SiRNA for inhibiting angiotensinogen expression and conjugate and application thereof

Abstract

The invention belongs to the field of biological medicine, and relates to siRNA for inhibiting AGT gene expression and a conjugate, the siRNA comprises a positive-sense strand and an antisense strand, the positive-sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II; each nucleotide in the nucleotide sequence I and the nucleotide sequence II is modified or unmodified nucleotide; the nucleotide sequence I and the nucleotide sequence II are at least partially reversely complementary to form a double-stranded region; the nucleotide sequence I is basically consistent with a first section of nucleotide sequence, and the first section of nucleotide sequence is a section of nucleotide sequence with the length of at least 15 nucleotides in mRNA expressed by AGT gene. The siRNA as well as the conjugate and the pharmaceutical composition thereof disclosed by the invention can effectively prevent and / or treat diseases related to AGT expression, such as hypertension and the like, and have a good patent medicine prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to siRNA for inhibiting angiotensinogen (AGT) expression, conjugates thereof and application. BACKGROUND

[0002] Hypertension is a common chronic disease, and is an important risk factor for leading to coronary heart disease, myocardial infarction, stroke and renal dysfunction. There are 245 million people with hypertension in China, and only 16.8% of the people with hypertension can effectively control their blood pressure. Globally, there are more than 1 billion people with hypertension, affecting one fifth of women and one fourth of men. With the aging of the population and the prevalence of risk factors such as obesity, lack of physical activity and unhealthy diet, the prevalence of hypertension is still increasing worldwide. Hypertension is divided into primary hypertension and secondary hypertension. Primary hypertension refers to chronic elevation of blood pressure (more than 140 / 90 mmHg or 130 / 80 mmHg according to different standards) without clear disease causes, and is a very common age-related disease.

[0003] The renin-angiotensin-aldosterone system (Renin-angiotensin-aldosterone system, RAAS) is a hormone cascade that plays a crucial role in blood pressure regulation. Angiotensinogen (Angiotensinogen, AGT) is the most upstream precursor protein in the RAAS pathway, and is a liver-specific protein. The renin-angiotensin system (Renin-angiotensin system, RAS) is an important humoral regulatory system composed of a series of peptide hormones and corresponding enzymes, which is involved in the metabolic balance of blood pressure, water and electrolytes in the human body. The renin-angiotensin system plays a key role in the long-term regulation of blood pressure, and AGT is the upstream molecule of RAS and the only substrate of RAS. Studies have confirmed that high levels of AGT are an important cause of hypertension. At the cellular level, AGT can be synthesized by hepatocytes, adipocytes, proximal tubular epithelial cells, astrocytes, etc., among which hepatocytes are the main source of plasma AGT, which makes it a potential target for nucleic acid drugs.

[0004] Hypertension is common in primary, but the pathogenesis is not fully clear, and AGT has important regulatory effect on hypertension, and even part of the primary hypertension patients appear AGT plasma concentration increase phenomenon. The existing hypertension commonly used antihypertensive drugs include five categories of calcium channel blockers, angiotensin converting enzyme inhibitors (ACEI), angiotensin receptor blockers (ARB), diuretics and beta-receptor blockers. Calcium channel blockers can cause facial flushing, palpitation, headache, hypotension and other adverse reactions. The use of diuretics in the treatment of hypertension can cause the side effects of hypokalemia and hyperuricemia and photosensitivity. The use of angiotensin converting enzyme inhibitors (ACEI) and angiotensin II type 1 receptor blockers (ARB) can inhibit RAAS, although it can be short-term benefit, but studies have found that long-term single drug therapy ACEI and ARB can cause up-regulation of renin and angiotensinase I through bypass and negative feedback mechanism, and "RAS escape" occurs, and can cause blood creatinine to rise, greatly increasing the risk of liver and kidney damage. In addition, the above existing hypertension drugs need to be taken daily, resulting in generally low drug compliance, greatly affecting the blood pressure control effect of the using population. SUMMARY

[0005] The purpose of the present application is to provide a kind of siRNA molecule capable of inhibiting AGT gene expression, to provide new treatment for AGT related diseases including hypertension and related diseases.

[0006] In a first aspect, the present disclosure provides a siRNA for inhibiting AGT gene expression, the siRNA comprising a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence I, and the antisense strand comprising a nucleotide sequence II; each nucleotide in the nucleotide sequence I and the nucleotide sequence II is a modified or unmodified nucleotide; the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region; the nucleotide sequence I is substantially identical to a first nucleotide sequence, the first nucleotide sequence being a nucleotide sequence of at least 15 nucleotides in length in the mRNA expressed by AGT gene, preferably the first nucleotide sequence being a nucleotide sequence of 15-25 nucleotides in length in the mRNA expressed by AGT gene, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides;

[0007] Preferably, the first nucleotide sequence is a nucleotide sequence of at least 15 nucleotides in length in the high activity interval of the mRNA expressed by AGT gene, preferably a nucleotide sequence of 15-25 nucleotides in length, and the high activity interval is the 85-404th, 805-1443rd, 1553-1899th position of the mRNA expressed by AGT gene;

[0008] Preferably, the mRNA expressed by the AGT gene is as shown in NCBI refseq ID NM_001384479.1; in particular, the mRNA sequence is as shown in SEQ ID NO: 1.

[0009] In some embodiments, in the siRNA described above, the nucleotide sequence II is substantially reverse-complementary, substantially reverse-complementary, or completely reverse-complementary to the first segment of nucleotide sequence.

[0010] In some embodiments, in any of the siRNAs described above, the sense strand and the antisense strand are the same length or different length, the length of the sense strand is 16-23 nucleotides, and the length of the antisense strand is 19-26 nucleotides. In some embodiments, the length ratio of the sense strand and the antisense strand of the siRNA is 19 / 21, 21 / 23, 19 / 24.

[0011] In some embodiments, in any of the siRNAs described above, the nucleotide sequence I has at least 70%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the first segment of nucleotide sequence.

[0012] In some embodiments, in any of the siRNAs described above, the nucleotide sequence I comprises at least 15 consecutive nucleotides, such as at least 15, 16, 17, 18, 19, 20, or 21 nucleotides, of SEQ ID NO: 2-126; preferably, the nucleotide sequence I is as shown in SEQ ID NO: 2-126.

[0013] In some embodiments, in any of the siRNAs described above, the nucleotide sequence II comprises at least 15 consecutive nucleotides, such as at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides, of SEQ ID NO: 128-252; preferably, the nucleotide sequence II is as shown in SEQ ID NO: 128-252.

[0014] In some embodiments, in any of the siRNAs described above, the sense strand comprises the sense strand of any one of the siRNAs shown in Table 1, and the antisense strand comprises the antisense strand of the corresponding siRNA in Table 1.

[0015] In some embodiments, in any of the siRNAs described above, each nucleotide in the nucleotide sequence I and the nucleotide sequence II is a modified nucleotide, which is a fluorine-modified nucleotide or a non-fluorine-modified nucleotide.

[0016] In some embodiments, the fluoro-modified nucleotide refers to a nucleotide having a hydroxyl group at the 2'-position of the ribosyl group of the nucleotide replaced by a fluorine to form a nucleotide having a structure shown in Formula (1); and the non-fluoro-modified nucleotide refers to a nucleotide or a nucleotide analog having a hydroxyl group at the 2'-position of the ribosyl group of the nucleotide replaced by a non-fluorine group. In some embodiments, each non-fluoro-modified nucleotide is independently selected from one of a nucleotide or a nucleotide analog having a hydroxyl group at the 2'-position of the ribosyl group of the nucleotide replaced by a non-fluorine group. These nucleotides or nucleotide analogs having a hydroxyl group at the 2'-position of the ribosyl group replaced by a non-fluorine group are well known to those skilled in the art, and can be selected from one of a 2'-alkoxy-modified nucleotide or a nucleotide analog, a 2'-substituted alkoxy-modified nucleotide or a nucleotide analog, a 2'-alkyl-modified nucleotide or a nucleotide analog, a 2'-substituted alkyl-modified nucleotide or a nucleotide analog, a 2'-amino-modified nucleotide or a nucleotide analog, a 2'-substituted amino-modified nucleotide or a nucleotide analog, and a 2'-deoxy nucleotide. In some embodiments, the 2'-alkoxy-modified nucleotide is a methoxy-modified nucleotide (2'-OMe), as shown in Formula (2). In some embodiments, the 2'-substituted alkoxy-modified nucleotide can be a 2'-O-methoxyethyl-modified nucleotide (2'-MOE), as shown in Formula (3). In some embodiments, the 2'-amino-modified nucleotide (2'-NH2) is as shown in Formula (4). In some embodiments, the 2'-deoxy nucleotide (DNA) is as shown in Formula (5).

[0017]

[0018] A nucleotide analog refers to a group that can replace a nucleotide in a nucleic acid, but is structurally different from an adenine ribonucleoside, a guanine ribonucleotide, a cytosine ribonucleotide, a uracil ribonucleotide, or a thymine ribonucleotide. In some embodiments, the nucleotide analog can be an iso-nucleotide, a bridged nucleotide, or an acyclic nucleotide.

[0019] A bridged nucleotide refers to a constrained or inaccessible nucleotide. The bridged nucleotide can contain a five-membered ring, a six-membered ring, or a seven-membered ring with a fixed C3-endo sugar conformation. In some embodiments, the bridged nucleotide can be an LNA, an ENA, a cET BNA, etc.; wherein the LNA is as shown in Formula (6), the ENA is as shown in Formula (7), and the cET BNA is as shown in Formula (8).

[0020]

[0021] Acyclic nucleotides are a class of nucleotides in which the sugar ring of the nucleotide is opened. In some embodiments, the acyclic nucleotide can be an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA), wherein the UNA is as shown in Formula (9) and the GNA is as shown in Formula (10).

[0022]

[0023] In the above Formula (9) and Formula (10), R is selected from H, OH, or alkoxy (O-alkyl).

[0024] Heteronucleotides refer to compounds in which the position of the base on the ribose ring of the nucleotide is changed. In some embodiments, the heteronucleotide can be a compound in which the base is moved from the 1' position to the 2' position or the 3' position on the ribose ring. As shown in Formula (11) or Formula (12).

[0025]

[0026] In Formula (11) and Formula (12), R is selected from H, OH, F, or a non-fluorine group as described above.

[0027] In Formula (1) to Formula (12), Base represents a base.

[0028] In some embodiments, in any of the above-described siRNAs, one or more of the nucleotides at positions 5, 7, 8, 9, 10, 11, 12 of the nucleotide sequence I in the direction from the 5' end to the 3' end is a fluorine-modified nucleotide; and one or more of the nucleotides at positions 2, 3, 5, 6, 7, 8, 9, 10, 14, 16 of the nucleotide sequence II in the direction from the 5' end to the 3' end is a fluorine-modified nucleotide.

[0029] Preferably, the nucleotides at positions 7, 9, 10, 11 of the nucleotide sequence I in the direction from the 5' end to the 3' end are fluorine-modified nucleotides; and the nucleotides at positions 2, 6, 8, 9, 14, 16 of the nucleotide sequence II in the direction from the 5' end to the 3' end are fluorine-modified nucleotides; or

[0030] Preferably, the nucleotides at positions 5, 7, 8, 9 of the nucleotide sequence I in the direction from the 5' end to the 3' end are fluorine-modified nucleotides; and the nucleotides at positions 2, 6, 8, 9, 14, 16 of the nucleotide sequence II in the direction from the 5' end to the 3' end are fluorine-modified nucleotides; or

[0031] Preferably, the nucleotides at positions 7, 9, and 11 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or

[0032] Preferably, the nucleotides at positions 5, 7, and 9 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or

[0033] Preferably, the nucleotides at positions 7, 9, 10, 11, and 12 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 3, 5, 7, 10, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or

[0034] Preferably, the nucleotides at positions 5, 7, 8, 9, and 10 of nucleotide sequence I are fluorinated nucleotides, in the direction from the 5' end to the 3' end; and the nucleotides at positions 2, 3, 5, 7, 10, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, in the direction from the 5' end to the 3' end.

[0035] In some embodiments, at least a portion of the phosphate ester groups in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands of the siRNA are phosphate ester groups with modifying groups. In some embodiments, the phosphate ester group with modifying groups is a thiophosphate ester group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom. In some embodiments, the phosphate ester group with modifying groups is a thiophosphate ester group having the structure shown in formula (13):

[0036]

[0037] In some embodiments, in any of the above-described siRNAs, the thiophosphate group linkage is present at least one of the following positions: between the first and second nucleotides of the sense and / or antisense strands; between the second and third nucleotides of the sense and / or antisense strands; between the 19th and 20th nucleotides of the antisense strand; between the 20th and 21st nucleotides of the antisense strand; between the 21st and 22nd nucleotides of the antisense strand; between the 22nd and 23rd nucleotides of the antisense strand; or any combination thereof.

[0038] In some embodiments, in any of the above-described siRNAs, the 5' terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide, as shown in formulas (14), (15), and (16):

[0039]

[0040] In a second aspect, this disclosure provides an siRNA conjugate comprising any of the siRNAs described above and a conjugating group conjugated to the siRNA. In some embodiments, the pharmaceutically acceptable conjugating group in the siRNA conjugate may be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule may be monovalent, divalent, trivalent, or tetravalent. In some embodiments, the conjugation site between the siRNA and the conjugating group may be at the 3' or 5' end of the sense strand of the siRNA, at the 3' end of the antisense strand, or within the internal sequence of the siRNA.

[0041] In some embodiments, the conjugating group in the above siRNA conjugate is L96, with the structure shown below:

[0042]

[0043] The siRNA conjugate has its sense strand selected from the nucleotide sequence shown in the following formula: 5'-XmsXmsXmXmXfXmXfXmXmXmXmXmXmXmXmXmXmXm-L96-3', or 5'-XmsXmsXmXmXmXmXfXmXmXmXmXmXmXmXmXmXm-L96-3', or 5'-XmsXmsXmXmXf ... XmXm-L96-3' or 5'-XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXmXm-L96-3' or 5'-XmsXmsXmXmXmXfXm XfXfXfXmXmXmXmXmXmXmXmXmXm-L96-3', or 5'-XmsXmsXmXmXmXmXfXmXfXfXfXfXmXmXmXmXmXmXmXmXmXm-L96-3';

[0044] The antisense strand is selected from the nucleotide sequence shown in the following formula: 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmsXmsXm-3', or 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmXmsXmsXm-3', or 5'-VPXmsXfsXmXmXmXf ... sXmsXm-3', or 5'-VPXms mXfXmXmXmXfXmXfXmXmXmsXmsXm-3', or 5'-VPXmsXfsXfXmXfXmXfXmXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm-3';

[0045] Wherein, Xm represents any nucleotide modified with 2'-methoxy, such as 2'-methoxy modified C, G, U, A, T; Xf represents any nucleotide modified with 2'-fluoride, such as 2'-fluoride modified C, G, U, A, T; the lowercase letter s indicates that the two nucleotides adjacent to the letter s on the left and right are linked by a thiophosphate subunit; VP indicates that the nucleotide adjacent to the right of the letter combination VP is a nucleotide modified with vinylphosphonate (5'-(E)-vinylphosphonate, E-VP).

[0046] In some implementations, the justice chain and the antisense chain are combined as follows:

[0047] M1 Modification Mode:

[0048] Chain of Justice: 5'-XmsXmsXmXmXmXmXfXmXfXfXfXfXmXmXmXmXmXmXmXmXmXm-L96-3';

[0049] Antonyms: 5'-VPXmsXfsXmXmXmXfXmXfXmXmXmXmXmXmXmXmXmXmXmXmXmXmXmsXmsXm-3'; or M1' modification mode:

[0050] Chain of Justice: 5'-XmsXmsXmXmXfXmXfXfXfXfXmXmXmXmXmXmXmXmXm-L96-3'

[0051] Antisense chain: 5'-VPXmsXfsXmXmXmXfXmXfXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm-3'; M2 modifier mode:

[0052] Chain of Justice: 5'-XmsXmsXmXmXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3';

[0053] Antonyms: 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmXmsXmsXm-3'; or M2' modification mode:

[0054] Chain of Justice: 5'-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3';

[0055] Antonym chain: 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmsXmsXm-3';

[0056] M5 Editing Mode:

[0057] Justice chain: 5'-XmsXmsXmXmXmXmXfXmXfXfXfXfXfXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXfXmXfXmXmXmXfXmXmXmXmXmXmXmXmXmXmXmXm-3'; or M5' modifier mode:

[0058] Chain of Justice: 5'-XmsXmsXmXmXfXmXfXfXfXfXfXmXmXmXmXmXmXmXm-L96-3';

[0059] Antonym chain: 5'-VPXmsXfsXfXmXfXmXmXfXmXmXmXmXfXmXmXmXmXmXmXmXmsXmsXm-3'.

[0060] In some embodiments, the siRNA conjugate comprises a sense strand and an antisense strand, wherein the sense strand comprises the sense strand of any of the siRNA conjugates shown in Tables 2, 3, 4, and 5 of this document, and the antisense strand comprises the antisense strand of the corresponding siRNA conjugate.

[0061] In a third aspect, this disclosure provides a composition comprising any of the siRNAs or siRNA conjugates described above.

[0062] In some embodiments, the composition is a pharmaceutical composition and further includes a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers or excipients involved in this disclosure include, but are not limited to, water for injection, sodium hydroxide, sodium dihydrogen phosphate monohydrate, sodium dihydrogen phosphate dihydrate, phosphoric acid, sodium chloride, potassium chloride, hydrochloric acid, anhydrous potassium dihydrogen phosphate, anhydrous disodium hydrogen phosphate, PEG2000, PEG6000, cholesterol, distearate, glyceryl 1,2-dimyristate, and dimethyl adipic acid.

[0063] In a fourth aspect, this disclosure provides the use of any of the foregoing siRNAs, siRNA conjugates, or compositions in the preparation of medicaments for the prevention and / or treatment of diseases associated with AGT expression.

[0064] In a fifth aspect, this disclosure provides a method for preventing and / or treating diseases associated with AGT expression, the method comprising administering to a subject the siRNA, siRNA conjugate, or pharmaceutical composition of this disclosure.

[0065] In some implementations, the diseases associated with AGT expression include hypertension and related diseases that may be induced by genetic, lifestyle, and environmental factors, such as borderline hypertension, primary hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, intractable hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, hypertension associated with low plasma renin activity or plasma renin concentration, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, and unstable hypertension. Hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, nocturnal hypertension, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, stroke, kidney disease, kidney failure, systemic sclerosis, intrauterine growth restriction (IUGR), fetal growth restriction, obesity, hepatic steatosis / fatty liver, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), glucose intolerance, type 2 diabetes, and metabolic syndrome.

[0066] The pharmaceutical compositions disclosed herein can be used alone for the treatment of hypertension or in combination with standard oral antihypertensive drugs, providing experimental support for diversified treatment options for clinical hypertension patients.

[0067] Based on the amount of siRNA contained therein, the generally suitable dosage range of the siRNA, siRNA conjugate or pharmaceutical composition relating to the present disclosure that inhibits AGT gene expression will be from about 0.1 mg / kg to about 10.0 mg / kg, preferably from about 0.3 mg / kg to about 3.0 mg / kg.

[0068] The routes of administration disclosed herein include intravenous administration, subcutaneous administration, intrathecal injection, intramuscular administration, transdermal administration, airway administration (aerosol), ocular administration, nasal administration, rectal administration, pulmonary administration, and local administration (including oral administration and sublingual administration).

[0069] The disclosed siRNA, its conjugates, and pharmaceutical compositions can specifically target the liver, complementary to the liver AGT mRNA sequence, induce AGT mRNA degradation, thereby inhibiting AGT synthesis in the liver, resulting in a sustained reduction of AGT protein, and ultimately reducing the vasoconstrictor angiotensin II, exerting a sustained blood pressure-lowering effect, while avoiding off-target effects, exhibiting low cytotoxicity, and fundamentally blocking the physiological effects of RAS, avoiding the "RAS escape" phenomenon caused by the bypass and negative feedback mechanisms of angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II type 1 receptor blockers that lead to the upregulation of renin and angiotensinase I.

[0070] In addition, compared with traditional small molecule drugs and antibody drugs, small nucleic acid drugs can directly regulate the expression of upstream genes and are relatively less likely to develop drug resistance; moreover, small nucleic acid drugs have a long half-life in the body, so the frequency of administration is low (they can be given once every six months), and patients have good compliance.

[0071] In summary, the siRNA, its conjugates, and pharmaceutical compositions disclosed herein exhibit strong inhibitory activity against the AGT gene, significantly reducing AGT mRNA expression levels, and possess low drug toxicity. Therefore, the siRNA, its conjugates, and pharmaceutical compositions disclosed herein can effectively prevent and / or treat diseases associated with AGT expression, such as hypertension, providing patients with more effective, safe, and convenient therapeutic drugs, and have promising prospects as pharmaceutical products. Attached Figure Description

[0072] Figure 1 The results of the cytotoxicity experiment of the siRNA conjugate of this invention are shown.

[0073] Figure 2 This is the result of the siRNA conjugate of the present invention inhibiting AGT expression in mice.

[0074] Figure 3The results show the dose-dependent effects of the siRNA conjugates YG-43M1, YG-44M1, YG-51M1, YG-55M1, YG-58M1, and YG-59M1 in mice.

[0075] Figure 4 This is a schematic diagram of the binding site of the siRNA of this invention to the human AGT gene.

[0076] Figure 5 This is the result of the siRNA conjugates YG-116M1, YG-117M1, and YG-118M1 inhibiting AGT expression in mice.

[0077] Figure 6 This is the result of the siRNA conjugate YG-43M1 of the present invention inhibiting AGT expression in mice.

[0078] Figure 7 The results show that the siRNA conjugates YG-120M1, YG-83M1 and YG-84M1 of this invention inhibit AGT expression in mice.

[0079] Figure 8 The results show that the siRNA conjugates YG-84M1, YG-124M5, YG-125M5, YG-98M5, YG-104M5, and YG-113M5 of this invention inhibited AGT expression in cynomolgus monkeys.

[0080] Figure 9 The results of detecting alanine aminotransferase (ALT) in rats using the siRNA conjugate of this invention are shown.

[0081] Figure 10 The results of detecting aspartate aminotransferase (AST) in rats using the siRNA conjugate of this invention are shown. Detailed Implementation

[0082] definition

[0083] Unless otherwise specified, in the preceding and following text, uppercase letters C, G, U, and A represent cytosine, guanine, uracil, and adenine nucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a fluorinated nucleotide; lowercase letter s indicates that the two nucleotides adjacent to the left and right of letter s are linked by a thiophosphate subunit; the letter combination VP indicates that the nucleotide adjacent to the right of letter combination VP is a vinylphosphonate (5'-(E)-vinylphosphonate, E-VP) modified nucleotide; L96 has the structure of formula (I) and is linked to the 3' end of the positive chain via a phosphate ester bond.

[0084] In the preceding and following text, "fluorinated nucleotides" refers to nucleotides formed by replacing the hydroxyl group at the 2' position of the ribosome with fluorine, and "non-fluorinated nucleotides" refers to nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribosome with a non-fluorinated group. "Nucleotide analogs" refer to groups that can replace nucleotides in nucleic acids but whose structure differs from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides. Examples include isonucleotides, bridged nucleic acids (BNAs), or acyclic nucleotides. "Methoxylated nucleotides" refers to nucleotides formed by replacing the hydroxyl group at the 2' position of the ribosome with a methoxy group.

[0085] In the context of this document, the terms "complementary" and "reverse complementary" are used interchangeably and have the meaning known to those skilled in the art: in a double-stranded nucleic acid molecule, the bases of one strand are paired complementaryly with the bases of the other strand. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair consists of one purine and one pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the complementary strand can be inferred from its sequence. Correspondingly, "mismatch" in the art means, in a double-stranded nucleic acid, that the bases at corresponding positions are not paired complementaryly.

[0086] Unless otherwise specified, in the foregoing and hereinafter, "substantially anticomplementary" means that there are no more than three base mismatches between the two nucleotide sequences involved; "substantially anticomplementary" means that there are no more than one base mismatch between the two nucleotide sequences; and "completely anticomplementary" means that there are no base mismatches between the two nucleotide sequences. In the foregoing and hereinafter, particularly in describing the methods for preparing siRNA, pharmaceutical compositions, or siRNA conjugates of this disclosure, unless otherwise specified, the nucleoside monomer refers to the modified or unmodified RNA phosphoramidites (sometimes also called nucleoside phosphoramidites) used in phosphoramidite solid-phase synthesis, depending on the type and sequence of nucleotides in the siRNA or siRNA conjugate to be prepared. Phosphoramidite solid-phase synthesis is a method known to those skilled in the art for the synthesis of siRNA. All nucleoside monomers used in this disclosure are commercially available.

[0087] As used in the context of this disclosure, the term "corresponding siRNA" refers to the same siRNA mentioned above. For example, when referring to "the positive strand comprising the positive strand of any of the siRNAs shown in Table 1 herein, and the antisense strand comprising the antisense strand of the corresponding siRNA in Table 1," it means that the included positive and antisense strands are from the same siRNA shown in Table 1 herein. For example, when the positive strand comprises 5'-GAGCCUGAGGGCCACCAUCCU-3' (SEQ ID NO: 2), the antisense strand comprises 5'-AGGAUGGUGGCCCUCAGGCUCAC-3' (SEQ ID NO: 128). Similarly, the term "corresponding siRNA conjugate" refers to the same siRNA conjugate mentioned above. For example, when referring to "the positive strand comprising the positive strand of any of the siRNA conjugates shown in Tables 2, 3, 4, and 5 herein, and the antisense strand comprising the antisense strand of the corresponding siRNA conjugate," it means that the included positive and antisense strands are from the same siRNA conjugates shown in Tables 2, 3, 4, and 5 herein. Additionally, in these contexts, "contains" includes cases where these sequences are composed of each other.

[0088] In Tables 1, 2, 3, 4, and 5, if the left side of the 5' terminal nucleotide of the positive strand and the modified positive strand with the connecting conjugation group is not marked with VP, it means that the 5' terminal nucleotide is not connected to a 5' phosphate group or a 5' phosphate derivative group, and its structure is shown in formula (II):

[0089]

[0090] Wherein, Base represents a base, such as A, U, G, C or T; R is a hydroxyl group or is substituted by various groups known to those skilled in the art, for example, R can be 2'-fluoro(2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, 2'-substituted amino, 2'-deoxynucleotide.

[0091] In Tables 1, 2, 3, 4, and 5, if the left side of the 5' terminal nucleotide of the antisense strand and the modified antisense strand is not marked with VP, it means that the 5' terminal nucleotide is not connected to a 5' phosphate group or a 5' phosphate derivative group, and its structure is as shown in Formula (II).

[0092] In Table 1, the 3' end nucleotide of the sense strand has a hydroxyl group at the 3' position. In Tables 1, 2, 3, 4, and 5, the 3' end nucleotide of the antisense strand and the modified antisense strand has a hydroxyl group at the 3' position.

[0093] Examples

[0094] Other objects, features, and advantages of this disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific embodiments (although illustrating specific implementations of this disclosure) are given for illustrative purposes only, as various changes and modifications that can be made within the spirit and scope of this disclosure will become apparent to those skilled in the art upon reading this detailed description.

[0095] Unless otherwise specified, the experimental techniques and methods used in this embodiment are conventional techniques and methods. For example, experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the embodiments can be obtained through legitimate commercial channels.

[0096] Example 1. siRNA Design and Synthesis

[0097] 1.1 siRNA Design

[0098] A set of siRNAs targeting the human AGT gene (human AGT gene: NCBI refseq ID NM_001384479.1; NCBI Gene ID: 183) was designed online using OligoWalk. The human NM_001384479.1 REFSEQ mRNA has a length of 2116 bases. Sequences similar to the human gene were also excluded to avoid any toxicity.

[0099]

[0100] 1.2 siRNA sequence synthesis

[0101] siRNA was synthesized according to a standard oligonucleotide solid-phase synthesis protocol, including a negative control siRNA (siCtrl).

[0102] Oligonucleotide solid-phase synthesis protocol: Commercially available 5'-DMT-2'-TBDMS-rU phosphoramide monomers, 5'-DMT-2'-TBDMS-rA(Bz) phosphoramide monomers, 5'-DMT-2'-TBDMS-rC(Ac) phosphoramide monomers, and 5'-DMT-2'-TBDMS-rG(iBu) phosphoramide monomers were used. RNA was synthesized at a synthesis scale of 500 nM. A phosphoramide solution was prepared at a concentration of 50 mM, and 0.3 M benzylthiotetrazole (BTT) acetonitrile solution was used as an activator. During synthesis, a 0.1 M oxidizing agent (pyridine:THF:water = 20:78:2) was used to convert trivalent phosphorus to pentavalent phosphorus to stabilize the phosphate backbone. After synthesis, the sequence was ammonolyzed from the solid support and precipitated. The 2'-2'-O-tert-butyldimethylsilyl protecting group was removed with triethylamine trihydrofluoric acid.

[0103] For the synthesized RNA sequence, ammonolysis was performed at 55°C for 40 minutes using an ammonia:methylamine ratio of 1:1. After ammonolysis, the solid support CPG powder was removed, and the supernatant was dried. A protecting group remover was added, and the reaction was carried out at 60°C for 2 hours. Then, n-butanol was added at a 1:5 ratio, and the mixture was allowed to stand at -20°C for 30 minutes. The precipitate was collected by centrifugation. The precipitate was dissolved in RNase-free water and purified using reversed-phase chromatography (0.1M triethylamineacetic acid (TEAA) and acetonitrile). The purified sample was desalted by ultrafiltration with PBS and annealed to obtain siRNA. Verification of the obtained siRNA confirmed successful preparation of the target siRNA.

[0104] 1.3 siRNA sequence modification and conjugate synthesis

[0105] Modified siRNAs are synthesized according to oligonucleotide solid-phase synthesis schemes. The modified nucleotide groups can be introduced into the siRNAs disclosed herein using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications are well known to those skilled in the art. L96 is conjugated to siRNA to synthesize siRNA conjugates, referring to the synthesis methods disclosed in WO2014025805A1 or WO2017015109A1.

[0106] The structure of the conjugation group L96 is shown below:

[0107]

[0108] Annealing of oligonucleotides to generate siRNA conjugates: The RNA oligomers to be annealed were prepared into a 200 μM solution using sterile RNase-free H2O (RNA hydrolase-free). The annealing reaction system was set up as follows: 100 μL of the above solution (double-strand concentration of 10 nM) was placed in a 95°C water bath for 10 minutes (≥100 nM requires 20 minutes at high temperature) → immediately cooled in a 60°C water bath → the annealed solution was stored at 4°C. The complementary strands were mixed by combining equimolar amounts of RNA solution. The siRNA conjugate was confirmed to be correctly constructed.

[0109] The siRNA and siRNA conjugate solution were prepared into a dry powder for later use.

[0110] The sequences of the synthesized siRNA molecules are shown in Table 1 below:

[0111] Table 1. siRNA sequence listing targeting AGT

[0112]

[0113]

[0114]

[0115]

[0116] The sequences of the synthesized siRNA conjugates are shown in Tables 2, 3, 4, and 5 below:

[0117] Table 2. Sequence listing of siRNA conjugates targeting AGT (M1 or M1' modification pattern)

[0118]

[0119]

[0120]

[0121] Note 1: YG-6, YG-44, YG-51, YG-52, YG-55, YG-58, YG-59, YG-64, YG-65, YG-66, YG in Table 2 -67, YG-68, YG-69, YG-70, YG-71, YG-72, YG-73, YG-74, YG-75, YG-76, YG-80, YG- 81, YG-82, YG-85, YG-95, YG-96, YG-97, YG-98, YG-99, YG-100, YG-101, YG-102, YG-103, YG-107, YG-108, YG-109, YG-113, and YG-114 are in M1' modification mode; the remaining sequences are in M1 modification mode. M1 modification mode:

[0122] Chain of Justice: 5'-XmsXmsXmXmXmXmXfXmXfXfXfXfXmXmXmXmXmXmXmXmXmXm-L96-3';

[0123] Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXfXmXmXmXmXmXmXfXmXmXmXmXmXmXmsXmsXm-3';

[0124] M1' Modification Mode:

[0125] Chain of Justice: 5'-XmsXmsXmXmXfXmXfXfXfXfXmXmXmXmXmXmXmXmXmXm-L96-3';

[0126] Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXfXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm-3';

[0127] Wherein, Xm represents any nucleotide modified with 2'-methoxy, such as 2'-methoxy modified C, G, U, A, T; Xf represents any nucleotide modified with 2'-fluoride, such as 2'-fluoride modified C, G, U, A, T; the lowercase letter s indicates that the two nucleotides adjacent to the letter s on the left and right are linked by a thiophosphate subunit; VP indicates that the nucleotide adjacent to the right of the letter combination VP is a nucleotide modified with vinylphosphonate (5'-(E)-vinylphosphonate, E-VP).

[0128] Note 2: siCtrlM1 Justice Chain: UmsUmsCmUmCmCmGfAmAfCfGfUmGmUmCmAmCmGmUmdTdT-L96 (SEQ ID NO:318);

[0129] siCtrlM1 antisense strand: VPAmsCfsGmUmGmAfCmAfCfGmUmUmCmGfGmAfGmAmAmsdTdT (SEQ IDNO: 319).

[0130] Table 3. Sequence listing of siRNA conjugates targeting AGT (M2 or M2' modification pattern)

[0131]

[0132]

[0133]

[0134]

[0135] Note 1: YG-6, YG-44, YG-51, YG-52, YG-55, YG-58, YG-59, YG-64, YG-65, YG-66, YG in Table 3 -67, YG-68, YG-69, YG-70, YG-71, YG-72, YG-73, YG-74, YG-75, YG-76, YG-80, YG- 81, YG-82, YG-85, YG-95, YG-96, YG-97, YG-98, YG-99, YG-100, YG-101, YG-102, YG-103, YG-107, YG-108, YG-109, YG-113, and YG-114 are in M2' modification mode, and the remaining sequences are in M2 modification mode; M2 modification mode:

[0136] Chain of Justice: 5'-XmsXmsXmXmXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3';

[0137] Antonym chain: 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmXmsXmsXm-3'; M2' modification mode:

[0138] Chain of Justice: 5'-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3';

[0139] Antonym chain: 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmsXmsXm-3';

[0140] Wherein, Xm represents any nucleotide modified with 2'-methoxy, such as 2'-methoxy modified C, G, U, A, T; Xf represents any nucleotide modified with 2'-fluoride, such as 2'-fluoride modified C, G, U, A, T; the lowercase letter s indicates that the two nucleotides adjacent to the letter s on the left and right are linked by a thiophosphate subunit; VP indicates that the nucleotide adjacent to the right of the letter combination VP is a nucleotide modified with vinylphosphonate (5'-(E)-vinylphosphonate, E-VP).

[0141] Note 2: siCtrlM2 Justice Chain: UmsUmsCmUmCmCmGfAmAfCmGfUmGmUmCmAmCmGmUmdTdT-L96 (SEQ ID NO:320);

[0142] siCtrlM2 antisense strand: VPAmsCfsGmUmGmAmCmAmCmGmUmUmCmGfGmAfGmAmAmsdTdT (SEQ IDNO: 321).

[0143] Table 4. Sequence listing of siRNA conjugates targeting AGT (M5 or M5' modification pattern)

[0144]

[0145]

[0146]

[0147]

[0148] Note 1: YG-6, YG-44, YG-51, YG-52, YG-55, YG-58, YG-59, YG-64, YG-65, YG-66, YG in Table 4 -67, YG-68, YG-69, YG-70, YG-71, YG-72, YG-73, YG-74, YG-75, YG-76, YG-80, YG- 81, YG-82, YG-85, YG-95, YG-96, YG-97, YG-98, YG-99, YG-100, YG-101, YG-102, YG-103, YG-107, YG-108, YG-109, YG-113, and YG-114 are in M5' modification mode; the remaining sequences are in M5 modification mode. M5 modification mode:

[0149] Chain of Justice: 5'-XmsXmsXmXmXmXmXfXmXfXfXfXfXfXmXmXmXmXmXmXmXm-L96-3';

[0150] Antonym chain: 5'-VPXmsXfsXfXmXfXmXmXfXmXmXmXmXfXmXmXmXmXmXmXmXmXmXmsXmsXm-3';

[0151] M5's Editing Mode:

[0152] Chain of Justice: 5'-XmsXmsXmXmXfXmXfXfXfXfXfXmXmXmXmXmXmXmXm-L96-3';

[0153] Antonym chain: 5'-VPXmsXfsXfXmXfXmXmXmXfXmXmXmXmXmXmXmXmXmXmXmXmXmsXmsXm-3';

[0154] Wherein, Xm represents any nucleotide modified with 2'-methoxy, such as 2'-methoxy modified C, G, U, A, T; Xf represents any nucleotide modified with 2'-fluoride, such as 2'-fluoride modified C, G, U, A, T; the lowercase letter s indicates that the two nucleotides adjacent to the letter s on the left and right are linked by a thiophosphate subunit; VP indicates that the nucleotide adjacent to the right of the letter combination VP is a nucleotide modified with vinylphosphonate (5'-(E)-vinylphosphonate, E-VP).

[0155] Note 2: siCtrlM5 Justice Chain: UmsUmsCmUmCfCmGfAfAfCfGmUmGmUmCmAmCmGmUmdTdT-L96 (SEQ ID NO:322);

[0156] siCtrlM5 antisense strand: VPAmsCfsGfUmGfAmCfAmCmGfUmUmCmGfGmAfGmAmAmsdTdT (SEQ IDNO: 323).

[0157] Table 5. Sequence listing of siRNA conjugates targeting AGT

[0158]

[0159]

[0160]

[0161] In this context, uppercase letters C, G, U, A, and T represent cytosine, guanine, uracil, adenine nucleotide, and thymine deoxynucleotide, respectively; lowercase letter m indicates that the nucleotide adjacent to the left of letter m is methoxy-modified; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is fluorinated; lowercase letter s indicates that the two nucleotides adjacent to the left and right of letter s are linked by thiophosphate subunits; VP indicates that the nucleotide adjacent to the right of the letter combination VP is a vinylphosphonate (5'-(E)-vinylphosphonate, E-VP) modified nucleotide; L96 has the structure of formula (I) and is linked to the 3' end of the positive chain via a phosphate ester bond; dT represents 2'-deoxythymine nucleotide.

[0162] Example 2. In vitro activity screening of siRNA HepG2 cell line

[0163] 2.1 Experimental Procedure

[0164] 2.1.1 Cell Culture

[0165] HepG2 cells (China Center for Type Culture Collection (CCTCC), catalog number: CVCL_0027) were cultured in MEM complete medium (Eallbio, with 10% FBS added) at 37°C and 5% CO2. When the confluence reached 80%-90%, the cells were digested with trypsin, counted, and transfected.

[0166] 2.1.2 Preparation of siRNA dilution buffer

[0167] (1) The dry powder of the siRNA to be tested was centrifuged at low temperature and high speed, and then dissolved in ultrapure distilled water to prepare a 100 μM siRNA stock solution.

[0168] (2) Prepare 200 nM siRNA dilution Y1, 20 nM siRNA dilution Y2, and 2 nM siRNA dilution Y3.

[0169] a) Take 50 μl of the 100 μM siRNA stock solution obtained in step (1) above, add 50 μl of ultrapure distilled water to obtain a siRNA dilution solution with a final concentration of 50 μM.

[0170] b) Take 2 μl of the 50 μM siRNA dilution solution prepared in step a), add 18 μl of ultrapure distilled water to obtain siRNA stock solution X1 with a final concentration of 5 μM. Take 2 μl of the prepared siRNA stock solution X1 and add 48 μl of Opti-medium (Opti-MEM I Medium, Gibco, catalog number: 31985070) to obtain 200 nM siRNA dilution solution Y1.

[0171] c) Take 2 μl of the 5 μM siRNA stock solution X1 prepared in step b), add 18 μl of ultrapure distilled water to obtain a final concentration of 0.5 μM siRNA stock solution X2. Take 2 μl of the prepared siRNA stock solution X2, add 48 μl of Opti-medium to obtain a 20 nM siRNA dilution Y2.

[0172] d) Take 2 μl of the 0.5 μM siRNA stock solution X2 prepared in step c), add 18 μl of ultrapure distilled water to obtain siRNA stock solution X3 with a final concentration of 0.05 μM. Take 2 μl of the prepared siRNA stock solution X3, add 48 μl of Opti-medium to obtain 2 nM siRNA dilution Y3.

[0173] 2.1.3 HepG2 cell transfection

[0174] Pick 0.6 μl of RNAiMAX transfection reagent (Thermo Fisher, catalog number: 13778150) was added to 10 μl of Opti-medium to obtain... RNAiMAX transfection reagent dilution solution; The RNAiMAX transfection reagent diluent was mixed with the 200nM siRNA diluent Y1, 20nM siRNA diluent Y2, or 2nM siRNA diluent Y3 prepared in step 2.1.2 at a 1:1 volume ratio to prepare a transfection mixture. After standing for 5 minutes, 10 μl of the transfection mixture was added to a 96-well plate, along with 90 μl of HepG2 cells cultured in step 2.1.1 (final volume 100 μl / well, cell number 30,000 / well, siRNA concentration in this system is 10nM, 1nM, and 0.1nM). The plate was then cultured for 24 hours after transfection.

[0175] 2.1.4 RNA Extraction

[0176] According to the instructions for the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number: CL132-01), total RNA was extracted from HepG2 cells obtained in step 2.1.3.

[0177] 2.1.5 Quantitative Real-Time PCR

[0178] The extracted total RNA was analyzed by reverse transcription and real-time PCR using the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit.

[0179] 2.1.6 Results Analysis

[0180] (1) The Ct value was automatically calculated using the software of the Thermo Fisher 7500 real-time fluorescence quantitative PCR instrument;

[0181] (2) Calculate the relative expression level of the gene using the following formula:

[0182] ΔCt1=Ct(AGT group)–Ct(AGT group's ACTIN)

[0183] ΔCt2=Ct(siCtrl group)–Ct(siCtrl group's ACTIN)

[0184] ΔCt = ΔCt1 - ΔCt2, where the siCtrl group is the negative control group;

[0185] mRNA expression relative to the siCtrl group = 2 -ΔΔCt

[0186] Inhibition rate (%) = (1 - mRNA expression relative to siCtrl group) × 100%.

[0187] 2.2 Experimental Results

[0188] The inhibitory effects of the siRNA of this invention are shown in Table 6 below:

[0189] Table 6. Results of in vitro screening of HepG2 cell lines using siRNA

[0190]

[0191]

[0192]

[0193]

[0194] The results showed that some of the siRNAs disclosed in this study could significantly inhibit the expression of the AGT gene in HepG2 cells at concentrations of 10 nM and 1 nM.

[0195] Example 3. In vitro activity screening of siRNA Hep3B cell line

[0196] 3.1 Experimental Procedure

[0197] 3.1.1 Cell Culture

[0198] Hep3B cells (Wuhan Pronosei Life Sciences Co., Ltd., catalog number: CL-0102) were cultured in MEM complete medium (Eallbio, with 10% FBS added) at 37℃ and 5% CO2. When the confluence reached 80%-90%, the cells were digested with trypsin, counted, and transfected.

[0199] 3.1.2 Preparation of siRNA dilution buffer

[0200] Prepare 200 nM siRNA dilution Y1 according to step 2.1.2 of Example 2.

[0201] 3.1.3 Hep3B cell transfection

[0202] Pick 0.6 μl of RNAiMAX transfection reagent was added to 10 μl of Opti-medium to obtain... RNAiMAX transfection reagent dilution solution, The RNAiMAX transfection reagent diluent and the 200 nM siRNA diluent Y1 prepared in step 3.1.2 were mixed at a 1:1 volume ratio to prepare a transfection mixture. After standing for 5 minutes, 10 μl of the transfection mixture was added to a 96-well plate, along with 90 μl of Hep3B cells cultured in step 3.1.1 (final volume 100 μl / well, cell number 20,000 / well, siRNA concentration in this system is 10 nM). The plate was then cultured for 24 hours after transfection.

[0203] Then, RNA extraction, quantitative PCR, and result analysis were performed according to steps 2.1.4, 2.1.5, and 2.1.6 of Example 2.

[0204] 3.2 Experimental Results

[0205] The inhibitory effects of the siRNA of this invention are shown in Table 7 below:

[0206] Table 7. Results of in vitro screening of Hep3B cell lines using siRNA

[0207]

[0208]

[0209]

[0210]

[0211] The results showed that some of the siRNAs disclosed in this study could significantly inhibit the expression of the AGT gene in Hep3B cells at a concentration of 10 nM.

[0212] Example 4. IC50 assay of siRNA HepG2 cell line

[0213] This embodiment examines the dose-response relationship between drug dosage and biological effect by calculating the half-maximal inhibitory concentration (IC50) of each siRNA, thereby quantitatively reflecting the ability of the drug to cause changes in this indicator.

[0214] 4.1 Experimental Procedure

[0215] Cell culture and transfection were performed using a method similar to that in Example 2. The concentrations of transfected siRNA were 50 nM, 10 nM, 2 nM, 0.4 nM, 0.08 nM, and 0.016 nM, respectively. The IC50 of siRNA inhibiting AGT gene expression was then measured.

[0216] 4.2 Results Analysis

[0217] (1) The Ct value was automatically calculated using the software of the Thermo Fisher 7500 real-time fluorescence quantitative PCR instrument;

[0218] (2) Calculate the relative expression level of the gene using the following formula:

[0219] ΔCt1=Ct(AGT group)–Ct(AGT group's ACTIN)

[0220] ΔCt2=Ct(siCtrl group)–Ct(siCtrl group's ACTIN)

[0221] ΔCt = ΔCt1 - ΔCt2, where the siCtrl group is the negative control group;

[0222] mRNA expression relative to the siCtrl group = 2 -ΔΔCt

[0223] Inhibition rate (%) = (1 - mRNA expression relative to siCtrl group) × 100%.

[0224] Using the log value of siRNA concentration as the X-axis and the percentage inhibition rate as the Y-axis, dose-response curves were fitted using data analysis software to obtain the IC50 value of each siRNA.

[0225] The fitting formula is: Y = 100 / (1 + 10^((LogIC50 - X) × HillSlope))

[0226] Where HillSlope represents the slope of the percentage inhibition rate curve.

[0227] 4.3 Experimental Results

[0228] The IC50 results of the siRNA of the present invention in the HepG2 cell line are shown in Table 8 below:

[0229] Table 8. Results of IC50 assay for siRNA-HepG2 cell line

[0230]

[0231]

[0232] IC50 assays using the HepG2 cell line showed that some of the siRNAs disclosed herein can significantly inhibit AGT gene expression, and these sequences all exhibit good in vitro activity.

[0233] Example 5. IC50 assay of siRNA Hep3B cell line

[0234] 5.1 Experimental Procedure

[0235] Cell culture and transfection were performed using a method similar to that in Example 3. The concentrations of transfected siRNA were 25 nM, 5 nM, 1 nM, 0.2 nM, 0.04 nM, 0.008 nM, and 0.0016 nM, respectively. Then, the IC50 of siRNA inhibiting AGT gene expression was measured.

[0236] 5.2 Results Analysis

[0237] The results were analyzed according to step 4.2 in Example 4.

[0238] 5.3 Experimental Results

[0239] The IC50 results of the siRNA of this invention in the Hep3B cell line are shown in Table 9 below:

[0240] Table 9. siRNA Hep3B cell line IC50 assay results

[0241]

[0242]

[0243] IC50 assays using the Hep3B cell line showed that some of the siRNAs disclosed herein can significantly inhibit AGT gene expression, and these sequences all exhibit good in vitro activity.

[0244] Example 6. Cytotoxicity of siRNA conjugates

[0245] 6.1 Experimental Procedure

[0246] 6.1.1 Cell Culture

[0247] Hep3B cells were cultured in MEM complete medium (Eallbio, with 10% FBS added) at 37°C and 5% CO2. When the confluence reached 80%-90%, the cells were digested with trypsin, counted, and transfected.

[0248] 6.1.2 Hep3B cell transfection

[0249] Based on the in vitro activity and IC50 data in HepG2 and Hep3B cells, 14 siRNA conjugates were selected for cytotoxicity assays. Hep3B cells were transfected using a method similar to that in Example 3, with 15,000 cells seeded per well. After 72 hours of culture, the cytotoxicity of each siRNA conjugate was measured by determining the cell viability / cytotoxicity ratio in each sample, with the transfected siRNA conjugate concentrations being 50 nM, 5 nM, and 0.5 nM, respectively. Cell viability was measured by determining intracellular ATP content using a CellTiter-Glo (Promega, catalog number G7570) assay, according to the manufacturer's protocol. ToxiLight was used according to the manufacturer's protocol. TM (Lonza, catalog number LT07-217) Measures cytotoxicity in the supernatant.

[0250] 6.2 Experimental Results

[0251] The cytotoxicity results of the siRNA conjugates are shown in [link to results]. Figure 1 .

[0252] The results showed that some of the siRNA conjugates disclosed in this paper have low cytotoxicity and good cell compatibility.

[0253] Example 7. In vivo activity screening of siRNA conjugates

[0254] Based on the in vitro screening results of Examples 2-6 above, some siRNA sequences with good in vitro activity were selected, and their conjugates were used to verify their in vivo activity.

[0255] 7.1 Experimental Procedure

[0256] Six- to eight-week-old male mice (C57BL / 6) were purchased from Spiford (Beijing) Biotechnology Co., Ltd., weighing approximately 20g. Each mouse was intravenously injected with 1×10 11A recombinant adeno-associated virus 8 (AAV8) vector containing one copy of the genome was used. The recombinant AAV8 vector consisted of an AAV8 capsid protein expression plasmid and a transfer plasmid carrying an AAV2 ITR. The transfer plasmid carried the human AGT sequence (NM_001384479.1) from positions 68 to 2116, controlled by the human thyroxine-binding globulin promoter (AAV8-TBG-hAGT) (purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd.). The AAV8-hAGT transgenic mouse model was established 14 days after injection. Mice were then administered a single subcutaneous dose of 3 mg / kg of the conjugate, with three mice in each group. Blood was collected from the orbital venous plexus of mice 1 day before administration (D0) and 3 (D3), 7 (D7), 14 (D14), 21 (D21), 28 (D28), 35 (D35), 42 (D42), and 58 (D58) after administration. Serum was separated and the expression level of the target protein hAGT in the serum was detected using an ELISA kit (IBL, catalog number: 27412).

[0257] 7.2 Experimental Results

[0258] The in vivo activity screening results of siRNA conjugates are shown in the figure. Figure 2 .

[0259] The results showed that the chemically modified siRNA sequence had good activity in mice and could significantly reduce the serum AGT expression level in mice.

[0260] Example 8. In vivo activity screening of siRNA conjugates

[0261] Based on the in vivo screening results of Example 7 above, some siRNA conjugate sequences with good in vivo activity were selected and their in vivo activity was verified at different dose concentrations.

[0262] 8.1 Experimental Procedure

[0263] Following step 7.1 of Example 7, the transgenic mouse model was constructed. Then, three mice were administered the conjugate at single subcutaneous doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg per mouse. Blood was collected from the orbital venous plexus of the mice one day before administration and seven days after administration. Serum was separated, and the expression level of the target protein hAGT in the serum was detected using an ELISA kit (IBL, catalog number: 27412).

[0264] 8.2 Experimental Results

[0265] The in vivo activity screening results of different doses of siRNA conjugates are shown in the figure. Figure 3 .

[0266] The results showed that the higher the dose of the siRNA conjugate, the better the inhibitory effect on AGT, which was dose-dependent.

[0267] Example 9. In vivo activity screening of siRNA conjugates

[0268] 9.1 Experimental Procedure

[0269] Following the experimental steps in Example 7, the siRNA conjugate sequences in Table 3 were screened for in vivo activity.

[0270] 9.2 Experimental Results

[0271] The inhibitory effects of the siRNA conjugates 14 days after administration are shown in Table 10 below:

[0272] Table 10. In vivo screening results of siRNA conjugates

[0273] siRNA name Inhibition rate (%) YG-6M2 97.75 YG-43M2 94.46 YG-52M2 92.50

[0274] The results showed that the siRNA conjugates YG-6M2, YG-43M2 and YG-52M2 could significantly inhibit AGT gene expression.

[0275] Based on embodiments of the present invention, the highly efficient activity region for inhibiting AGT mRNA was determined to be positions 85-404, 805-1443, and 1553-1899, calculated according to NCBIrefseqID NM_001384479.1. Specific results can be found in [link to specific data]. Figure 4 .

[0276] Example 10. In vivo activity screening of siRNA conjugates

[0277] 10.1 Experimental Procedure

[0278] Following step 7.1 of Example 7, the transgenic mouse model was constructed. Then, each mouse was administered a single subcutaneous dose of the conjugate at a dose of 3 mg / kg, with three mice in each group. Blood was collected from the orbital venous plexus of the mice one day before administration and on days 7 and 21 after administration. Serum was separated, and the expression level of the target protein hAGT in the serum was detected using an ELISA kit (IBL, catalog number: 27412).

[0279] 10.2 Experimental Results

[0280] The in vivo activity screening results of siRNA conjugates are shown in the figure. Figure 5 .

[0281] The results showed that the chemically modified siRNA sequences YG-116M1, YG-117M1, and YG-118M1 had good activity in mice and could significantly reduce the serum AGT expression level in mice.

[0282] Example 11. In vivo activity screening of siRNA conjugates

[0283] 11.1 Experimental Procedure

[0284] Following step 7.1 of Example 7, the transgenic mouse model was constructed. Then, three mice were administered YG-43M1 subcutaneously at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg of the conjugate, respectively. Blood was collected from the orbital venous plexus of the mice one day before administration and at 7, 14, 21, 42, 48, and 70 days after administration. Serum was separated, and the expression level of the target protein hAGT in the serum was detected using an ELISA kit (IBL, catalog number: 27412).

[0285] 11.2 Experimental Results

[0286] The in vivo activity screening results of siRNA conjugates are shown in the figure. Figure 6 .

[0287] The results showed that YG-43M1 exhibited good activity in mice and significantly and persistently reduced serum AGT expression levels. Furthermore, the higher the dose of the siRNA conjugate, the better the inhibitory effect on AGT, demonstrating a dose-dependent relationship.

[0288] Example 12. In vivo activity screening of siRNA conjugates

[0289] 12.1 Experimental Procedure

[0290] Following step 7.1 of Example 7, the transgenic mouse model was constructed. Then, each mouse was administered a single subcutaneous dose of 1 mg / kg of the conjugate, with three mice in each group. Blood was collected from the orbital venous plexus of the mice one day before administration and at 7, 14, 21, and 42 days after administration. Serum was separated, and the expression level of the target protein hAGT in the serum was detected using an ELISA kit (IBL, catalog number: 27412).

[0291] 12.2 Experimental Results

[0292] The in vivo activity screening results of different doses of siRNA conjugates are shown in the figure. Figure 7 .

[0293] The results showed that YG-83M1, YG-84M1, and YG-120M1 had good activity in mice and could significantly and continuously reduce the serum AGT expression level in mice.

[0294] Example 13. Results of in vivo activity of siRNA conjugates in cynomolgus monkeys

[0295] 13.1 Experimental Procedure

[0296] Twelve male cynomolgus macaques were selected and the experiment was conducted by Junke Zhengyuan (Guangxi) Biomedical Technology Co., Ltd. After acclimatization, they were randomly divided into 6 groups of 2 macaques each according to their body weight. Different siRNA conjugates were administered subcutaneously once at a dose of 3 mg / kg. Peripheral venous blood was collected 1 day before administration and at 7, 14, 21, 28, and 35 days after administration. Serum was separated, and the expression levels of target proteins in the serum were detected using an ELISA kit.

[0297] 13.2 Experimental Results

[0298] The in vivo activity screening results of different siRNA conjugates are shown in the figure. Figure 8 .

[0299] The siRNA conjugate of the present invention exhibits good activity in cynomolgus monkeys and can significantly and continuously reduce the expression level of AGT protein in cynomolgus monkeys.

[0300] Example 14. In vivo toxicity results of siRNA conjugates in rats

[0301] 14.1 Experimental Procedure

[0302] Male SD rats aged 5-7 weeks were purchased from Spiefol (Beijing) Biotechnology Co., Ltd. and divided into 5 groups of 10 rats each. Each rat was subcutaneously administered the conjugate at a dose of 30 mg / kg three times a week. The control group was given the same dose of Saline. The rats were injected subcutaneously one day before administration and on days 3 and 6 after administration. They were fasted for more than 12 hours. On day 7, they were anesthetized by intraperitoneal injection of tribromoethanol. Blood was collected from the portal vein using vacuum blood collection tubes until the rats died. Serum was separated to detect alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

[0303] 14.2 Experimental Results

[0304] In vivo toxicity results for different siRNA conjugates are shown in [link to relevant documentation]. Figure 9 and Figure 10 .

[0305] The results showed that some of the siRNA conjugates disclosed in this paper did not exhibit significant toxicity and had good safety.

Claims

1. An siRNA for inhibiting AGT gene expression, the siRNA comprising a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence I, the antisense strand comprising a nucleotide sequence II; each nucleotide in the nucleotide sequence I and the nucleotide sequence II is a modified or unmodified nucleotide; the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region; the nucleotide sequence I is essentially identical to a first nucleotide sequence of at least 15 nucleotides in length in a mRNA of AGT gene expression, preferably the first nucleotide sequence is a nucleotide sequence of 15-25 nucleotides in length in a mRNA of AGT gene expression, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides; Preferably, the first nucleotide sequence is a nucleotide sequence of at least 15 nucleotides in length in a high activity interval of a mRNA of AGT gene expression, preferably a nucleotide sequence of 15-25 nucleotides in length, the high activity interval is 85-404, 805-1443, 1553-1899 of a mRNA of AGT gene expression. Preferably, the mRNA of AGT gene expression is as shown in NCBI refseq ID NM_001384479.1; in particular, the mRNA sequence is as shown in SEQ ID NO:

1.

2. The siRNA of claim 1, wherein, The nucleotide sequence I has at least 70%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the first nucleotide sequence.

3. The siRNA of claim 1 or 2, wherein, The nucleotide sequence I comprises at least 15 consecutive nucleotides, such as at least 15, 16, 17, 18, 19, 20 or 21 nucleotides, of SEQ ID NO: 2-126; preferably the nucleotide sequence I is as shown in SEQ ID NO: 2-126. The nucleotide sequence II comprises at least 15 consecutive nucleotides, such as at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides, of SEQ ID NO: 128-252; preferably the nucleotide sequence II is as shown in SEQ ID NO: 128-252.

4. The siRNA according to any one of claims 1 to 3, wherein, The sense strand comprises the sense strand of any one of the siRNAs shown in Table 1 herein, and the antisense strand comprises the antisense strand of the corresponding siRNA in Table 1.

5. The siRNA according to any one of claims 1 to 4, wherein, Each nucleotide in the nucleotide sequence I and the nucleotide sequence II is a modified nucleotide, the modified nucleotide is a fluorinated modified nucleotide or a non-fluorinated modified nucleotide. Preferably, the fluorine-modified nucleotide refers to a nucleotide with the structure shown in formula (1) in which the hydroxyl group at the 2'-position of the ribose group of the nucleotide is replaced by fluorine; the non-fluorine-modified nucleotide refers to a nucleotide or nucleotide analogue in which the hydroxyl group at the 2'-position of the ribose group of the nucleotide is replaced by a non-fluorine group; preferably, the non-fluorine group is a 2'-alkoxy-modified nucleotide or nucleotide analogue, a 2'-substituted alkoxy-modified nucleotide or nucleotide analogue, a 2'-alkyl-modified nucleotide or nucleotide analogue, a 2'-substituted alkyl-modified nucleotide or nucleotide analogue, a 2'-amino-modified nucleotide or nucleotide analogue, a 2'-substituted amino-modified nucleotide or nucleotide analogue, or a 2'-deoxy nucleotide; more preferably, the non-fluorine-modified nucleotide has the structure shown in any one of formulae (2) to (12): In formulae (1) to (12), Base represents a base; In formulae (9) and (10), R is selected from H, OH or alkoxy (O-alkyl); In formulae (11) and (12), R is selected from H, OH, F or the non-fluorine group.

6. The siRNA of claim 5, wherein, Preferably, one or more of the nucleotides at positions 5, 7, 8, 9, 10, 11, 12 of the nucleotide sequence I in the direction from the 5' end to the 3' end is a fluorine-modified nucleotide; and one or more of the nucleotides at positions 2, 3, 5, 6, 7, 8, 9, 10, 14, 16 of the nucleotide sequence II in the direction from the 5' end to the 3' end is a fluorine-modified nucleotide; Preferably, the nucleotides at positions 7, 9, 10, 11 of the nucleotide sequence I in the direction from the 5' end to the 3' end are fluorine-modified nucleotides; and the nucleotides at positions 2, 6, 8, 9, 14, 16 of the nucleotide sequence II in the direction from the 5' end to the 3' end are fluorine-modified nucleotides; or Preferably, the nucleotides at positions 5, 7, 8, 9 of the nucleotide sequence I in the direction from the 5' end to the 3' end are fluorine-modified nucleotides; and the nucleotides at positions 2, 6, 8, 9, 14, 16 of the nucleotide sequence II in the direction from the 5' end to the 3' end are fluorine-modified nucleotides; or Preferably, the nucleotides at positions 7, 9, 11 of the nucleotide sequence I in the direction from the 5' end to the 3' end are fluorine-modified nucleotides; and the nucleotides at positions 2, 14, 16 of the nucleotide sequence II in the direction from the 5' end to the 3' end are fluorine-modified nucleotides; or Preferably, the nucleotides at positions 5, 7, 9 of the nucleotide sequence I in the direction from the 5' end to the 3' end are fluorine-modified nucleotides; and the nucleotides at positions 2, 14, 16 of the nucleotide sequence II in the direction from the 5' end to the 3' end are fluorine-modified nucleotides; or Preferably, the nucleotides at positions 7, 9, 10, 11, 12 of the nucleotide sequence I are fluorine-modified nucleotides in the direction from 5' end to 3' end; and the nucleotides at positions 2, 3, 5, 7, 10, 14, 16 of the nucleotide sequence II are fluorine-modified nucleotides in the direction from 5' end to 3' end; or Preferably, the nucleotides at positions 5, 7, 8, 9, 10 of the nucleotide sequence I are fluorine-modified nucleotides in the direction from 5' end to 3' end; and the nucleotides at positions 2, 3, 5, 7, 10, 14, 16 of the nucleotide sequence II are fluorine-modified nucleotides in the direction from 5' end to 3' end.

7. The siRNA of claim 5 or 6, wherein, At least one of the phosphates in the phosphate-sugar backbone of at least one of the single strands of the siRNA is a phosphate with a modification group, which is a phosphorothioate group formed by replacing at least one of the oxygen atoms in the phosphodiester bond of the phosphate with a sulfur atom; Preferably, the phosphate with a modification group is a phosphorothioate group with a structure as shown in formula (13): Preferably, the phosphorothioate group is linked at least at one of the following positions: between the 1st and 2nd nucleotides of the sense strand and / or the antisense strand; between the 2nd and 3rd nucleotides of the sense strand and / or the antisense strand; between the 19th and 20th nucleotides of the antisense strand; between the 20th and 21st nucleotides of the antisense strand; between the 21st and 22nd nucleotides of the antisense strand; between the 22nd and 23rd nucleotides of the antisense strand; or any combination thereof. Preferably, the 5' terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a 5'-phosphate analogue modified nucleotide, such as shown in formula (14), formula (15) and formula (16):

8. An siRNA conjugate comprising an siRNA according to any one of claims 5 to 7 and a conjugate group conjugated to the siRNA, wherein, The conjugate group is L96, with a structure as shown in formula (I): Preferably, the siRNA conjugate, the sense strand is selected from the following nucleotide sequences: 5'-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3', or 5'-XmsXmsXmXmXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3', or 5'-XmsXmsXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXmXm-L96-3', or 5'-XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXmXm-L96-3', or 5'-XmsXmsXmXmXfXmXfXfXfXfXmXmXmXmXmXmXmXmXm-L96-3', or 5'-XmsXmsXmXmXmXmXfXmXfXfXfXfXmXmXmXmXmXmXmXmXm-L96-3'; the nucleotide sequence of the following formula: 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmsXmsXm-3', or 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm-3', or 5'-VPXmsXfsXmXmXmXfXmXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm-3', or 5'-VPXmsXfsXmXmXmXfXmXfXfXmXmXmXmXfXmXfXmXmXmsXmsXm-3', or 5'-VPXmsXfsXfXmXfXmXfXmXmXfXmXmXmXfXmXfXmXmXmsXmsXm-3', or 5'-VPXmsXfsXfXmXfXmXfXmXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm-3'; wherein Xm represents any 2'-methoxy modified nucleotide, such as 2'-methoxy modified C, G, U, A, T; Xf represents any 2'-fluoro modified nucleotide, such as 2'-fluoro modified C, G, U, A, T; the lower case letter s represents a phosphorothioate subunit linkage between the two nucleotides adjacent to the left and right of the letter s; VP represents that the nucleotide adjacent to the right of the letter combination VP is a (5'-(E)-vinylphosphonate, E-VP) modified nucleotide; More preferably, the sense strand and the antisense strand in the conjugate are as follows: M1 modification pattern: sense strand: 5'-XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXmXm-L96-3'; antisense strand: 5'- VPXmsXfsXmXmXmXfXmXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm-3'; or M1' modification pattern: sense strand: 5'-XmsXmsXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXmXm-L96-3'; antisense strand: 5'-VPXmsXfsXmXmXmXfXmXfXfXmXmXmXmXfXmXfXmXmXmsXmsXm-3'; M2 modification pattern: sense strand: 5'-XmsXmsXmXmXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3'; antisense strand: 5'- VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm-3'; or M2' modification pattern: sense strand: 5'-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3'; antisense strand: 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmsXmsXm-3'; M5 modification pattern: sense strand: 5'-XmsXmsXmXmXmXmXfXmXfXfXfXfXmXmXmXmXmXmXmXmXm-L96-3'; antisense strand: 5'-VPXmsXfsXfXmXfXmXfXmXmXfXmXmXmXfXmXfXmXmXmsXmsXm-3'; or M5' modification pattern: sense strand: 5'-XmsXmsXmXmXfXmXfXfXfXfXmXmXmXmXmXmXmXmXm-L96-3'; antisense strand: 5'-VPXmsXfsXfXmXfXmXfXmXmXfXmXmXmXfXmXfXmXmXmsXmsXm-3'; further preferably, the siRNA conjugate comprises a sense strand comprising the sense strand of any one of the siRNA conjugates shown in Table 2, Table 3, Table 4, Table 5 herein and an antisense strand comprising the antisense strand of the corresponding siRNA conjugate.

9. A composition comprising the siRNA of any one of claims 1 to 7 or the siRNA conjugate of claim 8; preferably, the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier or excipient; preferably, the carrier or excipient includes but is not limited to water for injection, sodium hydroxide, sodium phosphate monobasic monohydrate, sodium phosphate monobasic dihydrate, phosphoric acid, sodium chloride, potassium chloride, hydrochloric acid, anhydrous potassium phosphate monobasic, anhydrous sodium phosphate dibasic, PEG2000, PEG6000, cholesterol, distearoylphosphatidylcholine, 1,2-dimyristoylglycerol, dimethyl adipate.

10. Use of the siRNA of any one of claims 1 to 7 or the siRNA conjugate of claim 8 or the composition of claim 9 in the manufacture of a medicament for the prevention and / or treatment of a disease associated with AGT expression. ​ ​ Preferably, the disease associated with AGT expression includes hypertension and related diseases that can be induced by genetic, lifestyle and environmental factors, such as borderline hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, low plasma renin activity or plasma renin concentration-related hypertension, ocular hypertension, glaucoma, pulmonary arterial hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, labile hypertension, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vasculopathy, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, nocturnal hypertension, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, stroke, kidney disease, renal failure, systemic sclerosis, intrauterine growth retardation (IUGR), fetal growth restriction, obesity, hepatic steatosis / fatty liver, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), glucose intolerance, type 2 diabetes, and metabolic syndrome. Preferably, the disease associated with AGT expression includes hypertension and related diseases that can be induced by genetic, lifestyle and environmental factors, such as borderline hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, low plasma renin activity or plasma renin concentration-related hypertension, ocular hypertension, glaucoma, pulmonary arterial hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, labile hypertension, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vasculopathy, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, nocturnal hypertension, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, stroke, kidney disease, renal failure, systemic sclerosis, intrauterine growth retardation (IUGR), fetal growth restriction, obesity, hepatic steatosis / fatty liver, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), glucose intolerance, type 2 diabetes, and metabolic syndrome.

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