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

Abstract

The invention provides double-stranded oligonucleotide and application thereof in inhibiting expression of an angiotensinogen gene (AGT). The double-stranded oligonucleotide comprises a positive-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 contiguous nucleotides in any one sequence of SEQ ID NO: 225 to SEQ ID NO: 448, or comprises a nucleotide sequence having 1, 2 or 3 nucleotide differences from the contiguous nucleotides. The double-stranded oligonucleotides of the present disclosure can inhibit the expression of an AGT gene, thereby contributing to the treatment or prevention of diseases or conditions mediated by an angiotensinogen (AGT) gene.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedicine. Specifically, the present disclosure relates to double-stranded oligonucleotides and their application in inhibiting the expression of angiotensinogen. 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 kidney secrete renin into the circulation. It is mainly produced in the liver and released into the circulation, where renin converts it into angiotensin I. Angiotensin I is subsequently converted into angiotensin II by angiotensin converting enzyme (ACE). Angiotensin II is a peptide hormone that causes vasoconstriction, which in turn can increase blood pressure. Angiotensin II also stimulates the secretion of the hormone aldosterone in the adrenal cortex. Aldosterone causes the kidneys to increase the reabsorption of sodium and water, leading to an increase in the volume of fluid in the body, which in turn can increase blood pressure. Overstimulation or activity of the RAAS pathway can lead to high blood pressure. Chronic high blood pressure is called hypertension. The high blood pressure of 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 the treatment of hypertension, more than two-thirds of subjects cannot be controlled with one 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.

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

[0006] The present disclosure aims to solve one of the technical problems existing in the prior art at least to a certain extent. To this end, the present disclosure provides a double-stranded oligonucleotide that can induce a silencing complex (RISC) The invention can inhibit the cleavage of RNA transcripts of the angiotensinogen (AGT) gene mediated by the gene, thereby inhibiting the expression of the AGT gene, thereby helping to treat or prevent diseases or disorders mediated by the angiotensinogen (AGT) gene.

[0007] In one aspect of the present disclosure, the present disclosure proposes 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 in any one of SEQ ID NO: 225 to SEQ ID NO: 448, or comprises a nucleotide sequence having 1, 2 or 3 nucleotide differences between 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 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.

[0008] 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.

[0009] According to the embodiments of the present disclosure, the above siRNA may also have at least one of the following additional technical features:

[0010] According to an embodiment of the present invention, in the direction from the 5' end to the 3' end, the 2nd to 19th nucleotides of the antisense strand comprise a complementary region to the 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 to 448, or comprises a nucleotide sequence having 1, 2 or 3 nucleotide differences with the consecutive nucleotides.

[0011] According to an embodiment of the present invention, the complementary region comprises any one of SEQ ID NOs. 225 to 448, or comprises 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; the substantially complementary means that the mismatch between the sense strand and the antisense strand in the duplex region does not exceed 3 nucleotides.

[0012] 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 with any one of SEQ ID NO: 225 to SEQ ID NO: 448, or comprises a nucleotide sequence having 1, 2 or 3 nucleotide differences with the consecutive nucleotides.

[0013] 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 with any one of SEQ ID NO: 225 to SEQ ID NO: 448, or comprises a nucleotide sequence having 1, 2 or 3 nucleotide differences with the consecutive nucleotides.

[0014] 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 with any one of SEQ ID NO: 225 to SEQ ID NO: 448, or comprises a nucleotide sequence having 1, 2 or 3 nucleotide differences with the consecutive nucleotides.

[0015] According to an embodiment of the present disclosure, the complementary region comprises any one of SEQ ID NOs. 225 to 448, or comprises a nucleotide sequence having 1, 2 or 3 nucleotides therefrom.

[0016] 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 to SEQ ID NO:448.

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

[0018] According to an embodiment of the present disclosure, the antisense strand is selected from or comprises any one of SEQ ID NO: 225 to SEQ ID NO: 448.

[0019] According to an embodiment of the present disclosure, the positive strand includes 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 includes a nucleotide sequence having 1, 2 or 3 nucleotide differences with the consecutive nucleotides.

[0020] 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 to SEQ ID NO:224.

[0021] 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.

[0022] 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:

[0023] 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

[0024] The antisense strand comprises one of the following nucleotide sequences, or has a difference of no more than 3 nucleotides therefrom:

[0025] 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.

[0026] 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, SEQID NO: 410 and SEQ ID NO: 413.

[0027] According to an embodiment of the present disclosure, the double-stranded oligonucleotide comprises one or more of the following groups:

[0028] A sense strand having the nucleotide sequence as shown in SEQ ID NO: 13, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and an antisense strand having the nucleotide sequence as shown in SEQ ID NO: 237, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom;

[0029] A sense strand having the nucleotide sequence as shown in SEQ ID NO: 117, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and an antisense strand having the nucleotide sequence as shown in SEQ ID NO: 341, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom;

[0030] A sense strand having the nucleotide sequence as shown in SEQ ID NO: 135, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and an antisense strand having the nucleotide sequence as shown in SEQ ID NO: 359, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom;

[0031] A sense strand having the nucleotide sequence as shown in SEQ ID NO: 179, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and an antisense strand having the nucleotide sequence as shown in SEQ ID NO: 403, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom;

[0032] A sense strand having the nucleotide sequence as shown in SEQ ID NO: 186, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and an antisense strand having the nucleotide sequence as shown in SEQ ID NO: 410, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom;

[0033] 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.

[0034] According to an embodiment of the present disclosure, the double-stranded oligonucleotide comprises one or more of the following groups:

[0035] A sense strand having the nucleotide sequence shown in SEQ ID NO: 13, and an antisense strand having the nucleotide sequence shown in SEQ ID NO: 237;

[0036] a sense strand having the nucleotide sequence shown in SEQ ID NO: 117, and an antisense strand having the nucleotide sequence shown in SEQ ID NO: 341;

[0037] a sense strand having the nucleotide sequence shown in SEQ ID NO: 135, and an antisense strand having the nucleotide sequence shown in SEQ ID NO: 359;

[0038] A sense strand having the nucleotide sequence shown in SEQ ID NO: 179, and an antisense strand having the nucleotide sequence shown in SEQ ID NO: 403;

[0039] A sense strand having the nucleotide sequence shown in SEQ ID NO: 186, and an antisense strand having the nucleotide sequence shown in SEQ ID NO: 410;

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] According to an embodiment of the present disclosure, the antisense strand comprises a 3' 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 2 nucleotides.

[0046] According to an embodiment of the present disclosure, the sense strand and / or the antisense strand independently comprises one or more phosphorothioate bonds.

[0047] According to an embodiment of the present disclosure, the sense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 5' end.

[0048] 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.

[0049] According to an embodiment of the present disclosure, all nucleotides in the double-stranded oligonucleotide are substantially independently selected from modified nucleotides.

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

[0051] According to an embodiment of the present disclosure, each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides:

[0052] 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);

[0053] 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;

[0054] Each R2 is independently selected from optionally substituted C 1-6 alkyl.

[0055] 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.

[0056] The structural formula of nucleotide 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.

[0057] The "2'-deoxy modified nucleotide" refers to a nucleotide in which the 2' hydroxyl group of the ribose group of the nucleotide is replaced by a hydrogen atom.

[0058] "2'-halogenated modified 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.

[0059] According to an embodiment of the present disclosure, the 2'-halogenated modified nucleotides are selected from 2'-fluoro-modified nucleotides.

[0060] "2'-O-optionally substituted C 1-6 "Alkyl modified nucleotide" refers to a nucleotide in which the hydrogen atom at the 2' hydroxyl group of the ribose group is optionally replaced by a C 1-6 The nucleotides formed by alkyl substitution. Exemplarily, 2'-O-methyl (also known as 2'-methoxy) modified nucleotides.

[0061] 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.

[0062] "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 formed nucleotide; 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.

[0063] 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.

[0064] 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.

[0065] "2'-O-Si(R2)3 modified nucleotide" refers to a nucleotide formed by replacing the hydrogen atom on the 2' hydroxyl group of the ribose group of the nucleotide with -Si(R2)3. Exemplary, 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

[0066] 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.

[0067] According to an embodiment of the present disclosure, each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides:

[0068] 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides or 2'-O-ethoxymethyl modified nucleotides.

[0069] According to an embodiment of the present disclosure, each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides:

[0070] 2'-Fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides.

[0071] 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.

[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.

[0073] According to an embodiment of the present disclosure, the duplex region has a structure as shown in the following formula (101):

[0074] SS: 5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3'

[0075] AS: 3'-(N)a-(X)p-(N)b-(X)q-(N)c-5' formula (101),

[0076] Among them, SS represents the sense strand and AS represents the antisense strand;

[0077] 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;

[0078] 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; 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;

[0079] 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.

[0080] 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.

[0081] 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.

[0082] According to an embodiment of the present disclosure, each X is independently selected from 2'-O-(CH2) n -O-R1 modified nucleotides.

[0083] 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.

[0084] 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.

[0085] 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.

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

[0087] 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.

[0088] According to an embodiment of the present disclosure, 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 positive strand are selected from 2'-fluoro modified nucleotides, and the nucleotides in the remaining positions are independently selected from 2'-O-methyl 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 at positions 7-10 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.

[0090] According to an embodiment of the present disclosure, in the direction from the 5' end to the 3' end, the nucleotides at positions 7-10 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, 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.

[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 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.

[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, 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.

[0094] 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.

[0095] 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):

[0096] (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;

[0097] (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;

[0098] (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;

[0099] (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;

[0100] (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;

[0101] (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;

[0102] (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;

[0103] (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;

[0104] (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;

[0105] (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;

[0106] (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;

[0107] (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;

[0108] (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;

[0109] (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.

[0110] According to an embodiment of the present disclosure, the double-stranded oligonucleotide comprises one of the following 45 groups:

[0111]

[0112]

[0113]

[0114] 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.

[0115] According to an embodiment of the present disclosure, the double-stranded oligonucleotide is selected from siRNA.

[0116] 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.

[0117] According to an embodiment of the present disclosure, the ligand specifically targets liver tissue.

[0118] According to an embodiment of the present disclosure, the ligand is selected from ligands having affinity to asialoglycoprotein receptors.

[0119] According to an embodiment of the present disclosure, the ligand comprises galactose or a derivative thereof.

[0120] According to an embodiment of the present disclosure, the derivative of galactose is selected from N-acetylgalactosamine.

[0121] 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.

[0122] According to an embodiment of the present disclosure, the number of the ligands is selected from one, and one of the ligands is conjugated to the 3' end of the sense strand.

[0123] 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 a stereoisomer thereof:

[0124]

[0125] Wherein, * represents the conjugation site of the ligand and the double-stranded oligonucleotide;

[0126] j is selected from 1, 2, 3 or 4;

[0127] Each Z is independently selected from hydroxyl or thiol;

[0128] Each h is independently selected from 1, 2 or 3;

[0129] Each i is independently selected from 1, 2 or 3;

[0130] Each R3 is independently selected from H, optionally substituted C 1-6 Alkyl or optionally substituted C 1-6 Alkoxy;

[0131] 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;

[0132] Each Y is independently selected from O, S or NH.

[0133] According to an embodiment of the present disclosure, j is selected from 1, 2 or 3.

[0134] According to an embodiment of the present disclosure, j is selected from 3.

[0135] According to an embodiment of the present disclosure, Z is selected from hydroxyl.

[0136] According to an embodiment of the present disclosure, h is selected from 1.

[0137] According to an embodiment of the present disclosure, i is selected from 1.

[0138] According to an embodiment of the present disclosure, R3 is selected from H.

[0139] 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.

[0140] According to an embodiment of the present disclosure, k is selected from 1.

[0141] According to an embodiment of the present disclosure, each L is independently selected from

[0142] According to an embodiment of the present disclosure, Y is selected from O.

[0143] 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:

[0144]

[0145] According to an embodiment of the present disclosure, the ligand is selected from any of the following structures or a pharmaceutically acceptable salt thereof or a stereoisomer thereof:

[0146]

[0147] According to an embodiment of the present disclosure, the ligand is selected from the following structures or pharmaceutically acceptable salts or stereoisomers thereof:

[0148]

[0149] 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:

[0150]

[0151] In another aspect of the present disclosure, the present disclosure proposes 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:

[0152] (I) the double-stranded oligonucleotide described above; and / or

[0153] (II) The conjugate described above.

[0154] As can be seen above, the aforementioned double-stranded oligonucleotides and conjugates can degrade AGT mRNA and inhibit the expression of AGT. Therefore, the pharmaceutical composition disclosed herein can effectively prevent and / or treat diseases or conditions mediated by the angiotensinogen (AGT) gene.

[0155] 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.

[0156] In yet another aspect of the present disclosure, the present disclosure provides a pharmaceutical composition.

[0157] According to an embodiment of the present disclosure, the pharmaceutical composition includes any of the following items and a pharmaceutically acceptable excipient or adjuvant:

[0158] (I) the double-stranded oligonucleotide described above; and / or

[0159] (II) The conjugate described above.

[0160] As can be seen above, the aforementioned double-stranded oligonucleotides and conjugates can degrade AGT mRNA and inhibit the expression of AGT. Therefore, the pharmaceutical composition disclosed herein can effectively prevent and / or treat diseases or conditions mediated by the angiotensinogen (AGT) gene.

[0161] According to an embodiment of the present disclosure, the pharmaceutical composition further includes: a pharmaceutically acceptable carrier or excipient.

[0162] In yet another aspect of the present disclosure, the present disclosure provides a kit.

[0163] According to an embodiment of the present disclosure, the kit includes any of the following items:

[0164] (I) the double-stranded oligonucleotide described above; and / or

[0165] (II) the conjugate described above; and / or

[0166] (III) The pharmaceutical composition described above.

[0167] The kit disclosed herein can effectively degrade AGT mRNA and inhibit the expression of AGT.

[0168] In yet another aspect of the present disclosure, the present disclosure provides a method for inhibiting the expression of AGT gene in a cell.

[0169] According to an embodiment of the present disclosure, the method comprises: administering the double-stranded oligonucleotide described above, and / or the conjugate described above, and / or the pharmaceutical composition described above to a subject. The method disclosed herein can be used to inhibit the expression of angiotensinogen. For example, the expression of angiotensinogen can be inhibited in an animal body; or the expression of angiotensinogen can be inhibited for non-disease treatment purposes, such as inhibiting the expression of angiotensinogen in vitro for subsequent research.

[0170] In another aspect of the present disclosure, the present disclosure proposes a method for alleviating, treating and / or preventing AGT-mediated diseases or conditions. 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 can be seen from the foregoing, the aforementioned double-stranded oligonucleotide and conjugate can degrade the mRNA of AGT, inhibit the expression of AGT, and contribute to AGT-related scientific research and clinical treatment and / or prevention of diseases or conditions mediated by the angiotensinogen gene.

[0171] According to an embodiment of the present disclosure, the AGT-mediated disease or condition includes a disease associated with the mRNA level of AGT gene expression.

[0172] 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.

[0173] Additional aspects and advantages of the present disclosure will be given in part in the following description and in part will be obvious from the following description or will be learned through practice of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0174] The above and / or additional aspects and advantages of the present disclosure will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0175] Figure 1 It shows the inhibitory activity of the target gene in Balb / c-HDI mice after administration of the siRNA conjugate described in this example;

[0176] Figure 2 The following table shows the changes in blood pressure in hREN×hAGT hypertensive mice after administration of the siRNA conjugate described in this example: (A) systolic blood pressure changes; (B) mean arterial pressure changes; (C) diastolic blood pressure changes;

[0177] Figure 3 It shows the change level of AGT protein in hREN×hAGT hypertensive mice after administration of the siRNA conjugate described in this example;

[0178] Figure 4 Typical histopathological images of each experimental group under 200-fold field of view of HE staining are shown;

[0179] Figure 5 The figure shows the change level of systolic blood pressure in hREN×hAGT hypertensive mice after administration of the siRNA conjugate described in this example;

[0180] Figure 6 It shows the change level of AGT protein in hREN×hAGT hypertensive mice after administration of the siRNA conjugate described in this example;

[0181] Figure 7 The graph shows the changes in blood pressure of SHR rats after administration of the siRNA conjugate described in this example; (A) changes in systolic blood pressure; (B) changes in mean arterial pressure; (C) changes in diastolic blood pressure. DETAILED DESCRIPTION

[0182] 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.

[0183] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present disclosure, unless otherwise specified, the meaning of "plurality" is two or more.

[0184] Herein, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present disclosure but not excluding other contents.

[0185] 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.

[0186] As used herein, the term "small interfering RNA (siRNA)" is a double-stranded RNA of 17 to 25 nucleotides in length, comprising a sense strand and an antisense strand. siRNA mediates targeted cleavage of RNA transcripts in the RISC pathway by forming a silencing complex (RISC). Specifically, siRNA guides the specific degradation of mRNA sequences through the known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and into proteins. For example, siRNA can regulate (e.g., inhibit) the expression of angiotensinogen (AGT) in cells.

[0187] In this article, the term "antisense strand (or guide strand)" includes a region that is substantially complementary to a target sequence, such as the mRNA of AGT. "Sense strand (or follower strand)" refers to an iRNA strand that contains a region 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 in the interior or terminal regions of the molecule, wherein the most tolerated mismatches exist in the terminal regions, for example, within 5, 4, 3 or 2 nucleotides at the 5'- and / or 3'-end of the iRNA.

[0188] It should be noted that "at least part of the antisense strand is substantially complementary to the mRNA" means that the antisense strand has a polynucleotide that is substantially complementary to a continuous portion of the mRNA of interest (e.g., the mRNA encoding angiotensinogen). Alternatively, if a polynucleotide is substantially non-interruptedly complementary to a portion of the mRNA encoding angiotensinogen, the antisense strand is complementary to at least a portion of the mRNA of angiotensinogen.

[0189] Herein, the term "target sequence" refers to a contiguous portion of a nucleotide sequence of an mRNA molecule formed during transcription of an angiotensinogen gene, including mRNA that is a product of RNA processing of a primary transcript. The angiotensinogen gene may be within a cell, for example, a cell in a subject.

[0190] As used herein, the term "subject" refers to any animal that is examined, studied, or treated, and it is not intended that the present disclosure be limited 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, cattle, sheep, goats, pigs, chickens, turkeys, dogs, cats, horses, and reptiles.

[0191] 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 evaluated based on the level of any variable associated with angiotensinogen gene expression, for example, the mRNA level or protein level of angiotensinogen. Inhibition can be evaluated by a reduction in 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 the art, e.g., a baseline level before administration, or a level measured from a similar subject, cell or sample that has never been treated or treated with a control (e.g., a control with only a buffer or a control without an active agent).

[0192] Double-stranded oligonucleotide

[0193] In one aspect of the present disclosure, the present disclosure proposes 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 in any one of SEQ ID NO: 225 to SEQ ID NO: 448, or comprises a nucleotide sequence having 1, 2 or 3 nucleotide differences between 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 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.

[0194] In this article, the term "nucleotide difference" and the term "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 of the former is changed compared with the latter. For example, when a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position of 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.

[0195] 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.

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202] The gene sequence of AGT is shown below, where the mRNA transcribed from the sequence marked with the downward line is used as the target sequence of the double-stranded oligonucleotide, which can induce the silencing complex (RISC) Mediates the cleavage of RNA transcripts of the angiotensinogen (AGT) gene, thereby inhibiting the expression of the AGT gene.

[0203]

[0204]

[0205] As used herein, the term "substantially complementary" means that the sense strand and the antisense strand 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.

[0206] In this article, "nucleotide difference" between one nucleotide sequence and another nucleotide sequence means that the base type of the nucleotide at the same position of the former is changed compared with the latter. For example, when a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position of the former is U, C, G or T, it is considered that there is a nucleotide difference at this position between the two nucleotide 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 nucleotide difference occurs at this position.

[0207] According to an embodiment of the present disclosure, in the direction from the 5' end to the 3' end, the 2nd to 19th nucleotides of the antisense strand contain a complementary region to the AGT RNA transcript; wherein the complementary region contains a nucleotide sequence of at least 17 consecutive nucleotides in any one of SEQ ID NOs. 225 to 448, or contains a nucleotide sequence with 1, 2 or 3 nucleotide differences between the consecutive nucleotides. In some embodiments, the complementary region contains any one of SEQ ID NOs. 225 to 448, or contains a nucleotide sequence with 1, 2 or 3 nucleotide differences therefrom; the sense strand contains a nucleotide sequence that is at least partially 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.

[0208] 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 with any one of SEQ ID NO: 225 to SEQ ID NO: 448, or comprises a nucleotide sequence with 1, 2 or 3 nucleotide differences between 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 with 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 with 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.

[0209] 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.

[0210] 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:

[0211] 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

[0212] The antisense strand comprises one of the following nucleotide sequences, or has a difference of no more than 3 nucleotides therefrom:

[0213] 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.

[0214] 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:

[0215] 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:

[0216] 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.

[0217] According to an embodiment of the present disclosure, the double-stranded oligonucleotide comprises one or more of the following groups:

[0218] A sense strand having the nucleotide sequence as shown in SEQ ID NO: 13, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and an antisense strand having the nucleotide sequence as shown in SEQ ID NO: 237, or a nucleotide sequence having 1, 2 or 3 nucleotides therefrom;

[0219] A sense strand having the nucleotide sequence shown in SEQ ID NO: 117, or a nucleotide sequence having 1, 2 or 3 nucleotides thereof, and an antisense strand having the nucleotide sequence shown in SEQ ID NO: 341, or a nucleotide sequence having 1, 2 or 3 nucleotides thereof;

[0220] A sense strand having the nucleotide sequence shown in SEQ ID NO: 135, or a nucleotide sequence having 1, 2 or 3 nucleotides therefrom, and an antisense strand having the nucleotide sequence shown in SEQ ID NO: 359, or a nucleotide sequence having 1, 2 or 3 nucleotides therefrom;

[0221] A sense strand having the nucleotide sequence shown in SEQ ID NO: 179, or a nucleotide sequence having 1, 2, or 3 nucleotides thereof, and an antisense strand having the nucleotide sequence shown in SEQ ID NO: 403, or a nucleotide sequence having 1, 2, or 3 nucleotides thereof;

[0222] A sense strand having the nucleotide sequence shown in SEQ ID NO: 186, or a nucleotide sequence having 1, 2 or 3 nucleotides therefrom, and an antisense strand having the nucleotide sequence shown in SEQ ID NO: 410, or a nucleotide sequence having 1, 2 or 3 nucleotides therefrom;

[0223] The sense strand has the nucleotide sequence shown in SEQ ID NO: 189 or a nucleotide sequence having 1, 2 or 3 nucleotides thereof, and the antisense strand has the nucleotide sequence shown in SEQ ID NO: 413 or a nucleotide sequence having 1, 2 or 3 nucleotides thereof.

[0224] According to an embodiment of the present disclosure, the double-stranded oligonucleotide comprises one or more of the following groups:

[0225] A sense strand having the nucleotide sequence shown in SEQ ID NO: 13, and an antisense strand having the nucleotide sequence shown in SEQ ID NO: 237;

[0226] a sense strand having the nucleotide sequence shown in SEQ ID NO: 117, and an antisense strand having the nucleotide sequence shown in SEQ ID NO: 341;

[0227] a sense strand having the nucleotide sequence shown in SEQ ID NO: 135, and an antisense strand having the nucleotide sequence shown in SEQ ID NO: 359;

[0228] A sense strand having the nucleotide sequence shown in SEQ ID NO: 179 or a nucleotide sequence having 1, 2 or 3 nucleotides thereof, and an antisense strand having the nucleotide sequence shown in SEQ ID NO: 403;

[0229] A sense strand having the nucleotide sequence shown in SEQ ID NO: 186, and an antisense strand having the nucleotide sequence shown in SEQ ID NO: 410;

[0230] 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.

[0231] 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.

[0232] 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.

[0233] 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.

[0234] As used herein, the term "overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded oligonucleotide double helix structure, and is also a nucleotide sequence in the siRNA structure other than the double-stranded region. For example, when the 3' end of one strand in the sense strand and / or the antisense strand exceeds the 5' end of the other strand, or when the 5' end of one strand in the sense strand and / or the antisense strand exceeds the 3' end of the other strand, there is a nucleotide overhang. The overhang may contain at least one nucleotide, at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more nucleotides. The nucleotide overhang may contain or may be composed of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang may be located in the sense strand, the antisense strand, or any combination thereof. In addition, the nucleotides of the overhang may appear at the 5'-end, 3'-end, or both ends of the antisense or sense strand.

[0235] According to an embodiment of the present disclosure, the antisense strand comprises a 3' overhang having at least 1 or at least 2 nucleotides.

[0236] According to an embodiment of the present disclosure, the antisense strand comprises a 3' overhang having 2 nucleotides.

[0237] 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 phosphorothioate bonds.

[0238] According to an embodiment of the present disclosure, the sense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 5' end.

[0239] 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.

[0240] According to an embodiment of the present disclosure, all nucleotides in the double-stranded oligonucleotide are substantially independently selected from modified nucleotides.

[0241] According to an embodiment of the present disclosure, all nucleotides in the double-stranded oligonucleotide are independently selected from modified nucleotides.

[0242] According to an embodiment of the present disclosure, each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides:

[0243] 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);

[0244] 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;

[0245] Each R2 is independently selected from optionally substituted C 1-6 alkyl;

[0246] 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.

[0247] According to an embodiment of the present disclosure, the 2'-halogenated modified nucleotides are selected from 2'-fluoro-modified nucleotides.

[0248] 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.

[0249] 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.

[0250] 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.

[0251] 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.

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

[0254] According to an embodiment of the present disclosure, each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides:

[0255] 2'-Fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides.

[0256] 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.

[0257] 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.

[0258] According to an embodiment of the present disclosure, the duplex region has a structure as shown in the following formula (101):

[0259] SS: 5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3'

[0260] AS: 3'-(N)a-(X)p-(N)b-(X)q-(N)c-5' formula (101),

[0261] Among them, SS represents the sense strand and AS represents the antisense strand;

[0262] 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;

[0263] 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 1-6Alkyl; 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;

[0264] 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.

[0265] 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.

[0266] 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.

[0267] According to an embodiment of the present disclosure, each X is independently selected from 2'-O-(CH2) n -O-R1 modified nucleotides.

[0268] 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.

[0269] 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.

[0270] According to an embodiment of the present disclosure, the duplex region comprises at least one 2'-O-methoxyethyl modified nucleotide.

[0271] 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.

[0272] According to an embodiment of the present disclosure, the sense strand is 19 nucleotides long, and the antisense strand is 21 nucleotides long.

[0273] 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.

[0274] According to an embodiment of the present disclosure, 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 positive strand are selected from 2'-fluoro modified nucleotides, and the nucleotides in the remaining positions are independently selected from 2'-O-methyl modified nucleotides.

[0275] According to an embodiment of the present disclosure, in the direction from the 5' end to the 3' end, at least 3 nucleotides at positions 7-10 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.

[0276] According to an embodiment of the present disclosure, in the direction from the 5' end to the 3' end, the nucleotides at positions 7-10 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.

[0277] 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 the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides.

[0278] 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.

[0279] 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.

[0280] 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.

[0281] 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):

[0282] (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;

[0283] (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;

[0284] (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;

[0285] (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;

[0286] (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;

[0287] (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;

[0288] (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;

[0289] (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;

[0290] (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;

[0291] (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;

[0292] (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;

[0293] (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;

[0294] (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;

[0295] (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.

[0296] According to an embodiment of the present disclosure, the double-stranded oligonucleotide comprises one of the following 45 groups:

[0297]

[0298]

[0299]

[0300] 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.

[0301] Conjugate

[0302] In another aspect of the present disclosure, the present disclosure proposes 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. The conjugate of the present disclosure can well deliver drugs to cells due to the addition of ligands, further improve the degradation of AGT mRNA, and can effectively prevent and / or treat diseases or conditions mediated by the angiotensinogen (AGT) gene.

[0303] According to an embodiment of the present disclosure, the ligand specifically targets liver tissue.

[0304] According to an embodiment of the present disclosure, the ligand is selected from ligands having affinity to asialoglycoprotein receptors.

[0305] According to an embodiment of the present disclosure, the ligand comprises galactose or a derivative thereof.

[0306] According to an embodiment of the present disclosure, the derivative of galactose is selected from N-acetylgalactosamine.

[0307] 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.

[0308] According to an embodiment of the present disclosure, the number of the ligands is selected from one, and one of the ligands is conjugated to the 3' end of the sense strand.

[0309] 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 a stereoisomer thereof:

[0310]

[0311] Wherein, * represents the conjugation site of the ligand and the double-stranded oligonucleotide;

[0312] j is selected from 1, 2, 3 or 4;

[0313] Each Z is independently selected from hydroxyl or thiol;

[0314] Each h is independently selected from 1, 2 or 3;

[0315] Each i is independently selected from 1, 2 or 3;

[0316] Each R3 is independently selected from H, optionally substituted C 1-6 Alkyl or optionally substituted C 1-6 Alkoxy;

[0317] 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;

[0318] Each Y is independently selected from O, S or NH.

[0319] According to an embodiment of the present disclosure, j is selected from 1, 2 or 3.

[0320] According to an embodiment of the present disclosure, j is selected from 3.

[0321] According to an embodiment of the present disclosure, Z is selected from hydroxyl.

[0322] According to an embodiment of the present disclosure, h is selected from 1.

[0323] According to an embodiment of the present disclosure, i is selected from 1.

[0324] According to an embodiment of the present disclosure, R3 is selected from H.

[0325] 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.

[0326] According to an embodiment of the present disclosure, k is selected from 1.

[0327] According to an embodiment of the present disclosure, each L is independently selected from

[0328] According to an embodiment of the present disclosure, Y is selected from O.

[0329] 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:

[0330]

[0331] According to an embodiment of the present disclosure, the ligand is selected from any of the following structures or a pharmaceutically acceptable salt thereof or a stereoisomer thereof:

[0332]

[0333]

[0334] According to an embodiment of the present disclosure, the ligand is selected from the following structures or pharmaceutically acceptable salts or stereoisomers thereof:

[0335]

[0336] 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:

[0337]

[0338] Pharmaceutical composition

[0339] In another aspect of the present disclosure, the present disclosure provides a pharmaceutical composition. According to an embodiment of the present disclosure, the pharmaceutical composition includes any of the following items and a pharmaceutically acceptable excipient or adjuvant:

[0340] (I) the double-stranded oligonucleotide described above; and / or

[0341] (II) The conjugate described above.

[0342] As can be seen above, the aforementioned double-stranded oligonucleotides and conjugates can degrade AGT mRNA and inhibit the expression of AGT. Therefore, the pharmaceutical composition disclosed herein can effectively prevent and / or treat diseases or conditions mediated by the angiotensinogen (AGT) gene.

[0343] According to an embodiment of the present disclosure, the pharmaceutical composition further includes: a pharmaceutically acceptable carrier or excipient.

[0344] In this article, "pharmaceutical composition" can refer to a composition used for the treatment of a disease, and can also be used for in vitro culture experiments of cells. When used for the treatment of 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 field. All methods include the step of combining the active ingredient with an excipient that constitutes one or more accessory ingredients. Typically, the composition is prepared by uniformly and fully combining the active siRNA with a liquid excipient, a finely divided solid excipient, or both.

[0345] In this article, 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 mammals treated therewith. Preferably, the "pharmaceutically acceptable" described in the present disclosure refers to those approved by federal regulatory agencies or national governments or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias for use in animals, especially humans.

[0346] In this article, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. Except for any conventional excipients that are incompatible with the siRNA of the present disclosure, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of the present disclosure.

[0347] In addition to any conventional excipients, to the extent that they are incompatible with the siRNA of the present disclosure, such as 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.

[0348] Pharmaceutical compositions of the present disclosure include preparations suitable for parenteral administration. Preparations can be conveniently present in unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of active ingredient that can be combined with auxiliary materials to prepare a single dose form is generally the amount of siRNA that produces a therapeutic effect. Generally speaking, in units of one percent, the amount is about 1% to about 99% active ingredient, preferably about 5% to about 70%, and most preferably about 10% to about 30%.

[0349] Reagent test kit

[0350] In another aspect of the present disclosure, the present disclosure provides a kit. According to an embodiment of the present disclosure, the kit includes any of the following items:

[0351] (I) the double-stranded oligonucleotide described above; and / or

[0352] (II) the conjugate described above; and / or

[0353] (III) The pharmaceutical composition described above.

[0354] The kit disclosed herein can effectively degrade AGT mRNA and inhibit the expression of AGT.

[0355] 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 described in detail here.

[0356] use

[0357] In another aspect of the present disclosure, the present disclosure proposes the use of any of the following items in the preparation of a drug for alleviating, treating and / or preventing AGT-mediated diseases or conditions: (I) the double-stranded oligonucleotide described above; and / or (II) the conjugate described above. As can be seen from the foregoing, both the double-stranded oligonucleotide and the conjugate can degrade AGT mRNA and inhibit the expression of AGT. Therefore, the pharmaceutical composition of the present disclosure can effectively prevent and / or treat diseases or conditions mediated by the angiotensinogen (AGT) gene.

[0358] 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.

[0359] As used herein, the term "treatment" refers to the use of drugs to obtain the desired pharmacological and / or physiological effects. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be 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, especially 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 blocking the development of the disease; or (c) alleviating the disease, such as alleviating 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 an individual's disease, including but not limited to administering a drug containing the siRNA or siRNA conjugate described herein to an individual in need.

[0360] The effective amount of the double-stranded oligonucleotide, conjugate or pharmaceutical composition disclosed herein may vary with 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 by 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, four times a day, three times a day, twice a day, once a day, or once every other day, or several doses administered daily may be proportionally reduced.

[0361] 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 injection.

[0362] 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.

[0363] method

[0364] In another aspect of the present disclosure, the present disclosure proposes a method for inhibiting the expression of AGT gene in cells. According to an embodiment of the present disclosure, the method comprises: administering the double-stranded oligonucleotide described above, and / or the conjugate described above, and / or the pharmaceutical composition described above to a subject. The method of the present disclosure can be used to inhibit the expression of angiotensinogen. For example, the expression of angiotensinogen can be inhibited in an animal body; or the expression of angiotensinogen can be inhibited for non-disease treatment purposes, such as inhibiting the expression of angiotensinogen in vitro for subsequent research.

[0365] In another aspect of the present disclosure, the present disclosure proposes a method for alleviating, treating and / or preventing AGT-mediated diseases or conditions. 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 can be seen from the foregoing, the aforementioned double-stranded oligonucleotide and conjugate can degrade the mRNA of AGT, inhibit the expression of AGT, and contribute to AGT-related scientific research and clinical treatment and / or prevention of diseases or conditions mediated by the angiotensinogen gene.

[0366] According to an embodiment of the present disclosure, the AGT-mediated disease or condition includes a disease associated with the mRNA level of AGT gene expression.

[0367] 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.

[0368] 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 described in detail here.

[0369] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will appreciate 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 indicated in the examples, the techniques or conditions described in the literature in this area or the product instructions are used. Where the manufacturers of reagents or instruments are not indicated, they are all conventional products that can be obtained commercially.

[0370] 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 of mice and rats in the present disclosure was measured using a full set of intelligent non-invasive blood pressure system; the serum samples of mice and rats in the present disclosure were detected using a human angiotensinogen kit.

[0371] In the context of the present disclosure, unless otherwise specified, the real-time PCR detection data of the activity experiments involved in the present disclosure are all calculated by the ΔΔCt method for relative quantitative calculation of the target gene mRNA in each test group. The calculation method is summarized as follows:

[0372] ΔCt(test group) = Ct(test group target gene) - Ct(test group reference gene)

[0373] ΔCt(control group) = Ct(control group target gene) - Ct(control group internal reference gene)

[0374] ΔΔCt(test group) = ΔCt(test group) - ΔCt(control group average)

[0375] ΔΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0376] The mRNA expression level of the target gene in the test group was normalized based on the control group, and the remaining expression level of the target gene mRNA in the control group was defined as 100%.

[0377] Relative residual expression level of target gene mRNA in the test group = 2 -ΔΔCt (Test group) × 100%

[0378] Test group target gene mRNA inhibition rate = 100% - test group target gene mRNA relative expression level

[0379] The blood pressure test data (blood pressure before the first administration (D0)) of the in vivo activity experiment involved in the present disclosure and the calculation method are summarized as follows:

[0380] Blood pressure reduction level (mmHg) = Dx-D0

[0381] The detection data of AGT and Ang II proteins at different time points in the in vivo activity experiment involved in the present disclosure and the calculation method are summarized as follows:

[0382]

[0383] 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.

[0384] In the context of the present disclosure, unless otherwise stated, the reagent ratios provided below are calculated on a volume basis (v / v).

[0385] Preparation of compounds

[0386] 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.

[0387] Table 1 Main reagents

[0388]

[0389] Here, CPG stands for controlled pore glass.

[0390] Preparation of compounds

[0391] Preparation Example 1: Synthesis of Compound CR01008

[0392]

[0393] (1-1) Synthesis of Compound 2

[0394] Compound 1 (trans-4-(Boc-amino)cyclohexylcarboxaldehyde, 10.0 g, 1.0 eq) and formaldehyde aqueous solution (8.9 g, 37 mass%, 2.4 eq) were dissolved in 33 ml of methanol, and 13 ml of 45.3 mass% KOH aqueous solution was added dropwise. After the addition was completed, the mixture was stirred at 25°C for 30 minutes, 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 product as a white solid. A small amount of water was added to the crude product for pulping, and the product was filtered to obtain compound 2 as a white solid (9 g, yield 78.9%). MS-ESI (m / z) = 260 [M+H] + .

[0395] (1-2) Synthesis of Compound 3

[0396] Compound 2 (9 g, 1 eq) prepared in step (1-1) was dissolved in 70 ml of 1,4-dioxane, and a solution of hydrogen chloride in 1,4-dioxane (45 ml, 4 M) was added, and the mixture was stirred at 25° C. for 1 hour. After the reaction, the reaction solution was evaporated under reduced pressure to obtain compound 3 (6.8 g, yield 100%) as a white solid.

[0397] (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. in a N2 atmosphere for 3 hours. After the reaction was completed, 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] + .

[0398] (1-4) Synthesis of Compound 6

[0399] The compound 5 (1.54 g, 1.0 eq) prepared in step (1-3) was dissolved in 15 ml of pyridine, the reaction system was cooled to 0°C with an ice-water bath, and DMTrCl (4,4'-dimethoxytriphenylmethane chloride, 1.32 g, 1.5 eq) was added at 0°C, and the reaction was carried out at 25°C for 3 hours. 15 ml of methanol was added to the reaction solution to quench the reaction. After the reaction was completed, the reaction solution was evaporated under reduced pressure and purified by column chromatography (C18 column, eluent: acetonitrile: water = 60 / 40, v / v) to obtain a yellow solid compound 6 (1 g, yield 42.7%). MS-ESI (m / z) = 891 [M+H] + .

[0400] (1-5) Synthesis of Compound CR01008

[0401] The compound 6 (1.08 g, 1.0 eq) prepared in step (1-4) was dissolved in 20 ml of anhydrous dichloromethane, and DCI (115 mg, 0.8 eq) and compound 7 (bis(diisopropylamino)(2-cyanoethoxy)phosphine, 732 mg, 2.1 eq) were added respectively, and the nitrogen was replaced 3 times. The reaction was stirred at 25°C for 2 hours. After the reaction was completed, 20 ml of saturated sodium bicarbonate aqueous solution was added to the reaction solution, and the mixture was extracted 3 times with 20 ml of dichloromethane (3×20 ml). The organic phases were combined, evaporated to dryness under reduced pressure, purified by reverse phase column chromatography (C18 column, eluent: acetonitrile: water = 72 / 28, v / v), and then vacuum dried for 12 hours to obtain compound CR01008 (1 g, yield 76.0%) as a white powder. MS-ESI (m / z) = 1091 [M+Na] +.

[0402] 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).

[0403] Preparation Example 2: Synthesis of Compound CR01008Z

[0404] The synthetic route of compound CR01008Z is as follows:

[0405]

[0406] (2-1) Synthesis of Compound 9

[0407] 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, and nitrogen was replaced 3 times. The mixture was stirred at 25°C for 16 hours, and purified by 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] + .

[0408] (2-2) Synthesis of Compound CR01008Z

[0409] 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 bottle and shaken for 16 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was first washed once with 10 ml of acetonitrile (1×10 ml) and then vacuum dried. 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 bottle and shaken for 6 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was first washed once with 10 ml of acetonitrile (1×10 ml) and then vacuum dried to obtain compound CR01008Z (1.03 g, loading 20-30 μmol / g).

[0410] Wherein, Cap1 and Cap2 are capping reagents, Cap1 is a 20 volume % N-methylimidazole mixed solution in pyridine / acetonitrile, and the volume ratio of pyridine to acetonitrile is 3:5; Cap2 is a 20 volume % acetic anhydride solution in acetonitrile.

[0411] Preparation Example 3: Synthesis of Compound CR01013

[0412] The synthetic route of compound CR01013 is as follows:

[0413]

[0414] (3-1) Synthesis of Compound 2

[0415] 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 mass %) and aqueous sodium hydroxide solution (6.5 ml, concentration of 45.3 mass %) were added dropwise. After the addition was completed, 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 product as a white solid. A small amount of water was added to the crude product for pulping, and the product was filtered and dried to obtain compound 2 as a white solid (4.8 g, yield 85.9%). ESI-MS (m / z) = 260.2 [M+H] + .

[0416] (3-2) Synthesis of Compound 3

[0417] Compound 2 (4.8 g) prepared in step (3-1) was dissolved in 25 ml of 1,4-dioxane, and 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 the reaction, the reaction solution was evaporated under reduced pressure to obtain compound 3 (3.6 g, yield 99.4%) as a white solid.

[0418] (3-3) Synthesis of Compound 11

[0419] The 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 the reaction, the reaction solution was added to 200 ml of saturated sodium bicarbonate aqueous 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 sodium chloride aqueous solution (1×50 ml), and then dried over anhydrous sodium sulfate. The organic phase was evaporated to dryness under reduced pressure and purified by 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] + .

[0420] (3-4) Synthesis of Compound 12

[0421] The compound 11 (2.3 g) prepared in step (3-3) was dissolved in 23 ml of methanol, wet palladium carbon (230 mg, loading 10 mass%) was added, and hydrogen was replaced three times. The reaction system was stirred at 25° C. in a hydrogen atmosphere (15 psi) for 16 hours. After the reaction was completed, the reaction solution was filtered to obtain a filtrate, and the filtrate was evaporated under reduced pressure to obtain a yellow oily compound 12 (1.48 g, yield 99.8%).

[0422] (3-5) Synthesis of Compound 13

[0423] The 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, and the mixture was stirred at 25°C for 16 hours. After the reaction, the reaction solution was added to 150 ml of saturated sodium bicarbonate aqueous solution, and extracted three times with 50 ml of ethyl acetate (3×50 ml), and the organic phases were combined. The organic phase was first washed once with 30 ml of saturated sodium chloride aqueous solution (1×30 ml), and then dried over anhydrous sodium sulfate. The organic phase was evaporated to dryness under reduced pressure, and purified by 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] + .

[0424] (3-6) Synthesis of Compound 14

[0425] The 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 with an ice-water bath, and DMTrCl (813 mg) was added in batches at 0°C, and 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 solvent was evaporated, and the reaction 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] + .

[0426] (3-7) Synthesis of Compound CR01013

[0427] At 25°C, compound 14 (550 mg) was dissolved in 5 ml of dichloromethane (DCM), 4,5-dicyanoimidazole (DCl, 53.2 mg) and compound 7 (2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite, 255.4 mg) were added, and the nitrogen atmosphere was replaced three times. The reaction system was stirred at 25°C for 1 hour in a nitrogen atmosphere. After the reaction was completed, the reaction solution was washed twice with 5 ml of saturated sodium bicarbonate aqueous solution (2×5 ml), and then washed once with 30 ml of saturated sodium chloride aqueous solution (1×30 ml), the organic phase was separated, dried over anhydrous sodium sulfate, and the organic phase solvent was evaporated under reduced pressure. The compound was purified by column chromatography (eluent: dichloromethane / methanol=20 / 1, v / v) to obtain compound CR01013 (532 mg, yield 80.4%) as a white solid. ESI-MS (m / z): 1176.7 [M+H] + .

[0428] 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).

[0429] Preparation Example 4: Synthesis of Compound CR01013Z

[0430] The synthetic route of compound CR01013Z is as follows:

[0431]

[0432] (4-1) Synthesis of Compound 15

[0433] At 25°C, compound 14 (100 mg, 0.10 mmol) was dissolved in 2 ml of dichloromethane, 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, and the reaction system was stirred at 25°C for 16 hours. After the reaction, the solvent in the reaction solution was evaporated, and column chromatography was performed for reverse phase purification (C18 column, eluent: water / acetonitrile = 2 / 1, v / v) to obtain yellow oily compound 15 (110 mg, 0.10 mmol, yield 100%). ESI-MS (m / z) = 1099.3 [M+Na] + .

[0434] (4-2) Synthesis of Compound CR01013Z

[0435] Compound 15 (50 mg, 0.04 mmol) was dissolved in 10 ml 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 the reaction, the reaction solution was filtered to obtain a filter cake, which was first washed twice with 50 ml dichloromethane (2×50 ml), washed three times with 50 ml acetonitrile (3×50 ml), and washed once with 50 ml ethyl acetate (1×50 ml), and then vacuum dried. Cap1 (4.8 ml), Cap2 (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 vacuum dried to obtain compound CR01013Z (900 mg, loading amount of 20-30 μmol / g).

[0436] Wherein, Cap1 and Cap2 are capping reagents, Cap1 is a 20 volume % N-methylimidazole mixed solution in pyridine / acetonitrile, and the volume ratio of pyridine to acetonitrile is 3:5; Cap2 is a 20 volume % acetic anhydride solution in acetonitrile.

[0437] Preparation of siRNA / siRNA conjugates

[0438] Preparation Example 5 Synthesis of siRNA

[0439] (5-1) Synthesize the positive strand (SS)

[0440] By the method of phosphoramidite nucleic acid solid phase synthesis, the compound of the solid phase carrier is connected to the nucleoside monomers one by one in the 3'-5' direction according to the nucleotide sequence. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. The synthesis conditions are given as follows:

[0441] The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0442] The conditions of the deprotection reaction in each step are the same. The conditions of the deprotection reaction are: temperature of 25° C., reaction time of 70 seconds, deprotection reagent of dichloroacetic acid in dichloromethane solution (3% by volume), and the molar ratio of dichloroacetic acid to 4,4'-dimethoxytrityl protecting group on the solid phase carrier is 5:1.

[0443] The conditions of each coupling reaction are the same. The coupling reaction conditions are: temperature is 25°C, the molar ratio of the nucleic acid sequence connected to the solid phase support to the nucleoside monomer is 1:10, the molar ratio of the nucleic acid sequence connected to the solid phase support to the coupling reagent is 1:65, the reaction time is 600 seconds, the coupling reagent is an acetonitrile solution of 5-ethylthio-1H-tetrazole with a concentration of 0.5M, and the thiolation reagent is an acetonitrile / pyridine mixed solution of hydrogenated xanthanol with a concentration of 0.2M (the volume ratio of acetonitrile and pyridine is 1:1).

[0444] The conditions of each capping reaction are the same. The conditions of the capping reaction are: temperature is 25°C; reaction time is 2 minutes; the capping reagent solution is a mixed solution of Cap1 and Cap2 with a molar ratio of 1:1, Cap1 is a pyridine / acetonitrile mixed solution of N-methylimidazole with a concentration of 20 volume%, the volume ratio of pyridine to acetonitrile is 3:5, Cap2 is an acetonitrile solution of acetic anhydride with a volume ratio of 20 volume%; the molar ratio of N-methylimidazole in Cap1 capping reagent, acetic anhydride in Cap2 capping reagent to the nucleic acid sequence connected to the solid phase carrier is 1:1:1.

[0445] 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; oxidation reagent concentration of 0.05M iodine water, the molar ratio of iodine to the nucleic acid sequence connected to the solid phase carrier in the coupling reaction is 30:1; oxidation reaction is carried out in a water / pyridine mixed solvent (the volume ratio of water to pyridine is 1:9). The conditions for the sulfidation reaction are: temperature of 25°C; reaction time of 360 seconds; thiolation reagent concentration of 0.2M hydrogenated xanthan gum pyridine solution, the molar ratio of thiolation reagent to the nucleic acid sequence connected to the solid phase carrier in the coupling reaction is 4:1; thiolation reaction is carried out in a water / pyridine mixed solvent (the volume ratio of water to pyridine is 1:9).

[0446] After the last nucleoside monomer is connected, the nucleic acid sequence connected to the solid phase carrier is cut, deprotected, purified, desalted, and then freeze-dried to obtain the positive chain, wherein:

[0447] The cleavage and deprotection conditions are as follows: the synthesized nucleotide sequence connected to the solid phase carrier is added to 25% by mass ammonia water, the amount of ammonia water is 0.5 ml / μmol, the reaction is carried out at 55°C for 16 hours, the solvent is removed, and the product is concentrated to dryness in vacuo. After the ammonia water treatment, the product is dissolved with 0.4 ml / μmol N-methylpyrrolidone relative to the amount of single-stranded nucleic acid, and then 0.3 ml / μmol triethylamine and 0.6 ml / μmol triethylamine trihydrofluoride are added to remove the 2'-O-TBDMS protection on the ribose.

[0448] Purification and desalting conditions: The nucleic acid was purified by gradient elution of NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, eluent 1 was 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixed solution (the volume ratio of water to acetonitrile was 9:1); eluent 2 was 1.5 M sodium chloride, 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixed solution (the volume ratio of water to acetonitrile was 9:1); the elution gradient was eluent 1: eluent 2 = (100:0)-(50:50). The product eluates were collected and combined, and desalted using a reverse chromatography purification column. The desalting conditions included desalting using a dextran gel column, the filler was dextran gel G25, and eluted with deionized water.

[0449] Detection: Purity was detected by ion exchange chromatography (IEX-HPLC); molecular weight was detected by liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value. The measured value was consistent with the theoretical value, indicating that the obtained compound was conjugated to the 3' end of the siRNA sense chain.

[0450] (5-2) Synthesis of antisense strand (AS)

[0451] The antisense chain was synthesized using a universal solid phase carrier. 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 (5-1) for synthesizing the sense chain.

[0452] Detection: Purity was detected by ion exchange chromatography (IEX-HPLC); molecular weight was detected by liquid chromatography-mass spectrometry (LC-MS), and the measured value of the molecular weight was compared with the theoretical value. The measured value was consistent with the theoretical value, indicating that the siRNA antisense chain was obtained.

[0453] (5-3) Synthesis of siRNA

[0454] The sense chain synthesized in step (5-1) and the antisense chain synthesized in step (5-2) are mixed in an equimolar ratio, dissolved in water for injection and heated to 95° C., slowly cooled to room temperature and kept at room temperature for 10 minutes, so that the sense chain and the antisense chain form a double-stranded structure through hydrogen bonds, thereby obtaining siRNA having the sense chain and antisense chain shown in Table 2.

[0455] 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 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; the 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.

[0456] Table 2 siRNA sequence information

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470] Preparation Example 6 Preparation of siRNA conjugate in which L96 vector is conjugated to the 3' end of the sense strand (L96 vector siRNA conjugate) Preparation

[0471] Compound L96-PS

[0472] 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:

[0473]

[0474] Wherein, PS represents polystyrene resin solid phase carrier.

[0475] (6-1) Synthesis of positive chain:

[0476] By the phosphoramidite nucleic acid solid phase synthesis method, the L96-PS compound connected to the solid phase carrier is used to start the cycle, and the nucleoside monomers are connected one by one in the 3'-5' direction according to the nucleotide sequence. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. The synthesis method and conditions refer to the positive chain synthesis method shown in step (5-1).

[0477] (6-2) Synthesis of antisense strand:

[0478] The siRNA antisense strand was synthesized according to the antisense strand synthesis method shown in step (5-2).

[0479] (6-3) Synthesis of L96 vector siRNA conjugate:

[0480] The L96 vector siRNA conjugate was synthesized according to the method shown in step (5-3).

[0481] Among them, the structural formula of L96 vector siRNA conjugate is as follows:

[0482]

[0483] in, L96 is conjugated to the 3' end of the sense strand of siRNA via a phosphodiester bond.

[0484] 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 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.

[0485] Table 3 siRNA conjugate information

[0486]

[0487]

[0488] Note: The structure of Tgn is as follows:

[0489] Preparation Example 7 Synthesis of siRNA conjugates in which (CR01008)×3 carriers are conjugated to the 3' end of the sense strand

[0490] (7-1) Sense Chain Synthesis

[0491] By the method of phosphoramidite nucleic acid solid phase synthesis, compound CR01008 and nucleoside monomers are connected one by one in a cycle using compound CR01008Z as the starting cycle (compound CR01008 is regarded as a nucleoside monomer during the synthesis process). Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cutting and deprotection conditions, purification and desalting conditions in the synthesis of the sense chain of this preparation example are the same as the synthesis conditions of the sense chain in step (5-1).

[0492] During the synthesis of the positive strand, three clusters of CR01008 vectors were obtained, which were recorded as (CR01008)×3 or (CR01008×3).

[0493] The structural formula of the three-cluster CR01008 is as follows:

[0494]

[0495] (7-2) Synthesis of antisense strand:

[0496] The siRNA antisense strand was synthesized according to the antisense strand synthesis method shown in step (5-2).

[0497] (7-3) (CR01008)×3 vector conjugated to the 3' end of the sense strand Synthesis of siRNA conjugates

[0498] The (CR01008)×3 vector-siRNA conjugate was synthesized according to the method shown in step (5-3).

[0499] in, (CR01008)×3 vector conjugated to the 3' end of the sense strand The structural formula of the siRNA conjugate is shown below:

[0500]

[0501] in, Represents siRNA. (CR01008×3) is conjugated to the 3' end of the sense strand of siRNA via a phosphodiester bond.

[0502] In the sequence disclosed herein, the base composition and modification meanings are as follows: capital letters A, U, G, C, and T represent the base composition of 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; the 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.

[0503] Table 4 siRNA conjugate information

[0504]

[0505]

[0506]

[0507] Biological detection experiments

[0508] Unless otherwise stated, the reagents and consumables (Table 5) and instruments and equipment (Table 6) used in the present disclosure are all commercially available products from the following manufacturers.

[0509] Table 5 Main reagents and consumables

[0510]

[0511]

[0512] Table 6 Main instruments and equipment

[0513] name Manufacturer Fully automatic nucleic acid extraction instrument Zhejiang Hanwei Technology Co., Ltd. High-speed refrigerated centrifuge Eppendorf Carbon dioxide incubator Thermo Fisher Scientific Biological Safety Cabinets Shanghai Lishen Constant temperature water bath Shanghai Boxun Automated cell counter Shanghai Ruiyu Inverted microscope Olympus NANODROP OneC Thermo Fisher Scientific Gradient PCR Amplifier Eppendorf CFX Opus 384 Bio-Rad LightCycler 480 Roche Real-time fluorescence quantitative (RT-PCR) instrument QuantStudio TM3 Thermo Fisher Scientific Gel Imager Shanghai Tianneng Life Sciences Co., Ltd. Electrophoresis Apparatus Beijing Liuyi Instrument Factory Tissuelyser II fully automatic tissue homogenizer Shanghai Jingxin Industrial Development Co., Ltd. Paraffin slicer Jinhua Yidi Tissue dehydrator Leica Fully automatic biological tissue embedding machine Jinhua Yidi Automatic modular blood and body fluid analyzer SYSMEX Automatic biochemical analyzer SYSMEX Automatic coagulation analyzer Mindray

[0514] Example 1. In vitro activity evaluation of angiotensinogen (AGT) siRNA

[0515] 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.

[0516] Test sample preparation:

[0517] After centrifugation of each siRNA test product, add an appropriate amount of DEPC H2O according to the specifications of each tube to dissolve it and prepare a 20μM stock solution. The stock solution is further diluted with DEPC H2O to 0.04μM and 0.004μM working solutions. The dose screening test is carried out with a final duplex concentration of 1nM and 0.1nM.

[0518] 96-well transfection and detection:

[0519] Huh7 cells grown to near confluence were digested with trypsin, and the cells were washed to prepare a cell suspension for plating in 96-well plates.

[0520] 5 μL of 0.04 μM and 0.004 μM working solutions were dispersed in 20 μL Opti-MEM to form siRNA mixtures, and 0.2 μL RNAiMAX was dispersed in 24.8 μL Opti-MEM. After incubation for 5 minutes, each siRNA mixture was mixed and incubated for 10 minutes to form transfection complexes. 150 μL of cell suspension (12,000 cells) was added to the transfection complexes in each well, and the 96-well plate was placed in an incubator for further 24 hours.

[0521] Among them, the Mock control group was prepared by dispersing 0.2 μL RNAiMAX in 24.8 μL Opti-MEM, incubating for 5 minutes, and then adding 25 μL Opti-MEM and incubating 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 continued to be cultured for 24 hours.

[0522] The 96-well plate was removed, the cells were lysed, and the lysate was diluted. A one-step qPCR kit (Thermo Fisher, Power SYBR Green RNA-to-CT TM 1-Step, 4389986) The expression of target gene mRNA in Huh7 cells was detected on a fluorescent quantitative PCR instrument (Roche, LightCycler480). In this real-time fluorescent quantitative PCR method, the TATA-box binding protein (TBP) gene was used as an internal reference gene, and primers for the target gene and the TBP internal reference gene were used to detect the target gene and the TBP internal reference gene, respectively. The sequences of the detection primers are shown in Table 7.

[0523] Table 7 Sequences of detection primers

[0524]

[0525] In the real-time fluorescence quantitative PCR method, the ΔΔCt method is used according to the technical method described in the embodiment to perform relative quantitative calculation of the expression level and inhibition rate of the target gene mRNA in each test group.

[0526] Table 8 Inhibitory activity of target genes in Huh7 cells after administration of siRNA described in this example

[0527]

[0528]

[0529]

[0530]

[0531]

[0532]

[0533]

[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 with L96 vector conjugates RZ003002 to RZ003023 on the target gene AGT, with RZ000002 used as a negative control.

[0536] Plasmid construction:

[0537] 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 a model established by rapidly injecting the pcDNA-CMV-RG003 plasmid solution into the mouse through the tail vein of the mouse. On the third day of the experiment, the mouse was injected with 10μg pcDNA-CMV-RG003 through the tail vein hydrodynamics within 5 seconds, and the injection volume was 8% of the mouse body weight. The plasmid DNA for injection was diluted with physiological saline, and the solution was prepared before 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 according to body weight (all female), with 5 mice in each group. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. The dose of all mice was calculated according to body weight, and a single dose was given by subcutaneous injection in the abdomen. Each drug conjugate was administered in the form of a PBS solution of 0.1 mg / mL (calculated as siRNA), 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 (without 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), and 5 mice in all groups were killed on the 4th day (recorded as D4). The mice that were killed were grossly dissected and the liver tissue of each mouse that was killed was collected, and the liver tissue was cut into about 2 mm 3 Small pieces were stored in RNA later.

[0542] For each mouse, an appropriate amount of liver tissue sample was taken from RNA later, and the liver tissue sample was broken in a Tissuelyser II fully automatic tissue homogenizer for 60 seconds, and then the 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 taken and reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) was used and Oligo(dT) was selected. 18 Reverse transcription primers, according to the method of reverse transcription kit specification sheet, configure 20 μ L reverse transcription system and complete reverse transcription reaction. After the reaction is completed, 60 μ L RNase-Free water is added to the reverse transcription system to obtain cDNA solution. Then the expression of target gene mRNA in animals is detected using real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific, TaqMan Fast Advanced Master Mix, 4444557) fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384). In this real-time fluorescence quantitative PCR method, Nero gene on plasmid backbone is used as internal reference gene, and primers for target gene and primers for Nero internal reference gene are used to detect target gene and Nero internal reference gene respectively. The sequence of detection primers is shown in Table 9.

[0544] Table 9 Sequences of detection primers

[0545]

[0546] According to the method described in the instruction manual 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 above 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 configured reaction system is placed on a real-time fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384), and a two-step method is used for Real-time PCR amplification. The amplification program is 50°C 2min, then 95°C pre-denaturation 20s, 95°C denaturation 3s, 60°C annealing and extension 30s, and the denaturation, annealing and extension process is repeated for 40 cycles. In the real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group are relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0547] 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).

[0548] Table 10. Inhibitory activity of target genes in Balb / c-HDI mice after administration of the siRNA conjugates described in this example

[0549]

[0550] Example 3. Evaluation of multiple dose activity of L96 vector-conjugated siRNA in Balb / c-HDI mice

[0551] In this example, the Balb / c mouse hydrodynamic injection model was used to evaluate the inhibitory activity of the same AGT target siRNA positive chain 3' end conjugated with L96 vector conjugates RZ003003, RZ003004, RZ003005, RZ003008, RZ003010, RZ003017, RZ003020, and RZ003021 on the target gene AGT at different doses, with Zilebesiran as a positive control.

[0552] Plasmid construction:

[0553] pcDNA-CMV-RG003 plasmid (ID: NM_001382817.3) was constructed by Sangon Biotech (Shanghai) Co., Ltd.

[0554] Mouse model construction:

[0555] The Balb / c mouse hydrodynamic injection model is a model established by rapidly injecting the pcDNA-CMV-RG003 plasmid solution into the mouse through the tail vein of the mouse. On the third day of the experiment, the mouse was injected with 10μg pcDNA-CMV-RG003 through the tail vein hydrodynamics within 5 seconds, and the injection volume was 8% of the mouse body weight. The plasmid DNA for injection was diluted with physiological saline, and the solution was prepared before injection and stored at 4°C.

[0556] Animal grouping, drug administration and tissue sample collection:

[0557] 6-8 week old Balb / c mice were randomly divided into groups according to body weight (all female), with 5 mice in each group. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. The drug dose of all mice was calculated according to body weight, and a single dose was given by subcutaneous injection in the abdomen. Each drug conjugate was administered in the form of 0.05mg / mL, 0.1mg / mL, and 0.3mg / mL (calculated as siRNA) PBS solution, and the administration volume was 10mL / kg mouse body weight, that is, the dosage of each drug conjugate was 0.5mg / kg, 1mg / kg, and 3mg / 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 plasmid injection was performed on the 3rd day after administration (recorded as D3), and 5 mice in all groups were killed on the 4th day (recorded as D4). The mice that were killed were grossly dissected and the liver tissue of each mouse that was killed was collected, and the liver tissue was cut into about 2mm 3 Small pieces were stored in RNA later.

[0558] For each mouse, an appropriate amount of liver tissue sample was taken from RNA later, and the liver tissue sample was broken in a Tissuelyser II fully automatic tissue homogenizer for 60 seconds, and then the 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.

[0559] For each mouse, 1 μg of total RNA was taken and reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) was used and Oligo(dT) was selected. 18 Reverse transcription primers, according to the method recorded in the reverse transcription kit specification, configure 20 μL reverse transcription system and complete the reverse transcription reaction. After the reaction is completed, 60 μL RNase-Free water is added to the reverse transcription system to obtain a cDNA solution. Then the expression of the target gene mRNA in the animal is detected using a real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific, TaqMan Fast Advanced Master Mix, 4444557) fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384). In this real-time fluorescence quantitative PCR method, the Nero gene on the plasmid backbone is used as an internal reference gene, and the primer for the target gene and the primer for the Nero internal reference gene are used to detect the target gene and the Nero internal reference gene respectively. The sequence of the detection primer is shown in Table 11.

[0560] Table 11. Sequences of detection primers

[0561]

[0562]

[0563] According to the method described in the instruction manual 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 cDNA solution obtained by the above reverse transcription reaction, 5 μL TaqManTM Fast Advanced Master Mix (2×), 0.15 μL of 10 μM upstream primer, 0.15 μL of 10 μM downstream primer (primer information is shown in Table X), 0.15 μL of 10 μM probe primer, and 0.55 μL of 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 min, followed by pre-denaturation at 95°C for 20 s, denaturation at 95°C for 3 s, annealing and extension at 60°C for 30 s, and the process of denaturation, annealing and extension was repeated for 40 cycles. In the real-time fluorescence quantitative PCR method, the ΔΔCt method is used according to the technical method described in the embodiment to perform relative quantitative calculation of the expression level and inhibition rate of the target gene mRNA in each test group.

[0564] The results of Example 3 show that RZ003003, RZ003004, RZ003005, RZ003008, RZ003010, RZ003017, RZ003020, and RZ003021 can inhibit AGT mRNA expression in a dose-dependent manner; at a dose of 3 mg / kg, RZ003003, RZ003017, RZ003020, and RZ003021 can reduce AGT mRNA by up to 95%, which is better than Zilebesiran ( Figure 1 , Table 12).

[0565] Table 12 Inhibitory activity of target genes in Balb / c-HDI mice after administration of the siRNA conjugates described in this example

[0566]

[0567] Example 4. Evaluation of the efficacy of L96 vector-conjugated siRNA in hREN×hAGT hypertensive mice

[0568] 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 regulating effects of RZ003008, RZ003017, RZ003020 and RZ0030021 in mice; the ELISA method was used to measure the AGT protein expression in the serum at different time points, with Zilebesiran as a control.

[0569] Animal grouping, drug administration and tissue sample collection:

[0570] 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 in each 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 given 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 dosage 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 3rd day (recorded as D3), 7th day (recorded as D7), 10th day (recorded as D10), 14th day (recorded as D14), 17th day (recorded as D17), 21st day (recorded as D21), 24th day (recorded as D24), 28th day (recorded as D28), 31st day (recorded as D31), 35th day (recorded as D35), and 38th day (recorded as D38) after administration were used. The 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 from all groups of mice was collected 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 the AGT protein expression was measured using a human angiotensinogen kit (IBL, 27412).

[0571] The results of Example 4 show that, at a single dose of 3 mg / kg, RZ003008, RZ003017, RZ003020, and RZ003021 can reduce the systolic blood pressure, mean arterial pressure, and diastolic blood pressure levels in hREN×hAGT hypertensive mice; as the observation time increases, the blood pressure level gradually recovers. Figure 2 , Table 13, Table 14, Table 15). Among them, RZ003021 reduced systolic blood pressure by more than 20 mmHg, and the effect lasted for 24 days; while Zilebesiran had no significant effect on lowering systolic blood pressure at the same time point ( Figure 2 , Table 13, Table 14, Table 15).

[0572] The results of Example 4 show that RZ003017, RZ003020, and RZ003021 can reduce the AGT protein level in hREN×hAGT hypertensive mice at a single dose of 3 mg / kg; the AGT protein level gradually increases with the extension of the observation time; among them, no obvious AGT protein inhibition effect was observed at a single dose of 3 mg / kg of Zilebesiran ( Figure 3, Table 16).

[0573]

[0574]

[0575]

[0576] Table 16 Changes in the level of human AGT protein in hREN×hAGT hypertensive mice after administration of the siRNA conjugates described in this example

[0577]

[0578] Example 5. Toxicological evaluation of L96 vector-conjugated siRNA in mice

[0579] In this example, a single subcutaneous injection of 300 mg / kg was used to evaluate the nature and extent of the toxicity of the carrier conjugates RZ003004, RZ003008, RZ003010, RZ003017, RZ003020, and RZ0030021 in ICR mice.

[0580] Animal grouping, drug administration and tissue sample collection:

[0581] 6-8 week old ICR mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were randomly divided into groups according to body weight, with 10 mice in each group, half male and half female. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. The drug dose of all mice was calculated according to body weight, and a single dose was given by subcutaneous injection in the abdomen. Each drug conjugate was administered in the form of a 30 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 300 mg / kg mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PBS solution (without drug conjugate) and observed for 14 days. The day of administration was recorded as day 0 (recorded as D0), and clinical observations were performed at least once a day after the first administration during the test period, and body weight and food intake were measured twice a week; blood was taken from all animals on the 14th day of the test (recorded as D14) (mice were fasted for at least 12 hours before sampling and testing, and water was not forbidden), and hematology and blood biochemistry tests were performed. Blood was collected and anticoagulated with EDTA-K2, and routine blood tests were performed using an automatic modular blood and body fluid analyzer (Sysmex XN-10B1); blood samples (without anticoagulation) were collected and placed at room temperature for about 30 minutes. After the blood coagulated, it was centrifuged at 2000g for 10 minutes at 4°C, and blood biochemistry tests were performed using a fully automatic biochemical analyzer (Sysmex BX-4000). All animals were dissected on the 14th day of the experiment (recorded as D14). The mice to be dissected were fasted but not watered for at least 12 hours before dissection, anesthetized with Zoltek 50 (Vick Co., France, 8G4VA), and euthanized by bleeding from the abdominal aorta after blood collection, and gross anatomical observations were performed; the livers and kidneys of all animals were subjected to histopathological examination (using hematoxylin-eosin staining). The results showed that the test substances had no significant effects on the general condition, body weight, and gross anatomical observations of food intake of ICR mice. The RZ003017 300 mg / 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 RZ003004 300 mg / kg group, increased mitosis was observed in the livers of some animals in the other groups. No pathological changes related to the test substances were observed in the kidneys.

[0582] Example 6. In vitro activity evaluation of CR01008 vector conjugate siRNA

[0583] 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 the CR01008 vector conjugate siRNA on the target gene AGT in cells.

[0584] Test sample preparation:

[0585] After centrifugation of each siRNA test product, add appropriate amount of PBS according to the specifications of each tube to dissolve it and prepare a 20μM stock solution, and then further dilute the stock solution with PBS to 0.1μM and 0.01μM working solutions. The dosage test was carried out with a final duplex concentration of 1nM and 0.1nM.

[0586] 96-well transfection and detection:

[0587] Huh7 cells grown to near confluence were digested with trypsin, washed and prepared into cell suspension, 100 μL of cell suspension was added to each well of a 96-well plate, 12,000 cells were added to each well, and cultured in a 37°C, 5% CO2 incubator. When the cells were attached for 24 hours, the DMEM medium in the 96-well plate was discarded, 80 μL of Opti-MEMTM medium was added to each well, and then the 96-well plate was placed in an incubator for further culture; 1 μL of 0.1 μM and 0.01 μM working solution was dispersed in 9 μL Opti-MEM to form an siRNA mixture, 0.3 μL of RNAiMAX was dispersed in 9.7 μL Opti-MEM, and mixed with each siRNA mixture to form a transfection complex. The transfection complex was incubated at room temperature for 10 minutes, and then the transfection complex was added to a 96-well plate, 20 μL / well. After 4 hours of culture, 100 μL of DMEM medium containing 20% ​​FBS was added to each well, and the 96-well plate was placed in an incubator for further culture for 24 hours. Among them, Mock control group: 0.3 μL RNAiMAX was dispersed in 9.7 μL Opti-MEM, and then 10 μL Opti-MEM was added, incubated at room temperature for 10 minutes; then added to the 96-well plate, 20 μL / well. After 4 hours of culture, 100 μL of DMEM medium containing 20% ​​FBS was added to each well, and the 96-well plate was placed in an incubator for further culture for 24 hours. Take out the 96-well plate, use the fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and the nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction to extract total RNA.

[0588] Use a reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) and select Oligo (dT) 18Reverse transcription primer, according to the method of reverse transcription kit specification sheet record configuration 20 μ L reverse transcription system 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 primer for target gene and primer for GAPDH internal reference gene to detect target gene and GAPDH internal reference gene respectively.The sequence of detection primer is shown in Table 17.

[0589] Table 17 Sequences of detection primers

[0590]

[0591]

[0592] According to the method described in the instruction manual 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 cDNA solution obtained by the above reverse transcription reaction, 5 μL TaqManTM Fast Advanced Master Mix (2×), 0.15 μL of 10 μM upstream primer, 0.15 μL of 10 μM downstream primer (primer information is shown in Table X), 0.15 μL of 10 μM probe primer, and 0.55 μL of 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 min, followed by pre-denaturation at 95°C for 20 s, denaturation at 95°C for 3 s, annealing and extension at 60°C for 30 s, and the process of denaturation, annealing and extension was repeated for 40 cycles. In the real-time fluorescence quantitative PCR method, the ΔΔCt method is used according to the technical method described in the embodiment to perform relative quantitative calculation of the expression level and inhibition rate of the target gene mRNA in each test group.

[0593] Table 18 Inhibitory activity of target genes in Huh7 cells after administration of the siRNA conjugates described in this example

[0594]

[0595]

[0596] The results of Example 6 showed that at the doses of 1 nM and 0.1 nM, the differently modified CR01008 vector conjugate siRNAs could significantly inhibit the expression of AGT mRNA in Huh7 cells (Table 18).

[0597] Example 7. Evaluation of the antihypertensive effect of CR01008 vector-conjugated siRNA in hREN×hAGT hypertensive mice

[0598] 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.

[0599] Animal grouping, drug administration and tissue sample collection:

[0600] 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 of all mice was calculated according to body weight, and a single dose was given 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 dosage 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 measuring instrument (Softlong, BP-2010A) before administration (recorded as pre-dose, pre-drug 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.

[0601] The results of Example 7 show that, at a single dose of 3 mg / kg, RZ003062, RZ003064, RZ003065, and RZ003066 can reduce the systolic blood pressure level of hREN×hAGT hypertensive mice; as the observation time increases, the blood pressure level gradually recovers ( Figure 5 , Table 19). Among them, RZ003064 and RZ003021 had the same systolic blood pressure lowering effect, while Zilebesiran had no significant systolic blood pressure lowering effect at the same time point ( Figure 5 , Table 19).

[0602] Table 19 Changes in systolic blood pressure in hREN×hAGT hypertensive mice after administration of the siRNA conjugates described in this example

[0603]

[0604] Example 8. Evaluation of the protein-lowering effect of CR01008 vector-conjugated siRNA in hREN×hAGT hypertensive mice

[0605] In this example, the enzyme-linked immunosorbent assay (ELISA) was used to determine the AGT protein expression in the serum of mice at different time points after a single administration of CR01008 vector conjugates RZ003064, RZ003065, RZ003066 and RZ0030021.

[0606] Animal grouping, drug administration and tissue sample collection:

[0607] 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 of all mice was calculated according to body weight, and a single dose was given 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 dosage 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 recorded as day 0 (denoted as D0), and 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, the serum of all groups of mice was collected, and the AGT protein expression was determined using the human angiotensinogen kit (IBL, 27412).

[0608] Table 20 Changes in the level of human AGT protein in transgenic mice after administration of the siRNA conjugates described in this example

[0609]

[0610] The results of Example 8 showed that RZ003064, RZ003065, RZ003066, and RZ003021 could significantly reduce the AGT protein level in hREN×hAGT hypertensive mice at a single dose of 3 mg / kg. It was observed that on day 28, the AGT protein level was still reduced by more than 70% ( Figure 6 , Table 20).

[0611] Example 9. Evaluation of the efficacy of L96 vector-conjugated siRNA in SHR rats

[0612] 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 regulating effect of RZR03001 and RZR03002 in SHR rats; an enzyme-linked immunosorbent assay (ELISA) was used to measure the AGT protein expression in serum at different time points, with valsartan (Beijing Novartis Pharmaceuticals Co., Ltd., X3113) as a control.

[0613] Animal grouping, drug administration and tissue sample collection:

[0614] 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 in each group. Each test group was given a predetermined dose of 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 mouse body weight. RZR03001 and RZR03002 were administered by subcutaneous injection in the abdomen, and each drug conjugate was administered in the form of a 0.3 mg / mL (calculated as siRNA) PBS solution, that is, the dosage of each drug conjugate was 3 mg / kg rat body weight (calculated as siRNA). The valsartan dose group was prepared with PBS to make a 2.16 mg / mL solution, and oral gavage was performed twice a day, that is, the drug dosage was 21.6 mg / kg; the PBS control group was given the same volume of PBS solution (without drug conjugate).

[0615] Before administration (denoted as pre-dose, pre-dose value), the day of administration was recorded as day 0 (denoted as D0), and 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. The serum of rats in all groups was collected 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, and the expression of AGT and AGT-Ⅱ proteins was measured 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 (recorded as D7), 4 rats in all groups were killed, and on the 28th day after administration (recorded as D28), 6 rats in all groups were killed. The rats were grossly dissected and the liver tissues of each rat were collected. The liver tissues were cut into approximately 2 mm 3The small pieces were stored in RNAlater and the AGT mRNA expression level was determined. In the real-time fluorescence quantitative PCR method, ActinB gene was used as the internal reference gene, and primers for the target gene and primers for the ActinB internal reference gene were used to detect the target gene and the ActinB internal reference gene respectively. The sequences of the detection primers are shown in Table 21.

[0616] Table 21 Sequences of detection primers

[0617]

[0618] In the real-time fluorescence quantitative PCR method, the ΔΔCt method is used according to the technical method described in the embodiment to perform relative quantitative calculation of the expression level and inhibition rate of the target gene mRNA in each test group.

[0619] The results of Example 9 showed that RZR03001 and RZR03002 could reduce the systolic, mean arterial and diastolic blood pressure levels of SHR rats at a single dose of 3 mg / kg; blood pressure levels continued to gradually decrease on day 28. From day 21, the blood pressure-lowering level of a single dose of RZR03001 was similar to that of valsartan ( Figure 7 , Table 22, Table 23, Table 24).

[0620] The results of Example 9 show that RZR03001 and RZR03002 can reduce the AGT mRNA and protein levels in SHR rats at a single dose of 3 mg / kg; it was observed that on the 28th day, the mRNA reduction effect was still greater than 90% (Table 25), and the AGT protein reduction effect was still greater than 70% (Table 26). It was also observed that on the 28th day, RZR03001 and RZR03002 had a certain effect of reducing Ang-Ⅱ protein (Table 27).

[0621]

[0622]

[0623] Table 25 Changes in AGT mRNA levels in SHR rats after administration of the siRNA conjugates described in this example

[0624]

[0625] Table 26 Changes in AGT protein levels in SHR rats after administration of the siRNA conjugates described in this example

[0626]

[0627] Table 27 Changes in Ang-Ⅱ protein levels in SHR rats after administration of the siRNA conjugates described in this example

[0628]

[0629] Although the embodiments of the present disclosure have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of 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 In the direction from the 5' end to the 3' end, the 2nd to 19th nucleotides of the antisense strand comprise a complementary region to the 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 to 448, or comprises a nucleotide sequence having 1, 2 or 3 nucleotide differences with 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 to 448; Optionally, the antisense strand comprises a sequence having 0, 1, 2 or 3 nucleotide differences from any one of SEQ ID NO:225 to 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 to SEQ ID NO:448; Optionally, the antisense strand is selected from or comprises any one of SEQ ID NO: 225 to 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 to 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 to SEQ ID NO: 224; Optionally, the sense strand is selected from or includes any one of SEQ ID NO: 1 to 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 45 groups: 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 10, characterized in that The double-stranded oligonucleotide is selected from siRNA.

12. A conjugate, characterized in that The conjugate comprises: The double-stranded oligonucleotide according to any one of claims 1 to 11 and one or more ligands capable of binding to a cell receptor; The ligand is conjugated to the double-stranded oligonucleotide.

13. The conjugate according to claim 12, 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 said ligands is selected from one, and one said ligand is conjugated to the 3' end of said sense strand.

14. The conjugate according to claim 12, 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 1, 2 or 3; Optionally, j is selected from 3; 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.

15. The conjugate according to claim 14, 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:

16. 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 11; and / or (II) the conjugate according to any one of claims 12 to 15; 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.

17. 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 11; and / or (II) The conjugate according to any one of claims 12 to 15.

18. 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 11; and / or (II) the conjugate according to any one of claims 12 to 15; and / or (III) The pharmaceutical composition according to claim 17.

19. A method for inhibiting the expression of AGT gene in cells, characterized in that: It includes: The double-stranded oligonucleotide according to any one of claims 1 to 11, and / or the conjugate according to any one of claims 12 to 15, and / or the pharmaceutical composition according to claim 17 is administered to a subject.

20. A method for alleviating, treating and / or preventing AGT-mediated diseases or conditions, characterized in that: It includes: Administering the double-stranded oligonucleotide according to any one of claims 1 to 11, and / or the conjugate according to any one of claims 12 to 15, and / or the pharmaceutical composition according to claim 17 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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