Double-stranded oligonucleotides, their conjugates and uses

Abstract

This invention provides methods for preparing and using modified double-stranded oligonucleotides, oligonucleotide conjugates, or compositions. By providing modified double-stranded oligonucleotides and performing specific modifications to one or more sites on specific sites of the sense strand and / or antisense strand of the oligonucleotide, the modified oligonucleotide can obtain good pharmaceutical activity and has high potential for application in the development of RNA interference (RNAi) drugs.

Description

Technical Field

[0001] The present disclosure belongs to the field of oligonucleotides, and specifically relates to a method for preparing and using modified double-stranded oligonucleotides, oligonucleotide conjugates or compositions. Cross-reference to Related Applications This application claims priority based on a Chinese application filed with the China National Intellectual Property Administration on May 19, 2023, with an application number of 202310574400.2 and a title of "Double-stranded Oligonucleotides, Their Conjugates and Uses", and all of its content is incorporated herein by reference.

Background Art

[0002] It is well-known in the art that double-stranded oligonucleotides (dsRNA) can induce gene-specific post-transcriptional silencing, and double-stranded oligonucleotides (dsRNA) are used as effective pharmaceutical active ingredients.

[0003] As is well-known, the development of drugs based on RNA interference (RNAi) requires double-stranded oligonucleotide molecules with good gene silencing properties or inhibitory activities, and generally, modified double-stranded oligonucleotide molecules are required. For example, it is often used to use siRNA duplexes containing alternative modifications and to stabilize siRNA in serum by different combinations of 2'-OMe, 2'-F and phosphorothioate modifications. However, different modifications may result in differences in the activity of double-stranded oligonucleotides, and the activity may even be lost. For example, Tai, W. ("Chemical modulation of siRNA lipophilicity for efficient delivery." J Control Release 307:98-107.) reported that the activity of siRNA decreased due to one or more MOE modifications. In the case of double-stranded oligonucleotide drugs, the design of modifications to double-stranded oligonucleotides with good pharmaceutical activity is an ongoing research topic in the art.

Summary of the Invention

[0004] The present disclosure aims to solve at least one of the technical problems in the prior art to some extent.

[0005] For this reason, in one aspect, the present disclosure provides a double-stranded oligonucleotide (dsRNA) that suppresses the expression of a target gene. The double-stranded oligonucleotide includes a sense strand and an antisense strand, each strand having 17 to 35 nucleotides, each nucleotide being a modified nucleotide or an unmodified nucleotide, the sense strand and the antisense strand being at least partially reverse complementary to form a double-stranded region, the double-stranded region including nucleotides having a sterically bulky modification and / or nucleotides having a disubstituted modification, and the composition of the double-stranded region being represented by the following formula (I): SS: 5’-(N)a’-(X)p’-(N)b’-(X)q’-(N)c’-(X)r’-(N)d’-3’ AS: 3’-(N)a-(X)p-(N)b-(X)q-(N)c-5’ (I), where SS represents the sense strand and AS represents the antisense strand, In formula (I), each X is independently a nucleotide in which the hydroxy group at the 2'-position in ribose is substituted with a sterically bulky group or a nucleotide in which the 2'-position is substituted with a disubstituted group (i.e., both the 2'-hydroxy group and hydrogen are substituted), the sterically bulky group at the 2'-position is selected from groups having a steric bulk larger than a 2'-methoxy group, and each substituent in the disubstituted group is independently selected from a C1-C6 alkyl group or a halogen. In some embodiments, when X represents a nucleotide in which the hydroxy group at the 2'-position in ribose is substituted with a sterically bulky group, it is independently a 2'-(O) m1 (CH2) n(O) m2 R1 is selected from, m1 or m2 is independently 0 or 1, n is selected from an integer between 0 and 6, R1 is selected from substituted or unsubstituted C1-C6 alkyl groups or -Si(R2)3, and R2 is independently selected from substituted or unsubstituted C1-C6 alkyl groups or substituted or unsubstituted C1-C6 alkoxy groups, the substitution includes one or more substituents selected from halogens, C1-C6 alkyl groups, C1-C6 alkoxy groups, hydroxyl groups, amino groups, cycloalkyl groups having 6 or less carbon atoms, aryl groups having 12 or less carbon atoms, or heteroaryl groups having 12 or less carbon atoms.

[0006] In some embodiments, m1=0, n=0, m2=1, and R1=Si(R2)3. In some embodiments, m1=1, m2=0, and n is a positive integer. In some embodiments, X is independently a modified nucleotide, 2′-O(CH2) n Selected from OR1 or 2′-R3-2′-R4. n is 1 or 2, and the definitions of substituents R1 and R2 are the same as above. R3 and R4 are each independently selected from a C1-C6 alkyl group or a halogen. Optionally, the halogen is fluorine. Optionally, the C1-C6 alkyl group is selected from a methyl group or an ethyl group.

[0007] In formula (I), each N independently represents an unmodified nucleotide or a nucleotide that is modified at the 2′ position of ribose and is not X (i.e., not the 2′ modification described above on X), and optionally the modified nucleotide is selected from the group consisting of nucleotides modified with 2′-O-alkyl groups, 2′-alkyl groups, 2′-substituted alkyl groups, 2′-halogens, 2′-deoxy (i.e., 2′-H modification), or nucleotide analogs, wherein the alkyl group has 1 to 6 carbon atoms, and the nucleotide analog is selected from ENA, BNA, LNA, GNA, or UNA, and optionally the 2′-substituted alkyl group has 1 to 6 total carbon atoms. In this disclosure, the alkyl group includes branched alkyl groups or linear alkyl groups.

[0008] Preferably, N is independently selected from 2′-OMe modified nucleotides, 2′-F modified nucleotides, or 2′-deoxy modified nucleotides.

[0009] In formula I, a, a', p, p', b, b', q, q', c, c', r', and d' in the general structural formula of the double-stranded region each independently represent the number of nucleotides, where, a' is selected from integers between 3 and 8, p' is selected from integers between 0 and 3, b' is selected from integers between 4 and 13, q' is selected from integers between 0 and 4, c' is selected from integers between 3 and 9, r' is selected from integers between 0 and 3, d' is selected from integers between 0 and 9, a is selected from integers between 4 and 7, p is selected from integers between 0 and 1, b is selected from integers between 4 and 8, q is selected from integers between 0 and 4, and c is selected from integers between 6 and 10, and p', q', r', p, and q are never simultaneously 0, and arbitrarily, q'+r' satisfies 0≦q'+r'≦4.

[0010] In some embodiments of the present disclosure, a double-stranded oligonucleotide molecule comprising a double-stranded region represented by formula (I) above has at least one modified nucleotide X at a specific position in the nucleotide sequence of the sense strand and / or antisense strand along the direction from the 5′ end to the 3′ end, i.e., a nucleotide having a sterically bulky modification or a nucleotide having a two-substituted modification.

[0011] In some embodiments of the present disclosure, the nucleotide having the sterically bulky modification and / or disubstituted modification is located at a specific position on the antisense strand in the double-stranded region represented by formula I, where the specific position is at least one of the 10th and 15th positions.

[0012] In some embodiments of this disclosure, the specific position further includes at least one of positions 8 through 10.

[0013] In some embodiments of the present disclosure, the nucleotide having the sterically bulky modification and / or disubstituted modification is located at a specific position on the sense strand in the double-stranded region represented by formula I, the specific position being complementary to at least one of the 2, 8, 12, or 15 positions (along the direction from the 5′ end to the 3′ end) of the antisense strand.

[0014] In some embodiments of the present disclosure, the modified nucleotide X in the sense strand is located at a complementary position to one of the 2nd, 8th, or 15th positions of the antisense strand complementary to the sense strand.

[0015] In some embodiments of the present disclosure, optionally, counting from the 5'-end, X is located at position 15 of the antisense chain, and at least one fluoromodification is present at position 9 or 12 of the antisense chain, and / or the position of the sense chain at a complementary position to position 10 of the antisense chain is a fluoromodification.

[0016] In some embodiments of this disclosure, (N)a contains at least one fluoromodified nucleotide.

[0017] In some embodiments of this disclosure, (N)c contains at least two fluoromodified nucleotides.

[0018] In some embodiments of this disclosure, when q'+c'+r'+d'=9, 0≦q'+r'≦4 is satisfied.

[0019] In some embodiments of the present disclosure, at least two fluoromodified nucleotides are included among the first four nucleotides of (N)b' in the sense strand, along the direction from the 5′ end to the 3′ end.

[0020] In some embodiments of this disclosure, X is independently selected from the group consisting of modified nucleotides modified with 2′-O-methoxyethyl (MOE), 2′-O-TBDMS, 2′-O-TOM, and 2′-O-CH2-O-R5, respectively. R5 is a substituted or unsubstituted C1-C3 alkyl group. Optionally, each substituent is independently selected from halogens, C1-C3 alkyl groups, C1-C3 alkoxy groups, and amino groups. Optionally, the halogen is fluorine. MOE is methoxyethyl, TBDMS is a tert-butyldimethylchlorosilane group, and TOM is a triisopropylsiloxymethyl group.

[0021] In some embodiments of this disclosure, the nucleotides substituted with two substituents at the 2′ position are selected from nucleotides modified with [2′-halogen-2′-halogen], [2′-halogen-2′-alkyl], or [2′-alkyl-2′-alkyl] substituents. Exemplarily, they are selected from nucleotides modified with [2′-F-2′-F] difluorinated substituents, wherein the alkyl group is a C1-C3 alkyl group.

[0022] In some embodiments of the present disclosure, the antisense chain further includes one or two phosphorothioate linkage modifications.

[0023] In some embodiments of the present disclosure, the double-stranded oligonucleotide comprises a double-stranded region and an overhang region, that is, the double-stranded oligonucleotide further includes one or more overhang regions at the 3'-end, 5'-end, or both ends of one strand, the length of which the overhang region is 1 to 6 nucleotides.

[0024] In some embodiments of this disclosure, a double-stranded oligonucleotide molecule including an overhang region is represented by the following formula (II): SS:5'-(T)t1-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-(T)t2-3' AS:3'-(T)t1-(N)a-(X)p-(N)b-(X)q-(N)c -(T)t2-5' (II), Here, SS represents the sense chain, AS represents the antisense chain, and T represents the overhang nucleotide. T1 and T2 are independently selected from integers between 0 and 6, and cannot be both 0. The definitions of other substituents are the same as in Formula I above. Each overhang nucleotide T independently represents either a modified nucleotide or an unmodified nucleotide. The modified nucleotide is selected from the group consisting of modified nucleotides modified with 2′-O-alkyl groups (2′-alkoxy groups), 2′-alkyl groups, 2′-substituted alkyl groups, 2′-halogens, 2′-deoxy groups, unbasic nucleotides, or antisense oligonucleotides. Optionally, the number of carbon atoms in the alkyl group is 1 to 6, preferably 1 to 3. The substituent is one or more selected from C1-C6 alkyl groups, C1-C6 alkoxy groups, halogens, and amino groups.

[0025] In some embodiments of this disclosure, the antisense strand of a double-stranded oligonucleotide has a nucleotide overhang at its 3' end and a blunt end at its 5' end, with the overhang containing 1 to 3 nucleotides. Here, the double-stranded oligonucleotide molecule is represented by the following formula (IIa): SS:5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3', AS:3'-(T)t1-(N)a-(X)p-(N)b-(X)q-(N)c-5' (IIa), Here, t1 is selected from integers between 1 and 3, and the definitions of the other substituents are the same as in Equation II above.

[0026] In some embodiments of the present disclosure, the overhang region (T)t1 has phosphorothioate groups between the nucleotides. In one embodiment, the double-stranded oligonucleotide according to the present disclosure further has two blunt ends at both ends of the double strand. In some embodiments of the present disclosure, the double-stranded oligonucleotides relating to the present disclosure have a double-stranded region having a length of 19 to 21 nucleotides (nt).

[0027] In some embodiments of the present disclosure, each strand of the double-stranded oligonucleotide contains 17 to 35 nucleotides. In some embodiments of this disclosure, each strand of the double-stranded oligonucleotide contains 17 to 25 nucleotides.

[0028] In some embodiments of this disclosure, each strand of the double-stranded oligonucleotide contains 17 to 21 nucleotides. In some embodiments of this disclosure, each strand of the double-stranded oligonucleotide contains 19 to 23 nucleotides.

[0029] In some embodiments of this disclosure, each strand of the double-stranded oligonucleotide contains 19 to 21 nucleotides.

[0030] In some embodiments of this disclosure, the modified nucleotide X in formulas I and II is represented by the following formula (A), or is a tautomer of formula (A), [ka] Here, B is selected from nucleotide bases and includes uracil or its derivatives, thymine or its derivatives, cytosine or its derivatives, 5-methylcytosine or its derivatives, adenine or its derivatives, guanine or its derivatives.

[0031] In some embodiments of the present disclosure, when the modified nucleotide X is a nucleotide modified with a 2′ sterically bulky group, W1 and W2 are each independently selected from the groups H, -O-MOE, -O-TBDMS, -O-TOM, and -O-CH2-OR, R is a substituted or unsubstituted C1-C3 alkyl group, and one of W1 and W2 is H, and W1 and W2 are different.

[0032] In some embodiments of this disclosure, when the modified nucleotide X is a 2′ disubstituted nucleotide, both W1 and W2 are halogens, or one of W1 and W2 is a halogen and the other is a C1-C6 alkyl group.

[0033] In some embodiments of this disclosure, the halogen is fluorine (F). In some embodiments of this disclosure, both W1 and W2 are F. In some embodiments of this disclosure, one of W1 and W2 is F and the other is a C1-C3 alkyl group.

[0034] In some embodiments of the present disclosure, the overhang (T)t1 of the AS chain comprises at least one nucleotide substituted with 2′-F-2′-Me. In some embodiments of the present disclosure, the double-stranded oligonucleotide further comprises at least one ligand.

[0035] In some embodiments of this disclosure, the ligand includes a liver-targeting ligand and a non-liver-targeting ligand. In some embodiments of the present disclosure, the ligand is a liver-targeted ASGPR ligand, and the ASGPR ligand comprises GalNAc or a derivative thereof conjugated via a branched linker.

[0036] In another embodiment, the disclosure further provides a double-stranded oligonucleotide comprising a sense strand and an antisense strand, each strand having 17 to 35 nucleotides, each nucleotide being either a modified or unmodified nucleotide, the sense strand and antisense strand forming a double-stranded region in at least partially reverse complementarity, the double-stranded oligonucleotide comprising at least one nucleotide having a 2'-disubstituted modification, each substituent in the 2'-disubstituted being independently selected from a methyl group or fluorine.

[0037] If the double-stranded region of the double-stranded oligonucleotide contains a nucleotide having a 2'-disubstituted modification, the nucleotide having the 2'-disubstituted modification is selected from nucleotides that are 2'-difluorosubstituted. Alternatively, if the double-stranded oligonucleotide contains a nucleotide having a 2'-disubstituted modification at a position other than the double-stranded region, the nucleotide having the 2'-disubstituted modification is selected from nucleotides that are substituted with 2'-F-2'-Me.

[0038] In some embodiments of this disclosure, the double-stranded oligonucleotide is (1) If the double-stranded oligonucleotide contains a 2'-difluorosubstituted nucleotide, the 2'-difluorosubstituted nucleotide is located at least one of the positions 7, 8, 9, or 10, counting from the 5' end of the sense strand. (2) If the double-stranded oligonucleotide includes a 2'-difluorosubstituted nucleotide, the 2'-difluorosubstituted nucleotide is located at least one of the following positions, counting from the 5' end of the antisense strand: (3) If the double-stranded oligonucleotide contains a nucleotide substituted with 2′-F-2′-Me, the nucleotide substituted with 2′-F-2′-Me is located outside the double-stranded region of the oligonucleotide. (4) If the double-stranded oligonucleotide contains a nucleotide substituted with 2′-F-2′-Me, the nucleotide substituted with 2′-F-2′-Me is located in the overhang at the 3′ end of the antisense strand. It possesses one or more of the following characteristics.

[0039] In some embodiments of the present disclosure, in feature (1), if the double-stranded oligonucleotide includes a 2'-difluorosubstituted nucleotide, the 2'-difluorosubstituted nucleotide is located at at least two or at least three of the positions 7, 8, 9, or 10, counting from the 5' end of the sense strand.

[0040] In some embodiments of the present disclosure, in feature (2), if the double-stranded oligonucleotide includes a 2'-difluorosubstituted nucleotide, the 2'-difluorosubstituted nucleotide is located at least two of the following positions, from the 5' end of the antisense strand: 2, 4, 6, 8, 9, 10, 12, 14, or 16.

[0041] In other embodiments, the Disclosure further provides oligonucleotide conjugates comprising a double-stranded oligonucleotide according to the Disclosure and a binding group attached to the double-stranded oligonucleotide, wherein the binding group comprises a linker and a pharmaceutically acceptable target-directing group and / or delivery auxiliary group, the target-directing group being selected from ligands capable of binding to cell surface receptors, and the delivery auxiliary group being selected from groups that enhance the biocompatibility of the oligonucleotide conjugate in organs or tissues of the delivery target.

[0042] In other embodiments, the Disclosure further provides pharmaceutical compositions comprising a double-stranded oligonucleotide or its conjugate relating to the Disclosure and a pharmaceutically acceptable carrier. In other embodiments, the Disclosure further provides the use of double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates relating to the Disclosure in the preparation of pharmaceuticals for the treatment and / or prevention of diseases or conditions relating to mRNA levels of target gene expression.

[0043] In other embodiments, the Disclosure further provides methods for treating and / or preventing diseases or conditions related to mRNA levels of target gene expression. The methods include administering to a subject a double-stranded oligonucleotide, pharmaceutical composition, and / or oligonucleotide conjugate relating to the Disclosure.

[0044] In other embodiments, the Disclosure further provides a method for delivering a double-stranded oligonucleotide according to the Disclosure to a specific target in a subject, the method including subcutaneous or intravenous administration. In other embodiments, the Disclosure further provides methods for modulating the expression level of a target gene in cells. The methods include contacting the cells with an effective amount of a double-stranded oligonucleotide, pharmaceutical composition, and / or oligonucleotide conjugate relating to the Disclosure.

[0045] Furthermore, this disclosure provides a kit comprising a double-stranded oligonucleotide, a pharmaceutical composition, and / or an oligonucleotide conjugate relating to this disclosure. Other aspects or advantages of this disclosure are partially set forth in the following description, become clear from the following description, or are understood through the practice of this disclosure.

[0046] Beneficial effects The double-stranded oligonucleotides and / or oligonucleotide conjugates relating to this disclosure can be made tolerable by specific modifications at one or more sites in the sense strand and / or antisense strand, maintain good pharmacokinetic activity of the oligonucleotide, and exhibit relatively high regulatory activity of target gene expression.

[0047] To more clearly explain the technical concepts of the embodiments in this application, the drawings necessary for describing the embodiments are briefly described below. The drawings described are only a selection of embodiments of this application and do not limit the scope. A person skilled in the art can obtain other relevant drawings based on these drawings without employing inventive ability. [Brief explanation of the drawing]

[0048] [Figure 1] Example 2 of this disclosure shows the relative expression levels of the target gene CC3 mRNA after transfection of HepG2 with siRNA modified at different sites of the sense strand using TBDMS. [Figure 2] Example 2 of this disclosure shows the relative expression levels of the target gene CC3 mRNA after transfection of HepG2 with siRNA modified at different sites of the antisense strand using TBDMS. [Figure 3] In Example 3 of this disclosure, the expression level of CC3 protein in the serum of mice after administration of an siRNA conjugate is shown. [Figure 4]Example 4 of this disclosure shows the relative expression levels of the target gene ANGPTL3 mRNA in primary mouse hepatocytes after ad libitum intake of siRNA modified at different sites of the sense strand using TBDMS. [Figure 5] Example 4 of this disclosure shows the relative expression levels of the target gene ANGPTL3 mRNA in primary mouse hepatocytes after ad libitum intake of siRNAs in which different sites of the antisense strand were modified using TBDMS. [Figure 6] Example 5 of this disclosure shows the expression level of ANGPTL3 mRNA in the mouse body after administration of an siRNA conjugate. [Figure 7] In Example 6 of this disclosure, primary mouse hepatocytes show the relative expression levels of the target gene FXI mRNA after ad libitum intake of siRNA modified at different sites of the sense strand using TBDMS. [Figure 8] In Example 6 of this disclosure, primary mouse hepatocytes show the relative expression levels of the target gene FXI mRNA after ad libitum intake of siRNA modified at different sites of the antisense strand using TBDMS. [Figure 9] Example 7 of this disclosure shows the expression level of CC3 mRNA in the mouse body after administration of an siRNA conjugate. [Figure 10] Example 8 of this disclosure shows the relative expression levels of the target gene CC3 mRNA after transfection of HepG2 with siRNA modified at different sites of the sense strand using MOE. [Figure 11] Example 8 of this disclosure shows the relative expression levels of the target gene CC3 mRNA after transfection of HepG2 with siRNA having MOE modifications at different sites of the antisense strand. [Figure 12] In Example 9 of this disclosure, the relative expression level of the target gene CC3 mRNA is shown after transfection of HepG2 with siRNA having multiple MOE modifications on the sense strand. [Figure 13]In Example 9 of this disclosure, the relative expression level of the target gene CC3 mRNA is shown after transfection of HepG2 with siRNA having multiple MOE modifications on the antisense strand. [Figure 14] Example 9 of this disclosure shows the relative expression levels of the target gene CC3 mRNA after transfection of HepG2 with siRNA having multiple MOE modifications on the sense and antisense strands. [Figure 15] In Example 10 of this disclosure, the expression levels of CC3 mRNA in mice after administration of siRNA conjugates (RZ502031, RZ502033, RZ502034, RZ502035, RZ502039) are shown. [Figure 16] In Example 10 of this disclosure, the expression levels of CC3 mRNA in mice after administration of siRNA conjugates (RZ502031, RZ502036, RZ502049, RZ502087) are shown. [Figure 17] Example 11 of this disclosure shows the expression level of SOD1 mRNA in the mouse body after administration of an siRNA conjugate. [Figure 18a] In Example 12 of this disclosure, the relative expression levels of the target gene CC3 mRNA are shown after transfection of HepG2 with siRNA conjugates (RZ002003, RZ002006, RZ002011, and RZ002031 have different modification patterns). [Figure 18b] In Example 12 of this disclosure, the relative expression levels of the target gene CC3 mRNA are shown after transfection of HepG2 with siRNA conjugates (RZ002003, RZ002006, RZ002011, and RZ002031 have different modification patterns). [Figure 18c] In Example 12 of this disclosure, the relative expression levels of the target gene CC3 mRNA are shown after transfection of HepG2 with siRNA conjugates (RZ002003, RZ002006, RZ002011, and RZ002031 have different modification patterns). [Figure 18d] In Example 12 of this disclosure, the relative expression levels of the target gene CC3 mRNA are shown after transfection of HepG2 with siRNA conjugates (RZ002003, RZ002006, RZ002011, and RZ002031 have different modification patterns). [Figure 19] In Example 13 of this disclosure, the relative expression levels of the target gene CC3 mRNA are shown after transfection of Huh7 with an siRNA conjugate (RZ003017 and RZ003020 have different modification designs). [Figure 20] In Example 14 of this disclosure, the expression levels of AGT protein in the serum of hREN×hAGT hypertensive mice after administration of siRNA conjugates (RZ003021, RZ003065, RZ003066) are shown. [Figure 21] In Example 15 of this disclosure, the relative expression levels of target gene mRNA in an LPA-targeted HDI mouse model are shown after administration of siRNA conjugates (RZ001032, RZ001034, RZ001037, RZ001039). [Figure 22] In Example 16 of this disclosure, the expression levels of LPA protein in hApo(a) transgenic mice after administration of siRNA conjugates (RZ001032, RZ001034) are shown. [Figure 23] In Example 17 of this disclosure, the relative expression levels of SOD1 mRNA in mice were shown after administration of siRNA conjugates in which different sites of the antisense strand were modified with NM062 and NM063. [Figure 24] In Example 18 of this disclosure, primary mouse hepatocytes show the relative expression levels of the target gene SOD1 mRNA after ad libitum intake of siRNA conjugates modified with antisense strands NM096, NM097, NM098, and NM099. [Figure 25]In Example 19 of this disclosure, primary mouse hepatocytes exhibit relative expression levels of the target gene SOD1 mRNA after ad libitum intake of an siRNA conjugate (RZ599051) in which the sense strand has been difluoromodified at the 2' position. [Figure 26] In Example 20 of this disclosure, the relative expression levels of the target gene SOD1 mRNA are shown after primary mouse hepatocytes ad libitum ingested siRNA conjugates (RZ599055, RZ599056) with 2' difluoromodification of the sense strand. [Figure 27] Example 21 of this disclosure shows the relative expression level of the target gene ANGPTL3 mRNA in primary mouse hepatocytes after ad libitum intake of an siRNA conjugate in which the sense strand has been difluoromodified at the 2' position. [Figure 28] Example 22 of this disclosure shows the relative expression level of the target gene CFB mRNA in primary mouse hepatocytes after ad libitum intake of an siRNA conjugate in which the sense strand has been difluoromodified at the 2' position. [Figure 29] In Example 23 of this disclosure, the relative expression level of the target gene CFB mRNA in a mouse body after administration of an siRNA conjugate in which the sense strand was difluoromodified at the 2' position is shown. [Figure 30] Example 24 of this disclosure shows the relative expression level of the target gene ANGPTL3 mRNA in primary mouse hepatocytes after ad libitum intake of an siRNA conjugate in which the antisense strand has been difluoromodified at the 2' position. [Figure 31] Example 25 of this disclosure shows the relative expression levels of target gene CFB mRNA in primary mouse hepatocytes after ad libitum intake of siRNA conjugates (RZM11504, RZM11505) in which the antisense strand is difluoromodified at the 2' position. [Figure 32] In Example 26 of this disclosure, primary mouse hepatocytes show the relative expression levels of the target gene CFB mRNA after ad libitum intake of an siRNA conjugate (RZM11507) in which the antisense strand has been difluoromodified at the 2' position. [Figure 33]In Example 27 of this disclosure, the relative expression level of the target gene CFB mRNA in a mouse body is shown after administration of an siRNA conjugate in which the antisense strand is difluoromodified at the 2' position. [Figure 34] In Example 28 of this disclosure, the relative expression level of the target gene C4B mRNA in a mouse body is shown after administration of an siRNA conjugate in which the antisense strand is difluoromodified at the 2' position. [Figure 35] In Example 29 of this disclosure, the relative expression level of the target gene SOD1 mRNA in the mouse body is shown after administration of an siRNA conjugate in which the antisense strand contains NM054 modification. [Figure 36] In Example 30 of this disclosure, the relative expression level of the target gene ANGPTL3 mRNA in the mouse body is shown after administration of an siRNA conjugate in which the antisense strand contains NM054 modification. [Figure 37] Example 31 of this disclosure shows the expression levels of CC3 protein in the serum of cynomolgus monkeys after administration of siRNA conjugates (RZ002099, RZ002101, RZ002106, RZ002113). [Figure 38] In Example 32 of this disclosure, the expression level of AGT protein in the serum of cynomolgus monkeys after administration of an siRNA conjugate (RZ003069) is shown. [Modes for carrying out the invention]

[0049] The embodiments of this disclosure are described below in detail. The embodiments described below are illustrative and intended to interpret this disclosure, and are not intended to limit it. Furthermore, the terms “First” and “Second” are for descriptive purposes only and do not express or imply relative importance or the number of technical features. Therefore, features limited by “First” and “Second” may expressly or imply include one or more such features. Moreover, in this disclosure, unless otherwise specified, “multiple” means two or more.

[0050] The endpoints and any values ​​of the ranges disclosed herein are not limited to those specific ranges or values, and these ranges or values ​​should be understood to include values ​​close to those ranges or values. In the case of numerical ranges, one or more new numerical ranges can be obtained by combinations of the endpoints of each range, combinations of the endpoints of each range and individual specific values, and combinations of individual specific values, and these numerical ranges should also be considered to be specifically disclosed herein.

[0051] To facilitate understanding of this disclosure, several technical and scientific terms are defined below. Unless otherwise noted, all other technical and scientific terms used herein have the meanings that are ordinarily understood by those skilled in the art.

[0052] In this specification, the terms “contains” or “includes” are open and include what is expressed in this disclosure, but do not exclude what is expressed in other aspects.

[0053] In this specification, the terms “optionally” or “optionally” usually mean that the event or situation described thereafter may occur, but is not necessarily so, and such description includes both the cases in which the event or situation occurs and the cases in which it does not occur.

[0054] As used herein, the term "and / or" describes a relationship between related objects, indicating that three types of relationships exist. For example, A and / or B describes three types of relationships: A exists alone, both A and B exist, and B exists alone.

[0055] In this specification, the term "complement component 3" (CC3) refers to the intrinsic component involved in the three complement activation pathways and is the most abundant complement component in plasma. When complement is activated, C3 is broken down by C3 convertase into small fragment C3a and large fragment C3b. C3a is released into the liquid phase and has anaphylatoxin activity. C3b is an important component of C5 convertase in the three activation pathways and C3 convertase in the secondary pathway, and mediates opsonization and immunoadhesion by binding to immune complexes, microorganisms, macromolecules, or cell membranes.

[0056] In this specification, the term “angiopoietin-like protein 3” (ANGPTL3) refers to a member of the family of angiopoietin-like proteins involved in the regulation of blood lipid metabolism, which further includes ANGPTL4 and ANGPTL8. The protein encoded by the ANGPTL3 gene is produced and secreted into the circulatory system only in the liver. In the liver, ANGPTL3 is entirely produced by hepatocytes, and its expression is controlled by the liver X receptor (LXR), which is activated by hydroxysterols.

[0057] In this specification, the term "coagulation factor XI" (FXI) refers to a plasma glycoprotein involved in the contact stage of the coagulation process (intrinsic coagulation pathway) and is important for normal hemostasis in the body.

[0058] In this specification, the term “oligonucleotide” refers to nucleic acid molecules (RNA or DNA) having a length of, for example, less than 100, 200, or 300 nucleotides.

[0059] In this specification, the term "LNA" refers to locked nucleic acid. LNA is a modified RNA nucleotide. The ribose portion of an LNA nucleotide is modified by linking the 2′ hydroxyl group to the 4′ carbon of the same ribose with an additional crosslink (e.g., a methylene crosslink or an ethylene crosslink). The term "ENA" refers to ethylene-crosslinked nucleic acid, which usually refers to restrained or inaccessible RNA. The term "BNA" refers to crosslinked nucleic acid, which comprises a crosslinked structure of "fixed" C3'-end sugar puckering of a 5-membered ring, 6-membered ring, and even a 7-membered ring, and the crosslink is usually introduced at the 2′- and 4′-positions of the ribose to provide a 2′,4′-BNA nucleotide (e.g., LNA or ENA). The term "GNA" refers to glycol nucleic acid, and "UNA" refers to acyclic nucleotide.

[0060] In this specification, unless otherwise specified, uppercase C, G, U, A, and T represent the base composition of a nucleotide; lowercase m indicates that the nucleotide to the left of the letter m is a methoxy-modified nucleotide; lowercase f indicates that the nucleotide to the left of the letter f is a fluoro-modified nucleotide; and lowercase s indicates that the two nucleotides adjacent to the left and right of the letter s are linked by a phosphorothioate group.

[0061] In this specification, "fluoromodified nucleotide" refers to a nucleotide formed by substituting the hydroxyl group at the 2′ position of the ribose group with fluorine. Unless otherwise specified, fluoromodification refers to monofluoromodification, and "nucleotide analogue" or "nucleotide analogue" refers to a group that can substitute for a nucleotide in nucleic acids but has a different structure from adenine-ribose nucleotide, guanine-ribose nucleotide, cytosine-ribose nucleotide, uracil-ribose nucleotide, or thymine, deoxyribonucleotide, such as isonucleotides, bridged nucleic acids (abbreviated as BNA), or acyclic nucleotides. "Methoxy-modified nucleotide" refers to a nucleotide formed by substituting the 2′-hydroxyl group of the ribose group with a methoxy group. In this specification, the 2-methoxy group may also be represented as 2′-OMe, and 2-methoxy group and 2′-OMe are interchangeable in this specification. Unless otherwise specified, throughout this text, "modified nucleotide" refers to a nucleotide in which the 2′ position of the ribose group of the nucleotide is modified.

[0062] In this specification, a nucleotide may refer to an individual nucleotide or to a nucleotide residue in an oligonucleotide. In this specification, a nucleotide may be singular or plural, and if plural, it refers to one type of nucleotide. In this specification, a double-stranded oligonucleotide is represented by dsRNA or siRNA, and double-stranded oligonucleotides, dsRNA, and siRNA are interchangeable in this specification.

[0063] In this specification, "alkyl group" refers to a linear or branched group having a predetermined number of carbon atoms (usually 1 to 20 carbon atoms). For example, C1-C6 alkyl groups include linear and branched alkyl groups having 1 to 6 carbon atoms.

[0064] In this specification, the siRNA molecule consists of two strands, the strand that binds to the target mRNA is called the antisense strand or guide strand, and the other strand is called the sense strand or passenger strand. The term “antisense strand” refers to the strand of siRNA that contains a region that is completely or essentially complementary to the target sequence. The term “sense strand” refers to the strand of siRNA that contains a region that is essentially complementary to the region of the antisense strand as defined herein. The term “complementary region” refers to a region in the antisense strand that is completely or essentially complementary to the target mRNA sequence. If the complementary region and the target sequence are not completely complementary, the mismatch is located in the interior or terminal region of the molecule. As used herein, the term “complementary” refers to the ability of a first polynucleotide to hybridize with a second polynucleotide under specific conditions, e.g., severe conditions.

[0065] Throughout this specification, the terms "double-stranded structure," "double-stranded region," and "double-stranded region" are interchangeable and have meanings familiar to those skilled in the art, namely, a double-stranded structure formed in a double-stranded nucleic acid molecule by the complementary pairing of a sense strand and an antisense strand. In this specification, "complementary" and "reverse complementary" are interchangeable and have meanings familiar to those skilled in the art, namely, in a double-stranded nucleic acid molecule, each base of one strand pairs with a base of the other strand in a complementary manner.

[0066] In this specification, unless otherwise specified, the term “complementary” includes “basically inversely complementary,” “substantially inversely complementary,” and “fully inversely complementary.” “Basically inversely complementary” means that there are three or fewer base mismatches between two nucleotide sequences. “Substantially inversely complementary” means that there is one or fewer base mismatches between two nucleotide sequences. “Fully inversely complementary” means that there are no base mismatches between two nucleotide sequences.

[0067] In this specification, unless otherwise specified, “conjugation” refers to the covalent linking of two or more chemical moieties, each having a specific function. Accordingly, “conjugate” refers to a compound formed by the covalent linking of these chemical moieties. Furthermore, “oligonucleotide conjugate” refers to a compound formed by the covalent linking of one or more chemical moieties, each having a specific function, to an oligonucleotide. Throughout this disclosure, “conjugating molecule” should be understood as a specific compound that reacts with an oligonucleotide to form an oligonucleotide conjugate according to this disclosure.

[0068] Through research and experimentation, the inventors have found that by making specific modifications to one or more sites on the sense strand and / or antisense strand of a double-stranded oligonucleotide, good tolerability can be achieved, the activity of the double-stranded oligonucleotide molecule can be well maintained, and ultimately, its activity can be further improved.

[0069] In a first embodiment, the disclosure provides modified double-stranded oligonucleotide molecules and reagents thereof that enhance inhibitory activity without reducing inhibitory activity, as well as pharmaceutical compositions suitable for therapeutic applications.

[0070] In a specific embodiment, the present disclosure provides a double-stranded oligonucleotide (dsRNA) for repressing the expression of a target gene. The double-stranded oligonucleotide comprises a sense strand and an antisense strand, each strand having 17 to 35 nucleotides, each nucleotide being either a modified or unmodified nucleotide, the sense strand and antisense strand forming a double-stranded region in at least partially reverse complementarity, and comprising nucleotides having sterically bulky modifications and / or disubstituted modifications at specific positions in the nucleotide sequences of the sense strand and / or antisense strand along the direction from the 5′ end to the 3′ end, wherein the sterically bulky nucleotide refers to a nucleotide in which the hydroxyl group at the 2′ position of the ribose of the nucleotide is substituted with a sterically bulky group, the sterically bulky group at the 2′ position is selected from groups having a sterically bulkier size than the 2′-O-methyl group, and the disubstituted nucleotide refers to a nucleotide in which both the hydroxyl group and hydrogen at the 2′ position of the ribose of the nucleotide are substituted. Optionally, each substituent in the two substituents is independently selected from C1-C6 alkyl groups or halogens.

[0071] In some embodiments, in the double-stranded oligonucleotide according to the Disclosure, at least one of the nucleotides at position 8, 9, 10, or 15 of the antisense strand along the direction from the 5′ end to the 3′ end is a nucleotide having a sterically bulky modification or a nucleotide having a disubstituted modification, and / or a modified nucleotide X is present or absent in the sense strand. If a modified nucleotide X is present in the sense strand, at least one nucleotide X is located at a complementary position to any position at position 2, 8, or 15 (in the direction from the 5′ end to the 3′ end) of the antisense strand.

[0072] In some embodiments, the antisense and sense strands of the double-stranded oligonucleotide are complementary and form a double-stranded region, the structure of which is represented by the following formula (I): SS: 5’-(N)a’-(X)p’-(N)b’-(X)q’-(N)c’-(X)r’-(N)d’-3’, AS: 3’-(N)a-(X)p-(N)b-(X)q-(N)c-5’ (I), Here, SS represents the sense strand and AS represents the antisense strand.

[0073] In formula (I), each X is independently a nucleotide in which the 2'-hydroxy group in ribose is substituted with a sterically bulky group, or a nucleotide in which the 2'-hydroxy group and hydrogen are substituted with a disubstituent. The sterically bulky group at the 2'-position is selected from groups having a steric bulk larger than a 2'-methoxy group. Each substituent in the disubstituent is independently selected from a C1-C6 alkyl group or a halogen.

[0074] In some embodiments, when X represents a nucleotide in which the 2'-hydroxy group in ribose is substituted with a sterically bulky group, it is independently a 2'-(O) m1 (CH2) n (O) m2 selected from R1, where m1 or m2 is independently 0 or 1, n is selected from integers from 0 to 6, R1 is a C1-C6 alkyl group substituted with a substituent or an unsubstituted C1-C6 alkyl group, or -Si(R2)3, and R2 is independently a substituted or unsubstituted C 1- C6 alkyl group, a substituted or unsubstituted C1-C6 alkoxy group. The substituent is one or more selected from a halogen, a C1-C6 alkyl group, a C1-C6 alkoxy group, a hydroxy group, an amino group, a cycloalkyl group having 6 or fewer carbon atoms, an aryl group having 12 or fewer carbon atoms, or a heteroaryl group having 12 or fewer carbon atoms.

[0075] In some embodiments, each X is independently 2'-O(CH2) which is a modified nucleotide nR1 is selected from OR1 or 2′-R3-2′-R4, where n is 1 or 2, R1 is selected from a substituted C1-C6 alkyl group or an unsubstituted C1-C6 alkyl group, or -Si(R2)3, and R2 is independently selected from a substituted or unsubstituted C1-C6 alkyl group or a substituted or unsubstituted C1-C6 alkoxy group, and the substituent is selected from a halogen, a C1-C6 alkyl group, a C1-C6 alkoxy group, a hydroxyl group, an amino group, a cycloalkyl group having 6 or fewer carbon atoms, an aryl group having 12 or fewer carbon atoms, or a heteroaryl group having 12 or fewer carbon atoms.

[0076] R3 and R4 are each independently selected from a C1-C6 alkyl group or a halogen. Optionally, the halogen is fluorine. Optionally, the C1-C6 alkyl group is selected from a methyl group or an ethyl group.

[0077] In formula I, each N independently represents an unmodified nucleotide or a nucleotide that is different from X and has a modified 2' position in the ribose, the modified nucleotide is selected from the group consisting of nucleotides modified with a 2'-O-alkyl group, a 2'-alkyl group, a 2'-substituted alkyl group, a 2'-halogen, a 2'-deoxy, or a nucleotide analog, the nucleotide analog is selected from ENA, BNA, LNA, GNA, or UNA, and optionally the alkyl group or alkoxy group has 1 to 6 carbon atoms.

[0078] In some embodiments, N is independently selected from 2′-OMe modified nucleotides, 2′-F modified nucleotides, or 2′-H modified nucleotides. 2′-H modified nucleotides and 2′-deoxy modified nucleotides have the same meaning, i.e., the 2′-hydroxyl group is substituted with H. In some other embodiments, N is independently selected from 2′-OMe modified nucleotides or 2′-F modified nucleotides.

[0079] In some embodiments, nucleotide X, in which the hydroxyl group at the 2′ position is substituted with a sterically bulky group, is independently selected from the group consisting of modified nucleotides modified with the groups 2′-O-methoxyethyl (MOE), 2′-O-TBDMS, 2′-O-TOM, and 2′-O-CH2-OR. R is a substituted or unsubstituted C1-C3 alkyl group. Optionally, each substituent is independently selected from halogens, C1-C3 alkyl groups, C1-C3 alkoxy groups, and amino groups, one or more of these.

[0080] In some embodiments, X is independently selected from modified nucleotides modified with 2′-O-methoxyethyl (MOE), 2′-O-CH2-O-CH2-CH3, and 2′-O-CH2-O-CH2-CF3, respectively.

[0081] In some embodiments, the nucleotides substituted with two substituents at the 2′ position are selected from nucleotides modified with [2′-halogen-2′-halogen], [2′-halogen-2′-alkyl], or [2′-alkyl-2′-alkyl], and optionally selected from nucleotides modified with [2′-F-2′-F] or [2′-fluoro-2′-alkyl]. Optionally, the alkyl group is a C1-C3 alkyl group.

[0082] In formula I, a, a', p, p', b, b', q, q', c, c', r', and d' each independently represent the number of nucleotides. For example, if a is 3 in (N)a, it represents three consecutive modified nucleotides NNN, where each N may be the same or different. a' is an integer from 3 to 8, p' is an integer from 0 to 3, b' is an integer from 4 to 13, q' is an integer from 0 to 4, c' is an integer from 3 to 9, r' is an integer from 0 to 3, d' is an integer from 0 to 9, a is an integer from 4 to 7, p is an integer from 0 to 1, b is an integer from 4 to 8, q is an integer from 0 to 4, and c is an integer from 6 to 10. p', q', r', p, and q cannot be 0 at the same time.

[0083] It is not possible for q' and r' to be 0 at the same time by any choice. We can choose to satisfy q'+c'+r'+d'=9 and 0≦q'+r'≦4. Selectively, at least one fluoromodified nucleotide is present in each of (N)a and (N)b, and at least two fluoromodified nucleotides are present in (N)c.

[0084] In some embodiments, the structure of nucleotide X according to the Disclosure is represented by the following formula (A), or is a tautomer of formula (A), [ka] Here, B is selected from nucleotide bases and includes uracil or its derivatives, thymine or its derivatives, cytosine or its derivatives, 5-methylcytosine or its derivatives, adenine or its derivatives, guanine or its derivatives.

[0085] In some embodiments of the present disclosure, when the modified nucleotide X is a nucleotide modified with a 2′ sterically bulky group, W1 and W2 are each independently selected from the groups H, -O-MOE, -O-TBDMS, -O-TOM, and -O-CH2-OR, R is a substituted or unsubstituted C1-C3 alkyl group, and one of W1 and W2 is H, and W1 and W2 are different.

[0086] In some embodiments of this disclosure, when the modified nucleotide X is a 2′ disubstituted nucleotide, both W1 and W2 are halogens, or one of W1 and W2 is a halogen and the other is a C1-C6 alkyl group.

[0087] In some embodiments of this disclosure, the halogen is fluorine (F). In some embodiments of this disclosure, both W1 and W2 are F. In some embodiments of this disclosure, one of W1 and W2 is F, and the other is a methyl group or an ethyl group.

[0088] Here, the wavy lines represent the bonds between nucleotides. In some embodiments, the double-stranded oligonucleotides of the present disclosure further include one or more overhang regions in addition to the double-stranded region, the length of which is 1 to 6 nucleotides. These overhangs may cause one strand to be longer than the other, or both strands to be longer but the same length. The overhangs may form a mismatch with the target mRNA, or may be complementary to the sequence of the target gene, or may be other sequences, for example, the sense strand and antisense strand may be linked by another base to form a hairpin structure, or by another non-base linker. In one example, each nucleotide in the overhang group may independently be a modified nucleotide or an unmodified nucleotide.

[0089] In one example, the overhang is located on the sense strand, the antisense strand, or at the 3' end of both strands. Optionally, the double-stranded oligonucleotide according to this disclosure contains only one overhang. In another example, this 3'-overhang is located on the antisense strand. In yet another example, this 3'-overhang is located on the sense strand.

[0090] In some embodiments of this disclosure, a double-stranded oligonucleotide molecule including an overhang region can be represented by the following formula II: SS:5'-(T)t1-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-(T)t2-3' AS:3'-(T)t1-(N)a-(X)p-(N)b-(X)q-(N)c-(T)t2-5' (II), Here, SS represents the sense strand, AS represents the antisense strand, T represents the overhang nucleotide, t1 and t2 are independently selected from integers between 0 and 6 and can never be 0 at the same time, and the definitions of the other substituents are the same as in formula (I) above.

[0091] Each of the overhang nucleotides T independently represents a modified nucleotide or an unmodified nucleotide, and the modified nucleotide is selected from the group consisting of a modified nucleotide modified with a 2'-alkoxy group, an alkoxy group substituted with 2'-C1-C6, a 2'-alkyl group, an alkyl group substituted with 2'-C1-C6, a 2'-halogen, a 2'-deoxy, a base-free nucleotide, or an antisense oligonucleotide. Optionally, the number of carbon atoms in the alkyl group and alkoxy group is 1 to 6, preferably 1 to 3.

[0092] In some embodiments of this disclosure, the antisense strand of a double-stranded oligonucleotide has one nucleotide overhang at its 3' end, and the overhang contains one to three nucleotides. Here, the double-stranded oligonucleotide molecule is represented by the following formula (IIa): SS:5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3' AS:3'-(T)t1-(N)a-(X)p-(N)b-(X)q-(N)c-5' (IIa), Here, t1 is selected from integers between 1 and 3, and the definitions of the other substituents are the same as in equation (II) above.

[0093] In some examples, the overhang group (T)t1 has a phosphorothioate group between the two nucleotides. In one embodiment, the double-stranded oligonucleotide according to the disclosure further has two blunt ends at both ends of the double strand.

[0094] In some embodiments, the double-stranded region of the double-stranded oligonucleotide according to the Disclosure has a length of 17 to 30 nucleotide pairs. For example, the double-stranded region has 17 to 25 nucleotide pairs, 17 to 21 nucleotide pairs, 17 to 19 nucleotide pairs, or 19 to 25 nucleotide pairs, 19 to 23 nucleotide pairs, 19 to 21 nucleotide pairs, or 21 to 25 nucleotide pairs, or 21 to 23 nucleotide pairs. In some embodiments, the length of the double-stranded region is selected from the lengths of 17, 18, 19, 20, 21, 22, and 23 nucleotide pairs.

[0095] In one embodiment, the length of the double-stranded region is the length of 19 or 21 nucleotide pairs. In some embodiments, the antisense strand and sense strand in the double-stranded oligonucleotide according to the disclosure are complementary in the double-stranded region represented by formula (I). This complementarity includes basically reverse complementarity, substantially reverse complementarity, or completely reverse complementarity. In one embodiment, the antisense strand and sense strand in the double-stranded region represented by formula (I) are completely reverse complementarity. In another embodiment, there are 1 to 3 base mismatches in the antisense strand and sense strand in the double-stranded region represented by formula (I), thereby achieving substantially reverse complementarity or essential reverse complementarity between the antisense strand and the sense strand.

[0096] In some embodiments, each strand of a double-stranded oligonucleotide represented by formula (II) of the present disclosure has a length of 17 to 35 nucleotides. For example, each strand of a double-stranded oligonucleotide may have 17 to 23 nucleotides, 17 to 21 nucleotides, 17 to 19 nucleotides, or 19 to 25 nucleotides, 19 to 23 nucleotides, 19 to 21 nucleotides, or 21 to 23 nucleotides.

[0097] In some embodiments, in the double-stranded oligonucleotide molecule according to the Disclosure, nucleotide X is a nucleotide modified with a sterically bulky group, and one or more nucleotide X are present and located only in the antisense strand. In some embodiments, if the antisense strand contains only one X along the direction from the 5′ end to the 3′ end, X is located at any one of the positions 8-10 or 15 of the antisense strand. Optionally, X is located at position 10 or 15 of the antisense strand.

[0098] In some embodiments, in the double-stranded oligonucleotide molecule according to the Disclosure, nucleotide X is a nucleotide modified with a sterically bulky group, and one or more X are present and located only on the sense strand. Optionally, the sense strand contains one or two X. Optionally, in the sense strand, X is located at a complementary position to any of the positions 2, 8, or 15 (counting from the 5' end) of the antisense strand. The complementary position refers to the corresponding position (i.e., complementary position) in the double-stranded region between a nucleotide on one strand and a corresponding complementary nucleotide on the other strand.

[0099] In some embodiments, nucleotide X is a nucleotide modified with a sterically bulky group. In a double-stranded oligonucleotide molecule comprising the double-stranded region structure represented by formula (I) above according to the present disclosure, if the double-stranded body has 19 nucleotide pairs (or the double-stranded oligonucleotide molecule has 19 / 21 nucleotides), at least one nucleotide X is located at positions 7-9 of the sense strand (counted from 5' to 3'), and the nucleotides at positions 7-9 of the sense strand are complementary to the nucleotides at positions 11-13 of the antisense strand (counted from 5' to 3').

[0100] In some embodiments, nucleotide X is a nucleotide modified with a sterically bulky group. In a double-stranded oligonucleotide molecule comprising the double-stranded structure represented by formula (I) above according to this disclosure, there are two or more nucleotide X located on the two strands, the antisense strand and the sense strand. In some embodiments, if the antisense strand contains only one X, the sense strand contains one or two X. In some embodiments, X is located at any position among positions 8 to 10 or 15 (counting from the 5' end) on the antisense strand, and on the sense strand, X is located at a complementary position to any position among positions 2, 8, 12 or 15 (counting from the 5' end) on the antisense strand.

[0101] In some embodiments, nucleotide X is a nucleotide modified with a sterically bulky group. In the double-stranded oligonucleotide molecule, there is at least one X located at position 15 (counting from the 5' end) of the antisense strand. In some embodiments, the 2′ sterically bulky group is 2′-O-methoxyethyl (MOE). In some other embodiments, the 2′ sterically bulky group is 2′-O-CH2-O-CH2-CH3 or 2′-O-CH2-O-CH2-CF3.

[0102] In some embodiments, in a double-stranded oligonucleotide represented by formula (II) or a double-stranded oligonucleotide comprising a double-stranded region structure represented by formula (I), N is independently selected from 2′-OMe-modified nucleotides, 2′-F-modified nucleotides, or 2′-deoxy-modified nucleotides. X is independently selected from the group consisting of modified nucleotides modified with 2′-O-MOE, 2′-O-TBDMS, 2′-O-TOM, 2′-O-CH2-O-CH2-CH3, and 2′-O-CH2-O-CH2-CF3, respectively. Here, the number of nucleotides independently represented by a, a', p, p', b, b', q, q', c, c', r', and d' is defined as follows: a' is an integer between 3 and 8, p' is an integer between 0 and 3, b' is an integer between 4 and 13, q' is an integer between 0 and 4, c' is an integer between 3 and 9, r' is an integer between 0 and 3, d' is an integer between 0 and 9, a is an integer between 4 and 7, p is an integer between 0 and 1, b is an integer between 4 and 8, q is an integer between 0 and 4, and c is an integer between 6 and 10. P', q', r', p, and q cannot all be 0 at the same time.

[0103] It is not possible for q' and r' to be 0 at the same time by any choice. We can choose to satisfy q'+c'+r'+d'=9 and 0≦q'+r'≦4. Selectively, at least one fluoromodified nucleotide is present in each of (N)a and (N)b, and at least two fluoromodified nucleotides are present in (N)c.

[0104] In one embodiment, if p'=0, i.e., (X)p' does not exist, then a'=6. In another embodiment, if the double-stranded oligonucleotide has SS / AS=21 / 23nt, then a'=8. In one embodiment, when p'=1, X is located on the sense chain and at one of the complementary positions (counting from the 5' end) to positions 14-16 of the antisense chain.

[0105] In one embodiment, there is at least one X in (X)p', and one X is located in a complementary position to the 15th position (counting from the 5' end) of the antisense chain. Optionally, when p'=1, X is located in a complementary position to the 15th position (counting from the 5' end) of the antisense chain. In one embodiment, when neither (X)q' nor (X)r' exists (i.e., both q' and r' are 0), the sense strand contains only (X)p', and the sense strand SS of the double-stranded region is 5'-(N)a'-(X)p'-(N)k'-(N)d'-3', where k'=7~22.

[0106] In one embodiment, the first four nucleotides of (N)b' in the sense strand (counting from the 5' end) are located in complementary positions to positions 10-13 of the antisense strand (counting from the 5' end), and these first four nucleotides include at least two fluoromodified nucleotides. In one embodiment, q' is preferably 0 to 2, and more preferably 0 to 1. In one embodiment, if neither r' nor q' is 0, then d' is not 0. For example, if r'=1 and q'=1, then b' and c' are 5, and d' is 1 (in this case, in the sense chain, counting from the 5' end, one X is at position 12 and the other X is at position 18).

[0107] In one embodiment, q' and r' are never 0 at the same time. Furthermore, if q' and r' are never 0 at the same time, in the sense chain, at least one X is located in a complementary position to the seed region of the antisense chain (positions 1 to 8, counting from the 5' end). In another embodiment, in the sense chain, there is at least one X located in a complementary position to the seed region of the antisense chain (positions 2 to 8, counting from the 5' end). In one embodiment, d' = 1 to 9. In one embodiment, q'+c'+r'+d'=9 and 0≦q'+r'≦4 is satisfied. In one embodiment, if (X)p is absent, a=5 to 7, for example, if dsRNA has SS / AS=21 / 23nt, a=7.

[0108] In one embodiment, (N)a contains at least one fluoromodified nucleotide, preferably at least one fluoromodified nucleotide located at position 16 (counting from the 5' end) of the antisense chain. In one embodiment, when p is not 0 (i.e., p=1), X is located at position 15 of the antisense chain (counting from the 5' end). In one embodiment, if (X)q does not exist, then b is 8 (i.e., if q=0, then b=8).

[0109] In one embodiment, if (X)q does not exist, then b+c=14. In a preferred embodiment, at least one fluoromodified nucleotide is present at (N)b, preferably at least one fluoromodified nucleotide located at position 14 (counting from the 5' end) of the antisense chain.

[0110] In one embodiment, if (X)q does not exist, then c is 10. In one preferred embodiment, at least two fluoromodified nucleotides are present at (N)c, and more preferably, at least two fluoromodified nucleotides are present and located at positions 2 and 6 (counting from the 5' end) of the antisense chain, respectively.

[0111] In some embodiments, in a double-stranded oligonucleotide represented by formula (II) or a double-stranded oligonucleotide comprising a double-stranded region represented by formula (I) above, the sequences of the antisense strand and the sense strand are sufficiently complementary to each other to form a double-stranded region having 17 to 23 base pairs. N is independently selected from 2′-OMe modified nucleotides, 2′-F modified nucleotides, or 2′-deoxy modified nucleotides. X is independently selected from the group consisting of modified nucleotides modified with 2′-O-MOE, 2′-O-TBDMS, 2′-O-TOM, 2′-O-CH2-O-CH2-CH3, and 2′-O-CH2-O-CH2-CF3, respectively. The number of nucleotides represented by a', p, p', b, b', q, q', c, c', r', and d' are further defined as follows.

[0112] a' is an integer between 3 and 8, p' is an integer between 0 and 2, b' is an integer between 4 and 12, q' is an integer between 0 and 2, c' is an integer between 3 and 8, r' is an integer between 0 and 2, d' is an integer between 1 and 9, a is an integer between 4 and 7, p is 0 or 1, b is an integer between 4 and 8, q is an integer between 0 and 2, and c is an integer between 6 and 10. Furthermore, the following conditions are met. (1) Each of (N)a and (N)b has at least one fluoromodified nucleotide, and (N)c has at least two fluoromodified nucleotides, such that q'+c'+r'+d'=9 and 0≦q'+r'≦4. (2) Of the first four nucleotides of (N)b' (counting from the 5' end), at least two fluoromodified nucleotides are included.

[0113] In some embodiments, in a double-stranded oligonucleotide represented by formula (II) or a double-stranded oligonucleotide comprising a double-stranded region represented by formula (I) above, the sequences of the antisense strand and the sense strand are complementary to each other and form a double-stranded region having 19 to 21 base pairs. N is independently selected from 2′-OMe modified nucleotides, 2′-F modified nucleotides, or 2′-deoxy modified nucleotides. X is independently selected from the group consisting of modified nucleotides modified with 2′-O-MOE, 2′-O-TBDMS, 2′-O-TOM, 2′-O-CH2-O-CH2-CH3, and 2′-O-CH2-O-CH2-CF3, respectively. The number of nucleotides represented by a', p, p', b, b', q, q', c, c', r', and d' are as follows: a' is an integer between 3 and 8, p' is 0 or 1, b' is an integer between 4 and 13, q' is 0 or 1, c' is an integer between 3 and 9, r' is 0 or 1, d' is an integer between 1 and 8, a is an integer between 4 and 7, p is 1, b is an integer between 4 and 8, q is 0 or 1, and c is an integer between 6 and 10. Furthermore, the following conditions are met.

[0114] (1) At least one fluoromodified nucleotide is present in (N)a, and the fluoromodified nucleotide is at position 16 of the antisense chain (counting from the 5' end). (2) At least one fluoromodified nucleotide is present in (N)b, and the fluoromodified nucleotide is at position 14 of the antisense strand (counting from the 5' end). (3) At least two fluoromodified nucleotides are present in (N)c, and both the 2nd and 6th positions (counting from the 5'-end) of the antisense strand are fluoromodified nucleotides. (4) q'+c'+r'+d'=9, and 0≦q'+r'≦2 is satisfied, so q' and r' cannot be 0 at the same time by any choice. (5) In the sense strand, there is at least one X located in a complementary position to the seed region of the antisense strand (positions 1 to 8, counting from the 5'-end). (6) The first four nucleotides of (N)b' (from the 5' to the 3' direction) are located at complementary positions (corresponding sites) to positions 10 to 13 of the antisense strand (counting from the 5' end), and these first four nucleotides include at least two fluoromodified nucleotides, and optionally include one or at least one non-fluoromodified nucleotide. The non-fluoromodified nucleotide is one or more selected from nucleotide X having a 2'-sterically bulky modification, a 2'-deoxy-modified nucleotide, and a 2'-methoxy-modified nucleotide. Selectively, the non-fluoromodified nucleotide is nucleotide X, which has a 2' sterically bulky modification, and X is located in the first three nucleotides of (N)b'. Furthermore, selectively, X is located in the first two nucleotides of (N)b'.

[0115] In one embodiment, the first four nucleotides of (N)b' (counted from the 5' end) include two or three fluoromodified nucleotides and one nucleotide X having a 2' sterically bulky modification or a disubstituted modification as described herein. In another embodiment, the first three nucleotides of (N)b' (counted from the 5' end) include two fluoromodified nucleotides and one nucleotide having a disubstituted modification or a 2'-deoxymodified nucleotide.

[0116] In one embodiment, the double-stranded region of the double-stranded oligonucleotide represented by formula (I) consists of 19 nucleotide pairs, with the first four nucleotides (counting from the 5' end) of the (N)b' region in the sense strand located at positions 7 through 10. In this case, the non-fluoromodified nucleotide is located at any one of positions 7, 8, 9, or 10. Exemplarily, a disubstituted nucleotide X or a 2'-deoxy-modified nucleotide (DNA) is located at any one of positions 7, 8, or 9 in the sense strand. Optionally, a disubstituted nucleotide X is located at position 8 in the sense strand.

[0117] In one embodiment, when q=0, b+c=14. In another embodiment, when q=0, b=8 and c=6. In yet another embodiment, when q=0, b=4 and c=10. In some embodiments, the double-stranded oligonucleotide molecule according to the Disclosure contains one or more X molecules, and X is located only on the antisense strand. In some embodiments, if the antisense strand contains only one X molecule along the direction from the 5' end to the 3' end, X is located at any one of the positions 8-10 or 15 of the antisense strand. Optionally, X is located at position 15 of the antisense strand.

[0118] In some embodiments, the double-stranded oligonucleotide molecule according to this disclosure contains one or more X molecules located only on the sense strand. Optionally, the sense strand may contain one or two X molecules. Exemplarily, in the sense strand, X is located at a complementary position to any of the positions 2, 8, or 15 (counting from the 5' end) of the antisense strand. The complementary position refers to the corresponding position in the double-stranded region between a nucleotide on one strand and its corresponding complementary nucleotide on the other strand. Exemplarily, in a structure consisting of 19 / 21 nucleotide pairs counted along the 5'-3' direction, the SS strand complementary to the nucleotide at position 15 of the AS strand is at position 5, and the SS strand complementary to the nucleotide at position 8 of the AS strand is at position 12. This is well known in the art.

[0119] In some embodiments, the double-stranded oligonucleotide molecule according to the Disclosure contains two or more X molecules, each X located on both the antisense and sense strands. In some embodiments, X is located at any position between positions 8, 10, or 15 (counting from the 5' end) of the antisense strand, and in the sense strand, X is located at a complementary position to any position between positions 2, 8, or 15 (counting from the 5' end) of the antisense strand. In some embodiments, in a double-stranded oligonucleotide represented by formula (II) of the present disclosure or a double-stranded oligonucleotide comprising a double-stranded region represented by formula (I) above, the sense strand has one or more or all of the following features.

[0120] (1) The sense strand has at least one X, and that one X is located in a complementary position to the 15th position (counting from the 5' end) of the antisense strand. (2) The sense strand contains at least two fluoromodified nucleotides among the four nucleotides located in complementary positions to positions 10-13 (counting from the 5' end) of the antisense strand. (3) Optionally, in the sense strand, at least one X is located in a complementary position to the seed region of the antisense strand (positions 1 to 8, counting from the 5' end).

[0121] and / or, the antisense strand of the double-stranded oligonucleotide has one or more or all of the following characteristics: (1) Positions 2, 6, 14, and 16 (counting from the 5' end) of the antisense chain are all fluoromodified nucleotides. (2) All nucleotides at other positions on the antisense strand are unfluoromodified nucleotides. (3) If X is not present in the antisense chain or if at least one X is present in the antisense chain, then X is located at any one of the positions 8, 9, 10, or 15 of the antisense chain.

[0122] The antisense strand optionally contains one X nucleotide, and X is located at position 15. Optionally, the double-stranded region of the double-stranded oligonucleotide contains 19 to 23 base pairs (exemplifiedly 19, 20, or 21 base pairs) in which the sequences of the antisense and sense strands are complementary.

[0123] In some other embodiments, a double-stranded oligonucleotide represented by formula (II) or a double-stranded oligonucleotide comprising a double-stranded region represented by formula (I) above has at least two, three or all of the following features. (1) The sense strand contains at least one X nucleotide, and at least one X is located in a complementary position to one of the following positions on the antisense strand: position 15, position 8, or position 2 (counting from the 5' end). (2) The sense strand contains at least two fluoromodified nucleotides among the four nucleotides located in complementary positions to positions 10 to 13 (counting from the 5' end) of the antisense strand. (3) In the antisense chain, positions 2, 6, 14, and 16 (counting from the 5' end) are all fluoromodified nucleotides, and the nucleotides at the other positions are all unfluoromodified nucleotides. (4) At least one of the 8th, 9th, 10th, and 15th positions of the antisense strand is an X nucleotide. (5) Optionally, in the sense strand, at least one X is located in a complementary position to the seed region of the antisense strand (positions 1 to 8, counting from the 5' end).

[0124] Optionally, the double-stranded region includes 19 to 21 base pairs (exemplifiedly 19, 20, or 21 base pairs) whose sequences complementarily form the antisense and sense strands. Exemplarily, the double-stranded oligonucleotide includes a sense strand having a length of 19 to 21 nucleotides and an antisense strand having a length of 19 to 23 nucleotides.

[0125] In another embodiment, a double-stranded oligonucleotide represented by formula (II) or a double-stranded oligonucleotide comprising a double-stranded region represented by formula (I) above has all of the following characteristics. (1) The sense strand contains one or two X nucleotides, and optionally, at least one X is located in a complementary position to any position on the antisense strand at position 15, position 8, or position 2 (counting from the 5' end). (2) Of the four nucleotides located in the sense strand that are complementary to positions 10 to 13 (counting from the 5' end) of the antisense strand, at least two fluoromodified nucleotides are present. (3) The nucleotides at positions 2, 6, 14, and 16 (counting from the 5' end) of the antisense chain are all fluoromodified nucleotides, and the nucleotides at the other positions are all unfluoromodified nucleotides. (4) At least one of the 8th, 9th, 10th, and 15th positions of the antisense strand is nucleotide X.

[0126] Selectively, the sense strand contains at least one nucleotide X, and at least one nucleotide X is located in a complementary position to one of the antisense strand's positions 15, 8, or 2 (counting from the 5' end). Optionally, the 15th position of the antisense strand is nucleotide X. In another embodiment, a double-stranded oligonucleotide represented by formula (II) according to the present disclosure or

[0127] A double-stranded oligonucleotide containing the double-stranded region shown by formula (I) above has all of the following characteristics. (1) In the sense chain, there is one X, and X is located in a complementary position to one of the 15th, 8th, or 2nd positions (counting from the 5' end) of the antisense chain. (2) Of the four nucleotides located in the sense strand that are complementary to positions 10 to 13 (counting from the 5' end) of the antisense strand, at least two fluoromodified nucleotides are present. (3) The nucleotides at positions 2, 6, 14, and 16 (counting from the 5' end) of the antisense chain are all fluoromodified nucleotides, and the nucleotides at the other positions are all unfluoromodified nucleotides. (4) At least one of the 10th and 15th positions of the antisense strand is an X nucleotide, and / or the antisense strand contains only one X nucleotide, and the X nucleotide is located at the 15th position (counting from the 5' end). (5) The double-stranded region may optionally contain 19 to 23 base pairs.

[0128] In one embodiment, in the double-stranded oligonucleotide molecule according to the present disclosure, the 15th nucleotide of the antisense strand in the double-stranded region is an X nucleotide along the direction from the 5' end to the 3' end, and the nucleotides located in the sense strand at complementary positions to the 2nd and 8th positions (counting from the 5' end) of the antisense strand are also all X nucleotides. In one embodiment, the double-stranded oligonucleotide molecule according to the present disclosure further includes phosphorothioate bond modifications. For example, it may have one or two phosphorothioate bond modifications at nucleotides 1 to 5 (counting from the 5' end) of the sense strand, and / or one or two phosphorothioate bond modifications at positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0129] In some embodiments, the double-stranded oligonucleotide according to the Disclosure is represented by formula (II), and each nucleotide in the double-stranded region is a modified nucleotide.

[0130] In some other embodiments, the double-stranded oligonucleotides of the Disclosure are represented by formula (IIa), where each nucleotide in the double-stranded region is a modified nucleotide, the overhang is located at the 3′ end of the antisense strand, and the length of the nucleotide sequence of the overhang is 1 to 3 nucleotides. Exemplary examples include the sense strand-to-antisense strand length ratios of the double-stranded oligonucleotides of the Disclosure, such as 19:19, 19:21, 19:22, 20:20, 21:21, 21:22, 21:23, 22:22, 22:24, 22:25, and 23:25. In some embodiments, the sense strand and antisense strand of the double-stranded oligonucleotides of the Disclosure have a length of 19 / 21 nucleotides or 21 / 23 nucleotides, respectively.

[0131] In some embodiments, the overhang consists of two nucleotides. Exemplarily, the overhang is dTdT (sequential thymine deoxyribose nucleotides), UU. In some embodiments, the double-stranded oligonucleotide represented by formula (II) above according to the present disclosure comprises a sense strand having a length of 19 to 21 nucleotides and an antisense strand having a length of 19 to 23 nucleotides. The sequences of the antisense strand and the sense strand are complementary to form a double-stranded region having 17 to 21 base pairs, and the double-stranded oligonucleotide has a nucleotide overhang at the 3′ end of the antisense strand. Optionally, the antisense strand or the sense strand each independently contains 1 to 4 phosphorothioate nucleotide interlinks, for example, 1, 2, 3, or 4, and / or the sense strand of the double-stranded oligonucleotide is bound to a ligand.

[0132] Regarding the nucleotide overhangs described above, as an example, the antisense strand has an overhang of 2 to 4 nucleotides at its 3′ end and a blunt end at its 5′ end.

[0133] In some embodiments, the double-stranded oligonucleotide represented by formula (II) above relating to this disclosure has the following characteristics along the direction from the 5' end to the 3' end: (1) At least one of the nucleotides at position 8, 9, 10, or 15 of the antisense strand is an X nucleotide, and positions 2, 6, 14, and 16 (counting from the 5' end) of the antisense strand are all fluoromodified nucleotides. (2) One or two X nucleotides are present in the sense strand, and at least one X nucleotide in the sense strand is located in a complementary position to any one of positions 2, 8, 12, or 15 (counting from the 5' end) of the antisense strand. At least two of the four nucleotides in the sense strand located in complementary positions to positions 10-13 (counting from the 5' end) of the antisense strand are fluoromodified nucleotides.

[0134] In some embodiments, at least one of the phosphate ester groups in the phosphate-sugar backbone of the sense and antisense strands of the double-stranded oligonucleotide according to this disclosure is a sulfur-modified phosphate ester group (i.e., a phosphorothioate group). The phosphorothioate group described herein is a phosphorothioate group formed by substituting at least one oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom. In some embodiments, the phosphate ester group having a modifying group is a phosphorothioate group formed by substituting one oxygen atom in the phosphodiester bond with a sulfur atom.

[0135] In some embodiments, in the double-stranded oligonucleotide according to the Disclosure, the phosphorothioate group is located in the terminal region of the sense strand and / or the terminal region of the antisense strand, the terminal region comprising three, four, or five consecutive nucleotides at the 5′ and 3′ ends. In some other embodiments, with the terminal nucleotide of the single strand being the first nucleotide, the phosphorothioate group is located between any two consecutive nucleotides in the first to third nucleotide sequences.

[0136] For example, if the terminal nucleotide of a single chain is the first nucleotide, the phosphorothioate group is located at one or more positions in the terminal region of the sense chain and / or antisense chain, between the first and second nucleotides of the 5′ end, between the second and third nucleotides of the 5′ end, between the first and second nucleotides of the 3′ end, and between the second and third nucleotides of the 3′ end.

[0137] In some embodiments, the 5′-terminal nucleotide of the antisense chain is a nucleotide modified with a 5′-phosphate nucleotide or a nucleotide modified with a 5′-phosphate analog. In one embodiment, the nucleotide modified with a 5′-phosphate analog is a nucleotide having vinyl phosphate modification (e.g., 5′-VP) or a nucleotide having phosphorothioate modification. Optionally, the 5′-VP is 5′-E-VP, 5′-Z-VP, or a composition thereof.

[0138] The double-stranded oligonucleotides relating to this disclosure may be a variety of double-stranded oligonucleotides that regulate gene expression. In some embodiments, they may be double-stranded oligonucleotides that repress or downregulate gene expression, such as siRNA. In some embodiments, they may be double-stranded oligonucleotides that activate or upregulate gene expression, such as saRNA.

[0139] In one embodiment, in the double-stranded oligonucleotide according to the present disclosure, (1) The antisense chain is such that, along the 5'-3' direction, at least one nucleotide at the 10th or 15th position of the antisense chain is selected from the group of modified nucleotides, each modified with 2'-O-MOE, 2'-O-TBDMS, 2'-O-TOM, 2'-O-CH2-O-CH2-CH3, and 2'-O-CH2-O-CH2-CF3, and the 2nd, 6th, 14th, and 16th positions of the antisense chain are all fluoromodified nucleotides, and the other positions of the antisense chain are methoxy-modified nucleotides. (2) In the sense strand, the complementary position to any one of the 2nd, 8th, or 15th positions (counting from the 5' end) of the antisense strand is selected from the group consisting of modified nucleotides modified with 2′-O-MOE, 2′-O-TBDMS, 2′-O-TOM, 2′-O-CH2-O-CH2-CH3, and 2′-O-CH2-O-CH2-CF3, and in the sense strand, at least two fluoromodified nucleotides are present among the four nucleotides located at the complementary positions to the 10th to 13th positions (counting from the 5' end) of the antisense strand. The other positions on the sense strand are methoxy-modified nucleotides. (3) Each sense strand and antisense strand contains at least one phosphorothioate group, and the phosphorothioate group is located in the terminal region of each single strand. The terminal region is in the range of 3 to 5 nucleotides in length.

[0140] In one embodiment, the double-stranded oligonucleotide further comprises one 5′-VP modified nucleotide. In one embodiment, each sense strand and antisense strand of the double-stranded oligonucleotide according to the Disclosure has 17 to 30 nucleotides. In one example, the sense strand has 19 to 21 nucleotides and the antisense strand has 19 to 25 nucleotides. In another example, the sense strand has 19 or 21 nucleotides and the antisense strand has 21 or 23 nucleotides.

[0141] In one embodiment, the nucleotide at the 5'-end position of the antisense strand in the double-stranded oligonucleotide is selected from A, dA, dU, U, or dT. In one embodiment, the antisense strand of the double-stranded oligonucleotide (dsRNA) according to the disclosure is 100% complementary to the target RNA, and its expression can be suppressed by RNA interference. In another embodiment, the antisense strand of the double-stranded oligonucleotide according to the disclosure and the target RNA are complementary by at least 95%, at least 90%, at least 85%, and at least 80% of base pairs. The double-stranded oligonucleotide dsRNA relating to this disclosure comprises a double-stranded region represented by formula (II) or formula (I).

[0142] In one embodiment, the double-stranded oligonucleotide according to the Disclosure comprises a sense strand and an antisense strand. (a) The sense chain has the following characteristics: (i) The sense strand has a length of 19 to 21 nucleotides. (ii) Having an ASGPR ligand bound to the 3′ end, wherein the ASGPR ligand is GalNAc or a derivative thereof. (iii) At least one complementary position to the 2nd, 8th, and 15th positions (counting from the 5' end) of the antisense chain is a nucleotide modified with a 2'-stereotactically bulky group. (iv) Of the four nucleotides located at complementary positions to positions 10 to 13 (counting from the 5′ end) of the antisense chain, at least two are 2′-fluoromodified nucleotides, and the other positions are independently selected from one or at least one nucleotide modified with a 2′-OMe modified nucleotide, a 2′-deoxy modified nucleotide, or a nucleotide modified with a 2′-stereotactically bulky group, wherein the nucleotide modified with a 2′-stereotactically bulky group is selected from the group consisting of nucleotides modified with 2′-O-MOE, 2′-O-TBDMS, 2′-O-TOM, 2′-O-CH2-O-CH2-CH3, and 2′-O-CH2-O-CH2-CF3, respectively. (b) The antisense chain has the following characteristics: (i) The antisense strand has a length of 21 to 23 nucleotides. (ii) At least one of the nucleotides at position 10 or 15 of the antisense chain is a nucleotide modified with a 2'-stereotactically bulky group. (iii) The 2, 6, 14 and 16 positions of the antisense chain are all 2′-fluoromodified nucleotides, and optionally, the other positions of the antisense chain are one or at least one selected from 2′-OMe modified nucleotides, 2′-deoxy modified nucleotides, or nucleotides modified with a 2′-stereotactically bulky group, and the nucleotides modified with a 2′-stereotactically bulky group are one or more selected from the group consisting of nucleotides modified with 2′-O-MOE, 2′-O-TBDMS, 2′-O-TOM, 2′-O-CH2-O-CH2-CH3, and 2′-O-CH2-O-CH2-CF3, respectively. (iv) The terminal region has at least one phosphorothioate group bond between nucleotides. (v) Optionally, the antisense strand has an overhang of 2 to 3 nucleotides located at the 3' end and a blunt end located at the 5' end. In one embodiment, the double-stranded oligonucleotide according to the present disclosure has phosphorothioate group bonds (these positions are counted from the 5′ end) between positions 1 and 2 of the nucleotide of the antisense strand, between positions 2 and 3 of the nucleotide, between positions 19 and 20 of the nucleotide, and between positions 20 and 21 of the nucleotide. In one embodiment, the antisense strand has an overhang of two nucleotides at the 3′ end, and the 5′ end of the antisense strand is a blunt end.

[0143] In one embodiment, the double-stranded oligonucleotide according to the present disclosure has a nucleotide modified with a 2'-sterically bulky group at position 15 of the antisense strand, counting from the 5'-end, and one of the complementary positions of position 2, 8, or 15 of the antisense strand in the sense strand is a nucleotide modified with a 2'-sterically bulky group, and the 2'-sterically bulky group provides the nucleotide in which it is located with a greater steric bulk than that of a 2'-OMe modification. In one embodiment, the antisense chain has a nucleotide modified with a 2'-stereotically bulky group at position 15, and in the sense chain, the position complementary to position 15 of the antisense chain is a nucleotide modified with a 2'-stereotically bulky group.

[0144] In one embodiment, the antisense chain has a nucleotide modified with a 2'-stereotically bulky group at position 15, and in the sense chain, the position complementary to position 8 of the antisense chain is a nucleotide modified with a 2'-stereotically bulky group. In one embodiment, the antisense chain has a nucleotide modified with a 2'-stereotically bulky group at position 15, and in the sense chain, the position complementary to position 2 of the antisense chain is a nucleotide modified with a 2'-stereotically bulky group.

[0145] In one embodiment, the antisense chain has a nucleotide modified with a 2'-stereotically bulky group at position 15, and in the sense chain, the positions complementary to positions 2 and 8 of the antisense chain are nucleotides modified with a 2'-stereotically bulky group. In an optional embodiment, the sense strand contains one nucleotide modified with a 2'-sterically bulky group located in a complementary position to the seed region of the antisense strand (counting from the 5' end, the seed region of the antisense strand is at positions 1 to 8).

[0146] The above 2′-sterically bulky group is one or more selected from 2′-O-MOE, 2′-O-TBDMS, 2′-O-TOM, 2′-O-CH2-O-CH2-CH3, and 2′-O-CH2-O-CH2-CF3.

[0147] For example, if the sense strand has 21 nucleotides, the nucleotides modified with the 2'-sterically bulky group are located at positions 7, 14, and 20 from the 5' end (corresponding to the complementary positions of positions 15, 8, and 2 of the antisense strand, respectively). For example, if the sense strand has 19 nucleotides, the nucleotides modified with the 2′-sterically bulky group are located at positions 5, 12, and 18 from the 5′ end (corresponding to the complementary positions of positions 15, 8, and 2 of the antisense strand, respectively).

[0148] The double-stranded oligonucleotides relating to this disclosure may be used alone, or may form pharmaceutical compositions with pharmacoagulably acceptable carriers, or may be bound to binding molecules to form oligonucleotide conjugates, or may be used in other forms. When used, the expression of a target gene can be regulated by contacting an effective amount of the double-stranded oligonucleotide, the pharmaceutical composition, or the oligonucleotide conjugate with cells, or the expression of a target gene can be regulated by administering the double-stranded oligonucleotide, the pharmaceutical composition, or the conjugate to a subject, thereby achieving the objective of treating a pathological condition or disease related to the expression level of the target gene.

[0149] In another embodiment, the disclosure provides a double-stranded oligonucleotide comprising a sense strand and an antisense strand, each strand having 17 to 35 nucleotides, each nucleotide being either modified or unmodified, and the sense strand and antisense strand being at least partially inversely complementary to form a double-stranded region. The double-stranded oligonucleotide comprises at least one nucleotide having a 2'-disubstituted modification, each substituent in the 2'-disubstituted being independently selected from a methyl group or fluorine.

[0150] If the double-stranded region of the double-stranded oligonucleotide contains a nucleotide having a 2'-disubstituted modification, the nucleotide having the 2'-disubstituted modification is selected from nucleotides that are 2'-difluorosubstituted. Alternatively, if the double-stranded oligonucleotide contains a nucleotide having a 2'-disubstituted modification at a position other than the double-stranded region, the nucleotide having the 2'-disubstituted modification is selected from nucleotides that are substituted with 2'-F-2'-Me.

[0151] In some embodiments, the double-stranded oligonucleotide has one or more of the following characteristics. (1) If the double-stranded oligonucleotide contains a 2'-difluorosubstituted nucleotide, the 2'-difluorosubstituted nucleotide is located at least one of the positions 7, 8, 9, or 10, counting from the 5' end of the sense strand. (2) If the double-stranded oligonucleotide includes a 2'-difluorosubstituted nucleotide, the 2'-difluorosubstituted nucleotide is located at least one of the positions 2, 4, 6, 8, 9, 10, 12, 14, or 16, counting from the 5' end of the antisense strand. (3) If the double-stranded oligonucleotide contains a nucleotide substituted with 2′-F-2′-Me, the nucleotide substituted with 2′-F-2′-Me is located outside the double-stranded region of the oligonucleotide. (4) If the double-stranded oligonucleotide contains a nucleotide substituted with 2′-F-2′-Me, the nucleotide substituted with 2′-F-2′-Me is located in the 3′ overhang of the antisense strand.

[0152] In some embodiments, in feature (1), if the double-stranded oligonucleotide includes a 2'-difluorosubstituted nucleotide, the 2'-difluorosubstituted nucleotide is located at at least two or at least three of the positions 7, 8, 9, or 10, counting from the 5' end of the sense strand.

[0153] In some embodiments, in feature (2), if the double-stranded oligonucleotide includes a 2'-difluorosubstituted nucleotide, the 2'-difluorosubstituted nucleotide is located at at least two of the following positions, from the 5' end of the antisense strand: 2, 4, 6, 8, 9, 10, 12, 14, or 16. In other embodiments, the disclosure provides a double-stranded oligonucleotide conjugate comprising a double-stranded oligonucleotide according to the disclosure or obtained by the method according to the disclosure, and a binding group attached to the double-stranded oligonucleotide. The disclosure can further improve the stability of the double-stranded oligonucleotide according to the disclosure in the blood and enhance its target-directivity by forming a pharmaceutical composition with a suitable carrier or by forming an oligonucleotide conjugate with a suitable binding molecule. The binding molecule is a binding molecule that targets tissues such as the liver, lungs, and kidneys.

[0154] In some embodiments, the binding group comprises a linker and a pharmacoagulably target-directing group, and the double-stranded oligonucleotide, the linker, and the target-directing group or the delivery auxiliary group are sequentially linked by covalent or non-covalent bonds, with each target-directing group selected from ligands capable of binding to cell surface receptors. Generally, there are 1 to 6 target-directing groups. In one embodiment, there are 2 to 4 target-directing groups. The binding site between the double-stranded oligonucleotide and the binding group may be located at the 3′ or 5′ end of the sense strand of the double-stranded oligonucleotide, at the 5′ end of the antisense strand, or in the internal sequence of the double-stranded oligonucleotide. In some specific embodiments, the binding site between the double-stranded oligonucleotide and the binding group is located at the 3′ end of the sense strand of the double-stranded oligonucleotide.

[0155] The above oligonucleotide conjugates can be synthesized by methods described in detail in the prior art. For example, WO2015006740A2 describes in detail methods for preparing several types of siRNA conjugates.

[0156] In other embodiments, the Disclosure provides conjugates or compounded double-stranded oligonucleotides comprising a double-stranded oligonucleotide as described herein and a target-directing group, providing tissue-specific targeting. In some embodiments, the conjugates according to the Disclosure are specifically targeted to receptors on the surface of hepatocytes and specifically targeted to liver tissue. In some embodiments, the target-directing group according to the Disclosure is a ligand that has affinity for asialoglycoprotein receptors, thereby specifically targeting asialoglycoprotein receptors (ASGPRs) on the surface of hepatocytes. Exemplarily, the target-directing group includes a ligand group derived from N-acetylgalactosamine (GalNAc) or a derivative thereof.

[0157] In one embodiment, the double-stranded oligonucleotide according to the Disclosure is linked to at least one ASGPR ligand. Exemplarily, the ASGPR ligand is GalNAc or a derivative thereof linked via a divalent or trivalent branched linker.

[0158] In some embodiments, the ASGPR ligand is bound to the 5'-end or 3'-end of the double-stranded oligonucleotide sense strand or antisense strand via a linker to form a conjugate. Exemplarily, the conjugate has the following structural formula (B). [ka] Here, Nu represents a double-stranded oligonucleotide or other nucleic acid-based pharmacoactive molecule (e.g., a single-stranded oligonucleotide) related to this disclosure. In one embodiment, the ligand is bound to the 3'-end or 5'-end of the sense strand of a double-stranded oligonucleotide.

[0159] This disclosure further provides the use of the double-stranded oligonucleotide and its reagents. In one embodiment, the use is used to suppress the expression of a target gene in a subject. The subject may be any animal, preferably a mammal, such as a mouse, rat, sheep, cattle, dog, cat, or human. In some embodiments, the present disclosure further provides pharmaceutical compositions comprising a double-stranded oligonucleotide according to the present disclosure and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any available carrier.

[0160] In some embodiments, the content of double-stranded oligonucleotides and pharmacoagulably acceptable carriers in the pharmaceutical composition is not particularly limited. The pharmaceutical composition may further contain other pharmacoagulably acceptable additives, which may be one or more of the common formulations or compounds of the art. For example, the other pharmacoagulably acceptable additives may include at least one of pH buffers, protective agents, and osmotic regulators.

[0161] In some embodiments, the present disclosure provides methods for the treatment and / or prevention of diseases or conditions relating to mRNA levels of target gene expression. The methods include administering to a subject an effective amount of a double-stranded oligonucleotide, pharmaceutical composition, and / or oligonucleotide conjugate relating to the present disclosure. In some embodiments, the diseases or conditions relating to mRNA levels of target gene expression are hepatitis, hepatic fibrosis, hepatoproliferative disorders, and / or dyslipidemia. In some embodiments, the dyslipidemia is hypercholesterolemia, hypertriglyceridemia, or atherosclerosis.

[0162] In some embodiments, the present disclosure provides the use of a double-stranded oligonucleotide, a pharmaceutical composition comprising a double-stranded oligonucleotide, or an oligonucleotide conjugate in the preparation of a pharmaceutically acceptable drug for the treatment and / or prevention of a pathological condition or disease (e.g., gene overexpression) relating to mRNA levels of target gene expression. In some embodiments, the target gene is a gene that is abnormally expressed in hepatocytes. In some embodiments, the target gene is an endogenous gene expressed in the liver. Endogenous genes expressed in the liver include, but are not limited to, genes such as ApoB, Ap℃, ANGPTL3, PCSK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HBV, and HCV.

[0163] In some embodiments, the disease is selected from chronic liver disease, hepatitis, hepatic fibrosis, hepatic proliferative disorders, and dyslipidemia. In some embodiments, the dyslipidemia in the blood is hypercholesterolemia, hypertriglyceridemia, or atherosclerosis. In some embodiments, the present disclosure provides methods for regulating gene expression. The methods include contacting a cell expressing the gene with an effective amount of the above-mentioned double-stranded oligonucleotide, the above-mentioned pharmaceutical composition, or the above-mentioned oligonucleotide conjugate.

[0164] In some embodiments, the present disclosure provides a method for treating a pathological condition or disease caused by abnormal gene expression. The method comprises administering to a subject an effective amount of the above-mentioned double-stranded oligonucleotide, the above-mentioned pharmaceutical composition, or the above-mentioned oligonucleotide conjugate. In some embodiments, the abnormal expression is overexpression, and accordingly, the regulation is suppression of the overexpression. In some embodiments, the regulation is suppression of the expression of a target gene in hepatocytes. The mRNA expressed by the target gene is selected from mRNA expressed by the hepatitis B virus gene (HBV), mRNA expressed by the angiopoietin-like protein 3 (ANGPTL3) gene, or mRNA expressed by the apolipoprotein C3 (Apoc3) gene.

[0165] In some embodiments, the double-stranded oligonucleotides, pharmaceutical compositions, or oligonucleotide conjugates described herein may also be used to treat cancer, hepatocellular carcinoma (HCC), liver metastases, or other liver diseases, including hepatoblastoma. In some embodiments, the drug is administered to the subject by any suitable route known in the art. The route includes, but is not limited to, oral or parenteral routes, such as intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, and topical administration (including oral buccal and sublingual administration). The frequency of administration may be once weekly, once monthly, once a year, or multiple times.

[0166] In some embodiments, the disclosure further provides a method for delivering the above-mentioned double-stranded oligonucleotides, the above-mentioned pharmaceutical compositions, or the above-mentioned oligonucleotide conjugates to specific targets of a subject by subcutaneous or intravenous administration.

[0167] This disclosure provides a kit comprising the above-mentioned double-stranded oligonucleotide, the above-mentioned pharmaceutical composition, and / or the above-mentioned oligonucleotide conjugate. In some embodiments, the kit described herein provides a double-stranded oligonucleotide in one container. In some embodiments, the kit described herein includes one container that provides a pharmaceutically acceptable excipient. In some embodiments, the kit further includes other components, such as stabilizers or preservatives. In some embodiments, the kit described herein includes at least one other therapeutic agent in a container different from the container that provides the double-stranded oligonucleotide described herein.

[0168] In the kits relating to this disclosure, the double-stranded oligonucleotide and a pharmaceutically acceptable carrier and / or additive, the double-stranded oligonucleotide composition and / or conjugate, and / or pharmaceutically acceptable additive can be provided in any form, such as liquid, dry, or lyophilized form. In some embodiments, the double-stranded oligonucleotide and a pharmaceutically acceptable carrier and / or additive, the double-stranded oligonucleotide composition and / or conjugate, and the optionally pharmaceutically acceptable additive are essentially pure and / or sterile. In some embodiments, sterile water is provided in the kits relating to this disclosure. Unless otherwise specified, the meanings of the base composition and modifications in each example of this disclosure are as follows: Uppercase A, U, G, C, and T represent the base composition of a nucleotide; lowercase m represents a nucleotide modified with a 2'-methoxy group; lowercase f represents a nucleotide modified with a 2'-fluoro group; lowercase d represents a nucleotide modified with a 2'-deoxyribonucleotide; uppercase TBDMS in parentheses, i.e., (TBDMS), represents a nucleotide modified with a 2'-O-tert-butyldimethyl group. The uppercase letter TOM in parentheses (TOM) indicates that the nucleotide represented by the preceding letter is modified with 2'-O-triisopropylmethoxysilane, the uppercase letter MOE in parentheses (MOE) indicates that the nucleotide represented by the preceding letter is modified with 2'-O-methoxyethyl, and the lowercase letter s indicates that the two nucleotides represented by the preceding and following letters are linked by a phosphorothioate bond.

[0169] In this disclosure, the structural formula of compound L96-PS is shown in formula (C). [ka] Here, PS represents a polystyrene resin solid support, and compound L96-PS was obtained from Asymchem Laboratories (Tianjin), with a supported weight of 120 ± 12 μmol / g (measurement method: UV / HPLC).

[0170] Unless otherwise specified, the reagents and consumables (Table 1) and equipment (Table 2) used in this disclosure are all commercially available products from the manufacturers listed below. [Table 1] [Table 2]

[0171] Unless otherwise specified, all dsRNA (indicated as siRNA in the examples) sequences used in this disclosure were synthesized by Suzhou Beixin Biotechnology Co., Ltd. All PCR primers were synthesized by Beijing Tsingke Biotech Co., Ltd. The experimental animals, C57BL / 6J mice and ICR mice, were obtained from SPF (Beijing) Biotechnology Co., Ltd. Blood biochemical indicators were measured by Beijing Xinuoyin Biotechnology Co., Ltd.

[0172] Unless otherwise specified, the Real-time PCR measurement data for in vivo / in vitro siRNA activity experiments in each example of this disclosure are all calculated relative to the target gene mRNA in each experimental group using the ΔΔCt method. The calculation method is as follows. ΔCt(experimental group) = Ct(experimental group target gene) - Ct(experimental group internal reference gene) ΔCt(control group) = Ct(control group target gene) - Ct(control group internal reference gene) ΔΔCt(experimental group) = ΔCt(experimental group) - ΔCt(control group mean) ΔΔCt(control group) = ΔCt(control group) - ΔCt(control group mean)

[0173] The mRNA expression level of the target gene in the experimental group is normalized using the control group as a baseline, and the residual mRNA expression level of the target gene in the control group is set to 100%. Relative residual mRNA expression level of the target gene in the experimental group = 2 -ΔΔCt (Experimental group) × 100% mRNA suppression rate of the target gene in the experimental group = 100% - relative mRNA expression level of the target gene in the experimental group The in vivo / in vitro activity experimental data are all The experimental data is represented as TIFF2026517427000007.tif23166 (or X±STDEV), and graphs are created and analyzed using the GraphPad prism 8.0 software. Here, both ±SD and ±STDEV values ​​represent the standard deviation. Unless otherwise specified, the reagent ratios in each example of this disclosure are calculated based on volume ratios (v / v).

[0174] The following examples will be used to interpret the proposed disclosure. As those skilled in the art will see, the following examples are for illustrative purposes only and do not limit the scope of the disclosure. Where specific techniques or conditions are not described in the examples, the techniques or conditions described in the literature in the art or the product specifications should be followed. Where the manufacturers of the reagents or equipment used are not specified, they are all commercially available general-purpose products. Preparation of nucleoside monomer analogs with sterically bulky modifications.

[0175] The reagents used in the preparation examples of this disclosure and their origins are shown in Table 3. [Table 3]

[0176] Preparation Example 1: Synthesis of Compound NM062 The synthesis route for compound NM062 is as follows: [ka] Here, "Exact Mass" refers to the calculated precise mass (the same applies below). (1-1) Synthesis of Compound 2

[0177] Compound 1 (10 g, 40.97 mmol, uridine) was dissolved in 100 ml of pyridine, and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (14.22 g, 45.07 mmol) was added. The mixture was stirred at 25°C for 16 hours. After the reaction was complete, the reaction mixture was concentrated to remove the solvent, diluted with 300 ml of dichloromethane, washed twice with 100 ml of saturated ammonium chloride aqueous solution (2 × 100 ml), and then washed twice with 100 ml of saturated sodium chloride aqueous solution (2 × 100 ml). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal-phase column chromatography (eluent gradient: petroleum ether / ethyl acetate = 3 / 1, volume ratio v / v) to obtain Compound 2 (11.5 g, yield: 57.7%). MS ESI (m / z) = 487.27 [M+H] + .

[0178] (1-2) Synthesis of Compound 3 Compound 2 (11 g, 22.62 mmol) was dissolved in 77 ml of dimethyl sulfoxide at 25°C, and 77 ml of acetic acid and 38 ml of acetic anhydride were added. The mixture was stirred at 25°C for 16 hours. After the reaction was complete, the mixture was diluted with 500 ml of ethyl acetate, washed twice with 100 ml of saturated sodium bicarbonate aqueous solution (2 × 100 ml), and washed twice again with 100 ml of saturated sodium chloride aqueous solution (2 × 100 ml). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal-phase column chromatography (eluent gradient: petroleum ether / ethyl acetate = 3 / 1, v / v) to obtain compound 3 (11.22 g, yield: 90.81%). MS ESI (m / z) = 546.23 [M+H] + .

[0179] (1-3) Synthesis of Compound 4 At 25 °C, compound 3 (5 g, 9.15 mmol) was dissolved in 50 ml of tetrahydrofuran, ethanol (4.21 g, 91.5 mmol) was added, the reaction system was purged with nitrogen gas three times, and the reaction system was cooled to -40 °C in a dry ice - acetonitrile bath. At -40 °C, a solution of N-iodosuccinimide (2.47 g, 10.98 mmol) in tetrahydrofuran (5 ml) and a solution of trifluoromethanesulfonic acid (2.75 g, 18.3 mmol) in tetrahydrofuran (5 ml) were added dropwise. Under a nitrogen gas atmosphere, the reaction solution was stirred at -40 °C for 1 hour. After the reaction was completed, 8 ml of triethylamine was added for quenching, an aqueous solution of 10% by mass sodium thiosulfate was added, and extraction was performed 4 times with 150 ml of dichloromethane (4 × 150 ml). The organic phases were combined, the organic phase was washed twice with 80 ml of saturated aqueous sodium hydrogen carbonate solution (2 × 80 ml), washed once with 100 ml of saturated aqueous sodium chloride solution (1 × 100 ml), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal phase column chromatography (elution gradient: petroleum ether / ethyl acetate = 4 / 1, v / v) to obtain compound 4 (4.78 g, yield: 95.96%). MS ESI (m / z) = 544.26 [M+H] + .

[0180] (1 - 4) Synthesis of compound 5 At 25 °C, compound 4 (4 g, 7.35 mmol) was dissolved in 32 ml of methanol, ammonium fluoride (1.36 g, 36.75 mmol) was added, and the mixture was stirred at 60 °C for 16 hours. After the reaction was completed, the solvent was removed by rotary evaporation and purified by normal phase column chromatography (elution gradient: dichloromethane / methanol = 20 / 1, v / v) to obtain compound 5 (2.05 g, yield: 92.34%). MS ESI (m / z) = 303.2 [M+H] + .

[0181] (1 - 5) Synthesis of compound 6 Compound 5 (2 g, 6.62 mmol) was dissolved in 20 ml of pyridine under an ice bath, and the ice bath was cooled to 0 °C. 4,4′-Dimethoxytrityl chloride (2.77 g, 7.94 mmol) was added in several portions, and the reaction mixture was stirred at 0 °C for 1 hour. After the reaction was completed, 2 ml of methanol was added to quench the reaction, and the solvent was removed by rotary evaporation. 50 ml of saturated ammonium chloride aqueous solution and 50 ml of ethyl acetate were added, and extraction was performed three times (3 × 50 ml). The combined organic phases were washed once with 100 ml of saturated sodium chloride aqueous solution (1 × 100 ml), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal-phase column chromatography (elution gradient: petroleum ether / ethyl acetate = 1 / 1, v / v) to obtain compound 6 (2.28 g, yield: 55.67%). MS ESI (m / z) = 604.3 [M+H] + .

[0182] (1 - 6) Synthesis of compound NM062 Compound 6 (1.5 g, 2.48 mmol) was azeotropically dehydrated three times (3 × 10 ml) with 10 ml of acetonitrile, then dissolved in 15 ml of dichloromethane. First, a dichloromethane (15 ml) solution of 3-{[bis(diisopropylamino)phosphoryl]oxy}propionitrile (899.6 mg, 2.98 mmol, azeotropically dehydrated three times (3 × 10 ml) with 10 ml of acetonitrile) was added, and then 1H-imidazole-4,5-dicarbonitrile (374.8 mg, 1.984 mmol) was added. Nitrogen gas substitution was performed three times, and the mixture was stirred at 25 °C for 1 hour under a nitrogen gas atmosphere. After the reaction was completed, the reaction mixture was diluted by adding 30 ml of saturated sodium bicarbonate aqueous solution, and extraction was performed three times with 20 ml of dichloromethane (3 × 20 ml). The combined organic phases were washed twice with 30 ml of saturated sodium chloride aqueous solution (2 × 30 ml), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse-phase column chromatography (elution gradient: water / acetonitrile = 1 / 9, v / v) to obtain compound NM062 (1.7 g, yield: 85.14%). MS ESI (m / z) = 604.3 [M+H] + .

[0183] 1 H NMR (400 MHz, DMSO-d6) δ = 11.41 (s, 1H), 7.75 (dd, J = 8.2, 10.6 Hz, 1H), 7.43 - 7.35 (m, 2H), 7.35 - 7.29 (m, 2H), 7.29 - 7.21 (m, 5H), 6.90 (dd, J = 6.1, 7.8 Hz, 4H), 5.88 (dd, J = 2.3, 4.2 Hz, 1H), 5.35 (dd, J = 8.5, 9.6 Hz, 1H), 4.81 - 4.68 (m, 2H), 4.48 - 4.33 (m, 2H), 4.19 - 4.04 (m, 1H), 3.74 (d, J = 2.0 Hz, 7H), 3.58 - 3.44 (m, 4H), 3.39 - 3.31 (m, 2H), 3.30 (br s, 1H), 2.78 (t, J = 5.9 Hz, 1H), 2.63 (t, J = 5.9 Hz, 1H), 1.16 - 1.03 (m, 12H), 0.98 (d, J = 6.7 Hz, 3H).

[0184] Preparation Example 2: Synthesis of Compound NM063 The synthesis route for compound NM063 is as follows: [ka]

[0185] (2-1) Synthesis of Compound 7 At 25°C, compound 3 (5g, 9.15 mmol) was dissolved in 50 ml of tetrahydrofuran, trifluoroethanol (9.15 g, 91.5 mmol) was added, nitrogen gas was purged three times, and the mixture was cooled to -40°C in a dry ice-acetonitrile bath. At -40°C, a solution of N-iodosuccinimide (2.47 g, 10.98 mmol) in tetrahydrofuran (5 ml) and a solution of trifluoromethanesulfonic acid (2.75 g, 18.3 mmol) in tetrahydrofuran (5 ml) were added dropwise, and the mixture was stirred at -40°C for 1 hour under a nitrogen atmosphere. After the reaction was complete, 80 ml of triethylamine was added to the reaction mixture to quench it, and a 10% by mass aqueous solution of sodium thiosulfate was added. Extraction was performed four times with 150 ml of dichloromethane (4 × 150 ml), the organic phase was combined, the organic phase was washed twice with 80 ml of saturated sodium bicarbonate aqueous solution (2 × 80 ml), and once with 100 ml of saturated sodium chloride aqueous solution (1 × 100 ml), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal-phase column chromatography (eluent gradient: petroleum ether / ethyl acetate = 4 / 1, v / v) to obtain compound 7 (3.04 g, yield: 55.51%). MS ESI (m / z) = 599.23 [M+H] + .

[0186] (2-2) Synthesis of Compound 8 At 25°C, compound 7 (3 g, 5.01 mmol) was dissolved in methanol (32 mL), ammonium fluoride (926.85 mg, 25.05 mmol) was added, and the reaction mixture was stirred at 60°C for 16 hours. After the reaction was complete, the solvent was removed by rotary evaporation, and the mixture was purified by normal-phase column chromatography (elution gradient: dichloromethane / methanol = 20 / 1, v / v) to obtain compound 8 (1.51 g, yield: 83.43%). MS ESI (m / z) = 356.26 [M+H] + .

[0187] (2-3) Synthesis of Compound 9 Compound 8 (1.51 g, 4.24 mmol) was dissolved in pyridine (15 mL) under ice bath conditions, cooled to 0°C in an ice bath, and 4,4′-dimethoxytrityl chloride (1.73 g, 5.09 mmol) was added in several portions. The reaction mixture was stirred at 0°C for 1 hour. After the reaction was complete, methanol (2 mL) was added to quench the mixture, the solvent was removed by rotary evaporation, saturated ammonium chloride aqueous solution (50 mL) was added, and extraction was performed with ethyl acetate (3 × 50 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution (100 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal-phase column chromatography (eluent gradient: petroleum ether / ethyl acetate = 1 / 1, v / v) to obtain compound 9 (2.08 g, yield: 74.53%). MS ESI (m / z) = 681.32 [M+Na] + .

[0188] (2-4) Synthesis of compound NM063 Compound 9 (1.9 g, 2.89 mmol) was repeatedly azeotropically dehydrated with acetonitrile (3 × 10 mL), dissolved in dichloromethane (15 mL), and azeotropically dehydrated with acetonitrile (3 × 10 mL) 3-{[bis(diisopropylamino)phosphoryl]oxy}propionitrile (1.307 g, 3.46 mmol) was added to a solution of dichloromethane (15 mL), 1H-imidazole-4,5-dicarbonitride (272.5 mg, 2.312 mmol) was added, nitrogen gas purging was performed three times, and the reaction mixture was stirred at 25°C for 1 hour under a nitrogen gas atmosphere. After the reaction was complete, the solution was diluted with saturated sodium bicarbonate aqueous solution (30 mL), extracted with dichloromethane (3 × 20 mL), combined with the organic phase, washed with saturated sodium chloride aqueous solution (2 × 30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse-phase column chromatography (C18 chromatography column, eluent gradient: water / acetonitrile = 1 / 9, v / v) to obtain compound NM063 (2.07 g, yield: 83.55%). MS ESI (m / z) = 859.43 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ = 11.41 (s, 1H), 7.77 (dd, J = 6.0, 7.9 Hz, 1H), 7.43 - 7.35 (m, 2H), 7.35 - 7.18 (m, 7H), 6.95 - 6.85 (m, 4H), 5.90 - 5.83 (m, 1H), 5.28 (d, J = 8.1 Hz, 1H), 4.99 - 4.86 (m, 2H), 4.53 - 4.40 (m, 2H), 4.26 - 4.02 (m, 3H), 3.85 - 3.59 (m, 8H), 3.52 (qd, J = 6.7, 10.4 Hz, 2H), 3.43 - 3.32 (m, 2H), 2.81 - 2.75 (m, 1H), 2.63 (t, J = 5.9 Hz, 1H), 1.16 - 1.04 (m, 9H), 0.97 (d, J = 6.7 Hz, 3H).

[0189] Preparation Example 3: Synthesis of Compound NM096 In this preparation example, the synthesis route for compound NM096 is as follows. [ka]

[0190] (3-1) Synthesis of Compound 10 At 25°C, compound 3 (3g, 5.49 mmol, 1 eq) was dissolved in tetrahydrofuran (30mL), n-butanol (4.06g, 54.92 mmol, 10 eq, CAS number: 71-36-3) was added, nitrogen gas was purged three times, and the reaction system was cooled to -40°C using dry ice. A solution of N-iodosuccinimide (1.48g, 6.59 mmol, 1.2 eq, abbreviation: NIS, CAS number: 516-12-1) in tetrahydrofuran (5mL) and a solution of trifluoromethanesulfonic acid (1.64g, 10.98 mmol, 2 eq, abbreviation: TfOH, CAS number: 1493-13-6) in tetrahydrofuran (5mL) were added dropwise, the reaction system was stirred at -40°C for 1 hour, and triethylamine (6mL) was added to quench the reaction. After the reaction was complete, the reaction mixture was concentrated, extracted with dichloromethane (2 × 50 mL), washed with saturated sodium bicarbonate aqueous solution (2 × 50 mL) and saturated sodium chloride aqueous solution (30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1, volume ratio v / v) to obtain compound 10 (3.05 g, yield: 96.95%) as a white solid. MS ESI (m / z) = 573.3 [M + H] + .

[0191] (3-2) Synthesis of Compound 11 At 25°C, compound 10 (3.05 g, 5.33 mmol, 1 eq) was dissolved in tetrahydrofuran (30 mL), and a solution of tetrabutylammonium fluoride (CAS number: 429-41-4) in tetrahydrofuran (10.66 mL, 1 M, 10.66 mmol, 2 eq) was added. The reaction mixture was stirred at 25°C for 3 hours. After the reaction was complete, the reaction mixture was concentrated and purified by column chromatography (eluent: dichloromethane / methanol = 10 / 1, v / v) to obtain compound 11 (1.7 g, yield: 98.27%) as a white solid. MS ESI (m / z) = 353.4 [M + Na] + .

[0192] (3-3) Synthesis of Compound 12 At 25°C, compound 11 (1.7g, 5.15 mmol, 1 eq) was dissolved in pyridine (17 mL), cooled to 0°C in an ice bath, and 4,4′-dimethoxytrityl chloride (2.27g, 6.69 mmol, 1.3 eq, abbreviation: DMTrCl, CAS number: 40615-36-9) was added in several portions. The reaction mixture was stirred at 25°C for 1 hour, and methanol was added to quench the mixture. After the reaction was complete, the reaction mixture was concentrated, diluted with ethyl acetate (50 mL), washed with saturated ammonium chloride aqueous solution (2 × 30 mL) and saturated sodium chloride aqueous solution (30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1, v / v) to obtain compound 12 (2.44 g, yield: 74.89%) as a yellow solid. MS ESI (m / z) = 633.3 [M + H] + .

[0193] (3-3) Synthesis of compound NM096 Compound 12 (2.44 g, 3.85 mmol, 1 eq) was repeatedly azeotropically dehydrated with acetonitrile (3 × 30 mL), then dissolved in dichloromethane (25 mL). A solution of bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.63 g, 5.78 mmol, 1.5 eq, CAS number: 102691-36-1), which had been azeotropically dehydrated with acetonitrile (3 × 10 mL), was added in dichloromethane (25 mL). 1H-imidazole-4,5-dicarbonitride (364.2 mg, 3.08 mmol, 0.8 eq, CAS number: 1122-28-7) was added, and the reaction system was stirred under a nitrogen atmosphere at 25°C for 1 hour after three nitrogen gas purgings. After the reaction was complete, the reaction mixture was diluted with saturated sodium bicarbonate aqueous solution (50 mL), extracted with dichloromethane (3 × 30 mL), combined with the organic phase, washed with saturated sodium chloride aqueous solution (2 × 30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 1, v / v) to obtain the white solid compound NM096 (2.7 g, yield: 84.11%). MS ESI (m / z) = 833.4 [M + H] + . 1 H NMR (400 MHz, DMSO-d6) δ 11.36 (s, 1H), 7.72 (dd, J = 11.8, 8.1 Hz, 1H), 7.42 - 7.29 (m, 4H), 7.29 - 7.20 (m, 5H), 6.89 (dd, J = 8.6, 5.8 Hz, 4H), 5.89 (dd, J = 5.3, 3.1 Hz, 1H), 5.37 (t, J = 7.7 Hz, 1H), 4.79 - 4.66 (m, 2H), 4.40 (dp, J = 19.0, 4.8 Hz, 2H), 4.12 (dq, J = 20.3, 3.8 Hz, 1H), 3.74 (d, J = 2.1 Hz, 7H), 3.71 - 3.58 (m, 1H), 3.53 (dq, J = 9.8, 6.9 Hz, 2H), 3.49 - 3.40 (m, 2H), 3.39 - 3.32 (m, 1H), 2.78 (t, J = 6.1 Hz, 1H), 2.63 (t, J = 5.9 Hz, 1H), 1.42 (dq, J = 10.1, 6.8 Hz, 2H), 1.26 (dq, J = 14.3, 7.3 Hz, 2H), 1.20-1.06 (m, 10H), 0.99 (d, J = 6.7 Hz, 3H), 0.83 (td, J = 7.4, 3.5 Hz, 3H).

[0194] Preparation Example 4: Synthesis of Compound NM097 This preparation example is a synthetic compound of compound NM097, and is described below.

change

[0195] (4--1) Synthesis of compound 13 At 25°C, compound 3 (3g, 5.49 mmol, 1 eq) was dissolved in tetrahydrofuran (30 mL), cyclopropanol (3.18 g, 54.92 mmol, 10 eq) was added, nitrogen gas was purged three times, and the reaction system was cooled to -40°C with dry ice. A solution of NIS (1.48 g, 6.59 mmol, 1.2 eq) in tetrahydrofuran (5 mL) and a solution of TfOH (1.64 g, 10.98 mmol, 2 eq) in tetrahydrofuran (5 mL) were added dropwise, the reaction system was stirred at -40°C for 1 hour, and triethylamine (6 mL) was added to quench the reaction. After the reaction was complete, the reaction mixture was concentrated, and dichloromethane (2 × 50 mL) was added for extraction. The organic phase was then combined with saturated sodium bicarbonate aqueous solution (2 × 50 mL) and saturated sodium chloride aqueous solution (30 mL). The mixture was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1, v / v) to obtain compound 13 (2.59 g, yield: 84.78%) as a white solid. MS ESI (m / z) = 557.3 [M + H] + .

[0196] (4-2) Synthesis of Compound 14 At 25°C, compound 13 (2.59 g, 4.66 mmol, 1 eq) was dissolved in tetrahydrofuran (30 mL), and a solution of tetrabutylammonium fluoride in tetrahydrofuran (9.32 mL, 1 M, 9.32 mmol, 2 eq) was added. The reaction mixture was stirred at 25°C for 3 hours. After the reaction was complete, the reaction mixture was concentrated and purified by column chromatography (eluent: dichloromethane / methyl--l = 20 / 1, v / v) to obtain compound 14 (1.45 g, yield: 99.3%) as a white solid. MS ESI (m / z) = 337.1 [M + Na] + .

[0197] (4-3) Synthesis of Compound 15 At 25°C, compound 14 (1.45 g, 4.62 mmol, 1 eq) was dissolved in pyridine (15 mL), cooled to 0°C in an ice bath, and DMTrCl (2.05 g, 6.00 mmol, 1.3 eq) was added in several portions. The reaction mixture was stirred at 25°C for 1 hour, and methanol was added to quench the reaction. After the reaction was complete, the reaction mixture was concentrated, diluted with ethyl acetate (50 mL), washed with saturated ammonium chloride aqueous solution (2 × 30 mL) and saturated sodium chloride aqueous solution (30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1, v / v) to obtain compound 15 (1.69 g, yield: 59.5%) as a yellow solid. MS ESI (m / z) = 617.2 [M + H] + .

[0198] (4-4) Synthesis of compound NM097 Compound 15 (1.69 g, 2.74 mmol, 1 eq) was repeatedly azeotropically dehydrated with acetonitrile (3 × 10 mL), then dissolved in dichloromethane (20 mL). A solution of bis(diisopropylamino)(2--cyanoethoxy)phosphine (1.24 g, 4.11 mmol, 1.5 eq), which had been azeotropically dehydrated with acetonitrile (3 × 10 mL), was added in dichloromethane (25 mL). Then, 1H--imidazo--l--4,5--dicarbonitride (258.9 mg, 2.19 mmol, 0.8 eq) was added, and the reaction system was stirred under a nitrogen atmosphere at 25°C for 1 hour after three nitrogen gas purgings. After the reaction was complete, the reaction mixture was diluted with saturated sodium bicarbonate aqueous solution (50 mL), extracted with dichloromethane (3 × 30 mL), combined with the organic phase, washed with saturated sodium chloride aqueous solution (2 × 30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1, v / v) to obtain the white solid compound NM097 (1.7 g, yield: 75.96%). MS ESI (m / z) = 817.2 [M + H] + .

[0199] 1H NMR (400 MHz, DMSO--d6) δ 11.40 (s, 1H), 7.76 (dd, J = 10.9, 8.1 Hz, 1H), 7.42 - 7.29 (m, 4H), 7.29 - 7.21 (m, 5H), 6.89 (td, J = 6.6, 3.2 Hz, 4H), 5.90 (dt, J = 7.1, 3.6 Hz, 1H), 5.40 - 5.24 (m, 1H), 4.94 - 4.68 (m, 2H), 4.56 - 4.37 (m, 2H), 4.13 (dt, J = 22.0, 4.1 Hz, 1H), 3.74 (d, J = 2.1 Hz, 7H), 3.71 - 3.44 (m, 4H), 3.35 (t, J = 3.6 Hz, 1H), 3.30 (d, J = 4.8 Hz, 1H), 2.78 (t, J = 5.9 Hz, 1H), 2.63 (t, J = 5.9 Hz, 1H), 1.15 - 1.08 (m, 9H), 0.98 (d, J = 6.8 Hz, 3H), 0.56 - 0.36 (m, 4H).

[0200] Preparation Example 5: Preparation of Compound NM098 In this preparation example, the synthesis route for compound NM098 is as follows. [ka]

[0201] (5-1) Synthesis of Compound 16 At 25°C, compound 3 (3g, 5.49 mmol, 1 eq) was dissolved in tetrahydrofuran (30mL), neopentyl alcohol (4.83g, 54.92 mmol, 10 eq, CAS number: 75-84-3) was added, nitrogen gas was purged three times, and the reaction system was cooled to -40°C using dry ice. A solution of NIS (1.48g, 6.59 mmol, 1.2 eq) in tetrahydrofuran (5mL) and a solution of TfOH (1.64g, 10.98 mmol, 2 eq) in tetrahydrofuran (5mL) were added dropwise, the reaction mixture was stirred at -40°C for 1 hour, and triethylamine (6mL) was added to quench the reaction. After the reaction was complete, the reaction mixture was concentrated, and dichloromethane (2 × 50 mL) was added for extraction. The organic phases were combined, washed with saturated sodium bicarbonate aqueous solution (2 × 50 mL) and saturated sodium chloride aqueous solution (30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1, v / v) to obtain compound 16 (3.06 g, yield: 95.03%) as a white solid. MS ESI (m / z) = 587.3 [M + H] + .

[0202] (5-2) Synthesis of Compound 17 At 25°C, compound 16 (3.06 g, 5.22 mmol, 1 eq) was dissolved in tetrahydrofuran (30 mL), and a solution of tetrabutylammonium fluoride in tetrahydrofuran (10.44 mL, 1 M, 10.44 mmol, 2 eq) was added. The reaction mixture was stirred at 25°C for 3 hours. After the reaction was complete, the reaction mixture was concentrated and purified by column chromatography (eluent: dichloromethane / methyl--l = 10 / 1, v / v) to obtain compound 17 (1.78 g, yield: 99.1%) as a white solid. MS ESI (m / z) = 367.1 [M + Na] + .

[0203] (5-3) Synthesis of Compound 18 At 25°C, compound 17 (1.78 g, 5.17 mmol, 1 eq) was dissolved in pyridine (15 mL), cooled to 0°C in an ice bath, and DMTrCl (2.28 g, 6.72 mmol, 1.3 eq) was added in several portions. The reaction mixture was stirred at 25°C for 1 hour, and quenched with methanol. After the reaction was complete, the reaction mixture was concentrated, diluted with ethyl acetate (50 mL), washed with saturated ammonium chloride aqueous solution (2 × 30 mL) and saturated sodium chloride aqueous solution (30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1, v / v) to obtain compound 18 (2.50 g, yield: 74.85%) as a yellow solid. MS ESI (m / z) = 647.3 [M + H] + .

[0204] (5-4) Synthesis of compound NM098 Compound 18 (2.50 g, 3.87 mmol, 1 eq) was repeatedly azeotropically dehydrated with acetonitrile (3 × 10 mL), then dissolved in dichloromethane (30 mL). A solution of bis(diisopropylamino)(2--cyanoethoxy)phosphine (1.75 g, 5.80 mmol, 1.5 eq) azeotropically dehydrated with acetonitrile (3 × 10 mL) was added in dichloromethane (25 mL), and 1H--imidazo--l--4,5--dicarbonitride (364.9 mg, 3.10 mmol, 0.8 eq) was added. The reaction system was purged with nitrogen gas three times and stirred at 25°C for 1 hour under a nitrogen atmosphere. After the reaction was complete, the reaction mixture was diluted with saturated sodium bicarbonate aqueous solution (50 mL), extracted with dichloromethane (3 × 30 mL), combined with the organic phase, washed with saturated sodium chloride aqueous solution (2 × 30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1, v / v) to obtain the white solid compound NM098 (2.84 g, yield: 86.87%). MS ESI (m / z) = 847.4 [M + H] + .

[0205] 1H NMR (400 MHz, DMSO--d6) δ 11.41 (s, 1H), 7.72 (dd, J = 9.6, 8.1 Hz, 1H), 7.43 - 7.30 (m, 4H), 7.30 - 7.20 (m, 5H), 6.89 (td, J = 6.8, 3.3 Hz, 4H), 5.89 (dd, J = 4.6, 2.4 Hz, 1H), 5.38 (d, J = 8.0 Hz, 1H), 4.85 - 4.66 (m, 2H), 4.38 (dp, J = 15.0, 4.9, 4.4 Hz, 2H), 4.12 (dd, J = 20.1, 3.7 Hz, 1H), 3.74 (d, J = 2.2 Hz, 7H), 3.70 - 3.46 (m, 3H), 3.42 - 3.32 (m, 1H), 3.28 (t, J = 5.4 Hz, 1H), 3.19 - 3.08 (m, 2H), 2.78 (td, J = 5.9, 2.1 Hz, 1H), 2.62 (t, J = 5.9 Hz, 1H), 1.15 - 1.08 (m, 9H), 0.98 (d, J = 6.7 Hz, 3H), 0.82 (d, J = 5.9 Hz, 9H).

[0206] Preparation Example 6: Preparation of Compound NM099 In this preparation example, the synthesis route for compound NM099 is as follows. [ka]

[0207] (6-1) Synthesis of Compound 19 At 25°C, compound 3 (3g, 5.49 mmol, 1 eq) was dissolved in tetrahydrofuran (30 mL), cyclopentanol (4.74 g, 54.92 mmol, 10 eq) was added, nitrogen gas was purged three times, and the reaction system was cooled to -40°C using dry ice. A solution of NIS (1.48 g, 6.59 mmol, 1.2 eq) in tetrahydrofuran (5 mL) and a solution of TfOH (1.64 g, 10.98 mmol, 2 eq) in tetrahydrofuran (5 mL) were added dropwise, the reaction system was stirred at -40°C for 1 hour, and triethylamine (6 mL) was added to quench the reaction. After the reaction was complete, the reaction mixture was concentrated, extracted with dichloromethane (2 × 50 mL), washed with saturated sodium bicarbonate aqueous solution (2 × 50 mL) and saturated sodium chloride aqueous solution (30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 4 / 1, v / v) to obtain compound 19 (3.20 g, yield: 99.6%) as a white solid. MS ESI (m / z) = 585.3 [M + H] + .

[0208] (6-2) Synthesis of Compound 20 At 25°C, compound 19 (3.20 g, 5.48 mmol, 1 eq) was dissolved in tetrahydrofuran (30 mL), and a tetrahydrofuran solution of tetrabutylammonium fluoride (10.95 mL, 1 M, 10.95 mmol, 2 eq) was added. The reaction mixture was stirred at 25°C for 3 hours. After the reaction was complete, the reaction mixture was concentrated and purified by column chromatography (eluent: dichloromethane / methyl = 20 / 1, v / v) to obtain compound 20 (1.87 g, yield: 99.3%) as a white solid. MS ESI (m / z) = 365.1 [M + Na] + .

[0209] (6-3) Synthesis of Compound 21 At 25°C, compound 20 (1.87 g, 5.47 mmol, 1 eq) was dissolved in pyridine (20 mL), cooled to 0°C in an ice bath, and DMTrCl (2.41 g, 7.11 mmol, 1.3 eq) was added in several portions. The reaction mixture was stirred at 25°C for 1 hour, and then quenched with methanol. After the reaction was complete, the reaction mixture was concentrated, diluted with ethyl acetate (50 mL), washed with saturated ammonium chloride aqueous solution (2 × 30 mL) and saturated sodium chloride aqueous solution (30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1) to obtain compound 21 (2.18 g, yield: 61.93%) as a yellow solid. MS ESI (m / z) = 645.3 [M + H] + .

[0210] (6-4) Synthesis of compound NM099 Compound 21 (2.18 g, 3.38 mmol, 1 eq) was azeotropically dehydrated three times with acetonitrile (10 ml each time), then dissolved in dichloromethane (20 mL). A solution of bis(diisopropylamino)(2--cyanoethoxy)phosphine (1.53 g, 5.08 mmol, 1.5 eq) azeotropically dehydrated with acetonitrile (3 × 10 mL) was added to dichloromethane (25 mL), then 1H--imidazo--l--4,5--dicarbonitride (319.7 mg, 2.71 mmol, 0.8 eq) was added, and the reaction system was purged with nitrogen gas three times and stirred at 25°C for 1 hour under a nitrogen atmosphere. After the reaction was complete, the reaction mixture was diluted with saturated sodium bicarbonate aqueous solution (50 mL), extracted with dichloromethane (3 × 30 mL), combined with the organic phase, washed with saturated sodium chloride aqueous solution (2 × 30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 1) to obtain the white solid compound NM099 (2.28 g, yield: 79.80%). MS ESI (m / z) = 845.3 [M + H] + .

[0211] 1H NMR (400 MHz, DMSO--d6) δ 11.40 (s, 1H), 7.72 (dd, J = 12.3, 8.1 Hz, 1H), 7.35 (dt, J = 21.9, 6.3 Hz, 4H), 7.26 (t, J = 8.9 Hz, 5H), 6.89 (dd, J = 8.6, 5.7 Hz, 4H), 5.90 (d, J = 5.6 Hz, 1H), 5.47 - 5.34 (m, 1H), 4.72 (dq, J = 14.7, 7.1 Hz, 2H), 4.49 - 4.31 (m, 2H), 4.18 - 4.07 (m, 2H), 3.74 (d, J = 2.0 Hz, 7H), 3.70 - 3.46 (m, 3H), 3.35 (q, J = 7.5, 4.9 Hz, 1H), 3.29 (d, J = 3.2 Hz, 1H), 2.78 (t, J = 6.0 Hz, 1H), 2.63 (t, J = 5.9 Hz, 1H), 1.72 - 1.35 (m, 8H), 1.12 (td, J = 6.7, 4.4 Hz, 9H), 0.99 (d, J = 6.7 Hz, 3H).

[0212] Preparation Example 7: Preparation of Compound NM054 In this preparation example, the synthesis route for compound NM054 is as follows. [ka]

[0213] (7-1) Synthesis of compound NM054-2 Compound NM054--1 (3g, 11.54 mmol, 1.0 eq, (2′R)--2′--deoxy--2′--fluoro--2′--methyluridine, CAS number: 863329--66--2) and pyridine (30 ml) were added to a 500 ml reaction vessel, the temperature was reduced to 0°C, and 4,4′--dimethoxytrityl chloride (4.29 g, 12.7 mmol, 1.1 eq) was added in several portions. Nitrogen gas purging was performed three times, and the reaction system was stirred at 25°C for 3 hours under a nitrogen atmosphere. HPLC showed that no starting materials were present. After the reaction was complete, the reaction mixture was concentrated, and extraction was performed by adding pure water (50 ml) and ethyl acetate (50 ml) to separate the organic phase. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM054-2 (2.7 g, yield: 41.7%). MS ESI (m / z) = 563.0 [M + H] + .

[0214] (7-2) Synthesis of compound NM054 Compound NM054--2 (2.7g, 4.8 mmol, 1.0 eq) was added to a 100 ml reaction vessel, bis(diisopropylamino)(2--cyanoethoxy)phosphine (1.74g, 5.76 mmol, 1.2 eq) was added in several batches, and then 4,5-dicyanoimidazole (0.45g, 3.8 mmol, 0.8 eq, CAS number: 1122--28--7) and dichloromethane (27 ml) were added. The reaction system was purged with nitrogen gas three times and stirred at 25°C for 3 hours under a nitrogen atmosphere. After the reaction was complete, 20 ml of aqueous sodium bicarbonate solution was added to the reaction mixture to separate the organic phase. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse-phase column chromatography (eluent: acetonitrile / water = 90 / 10, v / v) to obtain compound NM054 (3.0 g). MS ESI (m / z) = 763 [M + H] + . Preparation Example 8: Preparation of Compound CR01008 and Compound CR01008Z

[0215] (8-1) Synthesis of compound CR01008 In this preparation example, the synthesis route for compound CR01008 is as follows. [ka] Here, Molecular Weight refers to the molecular weight (the same applies below).

[0216] (8--1--1) Synthesis of compound CR01008--2 Compound CR01008--1 (10.0 g, 1.0 eq, trans-(4--formylcyclohexyl)carbamate tert-butyl ester, CAS number: 181308--57--6) and aqueous formaldehyde solution (8.9 g, 37% by mass, 2.4 eq) were dissolved in 33 ml of methanol. 13 ml of aqueous KOH solution with a concentration of 45.3% by mass was added dropwise. After the addition was complete, the mixture was stirred at 25°C and reacted for 30 minutes. The temperature was then raised to 60°C and the reaction was carried out under reflux at 60°C for 2 hours. After the reaction was complete, the reaction mixture was cooled to room temperature, and then dried under reduced pressure to obtain a white solid crude product. A small amount of water was added to the crude product to pulp it, and the mixture was filtered to obtain a white solid compound CR01008--2 (9 g, yield: 78.9%). MS-ESI (m / z) = 260 [M + H] + .

[0217] (8--1--2) Synthesis of compound CR01008--3 Compound CR01008-2 (9 g, 1 eq) was dissolved in 70 ml of 1,4-dioxane, and a 1,4-dioxane solution of hydrogen chloride (45 ml, 4 M) was added. The mixture was stirred at 25°C and reacted for 1 hour. After the reaction was complete, the reaction mixture was dried under reduced pressure to obtain the white solid compound CR01008-3 (6.8 g, yield: 100%).

[0218] (8--1--3) Synthesis of compound CR01008--5 Compound CR01008--3 (1.8g, 2.0eq), Compound CR01008--4 (2.1g, 1.0eq, 5--{[(2R,3R,4R,5R,6R)--3--acetamide--4,5--diacetoxy--6--(acetoxymethyl)--2--tetrahydropyran]oxy}pentanoic acid and N,N--diisopropylethylamine (3.5g, 6.0eq, abbreviation: DIEA) were dissolved in 15 ml of DMF, and O--(benzotriazol--) -1--yl)--N,N,N′,N′--tetramethyluronium hexafluorophosphate (1.9 g, 1.1 eq, abbreviation: HBTU, CAS number: 94790--37--1) was added and the mixture was stirred at 25°C under a nitrogen atmosphere for 3 hours. After the reaction was complete, the reaction mixture was dried under reduced pressure and reversed-phase purification (eluent: 22 vol% acetonitrile aqueous solution) was performed to obtain the white solid compound CR01008--5 (1.78 g, yield: 64.4%). MS--ESI (m / z) = 589[M + H] + .

[0219] (8--1--4) Synthesis of compound CR01008--6 Compound CR01008-5 (1.54 g, 1.0 eq) was dissolved in 15 ml of pyridine. The reaction system was cooled to 0°C using an ice bath. At 0°C, 4,4′-dimethoxytriphenylmethyl chloride (1.32 g, 1.5 eq, abbreviation: DMTrCl, CAS number: 40615-36-9) was added, and the reaction was carried out at 25°C for 3 hours. 15 ml of methanol was added to the reaction mixture to quench the reaction. After the reaction was complete, the reaction mixture was dried under reduced pressure and reversed-phase purification (eluent: 60 vol% aqueous solution of acetonitrile) yielded the yellow solid compound CR01008-6 (1 g, yield: 42.7%). MS-ESI (m / z) = 891 [M + H] + .

[0220] (8--1--5) Synthesis of compound CR01008 Compound CR01008--6 (1.08 g, 1.0 eq) was dissolved in 20 ml of anhydrous dichloromethane, and 4,5-dicyanoimidazole (115 mg, 0.8 eq, abbreviation: DCI, CAS number 1122--28--7) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (732 mg, 2.1 eq, CAS number: 102691--36--1) were added, respectively. The mixture was then purged with nitrogen gas three times and reacted with stirring at 25°C for 2 hours. After the reaction was complete, 20 ml of saturated sodium bicarbonate aqueous solution was added to the reaction mixture, and extraction was performed three times (3 × 20 ml) with 20 ml of dichloromethane. The organic phases were combined, the organic phase was dried under reduced pressure, and reverse-phase purification (eluent: 72 vol% acetonitrile aqueous solution) was performed. After vacuum drying for 12 hours, a white powdery compound CR01008 (1 g, yield: 76.0%) was obtained. MS-ESI (m / z) = 1091 [M + Na] + .

[0221] 1H NMR (400 MHz, DMSO--d6) δ 1.05 (d, J = 6.7 Hz, 6H).1.14 (d, J = 6.7 Hz, 6H), 1.37 - 1.17 (m, 5H), 1.60 - 1.40 (m, 6H), 1.68 - 1.62 (m, 1H), 1.80 (s, 3H), 1.80 (s, 3H), 1.92 (s, 3H), 2.02 (s, 5H), 2.13 (s, 3H), 2.71 (t, J = 5.9 Hz, 2H), 2.79 (d, J = 8.4 Hz, 1H), 2.87 (d, J = 8.4 Hz, 1H), 3.36 (s, 1H), 3.58 - 3.39 (m, 3H), 3.69 - 3.60 (m, 2H), 3.75 (s, 7H), 3.90 (dt, J = 11.2, 8.8 Hz, 1H), 4.05 (s, 3H), 4.51 (d, J = 8.4 Hz, 1H), 4.99 (dd, J = 11.3, 3.4 Hz, 1H), 5.24 (d, J = 3.4 Hz, 1H), 5.78 (s, 1H), 6.93 - 6.87 (m, 4H), 7.35 - 7.21 (m, 7H), 7.44 - 7.37 (m, 2H), 7.66 (d, J = 7.8 Hz, 1H), 7.84 (d, J = 9.2 Hz, 1H).

[0222] (8-2) Synthesis of compound CR01008Z In this preparation example, the synthesis route of compound CR01008Z is as follows: [ka]

[0223] (8--2--1) Synthesis of compound CR01008--7 Compound CR01008-6 (500 mg) was dissolved in 10 ml of dichloromethane, and compound 8 (succinic anhydride, 112 mg), 4-dimethylaminopyridine (6.8 mg, abbreviation: DMAP, CAS number: 1122-58-3) and triethylamine (226.2 mg) were added. The mixture was purged with nitrogen gas three times, stirred at 25°C for 16 hours, and then flash-purified to obtain compound CR01008-7 (300 mg, yield: 53.6%). MS-ESI (m / z) = 10¹³ [M + Na] + .

[0224] (1.2.2) Synthesis of compound CR01008Z Compound CR01008--7 (50 mg), amino-modified CPG (1.25 g, 80 μmol / g, 0.1 mmol), HBTU (27 mg), and DIEA (12 mg) were added to a 20 ml sample flask and reacted with a shaker for 16 hours. After the reaction was complete, the reaction mixture was filtered to obtain a filter cake. The filter cake was first washed once with 10 ml of acetonitrile (1 × 10 ml) and then vacuum dried. 20 ml The dried filtration cake, DMAP (3 mg), Cap 1 (10 ml), and Cap 2 (1 ml) were added to a 1 ml sample flask and reacted with a shaker for 6 hours. After the reaction was complete, the reaction mixture was filtered to obtain the filtration cake. The filtration cake was first washed once with 10 ml of acetonitrile (1 × 10 ml), and then vacuum dried to obtain compound CR01008Z (1.03 g, loading: 20-30 μmol / g). Here, Cap1 and Cap2 are capping reagents. Cap1 is a 20 vol% N-methylimidazole pyridine / acetonitrile mixture, with a volume ratio of pyridine to acetonitrile of 3:5. Cap2 is a 20 vol% anhydride acetonitrile solution.

[0225] Example 1 Synthesis of siRNA siRNA synthesis is similar to general phosphoramidite solid-phase synthesis methods. When synthesizing sense and antisense strands with sterically bulky groups such as TBDMS, TOM, and MOE, if it is necessary to introduce nucleotides modified with sterically bulky groups, the above-mentioned phosphoramidite monomers are used to replace the corresponding nucleotide monomers in the original sequence. The target siRNA sequence (siRNA single chain) was introduced into the ABI394 synthesizer, and synthesis was carried out using Universal CPG / PS or compound CR01008Z as a support, according to the configured synthesis process. During the synthesis process, compound CR01008 was treated as a single nucleoside monomer.

[0226] The introduction of each base involved a four-step reaction: de-DMTr, coupling, oxidation, and capping. The nucleoside phosphoramido monomers modified with 2'--OMe, 2'--F, 2'--TOM, 2'--TBDMS, and 2'--MOE were all obtained from Shanghai Zhaowei Technology Development Co., Ltd. Compound NM062 was synthesized by the method of Preparation Example 1, and compound NM063 was synthesized by the method of Preparation Example 2. As an activator, 5-ethylthio-1H-tetrazole (ETT) (0.6 M acetonitrile solution) was used; as a thiolation agent, a solution of 0.2 M xanthan hydride dissolved in acetonitrile and pyridine (Suzhou Kerama) in a 1:1 volume ratio was used; and as an oxidizing agent, a solution of 0.05 M iodine dissolved in pyridine and water (Suzhou Kerama) in a 9:1 volume ratio was used.

[0227] After the synthesis was complete, the support was removed and blow-dried, and deprotection was performed using concentrated ammonia water (50°C, 16 hours). After the reaction was complete, the mixture was cooled in a refrigerator at -20°C for 10 minutes. After centrifugation, the supernatant was transferred to another centrifuge tube, concentrated to dryness, and then purified by RP-HPLC, with liquid phase A being 0.1 M TEAA and liquid phase B being acetonitrile. The purified and recovered fractions were subjected to HPLC, MS, and UV measurements, and the fractions that passed the measurements were collected and freeze-dried to obtain siRNA single strands. The obtained siRNA single strands were annealed to obtain siRNA double strands.

[0228] The structural formula of the siRNA conjugate using (CR01008)(CR01008)(CR01008Z) (also written as (CR01008×3)) as a delivery carrier is as follows. [ka]

[0229] The structural formula of the siRNA conjugate using L96 as a delivery carrier is as follows. [ka]

[0230] The sequence and modification information of the siRNA compounds in each example of this disclosure are shown in Table 4. [Table 4] TIFF2026517427000021.tif235166TIFF2026517427000022.tif235166TIFF2026517427000023.tif235166TIFF20265174270000 24.tif235166TIFF2026517427000025.tif235166TIFF2026517427000026.tif235166TIFF2026517427000027.tif235166TIFF202 6517427000028.tif235166TIFF2026517427000029.tif235166TIFF2026517427000030.tif235166TIFF2026517427000031.tif2 35166TIFF2026517427000032.tif235166TIFF2026517427000033.tif235166TIFF2026517427000034.tif235166TIFF2026517427 000035.tif235166TIFF2026517427000036.tif235166TIFF2026517427000037.tif235166TIFF2026517427000038.tif235166TI FF2026517427000039.tif235166TIFF2026517427000040.tif235166TIFF2026517427000041.tif235166TIFF2026517427000042. tif235166TIFF2026517427000043.tif235166TIFF2026517427000044.tif235166TIFF2026517427000045.tif235166TIFF202651 7427000046.tif235166TIFF2026517427000047.tif235166TIFF2026517427000048.tif235166TIFF2026517427000049.tif89166 Here, Gemcitabine refers to gemcitabine.

[0231] (Gemcitabine) represents a single nucleotide in siRNA, and its structural formula is as follows. [ka]

[0232] (NM054) represents a single nucleotide in siRNA, and its structural formula is as follows. [ka]

[0233] (NM096) represents a single nucleotide in siRNA, and its structural formula is as follows. [ka]

[0234] (NM097) represents a single nucleotide in siRNA, and its structural formula is as follows. [ka]

[0235] (NM098) represents a single nucleotide in siRNA, and its structural formula is as follows. [ka]

[0236] (NM099) represents a single nucleotide, and its structural formula is as follows. [ka]

[0237] Example 2: In vitro inhibitory activity of complement component 3 (CC3) mRNA by siRNA modified at different sites using TBDMS. This example evaluated the inhibitory activity against the target gene CC3 in HepG2 cells using compounds RX502002-RX502041, which had different sites of the sense and antisense strands modified with TBDMS, as well as the control compound RX502001, which was not modified with TBDMS, using an in vitro cell screening method.

[0238] 1. Preparation of the test sample After centrifuging each of the above siRNA samples, an appropriate amount of 1×PBS was added according to the specifications of each tube to dissolve them, and a 20 μM mother liquor was prepared. This was then further diluted with 1×PBS to a 5 μM working solution. 2. Cell culture and seeding HepG2 cells were cultured and grown in DMEM medium containing 10% FBS in a 37°C, 5% CO2 incubator. Before seeding, the medium was removed, washed with 0.25% trypsin, and the cells were digested with trypsin. After digestion was stopped by adding the medium, the cells were suspended, centrifuged at 800 rpm for 5 minutes, resuspended in fresh DMEM medium containing 10% FBS, counted, and the cell density was set to 1 × 10⁶. 5 After diluting to cells / mL and thoroughly mixing to ensure uniformity, the cells were seeded onto a 24-well plate (500 μL / well), incubated for 24 hours, and then transfection was performed. 3. Cell transfection (1) Preparation of Solution 1 -- siRNA mixture: In each cell well, 49.4 μL of Opti-MEM medium and 0.6 μL of siRNA test working solution (5 μM) were uniformly mixed. Three cell replica wells were prepared for each siRNA test. (2) Preparation of Solution 2 - Lipofectamine 3000 mixture: The transfection reagent was gently mixed by inversion, and 2 μL of Lipo3000 reagent was diluted in 48 μL of Opti-MEM medium in each cell well. The mixture was then homogeneously mixed by gently pipetting 3 to 5 times and allowed to stand for 5 minutes. Three cell replica wells were prepared for each siRNA test. (3) Solution 3 -- Preparation of a mixture of Solution 1 and Solution 2: One part (50 μL) of solution 1 and one part (50 μL) of solution 2 were gently mixed to prepare solution 3 (100 μL) of the transfection complex, which was incubated at room temperature for 20 minutes. The cell culture plates awaiting transfection were removed, the old medium was removed, and 500 μL of Opti-MEM medium was added. Solution 3 (100 μL) of the prepared incubator was added dropwise to the cell culture wells to bring the concentration of the siRNA test in each cell transfection well to approximately 5 nM. The cell culture plates were incubated for a further 4 hours at 37°C in a 5% CO2 incubator, and 1 mL of DMEM medium containing 20% ​​FBS was added to each well. The cells were incubated for a further 24 hours at 37°C in a 5% CO2 incubator. The blank control group consisted of normally cultured HepG2 cells that had not undergone transfection. The mock control group consisted of cells to which only the Lipofectamine 3000 transfection reagent was added, without the addition of siRNA to the transfection complex. 4. Measurement RNA extraction: Total RNA was extracted from cells in each well using a fully automated nucleic acid extraction device and nucleic acid extraction kit manufactured by Zhejiang Hanwei Technology Co., Ltd., following the method described in the instructions. Reverse transcription reaction: 1 μg of total RNA extracted from cells in each well was taken and transcribed using a Promega reverse transcription kit (Reverse Transcription System, A3500) with Oligo (dT). 15 Using a reverse transcription primer, a 20 μL reverse transcription system was prepared according to the instructions in the kit, and the reverse transcription reaction was carried out. After the reaction was complete, 80 μL of RNase-free water was added to the reverse transcription system to obtain a cDNA solution for real-time PCR measurement.

[0239] Real-time PCR measurement: ABI SYBR TMUsing the Select Master Mix (Catalog number: 4472908) reagent, prepare a 20 μL Real-time PCR reaction system in each PCR measurement well according to the method described in the kit instructions. Add 5 μL of the cDNA template obtained from the reverse transcription reaction described above to each measurement system. TM The reaction system contains 10 μL of Select Master Mix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer (primer information is shown in Table 5), and 4 μL of RNase-free H2O. The prepared reaction system was placed in an ABI StepOnePlus PCR instrument, and real-time PCR amplification was performed using a three-step method. The amplification process involved pre-denaturation at 95°C for 10 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. This cycle of denaturation, annealing, and extension was repeated 40 times. After the process was completed, the difference in gene expression was calculated using the ΔΔCt method described above.

[0240] [Table 5]

[0241] The relative expression level of the target gene CC3 mRNA (Figure 1) and the residual activity of the target gene CC3 mRNA (Table 6) were statistically analyzed after transfection of HepG2 with siRNA modified at different sites on the sense strand using TBDMS. The results showed that the in vitro activity of siRNA sequences modified at positions 5 (RX502027), 6 (RX502028), 12 (RX502034), 13 (RX502035), and 18 (RX502040) of the sense strand was improved compared to the control sequence without TBDMS modification (RX502001). The attenuating effect of TBDMS modification at positions 7 (RX502029), 8 (RX502030), 9 (RX502031), and 10 (RX502032) of the sense strand on the in vitro activity of the siRNA sequence was relatively significant.

[0242] [Table 6]

[0243] After transfection of HepG2 with siRNA modified at different sites on the antisense strand using TBDMS, the relative expression level of the target gene CC3 mRNA (Figure 2) and the residual activity of the target gene CC3 zmRNA (Table 7) were statistically analyzed. Compared to the control sequence without TBDMS modification (RX502001), TBDMS modification at position 8 (RX502009) and position 15 (RX502016) of the antisense strand further enhanced the in vitro activity of the siRNA sequence.

[0244] [Table 7]

[0245] As can be seen from the results above, the effect of TBDMS modification on siRNA sequence activity is related to the modification site, and modification with large TBDMS groups at specific sites does not impair siRNA activity, but rather can even further enhance it.

[0246] Example 3. Inhibitory activity of siRNA conjugates modified at different sites using TBDMS against CC3 protein in mouse serum.

[0247] This example evaluated the inhibitory activity of compounds RZM02004, RZM02005, and RZM02006, which have TBDMS modifications at different sites of the antisense chain, and the control compound RZM02001, which is not TBDMS modified, on CC3 protein levels in mouse serum, based on the Elisa measurement method and principle.

[0248] 1. Animal grouping, administration, and blood collection. C57BL / 6j mice aged 6-8 weeks were randomly divided into groups of 5 mice each, based on body weight, for a total of 5 groups. Each mouse in each group was administered the above-mentioned siRNA conjugate subcutaneously to the abdomen, with a dose of 3 mg / kg per mouse and a volume of 5 mL / kg. The PBS control group was administered the same volume of PBS solution without the siRNA conjugate. Blood was collected before administration and labeled as pre-dose, with the day of administration designated as day 0 (D0). After administration, blood was collected from the retinal venous plexus of each mouse on D14, D28, D42, D56, D70, and D91 using a coagulation-activating tube (obtained from Kang Shifei, 220518), with a blood collection volume of approximately 0.1 mL / mouse / dose. Blood was collected using a blood coagulation-activating tube (obtained from Kang Shifei, 220518), the blood samples were left to stand overnight at 4°C, 1800g was centrifuged at 4°C for 10 minutes to separate the serum, and it was stored at -80°C. 2. Measurement of CC3 protein in serum Serum samples were diluted to 1:10000 in 10-fold serial dilutions, and the CC3 protein content in the serum of each mouse was measured using the Mouse Complement C3 ELISA Kit (obtained from abcam, ab157711). Standard working solutions were prepared according to the instructions in the kit, followed by sample placement and other procedures. The ELISA plates were then placed in a multi-functional microwell plate Retriever (obtained from Biotek, Biotek Synergy H1), and the absorbance at 450 nm was measured. Using Graphpad Prism 8.0, a 4-parameter logistic curve fitting was performed (Equation: Y = Bottom + (X^Hillslope) * (Top--Bottom) / (X^HillSlope + EC50^HillSlope)) to plot a standard curve, calculate the R-squared value, substitute the OD450 value into the standard curve, and calculate the protein content of each sample by multiplying by the appropriate dilution factor. Normalization was then performed based on the pre-dose detection value. 3.Result analysis After administration, the expression levels of CC3 protein in the serum of mice (Figure 3) and the residual activity of the target gene CC3 mRNA (Table 8) were statistically analyzed. The results showed that siRNAs modified with TBDMS at position 18 of the sense strand (RZM02004), position 15 of the antisense strand (RZM02005), and position 21 of the antisense strand (RXM02006) had equivalent activity to the control sequence without TBDMS modification (RZM02001), indicating that both sequences exhibit efficient and sustained inhibitory effects on the serum CC3 protein content. At D91, the inhibitory effect on serum CC3 protein levels by TBDMS-modified RZM02004 (remaining level: 61.69%), RZM02005 (remaining level: 45.29%), and RXM02006 (remaining level: 56.75%) was significantly higher than that of the control sequence RZM02001 (remaining level: 72.75%). This suggests that large TBDMS group modifications at specific sites of siRNA do not impair sequence activity and may even enhance it.

[0249] [Table 8]

[0250] Example 4. In vitro inhibitory activity of siRNA conjugates modified at different sites using TBDMS against angiopoietin-like protein 3 (ANGPTL3) mRNA in primary mouse hepatocytes. This example evaluated the inhibitory activity against the target gene ANGPTL3 in primary mouse hepatocytes using an in vitro mouse primary hepatocyte screening method. The compounds were modified using TBDMS at different sites on the sense strand, such as RZ597065; modified using TBDMS at different sites on the antisense strand, such as RZ597084; and the control compound RZ597024, which is not modified using TBDMS, as well as the negative control compound RZ000002, which has no known gene targeting properties.

[0251] 1. Preparation of the test sample After centrifuging each of the above siRNA conjugate samples, an appropriate amount of 1×PBS was added according to the specifications of each tube to dissolve them, and a 20 μM mother liquor was prepared. This was then further diluted with 1×PBS to a 5 μM working solution. 2. Isolation of primary mouse hepatocytes (1) Anesthesia: C57BL / 6j mice were intracavitarially injected with 0.04 mL / 10 g of 10% chloride hydrate solution. (2) Perfusion: The animals were fixed, their abdomen and chest were disinfected with 75% ethanol, and the abdominal cavity was opened to locate the hepatic portal vein and inferior vena cava. An indwelling needle was inserted from the lower end of the inferior vena cava, and perfusion of HBSS solution was started at a rate of 120 drops / min. The hepatic portal vein was incised, and after confirming that the perfusion fluid was flowing out of the incised hepatic portal vein, the infusion device was secured and perfusion was continued for 4 minutes. (3) Digestion of hepatocytes: Perfusion was carried out for 8 minutes at a rate of 120 drops / min using HBSS containing 0.08% type IV collagenase (Collagenase from Clostridium histolyticum, Sigma). (4) Isolation of primary hepatocytes: Perfused livers were removed from the animals and placed in sterile culture dishes containing 10 mL of DMEM, then loosened. Undigested liver tissue and connective tissue were filtered and removed using a cell filter (75 μm). After centrifugation, the cells were suspended in DMEM and washed twice. 10 mL of DMEM medium containing 10% FBS was added to the suspension, the cells were counted, and the cells were prepared for ad libitum administration of the drug. 3. Free intake of siRNA conjugates: Newly isolated primary mouse hepatocytes were seeded onto a type I collagen-coated 12-well cell culture plate (Corning), with 5 × 10 cells per well. 5 The sample contains live cells, 2 mL of DMEM medium containing 10% FBS. Two μL of the 5 μM siRNA conjugate working solution was added to each cell well and gently mixed. This resulted in a final siRNA conjugate concentration of 5 nM in each cell well. The blank control group consisted of original mouse primary hepatocytes that had not undergone the siRNA conjugate ad libitum procedure. The ad libitum-treated mouse primary hepatocytes were cultured for a further 24 hours in an incubator at 37°C and 5% CO2. 4. Measurement RNA extraction: Total RNA was extracted from each well cell using a fully automated nucleic acid extraction system and nucleic acid extraction kit manufactured by Zhejiang Hanwei Technology Co., Ltd., according to the method described in the specification. Reverse transcription reaction: 1 μg of total RNA extracted from cells in each well was taken and transcribed using a Promega reverse transcription kit (Reverse Transcription System, A3500) with Oligo (dT). 15 A reverse transcription primer was selected, and a 20 μL reverse transcription system was prepared according to the instructions in the kit, and the reverse transcription reaction was carried out. After the reaction was complete, 80 μL of RNase-free water was added to the reverse transcription system to obtain a cDNA solution for real-time PCR measurement.

[0252] Real-time PCR measurement: ABI SYBR TM Using Select Master Mix (Catalog number: 4472908), prepare a 20 μL Real-time PCR reaction system in each PCR measurement well according to the method described in the kit instructions. Add 5 μL of the cDNA template obtained from the reverse transcription reaction described above to each measurement system. TMThe reaction system contained 10 μL of Select Master Mix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer (primer information is shown in Table 9), and 4 μL of RNase-free H2O. The prepared reaction system was placed in an ABI StepOnePlus PCR instrument, and real-time PCR amplification was performed using a three-step method. The amplification process involved pre-denaturation at 95°C for 10 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. This cycle of denaturation, annealing, and extension was repeated 40 times. After the process was completed, the difference in gene expression was calculated using the ΔΔCt method described above.

[0253] [Table 9]

[0254] 5.Result analysis The relative expression levels (Figure 4) and residual activity (Table 10) of the target gene ANGPTL3 mRNA were statistically analyzed after primary mouse hepatocytes ad libitum ingested siRNA modified at different sites of the sense strand using TBDMS. The results showed that, compared to the control sequence without TBDMS modification (RZ597024), TBDMS modification at positions 5 (RZ597069), 6 (RZ597070), 12 (RZ597076), 13 (RZ597077), and 18 (RZ597082) of the sense strand contributed to improved in vitro activity of the siRNA sequence, while TBDMS modification at positions 2 (RZ597066), 3 (RZ597067), 7 (RZ597071), 8 (RZ597072), 9 (RZ597073), and 10 (RZ597074) of the sense strand had a relatively significant attenuating effect on the in vitro activity of the siRNA sequence.

[0255] [Table 10]

[0256] The relative expression levels (Figure 5) and residual activity (Table 11) of the target gene ANGPTL3 mRNA were statistically analyzed after primary mouse hepatocytes ad libitum-intake of siRNA modified at different sites of the antisense strand using TBDMS. The results showed that modifications at positions 8 (RZ597030), 9 (RZ597031), 10 (RZ597086), 15 (RZ597091), 20 (RZ597096), and 21 (RZ597097) of the antisense strand can contribute to improved in vitro activity of the siRNA sequence compared to a control sequence without TBDMS modification (RZ597024).

[0257] [Table 11]

[0258] The experimental conclusions shown in the experimental results of Example 4 are highly similar to those of Example 2, indicating that the effect of TBDMS modification on the activity of the siRNA sequence is related to the modification site, and that large TBDMS group modifications at specific sites do not impair siRNA activity and can even further enhance it. Example 5. Inhibitory activity of siRNA conjugates modified with TBDMS at different sites against ANGPTL3 mRNA in mice.

[0259] This example evaluated the inhibitory activity against the target gene ANGPTL3 in mice using siRNA conjugates RZ597026 and RZ597030, which had different TBDMS modifications on the antisense strand, and the control conjugate RZ597024, which was not modified by TBDMS, using a method for evaluating target gene inhibitory activity in mice.

[0260] C57BL / 6j mice aged 6-8 weeks were randomly divided into 4 groups of 25 mice each, based on body weight. Each mouse in each group was administered the siRNA conjugate subcutaneously via abdominal injection, with a dose of 3 mg / kg per mouse and a volume of 5 mL / kg. The PBS control group received the same volume of PBS solution without the siRNA conjugate. The day of administration was designated as day 0 (D0). After administration, 5 mice from each group were euthanized on days D7, D14, D28, and D56. The animals were dissected, liver tissue was collected, and several 2 mm cells were examined. 3 The liver tissue was cut into small pieces and stored in RNAlater. An appropriate amount of liver tissue sample was taken from RNAlater and disrupted for 60 seconds using a Tissuelyser II fully automated tissue homogenizer. RNA extraction, reverse transcription, and real-time PCR measurements were performed according to the measurement method described in Example 4, and relative quantification calculations were performed for the target gene mRNA in each experimental group according to the ΔΔCt method described above.

[0261] The expression level of ANGPTL3 mRNA in mice after administration (Figure 6) and the residual activity of ANGPTL3 mRNA (Table 12) were statistically analyzed. Similar to the experimental conclusions in in vitro primary hepatocytes in Example 4, the 8th position of the antisense strand (RZ597030) was well tolerated to TBDMS modification, and both the in vivo activity and duration of action of RZ597030 were superior to the control sequence without TBDMS modification (RZ597024). On the other hand, TBDMS modification at the 4th position of the antisense strand (RZ597026) had a relatively large impact on the in vivo activity of the sequence.

[0262] [Table 12]

[0263] The in vivo experimental results from Example 5 reaffirmed that the effect of TBDMS modification on the activity of siRNA sequences is related to the modification site, and that large TBDMS group modifications at specific sites do not impair siRNA activity, and on the contrary, can even further enhance it. Example 6. In vitro inhibitory activity of siRNA conjugates modified at different TOM sites against coagulation factor XI (FXI) mRNA in primary mouse hepatocytes.

[0264] Using the same experimental method as in Example 4, the inhibitory activity against the target gene FXI in primary mouse hepatocytes was evaluated using siRNA conjugates with TOM modifications at different sites on the antisense strand. The difference is that in this example, instead of the siRNA conjugate used in Example 4, siRNA conjugates with TOM modifications at different sites on the antisense strand were used. Also, in this example, instead of the target gene measurement primer used in Example 4, the target gene FXI measurement primer shown in Table 13 was used. Relative quantitative calculations were performed for target gene mRNA in each experimental group according to the ΔΔCt method described above.

[0265] [Table 13]

[0266] The relative expression levels (Figure 7) and residual activity (Table 14) of the target gene FXI mRNA were statistically analyzed after primary mouse hepatocytes ad libitum ingested siRNAs with TOM modifications at different sites on the sense strand. The results showed that, compared to the control sequence without TOM modification (RZ594001), TOM modifications at positions 5 (RZ594011), 6 (RZ594012), 12 (RZ594018), 13 (RZ594019), 18 (RZ594024), and 19 (RZ594025) of the sense strand contributed to improved in vitro activity of the siRNA sequence, while TBDMS modifications at positions 2 (RZ594008), 3 (RZ594009), 7 (RZ594013), 8 (RZ594014), 9 (RZ594015), and 10 (RZ594016) of the sense strand had a relatively significant attenuating effect on the in vitro activity of the siRNA sequence. [Table 14]

[0267] The relative expression levels (Figure 8) and residual activity (Table 15) of the target gene FXI mRNA were statistically analyzed after primary mouse hepatocytes ad libitum-intake of siRNAs with TOM modifications at different sites on the antisense strand. Compared to a control sequence without TOM modification (RZ594001), modifications at positions 7 (RZ594004), 8 (RZ594005), 9 (RZ594006), 10 (RZ594030), 15 (RZ594035), 20 (RZ594040), and 21 (RZ594041) of the antisense strand can contribute to improved in vitro activity of the siRNA sequence.

[0268] [Table 15]

[0269] According to the results of Example 6, the effect of TOM modification on the activity of the siRNA sequence is related to the modification site, and large TOM group modification at a specific site does not impair siRNA activity but can further improve it, and this effect is highly similar to that of TOM modification. Example 7. Inhibitory activity of siRNA conjugates modified at different sites using TOM to target CC3 mRNA in mice.

[0270] In this example, the inhibitory activity against the target gene CC3 in mice was evaluated using a method for evaluating target gene inhibitory activity in mice. The conjugate RZ502013 had a TOM modification at position 19 of the sense strand, the conjugate RZ502016 had a TOM modification at position 10 of the antisense strand, and the control conjugate RZ002001 did not have a TOM modification. C57BL / 6j mice aged 6-8 weeks were randomly divided into groups of 5 mice each, for a total of 4 groups. Each group of mice was administered the siRNA conjugate subcutaneously via abdominal administration, with a dose of 3 mg / kg per mouse and a volume of 5 mL / kg. The PBS control group received the same volume of PBS solution without the siRNA conjugate. The day of administration was designated as day 0 (D0). The mice were euthanized on day 7 after administration. The animals were dissected, liver tissue was collected, and several 2 mm cells were extracted. 3 The liver tissue was cut into small pieces and stored in RNAlater. An appropriate amount of liver tissue sample was taken from RNAlater and disrupted for 60 seconds in a Tissuelyser II fully automated tissue homogenizer. RNA extraction, reverse transcription, and real-time PCR measurement were performed according to the measurement method described in Example 2, and relative quantification calculations were performed for the target gene mRNA in each experimental group according to the ΔΔCt method described above. The difference is that in this example, TOM-modified siRNA conjugates were used instead of the siRNA conjugates used in Example 4. Relative quantification calculations were performed for the target gene mRNA in each experimental group according to the ΔΔCt method described above.

[0271] The expression levels of CC3 mRNA in mice after administration (Figure 9) and the residual activity of CC3 mRNA (Table 16) were statistically analyzed. The results showed that the in vivo activity (D7) of the siRNA conjugate RZ502013, in which the 19th position of the sense strand of the siRNA was modified with TOM, was higher than that of the control conjugate RZ002001, while the in vivo activity of RZ502016, in which the 10th position of the antisense strand of the siRNA was modified with TOM, was equivalent to that of the control conjugate RZ002001.

[0272] [Table 16]

[0273] According to the in vivo results of Example 7, large TOM group modification at specific sites does not impair siRNA activity and can even further enhance it. Example 8. Inhibitory activity of siRNA conjugates modified at different MOE sites against CC3 mRNA in HepG2 cells.

[0274] Using the same experimental method as in Example 2, the in vitro activity in HepG2 cells was evaluated for siRNA compounds with MOE modification at different sites on the sense strand, such as RX502165, siRNA compounds with MOE modification at different sites on the antisense strand, such as RX502172, and the control compound RX502001 without MOE modification. The difference is that in this example, instead of the siRNA compounds used in Example 2, siRNA compounds with MOE modification at different sites on the sense and antisense strands were used. Relative quantitative calculations were performed for target gene mRNA in each experimental group according to the ΔΔCt method described above.

[0275] According to the results of Example 8, the effect of MOE modification on the activity of the siRNA sequence is related to the modification site. MOE group modification at specific sites does not impair siRNA activity, but can even further enhance it, and this effect is highly similar to that of TBDMS modification and TOM modification. The relative expression levels of the target gene CC3 mRNA (Figure 10) and the residual activity of CC3 mRNA (Table 17) were statistically analyzed after transfection of HepG2 with siRNAs modified at different sites on the sense strand using the MOE method. The results showed that, compared to the control sequence without MOE modification (RX502001), MOE modifications at positions 5 (RZ594011), 12 (RX502115), 18 (RX502116), and 19 (RX502117) of the sense strand contributed to improved in vitro activity of the siRNA sequence, while MOE modifications at positions 7 (RX502166), 8 (RX502167), 9 (RX502168), and 10 (RX502169) of the sense strand had a relatively significant attenuating effect on the in vitro activity of the siRNA sequence.

[0276] [Table 17]

[0277] The relative expression level of the target gene CC3 mRNA (Figure 11) and the residual activity of CC3 mRNA (Table 18) were statistically analyzed after transfection of HepG2 with siRNAs modified at different MOE sites on the antisense strand. The results showed that modifications at positions 8 (RX502118), 10 (RX502119), and 15 (RX502120) of the antisense strand can contribute to improved in vitro activity of the siRNA sequence compared to a control sequence without MOE modification (RX502001).

[0278] [Table 18]

[0279] According to the results of Example 8, the effect of MOE modification on the activity of the siRNA sequence is related to the modification site. MOE group modification at specific sites does not impair siRNA activity, but can even further enhance it, and this effect is highly similar to that of TBDMS modification and TOM modification. Example 9. Inhibitory activity of a conjugate modified with multiple MOE sites against CC3 mRNA in HepG2 cells.

[0280] Using the same experimental method as in Example 2, the in vitro activity in HepG2 cells was evaluated for siRNA compounds with multiple MOE modifications on the sense strand, such as RX502123; siRNA compounds with multiple MOE modifications on the antisense strand, such as RX502130; compounds with simultaneous MOE modifications on both the sense and antisense strands, such as RX502146; as well as the control compound RX502001 (without MOE modification) and the negative control compound RZ000002 (not targeting a known target). The difference is that in this example, instead of the siRNA compounds used in Example 2, the above-mentioned siRNA compounds with MOE modifications on both the sense and / or antisense strands were used. Relative quantitative calculations were performed for target gene mRNA in each experimental group according to the ΔΔCt method described above. The relative expression level of the target gene CC3 mRNA (Figure 12) and the residual activity of CC3 mRNA (Table 19) were statistically analyzed after transfection of HepG2 with siRNA modified at multiple sites of the sense strand using the MOE method. The results showed that siRNA modified at multiple sites of the sense strand (positions 6, 12, 18, and 19) had in vitro activity equivalent to that of the unmodified siRNA control sequence (RX502001), while the in vitro activity of compound RX502126, which had simultaneous MOE modification at positions 12 and 18 of the sense strand, was slightly higher than that of the control sequence RX502001.

[0281] [Table 19]

[0282] The relative expression level of the target gene CC3 mRNA (Figure 13) and the residual activity of CC3 mRNA (Table 20) were statistically analyzed after transfection of HepG2 with siRNA modified at multiple sites of the antisense strand using the MOE method. siRNA modified at multiple sites of the antisense strand (positions 8, 10, 15, 20, and 21) showed in vitro activity equivalent to that of the unmodified siRNA control sequence (RX502001). The in vitro activity of multiple modified sequences, such as compound RX502130 with simultaneous MOE modification at positions 8 and 15 of the sense strand, was slightly higher than that of the control sequence RX502001.

[0283] [Table 20]

[0284] After transfection of HepG2 with siRNA modified at multiple MOE sites on both the sense and antisense strands, the relative expression level of the target gene CC3 mRNA (Figure 14) and the residual activity of CC3 mRNA (Table 21) were statistically analyzed. The results showed that compounds with MOE modifications at positions 6, 12, and 18 of the sense strand and positions 8, 10, 15, 20, and 21 of the antisense strand had in vitro activity equivalent to that of the unmodified siRNA control sequence (RX502001). The in vitro activity of compounds with multiple modified sequences, such as compound RX502151 (simultaneous MOE modification at position 18 of the sense strand and position 15 of the antisense strand), compound RX502154 (simultaneous MOE modification at positions 6 and 12 of the sense strand and positions 8 and 15 of the antisense strand), compound RX502156 (simultaneous MOE modification at positions 6 and 18 of the sense strand and positions 8 and 15 of the antisense strand), and compound RX502158 (simultaneous MOE modification at positions 12 and 18 of the sense strand and positions 8 and 15 of the antisense strand), was slightly higher than that of the control sequence RX502001. In the sequence (RX502164), in which eight sites—positions 6, 12, and 18 of the sense strand and positions 8, 10, 15, 20, and 21 of the antisense strand—were simultaneously modified by MOE, the in vitro activity of the sequence was still well maintained.

[0285] [Table 21]

[0286] According to the results of Example 9, the effect of MOE modification on the activity of the siRNA sequence is related to the modification site. By applying MOE modification to one or more sites at specific locations on the sense strand and / or antisense strand, good tolerability can be obtained, the activity of the siRNA sequence can be well maintained, and ultimately the activity can be improved.

[0287] Example 10. Inhibitory activity against CC3 mRNA in mice by siRNA conjugates with MOE modifications at different sites of the sense and / or antisense strands.

[0288] Using the same experimental method as in Example 7, the inhibitory activity against the target gene CC3 in mice was evaluated using siRNA conjugates with MOE modifications at different sites on the sense strand and / or antisense strand, as well as the control conjugate RZ502031 without MOE modification. The difference is that in this example, the above-mentioned siRNA compounds with MOE modifications on the sense strand and / or antisense strand were used instead of the siRNA compounds used in Example 7. Relative quantitative calculations were performed for the target gene mRNA in each experimental group according to the ΔΔCt method described above. RNA extraction, reverse transcription, and Real-time PCR measurement were performed according to the measurement method described in Example 2, and relative quantitative calculations were performed for the target gene mRNA in each experimental group according to the ΔΔCt method described above.

[0289] The expression levels of CC3 mRNA in mice after administration of the drug (siRNA conjugate according to this embodiment) (Figures 15, 16) and the residual activity of CC3 mRNA (Tables 22, 23) were statistically analyzed. The results showed that by performing multiple MOE modifications at specific sites on the sense strand and / or antisense strand, the in vivo activity of the siRNA sequence can still be well maintained, and even further improved. The in vivo activity (D21) of siRNA conjugate RZ502039, in which MOE modifications were simultaneously performed at positions 6 and 18 of the sense strand and positions 8 and 15 of the antisense strand, and siRNA conjugate RZ502036, in which MOE modifications were simultaneously performed at positions 12 and 18 of the sense strand and position 15 of the antisense strand, were both higher than that of the control conjugate RZ502031. In contrast, when MOE modification was performed at position 14 of the antisense strand (RZ502087), the in vivo activity of the siRNA was significantly impaired.

[0290] [Table 22]

[0291] [Table 23]

[0292] According to the results of Example 10, the effect of MOE modification on the activity of the siRNA sequence is related to the modification site. By applying MOE modification to one or more sites at specific sites on the sense strand and / or antisense strand, good tolerability can be obtained, the activity of the siRNA sequence can be well maintained, and even its activity can be further improved. However, at several other sites, applying MOE modification to a single site significantly impairs the activity of the siRNA sequence.

[0293] Example 11. Inhibitory activity against SOD1 mRNA in mice by siRNA conjugates with MOE modifications at different sites on the sense and / or antisense strands. Using the same experimental method as in Example 7, the inhibitory activity against the target gene SOD1 in mice was evaluated by siRNA conjugates with MOE modifications at different sites on the sense strand and / or antisense strand, as well as by the control conjugate RZ599001 without MOE modification. The difference is that in this example, instead of the siRNA compounds used in Example 7, the above-mentioned siRNA compounds with MOE modifications on the sense strand and / or antisense strand were used. Relative quantitative calculations were performed for the target gene mRNA in each experimental group according to the ΔΔCt method described above. RNA extraction, reverse transcription, and Real-time PCR measurement were performed according to the measurement method described in Example 2, and relative quantitative calculations were performed for the target gene mRNA in each experimental group according to the ΔΔCt method described above.

[0294] The expression levels of SOD1 mRNA in mice (Figure 17) and residual activity (Table 24) were statistically analyzed on days 7 and 21 after administration. The results showed that the effect of MOE modification on siRNA sequence activity is related to the modification site, and by applying single or multiple MOE modifications to specific sites on the sense strand and / or antisense strand, good tolerability can be obtained, siRNA sequence activity can be well maintained, and ultimately activity can be further improved.

[0295] [Table 24]

[0296] Example 12. Evaluation of in vitro activity of siRNA conjugates with different modifications on the target gene C3 in HepG2 cells. In this example, the inhibitory activity of each siRNA conjugate, formed by varying the modification patterns of RZ002003, RZ002006, RZ002011, and RZ002031, against the target gene C3 mRNA in human hepatocellular carcinoma (HepG2) cells was compared and evaluated.

[0297] Preparation of the test sample: After centrifuging each of the above siRNA samples, an appropriate amount of PBS was added according to the specifications of each tube to dissolve them and prepare a 20 μM mother liquor. The mother liquor was then serially diluted with PBS to 0.5 μM and 0.05 μM working solutions. Dose tests were performed at final double-stranded drug concentrations of 5 nM and 0.5 nM.

[0298] 96-well plate transfection and measurement: HepG2 cells, grown to near confluence using trypsin, were digested, washed, and prepared as a cell suspension. 100 μL of this cell suspension (12,000 cells per well) was added to each well of a 96-well plate, and the cells were cultured in a 37°C, 5% CO2 incubator. After the cells had adhered to the wall for 24 hours, DMEM medium from the 96-well plate was aspirated, and then 80 μL of Opti-MEM was added to each well. TMThe culture medium was added, and the 96-well plate was placed in an incubator to continue culturing. 1 μL of 0.1 μM working medium and 0.01 μM working medium were dispersed in 9 μL of Opti-MEM to form an siRNA mixture, and 0.3 μL of RNAiMAX was dispersed in 9.7 μL of Opti-MEM and homogeneously mixed with the various siRNA mixtures to form a transfection complex. The transfection complex was incubated at room temperature for 10 minutes, and then placed in a 96-well plate (20 μL / well). After culturing for 4 hours, 100 μL of DMEM medium containing 20% ​​FBS was added to each well, and the 96-well plate was placed in an incubator and cultured for a further 24 hours. Here, for the mock control group: 0.3 μL of RNAiMAX was dispersed in 9.7 μL of Opti-MEM, and an additional 10 μL of Opti-MEM was added. The mixture was incubated at room temperature for 10 minutes and then placed in a 96-well plate (20 μL / well). After 4 hours of incubation, 100 μL of DMEM medium containing 20% ​​FBS was added to each well, and the 96-well plate was placed in an incubator and incubated for a further 24 hours.

[0299] A 96-well plate was removed, and total RNA was extracted using a fully automated nucleic acid extraction system (obtained from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (GO-MNTR-100, obtained from Zhejiang Hanwei Technology Co., Ltd.) following the standard procedure for total RNA extraction. Oligo (dT)18 reverse transcription primers were selected using a reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622), and a 20 μL reverse transcription system was prepared according to the instructions in the reverse transcription kit specification, followed by the reverse transcription reaction. Then, the expression levels of target gene mRNA in HepG2 cells were measured using a real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific, TaqMan Fast Advanced Master Mix, 4444557) and a fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384). In this real-time fluorescence quantitative PCR method, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as the internal reference gene, and measurements were performed for both the target gene and the GAPDH internal reference gene using primers for both. The sequences of the measured primers are shown in Table 25.

[0300] [Table 25]

[0301] Prepare a 10 μL Real-time PCR reaction system in each PCR measurement well according to the method described in the specification of the Real-time Fluorescence Quantitative PCR Kit. Add 4 μL of the cDNA solution obtained by the reverse transcription reaction described above and 5 μL of TaqMan to each reaction system. TMIt contains Fast Advanced Master Mix(2×), 0.15 μL of 10 μM upstream primer--, 0.15 μL of 10 μM downstream primer--, 0.15 μL of 10 μM probe primer--, and 0.55 μL of RNase--Free H2O. The prepared reaction system was placed in a real-time P fluorescence quantitative CR instrument (Bio--Rad, CFX Opus 384), and real-time PCR amplification was performed by the two-step method. As the amplification process, pre-denaturation was carried out at 50 °C for 2 minutes and at 95 °C for 20 seconds, denaturation was carried out at 95 °C for 3 seconds, annealing and extension were carried out at 60 °C for 30 seconds, and the cycle consisting of denaturation, annealing, and extension was repeated 40 times. In this real-time fluorescence quantitative PCR method, relative quantitative calculations were performed on the expression levels and inhibition rates of the target gene C3 mRNA in each experimental group according to the above ΔΔCt method. According to the results of Example 12, at doses of 5 nM and 0.5 nM, the siRNAs formed by changing the modification schemes of RZ002003, RZ002006, RZ002011, and RZ002031 all significantly inhibited the expression of C3 gene mRNA in HepG2 cells and had higher in vitro activities than the original sequences of RZ002003, RZ002006, RZ002011, and RZ002031 (Figure 18a, 18b, 18c, 18d, Tables 26a, 26b, 26c, 26d).

[0302]

Table 26a

Table 26b

Table 26c

Table 26d

[0303] Example 13. Evaluation of in vitro activity of siRNA conjugates with different modifications against the target gene angiotensinogen (AGT) in Huh7 cells.

[0304] This study compared and evaluated the inhibitory activity of each siRNA conjugate, formed by altering the modification patterns of RZ003017 and RZ003020, against the target gene AGT mRNA in human hepatocellular carcinoma cells (Huh7). Preparation of the test sample: After centrifuging each of the above siRNA samples, an appropriate amount of PBS was added according to the specifications of each tube to dissolve them and prepare a 20 μM mother liquor. The mother liquor was then serially diluted with PBS to 0.1 μM and 0.01 μM working solutions. Dose tests were performed at the final double-stranded drug concentrations of 1 nM and 0.1 nM.

[0305] 96-well plate transfection and measurement: Huh7 cells, grown to near confluence using trypsin, were digested, washed, and prepared as a cell suspension. 100 μL of this cell suspension (12,000 cells per well) was added to each well of a 96-well plate, and the cells were cultured in a 37°C, 5% CO2 incubator. After the cells had adhered to the wall for 24 hours, DMEM medium from the 96-well plate was aspirated, and then 80 μL of Opti-MEM was added to each well. TMThe culture medium was added, and the 96-well plate was placed in an incubator to continue culturing. 1 μL of 0.1 μM working medium and 0.01 μM working medium were dispersed in 9 μL of Opti-MEM to form an siRNA mixture, and 0.3 μL of RNAiMAX was dispersed in 9.7 μL of Opti-MEM and homogeneously mixed with the various siRNA mixtures to form a transfection complex. The transfection complex was incubated at room temperature for 10 minutes, and then placed in a 96-well plate (20 μL / well). After culturing for 4 hours, 100 μL of DMEM medium containing 20% ​​FBS was added to each well, and the 96-well plate was placed in an incubator and cultured for a further 24 hours. Here, for the mock control group: 0.3 μL of RNAiMAX was dispersed in 9.7 μL of Opti-MEM, and 10 μL of Opti-MEM was added. The mixture was incubated at room temperature for 10 minutes and then placed in a 96-well plate (20 μL / well). After 4 hours of incubation, 100 μL of DMEM medium containing 20% ​​FBS was added to each well, and the 96-well plate was placed in an incubator and incubated for a further 24 hours. RNA extraction, reverse transcription, and fluorescence quantitative PCR procedures were performed as described in Example 12. In this real-time fluorescence quantitative PCR method, relative quantitative calculations were performed for the expression level and repression rate of target gene mRNA in each experimental group according to the ΔΔCt method described above. The sequences of the measured primers are shown in Table 27.

[0306] [Table 27]

[0307] According to the results of Example 13, siRNAs formed by altering the modification patterns of RZ003017 and RZ00302 at doses of 1 nM and 0.1 nM both significantly suppressed the expression of AGT gene mRNA in HepG2 cells and exhibited in vitro activity equivalent to or higher than the original sequences RZ003017 and RZ003020 (Figure 19, Table 28).

[0308] [Table 28]

[0309] Example 14. Evaluation of the effect of siRNA conjugates with different modifications on reducing protein expression in hREN × hAGT hypertensive mice. This example uses enzyme-linked immunosorbent assay (ELISA) to measure the effects of single-dose administration of the control siRNA conjugates RZ003021, RZ003065, and RZ003066 on serum AGT protein expression in hREN×hAGT hypertensive mice at different time points.

[0310] Animal grouping, administration, and tissue sample collection: hREN×hAGT hypertensive mice (obtained from Saiye (Suzhou) Biotechnology Co., Ltd.) aged 9-11 weeks were divided into groups based on systolic blood pressure levels (all male). Each experimental group was administered a predetermined dose of drug conjugate, and a PBS control group was also established. For all mice, the drug dose was calculated based on body weight and administered as a single subcutaneous injection in the abdomen. Each drug conjugate was administered in the form of a PBS solution at a concentration of 0.3 mg / mL (calculated based on siRNA), with an administration volume of 10 mL / kg relative to mouse body weight, meaning the dose of each drug conjugate was 3 mg / kg (calculated based on siRNA) relative to mouse body weight. The PBS control group was administered the same volume of PBS solution (without drug conjugate). Serum samples were collected from mice in all groups before administration (pre-dose, value before drug administration), on the day of administration (day 0 (D0)), and on days 7 (D7), 14 (D14), 21 (D21), 28 (D28), and 56 (D56) after administration. AGT protein expression was measured using a human angiotensinogen kit (IBL, 27412). According to the results of Example 14, when administered as a single dose of 3 mg / kg, RZ003021, RZ003065, and RZ003066 were all able to effectively reduce the level of AGT protein in hREN × hAGT hypertensive mice, and both the activity and duration of action of RZ003066 were superior to those of RZ003021 and RZ003065 (Figure 20, Table 29).

[0311] [Table 29]

[0312] Example 15. Effect of siRNA conjugate on the in vivo activity of lipoprotein(A) (LPA) mRNA in an HDI mouse model. In this example, the in vivo activity of LPA-targeted siRNA was compared and evaluated using a BALB / c mouse hydro-powered injection model, applying different modifications and GalNAc binding structures. Plasmid construction:

[0313] Sangon Biotech (Shanghai) was commissioned to incorporate the LPA gene sequence (NM_005577.4) into a pcDNA-CMV vector-plasmid to form a pcDNA-CMV-RG001 expression plasmid.

[0314] Mouse model construction: The BALB / c mouse hydrodynamic injection model was constructed by rapidly injecting a pcDNA-CMV-RG001 plasmid solution into the mouse body under high pressure via the tail vein. On the third day of the experiment, 10 μg of pcDNA-CMV-RG001 was injected into the mouse tail vein using a hydrodynamic method within 5 seconds, with an injection volume of 8% of the mouse's body weight. The plasmid DNA for injection was diluted with physiological saline, and the solution was prepared before injection and stored at 4°C.

[0315] Animal grouping, administration, and tissue sample collection: 6-8 week old BALB / c mice were randomly divided into groups based on body weight (all female), with 5 mice per group. Each experimental group was administered a predetermined dose of the drug conjugate, and a PBS control group was also established. For all mice, the drug dose was calculated based on body weight and administered as a single subcutaneous injection in the abdomen. Each drug conjugate was administered in the form of a PBS solution at a concentration of 0.1 mg / mL (calculated based on siRNA), with an administration volume of 10 mL / kg relative to mouse body weight, meaning the dose of each drug conjugate was 1 mg / kg (calculated based on siRNA) relative to mouse body weight. The PBS control group was administered the same volume of PBS solution (without the drug conjugate). The day of administration was designated as day 0 (D0). Plasmid injection was performed on day 3 (D3) after administration, and 5 mice from each group were euthanized on day 4 (D4). Each of the sacrificial mice was dissected to collect liver tissue, and the liver tissue was measured to approximately 2 mm. 3 It was cut into small pieces and stored in RNA later.

[0316] For each mouse, an appropriate amount of liver tissue sample was taken from the RNA later, the liver tissue sample was disrupted for 60 seconds using a Tissuelyser II fully automated tissue homogenizer, and total RNA was extracted using a fully automated nucleic acid extractor (obtained from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (GO-MNTR-100, obtained from Zhejiang Hanwei Technology Co., Ltd.) according to the standard procedure for total RNA extraction. Reverse transcription and fluorescence quantitative PCR were performed as described in Example 12. In this real-time fluorescence quantitative PCR method, the Nero gene in the plasmid backbone was used as the internal reference gene, and measurements were performed for the target gene and the internal reference gene Nero, respectively, using a primer for the target gene and a primer for the internal reference gene Nero. The sequences of the measured primers are shown in Table 30.

[0317] [Table 30]

[0318] Prepare a 10 μL Real-time PCR reaction system in each PCR measurement well according to the method described in the specification of the Real-time Fluorescence Quantitative PCR Kit. Add 4 μL of the cDNA solution obtained by the reverse transcription reaction described above and 5 μL of TaqMan to each reaction system. TM The reaction system contains Fast Advanced Master Mix (2×), 0.15 μL of 10 μM upstream primer, 0.15 μL of 10 μM downstream primer (primer information is shown in Table 11), 0.15 μL of 10 μM probe primer, and 0.55 μL of RNase-Free H2O. The prepared reaction system was placed in a real-time fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384) and real-time PCR amplification was performed using a two-step method. The amplification process involved 2 minutes of pre-denaturation at 50°C, followed by 20 seconds of pre-denaturation at 95°C, 3 seconds of denaturation at 95°C, annealing at 60°C for 30 seconds, and extension. This cycle of denaturation, annealing, and extension was repeated 40 times. In this real-time fluorescence quantitative PCR method, relative quantitative calculations were performed for the expression level and repression rate of target gene mRNA in each experimental group according to the ΔΔCt method, referring to the technical method described in the embodiment. According to the results of Example 15, at a dose of 1 mg / kg, the activity of RZ001032 was superior to that of RZ001003, the activity of RZ001034 was superior to that of RZ001005, the activity of RZ001037 was superior to that of RZ001008, the activity of RZ001039 was superior to that of RZ001012, and the activity of RZ001044 was superior to that of RZ001026 (Figure 21, Table 31).

[0319] [Table 31]

[0320] Example 16 Evaluation of inhibitory activity of siRNA conjugate against the target gene LPA in hApo(a) transgenic mice. The hApo(a) transgenic mouse is a conditional knock-in human Apo(a) transgenic mouse developed by Saiye (Suzhou) Biotechnology Co., Ltd. based on its proprietary TurboKnockout (trademarked) platform. The development process is as follows: (1) Design and construction of targeting vector: Incorporate the human-derived LPA gene into the vector, and use the CAG promoter --loxP--PGK--Neo--6 * The SV40 pA--loxP--Kozak--Human LPA CDS--rBG pA targeting vector can be formed, and this vector can insert the human-derived LPA gene (NM_005577.4) into the intergenetic region between the Eif4enif1 and Drg1 genes at the Hipp11 site of mouse chromosome 11. The targeting vector was validated by enzymatic cleavage, PCR, and sequencing, and after confirming that there were no problems, plasmid extraction and linearization were performed. (2) Electroporation of targeting vector into embryonic stem cells and selection of positive clones: Targeting vectors were electroporated into mouse embryonic stem cells (ES), and positive selection was performed using Neo and negative selection using DTA. Drug-resistant clones were selected, and the ES cells were verified by PCR, karyotype analysis, and Southern blot. (3) Production of TurboKnockout (trademark registered) mice: Positive ES cells were microinjected, and the injected blastocysts were transplanted into pseudopregnant female mice. The resulting mice were examined, and TurboKnockout mice capable of transmitting traits through the reproductive system were obtained. (4) Obtaining hApo(a) transgenic mice: Mutants expressing Cre specifically in the liver were crossed with TurboKnockout mice, drug resistance genes between loxP sites were removed, and the expression of human-derived LPA genes was activated to obtain hApo(a) transgenic mice. In this example, serum Apo(a) protein expression levels were measured at different time points in hApo(a) transgenic mice after single administration of siRNA conjugates RZ001032 and RZ001034 using enzyme-linked immunosorbent assay (ELISA).

[0321] Animal grouping, administration, and tissue sample collection: Apo(a) transgenic mice (prepared by Saiye (Suzhou) Biotechnology Co., Ltd.) were divided into groups based on serum Apo(a) levels. Each experimental group was administered a predetermined dose of a pharmaceutical conjugate, and a PBS control group was also established. For all mice, the drug dose was calculated based on body weight and administered as a single subcutaneous injection in the abdomen. Each pharmaceutical conjugate was administered in the form of a PBS solution at a concentration of 0.1 mg / mL (calculated based on siRNA), with an administration volume of 10 mL / kg relative to mouse body weight, meaning the dose of each pharmaceutical conjugate was 1 mg / kg (calculated based on siRNA) relative to mouse body weight. The PBS control group was administered the same volume of PBS solution, and serum was collected from mice in all groups on days 28 (D28), 35 (D35), 42 (D42), 49 (D49), and 56 (D56). Apo(a) protein expression was measured using the human Lipoprotein A ELISA kit (Abcam, ab212165).

[0322] According to the results of Example 16, when administered as a single dose of 1 mg / kg, both RZ001032 and RZ001034 can significantly reduce Apo(a) protein levels in Apo(a) transgenic mice (Figure 22, Table 32). [Table 32]

[0323] Example 17. Inhibitory activity against SOD1 mRNA in mice by siRNA conjugates modified at different sites of the antisense strand with NM062 and NM063. In this example, the inhibitory activity against the target gene SOD1 in mice was evaluated using the in vivo evaluation method shown in Example 7, by siRNA conjugates modified at different sites of the antisense strand with NM062 and NM063, as well as control conjugates RZ599001 and RZ599015 that lack NM062 and NM063 modifications. C57BL / 6j mice aged 6-8 weeks were randomly divided into 6 groups based on body weight, with 15 mice per group. Each mouse in each group was administered the siRNA conjugate via subcutaneous abdominal administration. In the PBS control group, the dose per mouse was 5 mL / kg (administered volume), while in the siRNA conjugate experimental group, the dose per mouse was 3 mg / kg (calculated based on siRNA), with an administered volume of 5 mL / kg. The day of administration was designated as day 0 (D0). After administration, 5 mice from each group were euthanized on days 7 (D7), 28 (D28), and 56 (D56). The animals were dissected, liver tissue was collected, and several 2 mm samples were taken. 3 The sample was cut into small pieces and stored in RNAlater. RNA extraction, reverse transcription, and fluorescence quantitative PCR procedures were performed as described above, and the difference in gene expression was calculated using the ΔΔCt method. The primers are shown in Table 33.

[0324] [Table 33]

[0325] The expression levels of SOD1 mRNA in mice at D7, D28, and D56 after administration (Figure 23) and residual activity (Table 34) were statistically analyzed. The results showed that good tolerability could be obtained by applying NM062 and NM063 modifications at specific sites on the antisense strand of the siRNA, and that NM062 and NM063 modifications at positions 8 and 15 of the antisense strand could further enhance the activity of the siRNA sequence.

[0326] [Table 34]

[0327] Example 18. Inhibitory activity of siRNA conjugates modified with NM096, NM098, and NM099 in the antisense strand against SOD1 mRNA in primary mouse hepatocytes. Using the experimental method described above for evaluating inhibitory activity against target genes in primary mouse hepatocytes, we evaluated the inhibitory activity against the target gene SOD1 in primary mouse hepatocytes of siRNA conjugates RZ599136 (modified with NM096 at position 15 of the antisense strand), RZ599138 (modified with NM098), RZ599139 (modified with NM099), and the control RZ599062.

[0328] Refer to the above for procedures such as isolation and cell culture of primary mouse hepatocytes. Primary mouse hepatocytes were extracted from fresh liver tissue of C56BL / 6j mice, and 2 × 10⁶ cells were cultured. 5 Cells were seeded into a 12-well culture plate at a cell / well rate. Double-stranded siRNA conjugates from each group were serially diluted with PBS to a 10 μM working solution (calculated relative to siRNA). 1 μL / well of each concentration of siRNA conjugate working solution was added to the 12-well culture plate, resulting in a final siRNA conjugate transfection concentration of 10 nM (calculated relative to siRNA). Two culture wells were prepared for each concentration of siRNA. Two additional culture wells were prepared as blank control wells (BLANK) with 1 μL / well of PBS added. The culture plates were shaken to ensure uniform mixing. The cells were then incubated for a further 24 hours in a cell incubator at 37°C and 5% CO2. Relative quantitative calculations were performed for target gene mRNA in each experimental group according to the ΔΔCt method described above. The primers used in this example are shown in Table 33.

[0329] According to the results of Example 18, siRNA conjugates modified at position 15 of the antisense chain with NM096, NM098, and NM099 exhibited equivalent inhibitory activity to the control RZ599062 in primary mouse hepatocytes (Figure 24, Table 35). [Table 35]

[0330] Example 19. Evaluation of inhibitory activity against the target gene SOD1 in primary mouse hepatocytes by an siRNA conjugate with a 2' difluoro substitution at a specific site of the sense strand. Example 19 evaluated the inhibitory activity of the siRNA conjugate RZ599051, in which the nucleotide at position 8 of the sense strand was difluoromodified at the 2' position, and the siRNA control conjugate RZ599001, in which the nucleotide at position 8 was fluorinated, against the target gene SOD1 in mouse primary hepatocytes, using the experimental method for evaluating the inhibitory activity against the target gene in mouse primary hepatocytes described above. Refer to the above for procedures such as isolation and cell culture of mouse primary hepatocytes. Mouse primary hepatocytes were extracted from fresh liver tissue of C56BL / 6j mice, and 2 × 10⁶ cells were collected. 5 Cells were seeded into a 12-well culture plate at a cell / well rate. Double-stranded siRNA conjugates from each group were serially diluted with PBS to a 5 μM working solution (calculated relative to siRNA). 1 μL / well of each concentration of siRNA conjugate working solution was added to the 12-well culture plate, resulting in a final siRNA conjugate transfection concentration of 5 nM (calculated relative to siRNA). Two culture wells were prepared for each concentration of siRNA. Two additional culture wells were prepared as blank control wells (BLANK) with 1 μL / well of PBS added. The culture plates were shaken to ensure uniform mixing. The cells were then incubated for a further 24 hours in a cell incubator at 37°C and 5% CO2. Relative quantitative calculations were performed for target gene mRNA in each experimental group according to the ΔΔCt method described above. The primers used in this example are shown in Table 34.

[0331] According to the results of Example 19, the siRNA conjugate RZ599051, in which the nucleotide at position 8 of the sense strand was difluoromodified at the 2' position, exhibited equivalent inhibitory activity to the control conjugate RZ599001 (Figure 25, Table 36). [Table 36]

[0332] Example 20. Evaluation of inhibitory activity against the target gene SOD1 in primary mouse hepatocytes by an siRNA conjugate with a 2' difluoro substitution at a specific site of the sense strand. Example 20 evaluated the inhibitory activity against the target gene SOD1 in primary mouse hepatocytes using the experimental method described above for evaluating inhibitory activity against the target gene in primary mouse hepatocytes. The siRNA conjugate RZ599055, in which the nucleotide at position 8 of the sense strand was difluoromodified at the 2' position, was difluoromodified at position 9 of the sense strand, was difluoromodified at the 2' position, was RZ599056, and the control conjugate RZ599001 was also evaluated. RZ599055 differs from RZ599051 in Example 19 in that the nucleotide modifications at position 10 of the sense strand and position 12 of the antisense strand were replaced from methoxy group modifications to fluoromodifications.

[0333] Mouse primary liver cells 2 × 10 5 Cells were seeded into a 12-well culture plate at a cell / well rate. Double-stranded siRNA conjugates from each group were serially diluted with PBS to a 5 μM working solution (calculated relative to siRNA). 1 μL / well of each concentration of siRNA conjugate working solution was added to the 12-well culture plate, resulting in a final siRNA conjugate transfection concentration of 5 nM (calculated relative to siRNA). Two culture wells were prepared for each concentration of siRNA. Two additional culture wells were prepared as blank control wells (BLANK) with 1 μL / well of PBS added. The primers used in this example are shown in Table 34.

[0334] According to the results of Example 20, the siRNA conjugate RZ599055, in which the nucleotide at position 8 of the sense strand was difluoromodified at the 2' position, and the siRNA conjugate RZ599056, in which the nucleotide at position 9 of the sense strand was difluoromodified at the 2' position, exhibited inhibitory activity equivalent to or better than that of the control conjugate RZ599001 (Figure 26, Table 37). [Table 37]

[0335] Example 21. Evaluation of inhibitory activity against the target gene ANGPTL3 in mouse primary hepatocytes by an siRNA conjugate with a 2' difluoro substitution at a specific site of the sense strand. Example 21 evaluated the inhibitory activity against the target gene ANGPTL3 in primary mouse hepatocytes using the experimental method described above for evaluating inhibitory activity against the target gene in primary mouse hepatocytes. The siRNA conjugate RZ597101, in which the nucleotide at position 8 of the sense strand was difluoromodified at the 2' position, and the control conjugate RZ597002 were used. RZ597101 differs from the control RZ597002 in that the nucleotide modifications at position 10 of the sense strand and position 12 of the antisense strand were replaced from methoxy group modifications to fluoro modifications, and the nucleotide modification at position 15 of the antisense strand was replaced from a 2' methoxy group to a moe modification.

[0336] Mouse primary liver cells 2 × 10 5Cells were seeded into a 12-well culture plate at a cell / well rate. Double-stranded siRNA conjugates from each group were serially diluted with PBS to a 10 μM working solution (calculated relative to siRNA). 1 μL / well of each concentration of siRNA conjugate working solution was added to the 12-well culture plate, resulting in a final siRNA conjugate transfection concentration of 10 nM (calculated relative to siRNA). Two culture wells were prepared for each concentration of siRNA. Two additional culture wells were prepared as blank control wells (BLANK) with 1 μL / well of PBS added. The primers used in this example are shown in Table 9. According to the results of Example 21, the siRNA conjugate RZ597101 exhibits superior inhibitory activity compared to the control conjugate RZ597002 (Figure 27, Table 38).

[0337] [Table 38]

[0338] Example 22. Evaluation of inhibitory activity against the target gene complement factor B (CFB) in primary mouse hepatocytes by an siRNA conjugate with a 2' difluoro substitution at a specific site of the sense strand. Example 22 evaluated the inhibitory activity of siRNA conjugates RZM11508, RZM11509, RZM11510, and the control conjugate RZM11015 against the target gene CFB in primary mouse hepatocytes using the experimental method for evaluating inhibitory activity against the target gene in primary mouse hepatocytes described above. The differences were that, compared to the control conjugate RZM11015, RZM11508 had nucleotide modifications at position 10 of the sense strand and position 12 of the antisense strand replaced from methoxy group modifications to fluoro modifications, and RZM11509 had the same modifications as RZM11508, but with the nucleotide modification at position 15 of the antisense strand replaced from a 2' methoxy group to a moe modification. RZM11510 differed from RZM11509 in that the nucleotide at position 7 of the sense strand was replaced with a 2' difluoro-modified nucleotide.

[0339] Mouse primary liver cells 2 × 10 5 Cells were seeded into a 12-well culture plate at a cell-to-well ratio. Double-stranded siRNA conjugates from each group were serially diluted with PBS to a 10 μM working solution (calculated relative to siRNA). 1 μL / well of each concentration of siRNA conjugate working solution was added to the 12-well culture plate, resulting in a final siRNA conjugate transfection concentration of 10 nM (calculated relative to siRNA). Two culture wells were prepared for each concentration of siRNA. Two additional culture wells were prepared as blank control wells (BLANK) with 1 μL / well of PBS added to each well.

[0340] [Table 39]

[0341] According to the results of Example 22, siRNA conjugates RZM11508, RZM11509, and RZM11510 exhibit superior inhibitory activity compared to the control conjugate RZ011015 (Figure 28, Table 40). [Table 40]

[0342] Example 23. Evaluation of inhibitory activity against the target gene CFB in mice by an siRNA conjugate with a 2' difluoro substitution at a specific site on the sense strand. This example evaluated the inhibitory activity of siRNA conjugates RZM11509, RZM11510, and the control conjugate RZM11015 against the target gene CFB in mice, using a method for evaluating target gene inhibitory activity in mice. C57BL / 6j mice aged 6-8 weeks were randomly divided into four groups based on body weight, with 10 mice per group. Each group of mice was administered the siRNA conjugate subcutaneously via abdominal injection. In the PBS control group, the dose was 5 ml / kg per mouse, while in the siRNA conjugate experimental group, the dose was 3 mg / kg per mouse (calculated based on siRNA), with a dose of 5 ml / kg. The day of administration was designated as D0. Five mice from each group were euthanized on day 7 (D7) and day 28 (D28). The animals were dissected, liver tissue was collected, and several 2 mm samples were taken. 3 The RNA was cut into small pieces and stored in RNAlater. RNA extraction, reverse transcription, and real-time PCR measurement methods were performed as described above, and relative quantification calculations were performed for target gene mRNA in each experimental group according to the ΔΔCt method described above. The primers are shown in Table 39.

[0343] According to the results of Example 23, siRNA conjugates RZM11509 and RZM11510 showed inhibitory activity against the target gene at D7 that was almost equivalent to that of the control conjugate RZM11015, and at D28, they showed superior inhibitory activity compared to the control conjugate (Figure 29, Table 41). [Table 41]

[0344] Example 24. Evaluation of inhibitory activity against the target gene ANGPTL3 in primary mouse hepatocytes by an siRNA conjugate with a 2' difluoro substitution at a specific site of the antisense chain. Example 24 evaluated the inhibitory activity of the siRNA conjugate RZ597060, in which the nucleotide at position 6 of the antisense strand was difluoromodified at the 2' position, and the control conjugate RZ597002, against the target gene ANGPTL3 in mouse primary hepatocytes, using the experimental method for evaluating inhibitory activity against the target gene in mouse primary hepatocytes described above. 2 × 10⁶ mouse primary hepatocytes were used. 5 Cells were seeded in a 12-well culture plate at a cell / well rate. Double-stranded siRNA conjugates from each group were serially diluted in PBS to a 10 μM working solution (calculated relative to siRNA). 1 μL / well of each concentration of siRNA conjugate working solution was added to the 12-well culture plate, resulting in a final siRNA conjugate transfection concentration of 10 nM (calculated relative to siRNA). Two culture wells were prepared for each concentration of siRNA. Two additional culture wells were prepared as blank control wells (BLANK) with 1 μL / well of PBS added. RNA extraction, reverse transcription, and real-time PCR measurement methods were performed as described above, and relative quantification calculations were performed for target gene mRNA in each experimental group according to the ΔΔCt method described above. The primers are shown in Table 9.

[0345] According to the results of Example 24, the siRNA conjugate RZ597060, in which the nucleotide at position 6 of the antisense chain was difluoromodified at the 2' position, exhibited equivalent inhibitory activity to the control conjugate RZ597002 (Figure 30, Table 42).

[0346] [Table 42]

[0347] Example 25. Evaluation of inhibitory activity against the target gene CFB in mouse primary hepatocytes by an siRNA conjugate with a 2' difluoro substitution at a specific site of the antisense chain. Example 25 evaluated the inhibitory activity of the siRNA conjugate RZM11504, in which the nucleotide at position 14 of the antisense strand was difluoromodified at the 2' position; the siRNA conjugate RZM11505, in which the nucleotide at position 16 of the antisense strand was difluoromodified at the 2' position; and the control conjugate RZM11001, against the target gene CFB in mouse primary hepatocytes, using the experimental method for evaluating inhibitory activity against the target gene in mouse primary hepatocytes described above. 2 × 10⁶ mouse primary hepatocytes were used. 5 Cells were seeded in a 12-well culture plate at a cell / well rate. Double-stranded siRNA conjugates from each group were serially diluted with PBS to a 10 μM working solution (calculated relative to siRNA). 1 μL / well of each concentration of siRNA conjugate working solution was added to the 12-well culture plate, resulting in a final siRNA conjugate transfection concentration of 10 nM (calculated relative to siRNA). Two culture wells were prepared for each concentration of siRNA. Two additional culture wells were prepared as blank control wells (BLANK) with 1 μL / well of PBS added. RNA extraction, reverse transcription, and real-time PCR measurement methods were performed as described above, and relative quantification calculations were performed for target gene mRNA in each experimental group according to the ΔΔCt method described above. The primers are shown in Table 39.

[0348] According to the results of Example 25, the siRNA conjugate RZM11504, in which the nucleotide at position 14 of the antisense strand was difluoromodified at the 2' position, and the siRNA conjugate RZM11505, in which the nucleotide at position 16 of the antisense strand was difluoromodified at the 2' position, exhibited superior inhibitory activity compared to the control conjugate RZM11001 (Figure 31, Table 43).

[0349] [Table 43]

[0350] Example 26. Evaluation of inhibitory activity against the target gene CFB in mouse primary hepatocytes by an siRNA conjugate with a 2' difluoro substitution at a specific site of the antisense chain. Example 26 evaluated the inhibitory activity against the target gene CFB in primary mouse hepatocytes using the experimental method described above for evaluating inhibitory activity against the target gene in primary mouse hepatocytes. This was done using the siRNA conjugate RZM11507, in which the nucleotide at position 14 of the antisense strand was difluoromodified at the 2' position, and the control conjugate RZM11502. This differs from Example 25 in that the nucleotide modification at position 15 of the antisense strand of the sequence was replaced from a methoxy group at the 2' position to a moe modification.

[0351] Mouse primary liver cells 2 × 10 5 Cells were seeded in a 12-well culture plate at a cell / well rate. Double-stranded siRNA conjugates from each group were serially diluted with PBS to a 10 μM working solution (calculated relative to siRNA). 1 μL / well of each concentration of siRNA conjugate working solution was added to the 12-well culture plate, resulting in a final siRNA conjugate transfection concentration of 10 nM (calculated relative to siRNA). Two culture wells were prepared for each concentration of siRNA. Two additional culture wells were prepared as blank control wells (BLANK) with 1 μL / well of PBS added. RNA extraction, reverse transcription, and real-time PCR measurement methods were performed as described above, and relative quantification calculations were performed for target gene mRNA in each experimental group according to the ΔΔCt method described above. The primers are shown in Table 39. According to the results of Example 26, the siRNA conjugate RZM11507, in which the nucleotide at position 14 of the antisense strand was difluoromodified at the 2' position, exhibited superior inhibitory activity compared to the control conjugate RZM11502 (Figure 32, Table 44).

[0352] [Table 44]

[0353] Example 27. Evaluation of inhibitory activity against the target gene CFB in mice by an siRNA conjugate with a 2' difluoro substitution at a specific site of the antisense strand. This example evaluated the inhibitory activity of siRNA conjugates RZM11502, RZM11507, and the control conjugate RZM11001 against the target gene CFB in mice, using a method for evaluating target gene inhibitory activity in mice. Differences between the conjugates include the replacement of methoxy group modifications from fluoromodifications at position 10 of the sense strand and position 12 of the antisense strand in RZM11502, and the replacement of the 2' methoxy group modification at position 15 of the antisense strand with a moe modification. RZM11507 differs from RZM11502 in that the nucleotide at position 14 of the antisense strand is a nucleotide with a 2' difluoromodification.

[0354] C57BL / 6j mice aged 6-8 weeks were randomly divided into four groups based on body weight, with 15 mice in each group. The siRNA conjugate was administered to each group of mice subcutaneously via abdominal injection. In the PBS control group, the dose per mouse was 5 ml / kg, while in the siRNA conjugate experimental group, the dose per mouse was 3 mg / kg (calculated based on siRNA), with a dose volume of 5 ml / kg. The day of administration was designated as D0. After administration, five mice from each group were euthanized on D7, D28, and D56. The animals were dissected, liver tissue was collected, and several 2 mm samples were taken. 3The RNA was cut into small pieces and stored in RNAlater. RNA extraction, reverse transcription, and real-time PCR measurement methods were performed as described above, and relative quantification calculations were performed for target gene mRNA in each experimental group according to the ΔΔCt method described above. The primers are shown in Table 39.

[0355] According to the results of Example 27, the inhibitory activity of siRNA conjugate RZM11502 was superior to that of the control conjugate RZM11001 at D7 and D28, and siRNA conjugate RZM11507 showed higher inhibitory effects and a longer duration of action at D7, D28, and D56 compared to the control conjugate RZM11001 (Figure 33, Table 45). [Table 45]

[0356] Example 28. Evaluation of inhibitory activity against the target gene complement component C4B (Complement C4B, C4B) in mice by an siRNA conjugate with a 2' difluoro substitution at a specific site of the antisense strand. This example evaluated the inhibitory activity of the siRNA conjugate RZM12503, in which the 9th position of the antisense strand of the C4B target is difluoromodified at the 2' position, and the control conjugate RZM12501, against the target gene C4B in mice, using a method for evaluating target gene inhibitory activity in mice. C57BL / 6j mice aged 6-8 weeks were randomly divided into three groups based on body weight, with 10 mice per group. The siRNA conjugates were administered to each group of mice by subcutaneous abdominal administration. In the PBS control group, the dose was 5 ml / kg per mouse, and in the siRNA conjugate experimental group, the dose was 3 mg / kg per mouse (calculated based on siRNA), with a dose of 5 ml / kg. The day of administration was designated as D0. After administration, 5 mice from each group were euthanized on D7 and D28, respectively. Dissecting animals, collecting liver tissue, and several 2mm 3The sample was cut into small pieces and stored in RNAlater. RNA extraction, reverse transcription, and real-time PCR measurement methods were performed as described above, and relative quantification calculations were performed for target gene mRNA in each experimental group according to the ΔΔCt method described above.

[0357] [Table 46]

[0358] According to the results of Example 28, the siRNA conjugate RZM12503, which was difluoromodified at the 2' position of the antisense chain at position 9, had comparable inhibitory effect and duration of action to the control conjugate RZM12501 (Figure 34, Table 47). [Table 47]

[0359] Example 29. Evaluation of inhibitory activity against the target gene SOD1 in mice by an siRNA conjugate containing an NM054 group in the antisense strand. This example evaluated the inhibitory activity against the target gene SOD1 in mice using a method for evaluating target gene inhibitory activity in mice. The conjugates RZ599060, which contains the NM054 group at position 20 of the SOD1 target siRNA antisense strand; RZ599061, which contains the NM054 group at positions 20 and 21; and RZ599001, which does not contain the NM054 group.

[0360] C57BL / 6j mice aged 6-8 weeks were randomly divided into four groups based on body weight, with 15 mice per group. Each mouse in each group was administered the siRNA conjugate via subcutaneous abdominal administration. In the PBS control group, the dose per mouse was 5 mL / kg, while in the siRNA conjugate experimental group, the dose per mouse was 3 mg / kg (calculated based on siRNA), with a dose of 5 mL / kg. The day of administration was designated as day 0 (D0). After administration, five mice from each group were euthanized on days 7 (D7), 28 (D28), and 49 (D49). The animals were dissected, liver tissue was collected, and several 2 mm samples were taken. 3 The sample was cut into small pieces and stored in RNAlater. RNA extraction, reverse transcription, and fluorescence quantitative PCR procedures were performed as described above, and the difference in gene expression was calculated using the ΔΔCt method. The primers are shown in Table 34.

[0361] According to the results of Example 29, both the conjugates RZ599060 and RZ599061, which contain the NM054 group, exhibited higher inhibitory activity at D7, D28, and D49 compared to the control conjugate RZ599001, which does not contain the NM054 group. At D49, the inhibitory activity of RZ599060 and RZ599061 was approximately 15% higher than that of the control conjugate RZ599001 (Figure 35, Table 48). [Table 48]

[0362] Example 30. Evaluation of inhibitory activity against the target gene ANGPTL3 in mice by an siRNA conjugate containing an NM054 group in the antisense strand. This example evaluated the inhibitory activity of conjugates RZ597115, which contains the NM054 group at position 20 of the siRNA antisense strand, RZ597116, which contains the NM054 group at positions 20 and 21, and RZ597114, which does not contain the NM054 group, against the target gene ANGPTL3 in mice using a method for evaluating target gene inhibitory activity in mice.

[0363] C57BL / 6j mice aged 6-8 weeks were randomly divided into four groups based on body weight, with 15 mice per group. Each group of mice was administered the siRNA conjugate subcutaneously via abdominal injection. In the PBS control group, the dose was 5 mL / kg per mouse, while in the siRNA conjugate experimental group, the dose was 3 mg / kg per mouse (calculated based on siRNA), with a dose of 5 mL / kg. The day of administration was designated as D0. After administration, five mice from each group were euthanized on D7, D28, and D56. The animals were dissected, liver tissue was collected, and several 2 mm samples were taken. 3 The sample was cut into small pieces and stored in RNAlater. RNA extraction, reverse transcription, and real-time PCR measurement were performed as described above, and the difference in gene expression was calculated using the ΔΔCt method. The primers are shown in Table 9.

[0364] According to the results of Example 30, conjugates RZ597115 and RZ597116 containing the NM054 group exhibited higher inhibitory activity at D7, D28, and D56 compared to the control conjugate RZ597114, which did not contain the NM054 group. At D56, the inhibitory activity of conjugate RZ597115, which contains the NM054 group at position 20 of the antisense chain, was approximately 25% higher than that of the control conjugate RZ597114. The inhibitory activity of conjugate RZ597116, which contains NM054 at positions 20 and 21 of the antisense chain, was approximately 35% higher than that of the control conjugate RZ597114 (Figure 36, Table 49).

[0365] [Table 49]

[0366] Example 31. Evaluation of the C3 protein reduction effect of siRNA conjugates in cynomolgus monkeys. In this example, the expression of C3 protein in cynomolgus monkey serum was measured at different time points after single administration of the (CR01008)×3 carrier conjugates RZ002099, RZ002101, RZ002106, and RZ002113 using the ELISA method. Animal grouping, administration, and tissue sample collection: Healthy cynomolgus monkeys weighing 3-5 kg ​​were divided into groups of 4 monkeys each, based on serum C3 protein levels, with 2 males and 2 females per group. Each experimental group was administered a predetermined dose of a pharmaceutical conjugate, and a PBS control group was also established. For all animals, the dosage was calculated based on body weight and administered as a single subcutaneous injection in the abdomen. Each pharmaceutical conjugate was administered in the form of a 9 mg / mL PBS solution, with a dosage volume of 1 mL / kg (relative to the monkey's body weight), meaning the dosage of each pharmaceutical conjugate was 9 mg (calculated based on siRNA) / kg (relative to the monkey's body weight). The PBS control group was administered a PBS solution without siRNA conjugate at a dose of 1 mL / kg (relative to the mouse's body weight). The pre-dose (value before drug administration) and the day of administration were defined as day 0 (D0). On days 7 (D7), 14 (D14), 21 (D21), 28 (D28), 35 (D35), 42 (D42), 49 (D49), and 56 (D56) after administration, cynomolgus monkey serum was collected, and C3 protein expression was measured using a human complement C3 ELISA kit (Hycult Biotech, HK366--01).

[0367] According to the results of Example 31, when administered as a single dose of 9 mg / kg, RZ002099, RZ002101, RZ002106, and RZ002113 can all significantly reduce the level of C3 protein in cynomolgus monkey serum. The C3 protein reduction effect of RZ002106 reached over 90% (Figure 37, Table 50). [Table 50]

[0368] Example 32. Evaluation of the AGT protein-reducing effect of siRNA conjugates in cynomolgus monkeys. In this example, the expression of AGT protein in cynomolgus monkey serum was measured at different time points after a single administration of the (CR01008)×3 carrier conjugate RZ003069 using the ELISA method.

[0369] Animal grouping, administration, and tissue sample collection: Healthy cynomolgus monkeys weighing 3-5 kg ​​were divided into groups of 4 based on serum AGT protein levels, with 2 males and 2 females per group. Each experimental group was administered a predetermined dose of the pharmaceutical conjugate, and a PBS control group was also established. For all animals, the dosage was calculated based on body weight and administered as a single subcutaneous injection to the abdomen. Each pharmaceutical conjugate was administered in the form of a 3 mg / mL PBS solution, with a dosage volume of 1 mL / kg (relative to the monkey's body weight), i.e., the dosage of each pharmaceutical conjugate was 3 mg / kg (relative to the monkey's body weight). The PBS control group was administered a PBS solution without siRNA conjugate at a dose of 1 mL / kg. The day of administration was designated as day 0 (D0). Cynomolgus monkey serum was collected on day 7 (D7), day 14 (D14), and day 21 (D21) after administration, and AGT protein expression was measured using the Human Angiotensinogen Elisa Measurement Kit (Immuno-Biological, 27412).

[0370] According to the results of Example 32, a single dose of 3 mg / kg of RZ003069 can significantly reduce AGT protein levels in cynomolgus monkey serum (Figure 38, Table 51). [Table 51]

[0371] Through research and experimentation, the inventors have found that by making specific modifications to one or more sites on the sense strand and / or antisense strand of an oligonucleotide, the modified oligonucleotide can achieve good tolerability, and consequently, the activity of the oligonucleotide molecule can be further improved, making it highly promising for application in the development of RNA interference (RNAi) drugs. (1) According to this disclosure, the effect of TBDMS modification on the activity of an siRNA sequence is related to the modification site, and large TBDMS group modification at a specific site does not impair the activity of the siRNA and can even further improve its activity. (2) According to the experimental results of this disclosure, the effect of MOE modification on the activity of the siRNA sequence is related to the modification site, and it has been verified that MOE group modification at a specific site does not impair the activity of the siRNA, and that the mechanism of effect shows a high degree of similarity to that of TBDMS modification and TOM modification. When an oligonucleotide with a double-stranded region having a length of 19 nucleotides (nt) is used as an example, MOE modifications at one or more sites (positions 5, 12, 18, and 19) of the sense strand contribute to improved in vitro activity of the siRNA sequence compared to a control sequence without MOE modifications, and MOE modifications at one or more sites (positions 8, 10, and 15) of the antisense strand contribute to improved in vitro activity of the siRNA sequence compared to a control sequence without MOE modifications. (3) According to the present disclosure, the effect of MOE modification on the activity of an siRNA sequence depends on the modification site, and by applying MOE modification to one or more sites at specific sites on the sense strand and / or antisense strand, good tolerability can be obtained, the activity of the siRNA sequence can be well maintained, and ultimately the activity can be further improved. In this specification, terms such as “one embodiment,” “some embodiments,” “example,” “specific example,” “some embodiments,” or “some examples” mean that the specific features, structures, materials, or properties described using such embodiments or examples are included in at least one embodiment or example in this disclosure. In this specification, exemplary expressions for the above terms do not necessarily refer to the same embodiment or example. The specific features, structures, materials, or properties described can be appropriately combined in any one or more embodiments or examples. Furthermore, to the extent that they do not conflict, those skilled in the art can combine or combine different embodiments or examples, or features of different embodiments or examples, described herein. While examples have been described and explained in this disclosure, these examples are merely illustrative and do not limit the disclosure. Those skilled in the art can modify, change, substitute, and transform these examples within the scope of this disclosure.

[0372] Industrial applicability This disclosure provides modified double-stranded oligonucleotides and oligonucleotide conjugates. The double-stranded oligonucleotides and / or oligonucleotide conjugates according to this disclosure can be made tolerable by specific modifications at one or more sites on the sense strand and / or antisense strand, maintain good pharmacokinetic activity of the oligonucleotide, and exhibit relatively high regulatory activity of target gene expression.

Claims

1. It is a double-stranded oligonucleotide, It comprises a sense strand and an antisense strand, each strand having 17 to 35 nucleotides, each nucleotide being either a modified or unmodified nucleotide, the sense strand and antisense strand forming a double-stranded region in at least partially reverse complementarity, and the double-stranded region containing nucleotides having sterically bulky modifications and / or nucleotides having disubstituted modifications. The aforementioned double-stranded region is represented by the following formula (I): SS: 5'--(N) a'--(X) p'--(N) b'--(X) q'--(N) c'--(X) r'--(N) d'--3' AS: 3'--(N)a--(X)p--(N)b--(X)q--(N)c--5' (I), Here, SS represents the sense chain and AS represents the antisense chain. Each of the above X independently represents a nucleotide in which the hydroxyl group at the 2' position of ribose is substituted with a sterically bulky group, or a nucleotide in which the 2' position is substituted with two substituents. Nucleotides in which the hydroxyl group at the 2′ position of ribose is replaced with a sterically bulky group are independently classified as 2′-modified nucleotides (2′--(O)). ｍ１ (CH ２ ) ｎ (O) ｍ２ R １ Selected from, where m1 or m2 is independently 0 or 1, and n is an integer selected from 0 to 6. R １ is a substituted or unsubstituted C １ --C ６ alkyl group, or --Si(R ２ ) ３ selected from, and R ２ are each independently a substituted or unsubstituted C １ --C ６ alkyl group, a C １ --C ６ alkoxy group, the substitution including one or more substituents selected from halogen, a C １ --C ６ alkyl group, a C １ --C ６ alkoxy group, a hydroxy group, an amino group, a cycloalkyl group having 6 or less carbon atoms, an aryl group having 12 or less carbon atoms, or a heteroaryl group having 12 or less carbon atoms. Each substituent in the two substituents is independently C １ --C ６ Selected from alkyl groups or halogens, Each of the aforementioned N independently represents a modified nucleotide or an unmodified nucleotide. When N represents a modified nucleotide, the 2' position of its ribose is modified and N is not X. Each of the aforementioned N independently represents one or more selected from the group consisting of nucleotides modified with a 2'-O-alkyl group, a 2'-alkyl group, a 2'-substituted alkyl group, a 2'-halogen, a 2'-deoxy, or a nucleotide analog, where the alkyl group has 1 to 6 carbon atoms, and the nucleotide analog is one or more selected from ENA, BNA, LNA, GNA, or UNA. The terms a, a', p, p', b, b', q, q', c, c', r', and d' each independently represent the number of nucleotides, where a' is selected from an integer between 3 and 8, p' from an integer between 0 and 3, b' from an integer between 4 and 13, q' from an integer between 0 and 4, c' from an integer between 3 and 9, r' from an integer between 0 and 3, d' from an integer between 0 and 9, a is selected from an integer between 4 and 7, p is selected from an integer between 0 and 1, b is selected from an integer between 4 and 8, q is selected from an integer between 0 and 4, and c is selected from an integer between 6 and 10. p', q', r', p, and q cannot all be zero at the same time, and 0 ≤ q' + r' ≤ 4. A double-stranded oligonucleotide characterized by the following features.

2. Each of the aforementioned X is independently a modified nucleotide, 2′--O(CH ２ ) ｎ OR １ Or 2'--R ３ --2'--R ４ Selected from, n is 1 or 2, R １ C is either substituted or unsubstituted. １ --C ６ Alkyl alkyl group, or --Si(R ２ ) ３ Selected from, R ３ and R ４ Each is independent of C １ --C ６ Selected from alkyl groups or halogens The double-stranded oligonucleotide according to claim 1.

3. N is independently selected from 2′--OMe modified nucleotides, 2′--F modified nucleotides, 2′--H modified nucleotides, or unmodified nucleotides. Selectively, X can be 2'--O--methoxyethyl, 2'--O--TBDMS, 2'--O--TOM, or 2'--O--CH ２ --OR-- ５ A selection is made from a group consisting of modified nucleotides modified by each of the following: ５ C is either unsubstituted or substituted with substituents. １ --C ３ It is an alkyl group, and optionally, the substituents are independently halogens, C １ --C ３ Alkyl alkyl group, C １ --C ３ One or more selected from alkoxy groups and amino groups, Optionally, the halogen is fluorine. A double-stranded oligonucleotide according to claim 1 or 2, characterized by the above.

4. Along the direction from the 5' end to the 3' end, at least one of the nucleotides at position 8, 9, 10, or 15 of the antisense strand in formula (I) is a modified nucleotide X, and / or the sense strand may or may not have a modified nucleotide X. If modified nucleotide X is optionally present in the sense strand, at least one nucleotide X is located in a complementary site to any of the positions 2, 8, or 15 of the antisense strand. Selectively, the modified nucleotide X is located at at least one of the nucleotides at position 10 or 15 of the antisense strand. Selectively, X can be 2'--O--methoxyethyl, 2'--O--TBDMS, 2'--O--TOM, or 2'--O--CH ２ --O--CH ２ --CH ３ , 2'--O--CH ２ --O--CH ２ --CF ３ Selected from a group consisting of modified nucleotides modified by each of the following. A double-stranded oligonucleotide according to any one of claims 1 to 3.

5. q' + c' + r' + d' = 9 and satisfying 0 ≤ q' + r' ≤ 4, If p = 0, then a = 5 to 7. Optionally, when p=1, along the direction from the 5' end to the 3' end, X is located at position 15 of the antisense chain. If we choose to set q = 0, then b + c = 14. Optionally, along the direction from the 5' end to the 3' end, at least one X in (X)p' is located in the sense strand at a complementary position to the 15th position of the antisense strand. If p'=1 optionally, then along the direction from the 5' end to the 3' end, X in (X)p' is located in the complementary region to position 15 of the antisense chain. Selectively, along the direction from the 5' end to the 3' end, include at least two fluoromodified nucleotides among the first four nucleotides of (N)b' in the sense strand, Selectively, q' is between 0 and 2. Selectively, q' is 0 or 1, If we choose to have r' = 1 and q' = 1, then b' and c' are 5 and d' is 1. If p' = 0, then a' = 6, If p' = 0, then a' = 8, If, optionally, q' and r' are never simultaneously zero, then along the direction from the 5' end to the 3' end, at least one X in the sense strand is located in a complementary position to positions 1-8 of the antisense strand. d' = 1 to 9 at will. A double-stranded oligonucleotide according to any one of claims 1 to 4.

6. a' is selected from integers between 3 and 8, p' is selected from integers between 0 and 2, b' is selected from integers between 4 and 12, q' is selected from integers between 0 and 2, c' is selected from integers between 3 and 8, r' is selected from integers between 0 and 2, d' is selected from integers between 1 and 9, a is selected from integers between 4 and 7, p is selected from 0 or 1, b is selected from integers between 4 and 8, q is selected from integers between 0 and 2, and c is selected from integers between 6 and 10. Selectively, (N)a and (N)b each contain at least one fluoromodified nucleotide, and (N)c contains at least two fluoromodified nucleotides. Selectively satisfy q' + c' + r' + d' = 9 and 0 ≤ q' + r' ≤ 4, Selectively, the first four nucleotides of (N)b', counting from the 5'--end, include at least two fluoromodified nucleotides. A double-stranded oligonucleotide according to any one of claims 1 to 5.

7. a' is selected from integers between 3 and 8, p' is selected from 0 or 1, b' is selected from integers between 4 and 13, q' is selected from 0 or 1, c' is selected from integers between 3 and 9, r' is selected from 0 or 1, d' is selected from integers between 1 and 8, a is selected from integers between 4 and 7, p is 1, b is selected from integers between 4 and 8, q is selected from 0 or 1, and c is selected from integers between 6 and 10. Selectively, (N)a has at least one fluoromodified nucleotide, and the 16th position of the antisense chain, counting from the 5'-- end, is a fluoromodified nucleotide. Selectively, at least one fluoromodified nucleotide is present at (N)b, and the 14th position of the antisense chain, counting from the 5'-- end, is a fluoromodified nucleotide. Selectively, at least two fluoromodified nucleotides are present at (N)c, and at positions 2 and 6 of the antisense chain, counting from the 5'-- end, both are fluoromodified nucleotides. We can choose to have q' + c' + r' + d' = 9, and satisfy 0 ≤ q' + r' ≤ 2. Optionally, in the sense chain, there is at least one X located at a complementary position to the seed region of the antisense chain, and the seed region of the antisense chain is at positions 1 to 8, counting from the 5' end. Optionally, counting from the 5'-- end, the first four nucleotides of (N)b' are located in complementary sites to positions 10-13 of the antisense strand, and of the first four nucleotides, at least two fluoromodified nucleotides are included. A double-stranded oligonucleotide according to any one of claims 1 to 6.

8. 5' -- Counting from the end, X in equation (I) is located at any position among positions 8 to 10 or 15 of the antisense chain. Optionally, in the sense chain, X is located in a complementary position to any of the positions 2, 8, or 15 of the antisense chain. Optionally, counting from the 5'-end, X is located at position 15 of the antisense chain, and at least one fluoromodification is present at position 9 or 12 of the antisense chain, and / or the position of the sense chain at a complementary position to position 10 of the antisense chain is a fluoromodification. A double-stranded oligonucleotide according to any one of claims 1 to 7.

9. The double-stranded oligonucleotide further includes one or more overhang regions in addition to the double-stranded region, the length of which is 1 to 6 nucleotides. A double-stranded oligonucleotide according to any one of claims 1 to 8.

10. The double-stranded oligonucleotide including the overhang region is represented by the following formula (II): SS: 5'--(T) t1--(N) a'--(X) p'--(N) b'--(X) q'--(N) c'--(X) r'--(N) d'--(T) t2--3', AS: 3'--(T)t1--(N)a--(X)p--(N)b--(X)q--(N)c--(T)t2--5' (II), Here, T represents an overhanging nucleotide, t1 and t2 are independently selected from integers between 0 and 6 and cannot be both 0, and the definitions of the other substituents are the same as in formula (I). The double-stranded oligonucleotide according to feature 9.

11. Each of the overhang nucleotides T is independently selected from modified nucleotides or unmodified nucleotides, and the modified nucleotide is one or more selected from the group consisting of modified nucleotides modified with 2'-O-alkyl groups, 2'-alkyl groups, 2'-substituted alkyl groups, 2'-halogens, 2'-deoxy groups, unbasic nucleotides, or antisense oligonucleotides. The double-stranded oligonucleotide according to claim 10.

12. The antisense strand of the double-stranded oligonucleotide has one nucleotide overhang at its 3' end, and the overhang contains one to three nucleotides, and the molecule of the double-stranded oligonucleotide is represented by the following formula (IIa): SS: 5'--(N) a'--(X) p'--(N) b'--(X) q'--(N) c'--(X) r'--(N) d'--3', AS: 3'--(T)t1--(N)a--(X)p--(N)b--(X)q--(N)c--5' (IIa), Here, t1 is selected from integers between 1 and 3. A double-stranded oligonucleotide according to any one of claims 1 to 8.

13. The aforementioned overhang region further has phosphorothioeth modification. A double-stranded oligonucleotide according to any one of claims 9 to 12.

14. Each strand of the double-stranded oligonucleotide contains 17 to 35 nucleotides. Selectively, each strand contains 17 to 23 nucleotides. Selectively, each strand contains 17 to 21 nucleotides. Selectively, each strand contains 17 to 19 nucleotides. Selectively, each strand contains 19 to 25 nucleotides. Selectively, each strand contains 19 to 23 nucleotides. Selectively, each strand contains 19 to 21 nucleotides. Selectively, each strand contains 21 to 23 nucleotides. A double-stranded oligonucleotide according to any one of claims 1 to 13.

15. The aforementioned double-stranded oligonucleotide is Optionally, the sense strand contains at least one nucleotide X, and counting from the 5'-- end, at least one X is located in a complementary position to any one of the 15, 8, or 2 positions of the antisense strand. Optionally, the sense strand contains at least two fluoromodified nucleotides among the four nucleotides located at complementary positions to positions 10-13 of the antisense strand, counting from the 5'-- end. Selectively, counting from the 5'-- end, positions 2, 6, 14, and 16 of the antisense chain are all fluoromodified nucleotides. Selectively, counting from the 5'-- end, at least one of the 8th, 9th, 10th, and 15th positions of the antisense strand is nucleotide X. Optionally, counting from the 5'-- end, there is at least one X located in a complementary position to positions 1 to 8 of the antisense chain in the sense chain. Selectively, the 15th position of the antisense strand, counting from the 5' end, is nucleotide X. Having at least one of the characteristics A double-stranded oligonucleotide according to any one of claims 1 to 12.

16. The nucleotide X is independently 2′--O--MOE, 2′--O--TBDMS, 2′--O--TOM, 2′--O--CH ２ --O--CH ２ --CH ３ , 2'--O-- CH ２ --O--CH ２ --CF ３ A group consisting of modified nucleotides modified by each of the following is selected: Optionally, each sense and antisense strand contains at least one phosphorothioate group, and the phosphorothioate group is located in the terminal region of each single strand. The double-stranded oligonucleotide according to claim 15, characterized by its features.

17. Overhang (T) of the aforementioned AS chain ｔ１ This includes at least one nucleotide substituted with 2′--F--2′--Me. The double-stranded oligonucleotide according to claim 12.

18. The double-stranded oligonucleotide further comprises at least one ligand, The ligand may optionally include a liver-targeting ligand and a non-liver-targeting ligand. The ligand is optionally an ASGPR ligand. Optionally, the ASGPR ligand includes GalNAc or a derivative thereof bound via a branched linker. A double-stranded oligonucleotide according to claim 1 or 2, characterized by the above.

19. It is a double-stranded oligonucleotide, The oligonucleotide comprises a sense strand and an antisense strand, each strand having 17 to 35 nucleotides, each nucleotide being either a modified or unmodified nucleotide, the sense strand and antisense strand forming a double-stranded region in at least partially reverse complementarity, the double-stranded oligonucleotide containing at least one nucleotide having a 2'--disubstituted modification, and each substituent in the 2'--disubstituted is independently selected from a methyl group or fluorine. If the double-stranded region of the double-stranded oligonucleotide contains a nucleotide having a 2'--disubstituted modification, the nucleotide having the 2'--disubstituted modification is selected from nucleotides that are 2'--difluorosubstituted. Alternatively, if the double-stranded oligonucleotide contains a nucleotide having a 2'--disubstituted modification at a position other than the double-stranded region, the nucleotide having the 2'--disubstituted modification is selected from nucleotides that are substituted with 2'--F--2'--Me. A double-stranded oligonucleotide characterized by the following features.

20. (1) If the double-stranded oligonucleotide contains a 2'-difluorosubstituted nucleotide, the 2'-difluorosubstituted nucleotide is located at least one of the positions 7, 8, 9, or 10 counting from the 5' end of the sense strand. The 2′--difluorosubstituted nucleotide is optionally located at at least two or at least three positions among the 7th, 8th, 9th, or 10th positions, counting from the 5′ end of the sense strand. (2) If the double-stranded oligonucleotide includes a 2'-difluorosubstituted nucleotide, the 2'-difluorosubstituted nucleotide is located at least one of the positions 2, 4, 6, 8, 9, 10, 12, 14, or 16, counting from the 5' end of the antisense strand. The 2′--difluorosubstituted nucleotide is optionally located at at least two of the following positions, counting from the 5' end of the antisense strand: 2, 4, 6, 8, 9, 10, 12, 14, or 16. (3) If the double-stranded oligonucleotide contains a nucleotide substituted with 2'--F--2'--Me, the nucleotide substituted with 2'--F--2'--Me is located outside the double-stranded region of the oligonucleotide. (4) If the double-stranded oligonucleotide contains a nucleotide substituted with 2'--F--2'--Me, the nucleotide substituted with 2'--F--2'--Me is located in the overhang of the 3' end of the antisense strand. Having one or more of the following characteristics A double-stranded oligonucleotide according to any one of claims 1 to 19.

21. Oligonucleotide conjugates, The oligonucleotide conjugate comprises a double-stranded oligonucleotide according to any one of claims 1 to 20 and a binding group attached to the double-stranded oligonucleotide. Optionally, the binding group may include a pharmacodynamically acceptable target-directing group and / or delivery-auxiliary group. Oligonucleotide conjugates characterized by the following features.

22. A pharmaceutical composition, The pharmaceutical composition comprises a double-stranded oligonucleotide according to any one of claims 1 to 20 or an oligonucleotide conjugate according to claim 21. A pharmaceutical composition characterized by the following features.

23. Use of a double-stranded oligonucleotide according to any one of claims 1 to 20, an oligonucleotide conjugate according to claim 21, or a pharmaceutical composition according to claim 22 in the preparation of a pharmaceutical for the treatment and / or prevention of a disease or condition relating to mRNA levels of target gene expression.

24. The method involves administering to a subject at least one of the following: a double-stranded oligonucleotide according to any one of claims 1 to 20, an oligonucleotide conjugate according to claim 21, or a pharmaceutical composition according to claim 22. A method for treating and / or preventing a disease or condition related to the mRNA level of target gene expression.

25. A method for regulating the expression level of a target gene in a cell, The process involves contacting the cells with at least one of the following: an effective amount of a double-stranded oligonucleotide according to any one of claims 1 to 20, an oligonucleotide conjugate according to claim 21, or a pharmaceutical composition according to claim 22. A method for regulating the expression level of target genes in cells.

26. A pharmaceutical composition comprising at least one of the following: a double-stranded oligonucleotide according to any one of claims 1 to 20, an oligonucleotide conjugate according to claim 21, or a pharmaceutical composition according to claim 22. kit.

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