Double-stranded oligonucleotide and use thereof in inhibiting expression of angiotensinogen

Abstract

A double-stranded oligonucleotide and a use thereof in inhibiting the expression of angiotensinogen (AGT). The double-stranded oligonucleotide comprises a sense strand and an antisense strand; the antisense strand contains a nucleotide sequence comprising at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleotides of any sequence in SEQ ID NO: 225-SEQ ID NO: 448, or contains a nucleotide sequence which differs from the contiguous nucleotides by 1, 2, or 3 nucleotide(s).

Description

Double-stranded oligonucleotides and their application in inhibiting angiotensinogen expression Technical Field

[0001] The present disclosure relates to the field of biomedicine. Specifically, the present disclosure relates to double-stranded oligonucleotides and their use in inhibiting angiotensinogen expression. Background Art

[0002] Angiotensinogen (AGT), also known as SERPINA8 or ANHU, is a member of the serpin family and a component of the renin-angiotensin-aldosterone system (RAAS), which plays a key role in blood pressure regulation. The juxtaglomerular cells of the kidneys secrete renin into the circulation. It is primarily produced in the liver and released into the circulation, where renin converts it to angiotensin I. Angiotensin I is subsequently converted to angiotensin II by angiotensin-converting enzyme (ACE). Angiotensin II is a peptide hormone that causes vasoconstriction, which can in turn increase blood pressure. Angiotensin II also stimulates the secretion of the hormone aldosterone by the adrenal cortex. Aldosterone causes the kidneys to increase the reabsorption of sodium and water, leading to an increase in the body's fluid volume, which can in turn increase blood pressure. Overstimulation or activity of the RAAS pathway can lead to high blood pressure. Chronically high blood pressure is called hypertension. The high blood pressure in hypertensive patients requires the heart to work harder to circulate blood through the blood vessels.

[0003] The World Health Organization (WHO) has identified hypertension as a major cause of cardiovascular morbidity. Hypertension is a major risk factor for a variety of diseases, disorders, and conditions, such as shortened life expectancy, chronic kidney disease, stroke, myocardial infarction, heart failure, vascular aneurysms (e.g., aortic aneurysms), peripheral arterial disease, heart damage (e.g., cardiac enlargement or hypertrophy), and other cardiovascular-related diseases, disorders, and / or conditions.

[0004] Despite the large number of antihypertensive drugs available for treating hypertension, more than two-thirds of subjects cannot be controlled with a single antihypertensive drug and require two or more antihypertensive drugs from different drug classes. This further reduces the number of subjects with controlled blood pressure, as compliance and side effects increase with increasing medication use.

[0005] Therefore, alternative and combination therapies for subjects with angiotensinogen-related diseases remain to be investigated.

[0006] Summary of the Invention

[0007] The present disclosure aims to at least partially address one of the technical problems existing in the prior art. To this end, the present disclosure provides a double-stranded oligonucleotide that can induce silencing complex (RISC)-mediated cleavage of RNA transcripts of the angiotensinogen (AGT) gene, inhibiting AGT gene expression, thereby contributing to the treatment or prevention of diseases or conditions mediated by the angiotensinogen (AGT) gene.

[0008] In one aspect of the present disclosure, the present disclosure provides a double-stranded oligonucleotide. According to an embodiment of the present disclosure, the double-stranded oligonucleotide includes a sense strand and an antisense strand; the antisense strand comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides from any one of SEQ ID NO: 225 to SEQ ID NO: 448, or comprises a nucleotide sequence having 1, 2, or 3 nucleotide differences from the consecutive nucleotides; the sense strand comprises at least 17 nucleotides and the sense strand is complementary or substantially complementary to the antisense strand to form a duplex region; the substantial complementarity means that the mismatch between the sense strand and the antisense strand in the duplex region does not exceed 3 nucleotides; each nucleotide in the double-stranded oligonucleotide is independently selected from a modified or unmodified nucleotide.

[0009] The inventors obtained the above-mentioned suitable double-stranded oligonucleotide through experimental design, which inhibits the expression of AGT in cells and effectively prevents and / or treats diseases or conditions mediated by the angiotensinogen (AGT) gene.

[0010] According to an embodiment of the present disclosure, the above siRNA may also have at least one of the following additional technical features:

[0011] According to an embodiment of the present invention, nucleotides 2 to 19 of the antisense strand comprise a complementary region to an AGT RNA transcript in the direction from the 5' end to the 3' end; wherein the complementary region comprises a nucleotide sequence of at least 17 consecutive nucleotides with any one of SEQ ID NOs. 225-448, or comprises a nucleotide sequence having 1, 2 or 3 nucleotide differences with the consecutive nucleotides.

[0012] According to an embodiment of the present invention, the complementary region comprises any one of SEQ ID NOs. 225-448, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; the sense strand comprises a nucleotide sequence that is at least partially complementary or substantially complementary to the antisense strand to form a duplex region; and the substantial complementarity means that the mismatch between the sense strand and the antisense strand in the duplex region does not exceed 3 nucleotides.

[0013] According to an embodiment of the present disclosure, the antisense strand comprises a nucleotide sequence of at least 17, at least 18, at least 19, at least 20 or at least 21 consecutive nucleotides compared to any one of SEQ ID NO: 225 to SEQ ID NO: 448, or comprises a nucleotide sequence having 1, 2 or 3 nucleotide differences from the consecutive nucleotides.

[0014] According to an embodiment of the present disclosure, the antisense strand comprises a nucleotide sequence of at least 19, at least 20 or at least 21 consecutive nucleotides compared to any one of SEQ ID NO: 225 to SEQ ID NO: 448, or comprises a nucleotide sequence having 1, 2 or 3 nucleotide differences from the consecutive nucleotides.

[0015] According to an embodiment of the present disclosure, the antisense strand comprises a nucleotide sequence of at least 20 or at least 21 consecutive nucleotides from any one of SEQ ID NO: 225 to SEQ ID NO: 448, or comprises a nucleotide sequence having 1, 2 or 3 nucleotide differences from the consecutive nucleotides.

[0016] According to an embodiment of the present disclosure, the complementary region comprises any one of SEQ ID NOs. 225-448, or a nucleotide sequence having 1, 2 or 3 nucleotides therefrom.

[0017] According to an embodiment of the present disclosure, the antisense strand comprises a sequence having 0, 1, 2 or 3 nucleotide differences from any one of SEQ ID NO: 225-SEQ ID NO: 448.

[0018] According to an embodiment of the present disclosure, the antisense strand comprises a sequence having 0 or 1 nucleotide difference from any one of SEQ ID NO: 225-SEQ ID NO: 448.

[0019] According to an embodiment of the present disclosure, the antisense strand is selected from or comprises any one of SEQ ID NO: 225-SEQ ID NO: 448.

[0020] According to an embodiment of the present disclosure, the positive strand comprises a nucleotide sequence of at least 17, at least 18 or at least 19 consecutive nucleotides with any one of SEQ ID NO: 1 to SEQ ID NO: 224, or comprises a nucleotide sequence having 1, 2 or 3 nucleotide differences with the consecutive nucleotides.

[0021] According to an embodiment of the present disclosure, the sense strand comprises a sequence having 0, 1, 2 or 3 nucleotide differences from any one of SEQ ID NO: 1-SEQ ID NO: 224.

[0022] According to an embodiment of the present disclosure, the sense strand is selected from or includes any one of SEQ ID NO: 1 to SEQ ID NO: 224.

[0023] According to an embodiment of the present disclosure, the sense strand comprises one of the following nucleotide sequences, or has a difference of no more than 3 nucleotides therefrom:

[0024] SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 77, SEQ ID NO: 102, SEQ ID NO: 117, SEQ ID NO: 132, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 164, SEQ ID NO: 169, SEQ ID NO: 176, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 185, SEQ ID NO: 186 and SEQ ID NO: 189; and / or

[0025] The antisense strand comprises one of the following nucleotide sequences, or differs therefrom by no more than 3 nucleotides:

[0026] SEQ ID NO: 237, SEQ ID NO: 240, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 301, SEQ ID NO: 326, SEQ ID NO: 341, SEQ ID NO: 356, SEQ ID NO: 359, SEQ ID NO: 360, SEQ ID NO: 364, SEQ ID NO: 366, SEQ ID NO:388, SEQ ID NO:393, SEQ ID NO:400, SEQ ID NO:403, SEQ ID NO:405, SEQ ID NO:409, SEQ ID NO:410 and SEQ ID NO:413.

[0027] According to an embodiment of the present disclosure, the sense strand comprises one of the following nucleotide sequences, or has a difference of no more than 3 nucleotides therefrom: SEQ ID NO: 13, SEQ ID NO: 117, SEQ ID NO: 135, SEQ ID NO: 179, SEQ ID NO: 186 and SEQ ID NO: 189; and / or, the antisense strand comprises one of the following nucleotide sequences, or has a difference of no more than 3 nucleotides therefrom: SEQ ID NO: 237, SEQ ID NO: 341, SEQ ID NO: 359, SEQ ID NO: 403, SEQ ID NO: 410 and SEQ ID NO: 413.

[0028] According to an embodiment of the present disclosure, the double-stranded oligonucleotide includes one or more of the following groups:

[0029] a sense strand having the nucleotide sequence set forth in SEQ ID NO: 13, or a nucleotide sequence differing therefrom by 1, 2, or 3 nucleotides; and an antisense strand having the nucleotide sequence set forth in SEQ ID NO: 237, or a nucleotide sequence differing therefrom by 1, 2, or 3 nucleotides;

[0030] a sense strand having the nucleotide sequence set forth in SEQ ID NO: 117, or a nucleotide sequence differing therefrom by 1, 2, or 3 nucleotides; and an antisense strand having the nucleotide sequence set forth in SEQ ID NO: 341, or a nucleotide sequence differing therefrom by 1, 2, or 3 nucleotides;

[0031] a sense strand having the nucleotide sequence set forth in SEQ ID NO: 135, or a nucleotide sequence differing therefrom by 1, 2, or 3 nucleotides, and an antisense strand having the nucleotide sequence set forth in SEQ ID NO: 359, or a nucleotide sequence differing therefrom by 1, 2, or 3 nucleotides;

[0032] a sense strand having the nucleotide sequence set forth in SEQ ID NO: 179, or a nucleotide sequence differing therefrom by 1, 2, or 3 nucleotides, and an antisense strand having the nucleotide sequence set forth in SEQ ID NO: 403, or a nucleotide sequence differing therefrom by 1, 2, or 3 nucleotides;

[0033] a sense strand having the nucleotide sequence set forth in SEQ ID NO: 186, or a nucleotide sequence differing therefrom by 1, 2, or 3 nucleotides, and an antisense strand having the nucleotide sequence set forth in SEQ ID NO: 410, or a nucleotide sequence differing therefrom by 1, 2, or 3 nucleotides;

[0034] The sense strand has the nucleotide sequence shown in SEQ ID NO: 189, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand has the nucleotide sequence shown in SEQ ID NO: 413, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom.

[0035] According to an embodiment of the present disclosure, the double-stranded oligonucleotide includes one or more of the following groups:

[0036] a sense strand having the nucleotide sequence set forth in SEQ ID NO: 13, and an antisense strand having the nucleotide sequence set forth in SEQ ID NO: 237;

[0037] a sense strand having the nucleotide sequence set forth in SEQ ID NO: 117, and an antisense strand having the nucleotide sequence set forth in SEQ ID NO: 341;

[0038] a sense strand having the nucleotide sequence set forth in SEQ ID NO: 135, and an antisense strand having the nucleotide sequence set forth in SEQ ID NO: 359;

[0039] a sense strand having the nucleotide sequence set forth in SEQ ID NO: 179, and an antisense strand having the nucleotide sequence set forth in SEQ ID NO: 403;

[0040] a sense strand having the nucleotide sequence set forth in SEQ ID NO: 186, and an antisense strand having the nucleotide sequence set forth in SEQ ID NO: 410;

[0041] The sense strand has the nucleotide sequence shown in SEQ ID NO: 189, and the antisense strand has the nucleotide sequence shown in SEQ ID NO: 413.

[0042] According to an embodiment of the present disclosure, the length of the sense strand does not exceed 21 nucleotides, and the length of the antisense strand does not exceed 23 nucleotides.

[0043] According to an embodiment of the present disclosure, the length of the sense strand is 19 nucleotides, and the length of the antisense strand is 21 nucleotides.

[0044] According to an embodiment of the present disclosure, the sense strand and / or antisense strand comprises a 3' overhang and / or a 5' overhang having at least 1 or at least 2 nucleotides.

[0045] According to an embodiment of the present disclosure, the antisense strand comprises a 3' overhang having at least 1 or at least 2 nucleotides.

[0046] According to an embodiment of the present disclosure, the antisense strand comprises a 3' overhang having 2 nucleotides.

[0047] According to an embodiment of the present disclosure, the sense strand and / or the antisense strand independently comprises one or more phosphorothioate bonds.

[0048] According to an embodiment of the present disclosure, the sense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 5' end.

[0049] According to an embodiment of the present disclosure, the antisense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 3' end and the 5' end, respectively.

[0050] According to an embodiment of the present disclosure, substantially all nucleotides in the double-stranded oligonucleotide are independently selected from modified nucleotides.

[0051] According to an embodiment of the present disclosure, all nucleotides in the double-stranded oligonucleotide are independently selected from modified nucleotides.

[0052] According to an embodiment of the present disclosure, each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides:

[0053] 2'-halogenated modified nucleotides, 2'-deoxy modified nucleotides, 2'-O- optionally substituted C 1-6 Alkyl-modified nucleotides, 2'-O-(CH2) n -O-R1 modified nucleotides, 2'-O-Si(R2)3 modified nucleotides, 2'-amino modified nucleotides, abasic nucleotides, or quasi-nucleotides; the quasi-nucleotides are selected from one or more of peptide nucleic acid (PNA), morpholino (MNA), bridged nucleic acid (BNA), locked nucleic acid (LNA), glycol nucleic acid / glycerol nucleic acid (GNA), threose nucleic acid (TNA), and unlocked nucleic acid (UNA);

[0054] wherein n is selected from 1 or 2, R1 is selected from optionally substituted C 1-6 Alkyl or optionally substituted C 1-6 alkoxy;

[0055] Each R2 is independently selected from optionally substituted C 1-6 alkyl.

[0056] The optional substitution means that one or more hydrogen atoms on the alkyl group are replaced by a substituent, and the substituent is independently selected from C 1-3 Alkyl, C 1-3 Alkoxy or halogen.

[0057] The structural formula of nucleotides is Wherein, Base represents a nucleoside base, and the nucleoside base on each nucleotide is independently selected from uracil U, thymine T, cytosine C, adenine A or guanine G.

[0058] "2'-deoxy-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group is replaced by a hydrogen atom.

[0059] "2'-halogenated nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribose group of the nucleotide is replaced by a halogen atom. For example, a 2'-fluoro-modified nucleotide.

[0060] According to an embodiment of the present disclosure, the 2'-halogenated modified nucleotides are selected from 2'-fluoro-modified nucleotides.

[0061] "2'-O-optionally substituted C 1-6 "Alkyl modified nucleotides" refer to nucleotides in which the hydrogen atom at the 2' hydroxyl group of the ribose group is optionally substituted with a C 1-6 The nucleotides formed by alkyl substitution are exemplified by 2'-O-methyl (also known as 2'-methoxy) modified nucleotides.

[0062] According to an embodiment of the present disclosure, the 2'-O-optionally substituted C 1-6 The alkyl-modified nucleotides are selected from 2'-O-methyl-modified nucleotides.

[0063] "2'-O-(CH2) n -R1 modified nucleotides refer to nucleotides in which the hydrogen atom on the 2' hydroxyl group of the ribose group is replaced by -(CH2) n -R1 replaces the nucleotide formed; illustratively, when n is selected from 1, 2'-O-(CH2) n -R1 can be selected from 2'-O-CH2-O-CH2-CH3 (2'-O-ethoxymethyl) modified nucleotides or 2'-O-CH2-O-CH2-CF3 (also known as 2'-O-(2,2,2-trifluoroethoxymethyl)) modified nucleotides; when n is selected from 2, 2'-O-(CH2) n -R1 can be selected from 2'-O-CH2-CH2-O-CH3 (also known as 2'-O-methoxyethyl, 2'-O-MOE) modified nucleotides. Exemplarily, the 2'-O-(CH2) nThe -O-R1 modified nucleotide is selected from 2'-O-methoxyethyl modified nucleotides.

[0064] According to an embodiment of the present disclosure, the 2'-O-(CH2) n The -O-R1 modified nucleotide is selected from a 2'-O-methoxyethyl modified nucleotide, a 2'-O-ethoxymethyl modified nucleotide or a 2'-O-2,2,2-trifluoroethoxymethyl modified nucleotide.

[0065] According to an embodiment of the present disclosure, the 2'-O-(CH2) n The -O-R1 modified nucleotide is selected from 2'-O-methoxyethyl modified nucleotides.

[0066] "2'-O-Si(R2)3 modified nucleotide" refers to a nucleotide in which the hydrogen atom on the 2' hydroxyl group of the ribose group of the nucleotide is replaced by -Si(R2)3. For example, 2'-O-TBDMS modified nucleotide, 2'-O-TIPS modified nucleotide or 2'-O-TOM modified nucleotide; wherein the structural formula of TBDMS is The structural formula of TIPS is The structural formula of TOM is

[0067] According to an embodiment of the present disclosure, the 2'-O-Si(R2)3 modified nucleotide is selected from a 2'-O-TBDMS modified nucleotide, a 2'-O-TIPS modified nucleotide or a 2'-O-TOM modified nucleotide.

[0068] According to an embodiment of the present disclosure, each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides:

[0069] 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides or 2'-O-ethoxymethyl modified nucleotides.

[0070] According to an embodiment of the present disclosure, each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides:

[0071] 2'-Fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides.

[0072] According to an embodiment of the present disclosure, the sense strand and / or the antisense strand contains at least one 2'-O-methoxyethyl-modified nucleotide or 2'-O-ethoxymethyl-modified nucleotide.

[0073] According to an embodiment of the present disclosure, the sense strand and / or the antisense strand contains at least one 2'-O-methoxyethyl-modified nucleotide.

[0074] According to an embodiment of the present disclosure, the duplex region has a structure as shown in the following formula (101):

[0075] SS: 5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3'

[0076] AS: 3'-(N)a-(X)p-(N)b-(X)q-(N)c-5' formula (101),

[0077] Among them, SS represents the sense strand and AS represents the antisense strand;

[0078] Each of the Ns is independently selected from the following modified nucleotides: 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides or 2'-deoxy modified nucleotides;

[0079] Each of the Xs is independently selected from 2'-O-(CH2) n -O-R1 modified nucleotides or 2'-O-Si(R2)3 modified nucleotides; wherein n is selected from 1 or 2, R1 is selected from optionally substituted C 1-6 Alkyl; each R2 is independently selected from optionally substituted C 1-6 Alkyl; the optional substitution refers to one or more hydrogen atoms on the alkyl or alkoxy group being replaced by a substituent, the substituent being independently selected from C 1-3 Alkyl, C 1-3 Alkoxy or halogen;

[0080] a, a', p, p', b, b', q, q', c, c', r', and d' independently represent the number of nucleotides; wherein a' is selected from an integer of 3-8, p' is selected from an integer of 0-3, b' is selected from an integer of 4-13, q' is selected from an integer of 0-4, c' is selected from an integer of 3-9, r' is selected from an integer of 0-3, d' is selected from an integer of 0-9, a is selected from an integer of 4-7, p is selected from an integer of 0-1, b is selected from an integer of 4-8, q is selected from an integer of 0-4, and c is selected from an integer of 6-10; and p', q', r', p, and q are not all 0 at the same time, and 0≤q'+r'≤4.

[0081] According to an embodiment of the present disclosure, a' is selected from an integer of 3-8, p' is selected from 0 or 1, b' is selected from an integer of 4-13, q' is selected from 0 or 1, and c' is selected from an integer of 3-9; r' is selected from 0 or 1, d' is selected from an integer of 1-8, a is selected from an integer of 4-7, p is selected from 1, b is selected from an integer of 4-8, q is selected from 0 or 1, and c is selected from an integer of 6-10.

[0082] According to an embodiment of the present disclosure, each N is independently selected from a 2'-fluoro-modified nucleotide or a 2'-O-methyl-modified nucleotide.

[0083] According to an embodiment of the present disclosure, each X is independently selected from 2'-O-(CH2) n -O-R1 modified nucleotides.

[0084] According to an embodiment of the present disclosure, each X is independently selected from a 2'-O-methoxyethyl modified nucleotide or a 2'-O-ethoxymethyl modified nucleotide.

[0085] According to an embodiment of the present disclosure, the duplex region comprises at least one 2'-O-methoxyethyl modified nucleotide or 2'-O-ethoxymethyl modified nucleotide.

[0086] According to an embodiment of the present disclosure, the sense strand is 17-21 nucleotides long, and the antisense strand is 19-23 nucleotides long.

[0087] According to an embodiment of the present disclosure, the sense strand is 19 nucleotides long, and the antisense strand is 21 nucleotides long.

[0088] According to an embodiment of the present disclosure, in the direction from the 5' end to the 3' end, at least 3 nucleotides among the nucleotides at positions 7-10 of the nucleotide sequence in the positive strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides.

[0089] According to an embodiment of the present disclosure, in the direction from the 5' end to the 3' end, at least 3 nucleotides in positions 7-10 of the nucleotide sequence in the positive strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides.

[0090] According to an embodiment of the present disclosure, in the direction from the 5' end to the 3' end, at least three nucleotides at positions 7 to 10 of the nucleotide sequence in the positive strand are selected from 2'-fluoro-modified nucleotides, the nucleotides at positions 12 and / or 18 are selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides.

[0091] According to an embodiment of the present disclosure, in the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the positive strand are selected from 2'-fluoro-modified nucleotides, the nucleotides at positions 12 and / or 18 are selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides.

[0092] According to an embodiment of the present disclosure, at least four nucleotides among the nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense chain are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides.

[0093] According to an embodiment of the present disclosure, at least four nucleotides among the nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at positions 2, 6, 14 and 16 are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides.

[0094] According to an embodiment of the present disclosure, at least four nucleotides among the nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at positions 2, 6, 14 and 16 are selected from 2'-fluoro-modified nucleotides, the nucleotides at positions 8 and / or 15 are selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0095] According to an embodiment of the present disclosure, in the direction from the 5' end to the 3' end, at least three nucleotides among the nucleotides at positions 7-10 of the nucleotide sequence in the sense chain are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides; at least four nucleotides among the nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense chain are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides.

[0096] According to an embodiment of the present disclosure, each nucleotide of the double-stranded oligonucleotide is independently selected from modified nucleotides, wherein the modifications of the sense strand and the antisense strand are selected from any one of the following (1)-(15):

[0097] (1) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0098] (2) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 10, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0099] (3) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 11, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (4) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 12, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0100] (5) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0101] (6) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 10, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0102] (7) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0103] (8) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 12, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0104] (9) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 12 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0105] (10) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 12 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 10, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0106] (11) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 12 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0107] (12) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 18 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 10, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0108] (13) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 18 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0109] (14) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 18 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 10, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 8 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0110] (15) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, the nucleotides at positions 12 and 18 are selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 10, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0111] According to an embodiment of the present disclosure, the double-stranded oligonucleotide includes one of the following groups 1-45:

[0112] Among them, C, G, U, A, and T represent cytidine-3'-phosphate, guanosine-3'-phosphate, uridine-3'-phosphate, adenosine-3'-phosphate, and thymidine-3'-phosphate, respectively; m indicates that the nucleotide adjacent to the left of the letter m is a 2'-O-methyl-modified nucleotide; f indicates that the nucleotide adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide; (moe) indicates that the nucleotide adjacent to the left of the combination mark (moe) is a 2'-O-methoxyethyl-modified nucleotide; and s indicates that the two nucleotides before and after the letter s are connected by a phosphorothioate bond.

[0113] According to an embodiment of the present disclosure, all nucleotides in the double-stranded oligonucleotide are independently selected from modified nucleotides; and each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides:

[0114] 2'-halogenated modified nucleotides, 2'-O-optionally substituted C 1-6 Alkyl-modified nucleotides, 2'-O-(CH2) n -O-R1 modified nucleotides, [2'-W1-2'-W2] modified nucleotides;

[0115] wherein n is selected from 1 or 2, R1 is selected from optionally substituted C 1-6 Alkyl or optionally substituted C 1-6 alkoxy;

[0116] Each R2 is independently selected from optionally substituted C 1-6 alkyl;

[0117] The optional substitution means that one or more hydrogen atoms on the alkyl group are replaced by a substituent, and the substituent is independently selected from C 1-3 Alkyl, C 1-3 Alkoxy or halogen;

[0118] The structural formula of the [2'-W1-2'-W2] modified nucleotide is W1 and W2 are each independently selected from halogen or C 1-6 Alkyl, Base represents uracil U, thymine T, cytosine C, adenine A or guanine G.

[0119] According to an embodiment of the present disclosure, the 2'-halogenated modified nucleotides are selected from 2'-fluoro-modified nucleotides.

[0120] According to an embodiment of the present disclosure, the 2'-O-optionally substituted C 1-6 The alkyl-modified nucleotides are selected from 2'-O-methyl-modified nucleotides.

[0121] According to an embodiment of the present disclosure, the 2'-O-(CH2) n The -O-R1 modified nucleotide is selected from 2'-O-methoxyethyl modified nucleotides.

[0122] According to an embodiment of the present disclosure, the [2'-W1-2'-W2] modified nucleotide is selected from [2'-F-2'-CH3] modified nucleotide.

[0123] According to an embodiment of the present disclosure, each nucleotide of the double-stranded oligonucleotide is independently selected from modified nucleotides, and each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides:

[0124] 2'-Fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides or [2'-F-2'-CH3]-modified nucleotides.

[0125] According to an embodiment of the present disclosure, the sense strand is 19 nucleotides long, and the antisense strand is 21 nucleotides long; and the modifications of the sense strand and the antisense strand are selected from any one of the following A1-A2:

[0126] A1. From the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0127] The nucleotides at positions 2, 6, 9, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotides at positions 20-21 are selected from [2'-F-2'-CH3]-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0128] A2. From the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0129] The nucleotides at positions 2, 6, 9, 14 and 16 of the nucleotide sequence in the antisense chain are selected from 2'-fluoro modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl modified nucleotides, the nucleotides at positions 20-21 are selected from [2'-F-2'-CH3] modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides.

[0130] According to an embodiment of the present disclosure, the sense strand is 19 nucleotides long, and the antisense strand is 20 nucleotides long; and the modifications of the sense strand and the antisense strand are selected from the following:

[0131] In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 19 is selected from [2'-F-2'-CH3]-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0132] The nucleotides at positions 2, 6, 9, 14 and 16 of the nucleotide sequence in the antisense chain are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, the nucleotide at position 20 is selected from [2'-F-2'-CH3]-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0133] According to an embodiment of the present disclosure, the double-stranded oligonucleotide includes one of the following groups 46-50:

[0134] Wherein, (NM054) represents a nucleotide, and its structural formula is When (NM054) is located at the 3' end of the sense strand or antisense strand, its structural formula is

[0135] According to an embodiment of the present disclosure, the double-stranded oligonucleotide is selected from siRNA.

[0136] In another aspect of the present invention, the present disclosure provides a conjugate. According to an embodiment of the present disclosure, the conjugate comprises: the double-stranded oligonucleotide described above and one or more ligands capable of binding to a cell receptor; the ligand is conjugated to the double-stranded oligonucleotide.

[0137] According to an embodiment of the present disclosure, the ligand specifically targets liver tissue.

[0138] According to an embodiment of the present disclosure, the ligand is selected from ligands having affinity for asialoglycoprotein receptors.

[0139] According to an embodiment of the present disclosure, the ligand comprises galactose or a derivative thereof.

[0140] According to an embodiment of the present disclosure, the derivative of galactose is selected from N-acetylgalactosamine.

[0141] According to an embodiment of the present disclosure, the ligand is conjugated to at least one of the 5' end of the sense strand, the middle position of the sense strand, the 3' end of the sense strand, the 5' end of the antisense strand, the middle position of the antisense strand and the 3' end of the antisense strand.

[0142] According to an embodiment of the present disclosure, the number of the ligands is selected from one, and one ligand is conjugated to the 3' end of the sense strand.

[0143] According to an embodiment of the present disclosure, the number of the ligands is selected from two, and the two ligands are conjugated to the 5' end of the sense strand and the 3' end of the antisense strand, respectively.

[0144] According to an embodiment of the present disclosure, the ligand is selected from the structure shown in formula (201) or a pharmaceutically acceptable salt or stereoisomer thereof:

[0145] Wherein, * represents the conjugation site between the ligand and the double-stranded oligonucleotide;

[0146] j is selected from 1, 2, 3 or 4;

[0147] Each Z is independently selected from hydroxyl or thiol;

[0148] Each h is independently selected from 1, 2 or 3;

[0149] Each i is independently selected from 1, 2 or 3;

[0150] Each R3 is independently selected from H, optionally substituted C 1-6 Alkyl or optionally substituted C1-6 alkoxy;

[0151] Each L is independently selected from optionally substituted C 2-20 Alkylene or R La and R La Independently selected from optionally substituted C 1-10 Alkylene, k is selected from 1, 2, 3, 4 or 5;

[0152] Each Y is independently selected from O, S or NH.

[0153] According to an embodiment of the present disclosure, j is selected from 2, 3 or 4.

[0154] According to an embodiment of the present disclosure, j is selected from 2.

[0155] According to an embodiment of the present disclosure, j is selected from 3.

[0156] According to an embodiment of the present disclosure, j is selected from 4.

[0157] According to an embodiment of the present disclosure, Z is selected from hydroxyl.

[0158] According to an embodiment of the present disclosure, h is selected from 1.

[0159] According to an embodiment of the present disclosure, i is selected from 1.

[0160] According to an embodiment of the present disclosure, R3 is selected from H.

[0161] According to an embodiment of the present disclosure, each L is independently selected from C 1-10 Alkylene or Among them, R La and R La Independently selected from C 1-5 Alkylene, k is 1, 2 or 3.

[0162] According to an embodiment of the present disclosure, k is selected from 1.

[0163] According to an embodiment of the present disclosure, each L is independently selected from

[0164] According to an embodiment of the present disclosure, Y is selected from O.

[0165] According to an embodiment of the present disclosure, the ligand is selected from the structure shown in formula (202) or a pharmaceutically acceptable salt or a stereoisomer thereof:

[0166] According to an embodiment of the present disclosure, the ligand is selected from any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof:

[0167] According to an embodiment of the present disclosure, the ligand is selected from the following structures or pharmaceutically acceptable salts or stereoisomers thereof:

[0168] According to an embodiment of the present disclosure, the ligand is selected from the structure shown in formula (L96) or a pharmaceutically acceptable salt thereof:

[0169] In another aspect of the present disclosure, the present disclosure provides the use of any of the following in the preparation of a medicament for alleviating, treating and / or preventing an AGT-mediated disease or condition:

[0170] (I) the double-stranded oligonucleotide described above; and / or

[0171] (II) The conjugate described above.

[0172] As can be seen above, the aforementioned double-stranded oligonucleotides and conjugates can degrade AGT mRNA and inhibit AGT expression. Therefore, the pharmaceutical composition disclosed herein can effectively prevent and / or treat diseases or conditions mediated by the angiotensinogen (AGT) gene.

[0173] According to an embodiment of the present disclosure, the diseases or conditions include hypertension, critical hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive emergency state, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm and ventricular fibrosis.

[0174] In yet another aspect of the present disclosure, the present disclosure provides a pharmaceutical composition.

[0175] According to an embodiment of the present disclosure, the pharmaceutical composition includes any of the following items and pharmaceutically acceptable excipients or adjuvants:

[0176] (I) the double-stranded oligonucleotide described above; and / or

[0177] (II) The conjugate described above.

[0178] As can be seen above, the aforementioned double-stranded oligonucleotides and conjugates can degrade AGT mRNA and inhibit AGT expression. Therefore, the pharmaceutical composition disclosed herein can effectively prevent and / or treat diseases or conditions mediated by the angiotensinogen (AGT) gene.

[0179] According to an embodiment of the present disclosure, the pharmaceutical composition further includes: a pharmaceutically acceptable carrier or excipient.

[0180] In yet another aspect of the present disclosure, the present disclosure provides a kit.

[0181] According to an embodiment of the present disclosure, the kit includes any of the following items:

[0182] (I) the double-stranded oligonucleotide described above; and / or

[0183] (II) the conjugate described above; and / or

[0184] (III) The pharmaceutical composition described above.

[0185] The kit disclosed herein can effectively degrade AGT mRNA and inhibit AGT expression.

[0186] In yet another aspect of the present disclosure, the present disclosure provides a method for inhibiting the expression of an AGT gene in a cell.

[0187] According to an embodiment of the present disclosure, the method comprises administering to a subject the aforementioned double-stranded oligonucleotide, and / or the aforementioned conjugate, and / or the aforementioned pharmaceutical composition. The method disclosed herein can be used to inhibit angiotensinogen expression. For example, angiotensinogen expression can be inhibited in vivo in an animal; or for non-disease therapeutic purposes, such as in vitro inhibition of angiotensinogen expression for subsequent research.

[0188] In yet another aspect of the present disclosure, a method for alleviating, treating, and / or preventing AGT-mediated diseases or conditions is provided. According to an embodiment of the present disclosure, the method comprises administering the aforementioned double-stranded oligonucleotide, and / or the aforementioned conjugate, and / or the aforementioned pharmaceutical composition to a subject. As previously noted, the aforementioned double-stranded oligonucleotide and conjugate can degrade AGT mRNA and inhibit AGT expression, thereby facilitating AGT-related scientific research and clinical treatment and / or prevention of diseases or conditions mediated by the angiotensinogen gene.

[0189] According to an embodiment of the present disclosure, the AGT-mediated disease or disorder includes a disease associated with the mRNA level of AGT gene expression.

[0190] According to an embodiment of the present disclosure, the diseases or conditions include hypertension, critical hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive emergency state, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm and ventricular fibrosis.

[0191] Additional aspects and advantages of the present disclosure will be set forth in part in the following description and, in part, will be obvious from the following description, or may be learned through practice of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0192] The above and / or additional aspects and advantages of the present disclosure will become apparent and readily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0193] FIG1 shows the inhibitory activity of target genes in Balb / c-HDI mice after administration of the siRNA conjugates described in this example;

[0194] Figure 2 shows the changes in blood pressure in hREN×hAGT hypertensive mice after administration of the siRNA conjugates described in this example, (A) changes in systolic blood pressure; (B) changes in mean arterial pressure; (C) changes in diastolic blood pressure;

[0195] FIG3 shows the changes in AGT protein levels in hREN×hAGT hypertensive mice after administration of the siRNA conjugates described in this example;

[0196] Figure 4 shows typical histopathological images of each experimental group under HE staining at 200 times magnification;

[0197] FIG5 shows the changes in systolic blood pressure in hREN×hAGT hypertensive mice after administration of the siRNA conjugates described in this example;

[0198] FIG6 shows the changes in AGT protein levels in hREN×hAGT hypertensive mice after administration of the siRNA conjugates described in this example;

[0199] Figure 7 shows the changes in blood pressure in SHR rats after administration of the siRNA conjugates described in this example; (A) changes in systolic blood pressure; (B) changes in mean arterial pressure; and (C) changes in diastolic blood pressure. DETAILED DESCRIPTION

[0200] The embodiments of the present disclosure are described in detail below. The embodiments described below are exemplary and are only used to explain the present disclosure, and should not be understood as limiting the present disclosure.

[0201] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of the features. Furthermore, in the description of this disclosure, unless otherwise specified, "plurality" means two or more.

[0202] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present disclosure, but not excluding other contents.

[0203] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0204] As used herein, the term "small interfering RNA (siRNA)" refers to a double-stranded RNA of 17 to 25 nucleotides in length, comprising a sense strand and an antisense strand. siRNA mediates the targeted cleavage of RNA transcripts in the RISC pathway by forming an RNA-induced silencing complex (RISC). Specifically, siRNA directs the specific degradation of mRNA sequences through the known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and protein. For example, siRNA can regulate (e.g., inhibit) the expression of angiotensinogen (AGT) in cells.

[0205] As used herein, the term "antisense strand (or guide strand)" includes a region that is substantially complementary to a target sequence, such as the mRNA of AGT. The "follower strand" refers to an iRNA strand that is substantially complementary to the antisense strand. The term "substantially complementary" refers to complete complementarity or at least partial complementarity, for example, the antisense strand is completely complementary to the target sequence or at least partially complementary. In the case of partial complementarity, mismatches can exist within the interior or terminal regions of the molecule, wherein the most tolerated mismatches exist within the terminal regions, for example, within 5, 4, 3 or 2 nucleotides at the 5'- and / or 3'-end of the iRNA.

[0206] It should be noted that "at least a portion of the antisense strand is substantially complementary to an mRNA" means that the antisense strand comprises a polynucleotide that is substantially complementary to a continuous portion of an mRNA of interest (e.g., an mRNA encoding angiotensinogen). Alternatively, if a polynucleotide is substantially non-interruptedly complementary to a portion of an mRNA encoding angiotensinogen, then the antisense strand is complementary to at least a portion of an mRNA encoding angiotensinogen.

[0207] As used herein, the term "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of an angiotensinogen gene, including mRNAs that are RNA processing products of the primary transcript. The angiotensinogen gene can be within a cell, for example, a cell in a subject.

[0208] As used herein, the term "subject" refers to any animal being examined, studied, or treated, and is not intended to limit the present disclosure to any particular type of subject. In some embodiments of the present disclosure, humans are preferred subjects, while in other embodiments, non-human animals are preferred subjects, including but not limited to mice, monkeys, ferrets, cows, sheep, goats, pigs, chickens, turkeys, dogs, cats, horses, and reptiles.

[0209] As used herein, the term "inhibiting the expression of angiotensinogen gene" includes any level of inhibition of angiotensinogen (abbreviated as AGT) gene, for example, at least partial inhibition of angiotensinogen gene expression, such as inhibition of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. The expression of angiotensinogen gene can be assessed based on the level of any variable associated with angiotensinogen gene expression, for example, angiotensinogen mRNA level or protein level. Inhibition can be evaluated by the reduction of the absolute or relative level of one or more of these variables compared to a control level. A control level can be any type of control level utilized in this area, for example, a baseline level before administration, or a level determined from a similar subject, cell or sample that has never been treated or treated with a control (e.g., a control or no active agent control using only a buffer).

[0210] Double-stranded oligonucleotides

[0211] In one aspect of the present disclosure, the present disclosure provides a double-stranded oligonucleotide. According to an embodiment of the present disclosure, the double-stranded oligonucleotide includes a sense strand and an antisense strand; the antisense strand comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides from any one of SEQ ID NO: 225 to SEQ ID NO: 448, or comprises a nucleotide sequence having 1, 2, or 3 nucleotide differences from the consecutive nucleotides; the sense strand comprises at least 17 nucleotides and the sense strand is complementary or substantially complementary to the antisense strand to form a duplex region; the substantial complementarity means that the mismatch between the sense strand and the antisense strand in the duplex region does not exceed 3 nucleotides; each nucleotide in the double-stranded oligonucleotide is independently selected from a modified or unmodified nucleotide.

[0212] In this article, the terms "nucleotide difference" and "nucleotide base difference" are used interchangeably. A "nucleotide difference" exists between a nucleotide sequence and another nucleic acid sequence, which means that the base type of the nucleotide at the same position in the former is changed compared to the latter. For example, when a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G or T, it is determined that there is a nucleic acid difference at that position between the two nucleic acid sequences. In some embodiments, when a nucleotide at the original position is replaced by an abasic nucleotide or its equivalent, it can also be considered that a nucleic acid difference has occurred at that position.

[0213] The inventors obtained the above-mentioned suitable double-stranded oligonucleotide through experimental design, which can inhibit the expression of AGT in cells and effectively prevent and / or treat diseases or conditions mediated by the angiotensinogen (AGT) gene.

[0214] The gene sequence of AGT is shown below, wherein the mRNA transcribed from the sequence marked with the underline serves as the target sequence of the above-mentioned double-stranded oligonucleotide, which can induce the silencing complex (RISC)-mediated cleavage of the RNA transcript of the angiotensinogen (AGT) gene, thereby inhibiting the expression of the AGT gene.

[0215] As used herein, the term "substantially complementary" means that the sense and antisense strands have no more than 3 nucleotides, no more than 2 nucleotides, or no more than 1 nucleotide mismatch in the duplex region, such as 3 nucleotides, 2 nucleotides, 1 nucleotide, or 0 nucleotides mismatch.

[0216] As used herein, a "nucleotide difference" between one nucleotide sequence and another nucleotide sequence refers to a change in the base type of the nucleotide at the same position in the former compared to the latter. For example, if a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G, or T, the two nucleotide sequences are considered to have a nucleotide difference at that position. In some embodiments, when a nucleotide at the original position is replaced by an abasic nucleotide or its equivalent, a nucleotide difference at that position can also be considered.

[0217] According to an embodiment of the present disclosure, nucleotides 2-19 of the antisense strand, in the 5' to 3' direction, comprise a region of complementarity with an AGT RNA transcript; wherein the region of complementarity comprises a nucleotide sequence of at least 17 consecutive nucleotides from any one of SEQ ID NOs. 225-448, or comprises a nucleotide sequence that differs from the consecutive nucleotides by 1, 2, or 3 nucleotides. In some embodiments, the region of complementarity comprises any one of SEQ ID NOs. 225-448, or comprises a nucleotide sequence that differs from the consecutive nucleotides by 1, 2, or 3 nucleotides; the sense strand comprises a nucleotide sequence that is at least partially complementary or substantially complementary to the antisense strand, forming a duplex region; and the term "substantial complementarity" means that the mismatch between the sense strand and the antisense strand in the duplex region does not exceed 3 nucleotides.

[0218] According to an embodiment of the present disclosure, the antisense strand comprises a nucleotide sequence of at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides from any one of SEQ ID NO: 225 to SEQ ID NO: 448, or comprises a nucleotide sequence with 1, 2, or 3 nucleotide differences from the consecutive nucleotides. In some embodiments, the complementary region comprises any one of SEQ ID NO. 225 to 448, or comprises a nucleotide sequence with 1, 2, or 3 nucleotide differences therefrom. In other embodiments, the antisense strand comprises a sequence with 0, 1, 2, or 3 nucleotide differences from any one of SEQ ID NO: 225 to SEQ ID NO: 448. In yet other embodiments, the antisense strand comprises a sequence with 0 or 1 nucleotide difference from any one of SEQ ID NO: 225 to SEQ ID NO: 448. In yet other embodiments, the antisense strand is selected from or comprises any one of SEQ ID NO: 225 to SEQ ID NO: 448.

[0219] According to an embodiment of the present disclosure, the sense strand comprises a nucleotide sequence of at least 17, at least 18, or at least 19 consecutive nucleotides from any one of SEQ ID NO: 1 to SEQ ID NO: 224, or comprises a nucleotide sequence having 1, 2, or 3 nucleotide differences from the consecutive nucleotides. In some embodiments, the sense strand comprises a sequence having 0, 1, 2, or 3 nucleotide differences from any one of SEQ ID NO: 1 to SEQ ID NO: 224. In other embodiments, the sense strand is selected from or comprises any one of SEQ ID NO: 1 to SEQ ID NO: 224.

[0220] According to an embodiment of the present disclosure, the sense strand comprises one of the following nucleotide sequences, or has a difference of no more than 3 nucleotides therefrom:

[0221] SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 77, SEQ ID NO: 102, SEQ ID NO: 117, SEQ ID NO: 132, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 164, SEQ ID NO: 169, SEQ ID NO: 176, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 185, SEQ ID NO: 186 and SEQ ID NO: 189; and / or

[0222] The antisense strand comprises one of the following nucleotide sequences, or differs therefrom by no more than 3 nucleotides:

[0223] SEQ ID NO: 237, SEQ ID NO: 240, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 301, SEQ ID NO: 326, SEQ ID NO: 341, SEQ ID NO: 356, SEQ ID NO: 359, SEQ ID NO: 360, SEQ ID NO: 364, SEQ ID NO: 366, SEQ ID NO:388, SEQ ID NO:393, SEQ ID NO:400, SEQ ID NO:403, SEQ ID NO:405, SEQ ID NO:409, SEQ ID NO:410 and SEQ ID NO:413.

[0224] According to an embodiment of the present disclosure, the sense strand comprises one of the following nucleotide sequences, or has a difference of no more than 3 nucleotides therefrom:

[0225] SEQ ID NO: 13, SEQ ID NO: 117, SEQ ID NO: 135, SEQ ID NO: 179, SEQ ID NO: 186 and SEQ ID NO: 189; and / or, the antisense strand comprises one of the following nucleotide sequences, or differs therefrom by no more than 3 nucleotides:

[0226] SEQ ID NO:237, SEQ ID NO:341, SEQ ID NO:359, SEQ ID NO:403, SEQ ID NO:410 and SEQ ID NO:413.

[0227] According to an embodiment of the present disclosure, the double-stranded oligonucleotide includes one or more of the following groups:

[0228] a sense strand having the nucleotide sequence set forth in SEQ ID NO: 13, or a nucleotide sequence differing therefrom by 1, 2, or 3 nucleotides, and an antisense strand having the nucleotide sequence set forth in SEQ ID NO: 237, or a nucleotide sequence differing therefrom by 1, 2, or 3 nucleotides;

[0229] A sense strand having the nucleotide sequence of SEQ ID NO: 117, or a nucleotide sequence having 1, 2, or 3 nucleotides thereof, and an antisense strand having the nucleotide sequence of SEQ ID NO: 341, or a nucleotide sequence having 1, 2, or 3 nucleotides thereof;

[0230] A sense strand having the nucleotide sequence of SEQ ID NO: 135, or a nucleotide sequence having 1, 2, or 3 nucleotides thereof, and an antisense strand having the nucleotide sequence of SEQ ID NO: 359, or a nucleotide sequence having 1, 2, or 3 nucleotides thereof;

[0231] A sense strand having the nucleotide sequence of SEQ ID NO: 179, or a nucleotide sequence having 1, 2, or 3 nucleotides thereof, and an antisense strand having the nucleotide sequence of SEQ ID NO: 403, or a nucleotide sequence having 1, 2, or 3 nucleotides thereof;

[0232] A sense strand having the nucleotide sequence of SEQ ID NO: 186, or a nucleotide sequence having 1, 2, or 3 nucleotides thereof, and an antisense strand having the nucleotide sequence of SEQ ID NO: 410, or a nucleotide sequence having 1, 2, or 3 nucleotides thereof;

[0233] The sense strand has the nucleotide sequence shown in SEQ ID NO: 189, or a nucleotide sequence having 1, 2 or 3 nucleotides therefrom, and the antisense strand has the nucleotide sequence shown in SEQ ID NO: 413, or a nucleotide sequence having 1, 2 or 3 nucleotides therefrom.

[0234] According to an embodiment of the present disclosure, the double-stranded oligonucleotide includes one or more of the following groups:

[0235] a sense strand having the nucleotide sequence set forth in SEQ ID NO: 13, and an antisense strand having the nucleotide sequence set forth in SEQ ID NO: 237;

[0236] a sense strand having the nucleotide sequence set forth in SEQ ID NO: 117, and an antisense strand having the nucleotide sequence set forth in SEQ ID NO: 341;

[0237] a sense strand having the nucleotide sequence set forth in SEQ ID NO: 135, and an antisense strand having the nucleotide sequence set forth in SEQ ID NO: 359;

[0238] a sense strand having the nucleotide sequence of SEQ ID NO: 179, or a nucleotide sequence having 1, 2, or 3 nucleotides thereof, and an antisense strand having the nucleotide sequence of SEQ ID NO: 403;

[0239] a sense strand having the nucleotide sequence set forth in SEQ ID NO: 186, and an antisense strand having the nucleotide sequence set forth in SEQ ID NO: 410;

[0240] The sense strand has the nucleotide sequence shown in SEQ ID NO: 189, and the antisense strand has the nucleotide sequence shown in SEQ ID NO: 413.

[0241] According to an embodiment of the present disclosure, the length of the sense strand does not exceed 21 nucleotides, and the length of the antisense strand does not exceed 23 nucleotides.

[0242] According to an embodiment of the present disclosure, the length of the sense strand is 19 nucleotides, and the length of the antisense strand is 21 nucleotides.

[0243] According to an embodiment of the present disclosure, the sense strand and / or antisense strand comprises a 3' overhang and / or a 5' overhang having at least 1 or at least 2 nucleotides.

[0244] As used herein, the term "overhang" refers to at least one unpaired nucleotide protruding from the double-stranded oligonucleotide duplex structure, and also refers to the nucleotide sequence in the siRNA structure outside the double-stranded region. For example, a nucleotide overhang exists when the 3' end of one of the sense and / or antisense strands extends beyond the 5' end of the other strand, or when the 5' end of one of the sense and / or antisense strands extends beyond the 3' end of the other strand. The overhang may comprise at least one nucleotide, at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. The nucleotide overhang may comprise or be composed of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang may be located on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotides in the overhang may appear at the 5' end, the 3' end, or both ends of the antisense or sense strand.

[0245] According to an embodiment of the present disclosure, the antisense strand comprises a 3' overhang having at least 1 or at least 2 nucleotides.

[0246] According to an embodiment of the present disclosure, the antisense strand comprises a 3' overhang having 2 nucleotides.

[0247] According to an embodiment of the present disclosure, the sense strand and / or the antisense strand independently comprises one or more phosphorothioate bonds, that is, two adjacent nucleotides are connected by a phosphorothioate bond.

[0248] According to an embodiment of the present disclosure, the sense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 5' end.

[0249] According to an embodiment of the present disclosure, the antisense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 3' end and the 5' end, respectively.

[0250] According to an embodiment of the present disclosure, substantially all nucleotides in the double-stranded oligonucleotide are independently selected from modified nucleotides.

[0251] According to an embodiment of the present disclosure, all nucleotides in the double-stranded oligonucleotide are independently selected from modified nucleotides.

[0252] According to an embodiment of the present disclosure, each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides:

[0253] 2'-halogenated modified nucleotides, 2'-deoxy modified nucleotides, 2'-O- optionally substituted C 1-6 Alkyl-modified nucleotides, 2'-O-(CH2) n-O-R1 modified nucleotides, 2'-O-Si(R2)3 modified nucleotides, 2'-amino modified nucleotides, abasic nucleotides, or quasi-nucleotides; the quasi-nucleotides are selected from one or more of peptide nucleic acid (PNA), morpholino (MNA), bridged nucleic acid (BNA), locked nucleic acid (LNA), glycol nucleic acid / glycerol nucleic acid (GNA), threose nucleic acid (TNA), or unlocked nucleic acid (UNA);

[0254] wherein n is selected from 1 or 2, R1 is selected from optionally substituted C 1-6 Alkyl or optionally substituted C 1-6 alkoxy;

[0255] Each R2 is independently selected from optionally substituted C 1-6 alkyl;

[0256] The optional substitution means that one or more hydrogen atoms on the alkyl group are replaced by a substituent, and the substituent is independently selected from C 1-3 Alkyl, C 1-3 Alkoxy or halogen.

[0257] According to an embodiment of the present disclosure, the 2'-halogenated modified nucleotides are selected from 2'-fluoro-modified nucleotides.

[0258] According to an embodiment of the present disclosure, the 2'-O-optionally substituted C 1-6 The alkyl-modified nucleotides are selected from 2'-O-methyl (also known as 2'-methoxy)-modified nucleotides.

[0259] According to an embodiment of the present disclosure, the 2'-O-(CH2) n The -O-R1 modified nucleotide is selected from a 2'-O-methoxyethyl modified nucleotide, a 2'-O-ethoxymethyl modified nucleotide or a 2'-O-2,2,2-trifluoroethoxymethyl modified nucleotide.

[0260] According to an embodiment of the present disclosure, the 2'-O-(CH2) n The -O-R1 modified nucleotide is selected from 2'-O-methoxyethyl modified nucleotides.

[0261] According to an embodiment of the present disclosure, the 2'-O-Si(R2)3 modified nucleotide is selected from a 2'-O-TBDMS modified nucleotide, a 2'-O-TIPS modified nucleotide or a 2'-O-TOM modified nucleotide.

[0262] According to an embodiment of the present disclosure, each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides:

[0263] 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides or 2'-O-ethoxymethyl modified nucleotides.

[0264] According to an embodiment of the present disclosure, each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides:

[0265] 2'-Fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides.

[0266] According to an embodiment of the present disclosure, the sense strand and / or the antisense strand contains at least one 2'-O-methoxyethyl-modified nucleotide or 2'-O-ethoxymethyl-modified nucleotide.

[0267] According to an embodiment of the present disclosure, the sense strand and / or the antisense strand contains at least one 2'-O-methoxyethyl-modified nucleotide.

[0268] According to an embodiment of the present disclosure, the duplex region has a structure as shown in the following formula (101):

[0269] SS: 5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3'

[0270] AS: 3'-(N)a-(X)p-(N)b-(X)q-(N)c-5' formula (101),

[0271] Among them, SS represents the sense strand and AS represents the antisense strand;

[0272] Each of the Ns is independently selected from the following modified nucleotides: 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides or 2'-deoxy modified nucleotides;

[0273] Each of the Xs is independently selected from 2'-O-(CH2) n -O-R1 modified nucleotides or 2'-O-Si(R2)3 modified nucleotides; wherein n is selected from 1 or 2, R1 is selected from optionally substituted C1-6 Alkyl; each R2 is independently selected from optionally substituted C 1-6 Alkyl; the optional substitution refers to one or more hydrogen atoms on the alkyl or alkoxy group being replaced by a substituent, the substituent being independently selected from C 1-3 Alkyl, C 1-3 Alkoxy or halogen;

[0274] a, a', p, p', b, b', q, q', c, c', r', and d' independently represent the number of nucleotides; wherein a' is selected from an integer of 3-8, p' is selected from an integer of 0-3, b' is selected from an integer of 4-13, q' is selected from an integer of 0-4, c' is selected from an integer of 3-9, r' is selected from an integer of 0-3, d' is selected from an integer of 0-9, a is selected from an integer of 4-7, p is selected from an integer of 0-1, b is selected from an integer of 4-8, q is selected from an integer of 0-4, and c is selected from an integer of 6-10; and p', q', r', p, and q are not all 0 at the same time, and 0≤q'+r'≤4.

[0275] According to an embodiment of the present disclosure, a' is selected from an integer of 3-8, p' is selected from 0 or 1, b' is selected from an integer of 4-13, q' is selected from 0 or 1, and c' is selected from an integer of 3-9; r' is selected from 0 or 1, d' is selected from an integer of 1-8, a is selected from an integer of 4-7, p is selected from 1, b is selected from an integer of 4-8, q is selected from 0 or 1, and c is selected from an integer of 6-10.

[0276] According to an embodiment of the present disclosure, each N is independently selected from a 2'-fluoro-modified nucleotide or a 2'-O-methyl-modified nucleotide.

[0277] According to an embodiment of the present disclosure, each X is independently selected from 2'-O-(CH2) n -O-R1 modified nucleotides.

[0278] According to an embodiment of the present disclosure, each X is independently selected from a 2'-O-methoxyethyl modified nucleotide or a 2'-O-ethoxymethyl modified nucleotide.

[0279] According to an embodiment of the present disclosure, the duplex region comprises at least one 2'-O-methoxyethyl modified nucleotide or 2'-O-ethoxymethyl modified nucleotide.

[0280] According to an embodiment of the present disclosure, the duplex region comprises at least one 2'-O-methoxyethyl modified nucleotide.

[0281] According to an embodiment of the present disclosure, the sense strand is 17-21 nucleotides long, and the antisense strand is 19-23 nucleotides long.

[0282] According to an embodiment of the present disclosure, the sense strand is 19 nucleotides long, and the antisense strand is 21 nucleotides long.

[0283] According to an embodiment of the present disclosure, in the direction from the 5' end to the 3' end, at least 3 nucleotides among the nucleotides at positions 7-10 of the nucleotide sequence in the positive strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides.

[0284] According to an embodiment of the present disclosure, in the direction from the 5' end to the 3' end, at least 3 nucleotides in positions 7-10 of the nucleotide sequence in the positive strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides.

[0285] According to an embodiment of the present disclosure, in the direction from the 5' end to the 3' end, at least three nucleotides at positions 7 to 10 of the nucleotide sequence in the positive strand are selected from 2'-fluoro-modified nucleotides, the nucleotides at positions 12 and / or 18 are selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides.

[0286] According to an embodiment of the present disclosure, in the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the positive strand are selected from 2'-fluoro-modified nucleotides, the nucleotides at positions 12 and / or 18 are selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides.

[0287] According to an embodiment of the present disclosure, at least four nucleotides among the nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense chain are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides.

[0288] According to an embodiment of the present disclosure, at least four nucleotides among the nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at positions 2, 6, 14 and 16 are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides.

[0289] According to an embodiment of the present disclosure, at least four nucleotides among the nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at positions 2, 6, 14 and 16 are selected from 2'-fluoro-modified nucleotides, the nucleotides at positions 8 and / or 15 are selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0290] According to an embodiment of the present disclosure, in the direction from the 5' end to the 3' end, at least three nucleotides among the nucleotides at positions 7-10 of the nucleotide sequence in the sense chain are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides; at least four nucleotides among the nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense chain are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides.

[0291] According to an embodiment of the present disclosure, each nucleotide of the double-stranded oligonucleotide is independently selected from modified nucleotides, wherein the modifications of the sense strand and the antisense strand are selected from any one of the following (1)-(15):

[0292] (1) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0293] (2) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 10, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0294] (3) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 11, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (4) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 12, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0295] (5) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0296] (6) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 10, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0297] (7) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0298] (8) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 12, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0299] (9) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 12 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0300] (10) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 12 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 10, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0301] (11) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 12 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0302] (12) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 18 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 10, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0303] (13) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 18 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0304] (14) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 18 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 10, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 8 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0305] (15) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, the nucleotides at positions 12 and 18 are selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 10, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0306] According to an embodiment of the present disclosure, the double-stranded oligonucleotide includes one of the following groups 1-45:

[0307] Among them, C, G, U, A, and T represent cytidine-3'-phosphate, guanosine-3'-phosphate, uridine-3'-phosphate, adenosine-3'-phosphate, and thymidine-3'-phosphate, respectively; m indicates that the nucleotide adjacent to the left of the letter m is a 2'-O-methyl-modified nucleotide; f indicates that the nucleotide adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide; (moe) indicates that the nucleotide adjacent to the left of the combination mark (moe) is a 2'-O-methoxyethyl-modified nucleotide; and s indicates that the two nucleotides before and after the letter s are connected by a phosphorothioate bond.

[0308] According to an embodiment of the present disclosure, all nucleotides in the double-stranded oligonucleotide are independently selected from modified nucleotides; and each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides:

[0309] 2'-halogenated modified nucleotides, 2'-O-optionally substituted C 1-6 Alkyl-modified nucleotides, 2'-O-(CH2) n -O-R1 modified nucleotides, [2'-W1-2'-W2] modified nucleotides;

[0310] wherein n is selected from 1 or 2, R1 is selected from optionally substituted C 1-6 Alkyl or optionally substituted C 1-6 alkoxy;

[0311] Each R2 is independently selected from optionally substituted C 1-6 alkyl;

[0312] The optional substitution means that one or more hydrogen atoms on the alkyl group are replaced by a substituent, and the substituent is independently selected from C 1-3 Alkyl, C 1-3 Alkoxy or halogen;

[0313] The structural formula of the [2'-W1-2'-W2] modified nucleotide is W1 and W2 are each independently selected from halogen or C 1-6 Alkyl, Base represents uracil U, thymine T, cytosine C, adenine A or guanine G.

[0314] According to an embodiment of the present disclosure, the 2'-halogenated modified nucleotides are selected from 2'-fluoro-modified nucleotides.

[0315] According to an embodiment of the present disclosure, the 2'-O-optionally substituted C 1-6 The alkyl-modified nucleotides are selected from 2'-O-methyl-modified nucleotides.

[0316] According to an embodiment of the present disclosure, the 2'-O-(CH2) n The -O-R1 modified nucleotide is selected from 2'-O-methoxyethyl modified nucleotides.

[0317] According to an embodiment of the present disclosure, the [2'-W1-2'-W2] modified nucleotide is selected from [2'-F-2'-CH3] modified nucleotide.

[0318] According to an embodiment of the present disclosure, each nucleotide of the double-stranded oligonucleotide is independently selected from modified nucleotides, and each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides:

[0319] 2'-Fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides or [2'-F-2'-CH3]-modified nucleotides.

[0320] According to an embodiment of the present disclosure, the sense strand is 19 nucleotides long, and the antisense strand is 21 nucleotides long; and the modifications of the sense strand and the antisense strand are selected from any one of the following A1-A2:

[0321] A1. From the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0322] The nucleotides at positions 2, 6, 9, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotides at positions 20-21 are selected from [2'-F-2'-CH3]-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0323] A2. From the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0324] The nucleotides at positions 2, 6, 9, 14 and 16 of the nucleotide sequence in the antisense chain are selected from 2'-fluoro modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl modified nucleotides, the nucleotides at positions 20-21 are selected from [2'-F-2'-CH3] modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides.

[0325] According to an embodiment of the present disclosure, the sense strand is 19 nucleotides long, and the antisense strand is 20 nucleotides long; and the modifications of the sense strand and the antisense strand are selected from the following:

[0326] In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 19 is selected from [2'-F-2'-CH3]-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0327] The nucleotides at positions 2, 6, 9, 14 and 16 of the nucleotide sequence in the antisense chain are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, the nucleotide at position 20 is selected from [2'-F-2'-CH3]-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0328] According to an embodiment of the present disclosure, the double-stranded oligonucleotide includes one of the following groups 46-50:

[0329] Wherein, (NM054) represents a nucleotide, and its structural formula is

[0330] Conjugate

[0331] In another aspect of the present disclosure, a conjugate is provided. According to an embodiment of the present disclosure, the conjugate comprises: the double-stranded oligonucleotide described above; and one or more ligands capable of binding to a cell receptor; the ligands are conjugated to the double-stranded oligonucleotide. Due to the addition of the ligands, the conjugate disclosed herein can effectively deliver the drug to cells, further enhancing the degradation of AGT mRNA, and effectively preventing and / or treating diseases or conditions mediated by the angiotensinogen (AGT) gene.

[0332] According to an embodiment of the present disclosure, the ligand specifically targets liver tissue.

[0333] According to an embodiment of the present disclosure, the ligand is selected from ligands having affinity for asialoglycoprotein receptors.

[0334] According to an embodiment of the present disclosure, the ligand comprises galactose or a derivative thereof.

[0335] According to an embodiment of the present disclosure, the derivative of galactose is selected from N-acetylgalactosamine.

[0336] According to an embodiment of the present disclosure, the ligand is conjugated to at least one of the 5' end of the sense strand, the middle position of the sense strand, the 3' end of the sense strand, the 5' end of the antisense strand, the middle position of the antisense strand and the 3' end of the antisense strand.

[0337] According to an embodiment of the present disclosure, the number of the ligands is selected from one, and one ligand is conjugated to the 3' end of the sense strand.

[0338] According to an embodiment of the present disclosure, the number of the ligands is selected from two, and the two ligands are conjugated to the 5' end of the sense strand and the 3' end of the antisense strand, respectively.

[0339] According to an embodiment of the present disclosure, the ligand is selected from the structure shown in formula (201) or a pharmaceutically acceptable salt or stereoisomer thereof:

[0340] Wherein, * represents the conjugation site between the ligand and the double-stranded oligonucleotide;

[0341] j is selected from 1, 2, 3 or 4;

[0342] Each Z is independently selected from hydroxyl or thiol;

[0343] Each h is independently selected from 1, 2 or 3;

[0344] Each i is independently selected from 1, 2 or 3;

[0345] Each R3 is independently selected from H, optionally substituted C 1-6 Alkyl or optionally substituted C 1-6 alkoxy;

[0346] Each L is independently selected from optionally substituted C 2-20 Alkylene or R La and R La Independently selected from optionally substituted C 1-10 Alkylene, k is selected from 1, 2, 3, 4 or 5;

[0347] Each Y is independently selected from O, S or NH.

[0348] According to an embodiment of the present disclosure, j is selected from 2, 3 or 4.

[0349] According to an embodiment of the present disclosure, j is selected from 2.

[0350] According to an embodiment of the present disclosure, j is selected from 3.

[0351] According to an embodiment of the present disclosure, j is selected from 4.

[0352] According to an embodiment of the present disclosure, Z is selected from hydroxyl.

[0353] According to an embodiment of the present disclosure, h is selected from 1.

[0354] According to an embodiment of the present disclosure, i is selected from 1.

[0355] According to an embodiment of the present disclosure, R3 is selected from H.

[0356] According to an embodiment of the present disclosure, each L is independently selected from C 1-10 Alkylene or Among them, R La and R La Independently selected from C 1-5 Alkylene, k is 1, 2 or 3.

[0357] According to an embodiment of the present disclosure, k is selected from 1.

[0358] According to an embodiment of the present disclosure, each L is independently selected from

[0359] According to an embodiment of the present disclosure, Y is selected from O.

[0360] According to an embodiment of the present disclosure, the ligand is selected from the structure shown in formula (202) or a pharmaceutically acceptable salt or a stereoisomer thereof:

[0361] According to an embodiment of the present disclosure, the ligand is selected from any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof:

[0362] According to an embodiment of the present disclosure, the ligand is selected from the following structures or pharmaceutically acceptable salts or stereoisomers thereof:

[0363] According to an embodiment of the present disclosure, the ligand is selected from the following structures or pharmaceutically acceptable salts or stereoisomers thereof:

[0364] According to an embodiment of the present disclosure, the ligand is selected from the following structures or pharmaceutically acceptable salts or stereoisomers thereof:

[0365] According to an embodiment of the present disclosure, the ligand is selected from the structure shown in Formula L96 or a pharmaceutically acceptable salt thereof:

[0366] Pharmaceutical composition

[0367] In another aspect of the present disclosure, a pharmaceutical composition is provided. According to an embodiment of the present disclosure, the pharmaceutical composition includes any of the following items and pharmaceutically acceptable excipients or adjuvants:

[0368] (I) the double-stranded oligonucleotide described above; and / or

[0369] (II) The conjugate described above.

[0370] As can be seen above, the aforementioned double-stranded oligonucleotides and conjugates can degrade AGT mRNA and inhibit AGT expression. Therefore, the pharmaceutical composition disclosed herein can effectively prevent and / or treat diseases or conditions mediated by the angiotensinogen (AGT) gene.

[0371] According to an embodiment of the present disclosure, the pharmaceutical composition further includes: a pharmaceutically acceptable carrier or excipient.

[0372] As used herein, "pharmaceutical composition" may refer to a composition for use in treating a disease or in vitro cell culture experiments. When used in treating a disease, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with an excipient that constitutes one or more adjunct ingredients. Typically, the composition is prepared by uniformly and thoroughly combining the active siRNA with a liquid excipient, a finely divided solid excipient, or both.

[0373] As used herein, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal to be treated therewith. Preferably, the "pharmaceutically acceptable" herein refers to a substance approved by a federal regulatory agency or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeia for use in animals, particularly humans.

[0374] As used herein, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the particular intended dosage form. Except to the extent that any conventional excipient is incompatible with the siRNA of the present disclosure, such as by producing any adverse biological effect or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present disclosure.

[0375] In addition to any conventional excipients, to the extent that they are incompatible with the siRNA of the present disclosure, such as by producing any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present disclosure.

[0376] The pharmaceutical compositions disclosed herein include formulations suitable for parenteral administration. The formulations can conveniently be in unit dosage form and can be prepared by any method known in the pharmaceutical art. The amount of active ingredient that can be combined with excipients to prepare a single dose is generally the amount of siRNA that produces a therapeutic effect. Generally speaking, in percent units, this amount is from about 1% to about 99% active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0377] Reagent test kit

[0378] In another aspect of the present disclosure, a kit is provided. According to an embodiment of the present disclosure, the kit includes any of the following items:

[0379] (I) the double-stranded oligonucleotide described above; and / or

[0380] (II) the conjugate described above; and / or

[0381] (III) The pharmaceutical composition described above.

[0382] The kit disclosed herein can effectively degrade AGT mRNA and inhibit AGT expression.

[0383] It should be noted that the features and advantages described above for the double-stranded oligonucleotide, conjugate and pharmaceutical composition are also applicable to the kit and will not be repeated here.

[0384] use

[0385] In yet another aspect, the present disclosure provides the use of any of the following in the preparation of a medicament for alleviating, treating, and / or preventing diseases or conditions mediated by AGT: (I) the double-stranded oligonucleotide described above; and / or (II) the conjugate described above. As previously noted, both the double-stranded oligonucleotide and conjugate can degrade AGT mRNA and inhibit AGT expression. Thus, the pharmaceutical compositions of the present disclosure can effectively prevent and / or treat diseases or conditions mediated by the angiotensinogen (AGT) gene.

[0386] According to an embodiment of the present disclosure, the diseases or conditions include hypertension, critical hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive emergency state, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm and ventricular fibrosis.

[0387] As used herein, the term "treatment" refers to a method for obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or siRNA to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing an siRNA or siRNA conjugate as described herein to an individual in need.

[0388] The effective amount of the double-stranded oligonucleotide, conjugate or pharmaceutical composition disclosed herein may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated for the patient, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment situation, several divided doses may be administered daily, for example, at a dose of four times a day, three times a day, twice a day, once a day, or every other day, or several doses administered daily may be proportionally reduced.

[0389] The drug can be administered to a subject by any suitable route known in the art, including but not limited to oral or parenteral routes, including intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration and topical administration (including buccal administration and sublingual administration), preferably intravenous administration.

[0390] It should be noted that the features and advantages described above for double-stranded oligonucleotides and conjugates are also applicable to this use and will not be repeated here.

[0391] method

[0392] In another aspect of the present disclosure, a method for inhibiting AGT gene expression in cells is provided. According to an embodiment of the present disclosure, the method comprises administering the aforementioned double-stranded oligonucleotide, and / or the aforementioned conjugate, and / or the aforementioned pharmaceutical composition to a subject. The method of the present disclosure can be used to inhibit angiotensinogen expression. For example, angiotensinogen expression can be inhibited in animals; or for non-disease treatment purposes, such as in vitro inhibition of angiotensinogen expression to facilitate subsequent research.

[0393] In yet another aspect of the present disclosure, a method for alleviating, treating, and / or preventing AGT-mediated diseases or conditions is provided. According to an embodiment of the present disclosure, the method comprises administering the aforementioned double-stranded oligonucleotide, and / or the aforementioned conjugate, and / or the aforementioned pharmaceutical composition to a subject. As previously noted, the aforementioned double-stranded oligonucleotide and conjugate can degrade AGT mRNA and inhibit AGT expression, thereby facilitating AGT-related scientific research and clinical treatment and / or prevention of diseases or conditions mediated by the angiotensinogen gene.

[0394] According to an embodiment of the present disclosure, the AGT-mediated disease or disorder includes a disease associated with the mRNA level of AGT gene expression.

[0395] According to an embodiment of the present disclosure, the diseases or conditions include hypertension, critical hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive emergency state, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm and ventricular fibrosis.

[0396] It should be noted that the features and advantages described above for the double-stranded oligonucleotide, conjugate and pharmaceutical composition are also applicable to this method and will not be repeated here.

[0397] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0398] In the context of the present disclosure, unless otherwise specified, the siRNA sequences used in the present disclosure were commissioned to Kunshan Aotai Biotechnology Co., Ltd. for synthesis; the synthesis of PCR primers used in the present disclosure was commissioned to Sangon Biotechnology (Shanghai) Co., Ltd.; the human liver cancer cell line Huh7 used in the present disclosure was purchased from Wuhan Pronocell Life Science Co., Ltd.; the experimental animal C57BL / 6J mice used in the present disclosure were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.; the hREN×hAGT transgenic mice used in the present disclosure were purchased from Saiye (Suzhou) Biotechnology Co., Ltd.; the SHR rats used in the present disclosure were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.; the blood pressure measurements of mice and rats in the present disclosure were performed using a complete set of intelligent non-invasive blood pressure systems; the serum samples of mice and rats in the present disclosure were tested using a human angiotensinogen kit.

[0399] In the context of this disclosure, unless otherwise stated, the real-time PCR detection data of the activity experiments involved in this disclosure are all calculated using the ΔΔCt method to perform relative quantitative calculation of the target gene mRNA in each test group. The calculation method is summarized as follows:

[0400] ΔCt(test group) = Ct(test group target gene) - Ct(test group reference gene)

[0401] ΔCt(control group) = Ct(control group target gene) - Ct(control group internal reference gene)

[0402] ΔΔCt(test group)=ΔCt(test group)-ΔCt(control group average)

[0403] ΔΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0404] The mRNA expression level of the target gene in the test group was normalized with the control group as the benchmark, and the remaining expression level of the target gene mRNA in the control group was defined as 100%.

[0405] Relative residual expression level of target gene mRNA in the test group = 2 -ΔΔCt (Test group) × 100%

[0406] Test group target gene mRNA inhibition rate = 100% - test group target gene mRNA relative expression level

[0407] The blood pressure test data (blood pressure before the first administration (D0)) of the in vivo activity experiment involved in the present disclosure are calculated as follows: Blood pressure reduction level (mmHg) = Dx-D0

[0408] The calculation method for the detection data of AGT and Ang II proteins at different time points in the in vivo activity experiment involved in the present disclosure is summarized as follows:

[0409] In the context of this disclosure, unless otherwise stated, in vivo activity experimental data are presented as The experimental data were plotted and analyzed using GraphPad prism 8.0 software.

[0410] In the context of the present disclosure, unless otherwise stated, the reagent ratios provided below are calculated on a volume basis (v / v).

[0411] Preparation of compounds

[0412] Unless otherwise stated, all reagents used in the preparation of the compounds disclosed herein were purchased from Beijing Coupling Technology Co., Ltd., wherein the information of the main reagents is shown in Table 1.

[0413] Table 1 Main reagents

[0414] Here, CPG stands for controlled pore glass (CPG) carrier.

[0415] Preparation of compounds

[0416] Preparation Example 1: Synthesis of Compound CR01008

[0417] (1-1) Synthesis of Compound 2

[0418] Compound 1 (trans-4-(Boc-amino)cyclohexylcarboxaldehyde, 10.0 g, 1.0 eq) and formaldehyde solution (8.9 g, 37 mass %, 2.4 eq) were dissolved in 33 ml of methanol, and 13 ml of a 45.3 mass % KOH aqueous solution was added dropwise. After the addition was complete, the mixture was stirred at 25°C for 30 minutes, then heated to 60°C and refluxed at 60°C for 2 hours. After the reaction was completed, the reaction solution was cooled to 25°C and evaporated to dryness under reduced pressure to obtain a crude white solid. A small amount of water was added to the crude product to slurry it, and the mixture was filtered to obtain compound 2 (9 g, yield 78.9%) as a white solid. MS-ESI (m / z) = 260 [M+H] + .

[0419] (1-2) Synthesis of Compound 3

[0420] Compound 2 (9 g, 1 eq) prepared in step (1-1) was dissolved in 70 ml of 1,4-dioxane. A 45 ml, 4 M solution of hydrogen chloride in 1,4-dioxane was added, and the mixture was stirred at 25°C for 1 hour. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure to obtain compound 3 (6.8 g, 100% yield) as a white solid.

[0421] (1-3) Synthesis of Compound 5: Compound 3 (1.8 g, 2.0 eq) prepared in step (1-2), compound 4 (5-[[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)-2-tetrahydropyranyl]oxy]pentanoic acid, 2.1 g, 1.0 eq), and DIEA (N,N-diisopropylethylamine, 3.5 g, 6.0 eq) were dissolved in 15 ml of DMF. HBTU (1.9 g, 1.1 eq) was added, and the mixture was stirred at 25°C under a N2 atmosphere for 3 hours. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and purified by reverse phase column chromatography (C18 column, eluent: acetonitrile / water = 22 / 78, v / v) to obtain compound 5 (1.78 g, yield 64.4%) as a white solid. MS-ESI (m / z) = 589 [M+H] + .

[0422] (1-4) Synthesis of Compound 6

[0423] Compound 5 (1.54 g, 1.0 eq) prepared according to step (1-3) was dissolved in 15 ml of pyridine. The reaction system was cooled to 0°C using an ice-water bath and DMTrCl (4,4'-dimethoxytriphenylmethane chloride, 1.32 g, 1.5 eq) was added at 0°C. The reaction was allowed to react at 25°C for 3 hours. 15 ml of methanol was added to the reaction solution to quench the reaction. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and purified by reverse phase column chromatography (C18 column, eluent: acetonitrile:water = 60 / 40, v / v) to obtain compound 6 (1 g, yield 42.7%) as a yellow solid. MS-ESI (m / z) = 891 [M+H] + .

[0424] (1-5) Synthesis of Compound CR01008

[0425] Compound 6 (1.08 g, 1.0 eq) prepared according to step (1-4) was dissolved in 20 ml of anhydrous dichloromethane. DCI (115 mg, 0.8 eq) and compound 7 (bis(diisopropylamino)(2-cyanoethoxy)phosphine, 732 mg, 2.1 eq) were added, respectively. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 25°C for 2 hours. After completion of the reaction, 20 ml of saturated aqueous sodium bicarbonate solution was added to the reaction solution, and the mixture was extracted three times with 20 ml of dichloromethane (3 × 20 ml). The organic phases were combined and evaporated to dryness under reduced pressure. The organic phases were purified by reverse phase column chromatography (C18 column, eluent: acetonitrile:water = 72 / 28, v / v), and then dried in vacuo for 12 hours to obtain compound CR01008 (1 g, yield 76.0%) as a white powder. MS-ESI (m / z) = 1091 [M+Na]+.

[0426] 1H NMR(400MHz, DMSO-d6)δ1.05(d,J=6.7Hz,6H).1.14(d,J=6.7Hz,6H),1.37-1.17(m,5H),1.60-1.40(m,6H),1.68-1.62(m,1H),1.80(s,3H), 1.80(s,3H),1.92(s,3H),2.02(s,5H),2.13(s,3H),2.71(t,J=5.9Hz,2H),2.79(d,J=8.4Hz,1H),2.87(d,J=8.4Hz,1H),3.36(s,1H),3.58- 3.39(m,3H),3.69-3.60(m,2H),3.75(s,7H),3.90(dt,J=11.2,8.8Hz,1H),4.05(s,3H),4.51(d,J=8.4Hz,1H),4.99(dd,J=11.3,3.4Hz,1H) ,5.24(d,J=3.4Hz,1H),5.78(s,1H),6.93-6.87(m,4H),7.35-7.21(m,7H),7.44-7.37(m,2H),7.66(d,J=7.8Hz,1H),7.84(d,J=9.2Hz,1H).

[0427] Preparation Example 2: Synthesis of Compound CR01008Z

[0428] The synthetic route of compound CR01008Z is as follows:

[0429] (2-1) Synthesis of Compound 9

[0430] Compound 6 (500 mg) prepared in step (1-4) was dissolved in 10 ml of dichloromethane, and compound 8 (succinic anhydride, 112 mg), DMAP (6.8 mg), and TEA (226.2 mg) were added. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 25°C for 16 hours. The mixture was then purified by reverse phase column chromatography (C18 column, eluent: acetonitrile:water = 72 / 28, v / v) to obtain compound 9 (300 mg, yield 53.6%). MS-ESI (m / z) = 1013 [M+Na] + .

[0431] (2-2) Synthesis of Compound CR01008Z

[0432] Compound 9 (50 mg), aminoCPG (1.25 g, 80 μmol / g, 0.1 mmol), HBTU (27 mg), and DIEA (12 mg) prepared in step (2-1) were added to a 20 ml sample vial and shaken for 16 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was washed once with 10 ml of acetonitrile (1×10 ml) and then dried in vacuo. The dried filter cake, DMAP (3 mg), Cap1 (10 ml, 200 V), and Cap2 (1 ml, 20 V) were added to a 20 ml sample vial and shaken for 6 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was washed once with 10 ml of acetonitrile (1×10 ml) and then dried in vacuo to obtain compound CR01008Z (1.03 g, loading 20-30 μmol / g).

[0433] Cap1 and Cap2 are capping reagents, Cap1 is a 20% by volume N-methylimidazole mixed solution in pyridine / acetonitrile, with a volume ratio of pyridine to acetonitrile of 3:5; Cap2 is a 20% by volume acetic anhydride solution in acetonitrile.

[0434] Preparation Example 3: Synthesis of Compound CR01013

[0435] The synthetic route of compound CR01013 is as follows:

[0436] (3-1) Synthesis of Compound 2

[0437] Compound 1 (trans-4-(Boc-amino)cyclohexylcarboxaldehyde, 4.9 g) was dissolved in 17 ml of methanol, and aqueous formaldehyde solution (4.21 g, concentration of 37% by mass) and aqueous sodium hydroxide solution (6.5 ml, concentration of 45.3% by mass) were added dropwise. After the addition was complete, the temperature was raised to 60°C and stirred at 60°C for 2 hours. After the reaction was completed, the reaction solution was cooled to 25°C and evaporated to dryness under reduced pressure to obtain a crude white solid. A small amount of water was added to the crude product to make a pulp, filtered and dried to obtain compound 2 (4.8 g, yield 85.9%) as a white solid. ESI-MS (m / z) = 260.2 [M+H] + .

[0438] (3-2) Synthesis of Compound 3

[0439] Compound 2 (4.8 g) prepared in step (3-1) was dissolved in 25 ml of 1,4-dioxane. A 1,4-dioxane solution of hydrochloric acid (25 ml, 4 M) was added, and the mixture was stirred at 25°C for 2 hours. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure to obtain compound 3 (3.6 g, 99.4% yield) as a white solid.

[0440] (3-3) Synthesis of Compound 11

[0441] Compound 3 (3.6 g) prepared in step (3-2) was dissolved in 36 ml of DMF, and TEA (5.62 g), compound 10 (N-benzyloxycarbonyl-4-aminobutyric acid, 5.28 g), and HBTU (8.43 g) were added, and the mixture was stirred at 25°C for 16 hours. After completion of the reaction, the reaction solution was added to 200 ml of saturated aqueous sodium bicarbonate solution and extracted three times with 100 ml of ethyl acetate (3×100 ml). The organic phases were combined, washed once with 50 ml of saturated aqueous sodium chloride solution (1×50 ml), and then dried over anhydrous sodium sulfate. The organic phase was evaporated to dryness under reduced pressure and purified by normal phase column chromatography (eluent: dichloromethane / methanol = 10 / 1, v / v) to obtain compound 11 (2.3 g, yield 33.0%) as a white solid. ESIMS (m / z) = 379.5 [M+H] + .

[0442] (3-4) Synthesis of Compound 12

[0443] Compound 11 (2.3 g) prepared in step (3-3) was dissolved in 23 ml of methanol, and wet palladium on carbon (230 mg, 10% loading) was added. The atmosphere was replaced with hydrogen three times, and the reaction system was stirred at 25°C under a hydrogen atmosphere (15 psi) for 16 hours. After completion of the reaction, the reaction solution was filtered to obtain a filtrate, which was evaporated to dryness under reduced pressure to obtain compound 12 (1.48 g, 99.8% yield) as a yellow oil.

[0444] (3-5) Synthesis of Compound 13

[0445] Compound 12 (1.48 g) prepared in step (3-4) was dissolved in 15 ml of DMF, and triethylamine (TEA, 1.22 g), compound 4 (1.35 g), and HBTU (3.45 g) were added. The mixture was stirred at 25°C for 16 hours. After completion of the reaction, the reaction solution was added to 150 ml of saturated aqueous sodium bicarbonate solution and extracted three times with 50 ml of ethyl acetate (3 × 50 ml). The organic phases were combined, washed once with 30 ml of saturated aqueous sodium chloride solution (1 × 30 ml), and then dried over anhydrous sodium sulfate. The organic phase was evaporated to dryness under reduced pressure and purified by reverse phase column chromatography (C18 column, eluent: water / acetonitrile = 5 / 1, v / v) to obtain compound 13 (1.3 g, yield 31.8%) as a white solid. ESI-MS (m / z): 674.3 [M+H] + .

[0446] (3-6) Synthesis of Compound 14

[0447] Compound 13 (1.1 g) prepared in step (3-5) was dissolved in 11 ml of pyridine. The reaction system was cooled to 0°C using an ice-water bath and DMTrCl (813 mg) was added portionwise at 0°C. The reaction system was stirred at 0°C for 1 hour. After the reaction was completed, methanol was added to the reaction solution to quench the reaction. The solvent was evaporated and the solution was purified by reverse phase column chromatography (eluent: water / acetonitrile = 1 / 4, v / v) to obtain compound 14 (800 mg, yield 50.3%) as a white solid. ESI-MS (m / z): 976.5 [M+H] + .

[0448] (3-7) Synthesis of Compound CR01013

[0449] At 25°C, compound 14 (550 mg) was dissolved in 5 ml of dichloromethane (DCM), and 4,5-dicyanoimidazole (DCl, 53.2 mg) and compound 7 (2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite, 255.4 mg) were added. The atmosphere was purged with nitrogen three times, and the reaction system was stirred at 25°C for 1 hour. After completion of the reaction, the reaction solution was washed twice with 5 ml of saturated sodium bicarbonate solution (2 × 5 ml) and once with 30 ml of saturated sodium chloride solution (1 × 30 ml). The organic phase was separated and dried over anhydrous sodium sulfate. The organic phase solvent was evaporated to dryness under reduced pressure and purified by normal phase column chromatography (eluent: dichloromethane / methanol = 20 / 1, v / v) to obtain compound CR01013 (532 mg, 80.4% yield) as a white solid. ESI-MS (m / z): 1176.7 [M+H]+ .

[0450] 1H NMR (400MHz, DMSO-d6) δ0.95-1.05 (d, J=6.7Hz, 5H), 1.06-1.15 (q, J=7.6Hz, 8H), 1.15-1.21 (t, J=7.2Hz, 14H), 1.72-1 .80(s,3H),1.84-1.92(s,3H),1.94-2.07(d,J=16.0Hz,7H),2.07-2.14(s,3H),2.64-2.72(q,J=5.8Hz,2H),2.74-2.89 (d,J=8.5Hz,2H),3.35-3.56(m,4H),3.57-3.70(m,4H),3.71-3.77(s,6H),3.81-3.93(m,1H),3.96-4.09(d,J=6.4Hz, 3H), 6.82-6.97 (d, J=8.7Hz, 4H), 7.17-7.27 (t, J=8.7Hz, 5H), 7.27-7.34 (t, J=7.6Hz, 2H), 7.34–7.43 (d, J=7.5Hz, 2H).

[0451] Preparation Example 4: Synthesis of Compound CR01013Z

[0452] The synthetic route of compound CR01013Z is as follows:

[0453] (4-1) Synthesis of Compound 15

[0454] Compound 14 (100 mg, 0.10 mmol) was dissolved in 2 ml of dichloromethane at 25°C, and triethylamine (25.9 mg, 0.25 mmol), DMAP (1.25 mg, 0.01 mmol), and compound 8 (succinic anhydride, 15.4 mg, 0.15 mmol) were added. The reaction system was stirred at 25°C for 16 hours. After completion of the reaction, the solvent was evaporated from the reaction solution and the product was purified by reverse phase column chromatography (C18 column, eluent: water / acetonitrile = 2 / 1, v / v) to obtain compound 15 (110 mg, 0.10 mmol, yield 100%) as a yellow oil. ESI-MS (m / z) = 1099.3 [M+Na] + .

[0455] (4-2) Synthesis of Compound CR01013Z

[0456] Compound 15 (50 mg, 0.04 mmol) was dissolved in 10 ml of acetonitrile, and HBTU (24.2 mg, 0.06 mmol), DIEA (11.0 mg, 0.08 mmol), and amino-CPG (1.06 g, loading 80 μmol / g) were added. The reaction system was stirred at 25°C for 16 hours. After completion of the reaction, the reaction solution was filtered to obtain a filter cake, which was washed twice with 50 ml of dichloromethane (2 × 50 ml), three times with 50 ml of acetonitrile (3 × 50 ml), and once with 50 ml of ethyl acetate (1 × 50 ml), and then vacuum dried. Cap 1 (4.8 ml), Cap 2 (0.54 ml), and DMAP (2.59 mg) were added to the dried filter cake, and the reaction system was stirred at 25°C for 5 hours. After the reaction was completed, the reaction solution was filtered to obtain a filter cake, which was washed three times with 50 ml of acetonitrile (3×50 ml) and dried in vacuo to obtain compound CR01013Z (900 mg, loading amount of 20-30 μmol / g).

[0457] Cap1 and Cap2 are capping reagents, Cap1 is a 20% by volume N-methylimidazole mixed solution in pyridine / acetonitrile, with a volume ratio of pyridine to acetonitrile of 3:5; Cap2 is a 20% by volume acetic anhydride solution in acetonitrile.

[0458] Preparation Example 5: Preparation of nucleoside monomer NM054

[0459] In this preparation example, the synthetic route of nucleoside monomer NM054 is as follows:

[0460] (5-1) Synthesis of Compound NM054-2

[0461] Compound NM054-1 (3 g, 11.54 mmol, 1.0 eq, (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine, CAS No. 863329-66-2) and pyridine (30 ml) were added to a 500 ml reactor. The temperature was lowered to 0°C, and 4,4'-bismethoxytrityl chloride (4.29 g, 12.7 mmol, 1.1 eq) was added portionwise. The atmosphere was purged with nitrogen three times. The reaction system was stirred at 25°C under a nitrogen atmosphere for 3 hours. HPLC indicated the absence of starting material. After completion of the reaction, the reaction solution was concentrated and extracted with purified water (50 ml) and ethyl acetate (50 ml). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to yield compound NM054-2 (2.7 g, 41.7% yield). MS ESI (m / z) = 563.0 [M+H] + .

[0462] (5-2) Synthesis of Compound NM054

[0463] Compound NM054-2 (2.7 g, 4.8 mmol, 1.0 eq) was added to a 100 ml reactor. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.74 g, 5.76 mmol, 1.2 eq) was added portionwise. 4,5-Dicyanoimidazole (0.45 g, 3.8 mmol, 0.8 eq, CAS No. 1122-28-7) and dichloromethane (27 ml) were then added. The atmosphere was purged with nitrogen three times. The reaction system was stirred at 25°C under a nitrogen atmosphere for 3 hours. After completion of the reaction, aqueous sodium bicarbonate solution (20 ml) was added to the reaction mixture. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (eluent: acetonitrile / water = 90 / 10, v / v) to afford compound NM054 (3.0 g). MS ESI (m / z) = 763 [M+H] + .

[0464] Preparation of siRNA / siRNA conjugates

[0465] Preparation Example 6: Synthesis of siRNA

[0466] (6-1) Synthetic sense chain (SS)

[0467] Using the phosphoramidite nucleic acid solid-phase synthesis method, the solid-phase support compound is initially linked to nucleoside monomers one by one in the 3'-5' direction according to the nucleotide sequence. Each linking of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfurization. The synthesis conditions are given below:

[0468] The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0469] The deprotection reaction conditions for each step were identical: 25°C, 70 seconds, a 3% vol. dichloroacetic acid solution in dichloromethane as the deprotection reagent, and a 5:1 molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid support.

[0470] The conditions for each coupling reaction were the same. The coupling reaction conditions were: temperature 25°C, a molar ratio of the nucleic acid sequence attached to the solid support to the nucleoside monomer of 1:10, a molar ratio of the nucleic acid sequence attached to the solid support to the coupling reagent of 1:65, a reaction time of 600 seconds, a 0.5 M solution of 5-ethylthio-1H-tetrazole in acetonitrile as the coupling reagent, and a 0.2 M solution of hydrogenated xanthan gum in acetonitrile / pyridine (1:1 volume ratio of acetonitrile to pyridine) as the thiolation reagent.

[0471] The capping reaction conditions were identical for each step. The capping reaction conditions were: 25°C; 2 minutes; a 1:1 molar ratio of Cap1 and Cap2; Cap1: a 20% by volume N-methylimidazole solution in pyridine / acetonitrile (with a 3:5 volume ratio of pyridine to acetonitrile); and Cap2: a 20% by volume solution of acetic anhydride in acetonitrile. The molar ratio of the N-methylimidazole in Cap1 to the acetic anhydride in Cap2 to the nucleic acid sequence attached to the solid support was 1:1:1.

[0472] The conditions for each step of the oxidation reaction are the same. The conditions for the oxidation reaction are: temperature of 25°C; reaction time of 3 seconds; oxidizing agent concentration of 0.05M iodine water, the molar ratio of iodine to the nucleic acid sequence connected to the solid phase support in the coupling reaction is 30:1; the oxidation reaction is carried out in a water / pyridine mixed solvent (the volume ratio of water and pyridine is 1:9). The conditions for the sulfidation reaction are: temperature of 25°C; reaction time of 360 seconds; thiolation agent concentration of 0.2M hydrogenated xanthan gum pyridine solution, the molar ratio of thiolation agent to the nucleic acid sequence connected to the solid phase support in the coupling reaction is 4:1; the sulfidation reaction is carried out in a water / pyridine mixed solvent (the volume ratio of water and pyridine is 1:9). After the last nucleoside monomer is connected, the nucleic acid sequence connected to the solid phase support is cut, deprotected, purified, desalted, and then freeze-dried to obtain the positive chain, wherein:

[0473] Cleavage and deprotection conditions were as follows: the synthesized nucleotide sequence attached to the solid phase support was added to 0.5 ml / μmol of 25% by mass ammonia water at 55°C for 16 hours, the solvent was removed, and the product was concentrated to dryness in vacuo. After the ammonia treatment, the product was dissolved in 0.4 ml / μmol of N-methylpyrrolidone relative to the amount of single-stranded nucleic acid, followed by the addition of 0.3 ml / μmol of triethylamine and 0.6 ml / μmol of triethylamine trihydrofluoride to remove the 2'-O-TBDMS protection from the ribose.

[0474] Purification and desalting conditions: Nucleic acid purification was achieved using a preparative ion chromatography column (Source 15Q) with a NaCl gradient elution. Specifically, eluent 1 consisted of 20 mM sodium phosphate (pH 8.1) in a water / acetonitrile mixture (9:1 volume ratio of water to acetonitrile); eluent 2 consisted of 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1) in a water / acetonitrile mixture (9:1 volume ratio of water to acetonitrile); the elution gradient was eluent 1:eluent 2 = (100:0) to (50:50). The product eluates were collected and combined, and desalted using a reversed-phase chromatography column. Desalting conditions included using a Sephadex column with Sephadex G25 as the filler and eluting with deionized water.

[0475] Detection: Purity was determined using ion exchange chromatography (IEX-HPLC); molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value. The agreement between the measured and theoretical values ​​indicated that the compound was conjugated to the 3' end of the siRNA sense strand.

[0476] (6-2) Synthesis of antisense strand (AS)

[0477] The antisense chain was synthesized using a universal solid phase support. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection conditions, purification and desalting conditions in the solid phase synthesis method of the antisense chain are the same as those in step (6-1) for synthesizing the sense chain.

[0478] Detection: Purity was determined by ion exchange chromatography (IEX-HPLC); molecular weight was determined by liquid chromatography-mass spectrometry (LC-MS). The molecular weight was compared with the theoretical value. Consistency between the measured and theoretical values ​​indicated that the siRNA antisense strand was obtained.

[0479] (6-3) Synthesis of siRNA

[0480] The sense chain synthesized in step (6-1) and the antisense chain synthesized in step (6-2) were mixed in an equimolar ratio, dissolved in water for injection and heated to 95° C., slowly cooled to room temperature and maintained at room temperature for 10 minutes to allow the sense chain and antisense chain to form a double-stranded structure through hydrogen bonds, thereby obtaining siRNA having the sense chain and antisense chain shown in Table 2.

[0481] In the sequences disclosed herein, the base composition and modification meanings are as follows: capital letters A, U, G, C, and T represent the base composition of the nucleotides; lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide; lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide; combination mark (moe) represents that the nucleotide adjacent to the left of the combination mark (moe) is a 2'-O-methoxyethyl-modified nucleotide; lowercase letter s represents that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate bond.

[0482] Table 2 siRNA sequence information

[0483] Preparation Example 7 Preparation of siRNA Conjugate Conjugated to the 3' End of the Sense Strand (L96 Vector siRNA Conjugate)

[0484] Compound L96-PS

[0485] Compound L96-PS was purchased from Ailiying Pharmaceutical Group (Tianjin) Co., Ltd. with a loading of 120±12 μmol / g (detection method: UV / HPLC). The structural formula of compound L96-PS is as follows:

[0486] Wherein, PS represents polystyrene resin solid phase carrier.

[0487] (7-1) Synthesis of the positive chain:

[0488] Using the phosphoramidite nucleic acid solid-phase synthesis method, the L96-PS compound attached to the solid support is used as the initial cycle, and nucleoside monomers are attached one by one in the 3'-5' direction according to the nucleotide sequence. Each nucleoside monomer attachment involves four steps: deprotection, coupling, capping, and oxidation or sulfurization. The synthesis method and conditions refer to the positive strand synthesis method shown in step (6-1).

[0489] (7-2) Synthesis of antisense strand:

[0490] The siRNA antisense strand was synthesized according to the antisense strand synthesis method shown in step (6-2).

[0491] (7-3) Synthesis of L96 vector siRNA conjugates:

[0492] The L96 vector siRNA conjugate was synthesized according to the method shown in step (6-3).

[0493] The structural formula of the L96 vector siRNA conjugate is as follows:

[0494] in, L96 is conjugated to the 3' end of the sense strand of siRNA via a phosphodiester bond.

[0495] In the sequences disclosed herein, the base composition and modification meanings are as follows: capital letters A, U, G, C, and T represent the base composition of the nucleotides; lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide; lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide; lowercase letter d represents that the nucleotide adjacent to the left of the letter d is a 2'-deoxy-modified nucleotide; combination mark (moe) represents that the nucleotide adjacent to the left of the combination mark (moe) is a 2'-O-methoxyethyl-modified nucleotide; lowercase letter s represents that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate bond.

[0496] Table 3 siRNA conjugate information

[0497] Note: (Tgn) represents a nucleotide, and its structural formula is:

[0498] Preparation Example 8 Synthesis of siRNA Conjugate

[0499] (8-1) Sense and antisense strand synthesis

[0500] Using phosphoramidite solid-phase nucleic acid synthesis, starting with a carrier compound (e.g., Universal CPG carrier, Universal PS carrier, compound CR01008Z), nucleoside monomers are linked one by one along the nucleotide sequence from the 3' end to the 5' end. During the synthesis, compound CR01008 and compound NM054 are each considered a nucleoside monomer.

[0501] Each linking of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfurization. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection conditions, and purification and desalting conditions in the synthesis of the sense chain of this preparation example are the same as those for the synthesis of the sense chain in step (6-1).

[0502] During the synthesis of the positive chain, two clusters of CR01008 vectors (denoted as (CR01008×2) or (CR01008)×2), three clusters of CR01008 vectors (denoted as (CR01008×3) or (CR01008)×3), and four clusters of CR01008 vectors (denoted as (CR01008×4) or (CR01008)×4) were obtained.

[0503] The structural formula of the two-cluster CR01008 is:

[0504] The structural formula of the three-cluster CR01008 is

[0505] The structural formula of the four-cluster CR01008 is

[0506] (8-2) Synthesis of siRNA Conjugates

[0507] The siRNA conjugate was synthesized according to the method shown in step (6-3).

[0508] The structural formula of the siRNA conjugate in which the (CR01008)×3 vector is conjugated to the 3' end of the sense strand is as follows:

[0509] in, denoted as siRNA, SS represents the sense strand of the siRNA, and AS represents the antisense strand of the siRNA. (CR01008×3) is conjugated to the 3' end of the sense strand of the siRNA via a phosphodiester bond.

[0510] The structural formula of the siRNA conjugate with (CR01008)×4 vector conjugated to the 3' end of the sense strand is shown below:

[0511] in, denoted as siRNA, SS represents the sense strand of the siRNA, and AS represents the antisense strand of the siRNA. (CR01008×4) is conjugated to the 3' end of the sense strand of the siRNA via a phosphodiester bond.

[0512] The structural formula of the siRNA conjugate with two (CR01008) × 2 vectors conjugated to the 5' end of the sense strand and the 3' end of the antisense strand is shown below:

[0513] in, denoted as siRNA, SS represents the sense strand of siRNA, and AS represents the antisense strand of siRNA. Two (CR01008×2) were conjugated to the 5' end of the sense strand and the 3' end of the antisense strand of siRNA, respectively, through a phosphodiester bond.

[0514] In the sequences disclosed herein, the base composition and modification meanings are as follows: capital letters A, U, G, C, and T represent the base composition of the nucleotides; lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide; lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide; combination mark (moe) represents that the nucleotide adjacent to the left of the combination mark (moe) is a 2'-O-methoxyethyl-modified nucleotide; lowercase letter s represents that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate bond.

[0515] Wherein, (NM054) represents a nucleotide, and its structural formula is

[0516] Table 4-1 siRNA conjugate information

[0517] Table 4-2 siRNA conjugate information

[0518] Biological detection experiments

[0519] Unless otherwise stated, the reagents and consumables (Table 5) and instruments and equipment (Table 6) used in this disclosure were all commercially available products from the following manufacturers.

[0520] Table 5 Main reagents and consumables

[0521] Table 6 Main instruments and equipment

[0522] Example 1. In vitro activity evaluation of angiotensinogen (AGT) siRNA

[0523] In this example, the target gene inhibitory activity evaluation method in the human liver cancer cell line Huh7 was used to evaluate the inhibitory activity of RX003001 to RX003224 targeting the AGT target, and the negative control compound RX000001 on the target gene AGT.

[0524] Test sample preparation:

[0525] After centrifugation, dissolve each siRNA sample in DEPC H2O according to the specifications of each tube to prepare a 20 μM stock solution. This stock solution was then serially diluted with DEPC H2O to produce 0.04 μM and 0.004 μM working solutions. Dose screening experiments were performed at final duplex concentrations of 1 nM and 0.1 nM.

[0526] 96-well transfection and detection:

[0527] Huh7 cells grown to near confluence were trypsinized, washed, and a cell suspension was prepared for plating in 96-well plates.

[0528] Disperse 5 μL of 0.04 μM and 0.004 μM working solutions in 20 μL of Opti-MEM to form an siRNA mixture. Disperse 0.2 μL of RNAiMAX in 24.8 μL of Opti-MEM. After incubation for 5 minutes, mix with each siRNA mixture and incubate for 10 minutes to form a transfection complex. Add 150 μL of cell suspension (12,000 cells) per well to the transfection complex, and place the 96-well plate in an incubator for an additional 24 hours.

[0529] For the mock control group, 0.2 μL RNAiMAX was dispersed in 24.8 μL Opti-MEM and incubated for 5 minutes. Then, 25 μL Opti-MEM was added and incubated for 10 minutes to form a transfection complex. 150 μL of cell suspension (12,000 cells) was added to the transfection complex per well, and the 96-well plate was placed in an incubator and incubated for 24 hours.

[0530] Remove the 96-well plate, lyse the cells, and dilute the lysate. Use a one-step qPCR kit (Thermo Fisher, Power SYBR Green RNA-to-CT TM 1-Step, 4389986) Target gene mRNA expression in Huh7 cells was measured using a fluorescent quantitative PCR instrument (Roche, LightCycler 480). In this real-time fluorescent quantitative PCR method, the TATA-box binding protein (TBP) gene was used as an internal reference gene. Primers targeting the target gene and primers targeting the TBP internal reference gene were used to detect the target gene and TBP internal reference gene, respectively. See Table 7 for the sequences of the detection primers.

[0531] Table 7 Sequences of detection primers

[0532] In the real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0533] Table 8 Inhibitory activity of target genes in Huh7 cells after administration of the siRNA described in this example

[0534] Example 2. Evaluation of the activity of L96 vector-conjugated siRNA in Balb / c-HDI mice

[0535] In this example, the Balb / c mouse hydrodynamic injection model was used to evaluate the inhibitory activity of the same AGT target siRNA sense strand 3' end conjugated to L96 vector conjugates RZ003002 to RZ003023 on the target gene AGT, with RZ000002 used as a negative control.

[0536] Plasmid construction:

[0537] The pcDNA-CMV-RG003 plasmid (ID: NM_001382817.3) was constructed by Sangon Biotech (Shanghai) Co., Ltd.

[0538] Mouse model construction:

[0539] The Balb / c mouse hydrodynamic injection model is established by rapidly injecting a pcDNA-CMV-RG003 plasmid solution through the tail vein at high pressure. On day 3 of the experiment, mice were injected with 10 μg of pcDNA-CMV-RG003 via the tail vein hydrodynamic injection over 5 seconds, at an injection volume of 8% of their body weight. The plasmid DNA for injection was diluted with normal saline. The solution was prepared immediately prior to injection and stored at 4°C.

[0540] Animal grouping, drug administration and tissue sample collection:

[0541] 6-8 week old Balb / c mice were randomly divided into groups (all female) by body weight, with 5 mice in each group. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. All mice were dosed according to body weight, and a single dose was administered by subcutaneous injection in the abdomen. Each drug conjugate was administered in the form of a 0.1 mg / mL (calculated as siRNA) PBS solution, and the administration volume was 10 mL / kg mouse body weight. That is, the dosage of each drug conjugate was 1 mg / kg mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PBS solution (not containing drug conjugate). The administration day was recorded as day 0 (recorded as D0), and plasmid injection was performed on the 3rd day after administration (recorded as D3). On the 4th day (recorded as D4), 5 mice of all groups were killed. The mice were subjected to gross dissection and the liver tissue of each mouse was collected. The liver tissue was cut into about 2 mm 3 Small pieces were stored with RNA later.

[0542] For each mouse, an appropriate amount of liver tissue sample was taken from RNA later, and the liver tissue sample was disrupted in a Tissuelyser II fully automatic tissue homogenizer for 60 seconds. Then, total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction.

[0543] For each mouse, 1 μg of total RNA was collected and reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) was used to select Oligo (dT) 18 Reverse transcription primers were prepared according to the method described in the reverse transcription kit instructions to configure 20 μL reverse transcription system and complete the reverse transcription reaction. After the reaction was completed, 60 μL RNase-Free water was added to the reverse transcription system to obtain a cDNA solution. The expression of the target gene mRNA in the animal was then detected 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 Nero gene on the plasmid backbone was used as an internal reference gene, and primers for the target gene and the Nero internal reference gene were used to detect the target gene and the Nero internal reference gene, respectively. The sequences of the detection primers are shown in Table 9.

[0544] Table 9 Sequences of detection primers

[0545] According to the instructions of the real-time fluorescence quantitative PCR kit, 10 μL of real-time PCR reaction system was prepared for each PCR detection well. Each reaction system contained 4 μL of the cDNA solution obtained by the reverse transcription reaction, 5 μL of TaqMan TMFast Advanced Master Mix (2×), 0.15 μL 10 μM upstream primer, 0.15 μL 10 μM downstream primer (primer information see Table X), 0.15 μL 10 μM probe primer, 0.55 μL RNase-Free H2O. The prepared reaction system was placed on 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 program was 50°C for 2 minutes, followed by pre-denaturation at 95°C for 20 seconds, denaturation at 95°C for 3 seconds, annealing and extension at 60°C for 30 seconds, and the denaturation, annealing and extension process was repeated for 40 cycles. In this real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0546] The results of Example 2 showed that at a dose of 1 mg / kg, RZ003003, RZ003004, RZ003005, RZ003008, RZ003010, RZ003017, RZ003020, and RZ003021 could significantly inhibit AGT mRNA expression (Table 10).

[0547] Table 10. Inhibitory activity of target genes in Balb / c-HDI mice after administration of the siRNA conjugates described in this example

[0548] Example 3. Evaluation of Multiple-Dose Activity of L96 Vector-Conjugated siRNA in Balb / c-HDI Mice

[0549] In this example, the Balb / c mouse hydrodynamic injection model was used to evaluate the inhibitory activity of the same AGT target siRNA sense chain 3' end conjugated to the L96 carrier conjugates RZ003003, RZ003004, RZ003005, RZ003008, RZ003010, RZ003017, RZ003020, and RZ003021 on the target gene AGT at different doses, with Zilebesiran used as a positive control.

[0550] Plasmid construction:

[0551] The pcDNA-CMV-RG003 plasmid (ID: NM_001382817.3) was constructed by Sangon Biotech (Shanghai) Co., Ltd.

[0552] Mouse model construction:

[0553] The Balb / c mouse hydrodynamic injection model is established by rapidly injecting a pcDNA-CMV-RG003 plasmid solution through the tail vein at high pressure. On day 3 of the experiment, mice were injected with 10 μg of pcDNA-CMV-RG003 via the tail vein hydrodynamic injection over 5 seconds, at an injection volume of 8% of their body weight. The plasmid DNA for injection was diluted with normal saline. The solution was prepared immediately prior to injection and stored at 4°C.

[0554] Animal grouping, drug administration and tissue sample collection:

[0555] 6-8 week old Balb / c mice were randomly divided into groups (all female) according to body weight, with 5 mice in each group. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. All mice were dosed according to body weight and administered subcutaneously in the abdomen. Each drug conjugate was administered in the form of 0.05 mg / mL, 0.1 mg / mL, 0.3 mg / mL (calculated as siRNA) PBS solution, and the administration volume was 10 mL / kg mouse body weight, that is, the dosage of each drug conjugate was 0.5 mg / kg, 1 mg / kg, 3 mg / kg mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PBS solution (not containing drug conjugate). The day of administration was recorded as day 0 (recorded as D0), and plasmid injection was performed on the 3rd day after administration (recorded as D3). On the 4th day (recorded as D4), 5 mice of all groups were killed. The mice were subjected to gross anatomy and the liver tissue of each mouse was collected. The liver tissue was cut into about 2 mm 3 Small pieces were stored with RNA later.

[0556] For each mouse, an appropriate amount of liver tissue sample was taken from RNA later, and the liver tissue sample was disrupted in a Tissuelyser II fully automatic tissue homogenizer for 60 seconds. Then, total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction.

[0557] For each mouse, 1 μg of total RNA was collected and reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) was used to select Oligo (dT) 18Reverse transcription primers were prepared according to the method described in the reverse transcription kit instructions to configure 20 μL reverse transcription system and complete the reverse transcription reaction. After the reaction was completed, 60 μL RNase-Free water was added to the reverse transcription system to obtain a cDNA solution. The expression of the target gene mRNA in the animal was then detected 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 Nero gene on the plasmid backbone was used as an internal reference gene, and primers for the target gene and the Nero internal reference gene were used to detect the target gene and the Nero internal reference gene, respectively. The sequences of the detection primers are shown in Table 11.

[0558] Table 11. Sequences of detection primers

[0559] According to the instructions of the real-time fluorescence quantitative PCR kit, 10 μL of real-time PCR reaction system was prepared for each PCR detection well. Each reaction system contained 4 μL of the cDNA solution obtained by the reverse transcription reaction, 5 μL of TaqMan TM Fast Advanced Master Mix (2×), 0.15 μL 10 μM upstream primer, 0.15 μL 10 μM downstream primer (primer information see Table X), 0.15 μL 10 μM probe primer, 0.55 μL RNase-Free H2O. The prepared reaction system was placed on 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 program was 50°C for 2 minutes, followed by pre-denaturation at 95°C for 20 seconds, denaturation at 95°C for 3 seconds, annealing and extension at 60°C for 30 seconds, and the denaturation, annealing and extension process was repeated for 40 cycles. In this real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0560] The results of Example 3 showed that RZ003003, RZ003004, RZ003005, RZ003008, RZ003010, RZ003017, RZ003020, and RZ003021 could dose-dependently inhibit AGT mRNA expression; among them, at a dose of 3 mg / kg, RZ003003, RZ003017, RZ003020, and RZ003021 reduced AGT mRNA expression by up to 95%, which was superior to zilebesiran ( Figure 1 , Table 12).

[0561] Table 12 Inhibitory activity of target genes in Balb / c-HDI mice after administration of the siRNA conjugates described in this example

[0562] Example 4. Evaluation of the efficacy of L96 vector-conjugated siRNA in hREN×hAGT hypertensive mice

[0563] In this example, a small animal non-invasive blood pressure measuring instrument was used to measure the systolic blood pressure, diastolic blood pressure, and mean arterial pressure of mice to evaluate the blood pressure regulatory effects of RZ003008, RZ003017, RZ003020, and RZ0030021 in mice. An ELISA method was used to determine the expression of AGT protein in serum at different time points, using zilebesiran as a control.

[0564] Animal grouping, drug administration and tissue sample collection:

[0565] 9-11 week old hREN×hAGT hypertensive mice (purchased from Saiye (Suzhou) Biotechnology Co., Ltd.) were grouped according to systolic blood pressure levels (all male), with 7 mice per group. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. The drug dose for all mice was calculated based on body weight, and a single dose was administered by subcutaneous injection in the abdomen. Each drug conjugate was administered in the form of a 0.3 mg / mL (calculated as siRNA) PBS solution, and the administration volume was 10 mL / kg mouse body weight, that is, the administration dose of each drug conjugate was 3 mg / kg mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PBS solution (without drug conjugate). The day of administration was recorded as day 0 (recorded as D0), and the day after administration was recorded as day 3 (recorded as D3), day 7 (recorded as D7), day 10 (recorded as D10), day 14 (recorded as D14), day 17 (recorded as D17), day 21 (recorded as D21), day 24 (recorded as D24), day 28 (recorded as D28), day 31 (recorded as D31), day 35 (recorded as D35), day 38 (recorded as D38) Systolic blood pressure (SBP), mean arterial pressure (MAP), and diastolic blood pressure (DBP) were measured using a small animal non-invasive blood pressure meter (Softlon, BP-2010A); serum was collected from all groups of mice on the 8th day (denoted as D8), 15th day (denoted as D15), 29th day (denoted as D29), and 43rd day (denoted as D43) after administration, and AGT protein expression was determined using a human angiotensinogen kit (IBL, 27412).

[0566] The results of Example 4 demonstrate that, at a single 3 mg / kg dose, RZ003008, RZ003017, RZ003020, and RZ003021 reduced systolic, mean arterial, and diastolic blood pressure in hREN×hAGT hypertensive mice; blood pressure gradually recovered over time (Figure 2, Table 13, Table 14, Table 15). RZ003021 reduced systolic blood pressure by more than 20 mmHg, an effect that persisted for 24 days. Zilebesiran, however, had no significant systolic blood pressure-lowering effect at the same time point (Figure 2, Table 13, Table 14, Table 15).

[0567] The results of Example 4 showed that, at a single dose of 3 mg / kg, RZ003017, RZ003020, and RZ003021 could reduce AGT protein levels in hREN×hAGT hypertensive mice; AGT protein levels gradually recovered with prolonged observation; no significant AGT protein inhibitory effect was observed with a single dose of 3 mg / kg of zilebesiran (Figure 3, Table 16).

[0568] Table 16 Changes in human AGT protein levels in hREN×hAGT hypertensive mice after administration of the siRNA conjugates described in this example

[0569] Example 5. Toxicological evaluation of L96 vector-conjugated siRNA in mice

[0570] In this example, a single subcutaneous injection of 300 mg / kg was used to evaluate the nature and extent of the toxic reactions of the carrier conjugates RZ003004, RZ003008, RZ003010, RZ003017, RZ003020, and RZ0030021 in ICR mice.

[0571] Animal grouping, drug administration and tissue sample collection:

[0572] 6-8 week old ICR mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were randomly divided into groups according to body weight, with 10 mice per group, half male and half female. Each test group was given a predetermined dose of the drug conjugate and a PBS control group was added. The drug dose was calculated based on the body weight of all mice and administered as a single subcutaneous injection in the abdomen. Each drug conjugate was administered as a 30 mg / mL (calculated as siRNA) PBS solution, with a dosing volume of 10 mL / kg mouse body weight, i.e., the dose of each drug conjugate was 300 mg / kg mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PBS solution (without the drug conjugate) and observed for 14 days. The day of administration was designated as day 0 (denoted as D0). Clinical observations were performed at least once a day after the first dose during the experimental period, and body weight and food intake were measured twice a week. On the 14th day of the experiment (denoted as D14), blood was drawn from all animals (mice were fasted for at least 12 hours before sampling and testing, but water was not allowed) for hematological and blood biochemical tests. Blood was collected and anticoagulated with EDTA-K2, and routine blood tests were performed using an automated modular blood and body fluid analyzer (Sysmex XN-10B1). Blood samples (without anticoagulation) were collected and allowed to stand at room temperature for approximately 30 minutes. After blood coagulation, they were centrifuged at 2000 g for 10 minutes at 4°C, and blood biochemical analysis was performed using a fully automated biochemical analyzer (Sysmex BX-4000). All animals were dissected on day 14 of the experiment (designated D14). Mice were fasted for at least 12 hours prior to dissection and anesthetized with Zotal 50 (Vicco, 8G4VA, France). After blood collection, the animals were euthanized by exsanguination of the abdominal aorta for gross anatomical examination. Histopathological examination of the liver and kidneys of all animals was performed using hematoxylin-eosin staining. The results showed that none of the test substances had significant effects on the general condition, body weight, and gross anatomical observations of ICR mice. The RZ003017 300mg / kg group induced an increase in megakaryocytes in the liver, and central venous inflammatory cell infiltration was observed in the livers of some animals. Except for the RZ00300 4300mg / kg group, increased mitosis was observed in the livers of some animals in all other groups. No pathological changes related to the test substances were observed in the kidneys.

[0573] Example 6. In vitro activity evaluation of CR01008 vector-conjugated siRNA

[0574] This example uses the target gene inhibitory activity assessment method in the human liver cancer cell line Huh7 to evaluate the inhibitory activity of the CR01008 vector conjugate siRNA on the target gene AGT in cells.

[0575] Test sample preparation:

[0576] After centrifugation, dissolve each siRNA sample in PBS according to the specifications of each tube to prepare a 20 μM stock solution. This stock solution was then serially diluted with PBS to produce 0.1 μM and 0.01 μM working solutions. Dosage testing was performed at final duplex concentrations of 1 nM and 0.1 nM.

[0577] 96-well transfection and detection:

[0578] Huh7 cells grown to near confluence were trypsinized and washed to prepare a cell suspension. 100 μL of this suspension was added to each well of a 96-well plate, with a cell count of 12,000 cells per well. The cells were cultured at 37°C in a 5% CO2 incubator. After 24 hours of cell attachment, the DMEM medium in the 96-well plate was aspirated and 80 μL of Opti-MEM™ medium was added to each well. The 96-well plate was then placed in the incubator and continued to be cultured. 1 μL of the 0.1 μM and 0.01 μM working solutions were diluted in 9 μL of Opti-MEM to form the siRNA mixture. 0.3 μL of RNAiMAX was diluted in 9.7 μL of Opti-MEM and mixed with each siRNA mixture to form the transfection complex. The transfection complex was incubated at room temperature for 10 minutes, and then 20 μL of the transfection complex was added to each well of the 96-well plate. After 4 hours of incubation, each well was supplemented with 100 μL of DMEM medium containing 20% ​​FBS, and the 96-well plate was placed in an incubator for a further 24 hours. For the mock control group, 0.3 μL of RNAiMAX was dispersed in 9.7 μL of Opti-MEM, followed by 10 μL of Opti-MEM, and incubated at room temperature for 10 minutes; this was then added to the 96-well plate at 20 μL / well. After 4 hours of incubation, each well was supplemented with 100 μL of DMEM medium containing 20% ​​FBS, and the 96-well plate was placed in an incubator for a further 24 hours. The 96-well plate was removed, and total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to standard total RNA extraction procedures.

[0579] Use reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) and select Oligo (dT) 18Reverse transcription primers, according to the method configuration 20 μ L reverse transcription system of reverse transcription kit specification sheets and complete reverse transcription reaction.Then use real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific company, TaqMan Fast Advanced Master Mix, 4444557) to detect the expression of target gene mRNA in Hela cells on fluorescence quantitative PCR instrument (Roche company, LightCycler 480).In this real-time fluorescence quantitative PCR method, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as internal reference gene, use the primer for target gene and the primer for GAPDH internal reference gene to detect target gene and GAPDH internal reference gene respectively.The sequence of detection primer is referring to Table 17.

[0580] Table 17 Sequences of detection primers

[0581] According to the instructions of the real-time fluorescence quantitative PCR kit, 10 μL of real-time PCR reaction system was prepared for each PCR detection well. Each reaction system contained 4 μL of the cDNA solution obtained by the reverse transcription reaction, 5 μL of TaqMan TM Fast Advanced Master Mix (2×), 0.15 μL 10 μM upstream primer, 0.15 μL 10 μM downstream primer (primer information see Table X), 0.15 μL 10 μM probe primer, 0.55 μL RNase-Free H2O. The prepared reaction system was placed on 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 program was 50°C for 2 minutes, followed by pre-denaturation at 95°C for 20 seconds, denaturation at 95°C for 3 seconds, annealing and extension at 60°C for 30 seconds, and the denaturation, annealing and extension process was repeated for 40 cycles. In this real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0582] Table 18 Inhibitory activity of target genes in Huh7 cells after administration of the siRNA conjugates described in this example

[0583] The results of Example 6 showed that at doses of 1 nM and 0.1 nM, the differently modified CR01008 vector-conjugated siRNAs could significantly inhibit the expression of AGT mRNA in Huh7 cells (Table 18).

[0584] Example 7. Evaluation of the antihypertensive effect of CR01008 vector-conjugated siRNA in hREN×hAGT hypertensive mice

[0585] In this example, a small animal non-invasive blood pressure measuring instrument was used to measure the systolic blood pressure of mice to evaluate the blood pressure regulating effects of RZ003062, RZ003064, RZ003065, RZ003066, and RZ0030021 in mice, with Zilebesiran used as a positive control.

[0586] Animal grouping, drug administration and tissue sample collection:

[0587] 9-11 week old hREN×hAGT hypertensive mice (purchased from Saiye (Suzhou) Biotechnology Co., Ltd.) were grouped according to systolic blood pressure levels (all male). Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. The drug dose for all mice was calculated based on body weight and administered as a single dose by subcutaneous injection in the abdomen. Each drug conjugate was administered in the form of a 0.3 mg / mL (calculated as siRNA) PBS solution at a dosing volume of 10 mL / kg mouse body weight, i.e., the dose of each drug conjugate was 3 mg / kg mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PBS solution (without drug conjugate). Systolic blood pressure (SBP) was measured using a small animal non-invasive blood pressure meter (Softlong, BP-2010A) before administration (recorded as pre-dose, pre-dose value), the day of administration was recorded as day 0 (recorded as D0), and on the 3rd day (recorded as D3), 6th day (recorded as D6), 10th day (recorded as D10), 13th day (recorded as D13), 17th day (recorded as D17), 20th day (recorded as D20), 24th day (recorded as D24), 27th day (recorded as D27), and 31st day (recorded as D31) after administration.

[0588] The results of Example 7 demonstrate that, at a single 3 mg / kg dose, RZ003062, RZ003064, RZ003065, and RZ003066 reduced systolic blood pressure in hREN×hAGT hypertensive mice; blood pressure gradually recovered over time ( Figure 5 , Table 19 ). RZ003064 and RZ003021 achieved comparable systolic blood pressure-lowering effects, while zilebesiran had no significant systolic blood pressure-lowering effect at the same time point ( Figure 5 , Table 19 ).

[0589] Table 19 Changes in systolic blood pressure in hREN×hAGT hypertensive mice after administration of the siRNA conjugates described in this example

[0590] Example 8. Evaluation of the protein-lowering effect of CR01008 vector-conjugated siRNA in hREN×hAGT hypertensive mice

[0591] In this example, enzyme-linked immunosorbent assay (ELISA) was used to determine the expression of AGT protein in the serum of mice at different time points after a single administration of CR01008 vector conjugates RZ003064, RZ003065, RZ003066, and RZ0030021.

[0592] Animal grouping, drug administration and tissue sample collection:

[0593] 9-11 week old hREN×hAGT hypertensive mice (purchased from Saiye (Suzhou) Biotechnology Co., Ltd.) were grouped according to systolic blood pressure levels (all male). Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. The drug dose for all mice was calculated based on body weight and administered as a single dose by subcutaneous injection in the abdomen. Each drug conjugate was administered in the form of a 0.3 mg / mL (calculated as siRNA) PBS solution at a dosing volume of 10 mL / kg mouse body weight, i.e., the dose of each drug conjugate was 3 mg / kg mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PBS solution (without drug conjugate). Before administration (denoted as pre-dose, pre-drug value), the day of administration was designated as day 0 (denoted as D0), and on the 7th day (denoted as D7), 14th day (denoted as D14), 21st day (denoted as D21), and 28th day (denoted as D28) after administration, sera were collected from all groups of mice, and AGT protein expression was determined using a human angiotensinogen kit (IBL, 27412).

[0594] Table 20: Changes in human AGT protein levels in transgenic mice after administration of the siRNA conjugates described in this example

[0595] The results of Example 8 showed that at a single dose of 3 mg / kg, RZ003064, RZ003065, RZ003066, and RZ003021 could significantly reduce the AGT protein level in hREN×hAGT hypertensive mice; it was observed that on day 28, the AGT protein level was still reduced by more than 70% (Figure 6, Table 20).

[0596] Example 9. Evaluation of the efficacy of L96 vector-conjugated siRNA in SHR rats

[0597] In this example, a small animal non-invasive blood pressure measuring instrument was used to measure the systolic blood pressure, diastolic blood pressure, and mean arterial pressure of mice to evaluate the blood pressure regulatory effects of RZR03001 and RZR03002 in SHR rats. An enzyme-linked immunosorbent assay (ELISA) was used to determine the expression of AGT protein in serum at different time points, using valsartan (Beijing Novartis Pharmaceuticals Co., Ltd., X3113) as a control.

[0598] Animal grouping, drug administration and tissue sample collection:

[0599] 11-12 week old SHR rats (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were grouped according to systolic blood pressure levels (all male), with 10 rats per group. Each test group was given a predetermined dose of the drug conjugate and a PBS control group was added. The drug dose for all rats was calculated based on body weight, and the administration volume was 10 mL / kg of mouse body weight. RZR03001 and RZR03002 were administered as a single subcutaneous injection in the abdomen, with each drug conjugate administered in the form of a 0.3 mg / mL (calculated as siRNA) PBS solution, i.e., the dosage of each drug conjugate was 3 mg / kg of rat body weight (calculated as siRNA). The valsartan dose group was prepared with PBS to a 2.16 mg / mL solution and orally administered twice a day, i.e., the drug dosage was 21.6 mg / kg; the PBS control group was given the same volume of PBS solution (without the drug conjugate).

[0600] Before administration (denoted as pre-dose, pre-dose value), the day of administration was designated as day 0 (denoted as D0). On the 7th day (denoted as D7), the 14th day (denoted as D14), the 21st day (denoted as D21), and the 28th day (denoted as D28) after administration, blood pressure (SBP, DBP, MAP) was measured using a small animal non-invasive blood pressure measuring instrument (Ruanlong, BP-2010A). On the 7th day (denoted as D7), the 14th day (denoted as D14), the 21st day (denoted as D21), and the 28th day (denoted as D28) after administration, serum was collected from all groups of rats using a small animal non-invasive blood pressure measuring instrument (Ruanlong, BP-2010A), and the expression of AGT and AGT-Ⅱ proteins was determined using a human angiotensinogen kit (IBL, 27412) and a rat Ang-Ⅱ kit (Shanghai Zhuocai, E-EL-R1430c), respectively. On the 7th day after administration (denoted as D7), 4 rats in all groups were killed, and on the 28th day after administration (denoted as D28), 6 rats in all groups were killed. The rats were subjected to gross anatomy and the liver tissue of each rat was collected. The liver tissue was cut into approximately 2 mm 3Small pieces were stored in RNA later and AGT mRNA expression levels were measured. In this real-time fluorescence quantitative PCR method, ActinB was used as an internal reference gene. Primers targeting the target gene and primers targeting the ActinB internal reference gene were used to detect the target gene and ActinB internal reference gene, respectively. See Table 21 for the sequences of the detection primers.

[0601] Table 21 Sequences of detection primers

[0602] In the real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0603] The results of Example 9 show that a single dose of 3 mg / kg of RZR03001 and RZR03002 can reduce systolic, mean arterial, and diastolic blood pressure in SHR rats; blood pressure levels continue to decrease gradually through day 28. Starting from day 21, a single dose of RZR03001 achieved a blood pressure-lowering effect similar to that of valsartan ( Figure 7 , Tables 22 , 23 , and 24 ).

[0604] The results of Example 9 demonstrate that, at a single 3 mg / kg dose, RZR03001 and RZR03002 were able to reduce AGT mRNA and protein levels in SHR rats. On day 28, the mRNA reduction effect was still greater than 90% (Table 25), and the AGT protein reduction effect was still greater than 70% (Table 26). Furthermore, RZR03001 and RZR03002 demonstrated a modest effect on reducing Ang-II protein on day 28 (Table 27).

[0605] Table 25: Changes in AGT mRNA levels in SHR rats after administration of the siRNA conjugates described in this example

[0606] Table 26 Changes in AGT protein levels in SHR rats after administration of the siRNA conjugates described in this example

[0607] Table 27 Changes in Ang-II protein levels in SHR rats after administration of the siRNA conjugates described in this example

[0608] Example 10 Evaluation of the efficacy of CR01008 vector-conjugated siRNA in normal cynomolgus monkeys

[0609] In this example, the ELISA method was used to determine the expression of AGT protein in the serum of cynomolgus monkeys at different time points after a single administration of RZ003069, RZ003073, RZ003076, RZ003089, and RZ003093.

[0610] Animal grouping, drug administration and tissue sample collection:

[0611] Healthy cynomolgus monkeys weighing 2.5-5 kg ​​were grouped according to serum AGT protein levels, with 3 males in each group. Each test group was given a predetermined dose of drug conjugate and a vehicle control group was added. The dosage of all animals was calculated based on body weight, and a single dose was administered by subcutaneous injection in the interscapular region of the back. Each drug conjugate was administered in the form of 6 mg (calculated as siRNA) / mL of normal saline (i.e., 0.9% sodium chloride injection), and the administration volume was 1 mL / kg (cynomolgus monkey body weight), that is, the dosage of each drug conjugate was 6 mg (calculated as siRNA) / kg (cynomolgus monkey body weight). The vehicle control group was given 1 mL / kg (cynomolgus monkey body weight) of 0.9% sodium chloride injection without siRNA conjugate. Serum was collected from cynomolgus monkeys on the day of administration (recorded as day 1, before administration), the 8th day (recorded as D8), the 15th day (recorded as D15), the 22nd day (recorded as D22), the 29th day (recorded as D29), the 36th day (recorded as D36), the 43rd day (recorded as D43), the 50th day (recorded as D50), and the 57th day (recorded as D57) after administration, and the AGT protein expression was determined using a human angiotensinogen (AGT) kit (IBL, 27412). Among them, the vehicle control group and the RZ003089 group continued to collect crab-eating macaque serum on the 64th day (recorded as D64), the 71st day (recorded as D71), the 78th day (recorded as D78), the 85th day (recorded as D85), the 92nd day (recorded as D92), the 99th day (recorded as D99), the 106th day (recorded as D106), the 132nd day (recorded as D132), the 146th day (recorded as D146), the 160th day (recorded as D160), the 174th day (recorded as D174), and the 188th day (recorded as D188) to determine the AGT protein expression.

[0612] Table 28 Changes in AGT protein levels in cynomolgus monkey serum after administration of the siRNA conjugates described in this example

[0613] The results of Example 10 are shown in Table 28 and Figure 8. The results indicate that, at a single dose of 6 mg / kg, RZ003069, RZ003073, RZ003076, RZ003089, and RZ003093 can all significantly reduce the AGT protein level in the serum of cynomolgus monkeys. At different time points, RZ003089 has the best effect on serum AGT protein, with the maximum inhibitory effect reaching more than 90%. It was also observed that D188 still had a protein-lowering effect of about 50%.

[0614] Example 11 Comparison of the efficacy of RZ003089 modified and optimized compounds in normal cynomolgus monkeys

[0615] In this example, the ELISA method was used to determine the expression of AGT protein in the serum of cynomolgus monkeys at different time points after a single administration of RZ003089, R303079, R303082, R303086, R303087, R303088, and R303089.

[0616] Animal grouping, drug administration and tissue sample collection:

[0617] Healthy cynomolgus monkeys weighing 3-5 kg ​​were grouped according to serum AGT protein levels, with 3 males in each group. Each test group was given a predetermined dose of drug conjugate and a vehicle control group was added. The dosage of all animals was calculated based on body weight, and a single dose was administered by subcutaneous injection in the interscapular region of the back. Each drug conjugate was administered in the form of 6 mg (calculated as siRNA) / mL of 0.9% sodium chloride injection, and the administration volume was 1 mL / kg (cynomolgus monkey body weight), that is, the dosage of each drug conjugate was 6 mg (calculated as siRNA) / kg (cynomolgus monkey body weight). The vehicle control group was given 1 mL / kg (cynomolgus monkey body weight) of 0.9% sodium chloride injection without siRNA conjugate. Serum was collected from cynomolgus monkeys on the day of dosing (designated as Day 1, pre-dose) (designated as D0), and on the 7th day (designated as D7), 14th day (designated as D14), 21st day (designated as D21), 28th day (designated as D28), 35th day (designated as D35), and 42nd day (designated as D42) after dosing. AGT protein expression was measured using a human angiotensinogen (AGT) kit (IBL, 27412). This trial is ongoing.

[0618] Table 29 Changes in AGT protein levels in cynomolgus monkey serum after administration of the siRNA conjugates described in this example

[0619] The results of Example 11 are shown in Table 29 and Figure 9. The results indicate that, at a single dose of 6 mg / kg, RZ003069, RZ003073, RZ003076, RZ003089, and RZ003093 can significantly reduce the AGT protein level in the serum of cynomolgus monkeys. As the observation time increases, the effects of R303082 and R303089 on serum AGT protein are slightly better than those of RZ003089.

[0620] Although the embodiments of the present disclosure have been shown and described above, it is understood that the above embodiments are illustrative and are not to be construed as limitations on the present disclosure. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present disclosure.

Claims

1. A double-stranded oligonucleotide, characterized in that The double-stranded oligonucleotide comprises a sense strand and an antisense strand; The antisense strand comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides from any one of SEQ ID NO: 225 to SEQ ID NO: 448, or comprises a nucleotide sequence having 1, 2, or 3 nucleotide differences from the consecutive nucleotides; The sense strand contains at least 17 nucleotides and the sense strand is complementary or substantially complementary to the antisense strand to form a duplex region; the substantially complementary means that the mismatch between the sense strand and the antisense strand in the duplex region does not exceed 3 nucleotides; Each nucleotide in the double-stranded oligonucleotide is independently selected from modified or unmodified nucleotides.

2. The double-stranded oligonucleotide according to claim 1, characterized in that From the 5' end to the 3' end, nucleotides 2 to 19 of the antisense strand comprise a complementary region to an AGT RNA transcript; wherein the complementary region comprises a nucleotide sequence of at least 17 consecutive nucleotides in any one of SEQ ID NOs. 225-448, or comprises a nucleotide sequence having 1, 2 or 3 nucleotide differences from the consecutive nucleotides; Optionally, the complementary region comprises a nucleotide sequence having 0, 1, 2 or 3 nucleotide differences from any one of SEQ ID NOs. 225-448; Optionally, the antisense strand comprises a sequence having 0, 1, 2, or 3 nucleotide differences from any one of SEQ ID NO:225-SEQ ID NO:448; Optionally, the antisense strand comprises a sequence having 0 or 1 nucleotide difference from any one of SEQ ID NO:225-SEQ ID NO:448; Optionally, the antisense strand is selected from or comprises any one of SEQ ID NO:225-SEQ ID NO:

448.

3. The double-stranded oligonucleotide according to claim 1 or 2, characterized in that The sense strand comprises a nucleotide sequence of at least 17, at least 18, or at least 19 consecutive nucleotides from any one of SEQ ID NO: 1 to SEQ ID NO: 224, or comprises a nucleotide sequence having 1, 2, or 3 nucleotide differences from the consecutive nucleotides; Optionally, the sense strand comprises a sequence having 0, 1, 2, or 3 nucleotide differences from any one of SEQ ID NO: 1-SEQ ID NO: 224; Optionally, the sense strand comprises a sequence having 0 or 1 nucleotide difference from any one of SEQ ID NO: 1-SEQ ID NO: 224; Optionally, the sense strand is selected from or includes any one of SEQ ID NO: 1-SEQ ID NO:

224.

4. The double-stranded oligonucleotide according to claim 1, characterized in that The sense strand comprises one of the following nucleotide sequences, or has a difference of no more than 3 nucleotides therefrom: SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 77, SEQ ID NO: 102, SEQ ID NO: 117, SEQ ID NO: 132, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 164, SEQ ID NO: 169, SEQ ID NO: 176, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 185, SEQ ID NO: 186 and SEQ ID NO: 189; and / or The antisense strand comprises one of the following nucleotide sequences, or has a difference of no more than 3 nucleotides therefrom: SEQ ID NO: 237, SEQ ID NO: 240, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 301, SEQ ID NO: 326, SEQ ID NO: 341, SEQ ID NO: 356, SEQ ID NO: 359, SEQ ID NO: 360, SEQ ID NO: 364, SEQ ID NO: 366, SEQ ID NO: 388, SEQ ID NO: 393, SEQ ID NO: 400, SEQ ID NO: 403, SEQ ID NO: 405, SEQ ID NO: 409, SEQ ID NO: 410 and SEQ ID NO: 413; Optionally, the sense strand comprises one of the following nucleotide sequences, or differs therefrom by no more than 3 nucleotides: SEQ ID NO: 13, SEQ ID NO: 117, SEQ ID NO: 135, SEQ ID NO: 179, SEQ ID NO: 186 and SEQ ID NO: 189; and / or, the antisense strand comprises one of the following nucleotide sequences, or differs therefrom by no more than 3 nucleotides: SEQ ID NO:237, SEQ ID NO:341, SEQ ID NO:359, SEQ ID NO:403, SEQ ID NO:410 and SEQ ID NO:

413.

5. The double-stranded oligonucleotide according to claim 1, characterized in that The double-stranded oligonucleotides include one or more of the following groups: A sense strand having the nucleotide sequence as shown in SEQ ID NO: 13 or a nucleotide sequence having no more than 3 nucleotide differences therefrom, and an antisense strand having the nucleotide sequence as shown in SEQ ID NO: 237 or a nucleotide sequence having no more than 3 nucleotide differences therefrom; A sense strand having the nucleotide sequence as shown in SEQ ID NO: 117 or a nucleotide sequence having no more than 3 nucleotide differences therefrom, and an antisense strand having the nucleotide sequence as shown in SEQ ID NO: 341 or a nucleotide sequence having no more than 3 nucleotide differences therefrom; A sense strand having the nucleotide sequence as shown in SEQ ID NO: 135 or a nucleotide sequence having no more than 3 nucleotide differences therefrom, and an antisense strand having the nucleotide sequence as shown in SEQ ID NO: 359 or a nucleotide sequence having no more than 3 nucleotide differences therefrom; A sense strand having the nucleotide sequence as shown in SEQ ID NO: 179 or a nucleotide sequence having no more than 3 nucleotide differences therefrom, and an antisense strand having the nucleotide sequence as shown in SEQ ID NO: 403 or a nucleotide sequence having no more than 3 nucleotide differences therefrom; A sense strand having the nucleotide sequence as shown in SEQ ID NO: 186 or a nucleotide sequence having no more than 3 nucleotide differences therefrom, and an antisense strand having the nucleotide sequence as shown in SEQ ID NO: 410 or a nucleotide sequence having no more than 3 nucleotide differences therefrom; A sense strand having the nucleotide sequence as shown in SEQ ID NO: 189 or a nucleotide sequence having no more than 3 nucleotide differences therefrom, and an antisense strand having the nucleotide sequence as shown in SEQ ID NO: 413 or a nucleotide sequence having no more than 3 nucleotide differences therefrom; Optionally, the sense strand is no longer than 21 nucleotides and the antisense strand is no longer than 23 nucleotides.

6. The double-stranded oligonucleotide according to claim 1, characterized in that The sense strand and / or antisense strand comprises a 3' overhang and / or a 5' overhang having at least 1 or at least 2 nucleotides; Optionally, the antisense strand comprises a 3' overhang of at least 1 or at least 2 nucleotides; Optionally, the antisense strand comprises a 3' overhang of 2 nucleotides.

7. The double-stranded oligonucleotide according to claim 1, characterized in that The sense strand and / or the antisense strand independently comprises one or more phosphorothioate bonds; Optionally, the sense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 5' end; Optionally, the antisense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 3' end and the 5' end, respectively.

8. The double-stranded oligonucleotide according to any one of claims 1 to 7, characterized in that All nucleotides in the double-stranded oligonucleotide are substantially independently selected from modified nucleotides; Optionally, all nucleotides in the double-stranded oligonucleotide are independently selected from modified nucleotides; Optionally, each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides: 2'-halogenated modified nucleotides, 2'-deoxy modified nucleotides, 2'-O-optionally substituted C 1-6 Alkyl-modified nucleotides, 2'-O-(CH2) n -O-R1 modified nucleotides, 2'-O-Si(R2)3 modified nucleotides, 2'-amino modified nucleotides, abasic nucleotides, or quasi-nucleotides; the quasi-nucleotides are selected from one or more of PNA, MNA, BNA, LNA, GNA, TNA and UNA; wherein n is selected from 1 or 2, R1 is selected from optionally substituted C 1-6 Alkyl or optionally substituted C 1-6 Alkoxy; Each R2 is independently selected from optionally substituted C 1-6 alkyl; The optional substitution means that one or more hydrogen atoms on the alkyl group are replaced by a substituent, and the substituent is independently selected from C 1-3 Alkyl, C 1-3 Alkoxy or halogen; Optionally, the 2'-halogenated modified nucleotides are selected from 2'-fluoro-modified nucleotides; Optionally, the 2'-O-optionally substituted C 1-6 The alkyl-modified nucleotides are selected from 2'-O-methyl-modified nucleotides; Optionally, the 2'-O-(CH2) n -O-R1 modified nucleotide is selected from 2'-O-methoxyethyl modified nucleotide, 2'-O-ethoxymethyl modified nucleotide or 2'-O-2,2,2-trifluoroethoxymethyl modified nucleotide; Optionally, the 2'-O-(CH2) n -O-R1 modified nucleotides are selected from 2'-O-methoxyethyl modified nucleotides; Optionally, the 2'-O-Si(R2)3 modified nucleotide is selected from a 2'-O-TBDMS modified nucleotide, a 2'-O-TIPS modified nucleotide or a 2'-O-TOM modified nucleotide; Optionally, each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides: 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides or 2'-O-ethoxymethyl-modified nucleotides; Optionally, each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides: 2'-Fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides; Optionally, the sense strand and / or the antisense strand contains at least one 2'-O-methoxyethyl modified nucleotide or 2'-O-ethoxymethyl modified nucleotide; Optionally, the sense strand and / or the antisense strand contains at least one 2'-O-methoxyethyl modified nucleotide.

9. The double-stranded oligonucleotide according to claim 8, characterized in that The duplex region has a structure as shown in the following formula (101): 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' formula (101), Among them, SS represents the sense strand and AS represents the antisense strand; Each of the Ns is independently selected from the following modified nucleotides: 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides or 2'-deoxy modified nucleotides; Each of the X is independently selected from 2'-O-(CH2) n -O-R1 modified nucleotides or 2'-O-Si(R2)3 modified nucleotides; wherein n is selected from 1 or 2, R1 is selected from optionally substituted C 1-6 Alkyl or optionally substituted C 1-6 Alkoxy; each R2 is independently selected from optionally substituted C 1-6 Alkyl; the optional substitution means that one or more hydrogen atoms on the alkyl or alkoxy group are replaced by a substituent, and the substituent is independently selected from C 1-3 Alkyl, C 1-3 Alkoxy or halogen; a, a', p, p', b, b', q, q', c, c', r', d' independently represent the number of nucleotides; wherein a' is selected from an integer of 3-8, p' is selected from an integer of 0-3, b' is selected from an integer of 4-13, q' is selected from an integer of 0-4, c' is selected from an integer of 3-9, r' is selected from an integer of 0-3, d' is selected from an integer of 0-9, a is selected from an integer of 4-7, p is selected from an integer of 0-1, b is selected from an integer of 4-8, q is selected from an integer of 0-4, and c is selected from an integer of 6-10; and p', q', r', p, q are not 0 at the same time, and 0≤q'+r'≤4; Preferably, a' is selected from an integer of 3-8, p' is selected from 0 or 1, b' is selected from an integer of 4-13, q' is selected from 0 or 1, c' is selected from an integer of 3-9; r' is selected from 0 or 1, d' is selected from an integer of 1-8, a is selected from an integer of 4-7, p is selected from 1, b is selected from an integer of 4-8, q is selected from 0 or 1, and c is selected from an integer of 6-10; Optionally, each N is independently selected from a 2'-fluoro modified nucleotide or a 2'-O-methyl modified nucleotide; Optionally, each X is independently selected from 2'-O-(CH2) n -O-R1 modified nucleotides; Optionally, each X is independently selected from a 2'-O-methoxyethyl modified nucleotide or a 2'-O-ethoxymethyl modified nucleotide; Optionally, the duplex region comprises at least one 2'-O-methoxyethyl modified nucleotide or 2'-O-ethoxymethyl modified nucleotide; Optionally, the sense strand is 17-21 nucleotides long, and the antisense strand is 19-23 nucleotides long; Optionally, the sense strand is 19 nucleotides long and the antisense strand is 21 nucleotides long; Optionally, in the direction from the 5' end to the 3' end, at least three nucleotides among the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides; Optionally, in the direction from the 5' end to the 3' end, at least 3 nucleotides in the 7th to 10th positions of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides in the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; Optionally, in the direction from the 5' end to the 3' end, at least three nucleotides in the 7th to 10th positions of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, the nucleotides at positions 12 and / or 18 are selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; Optionally, in the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, the nucleotides at positions 12 and / or 18 are selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides; Optionally, at least four nucleotides among the nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides; Optionally, at least four nucleotides among the nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at positions 2, 6, 14 and 16 are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides; Optionally, at least four nucleotides among the nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at positions 2, 6, 14 and 16 are selected from 2'-fluoro-modified nucleotides, the nucleotides at positions 8 and / or 15 are selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; Optionally, in the direction from the 5' end to the 3' end, at least three nucleotides among the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides; at least four nucleotides among the nucleotides at positions 2, 6, 9 to 12, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides; Optionally, each nucleotide of the double-stranded oligonucleotide is independently selected from modified nucleotides, wherein the modifications of the sense strand and the antisense strand are selected from any one of the following (1)-(15): (1) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 9, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (2) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 10, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (3) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 11, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (4) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 12, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (5) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 9, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (6) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 10, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (7) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 11, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (8) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 12, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (9) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro modified nucleotides, the nucleotide at position 12 is selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; (10) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro modified nucleotides, the nucleotide at position 12 is selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 10, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; (11) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro modified nucleotides, the nucleotide at position 12 is selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 11, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; (12) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro modified nucleotides, the nucleotide at position 18 is selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 10, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; (13) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro modified nucleotides, the nucleotide at position 18 is selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 11, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; (14) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro modified nucleotides, the nucleotide at position 18 is selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 10, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro modified nucleotides, the nucleotide at position 8 is selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; (15) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro modified nucleotides, the nucleotides at positions 12 and 18 are selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 10, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides.

10. The double-stranded oligonucleotide according to any one of claims 1 to 9, characterized in that The double-stranded oligonucleotide comprises one of the following groups 1-45: Among them, C, G, U, A, and T represent cytidine-3'-phosphate, guanosine-3'-phosphate, uridine-3'-phosphate, adenosine-3'-phosphate, and thymidine-3'-phosphate, respectively; m represents that the nucleotide adjacent to the left of the letter m is a 2'-O-methyl-modified nucleotide; f represents that the nucleotide adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide; (moe) represents that the nucleotide adjacent to the left of the combination mark (moe) is a 2'-O-methoxyethyl-modified nucleotide; and s represents that the two nucleotides before and after the letter s are connected by a phosphorothioate bond.

11. The double-stranded oligonucleotide according to any one of claims 1 to 7, characterized in that All nucleotides in the double-stranded oligonucleotide are independently selected from modified nucleotides; and each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides: 2'-halogenated modified nucleotides, 2'-O-optionally substituted C 1-6 Alkyl-modified nucleotides, 2'-O-(CH2) n -O-R1 modified nucleotides, [2'-W1-2'-W2] modified nucleotides; wherein n is selected from 1 or 2, R1 is selected from optionally substituted C 1-6 Alkyl or optionally substituted C 1-6 Alkoxy; Each R2 is independently selected from optionally substituted C 1-6 alkyl; The optional substitution means that one or more hydrogen atoms on the alkyl group are replaced by a substituent, and the substituent is independently selected from C 1-3 Alkyl, C 1-3 Alkoxy or halogen; The structural formula of the [2'-W1-2'-W2] modified nucleotide is W1 and W2 are each independently selected from halogen or C 1-6 Alkyl, Base represents uracil U, thymine T, cytosine C, adenine A or guanine G; Optionally, the 2'-halogenated modified nucleotides are selected from 2'-fluoro-modified nucleotides; Optionally, the 2'-O-optionally substituted C 1-6 The alkyl-modified nucleotides are selected from 2'-O-methyl-modified nucleotides; Optionally, the 2'-O-(CH2) n -O-R1 modified nucleotides are selected from 2'-O-methoxyethyl modified nucleotides; Optionally, the [2'-W1-2'-W2] modified nucleotides are selected from [2'-F-2'-CH3] modified nucleotides; Optionally, each nucleotide of the double-stranded oligonucleotide is independently selected from modified nucleotides, and each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides: 2'-Fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides or [2'-F-2'-CH3]-modified nucleotides; Optionally, the sense strand is 19 nucleotides long, and the antisense strand is 21 nucleotides long; and the modifications of the sense strand and the antisense strand are selected from any one of the following A1-A2: A1. In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; The nucleotides at positions 2, 6, 9, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro modified nucleotides, the nucleotides at positions 20-21 are selected from [2'-F-2'-CH3] modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; A2, in the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; The nucleotides at positions 2, 6, 9, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl modified nucleotides, the nucleotides at positions 20-21 are selected from [2'-F-2'-CH3] modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; Optionally, the sense strand is 19 nucleotides long, and the antisense strand is 20 nucleotides long; and the modifications of the sense strand and the antisense strand are selected from the following: In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the positive strand are selected from 2'-fluoro modified nucleotides, the nucleotide at position 19 is selected from [2'-F-2'-CH3] modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; The nucleotides at positions 2, 6, 9, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl modified nucleotides, the nucleotide at position 20 is selected from [2'-F-2'-CH3] modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; Optionally, the double-stranded oligonucleotide comprises one of the following groups 46-50: Wherein, (NM054) represents a nucleotide, and its structural formula is 12. The double-stranded oligonucleotide according to any one of claims 1 to 11, characterized in that The double-stranded oligonucleotide is selected from siRNA.

13. A conjugate, characterized in that The conjugate comprises: The double-stranded oligonucleotide according to any one of claims 1 to 12 and one or more ligands capable of binding to a cell receptor; The ligand is conjugated to the double-stranded oligonucleotide.

14. The conjugate according to claim 13, characterized in that The ligand specifically targets liver tissue; Optionally, the ligand is selected from ligands with affinity for asialoglycoprotein receptors; Optionally, the ligand comprises galactose or a derivative thereof; Optionally, the derivative of galactose is selected from N-acetylgalactosamine; Optionally, at least one of the 5' end of the sense strand, the middle position of the sense strand, the 3' end of the sense strand, the 5' end of the antisense strand, the middle position of the antisense strand, and the 3' end of the antisense strand is conjugated with the ligand; Optionally, the number of the ligands is selected from one, one ligand is conjugated to the 3' end of the sense strand; Optionally, the number of the ligands is selected from two, and the two ligands are conjugated to the 5' end of the sense strand and the 3' end of the antisense strand, respectively.

15. The conjugate according to claim 13, characterized in that The ligand is selected from the structure shown in formula (201) or a pharmaceutically acceptable salt thereof: Wherein, * represents the conjugation site of the ligand and the double-stranded oligonucleotide; j is selected from 1, 2, 3 or 4; Each Z is independently selected from hydroxyl or thiol; Each h is independently selected from 1, 2 or 3; Each i is independently selected from 1, 2 or 3; Each R3 is independently selected from H, optionally substituted C 1-6 Alkyl or optionally substituted C 1-6 Alkoxy; Each L is independently selected from optionally substituted C 2-20 Alkylene or R La and R La are independently selected from optionally substituted C 1-10 Alkylene, k is selected from 1, 2, 3, 4 or 5; Each Y is independently selected from O, S or NH; Optionally, j is selected from 2, 3 or 4; Optionally, Z is selected from hydroxyl; Optionally, h is selected from 1; Optionally, i is selected from 1; Optionally, R3 is selected from H; Optionally, each L is independently selected from C 1-10 Alkylene or Among them, R La and R La Independently selected from C 1-5 Alkylene, k is 1, 2 or 3; Optionally, k is selected from 1; Optionally, each L is independently selected from Optionally, Y is selected from O.

16. The conjugate according to claim 15, characterized in that The ligand is selected from the structure shown in formula (202) or a pharmaceutically acceptable salt thereof: Optionally, the ligand is selected from any of the following structures or a pharmaceutically acceptable salt thereof:

17. Use of any of the following in the preparation of a medicament for alleviating, treating and / or preventing an AGT-mediated disease or condition: (I) the double-stranded oligonucleotide according to any one of claims 1 to 12; and / or (II) the conjugate according to any one of claims 13 to 16; Optionally, the disease or condition comprises hypertension, critical hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive emergency state, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm and ventricular fibrosis.

18. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises any of the following items and pharmaceutically acceptable excipients or adjuvants: (I) the double-stranded oligonucleotide according to any one of claims 1 to 12; and / or (II) The conjugate according to any one of claims 13 to 16.

19. A kit, characterized in that: The kit includes any of the following items: (I) the double-stranded oligonucleotide according to any one of claims 1 to 12; and / or (II) the conjugate according to any one of claims 13 to 16; and / or (III) The pharmaceutical composition according to claim 17.

20. A method for inhibiting the expression of AGT gene in a cell, characterized in that: It includes: The double-stranded oligonucleotide according to any one of claims 1 to 12, and / or the conjugate according to any one of claims 13 to 16, and / or the pharmaceutical composition according to claim 18 is administered to a subject.

21. A method for alleviating, treating and / or preventing AGT-mediated diseases or conditions, characterized in that: It includes: Administering the double-stranded oligonucleotide of any one of claims 1 to 12, and / or the conjugate of any one of claims 13 to 16, and / or the pharmaceutical composition of claim 18 to a subject; Optionally, the AGT-mediated disease or condition comprises a disease associated with the level of mRNA expressed by the AGT gene; Optionally, the disease or condition comprises hypertension, critical hypertension, essential hypertension, secondary hypertension, hypertensive crisis, hypertensive emergency state, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm and ventricular fibrosis.

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