleotide, i.e., no protruding nucleotide; that is, "protruding nucleotide" means that in a double-stranded nucleotide, one strand has more nucleotides than the other strand, resulting in unpaired nucleotides at the 3' and 5' ends of the two strands.

[0230] As used in this application, the terms "3' end" and "5' end" have the common meaning in the art. Where no specific direction is explicitly indicated (i.e., "3' end direction" and "5' end direction"), they encompass the meanings of "3' end" and "5' end," with "3' end" and "5' end" emphasizing the specific end position. In this application, unless it would cause ambiguity to those skilled in the art, no distinction is made between "5' end" and "5' end," nor between "3' end" and "3' end."

[0231] As used in this application, the terms “close,” “near,” or “far” when describing nucleotide positions on the same sequence or nucleoside chain refer to the number of nucleotides separating the two nucleotide positions.

[0232] As used in this application, based on the location of the chemical modification, nucleotide chemical modification methods are divided into phosphate ester modification, base modification, and ribose modification. Chemical modification methods have accelerated the development of siRNA drugs. Due to the uniqueness of drugs, the specific application of chemical modification methods still needs to be explored in different specific scenarios. "Ribose modification" refers to chemical modification at various positions of the sugar ring of ribonucleic acid, including substituent modification, such as 2'-modification (e.g., 2'-OMe, 2'-F, 2'-O-MOE), 5' modification (e.g., 5'-Mo, 5'-VP), and isomer modification (e.g., LNA, GNA modification). "Phosphate ester" modification mainly refers to the modification of the phosphate ester backbone between ribose on siRNA, mainly including thiophosphate (PS), dithiophosphate (PS2), methylphosphate (MP), aminophosphate, methoxypropyl phosphate (MOP), and peptide nucleic acid (PNA), etc. In this field and in this application, phosphate ester bond and phosphodiester bond have the same meaning, and thiophosphate bond and thiophosphodiester bond have the same meaning and can be used interchangeably. Base modification refers to the chemical modification of the bases of siRNA, which is mainly divided into three forms: purine modification (such as N6-methyladenosine), pyrimidine modification (such as 5-methylcytidine), and base substitution.

[0233] The specific modification rules for a particular siRNA sequence are referred to as "motifs" in this application. The motifs involved in the embodiments are described in Table 1 below:

[0234] Table 1

[0235] Although phosphate thioester bonds are also a type of modification to siRNA sequences, the description of motif numbers and their corresponding motifs in this application (as shown in Table 1 above) does not cover the description of phosphate thioesters, but only the modifications of ribose and bases; otherwise, the types of motifs would appear too varied. The portions linked by phosphate thioester bonds will be described or represented separately. In specific modified sequences, if no 's' is marked between two adjacent nucleotides, it indicates that the nucleotides are linked by a phosphate thioester bond; if 's' is marked, it indicates that the nucleotides are linked by a phosphate thioester bond. Furthermore, dN can be described as either a 2'-deoxy modification of a ribonucleotide or as the deoxyribonucleotide itself; those skilled in the art should understand that this is merely a difference in descriptive method. Unless it would cause confusion among those skilled in the art, this application will not specifically emphasize that siRNA containing 2'-deoxy modifications is a hybrid molecule of DNA and RNA, but will only describe it as siRNA.

[0236] As used in this application, This is merely an illustration of a thiophosphate bond; other possible structures include the following: The above structures are interchangeable in this application; This is a schematic diagram of a phosphate ester bond, which also includes... The above structures are interchangeable in this application. When O - or S - The corresponding formation is like O. - Na + or S - Na + Form, that is, the salt corresponding to that nucleic acid molecule.

[0237] As used herein, “5’-vp modification” or “5’-VP modification” has the common meaning in the art, namely, 5’-vinylphosphonate modification. In the art, the E-configuration of 5’-vinylphosphonate is far superior to that of the Z-configuration, therefore, unless otherwise specified, 5’-vinylphosphonate modification in the art refers to 5’-(E)-vinylphosphonate (i.e., 5’-(E)-VP).

[0238] As used in this application, "naked sequence" is relative to the chemically modified nucleotide sequence described above. In this application, for the sake of simplicity, when a nucleotide sequence has not undergone any chemical modification, it is referred to as a naked sequence.

[0239] As used in this application, "inhibit gene expression" includes inhibition of any level of a gene, such as at least partial inhibition of the expression of the ANG-2 gene, such as inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0240] "Suppressing gene expression" includes suppressing the expression of any gene (e.g., the gene in mice, rats, monkeys, or humans) as well as variants (e.g., naturally occurring variants) or mutants of that gene. Therefore, the gene can be a wild-type gene, a mutant gene, or a transgene in the case of genetically manipulated cells, cell groups, or organisms.

[0241] As used in this application, the following abbreviations have the following corresponding meanings:

[0242] “G”, ​​“C”, “A”, “T” and “U” usually represent nucleotides with guanine, cytosine, adenine, thymine and uracil as bases, respectively.

[0243] REL (Relative expression level): relative mRNA expression level

[0244] Modifications: N = ribonucleotide; dN = deoxyribonucleotide; Nm = 2'-O-methyl modified ribonucleotide; Nf = 2'-F modified ribonucleotide; s = adjacent nucleotides linked by a phosphate thioester bond; N(hd) = 2'-O-hexadecyl modified ribonucleotide; N(hdt) = 2'-S-hexadecyl modified ribonucleotide; N(dat) = 2'-S-dodecyl modified ribonucleotide

[0245] As used in this application, the term "about" refers to the general range of error for various values ​​that is readily known to those skilled in the art. References to "about" values ​​or parameters in this application include (and describe) embodiments for that value or parameter itself. As used in this application, when the term "about" precedes a numerical value, it indicates a range of 10% above or below that value. For example, "about 100" encompasses both 90 and 110.

[0246] As used in this application, unless otherwise indicated, the singular forms “a,” “an,” and “the” include the plural forms.

[0247] Unless otherwise defined in this application, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0248] It should be understood that this application includes the various aspects, embodiments, and combinations of said aspects and / or embodiments described herein. The above description and the following embodiments are intended to illustrate, not limit, the scope of this application. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solutions of this application, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in this application and are all within the protection scope of this application.

[0249] Unless otherwise stated, the practice of this application will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. These conventional techniques are described in existing art literature. All cells were obtained from the Chinese Academy of Sciences Type Culture Collection Committee, or from other publicly available sources; other reagents are commercially available.

[0250] It should be understood that this application includes the various aspects, embodiments, and combinations of said aspects and / or embodiments described herein. The above description and the following examples are intended to illustrate, not limit, the scope of this application. Other aspects, improvements, and modifications within the scope of this application will be apparent to those skilled in the art to which this application pertains. Therefore, those skilled in the art should recognize that the scope of this application also includes the improvements and modifications to the said aspects and embodiments. Detailed Implementation

[0251] Example 1: Activity screening of naked sequences of vegfa siRNA and ang-2 siRNA

[0252] First, determine the naked vegfa siRNA sequence and ang-2 siRNA sequence that will be used in subsequent experiments.

[0253] 1. siRNA design

[0254] Referring to the target genes (as shown in Table 2), the mRNA sequence of the human vegfa gene (NM_001171626.2) or the mRNA sequence of the human ang-2 gene (NM_001147.3), multiple vegfa or ang-2 siRNAs were designed at different sites (as shown in Tables 3 and 4, respectively). All designed individual siRNAs can target all transcripts of the target genes. The above sequences showed the lowest homology with all other non-target gene sequences after sequence similarity comparison with sequence similarity software.

[0255] Table 2. Target gene information

[0256] Table 3. VEGFA siRNA sequence design

[0257] Table 4. Ang-2 sequence design

[0258] 2. siRNA synthesis (natural RNA / modified RNA)

[0259] Both unmodified naked siRNA and modified siRNA were prepared according to this method, the difference being the addition of different ribonucleoside monomers or deoxyribonucleoside monomers.

[0260] In this application, ribonucleoside monomers, deoxyribonucleoside monomers, modified ribonucleoside monomers, or modified deoxyribonucleoside monomers were synthesized according to the theoretical yield of 1 μmol, using a 1 μmol general-purpose Frit solid support. All siRNA nucleotide sequences were prepared using an LK-192X synthesizer. According to sequence requirements, the phosphoramidite monomers corresponding to the nucleosides were diluted 1:40 (g / mL) with anhydrous acetonitrile, and coupled twice for 3 min. Activation was performed using 0.3 M benzylthiotetrazole acetonitrile solution, followed by deprotection with 3% TCA, and capping and oxidation with CAPA / CAPB and 50 mM I2 solutions, respectively. After trityl-off synthesis, the solid-phase support was transferred to a 2 mL centrifuge tube, 1.2 mL of ammonia was added, and the tube was heated in a 65 °C oven for 3 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 10 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 final product was obtained by UV concentration determination using a microplate reader, mixing equimolar amounts of the sense and antisense strands, heating at 95°C for 5 min, slowly annealing to room temperature, and finally evaporating to dryness at room temperature using a vacuum concentrator.

[0261] 3. Transfect cells with naked vegfa / ang-2 siRNA sequences

[0262] ARPE-19 cells (human retinal pigment epithelial cell line) were placed in 1640 medium containing 10% fetal bovine serum, and U87MG cells (human glioblastoma of the brain) were placed in DMEM medium containing 10% fetal bovine serum. Both were cultured in a 5% CO2, 37°C incubator. Cells were seeded and transfected when they were in the logarithmic growth phase and in good condition (70% confluence). The cell density was adjusted to 2 × 10⁶ cells / year. 5 Add 1 ml of cell solution to each well of a 24-well plate and incubate overnight at 37°C with 5% CO2. Prepare the transfection complex: Mix 250 μL of Opti-MEM and 5 μL of 1 nM or 10 nM siRNA naked sequence to form a mixture. Mix 250 μL of Opti-MEM and 2.5 μL of Lipofectamine 2000 transfection reagent to form a mixture. Let stand for 5 min. Then mix the two mixtures and let stand for 20 min to form the transfection complex. Add the transfection complex to the 24-well plate and incubate at 37°C with 5% CO2 for 6 h. Aspirate the supernatant, add 1 ml of the corresponding complete culture medium to each well, and incubate for 24 h at 37°C with 5% CO2.

[0263] 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.

[0264] 4. Real-time quantitative PCR analysis of target mRNA levels

[0265] 1) After transfection for 24 hours in step 3, cells were lysed and total RNA was extracted from the cells using a column extraction kit (Novizan).

[0266] 2) One-step real-time PCR:

[0267] 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. Primers were designed for real-time quantitative PCR analysis of target mRNA levels in human, mouse, and rabbit cells. The first letter in the primer name indicates the cell origin: H for human, m for mouse, and Rabbit for rabbit.

[0268] Table 5. Primer sequence information

[0269] 5. Data Analysis

[0270] 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. Tables 5 and 6 show the average expression levels of the screened siRNA naked sequences relative to the target gene expression levels of the NC group (with the relative mRNA expression level of the NC group as 1).

[0271] Cell viability screening of naked vegfa siRNA sequences was performed in ARPE-19 cells. Twenty-three siRNA molecules were tested at concentrations of 1 nM and 10 nM (i.e., cells were transfected according to step 3 of Example 1). The target gene fragments of the 23 siRNA molecules (Table 3) are homologous to those of humans, mice, rabbits, and cynomolgus monkeys. The expression of the vegfa gene in cells after transfection with different concentrations of candidate naked vegfa siRNA sequences is shown in Figures 1a and 1b. The average relative expression levels of the target gene relative to the NC group in Figures 1a and 1b are listed in Table 6.

[0272] Cellular viability screening of naked ang-2 siRNA sequences was performed in ARPE-19 and U87MG cells. Twenty-two siRNA molecules were tested at 10 nM (Table 4). The target gene fragments of these 22 siRNA molecules were homologous to those of humans, mice, rabbits, and cynomolgus monkeys. The expression of the ang-2 gene in different cells after transfection with the candidate naked ang-2 siRNA sequences is shown in Figures 2a and 2b. The average relative expression levels of the target gene relative to the NC group in Figures 2a and 2b are listed in Table 7.

[0273] Table 6. Average values ​​of real-time quantitative PCR detection results for vegfa gene expression

[0274] Table 7. Average values ​​of ang-2 real-time quantitative PCR detection results

[0275] Example 2: In vivo efficacy detection of motif-modified siRNA molecules in mice and rabbits - vegfa mRNA detection

[0276] Six siRNA naked sequence molecules with high inhibitory activity against the Vegfa target gene were selected from Example 1. We performed the same or essentially the same motif modifications on them to further verify the efficacy in in vivo experiments (the modification details are shown in Table 8. The names of the motif-modified siRNA molecules are derived from the names of the siRNA naked sequences in Example 1. For example, the V3.1 molecule is derived from the V3 naked sequence molecule after modification, and V22.1 and V22.2 represent two different modifications to the V22 naked sequence molecule, respectively. Different types of modifications exist in the motifs involved in Table 8, including: m, f, 5'-VP, hdt, and hd modifications. Although thiophosphate bonds can also be considered as a modification of the siRNA naked sequence, the description of thiophosphate bonds is not covered in this application and in the description of the motif numbers and their corresponding motifs in Table 8. Otherwise, the types of motifs would appear too chaotic. The parts connected by thiophosphate bonds will be described or represented separately).

[0277] Male, 6-8 week old SPF-grade C57BL / 6J mice (Guangzhou Cyagen Biotech Co., Ltd.) were used in the mouse experiments. An ophthalmological examination was performed before the experiment to exclude mice with obvious ocular abnormalities. Intravitreal injection (IVT) was administered (dosage groups are shown in Table 9, and the naked sequence and modification status of the siRNA molecules administered to each group are shown in Table 8). Retinal tissues were collected on day 14, and the mice were subsequently euthanized. The collected tissues were used to detect vegfa-mRNA levels: frozen mouse retinal tissue was ground using an automated grinder, and total RNA was extracted from the mouse retinal tissue using the Trizol method. RNA purity and concentration were determined. The quantitative fluorescence method described in steps 4 and 5 of Example 1 was used to detect the mRNA expression level of the vegfa gene, and inter-group comparisons were performed.

[0278] The rabbit experiments used male New Zealand rabbits weighing 2-2.5 kg (Guangzhou Cyagen Biotech Co., Ltd.). Healthy rabbits were selected and isolated for rearing. Basic physiological parameters of the experimental animals, such as weight, age, and sex, were determined. An ophthalmological examination was performed before the experiment to exclude rabbits with obvious ocular abnormalities. IVT was administered (dosage groups are shown in Table 10, and the naked sequence and modification status of the siRNA molecules administered to each group are shown in Table 8). Retinal tissue was collected from the rabbits on day 14, and the rabbits were subsequently euthanized. The collected tissue was used for vegfa-mRNA level detection: frozen rabbit retinal tissue was ground using an automated grinder, and total RNA was extracted from the rabbit retinal tissue using the Trizol method. RNA purity and concentration were determined using a K550 analyzer. The quantitative real-time PCR method described in steps 4 and 5 of Example 1 was used to detect the mRNA expression level of the vegfa gene, and inter-group comparisons were performed.

[0279] Table 8. New modified sequences of vegfa siRNA

[0280] In Table 8, all siRNA molecules are linked by phosphate thioester bonds between the first to third nucleotides at the 3' end and between the first to third nucleotides at the 5' end of the sense and antisense strands.

[0281] Table 9. IVT-administered siRNA drug – mouse experimental protocol

[0282] Table 10. IVT-administered siRNA drug – rabbit experimental protocol

[0283] Mouse experimental results: As shown in Figure 3a, the vegfa gene mRNA expression level in the test group was significantly lower than that in the PBS control group, and the difference was statistically significant (P<0.01). Specifically, compared with the PBS control group, after 14 days of administration of V3.1, V5.1, V10.1, V14.1, and V22.1, the vegfa mRNA level was downregulated by 18%, 24%, 30%, 45%, and 53%, respectively. The experimental results indicate that the above-mentioned vegfa siRNAs can significantly downregulate the vegfa gene mRNA expression level in mice.

[0284] Rabbit experimental results: As shown in Figure 3b, the vegfa gene mRNA expression level in the test group was significantly lower than that in the PBS control group, and the difference was statistically significant (P<0.01). Specifically, compared with the PBS control group, after 14 days of V22.2 administration, the vegfa mRNA level was downregulated by 30.84%, indicating that the above-mentioned vegfa siRNA can significantly downregulate the vegfa gene mRNA expression level in rabbits.

[0285] Based on the experimental results in mice and rabbits, V22.1 showed the best effect in downregulating the expression level of the vegfa gene mRNA in mice, while V22.2 also showed a good effect in downregulating the expression level of the vegfa gene mRNA in rabbits. Subsequent experiments will use the naked V22 sequence.

[0286] Example 3: In vivo efficacy detection of motif-modified siRNA molecules in mice and rabbits - ang-2 mRNA detection

[0287] Five siRNA naked sequence molecules with high inhibitory activity against the ang-2 target gene were selected from Example 1. We performed the same motif modifications on each of them to further verify the efficacy in in vivo experiments (the modifications are shown in Table 11. The names of the motif-modified siRNA molecules are derived from the names of the siRNA naked sequences in Example 1, such as A1.1 molecule, which is derived from the A1 naked sequence molecule. Different types of modifications exist among the motifs mentioned in Table 11, including m, f, 5'-VP, and hd modifications. Although thiophosphate bonds can also be considered a type of modification to the siRNA naked sequence, the description of thiophosphate bonds is not included in the description of the motif numbers and their corresponding motifs in this application and Table 11. Otherwise, the types of motifs would appear too chaotic. The parts connected by thiophosphate bonds will be described or represented separately).

[0288] Male, 6-8 week old SPF-grade C57BL / 6J mice (Guangzhou Cyagen Biotech Co., Ltd.) were used in the mouse experiments. An ophthalmological examination was performed before the experiment to exclude mice with obvious ocular abnormalities. Intravenous transurethral resection (IVT) was administered (dosage groups are shown in Table 12, and the naked sequence and modification status of the siRNA molecules administered to each group are shown in Table 11). Retinal tissues were collected from the mice on day 14, and the mice were subsequently euthanized. The collected tissues were used to detect ang-2 mRNA levels: frozen mouse retinal tissue was ground using an automated grinder, and total RNA was extracted from the mouse retinal tissue using the Trizol method. RNA purity and concentration were determined using a K550 analyzer. The quantitative fluorescence method described in steps 4 and 5 of Example 1 was used to detect the mRNA expression level of the ang-2 gene, and intergroup comparisons were performed.

[0289] The rabbit experiments used male New Zealand rabbits weighing 2-2.5 kg (Guangzhou Cyagen Biotech Co., Ltd.). Healthy rabbits were selected and isolated for rearing. Basic physiological parameters of the experimental animals, such as weight, age, and sex, were determined. An ophthalmological examination was performed before the experiment to exclude rabbits with obvious ocular abnormalities. IVT was administered (dosage groups are shown in Table 13, and the naked sequence and modification status of the siRNA molecules administered to each group are shown in Table 11). Retinal tissue was collected from the rabbits on day 14, and the rabbits were subsequently euthanized. The collected tissue was used for ang-2 mRNA level detection: frozen rabbit retinal tissue was ground using an automated grinder, and total RNA was extracted from the rabbit retinal tissue using the Trizol method. RNA purity and concentration were determined using a K550 analyzer. The quantitative fluorescence method described in steps 4 and 5 of Example 1 was used to detect the mRNA expression level of the ang-2 gene, and inter-group comparisons were performed.

[0290] Table 11. New modified sequences of ang-2 siRNA

[0291] In Table 11, the first to third nucleotides at the 3' end and the first to third nucleotides at the 5' end of the sense and antisense strands of all siRNA molecules are linked by phosphate thioester bonds.

[0292] Table 12. IVT-administered siRNA drug-mouse experimental protocol

[0293] Table 13. IVT-administered siRNA drug – rabbit experimental protocol

[0294] Mouse experimental results: As shown in Figure 4a, the mRNA expression levels of the ang-2 gene in the test group were significantly lower than those in the PBS control group, and the differences were statistically significant (P<0.01). Specifically, compared with the PBS control group, after 14 days of administration of A1.1, A7.1, A8.1, A9.1, and A12.1, the mRNA levels of ang-2 were downregulated by 53%, 42%, 44%, 56%, and 58%, respectively. The experimental results indicate that the above-mentioned ang-2 siRNAs can significantly downregulate the mRNA expression level of the mouse ang-2 gene.

[0295] Rabbit experimental results: As shown in Figure 4b, the mRNA expression level of the ang-2 gene in the test group was significantly lower than that in the PBS control group, and the difference was statistically significant (P<0.01). Specifically, compared with the PBS control group, the mRNA level of ang-2 was downregulated by 34.12% after 14 days of A12.1 administration. The experimental results indicate that the above-mentioned ang-2 siRNA can significantly downregulate the mRNA expression level of the rabbit ang-2 gene.

[0296] Based on the experimental results in mice and rabbits, A12.1 showed the best effect in downregulating the expression level of the ang-2 gene mRNA in mice, and also had a good effect in downregulating the expression level of the vegfa gene mRNA in rabbits. Subsequent experiments will use the naked A12 sequence.

[0297] Example 4: Effectiveness test of vegfa siRNAs modified with different delivery ligands in mice to select delivery ligand modification structures.

[0298] The motif of this application involves different modifications of nucleotides, wherein: 2'-OC 16 Modification (hd modification) and 2'-SC 16Modification (hdt modification) is a modification that enables the delivery of ligands. In this example, which is parallel to Examples 2-3, to further confirm the effects of hd and hdt modifications, which act as ligand delivery agents, on the activity of Vegfa siRNA, we introduced either no hd or hdt ligand delivery modifications at the same position (5'->3', position 7) on the sense strand of the same Vegfa siRNA naked sequence. The modifications at other positions on the sense and antisense strands were completely identical (the specific modifications are shown in Table 14. The names of the modified siRNAs are derived from the names of the naked siRNA sequences in Example 1. For example, V14-unconjugated is derived from the modification of the V14 molecule without introducing ligand delivery agents, V14(7-C16-O) is derived from the hd modification of the V14 molecule at position 7 on the sense strand, and V14(7-C16-S) is derived from the hdt modification of the V14 molecule at position 7 on the sense strand. The difference between motifs 3, 4, and 5 is only in the modification at position 7 on the sense strand from 5'). The efficacy of the compounds was verified in vivo.

[0299] Male, 6-8 week old SPF-grade C57BL / 6J mice (Guangzhou Cyagen Biotech Co., Ltd.) were used for in vitro transurethral resection (IVT) administration (dosing groups are shown in Table 14, and the naked sequence and modification status of the siRNA molecules administered to each group are shown in Table 13). Retinal tissue was collected from the mice on day 14 post-administration, and the mice were subsequently euthanized. The collected tissue was used for vegfa-mRNA level detection: frozen mouse retinal tissue was picked and ground using an automated grinder. 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 vegfa gene mRNA expression level was detected using the quantitative fluorescence method described in steps 4 and 5 of Example 1, and inter-group comparisons were performed.

[0300] Table 14. Information on siRNA molecules used in Example 4

[0301] In Table 14, all the 3' end nucleotides of the sense and antisense strands are linked by phosphate thioester bonds to each other.

[0302] Table 15. Experimental protocol for IVT-treated C57BL / 6J mice

[0303] Mouse experimental results: As shown in Figure 5, the expression levels of vegfa gene mRNA in the test groups were lower than those in the PBS control group to varying degrees, and the differences were statistically significant (P<0.01). Specifically, compared with the PBS control group, after 14 days of drug administration, the vegfa mRNA levels in groups G2, G3, and G4 were downregulated by 12.18%, 28.66%, and 36.11%, respectively. This indicates that both hd and hdt modifications can improve the performance of vegfa siRNA in reducing the expression levels of its target gene mRNA; comparatively, hdt modification is more effective. Subsequent experiments will use hdt modification for vegfa siRNA.

[0304] Example 5: Effectiveness test of ang-2 siRNAs with different delivery ligand modifications in mice to select delivery ligand modification structures.

[0305] Similar to Example 4, in this example, which was conducted in parallel with Examples 2-3, to further confirm the effects of hd and hdt modifications, which act as ligand delivery agents, on the activity of ang-2 siRNA, we either did not introduce hd or hdt ligand delivery modifications at the same position (position 7, 5'->3') on the sense strand of the same ang-2 siRNA naked sequence. The modifications at other positions on the sense and antisense strands were completely identical (the specific modifications are shown in Table 16; the names of the modified siRNAs are derived from the names of the siRNA naked sequences in Example 1, such as A12-unconjugated, which is derived from the modification of A12 molecules without introducing ligand delivery agents; A12(7-C16-O), which is derived from the hd modification of position 7 on the sense strand of A12 molecules; and A12(7-C16-S), which is derived from the hdt modification of position 7 on the sense strand of A12 molecules. The difference between motifs 3, 4, and 5 is only in the modification at position 7 on the sense strand from 5'). The efficacy of the compounds was verified in vivo.

[0306] Male, 6-8 week old SPF-grade C57BL / 6J mice (Guangzhou Cyagen Biotech Co., Ltd.) were used for in vitro transurethral resection (IVT) administration (dosing groups are shown in Table 17, and the naked sequence and modification status of the siRNA molecules administered to each group are shown in Table 16). 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: frozen mouse retinal tissue was picked and ground using an automated grinder, and total RNA was extracted from the mouse retinal tissue using the Trizol method. RNA purity and concentration were determined using a K550 assay. The quantitative fluorescence method described in steps 4 and 5 of Example 1 was used to detect the mRNA expression level of the ang-2 gene, and inter-group comparisons were performed.

[0307] Table 16. Information on siRNA molecules used in Example 5

[0308] In Table 16, all the 3' terminal nucleotides of the sense and antisense strands are linked by phosphate thioester bonds between the first and third nucleotides at the 3' end and between the first and third nucleotides at the 5' end.

[0309] Table 17. IVT-induced experimental protocol in C57BL / 6J mice

[0310] Mouse experimental results: As shown in Figure 6, the expression levels of ang-2 gene mRNA in the test groups were lower than those in the PBS control group to varying degrees, and the differences were statistically significant (P<0.01). Specifically, compared with the PBS control group, after 14 days of drug administration, the ang-2 mRNA levels in groups G2, G3, and G4 (Table 17) were downregulated by 49.82%, 57.33%, and 59.14%, respectively. This indicates that both hd and hdt modifications can improve the performance of ang-2 siRNA in reducing the expression level of its target gene mRNA. Since hdt modification was chosen for the VEGFA target, subsequent experiments with ang-2 siRNA also used hdt modification.

[0311] Example 6: Efficacy testing of dual-target siRNA in mice using different drug forms.

[0312] In the process of product development, siRNA targeting two targets can be either linked together using linkers to obtain a single-target drug, or the siRNAs targeting each target can be mixed to obtain a dual-target drug combination. To determine the effect of different drug forms of dual-target siRNA on efficacy, we attempted to link siRNAs targeting vegfa and ang-2 together using linkers to obtain the dual-target single-target molecule VA1 (its sequence and modification details are shown in Table 18). We also attempted to mix siRNAs targeting vegfa and ang-2 in a 1:1 molar ratio to form a dual-target drug combination (V22.3; A12.2), and compared the efficacy of the two.

[0313] In this embodiment, the naked vegfa siRNA sequence V22 and ang-2 siRNA sequence A12, which were confirmed by the inventors in previous experiments, were used. The hdt modification, which was confirmed by the inventors in previous experiments, was used to promote delivery. After comprehensively considering the f modification, m modification and hdt modification, the modification situation is shown in Table 18. The efficacy of the compound was verified in vivo.

[0314] The mouse experiments used male, 6-8 week old SPF-grade C57BL / 6J mice (Guangzhou Cyagen Biotech Co., Ltd.). Intravenous transfusion (IVT) was administered (drug administration groups are shown in Table 18, and the naked sequence and modification status of the siRNA molecules administered to each group are also shown in Table 18). 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 the expression levels of vegfa and ang-2 proteins. The specific procedures are as follows:

[0315] 1) Sample preparation: Add 200 μL of 1X PBS (containing 1 μM PMSF) to each sample, homogenize, centrifuge at 12000 rpm for 10 min, and collect the supernatant as the sample to be tested. Then, dilute the mouse eyeball sample 5 times for subsequent ELISA operations, and at the same time measure the protein content of each sample for subsequent calibration.

[0316] 2) Sample addition: Add 100 μL of standard and sample to each well, with two replicates for each sample, and incubate at 37°C for 2 h.

[0317] 3) Discard the liquid; do not wash the plate.

[0318] 4) Add the prepared Biotin-antibody (1X) to the strip at a rate of 100 μL / well and incubate at 37°C for 1 h.

[0319] 5) Washing the plate: Add 300 μL of 1x Wash buffer to each well of the 96-well plate, pour out the liquid and wash three times in this way. After the last wash, pat the plate dry on paper to ensure that there is no washing liquid residue after each pat.

[0320] 6) HRP: Add 100 μL of HRP-avidin(1X) to each well and incubate at 37°C for 1 h.

[0321] 7) Repeat step 5 to wash the plate 5 times.

[0322] 8) Color development: Add 90 μL of TMB Substrate equilibrated to room temperature to each well and develop color at room temperature in the dark for 15 min.

[0323] 9) Stop the color development: Add 50 μL of Stop Solution per well, following the order in which the color development solution was added.

[0324] 10) Reading: Within 5 minutes after the color development is terminated, read the absorbance at 450nm. 540nm or 570nm is the correction wavelength.

[0325] 11) Data Processing: After subtracting the OD values ​​of blank wells from the OD values ​​of all wells, a standard curve was plotted. The average of the two replicates was taken, with the OD value as the ordinate and the standard curve concentration as the abscissa. The sample concentration was calculated by substituting the sample OD values ​​into the curve, and finally corrected using mouse ocular protein content.

[0326] Table 18. New modified sequences of vegfa / ang-2 siRNA

[0327] The names of the modified siRNA molecules are derived from the naked siRNA sequence names in Example 1, such as V22.3 derived from the third modification method of the V22 molecule in this application, and A12.2 derived from the second modification method of the A12 molecule in this application. Furthermore, in Table 17, the first to third nucleotides at the 3' end and the first to third nucleotides at the 5' end of all the sense and antisense strands are linked by thiophosphate bonds. In VA1, for the two target siRNAs linked by linkers, the first to third nucleotides at the 3' end and the first to third nucleotides at the 5' end of the sense strand for each target are also linked by thiophosphate bonds.

[0328] Table 19. IVT-induced experimental protocol in C57BL / 6J mice

[0329] Mouse experimental results: As shown in Figures 7a and 7b, compared with the G1 (PBS group), the expression levels of vegfa and ang-2 proteins in the test group were lower than those in the PBS control group to varying degrees, and the differences were statistically significant (P<0.01). Specifically, compared with the PBS control group, after 14 days of administration of the dual-target two-drug combination and the dual-target single-drug compound, the expression levels of vegfa protein were reduced by 37.53% and 51.42%, respectively, and the expression levels of ang-2 protein were reduced by 16.75% and 56.96%, respectively. The results indicate that, targeting both vegfa and ang-2, the efficacy of the dual-target single-drug compound was significantly better than that of the dual-target two-drug combination.

[0330] Example 7: Selection of Delivery Ligand Modification Site in Dual-Target Single-Drug Compounds

[0331] Based on the dual-target single-drug molecule VA1, the inventors attempted to optimize the dual-target single-drug molecule and validated the efficacy of their compound using a rabbit retinal angiogenesis and leakage (RNV) model induced by DL-AAA. DL-AAA is a selective glial cytotoxic agent that irreversibly damages Müller cells after intravitreal injection, leading to disruption of the retinal vascular barrier function. Stable retinal angiogenesis and leakage were formed 8–10 weeks after model induction.

[0332] Male Dutch rabbits, weighing 1.3-2.5 kg, were used. One rabbit per cage was housed in a stainless steel cage (900mm×600mm×500mm). The room for the rabbits was well-ventilated with a filter and ventilation system, providing 10-20 air changes per hour. The temperature was maintained between 16-26℃, and the relative humidity between 40-70%. Lighting consisted of 12 hours of fluorescent illumination and 12 hours of no illumination per day. Two to four months after DL-α-aminoadipic acid (DL-AAA) intravitreal injection to induce modeling, retinal angiogenesis and leakage were assessed using FFA before administration. Sufficient successfully modeled eyes without severe inflammation or hemorrhage were selected for inclusion in the group. The leakage fluorescence area before administration was used as the baseline. The leakage inhibition rate was calculated as (baseline leakage area - leakage area at time T) / baseline leakage area * 100%. The drug administration groups are detailed in Table 21, and the naked sequences and modifications of the siRNA molecules administered to each group are shown in Table 20.

[0333] Table 20. New modified sequences of vegfa / ang-2 siRNA

[0334] In the table above, antisense chain 1 refers to the antisense chain targeting the vegfa target, and antisense chain 2 refers to the antisense chain targeting the ang-2 target.

[0335] Table 21. Rabbit experimental protocol for IVT-administered novel modified siRNA drugs.

[0336] Table 22. Inhibition rate of each group on leakage area

[0337] The experimental results are shown in Figure 8 and Table 22. Figure 8 shows the leakage area size of each group at each time point, and Table 22 shows the inhibition rate of each group relative to the pre-drug baseline, calculated based on the leakage area size at each time point. Different efficacy was observed in the leakage area statistics of the disease model at Day 14, Day 28, Day 42, Day 56, and Day 69 after administration of the dual-target single-drug molecules. Based on comprehensive data evaluation, the long-term efficacy of VA4 and VA5 was superior to that of the Yangshen drug Eylea. Among VA1-VA5, only the sense chain hdt modification position of VA4 and VA5 was located on the linker. Compared with VA2, and VA5 with VA3, the difference between the dual-target single-drug molecules was only that the sense chain hdt modification position was located on the linker. VA4 was more effective than VA2, and VA5 was more effective than VA3. This indicates that although different dual-target single-drug molecules can effectively inhibit leakage area, the effect is better when the sense chain hdt modification position is located on the linker.

[0338] Example 8: Further Selection of Modifications in Dual-Target Monotherapy Compounds

[0339] In previous experiments, the inventors explored using hdt modification to promote siRNA delivery, experimented with dual-target monotherapy compounds, and modified the position of hdt modification in dual-target monotherapy compounds. In this embodiment, the inventors further explored novel modifications to the nucleotides in the sense and antisense strands of the dual-target monotherapy compound after designing the hdt modification position on the linker. Specific modifications are shown in Table 23. The inventors validated the efficacy of their new compound using a rabbit disease model. The rabbit disease model was established as described in Example 7, and the drug administration groups are detailed in Table 24. The naked sequences and modifications of the siRNA molecules administered to each group are shown in Table 23.

[0340] Table 23. New modified sequences of vegfa / ang-2 siRNA

[0341] The first to third nucleotides of the 5' end of the positive strand of vegfa siRNA and the first to third nucleotides of the 3' end of the positive strand of ang-2 siRNA are linked by phosphate thioester bonds. The vegfa siRNA and ang-2 siRNA are linked to the adjacent ends of the linker (i.e., the first to third nucleotides of the 3' end of the positive strand of vegfa siRNA and the first to third nucleotides of the 5' end of the positive strand of ang-2 siRNA) by phosphate ester bonds.

[0342] The antisense strands of vegfa siRNA and ang-2 siRNA are linked by phosphate thioester bonds between the first and third nucleotides at the 3' end and between the first and third nucleotides at the 5' end.

[0343] Table 24. Rabbit experimental protocol for IVT-administered novel modified siRNA drugs.

[0344] The experimental results are shown in Figure 9 and Table 25. Figure 9 shows the leakage area size of each group at each time point, and Table 25 shows the inhibition rate of leakage area relative to the baseline before administration, calculated based on the leakage area size of each group at each time point. Different efficacy was observed in the leakage area statistics of the disease model on Day 14, D28, D42, and D54 after administration of the dual-target single-drug molecule in each group. Based on comprehensive data evaluation, the long-term efficacy of G2-G7 was superior to that of the Yangshen drug Eylea.

[0345] Table 25. Rabbit experimental results of IVT-administered novel modified siRNA drugs

[0346] Preparation Example 1: Synthesis of Modified Nucleoside Monomer Compounds

[0347] Synthesis of compound 2:

[0348] 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.

[0349] Synthesis of compound 3:

[0350] 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.

[0351] Synthesis of compound 4:

[0352] 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 value of m / z is 987.54, and the measured value is 987.6.

[0353] Synthesis of compound 6:

[0354] 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.

[0355] Synthesis of compound 7:

[0356] 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, then 8.09 g of trimethylchlorosilane was added, and stirring continued for 2 hours at room temperature. The reaction mixture was cooled to 0 °C, and 3.14 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 7.30 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.

[0357] Synthesis of compound 8:

[0358] 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.

[0359] Synthesis of compound 9:

[0360] 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.

[0361] Synthesis of compound 12:

[0362] 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%.

[0363] Synthesis of compound 13:

[0364] 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.

[0365] 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.

[0366] Synthesis of compound 14:

[0367] 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.

[0368] Synthesis of compound 15:

[0369] 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.

[0370] Synthesis of compound 16:

[0371] 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.

[0372] Synthesis of compound 17:

[0373] 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.

[0374] Synthesis of compound 20:

[0375] 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%.

[0376] Synthesis of compound 21:

[0377] 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.

[0378] 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 value of m / z is 766.48, and the measured value is 766.5.

[0379] Synthesis of compound 22:

[0380] 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.

[0381] Synthesis of compound 23:

[0382] 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 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.

[0383] Synthesis of compound 24:

[0384] 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 value of m / z is 881.50, and the measured value is 881.5.

[0385] Synthesis of compound 25:

[0386] 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.

[0387] Synthesis of compound 26:

[0388] 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.

[0389] Synthesis of compound 27:

[0390] 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'-bismethoxytriphenylmethyl 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.

[0391] Synthesis of compound 28:

[0392] 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] + Theoretical m / z value: 1070.63; Measured value: 1070.9

[0393] Synthesis of compound 29:

[0394] 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.

[0395] Synthesis of compound 30:

[0396] 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.

[0397] Synthesis of compound 31:

[0398] 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.

[0399] Synthesis of compound 32:

[0400] 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.

[0401] Synthesis of compound 12:

[0402] 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%.

[0403] Synthesis of compound 33:

[0404] 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.

[0405] 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.

[0406] Synthesis of compound 34:

[0407] 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.

[0408] Synthesis of compound 35:

[0409] 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.

[0410] Synthesis of compound 36:

[0411] 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.

[0412] Synthesis of compound 37:

[0413] 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.

[0414] Synthesis of compound 20:

[0415] 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%.

[0416] Synthesis of compound 38:

[0417] 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.

[0418] 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.

[0419] Synthesis of compound 39:

[0420] 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.

[0421] Synthesis of compound 40:

[0422] 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.

[0423] Synthesis of compound 41:

[0424] 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'-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 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.

[0425] Synthesis of compound 42:

[0426] 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.

[0427] 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.

[0428] Table 26. Representative modified nucleoside monomers prepared and used in this invention 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.

[0429] The sequences used in the above embodiments of this application are shown in the following sequence listing. It should be understood that the following sequences are merely exemplary sequences for the embodiments of this application and are not intended to limit the scope of this application. The nucleic acid sequences in the following sequence listing may represent DNA sequences or RNA sequences. When they represent RNA sequences, "T" represents uridine. Furthermore, in the context of RNA, unless otherwise specified, "T" and "U" refer to uracil or uridine.

Claims

A dual-target nucleic acid molecule or a pharmaceutically acceptable salt thereof for inhibiting the expression of the vegfa gene and the ang-2 gene, comprising a vegfa siRNA for inhibiting vegfa gene expression and an ang-2 siRNA for inhibiting ang-2 gene expression linked by a linker, wherein the vegfa siRNA comprises or consists of complementary sense and antisense strands, and the ang-2 siRNA comprises or consists of complementary sense and antisense strands, wherein the linker comprises 2-10 nucleotides or a baseless nucleotide for linking two independent oligonucleotides, and one or more nucleotides or a baseless nucleotides in the linker comprise a lipophilic moiety, wherein the lipophilic moiety is C 12-26 Saturated or unsaturated hydrocarbon chains; in, One or more alkylene groups on the saturated or unsaturated hydrocarbon chain may be independently and optionally replaced with -O-, -S-, -C(O)-, -C(S)-, -S(O)-, -S(O)2-, -SS-, -N(G)C(O)-, -C(O)N(G)-, -N(G)-, -P(O)(OH)-, -P(O)(SH)-, -P(S)(SH)-, In this context, G is independently selected from hydrogen, deuterium, halogen, hydroxyl, amino, and C each time it appears. 1-6 Alkyl, C 1-6 Alkoxy, C 3-10 Cycloalkyl, or C substituted with one or more of deuterium, halogen, hydroxyl, or amino groups. 1-6 Alkyl, C 1-6 Alkoxy, C 3-10 cycloalkyl; Wherein, one or more alkylene groups on the saturated or unsaturated hydrocarbon chain may also be independently and optionally substituted by one or more of the following: deuterium, halogen, hydroxyl, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-10 Cycloalkyl, or C substituted with one or more of deuterium, halogen, hydroxyl, or amino groups. 1-6 Alkyl, C 1-6 Alkoxy and C 3-10 Cycloalkyl. According to claim 1, one or more alkylene groups on the saturated or unsaturated hydrocarbon chain may be independently and optionally replaced with -O-, -S-, -C(O)-, -N(G)C(O)-, -C(O)N(G)-; optionally, they may be independently and optionally replaced with -O-, -S-; optionally, one or more alkylene groups on the saturated or unsaturated hydrocarbon chain may be independently and optionally replaced with -C(O)-, -N(G)C(O)-, -C(O)N(G)-; And / or, one or more alkylene groups on the saturated or unsaturated hydrocarbon chain may also be independently, optionally, substituted with one or more of the following: halogen, hydroxyl, amino, C 1-6 alkyl; And / or, G is independently selected from hydrogen, C each time it appears. 1-6 alkyl; And / or, at least one nucleotide or abasic nucleotide in the linker has a lipophilic moiety at the 1' and / or 2' positions of its sugar ring; optionally, the 1' position of the one or more abasic nucleotides contains a lipophilic moiety; optionally, the 2' position of the one or more nucleotides or abasic nucleotides contains a lipophilic moiety. According to claim 1, the dual-target nucleic acid molecule or its pharmaceutically acceptable salt, wherein the lipophilic moiety is C 14-24 A saturated or unsaturated hydrocarbon chain; optionally, the lipophilic moiety is C. 16-22 Saturated or unsaturated hydrocarbon chains. According to claim 1, the dual-target nucleic acid molecule or its pharmaceutically acceptable salt, wherein the lipophilic moiety is C 12-26 A saturated hydrocarbon chain; optionally, the lipophilic moiety is C. 14-24 A saturated hydrocarbon chain; further optionally, the lipophilic moiety is C. 16-22 saturated hydrocarbon chains. According to claim 1, the dual-target nucleic acid molecule or its pharmaceutically acceptable salt thereof, the saturated or unsaturated hydrocarbon chain of the lipophilic moiety is a straight chain or a branched chain; optionally, the saturated or unsaturated hydrocarbon chain of the lipophilic moiety is a straight chain. According to claim 1, the lipophilic moiety is linked to a nucleotide or a non-basic nucleotide via -O-, -S-, -C(O)-, -N(G)C(O)-, or -C(O)N(G)-; optionally, the lipophilic moiety is linked to a nucleotide or a non-basic nucleotide via -O- or -S-; optionally, the lipophilic moiety is linked to a nucleotide or a non-basic nucleotide via -C(O)-, -N(G)C(O)-, or -C(O)N(G)-. According to claim 1, the dual-target nucleic acid molecule or a pharmaceutically acceptable salt thereof, wherein the lipophilic portion of one or more nucleotides or abase-free nucleotides in the linker is independently selected from: 2'-SC 14-22 Alkyl, 2'-OC 14-22 Alkyl, 2'-C(O)NH-C 14-22 Alkyl, 2'-C(O)NH-C 2-6 Alkyl-C(O)NH-C 10-18 Alkyl, 2'-C(O)NH-C 2-6 Alkyl-NHC(O)-C 10-18 Alkyl, 2'-C(O)NH-C 10-18 Alkyl-C(O)NH-C 2-6 Alkyl, 2'-C(O)NH-C 10-18 Alkyl-NHC(O)-C 2-6 Alkyl, 2'-NHC(O)-C 14-22 Alkyl, 2'-NHC(O)-C 2-6 Alkyl-NHC(O)-C 10-18 Alkyl, 2'-NHC(O)-C 2-6 Alkyl-NHC(O)-C 10-18 Alkyl, 2'-NHC(O)-C 10-18 Alkyl-C(O)NH-C 2-6 Alkyl, 2'-NHC(O)-C 10-18 Alkyl-NHC(O)-C 2-6 Alkyl, 2'-OC 2-6 Alkyl-C(O)NH-C 10-18 Alkyl, 2'-OC 2-6 Alkyl-NHC(O)-C 10-18 Alkyl, 2'-OC 10-18 Alkyl-C(O)NH-C 2-6 Alkyl, 2'-OC 10-18 Alkyl-NHC(O)-C 2-6 Alkyl, 2'-SC 2-6 Alkyl-C(O)NH-C 10-18 Alkyl, 2'-SC 2-6 Alkyl-NHC(O)-C 10-18 Alkyl, 2'-SC 10-18 Alkyl-C(O)NH-C 2-6 Alkyl and 2'-SC 10-18 Alkyl-NHC(O)-C 2-6 alkyl. According to claim 1, the dual-target nucleic acid molecule or its pharmaceutically acceptable salt thereof, wherein the lipophilic portions of one or more nucleotides or non-base nucleotides in the linker are independently selected from: 2'-SC 14 Alkyl modification, 2'-SC 15 Alkyl modification, 2'-SC 16 Alkyl modification, 2'-SC 17 Alkyl modification, 2'-SC 18 Alkyl, 2'-OC 14 Alkyl modification, 2'-OC 15 Alkyl modification, 2'-OC 16 Alkyl modification, 2'-OC 17 Alkyl modification, 2'-OC 18 Alkyl; optionally, the lipophilic portion of one or more nucleotides or baseless nucleotides in the linker is 2'-SC. 16 Alkyl modification; further optionally, one, two, or three nucleotides or baseless nucleotides in the linker have a lipophilic moiety of 2'-SC. 16 Alkyl modification; more preferably, one, two, or three nucleotides or abase-free nucleotides in the linker are independently 2'-SC. 16 Straight-chain alkyl-modified uridine (U) or 2'-SC 16 Straight-chain alkyl-modified thymidine (T). According to claim 1, each nucleotide in the linker is independently selected from deoxyribonucleotides or ribonucleotides; optionally, the linker is a hybrid molecule of DNA and RNA; and / or, the length of the linker is 2-7; optionally, it is 3, 4, 5, 6 or 7. According to claim 1, the dual-target nucleic acid molecule or its pharmaceutically acceptable salt, the linker has the structure shown in Formula I, where X = 0. - or S - : Alternatively, X = O - ; and / or, -C 16 H 33 The group is a straight-chain alkyl group; further optionally, X = O - And -C 16 H 33 The group is a straight-chain alkyl group. According to claim 1, the dual-target nucleic acid molecule or its pharmaceutically acceptable salt thereof, the linker connects the vegfa siRNA and ang-2 siRNA by linking them to the positive strand of the vegfa siRNA and the positive strand of the ang-2 siRNA, respectively. According to claim 11, the connection direction of the positive strand of vegfa siRNA and the positive strand of ang-2 siRNA is as follows: (5')vegfa siRNA positive strand (3')-(5')linker (3')-(5')ang-2 siRNA positive strand (3'). According to claim 1, the dual-target nucleic acid molecule or its pharmaceutically acceptable salt thereof, the antisense strand of the vegfa siRNA has a nucleotide length of 18-23 nt, and the antisense strand comprises or is a sequence of 18-21 consecutive nucleotides, which are no more than 3 nucleotides distinct from the 1st to 21st nucleotides from the 5' end of any one of the sequences SEQ ID NO: 6, 10, 20, 28, 44, 46. And / or, the antisense strand of the ang-2 siRNA has a nucleotide length of 18-23 nt, and the antisense strand comprises or is a continuous nucleotide sequence of 18-21 nucleotides, which are no more than 3 nucleotides different from the nucleotide sequence of positions 1-21 from the 5' end of any one of the sequences of SEQ ID NO:48, 60, 62, 64, 70, 90. According to claim 13, the dual-target nucleic acid molecule or its pharmaceutically acceptable salt thereof, wherein the difference of no more than 3 nucleotides is a difference of 3 nucleotides, a difference of 2 nucleotides, a difference of 1 nucleotide, or complete sameness. According to claim 13, the dual-target nucleic acid molecule or its pharmaceutically acceptable salt thereof, the 18-21 consecutive nucleotide sequence is an 18, 19, 20 or 21 consecutive nucleotide sequence. According to claim 1, the dual-target nucleic acid molecule or its pharmaceutically acceptable salt thereof, wherein the antisense strand of the vegfa siRNA and / or the antisense strand of the ang-2 siRNA further comprises 1-6 overhanging nucleotides at its 3' and / or 5' ends; optionally, the overhangs are 2-6 nucleotides, 1-5 nucleotides, 2-5 nucleotides, 1-4 nucleotides, 2-4 nucleotides, 1-3 nucleotides, 2-3 nucleotides, 1-2 nucleotides, or 2 nucleotides. According to claim 13, the dual-target nucleic acid molecule or a pharmaceutically acceptable salt thereof, wherein the antisense strands of the vegfa siRNA and ang-2 siRNA differ in length by more than 2 nucleotides; Optionally, the complementary region formed by the sense and antisense strands of the vegfa siRNA is 21 bp, and the 3' end of the antisense strand of the vegfa siRNA has two protruding ends; the complementary region formed by the sense and antisense strands of the ang-2 siRNA is 21 bp, and the antisense strand of the ang-2 siRNA does not have protruding ends. In the dual-target nucleic acid molecule or its pharmaceutically acceptable salt according to claim 1, the antisense strand of the vegfa siRNA is a polynucleotide sequence shown in SEQ ID NO: 6, 10, 20, 28, 44 or 46, or the antisense strand is a polynucleotide sequence shown in positions 1-21 of SEQ ID NO: 6, 10, 20, 28, 44 or 46; And / or, in the ang-2 siRNA, the antisense strand is a polynucleotide sequence as shown in SEQ ID NO:48, 60, 62, 64, 70 or 90, or the antisense strand is a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO:48, 60, 62, 64, 70 or 90. According to claim 18, the dual-target nucleic acid molecule or a pharmaceutically acceptable salt thereof, wherein: In vegfa siRNA: The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 5, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 6; The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 9, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 10; The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 19, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 20; The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 27, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 28; The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 43, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 44; Alternatively, the sense strand may be a polynucleotide sequence as shown in SEQ ID NO: 45, and the antisense strand may be a polynucleotide sequence as shown in SEQ ID NO: 46; And / or, in ang-2 siRNA: The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 47, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 48 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 48; The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 59, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 60 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 60; The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 61, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 62 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 62; The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 63, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 64 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 64; The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 69, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 70 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 70; Alternatively, the sense strand may be a polynucleotide sequence as shown in SEQ ID NO: 89, and the antisense strand may be a polynucleotide sequence as shown in SEQ ID NO: 90 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO:

90. According to claim 19, the dual-target nucleic acid molecule or a pharmaceutically acceptable salt thereof, wherein: In vegfa siRNA: the sense strand is a polynucleotide sequence as shown in SEQ ID NO: 43, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 44; In the ang-2 siRNA: the sense strand is a polynucleotide sequence as shown in SEQ ID NO: 69, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 70 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO:

70. According to claim 18, the dual-target nucleic acid molecule or a pharmaceutically acceptable salt thereof, wherein the motifs of the positive strand of the vegfa siRNA and the positive strand of the ang-2 siRNA independently comprise or are one of the following motifs: (1) The 2' position of the 7th and 9th-11th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methyl. (2) The 2' position of the 7th, 9th and 11th nucleotides starting from the 5' end of the positive strand is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the positive strand is 2'-O-methyl. (3) The 2' position of the 9th and 11th nucleotides starting from the 5' end of the positive strand is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the positive strand is 2'-O-methyl. (4) The 2' position of the 7th and 9th-12th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methyl. in, The corresponding deoxyribonucleotides for ribonucleotides A, C, and G are dA, dC, and dG, respectively, and the corresponding deoxyribonucleotide for ribonucleotide U is dT. According to claim 18, the dual-target nucleic acid molecule or a pharmaceutically acceptable salt thereof, wherein the antisense strands of the vegfa siRNA and the ang-2 siRNA independently contain or are one of the following motifs: (1) The 2' position of the nucleotides at positions 2, 14 and 16 of the antisense strand starting from the 5' end is 2'-fluorinated, and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methyl. (2) The 2' positions of the nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, are 2'-fluorinated, and the nucleotides at positions 5 and 7 are the corresponding deoxyribonucleotides (dN). The 2' positions of the remaining nucleotides in the antisense strand are all 2'-O-methyl modified. The corresponding deoxyribonucleotides for ribonucleotides A, C, and G are dA, dC, and dG, respectively, and the corresponding deoxyribonucleotide for ribonucleotide U is dT. Optionally, the antisense strands of the vegfa siRNA and the ang-2 siRNA also independently contain a 5'-VP modification at the 5' position of the first nucleotide starting from the 5' end. According to claim 18, the dual-target nucleic acid molecule or a pharmaceutically acceptable salt thereof, wherein the motif combination consisting of the sense strand motif and the antisense strand motif of the vegfa siRNA and ang-2 siRNA independently comprises or is one of the following motif combinations: (1) Sensitive strand motif: The 2' position of the 7th and 9th-11th nucleotides starting from the 5' end is 2'-fluorinated, and the 2' position of the remaining nucleotides in the sense strand is 2'-O-methylated; Antisense strand motif: The 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end is 2'-fluorinated, and the 2' position of the remaining nucleotides in the antisense strand is 2'-O-methylated. (2) Sensitive strand motif: The 2' position of the 7th, 9th and 11th nucleotides starting from the 5' end is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the positive strand is 2'-O-methylated; Antisense strand motif: The 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end is 2'-fluorinated, the 5th and 7th nucleotides are the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methylated. (3) Sensitive strand motif: The 2' position of the 9th and 11th nucleotides starting from the 5' end is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the positive strand is 2'-O-methylated; Antisense strand motif: The 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end is 2'-fluorinated, the 5th and 7th nucleotides are the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methylated. (4) Sensitive strand motif: The 2' position of the 7th and 9th-12th nucleotides starting from the 5' end is 2'-fluorinated, and the 2' position of the remaining nucleotides in the sense strand is 2'-O-methylated; Antisense strand motif: The 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end is 2'-fluorinated, and the 2' position of the remaining nucleotides in the antisense strand is 2'-O-methylated. in, The corresponding deoxyribonucleotides for ribonucleotides A, C, and G are dA, dC, and dG, respectively, and the corresponding deoxyribonucleotide for ribonucleotide U is dT. Optionally, the motif combination consisting of the sense strand motif and the antisense strand motif of the vegfa siRNA and ang-2 siRNA independently also includes: the 5' position of the first nucleotide of the antisense strand starting from the 5' end is 5'-VP modified. According to claim 12, the dual-target nucleic acid molecule or its pharmaceutically acceptable salt thereof, the vegfa siRNA antisense strand and the ang-2 siRNA antisense strand are linked by phosphate thioester bonds between the first 3 nucleotides starting from their 5' ends and between the first 3 nucleotides starting from their 3' ends. According to claim 12, the structure of the positive strand of vegfa siRNA and the positive strand of ang-2 siRNA linked by a linker (5')vegfa siRNA positive strand (3')-(5')linker (3')-(5')ang-2 siRNA positive strand (3'): The positive strands of vegfa siRNA and ang-2 siRNA are linked by phosphate thioester bonds between the first three nucleotides starting from their 5' ends and between the first three nucleotides starting from their 3' ends. Alternatively, the first three nucleotides of the positive strand of vegfa siRNA are linked by thiophosphate bonds starting from its 5' end, and the first three nucleotides of the positive strand of ang-2 siRNA are linked by thiophosphate bonds starting from its 3' end, and the first three nucleotides of the positive strand of vegfa siRNA are linked by phosphate bonds starting from its 3' end, and the first three nucleotides of the positive strand of ang-2 siRNA are linked by phosphate bonds starting from its 5' end. According to claim 1, the dual-target nucleic acid molecule or a pharmaceutically acceptable salt thereof, wherein the dual-target nucleic acid molecule is selected from the following molecules: VA4, VA4-17, VA4-18, VA4-20, VA5, VA5-17, VA5-18, VA5-20: Wherein: Am Um, Cm, and Gm represent ribonucleotides A, U, C, and G modified with 2′-O-methyl, respectively; Af, Uf, Cf, and Gf represent ribonucleotides A, U, C, and G modified with 2′-fluorine, respectively; 's' between adjacent nucleotides indicates that adjacent nucleotides are linked by a thiophosphate bond, and the absence of 's' between adjacent nucleotides indicates that adjacent nucleotides are linked by a phosphate bond; dC and dG represent the deoxyribonucleotides corresponding to ribonucleotides C and G, respectively, and dT represents deoxyribonucleotide T; 5'VP represents 5'-vinylphosphonate modification; hdt represents 2'-S-linear C16 alkyl modification. According to claim 26, the dual-target nucleic acid molecule or its pharmaceutically acceptable salt, wherein the pharmaceutically acceptable salt is a pharmaceutically acceptable salt of the dual-target nucleic acid molecule selected from the following molecules: VA4, VA4-17, VA4-18, VA4-20, VA5, VA5-17, VA5-18, VA5-20; optionally, the pharmaceutically acceptable salt is a sodium salt, potassium salt, or ammonium salt. A nucleic acid delivery system comprising a dual-target nucleic acid molecule or a pharmaceutically acceptable salt thereof that inhibits the expression of the vegfa gene and the ang-2 gene as described in any one of claims 1-27. The nucleic acid delivery body according to claim 28 is a liposome, lipid nanoparticle or other polymer, endosome, exosome or vesicle. A cell comprising a dual-target nucleic acid molecule or a pharmaceutically acceptable salt thereof that inhibits the expression of the vegfa gene and the ang-2 gene as described in any one of claims 1-27. A pharmaceutical composition comprising a dual-target nucleic acid molecule or a pharmaceutically acceptable salt thereof that inhibits the expression of the vegfa gene and the ang-2 gene as described in any one of claims 1-27, and a pharmaceutically acceptable excipient. Use in the preparation of a medicament of a dual-target nucleic acid molecule for inhibiting the expression of the vegfa gene and the ang-2 gene as described in any one of claims 1-27, or a pharmaceutically acceptable salt thereof, a nucleic acid delivery body as described in claim 28 or 29, a cell as described in claim 30, or a pharmaceutical composition as described in claim 31. According to the use of claim 32, the drug is a drug that inhibits the expression of the vegfa gene and / or ang-2 gene in the subject; optionally, inhibiting the expression of the vegfa gene and / or ang-2 gene in the subject is for the prevention and / or treatment of ocular disorders; further optionally, the ocular disorder is age-related macular degeneration and / or diabetic macular edema; even more optionally, the ocular disorder is wet age-related macular degeneration and / or diabetic macular edema. A method for inhibiting the expression of the vegfa gene and / or ang-2 gene in a subject, comprising the following steps: The administration of an effective dose of any one of claims 1-27 of a dual-target nucleic acid molecule or a pharmaceutically acceptable salt thereof that inhibits the expression of the vegfa gene and / or ang-2 gene, the nucleic acid delivery body of claim 28 or 29, the cell of claim 30, or the pharmaceutical composition of claim 31 to a subject who requires inhibition of the expression of the vegfa gene and / or ang-2 gene in the subject. According to the method of claim 34, inhibiting the expression of the vegfa gene and / or ang-2 gene in the subject is used to prevent or treat ocular disorders in the subject; optionally, the ocular disorder is age-related macular degeneration and / or diabetic macular edema; further optionally, the ocular disorder is wet age-related macular degeneration and / or diabetic macular edema. A vegfa siRNA or a pharmaceutically acceptable salt thereof that inhibits the expression of the vegfa gene, comprising or composed of complementary sense and antisense strands, wherein the antisense strand of the vegfa siRNA has a nucleotide length of 18-23 nt, and the antisense strand comprises or is a sequence of 18-21 consecutive nucleotides, consisting of no more than 3 nucleotides distinct from the 1st to 21st nucleotides from the 5' end of any one of the sequences SEQ ID NO: 6, 10, 20, 28, 44, 46. According to claim 36, the vegfa siRNA or a pharmaceutically acceptable salt thereof, wherein the difference of no more than 3 nucleotides is a difference of 3 nucleotides, a difference of 2 nucleotides, a difference of 1 nucleotide, or complete sameness. The vegfa siRNA or a pharmaceutically acceptable salt thereof according to claim 37, wherein the 18-21 consecutive nucleotide sequence is an 18, 19, 20 or 21 consecutive nucleotide sequence. The vegfa siRNA or a pharmaceutically acceptable salt thereof according to claim 36, wherein the positive strand of the vegfa siRNA further comprises 1-6 overhanging nucleotides at its 3' end and / or 5' end; optionally, the overhangs may be 2-6 nucleotides, 1-5 nucleotides, 2-5 nucleotides, 1-4 nucleotides, 2-4 nucleotides, 1-3 nucleotides, 2-3 nucleotides, 1-2 nucleotides, or 2 nucleotides; And / or, wherein the vegfa siRNA antisense strand further comprises 1-6 overhanging nucleotides at its 3' end and / or 5' end; optionally, the overhangs may be 2-6 nucleotides, 1-5 nucleotides, 2-5 nucleotides, 1-4 nucleotides, 2-4 nucleotides, 1-3 nucleotides, 2-3 nucleotides, 1-2 nucleotides, or 2 nucleotides. The vegfa siRNA or a pharmaceutically acceptable salt thereof according to claim 36, wherein the antisense strand of the vegfa siRNA is a polynucleotide sequence as shown in SEQ ID NO:6, 10, 20, 28, 44, 46 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO:6, 10, 20, 28, 44, 46. The vegfa siRNA or a pharmaceutically acceptable salt thereof according to claim 40, wherein: The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 5, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 6 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 6; The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 9, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 10 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 10; The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 19, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 20 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 20; The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 27, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 28 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 28; The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 43, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 44 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 44; Alternatively, the sense strand may be a polynucleotide sequence as shown in SEQ ID NO: 45, and the antisense strand may be a polynucleotide sequence as shown in SEQ ID NO: 46 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO:

46. The vegfa siRNA of claim 41 or a pharmaceutically acceptable salt thereof, wherein the motif of the sense strand of the vegfa siRNA comprises or is one of the following motifs: (1) The 2' position of the 7th and 9th-11th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methyl. (2) The 2' position of the 7th, 9th and 11th nucleotides starting from the 5' end of the positive strand is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the positive strand is 2'-O-methyl. (3) The 2' position of the 9th and 11th nucleotides starting from the 5' end of the positive strand is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the positive strand is 2'-O-methyl. (4) The 2' position of the 7th and 9th-12th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methyl. (5) The 6th position of the justice chain starting from the 5' end is 2'-S-straight chain C. 16 Alkyl modification or 2'-O-straight-chain C 16 Alkyl modification: the 2' position of the 7th and 9th-11th nucleotides starting from the 5' end of the sense chain is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense chain is 2'-O-methyl. (6) The 7th position of the justice chain starting from the 5' end is 2'-S-straight chain C. 16 Alkyl modification or 2'-O-straight-chain C 16 Alkyl modification: the 2' position of the nucleotides at positions 9-11 starting from the 5' end of the sense chain is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense chain is 2'-O-methyl. in, The corresponding deoxyribonucleotides for ribonucleotides A, C, and G are dA, dC, and dG, respectively, and the corresponding deoxyribonucleotide for ribonucleotide U is dT. The vegfa siRNA of claim 41 or a pharmaceutically acceptable salt thereof, wherein the motif of the antisense strand of the vegfa siRNA comprises or is one of the following motifs: (1) The 2' position of the nucleotides at positions 2, 14 and 16 of the antisense strand starting from the 5' end is 2'-fluorinated, and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methyl. (2) The 2' positions of the nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, are 2'-fluorinated, and the nucleotides at positions 5 and 7 are the corresponding deoxyribonucleotides (dN). The 2' positions of the remaining nucleotides in the antisense strand are all 2'-O-methyl modified. The corresponding deoxyribonucleotides for ribonucleotides A, C, and G are dA, dC, and dG, respectively, and the corresponding deoxyribonucleotide for ribonucleotide U is dT. Optionally, the antisense motif also includes a 5'-VP modification at the 5' position of the first nucleotide starting from the 5' end of the antisense strand. The vegfa siRNA of claim 41 or a pharmaceutically acceptable salt thereof, wherein the motif combination consisting of the sense strand motif and the antisense strand motif of the vegfa siRNA comprises or is one of the following motif combinations: (1) The 2' position of the 7th and 9th-11th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methylated; the 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end of the antisense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methylated. (2) The 2' position of the 7th, 9th and 11th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methylated; The 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end of the antisense strand is 2'-fluorinated, the 5th and 7th nucleotides are the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methylated. (3) The 2' position of the 9th and 11th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methylated; The 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end of the antisense strand is 2'-fluorinated, the 5th and 7th nucleotides are the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methylated. (4) The 2' position of the 7th and 9th-12th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methylated; the 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end of the antisense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methylated. in, The corresponding deoxyribonucleotides for ribonucleotides A, C, and G are dA, dC, and dG, respectively, and the corresponding deoxyribonucleotide for ribonucleotide U is dT. Optionally, the motif assembly also includes a 5'-VP modification at the 5' position of the first nucleotide of the antisense strand starting from the 5' end. According to claim 44, the vegfa siRNA or a pharmaceutically acceptable salt thereof, wherein the antisense strand of the vegfa siRNA is linked by a phosphate thioester between the first 1-3 nucleotides starting from its 5' end and / or between the first 1-3 nucleotides starting from its 3' end. According to claim 44, the vegfa siRNA or a pharmaceutically acceptable salt thereof, wherein the first to third nucleotides of the positive strand of the vegfa siRNA are linked by phosphate thioesters, starting from its 5' end and / or starting from its 3' end. The vegfa siRNA according to claim 44, or a pharmaceutically acceptable salt thereof, wherein the vegfa siRNA is selected from the following molecules, the motifs of which are described in Table 1: The sense strand of the nucleic acid molecule is SEQ ID NO: 43 with modification motif 5, and the antisense strand is SEQ ID NO: 44 with modification motif 7. The sense strand of the nucleic acid molecule is SEQ ID NO: 43 with modification motif 11, and the antisense strand is SEQ ID NO: 44 with modification motif 12. The sense strand of the nucleic acid molecule is SEQ ID NO: 43 with modification motif 14, and the antisense strand is SEQ ID NO: 44 with modification motif 12. The sense strand of the nucleic acid molecule is SEQ ID NO: 43 with modification motif 15, and the antisense strand is SEQ ID NO: 44 with modification motif 7. The sense strand of the nucleic acid molecule is SEQ ID NO: 43 with modification motif 5, and the antisense strand is nucleotides 1-21 from 5' of SEQ ID NO: 44 with modification motif 10. The sense strand of the nucleic acid molecule is SEQ ID NO: 43 modified with motif 11, and the antisense strand is nucleotides 1-21 from 5' of SEQ ID NO: 44 modified with motif 13. The sense strand of the nucleic acid molecule is SEQ ID NO: 43 modified with motif 14, and the antisense strand is nucleotides 1-21 from 5' of SEQ ID NO: 44 modified with motif 13. The sense strand of the nucleic acid molecule is SEQ ID NO: 43 with modification motif 15, and the antisense strand is nucleotides 1-21 from 5' of SEQ ID NO: 44 with modification motif 10. Optionally, the three nucleotides at the 3' end and the three nucleotides at the 5' end of the sense strand of the nucleic acid molecule are linked by phosphate thioester bonds, and / or, the three nucleotides at the 3' end and the three nucleotides at the 5' end of the antisense strand of the nucleic acid molecule are linked by phosphate thioester bonds. The vegfa siRNA or a pharmaceutically acceptable salt thereof according to claim 47, wherein the pharmaceutically acceptable salt is a sodium salt, potassium salt, or ammonium salt. An ang-2 siRNA or a pharmaceutically acceptable salt thereof that inhibits ang-2 gene expression, comprising or composed of complementary sense and antisense strands, wherein the antisense strand of the ang-2 siRNA has a nucleotide length of 18-23 nt, and the antisense strand comprises or is a sequence of 18-21 consecutive nucleotides, consisting of no more than 3 nucleotides distinct from the 1st to 21st nucleotides from the 5' end of any one of the sequences SEQ ID NO:48, 60, 62, 64, 70, 90. The ang-2 siRNA or a pharmaceutically acceptable salt thereof according to claim 49, wherein the difference of no more than 3 nucleotides is a difference of 3 nucleotides, a difference of 2 nucleotides, a difference of 1 nucleotide, or complete sameness. The ang-2 siRNA or a pharmaceutically acceptable salt thereof according to claim 49, wherein the 18-21 consecutive nucleotide sequence is an 18, 19, 20 or 21 consecutive nucleotide sequence. The ang-2 siRNA or a pharmaceutically acceptable salt thereof according to claim 49, wherein the positive strand of the ang-2 siRNA further comprises 1-6 overhanging nucleotides at its 3' end and / or 5' end; optionally, the overhangs may be 2-6 nucleotides, 1-5 nucleotides, 2-5 nucleotides, 1-4 nucleotides, 2-4 nucleotides, 1-3 nucleotides, 2-3 nucleotides, 1-2 nucleotides, or 2 nucleotides; And / or, wherein the ang-2 siRNA antisense strand further comprises 1-6 overhanging nucleotides at its 3' end and / or 5' end; optionally, the overhangs may be 2-6 nucleotides, 1-5 nucleotides, 2-5 nucleotides, 1-4 nucleotides, 2-4 nucleotides, 1-3 nucleotides, 2-3 nucleotides, 1-2 nucleotides, or 2 nucleotides. The ang-2 siRNA of claim 49 or a pharmaceutically acceptable salt thereof, wherein the antisense strand is a polynucleotide sequence as shown in SEQ ID NO:48, 60, 62, 64, 70, 90 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO:48, 60, 62, 64, 70, 90. The ang-2 siRNA or a pharmaceutically acceptable salt thereof according to claim 53, wherein: The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 47, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 48 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 48; The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 59, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 60 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 60; The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 61, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 62 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 62; The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 63, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 64 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 64; The sense strand is a polynucleotide sequence as shown in SEQ ID NO: 69, and the antisense strand is a polynucleotide sequence as shown in SEQ ID NO: 70 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO: 70; Alternatively, the sense strand may be a polynucleotide sequence as shown in SEQ ID NO: 89, and the antisense strand may be a polynucleotide sequence as shown in SEQ ID NO: 90 or a polynucleotide sequence as shown in positions 1-21 of SEQ ID NO:

90. The ang-2 siRNA of claim 54 or a pharmaceutically acceptable salt thereof, wherein the motif of the sense strand of the ang-2 siRNA comprises or is one of the following motifs: (1) The 2' position of the 7th and 9th-11th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methyl. (2) The 2' position of the 7th, 9th and 11th nucleotides starting from the 5' end of the positive strand is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the positive strand is 2'-O-methyl. (3) The 2' position of the 9th and 11th nucleotides starting from the 5' end of the positive strand is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the positive strand is 2'-O-methyl. (4) The 2' position of the 7th and 9th-12th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methyl. (5) The 6th position of the justice chain starting from the 5' end is 2'-S-straight chain C. 16 Alkyl modification or 2'-O-straight-chain C 16 Alkyl modification: the 2' position of the 7th and 9th-11th nucleotides starting from the 5' end of the sense chain is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense chain is 2'-O-methyl. (6) The 7th position of the justice chain starting from the 5' end is 2'-S-straight chain C. 16 Alkyl modification or 2'-O-straight-chain C 16 Alkyl modification: the 2' position of the nucleotides at positions 9-11 starting from the 5' end of the sense chain is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense chain is 2'-O-methyl. in, The corresponding deoxyribonucleotides for ribonucleotides A, C, and G are dA, dC, and dG, respectively, and the corresponding deoxyribonucleotide for ribonucleotide U is dT. The ang-2 siRNA of claim 54 or a pharmaceutically acceptable salt thereof, wherein the motif of the antisense strand of the ang-2 siRNA comprises or is one of the following motifs: (1) The 2' position of the nucleotides at positions 2, 14 and 16 of the antisense strand starting from the 5' end is 2'-fluorinated, and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methyl. (2) The 2' positions of the nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, are 2'-fluorinated, and the nucleotides at positions 5 and 7 are the corresponding deoxyribonucleotides (dN). The 2' positions of the remaining nucleotides in the antisense strand are all 2'-O-methyl modified. The corresponding deoxyribonucleotides for ribonucleotides A, C, and G are dA, dC, and dG, respectively, and the corresponding deoxyribonucleotide for ribonucleotide U is dT. Optionally, the antisense motif also includes a 5'-VP modification at the 5' position of the first nucleotide starting from the 5' end of the antisense strand. The ang-2 siRNA of claim 54 or a pharmaceutically acceptable salt thereof, wherein the motif combination consisting of the sense strand motif and the antisense strand motif of the ang-2 siRNA comprises or is one of the following motif combinations: (1) The 2' position of the 7th and 9th-11th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methylated; the 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end of the antisense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methylated. (2) The 2' position of the 7th, 9th and 11th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methylated; The 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end of the antisense strand is 2'-fluorinated, the 5th and 7th nucleotides are the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methylated. (3) The 2' position of the 9th and 11th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, the 10th nucleotide is the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methylated; The 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end of the antisense strand is 2'-fluorinated, the 5th and 7th nucleotides are the corresponding deoxyribonucleotide (dN), and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methylated. (4) The 2' position of the 7th and 9th-12th nucleotides starting from the 5' end of the sense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the sense strand is 2'-O-methylated; the 2' position of the 2nd, 14th and 16th nucleotides starting from the 5' end of the antisense strand is 2'-fluorinated, and the 2' position of the remaining nucleotides of the antisense strand is 2'-O-methylated. in, The corresponding deoxyribonucleotides for ribonucleotides A, C, and G are dA, dC, and dG, respectively, and the corresponding deoxyribonucleotide for ribonucleotide U is dT. Optionally, the motif assembly also includes a 5'-VP modification at the 5' position of the first nucleotide of the antisense strand starting from the 5' end. The ang-2 siRNA of claim 57 or a pharmaceutically acceptable salt thereof, wherein the antisense strand of the ang-2 siRNA is linked by a phosphate thioester between the first 1-3 nucleotides starting from its 5' end and / or between the first 1-3 nucleotides starting from its 3' end. The ang-2 siRNA of claim 57 or a pharmaceutically acceptable salt thereof, wherein the first to third nucleotides of the positive strand of the ang-2 siRNA, starting from its 5' end, are linked by phosphate thioesters. The ang-2 siRNA according to claim 57 or a pharmaceutically acceptable salt thereof, wherein the nucleic acid molecule is selected from the following molecules, the motifs of which are described in Table 1: The sense strand of the nucleic acid molecule is SEQ ID NO: 69 with modification motif 5, and the antisense strand is SEQ ID NO: 70 with modification motif 7. The sense strand of the nucleic acid molecule is SEQ ID NO: 69 with modification motif 11, and the antisense strand is SEQ ID NO: 70 with modification motif 12. The sense strand of the nucleic acid molecule is SEQ ID NO: 69 with motif 14 modified, and the antisense strand is SEQ ID NO: 70 with motif 12 modified. The sense strand of the nucleic acid molecule is SEQ ID NO: 69 with modification motif 15, and the antisense strand is SEQ ID NO: 70 with modification motif 7. The sense strand of the nucleic acid molecule is SEQ ID NO: 69 with modification motif 5, and the antisense strand is nucleotides 1-21 from 5' of SEQ ID NO: 70 with modification motif 10. The sense strand of the nucleic acid molecule is SEQ ID NO: 69 with modification motif 11, and the antisense strand is nucleotides 1-21 from 5' of SEQ ID NO: 70 with modification motif 13. The sense strand of the nucleic acid molecule is SEQ ID NO: 69 with motif 14 modified, and the antisense strand is nucleotides 1-21 from 5' of SEQ ID NO: 70 with motif 13 modified. The sense strand of the nucleic acid molecule is SEQ ID NO: 69 with modification motif 15, and the antisense strand is nucleotides 1-21 from 5' of SEQ ID NO: 70 with modification motif 10. Optionally, the three nucleotides at the 3' end and the three nucleotides at the 5' end of the sense strand of the nucleic acid molecule are linked by phosphate thioester bonds, and / or, the three nucleotides at the 3' end and the three nucleotides at the 5' end of the antisense strand of the nucleic acid molecule are linked by phosphate thioester bonds. The ang-2 siRNA or a pharmaceutically acceptable salt thereof according to claim 60, wherein the pharmaceutically acceptable salt is a sodium salt, a potassium salt, or an ammonium salt. A vegfa siRNA or a pharmaceutically acceptable salt thereof for inhibiting vegfa gene expression as described in any one of claims 36-47, or an ang-2 siRNA or a pharmaceutically acceptable salt thereof for inhibiting ang-2 gene expression as described in any one of claims 49-61, is used to prepare a medicament; optionally, the medicament is a medicament for inhibiting vegfa gene or ang-2 gene expression in a subject; further optionally, inhibiting vegfa gene or ang-2 gene expression in a subject is for the prevention and / or treatment of an ocular disorder; even further optionally, the ocular disorder is age-related macular degeneration and / or diabetic macular edema; even further optionally, the ocular disorder is wet age-related macular degeneration and / or diabetic macular edema.

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