Oligonucleotides targeting angiotensinogen and uses thereof

Abstract

Relates to oligonucleotide targeting angiotensinogen and application thereof, the oligonucleotide obviously inhibits AGT gene expression level, and the drug effect is lasting.

Description

Oligonucleotides targeting angiotensinogen and uses thereof Technical Field

[0001] The present disclosure relates to an oligonucleotide, in particular to a targeted oligonucleotide for treating hypertension or related diseases. Background Art

[0002] In 1998, two American scientists, Andrew Fire and Craig Mello, discovered a biological mechanism by which small interfering RNA (siRNA) molecules can mediate the degradation of specific mRNAs (Fire, Andrew, et al. Nature 391, 6669 (1998): 806-811). This mechanism is activated when double-stranded RNA molecules are present in cells, resulting in RNA interference (RNA interference). This discovery heralded the beginning of a new research field, and the two scientists were awarded the 2006 Nobel Prize in Physiology or Medicine. When double-stranded RNA binds to the protein complex Dicer, Dicer cleaves the dsRNA into fragments. Another protein complex, RISC, then binds to these fragments. One strand of the siRNA double strand is removed, but the other strand remains bound to the RISC complex. Guided by the single-stranded RNA, RISC recognizes and degrades the target gene's mRNA, inhibiting the expression of a specific protein and thus specifically silencing the gene.

[0003] RNA interference has opened up a new area of ​​application for genetic technology. Double-stranded RNA (dsRNA) molecules have been artificially designed to silence specific genes in humans, animals, or plants. These artificially designed, synthesized double-stranded small interfering RNA molecules (siRNA) for gene silencing are introduced into cells and activate the RNA interference mechanism to degrade the corresponding mRNA. Currently, this method is an important research tool in biology and biomedicine. In addition, a large number of siRNA drugs have been developed to treat viral infections, cardiovascular diseases, cancer, endocrine disorders, and many other diseases. Most siRNA therapies are in the research and development stage or have been approved for marketing, showing excellent therapeutic effects. Since the first siRNA drug was launched in 2018, at least six siRNAs have been approved for marketing in the European Union or the United States. Therefore, using RNA interference technology to inhibit the expression of specific target genes has become an effective way to treat diseases.

[0004] The asialoglycoprotein receptor (ASGPR) in the liver is a receptor specifically expressed on hepatocytes and is a highly efficient endocytic receptor. Because galactose residues are exposed at the penultimate end of various glycoproteins after enzymatic or acidic hydrolysis of sialic acid in the body, ASGPR specifically binds to galactosyl groups, hence the name galactose-specific receptor. Monosaccharides and polysaccharides such as galactose, galactosamine, and N-acetylgalactosamine (GalNAc) all have a high affinity for ASGPR. The primary physiological function of ASGPR is to mediate the clearance of substances such as asialoglycoproteins and lipoproteins from the blood, and it is closely associated with the development and progression of liver diseases such as viral hepatitis, cirrhosis, and liver cancer. The discovery of this characteristic of ASGPR has played a crucial role in the diagnosis and treatment of liver diseases (Ashwell G, Harford J, Carbohydrate specific Receptors of the Liver, Ann Rev Biochem 1982 51:531-554). Liver-derived disease therapeutic drugs containing galactose or galactosamine and their derivatives in their structures can specifically bind to ASGPR, thereby having active liver targeting and requiring no other carrier system for delivery.

[0005] Blood pressure refers to the force of blood against the walls of blood vessels in the circulatory system. Blood pressure is primarily due to the beating of an animal's heart. During each heartbeat, blood pressure varies between a maximum (systolic) blood pressure (SBP) and a minimum (diastolic) blood pressure (DBP). Mean arterial pressure (MAP) is the average arterial pressure during the cardiac cycle. Blood pressure can be measured using a sphygmomanometer (i.e., a blood pressure measuring device). Normal blood pressure at rest is in the range of 100-140 mmHg systolic and 60-90 mmHg diastolic, and is usually expressed as systolic (top reading) / diastolic (bottom reading) mmHg.

[0006] The Chinese Clinical Practice Guidelines for Hypertension (2022 edition) recommends lowering the diagnostic threshold for hypertension in adults from SBP ≥ 140 mmHg and / or DBP ≥ 90 mmHg to SBP ≥ 130 mmHg and / or DBP ≥ 80 mmHg (1B).

[0007] It is recommended that adult hypertensive patients in my country be divided into the following categories according to their blood pressure levels:

[0008] Grade 1 (SBP 130–139 mmHg and / or DBP 80–89 mmHg)

[0009] The absolute risk of cardiovascular disease is significantly increased only when SBP is 130-139 mmHg and / or DBP is 80-89 mmHg and accompanied by clinical comorbidities, target organ damage or ≥3 cardiovascular risk factors.

[0010] Grade 2 (SBP ≥140 mmHg and / or DBP ≥90 mmHg) (1B)

[0011] Research evidence shows that for those with SBP ≥ 140 mmHg and / or DBP ≥ 90 mmHg, the cumulative risk of cardiovascular disease over 10 years has reached 15%, and ≥ 80% of them have more than two cardiovascular risk factors. The vast majority of patients with SBP ≥ 140 mmHg and / or DBP ≥ 90 mmHg belong to the high-risk cardiovascular group.

[0012] Based on the cause of the disease, hypertension can be divided into primary hypertension and secondary hypertension. Primary hypertension is hypertension caused by multiple factors, or hypertension caused by unknown reasons. There are various reasons such as genetics, region, sodium and water retention, sympathetic excitation, RAS activation, etc. Therefore, primary hypertension can only be controlled but not cured. Secondary hypertension refers to increased blood pressure caused by a certain disease. Hypertension is one of the clinical symptoms of the primary disease, accounting for 95%. Generally, secondary hypertension is common in renal hypertension, renal artery stenosis, primary aldosteronism, pheochromocytoma, polyarteritis, etc.

[0013] The renin-angiotensin-aldosterone system (RAAS or RAS (renin-angiotensin system)) is a system in the human body that regulates cardiovascular function. It is led by the sympathetic nervous system and secretes angiotensin, which has a vasoconstrictive effect. Excessive stimulation or activity of the RAS pathway is one of the causes of hypertension. Angiotensinogen (AGT), a member of the serpin family, also known as SERPINA8, is encoded by the AGT gene and is the sole precursor of all angiotensin peptides in the RAS. Human AGT consists of 485 amino acids, including a 33-amino acid signal peptide. It is primarily produced in the liver and released into the systemic circulation, where it is converted by renin into angiotensin I. Angiotensin I is then converted to angiotensin II by angiotensin-converting enzyme (ACE). Angiotensin I can stimulate the adrenal medulla to secrete adrenaline, but its direct effect on constricting blood vessels is not obvious; angiotensin II can constrict small arteries throughout the body and increase blood pressure. In addition, it can also promote the adrenal cortex to secrete aldosterone. Aldosterone acts on the renal tubules to retain sodium, water, and excrete potassium, thereby causing an increase in blood volume and increased blood pressure.

[0014] The Chinese Clinical Practice Guidelines for Hypertension (2022 Edition) recommends ACEI, ARB, CCB, and diuretics as the first-line initial antihypertensive drugs for hypertensive patients without clinical complications (1B). For hypertensive patients with blood pressure ≥140 / 90 mmHg, initial combined antihypertensive drug therapy is recommended (1B). For hypertensive patients who require combined antihypertensive drug treatment, SPC (single-pill combination, SPC, 2C) is recommended as a priority. When choosing SPC, it is recommended to give priority to the combination of renin-angiotensin system inhibitor (RASI) + CCB or RASI + diuretic (2C). On March 11, 2023, at the 2022 China Hypertension Annual Meeting and the 24th International Academic Symposium on Hypertension and Related Diseases, the expert group of the "Guidelines for the Prevention and Treatment of Hypertension in China" Committee looked forward to the revised "Guidelines for the Prevention and Treatment of Hypertension in China 2023" and discussed the update points, focusing mainly on new drug recommendations, hypertension epidemiological survey data, and risk factor refinement. As one of the key points of the outlook, angiotensin receptor neprilysin inhibitors (ARNI) were included in the guideline recommendations as a commonly used hypertension drug for the first time. The mechanism of action is shown in Figure 1.

[0015] The six types of drugs currently commonly used in clinical practice require long-term, daily, uninterrupted use. Some patients have poor compliance, and side effects are the main reason for poor compliance. ACEIs and ARBs can cause dry coughs and edema, and in severe cases can lead to renal insufficiency; calcium channel antagonists (CCBs) and beta-blockers have side effects such as rapid heartbeat, flushing, headache, and swollen feet; beta-blockers can also cause fatigue and affect blood sugar and blood lipid metabolism; diuretics can cause body weakness and cramps, and in severe cases can lead to gout. In addition, all targets of current oral antihypertensive drugs often experience RAAS system drug escape due to the existence of alternative pathways. Therefore, there is a need in this field for more effective treatments with new mechanisms of action that are different from existing clinical drugs and with fewer side effects.

[0016] One of the off-target effects of siRNA is the miRNA-like effect - the argonaute protein, a core effector in RNA interference, treats artificially introduced siRNAs to induce RNA interference as miRNAs (microRNAs) (Lam et al. (2015) Molecular Therapy Nucleic Acids [Molecular Therapy - Nucleic Acids] (2015) 4, e252). MiRNAs recognize target genes primarily through base pairing between the seed region (positions 2-9 from the 5' end) and the target mRNA for gene suppression. The off-target effects caused by siRNAs originate from the base complementarity of the seed region of the RISC-loaded antisense strand of the iRNA with one or more mRNAs. MiRNA-like off-target effects in siRNAs have been reported in several studies and affect the expression of multiple genes depending on the sequence of the seed region and are severe enough to cause up to 30% of positive hits in siRNA-based phenotypic screens. In addition, in the case of miRNAs, when the interaction between the seed region and the target becomes weak, they have also been reported to silence the target gene through compensatory pairing (3'-compensatory pairing) in their 3' terminal region, suggesting that miRNA-like off-target effects may be regulated by this mechanism. It is known that the addition of chemically modified siRNA designs at different positions of the antisense strand of siRNA can not impair the gene silencing efficacy of siRNA gene therapy and eliminate or reduce the miRNA-like off-target effects of siRNA. For example, the bases modified with glycerol nucleic acid (GNA) at different positions of the antisense strand of siRNA are replaced. Generally, diol nucleic acids include Tgn, Cgn, Agn and Ggn, or the miRNA-like off-target effects of siRNA are eliminated or reduced by connecting the 2' phosphate to the 5' phosphate of the next nucleic acid (Schlegel et al. (2021), CN 110582283 A).

[0017] Summary of the Invention

[0018] In order to solve the problems existing in the prior art, the purpose of the present disclosure is to provide an inhibitor targeting AGT with good efficacy, high safety and long-lasting efficacy.

[0019] In one aspect, the present disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof for reducing the expression of AGT, wherein the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand has a sequence having at least 80% sequence identity to the sequence shown in any one of SEQ ID NOs: 2-219, 789-804, 868-871, or a fragment thereof, or a modified sequence of the aforementioned sequence or fragment thereof, and preferably has a sequence identity of 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more; and the antisense strand has a sequence having at least 80% sequence identity to the sequence shown in any one of SEQ ID NOs: 221-424, 805-821, 872-875, or a fragment thereof, or a modified sequence of the aforementioned sequence or fragment thereof, and preferably has a sequence identity of 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more.

[0020] In another aspect, the present disclosure provides a composition comprising the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

[0021] In another aspect, the present disclosure provides use of the aforementioned oligonucleotide or a pharmaceutically acceptable salt or composition thereof in the preparation of a medicament for treating and / or preventing AGT-related diseases.

[0022] Experiments have demonstrated that the candidate compounds disclosed herein have a significant inhibitory effect on AGT gene expression in the human liver cancer cell line Hep3B. Some of these candidate compounds achieved an inhibitory effect of over 90% on AGT gene expression in Hep3B cell lines, while some compounds achieved 70%, 80%, and even over 90% inhibition of AGT gene expression in cynomolgus monkey hepatocytes, demonstrating excellent inhibitory effects. Some of these compounds maintained a reduction in AGT protein of around 60% after approximately two months (Day 29) of administration to hAGT transgenic mice. Some compounds reduced AGT protein by over 80% in mice for four consecutive weeks (Day 28), demonstrating sustained efficacy. These compounds can be used to treat essential and secondary hypertension and related diseases.

[0023] Inhibition of AGT gene expression can be represented by a decrease in the amount of mRNA expressed by a cell line (such cells can be present, for example, in a sample derived from a subject) in which the AGT gene is transcribed and which is treated (e.g., by contacting one or more cells with an iRNA of the disclosure, or by administering an iRNA of the disclosure to a subject in which cells are or were present) such that expression of the AGT gene is inhibited compared to a cell line that is substantially identical to the cell line but is not treated (control cells that are not treated with the iRNA or that are not treated with an iRNA targeting the gene of interest). In a preferred embodiment, inhibition is assessed using 0.5 nM and 0.05 nM siRNA concentrations in species-matched cell lines as described in Example 1, and is represented by the mRNA expression level in the treated cells relative to the mRNA level in the control cells using the following formula, wherein a housekeeping gene (e.g., hTBP or GAPDH) is used as an internal reference for normalization:

[0024] △CT=CT AGT -CT 管家基因

[0025] △△CT=△CT 处理细胞 -△CT 对照细胞

[0026] mRNA level = 2^-△△CT

[0027] In other embodiments, inhibition of AGT gene expression can be assessed based on a decrease in a parameter functionally associated with AGT gene expression, e.g., AGT protein levels in blood or serum from a subject. AGT gene silencing can be determined in any cell expressing AGT, whether endogenous or heterologous from an expression construct, and by any assay known in the art.

[0028] Inhibition of AGT protein expression can be manifested by a decrease in the level of AGT protein expressed by a cell or cell population or in a subject sample (e.g., the level of protein in a blood sample from a subject). As described above, to assess mRNA inhibition, inhibition of protein expression levels in treated cells or cell populations can be similarly expressed as a percentage of the protein level in a control cell or cell population, or as a change in protein level in a subject sample (e.g., blood or serum thereof), inhibition is assessed by the methods provided in Example 3, Example 4, Example 5, Example 6, or Example 7, using the following formula, expressed as a percentage of the amount of AGT expressed in the treated sample (e.g., blood or serum thereof) relative to the amount of AGT expressed in the control or relative to the amount of AGT expressed before administration.

[0029] mRNA inhibition percentage = (protein expression处理细胞 -Protein expression 对照细胞 ) / protein expression 对照细胞 *100%

[0030] Control cells, cell populations, or subject samples that can be used to assess inhibition of AGT gene expression include cells, cell populations, or subject samples that have not been contacted with the RNAi agents of the present disclosure. For example, control cells, cell lines, or subject samples can be derived from individual subjects (e.g., human or animal subjects) prior to treating the subject or an appropriately matched group control with an RNAi agent.

[0031] In some embodiments of the disclosed methods, the iRNA is administered to a subject so that the iRNA is delivered to a specific site within the subject. Inhibition of AGT expression can be assessed by measuring the level or change of AGT mRNA or AGT protein in a sample of fluid or tissue from a specific site (e.g., liver or blood) of the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 shows the sites of action of inhibitors of the renin-angiotensin-aldosterone system, where the solid line represents the primary pathway, the dashed line represents the alternative pathway, and the dotted line represents the blocked pathway. ACE: angiotensin-converting enzyme; ARB: angiotensin receptor blocker; AT-R: angiotensin receptor; DRI: direct renin inhibitor; LVH: left ventricular hypertrophy.

[0033] FIG2 shows the steps of solid phase synthesis of siRNA.

[0034] FIG3 shows the in vivo efficacy of AGT siRNA in hAGT transgenic mice.

[0035] FIG. 4 shows the in vivo efficacy of AGT siRNA in hAGT transgenic mice (II).

[0036] FIG5 and FIG6 show the in vivo efficacy of AGT siRNA in hAGT transgenic mice III.

[0037] FIG7 and FIG8 show the in vivo efficacy of AGT siRNA in hAGT transgenic mice IV.

[0038] FIG9 , FIG10 and FIG11 show the in vivo efficacy of AGT siRNA in non-human primate (NHP) animals. DETAILED DESCRIPTION

[0039] In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are common procedures. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0040] As used herein, the term "approximately" or "approximately" as applied to one or more target values ​​refers to a value similar to a reference value. In certain embodiments, unless otherwise indicated or in addition apparent from context, the term "approximately" or "approximately" refers to a value falling within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in either direction (greater than or less than) or less (unless such numerals will exceed 100% of possible values).

[0041] As used herein, the term "angiotensinogen" is used interchangeably with the term "AGT" and refers to the well-known gene and polypeptide, also known in the art as Serpin peptidase inhibitor, clade A, member 8; alpha-1 antiproteinase; antitrypsin; SERPINA8; angiotensin I; Serpin A8; angiotensin II; alpha-1 antiproteinase angiotensinogen; antitrypsin; proangiotensinogen 2; ANHU; serine protease inhibitors; and cysteine ​​protease inhibitors.

[0042] As used herein, term " complementary " refers to the structural relationship that allows nucleotide to form base pairs with each other between nucleotide (for example, on relative nucleic acid or on two nucleotides on the relative region of single nucleic acid chain).For example, the purine nucleotides complementary to the pyrimidine nucleotides of a nucleic acid can be base paired together by forming hydrogen bonds with each other.In some embodiments, complementary nucleotides can be base paired in Watson-Crick (Watson-Crick) mode or in any other manner that allows to form a stable duplex.In some embodiments, two nucleic acids can have and be complementary to each other to form the nucleotide sequence of complementary region, as described herein.

[0043] As used herein, the term "strand" refers to a single continuous sequence of nucleotides linked together by internucleotide bonds (e.g., phosphodiester bonds, phosphorothioate bonds). In some embodiments, the strand has two free ends, e.g., a 5'-end and a 3'-end.

[0044] As used herein, the term "deoxyribonucleotide" refers to a nucleotide that has a hydrogen at the 2' position of its pentose sugar compared to a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide that has a modification or substitution of one or more atoms other than the 2' position, including a modification or substitution in or of a sugar, a phosphate group, or a base.

[0045] As used herein, the term "double-stranded RNA" or "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel and substantially complementary nucleic acid chains having "sense" and "antisense" orientations relative to the target RNA (i.e., the AGT gene). In some embodiments of the present disclosure, double-stranded RNA (dsRNA) triggers the degradation of the target RNA (e.g., mRNA) through a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi. Generally speaking, the majority of the nucleotides of each chain of the dsRNA molecule are ribonucleotides, but as described in detail herein, each chain or both chains may also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. Additionally, as used herein, "iRNA" may contain ribonucleotides having chemical modifications; iRNA may contain substantial modifications at multiple nucleotides.

[0046] As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide linkage, or a modified nucleobase, or any combination thereof. Thus, the term "modified nucleotide" encompasses substitutions, additions, or removals of internucleoside linkages, sugar moieties, or nucleobases, such as functional groups or atoms. Modifications suitable for use with the agents of the present disclosure include all types of modifications disclosed herein or known in the art.

[0047] As used herein, the term "oligonucleotide" refers to a short nucleic acid, for example, a short nucleic acid of less than 100 nucleotides in length. An oligonucleotide may comprise ribonucleotides, deoxyribonucleotides and / or modified nucleotides, including, for example, modified ribonucleotides. An oligonucleotide may be single-stranded or double-stranded. An oligonucleotide may or may not have a duplex region. As a set of non-limiting examples, an oligonucleotide may be, but is not limited to, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a Dicer substrate interfering RNA (dsiRNA), an antisense oligonucleotide, a short siRNA, or a single-stranded siRNA. In some embodiments, the double-stranded oligonucleotide is an RNAi oligonucleotide. As used herein, "conjugation" refers to the covalent attachment of two or more chemical moieties, each of which has a specific function, to each other; accordingly, a "conjugate" refers to a compound formed by covalent attachment of the chemical moieties. Further, an "siRNA conjugate" refers to a compound formed by covalent attachment of one or more chemical moieties having a specific function to an siRNA. Hereinafter, the siRNA conjugates of the present disclosure may sometimes be referred to as "conjugates." The term "siRNA conjugate" should be understood as a general term for siRNA conjugates, the first siRNA conjugate or the second siRNA conjugate, or the siRNA sense strand conjugate or the siRNA antisense strand conjugate, depending on the context.

[0048] As used herein, the term "double-stranded oligonucleotide" refers to an oligonucleotide that is substantially in duplex form. In some embodiments, the complementary base pairing of one or more duplex regions of a double-stranded oligonucleotide is formed between the antiparallel sequence of the nucleotides of the covalently separated nucleic acid chains. In some embodiments, the complementary base pairing of one or more duplex regions of a double-stranded oligonucleotide is formed between the antiparallel sequence of the nucleotides of the covalently attached nucleic acid chains. In some embodiments, the complementary base pairing of one or more duplex regions of a double-stranded oligonucleotide is formed from a single nucleic acid chain, and the single nucleic acid chain is folded (for example, via a hairpin) to provide the complementary antiparallel sequence of the nucleotides of base pairing together. In some embodiments, a double-stranded oligonucleotide comprises two covalently separated nucleic acid chains that are completely duplexed from each other. However, in some embodiments, a double-stranded oligonucleotide comprises partially duplexed, for example, two covalently separated nucleic acid chains with an overhang at one or both ends. In some embodiments, a double-stranded oligonucleotide comprises the antiparallel sequence of nucleotides, which are partially complementary, and therefore, can have one or more mispairings, and the mispairings can include internal mispairings or terminal mispairings.

[0049] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of a double-stranded iRNA. For example, a nucleotide overhang exists when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. A dsRNA may include an overhang of at least one nucleotide; alternatively, the overhang may include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. The nucleotide overhang may include or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. In addition, the nucleotides of the overhang may be present on the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of the dsRNA.

[0050] As used herein, the term "naked sequence" refers to an unmodified nucleotide sequence.

[0051] As used herein, the term "subject" refers to an animal that expresses the target gene endogenously or heterologously, such as a mammal, including primates (such as humans, non-human primates, such as monkeys and chimpanzees), non-primates (such as cows, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats or mice) or birds. In one embodiment, the subject is a human.

[0052] As used herein, the term "treating" or "treatment" refers to a beneficial or desired result, such as reducing at least one sign or symptom of an AGT-related disorder in a subject. Treatment also includes reducing one or more signs or symptoms associated with undesirable AGT expression; reducing the degree of undesirable AGT activation or stabilization; ameliorating or alleviating undesirable AGT activation or stabilization. Treatment also includes reducing one or more signs or symptoms associated with undesirable AGT expression. "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment.

[0053] As used herein, the terms "prevention" or "preventing" when used in reference to a disease or condition that would benefit from a decrease in AGT gene expression or agt protein production.

[0054] As used herein, the term "therapeutically effective amount" is intended to encompass an amount of a RNAi agent that, when administered to a subject with an AGT-related disorder, is sufficient to affect treatment of the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more disease symptoms). A "therapeutically effective amount" may vary depending on the RNAi agent, how the agent is administered, the disease and its severity, as well as medical history, age, weight, family history, genetic makeup, type of previous or concomitant treatment (if any), and other individual characteristics of the subject to be treated.

[0055] As used herein, the term "prophylactically effective amount" is intended to encompass an amount of an RNAi agent that, when administered to a subject suffering from an AGT-related disorder, is sufficient to prevent or ameliorate the disorder or one or more symptoms of the disorder. Amelioration of the disease includes slowing the progression of the disease or reducing the severity of the disease that develops later. A "prophylactically effective amount" may vary depending on the RNAi agent, how the agent is administered, the degree of disease risk, and the patient's medical history, age, weight, family history, genetic makeup, type of previous or concomitant therapy (if any), and other individual characteristics of the patient being treated.

[0056] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material (involved in carrying or transporting the subject compound from one organ or part of the body to another organ or part of the body). Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject being treated. Such carriers are known in the art. Pharmaceutically acceptable carriers include carriers for administration by injection.

[0057] In one aspect, the present disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof for reducing the expression of AGT, wherein the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand has a sequence having at least 80% sequence identity with the sequence shown in any one of SEQ ID NOs: 2-219, 789-804, 868-871, or a fragment thereof, or a modified sequence of the aforementioned sequence or fragment thereof, and preferably has a sequence identity of 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more; and the antisense strand has a sequence having at least 80% sequence identity with the sequence shown in any one of SEQ ID NOs: 221-424, 805-821, 872-875, or a fragment thereof, or a modified sequence of the aforementioned sequence or fragment thereof, and preferably has a sequence identity of 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more.

[0058] In some embodiments, wherein each strand is independently 19 to 25 nucleotides in length.

[0059] In some embodiments, the antisense strand is 19 to 23 nucleotides in length.

[0060] In some embodiments, the sense strand is 19 to 23 nucleotides in length.

[0061] In some embodiments, the aforementioned oligonucleotides include 5' and / or 3'-overhang sequences of one or more nucleotides in length, wherein the aforementioned 5' and / or 3'-overhang sequences are present on the antisense strand and / or sense strand. In one embodiment, the antisense strand of the aforementioned oligonucleotides has 1 to 10 nucleotides at the 3' end or 5' overhang, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has 1 to 10 nucleotides at the 3' end or 5' overhang, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. In another embodiment, one or more nucleotides in the overhang are replaced by nucleoside thiophosphates.

[0062] In some embodiments, the antisense strand has an overhang.

[0063] In some embodiments, the sense strand has an overhang.

[0064] In some embodiments, the aforementioned oligonucleotide comprises a 3'-overhang sequence of two nucleotides in length.

[0065] In some embodiments, the aforementioned 3'-overhang sequence is present on the aforementioned sense strand; preferably, the aforementioned overhang sequence is selected from: GG, GA, GC, UC, UG, UU, UA, CA, CC, CG, CU, AA, AG, AU, AC.

[0066] In some embodiments, the aforementioned 3'-overhang sequence is present on the aforementioned antisense strand; preferably, the aforementioned overhang sequence is selected from: UU, UC, UA, UG, GA, GG, GU, GC, TT, AG, AU, AA, AC, CA, CC, U; more preferably, the aforementioned overhang sequence is UU.

[0067] In some embodiments, the aforementioned oligonucleotides comprise an antisense strand and a sense strand each ranging from 19 to 23 nucleotides in length.

[0068] In some embodiments, the aforementioned sense strand and the aforementioned antisense strand form a duplex region.

[0069] In some embodiments, the sense strand and the antisense strand are respectively in a 19 / 21 pairing, a 21 / 21 pairing, a 21 / 23 pairing, or a 23 / 23 pairing duplex structure.

[0070] In some embodiments, the oligonucleotide comprises a 3'-overhang sequence of two nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand, and wherein the sense strand is 19 nucleotides in length and the antisense strand is 21 nucleotides in length, such that the sense strand and the antisense strand form a duplex of 19 nucleotides in length.

[0071] In some embodiments, the oligonucleotide comprises a 3'-overhang sequence of two nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand and the sense strand, and wherein the sense strand is 21 nucleotides in length and the antisense strand is 21 nucleotides in length, such that the sense strand and the antisense strand form a duplex of 19 nucleotides in length.

[0072] In some embodiments, the oligonucleotide comprises a 3'-overhang sequence of two nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand, and wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense strand and the antisense strand form a duplex of 21 nucleotides in length.

[0073] In some embodiments, the oligonucleotide comprises a 3'-overhang sequence of two nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand and the sense strand, and wherein the sense strand is 23 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense strand and the antisense strand form a duplex of 21 nucleotides in length.

[0074] In some embodiments, the modified sequence of the sense strand comprises a sequence selected from any one of SEQ ID NOs: 426-598, 822-838, 876-886;

[0075] In some embodiments, the modified sequence of the antisense strand comprises a sequence selected from any one of SEQ ID NOs: 600-676, 678-788, 839-867, and 887-911.

[0076] In some embodiments, the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof is preferably prepared or synthesized in the form of a carboxylate salt, a sodium salt, a triethylamine salt or other pharmaceutically acceptable salts.

[0077] In some embodiments, the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof is more preferably a sodium salt or a triethylamine salt thereof.

[0078] In some embodiments, the sense strand comprises an unmodified oligonucleotide selected from any one of SEQ ID NOs: 471, 477, 478, 481-483, 518, 536, 548, 554, 592-598, 822, 827, 831, 832, 833, 834, 835, 836, 837, and 838.

[0079] In some embodiments, the antisense strand comprises an unmodified oligonucleotide selected from any one of SEQ ID NOs: 259, 281, 286, 311, 317, 318, 319, 334, 338, 340, 342, 353, 371, 383, 389, 805, 808, 811, 812, 813, 814, 815, 816, 817, 818, 874, or SEQ ID NOs: The modified oligonucleotide of any one of NO: 643, 644, 645, 647, 648, 681, 678, 679, 680, 764, 610, 773, 774, 775, 776, 777, 778, 779, 780, 782, 783, 785, 839, 854, 857, 858, 859, 860, 861, 862, 863, 864.

[0080] In some preferred embodiments, the aforementioned oligonucleotide has a sense strand and an antisense strand, wherein the aforementioned oligonucleotide is selected from any one of the following sense strand and antisense strand combinations:

[0081] (1) the sense strand comprises the sequence shown in SEQ ID NO: 101, and the antisense strand comprises the sequence shown in SEQ ID NO: 311;

[0082] (2) the sense strand comprises the sequence set forth in SEQ ID NO: 107, and the antisense strand comprises the sequence set forth in SEQ ID NO: 281;

[0083] (3) the sense strand comprises the sequence shown in SEQ ID NO: 108, and the antisense strand comprises the sequence shown in SEQ ID NO: 286;

[0084] (4) the sense strand comprises the sequence shown in SEQ ID NO: 111, and the antisense strand comprises the sequence shown in SEQ ID NO: 317;

[0085] (5) the sense strand comprises the sequence shown in SEQ ID NO: 112, and the antisense strand comprises the sequence shown in SEQ ID NO: 318;

[0086] (6) the sense strand comprises the sequence shown in SEQ ID NO: 113, and the antisense strand comprises the sequence shown in SEQ ID NO: 319;

[0087] (7) the sense strand comprises the sequence shown in SEQ ID NO: 129, and the antisense strand comprises the sequence shown in SEQ ID NO: 334;

[0088] (8) the sense strand comprises the sequence shown in SEQ ID NO: 133, and the antisense strand comprises the sequence shown in SEQ ID NO: 338;

[0089] (9) the sense strand comprises the sequence shown in SEQ ID NO: 135, and the antisense strand comprises the sequence shown in SEQ ID NO: 340;

[0090] (10) the sense strand comprises the sequence shown in SEQ ID NO: 137, and the antisense strand comprises the sequence shown in SEQ ID NO: 342;

[0091] (11) the sense strand comprises the sequence shown in SEQ ID NO: 148, and the antisense strand comprises the sequence shown in SEQ ID NO: 353;

[0092] (12) the sense strand comprises the sequence shown in SEQ ID NO: 166, and the antisense strand comprises the sequence shown in SEQ ID NO: 371;

[0093] (13) the sense strand comprises the sequence shown in SEQ ID NO: 178, and the antisense strand comprises the sequence shown in SEQ ID NO: 383;

[0094] (14) the sense strand comprises the sequence shown in SEQ ID NO: 184, and the antisense strand comprises the sequence shown in SEQ ID NO: 389;

[0095] (15) the sense strand comprises the sequence shown in SEQ ID NO: 113, and the antisense strand comprises the sequence shown in SEQ ID NO: 808;

[0096] (16) the sense strand comprises the sequence shown in SEQ ID NO: 113, and the antisense strand comprises the sequence shown in SEQ ID NO: 811;

[0097] (17) the sense strand comprises the sequence shown in SEQ ID NO: 113, and the antisense strand comprises the sequence shown in SEQ ID NO: 818;

[0098] (18) the sense strand comprises the sequence shown in SEQ ID NO: 789, and the antisense strand comprises the sequence shown in SEQ ID NO: 805;

[0099] (19) the sense strand comprises the sequence shown in SEQ ID NO: 794, and the antisense strand comprises the sequence shown in SEQ ID NO: 805;

[0100] (20) the sense strand comprises the sequence shown in SEQ ID NO: 798, and the antisense strand comprises the sequence shown in SEQ ID NO: 805;

[0101] (21) the sense strand comprises the sequence shown in SEQ ID NO: 799, and the antisense strand comprises the sequence shown in SEQ ID NO: 812;

[0102] (22) the sense strand comprises the sequence shown in SEQ ID NO: 800, and the antisense strand comprises the sequence shown in SEQ ID NO: 813;

[0103] (23) the sense strand comprises the sequence shown in SEQ ID NO: 801, and the antisense strand comprises the sequence shown in SEQ ID NO: 814;

[0104] (24) the sense strand comprises the sequence shown in SEQ ID NO: 802, and the antisense strand comprises the sequence shown in SEQ ID NO: 815;

[0105] (25) the sense strand comprises the sequence shown in SEQ ID NO: 803, and the antisense strand comprises the sequence shown in SEQ ID NO: 816;

[0106] (26) the sense strand comprises the sequence shown in SEQ ID NO: 804, and the antisense strand comprises the sequence shown in SEQ ID NO: 817;

[0107] (27) the sense strand comprises the sequence shown in SEQ ID NO: 868, and the antisense strand comprises the sequence shown in SEQ ID NO: 872;

[0108] (28) the sense strand comprises the sequence shown in SEQ ID NO: 869, and the antisense strand comprises the sequence shown in SEQ ID NO: 873;

[0109] (29) the sense strand comprises the sequence shown in SEQ ID NO: 870, and the antisense strand comprises the sequence shown in SEQ ID NO: 874;

[0110] (30) the sense strand comprises the sequence shown in SEQ ID NO: 871, and the antisense strand comprises the sequence shown in SEQ ID NO: 875; and

[0111] (31) the sense strand comprises the sequence shown in SEQ ID NO: 74, and the antisense strand comprises the sequence shown in SEQ ID NO: 259;

[0112] wherein each strand is independently 19 to 25 nucleotides in length.

[0113] In some embodiments, the aforementioned oligonucleotides comprise at least one modified nucleotide.

[0114] In some embodiments, at least one of the aforementioned modified nucleotides is selected from the group consisting of deoxynucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, 2'-5'-linked ribonucleotides (3'-RNA), unlocked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-O-alkyl modified nucleosides Acids, 2'-hydroxy modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides comprising unnatural bases, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides comprising phosphorothioate groups, nucleotides comprising methylphosphonate groups, nucleotides comprising 5'-phosphates, nucleotides comprising 5'-phosphate mimetics, vinyl-phosphonate nucleotides, thermolabile nucleotides, glycol modified nucleotides, nucleotides comprising 2' phosphates, and 2-O-(N-methylacetamide) modified nucleotides; and combinations thereof.

[0115] In some embodiments, at least one of the aforementioned modified nucleotides is selected from the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl, and ethylene glycol; and combinations thereof.

[0116] In some embodiments, at least one of the aforementioned modified nucleotides is selected from the group consisting of deoxynucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, ethylene glycol modified nucleotides (GNA), nucleotides comprising a 2' phosphate, and nucleotides comprising a phosphorothioate group; and combinations thereof.

[0117] In some embodiments, the aforementioned oligonucleotides include at least one 2'-modified nucleotide. For example, these nucleotides can be selected from 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-acylamino modified nucleotides, 2'-deoxy modified nucleotides, 2'-O-allyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-hydroxy modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, 2'-deoxy nucleotides, nucleotides including 2' phosphates, and one or more nucleotides modified with 2'-O-(N-methylacetamide). For example, C1-C3 alkoxy (such as methoxy); substituted alkoxy (such as C1-C3 alkoxy substituted by C1-C3 alkoxy, such as methoxyethoxy); alkyl (such as C1-C3 alkyl, such as methyl), substituted alkyl (such as C1-C3 alkyl substituted by C1-C3 alkoxy, such as methoxymethyl, methoxyethyl); amino (-NH2), substituted amino (such as C1-C3 alkyl mono- or di-substituted amino, such as methylamino, ethylamino), but not limited thereto.

[0118] In some embodiments, the aforementioned 2'-modified nucleotides are selected from one or more of 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, and 2'-deoxy nucleotides.

[0119] In some embodiments, the aforementioned 2'-modification is a modification selected from the group consisting of 2'-methoxy, 2'-acetylamino, 2'-aminoethyl, 2'-fluoro, 2'-O-methoxyethyl, and 2'-fluoro-β-d-arabinorubicin.

[0120] In some embodiments, the aforementioned modification is a modification selected from the group consisting of a 5'-phosphate analog modification, a 2'-methoxy (CH3O-, m), a 2'-fluoro (f), a 2'-acetamido (CH3CO-NH-), and a phosphorothioate (s).

[0121] In some embodiments, the aforementioned 2'-modification is a 2'-methoxy modification.

[0122] In some embodiments, the aforementioned 2'-modification is 2'-acetamido.

[0123] In some embodiments, all nucleotides of the aforementioned oligonucleotides are modified.

[0124] In some embodiments, the aforementioned oligonucleotides may comprise a glycol nucleic acid (GNA) modification.

[0125] In some embodiments, the aforementioned diol nucleic acid (GNA) modification is selected from adenosine-diol nucleic acid, cytidine-diol nucleic acid, thymidine-diol nucleic acid, and guanosine-diol nucleic acid.

[0126] In some embodiments, the aforementioned diol nucleic acid (GNA) modification is selected from the group consisting of thymidine-diol nucleic acid S-isomer (Tgn) as shown in formula (I), cytidine-diol nucleic acid S-isomer (Cgn) as shown in formula (II), adenosine-diol nucleic acid S-isomer (Agn) as shown in formula (III), and guanosine-diol nucleic acid S-isomer (Ggn) as shown in formula (IV);

[0127] In some embodiments, the aforementioned oligonucleotides may comprise a 2'-5'-phosphodiester bond.

[0128] In some embodiments, the aforementioned oligonucleotide comprises a uridine-2'-phosphate (U-2'5') represented by formula (V), a guanosine-2'-phosphate (G-2'5') represented by formula (VI); a cytidine-2'-phosphate (C-2'5') represented by formula (VII); an adenosine-2'-phosphate (A-2'5') represented by formula (VIII); and a thymidine-2'-phosphate (T-2'5') represented by formula (IX);

[0129] In some embodiments, the aforementioned oligonucleotide further comprises a 5'-phosphate analog modified nucleotide.

[0130] In some embodiments, the aforementioned phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.

[0131] In some embodiments, the 4'-carbon of the sugar of the 5'-nucleotide of the aforementioned antisense strand comprises a phosphate analog.

[0132] In some embodiments, the aforementioned 5'-phosphate analog modified nucleotide is APU represented by formula (X). In some embodiments, the aforementioned 5'-phosphate analog modified nucleotide is VPUm represented by formula (XI);

[0133] In some embodiments, the aforementioned oligonucleotides comprise at least one modified internucleotide linkage.

[0134] In some embodiments, at least one of the aforementioned modified internucleotide bonds is a phosphorothioate bond. The phosphorothioate internucleotide bond modification can occur on any nucleotide of the sense strand, antisense strand, or both strands at any position in the chain. For example, the internucleotide bond modification can occur on each nucleotide on the sense strand or antisense strand; each internucleotide bond modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand can contain two internucleotide bond modifications in an alternating pattern. The alternating pattern of the internucleotide bond modification on the sense strand can be the same or different from the antisense strand, and the alternating pattern of the internucleotide bond modification on the sense strand can have an offset relative to the alternating pattern of the internucleotide bond on the antisense strand. In one embodiment, the double-stranded RNAi agent includes 6 to 8 phosphorothioate internucleotide bonds. In some embodiments, the antisense strand includes two phosphorothioate internucleotide bonds at the 5' end and two phosphorothioate internucleotide bonds at the 3' end, and the sense strand includes at least two phosphorothioate internucleotide bonds at the 5' end or the 3' end.

[0135] In some preferred embodiments, the aforementioned oligonucleotide comprises any one selected from the following sense strand and antisense strand combinations:

[0136] (1) the sense strand comprises the sequence shown in SEQ ID NO: 477, and the antisense strand comprises the sequence shown in SEQ ID NO: 643;

[0137] (2) the sense strand comprises the sequence set forth in SEQ ID NO:471, and the antisense strand comprises the sequence set forth in SEQ ID NO:644;

[0138] (3) the sense strand comprises the sequence set forth in SEQ ID NO:478, and the antisense strand comprises the sequence set forth in SEQ ID NO:645;

[0139] (4) the sense strand comprises the sequence shown in SEQ ID NO: 482, and the antisense strand comprises the sequence shown in SEQ ID NO: 647;

[0140] (5) the sense strand comprises the sequence shown in SEQ ID NO: 483, and the antisense strand comprises the sequence shown in SEQ ID NO: 648;

[0141] (6) the sense strand comprises the sequence shown in SEQ ID NO: 481, and the antisense strand comprises the sequence shown in SEQ ID NO: 681;

[0142] (7) the sense strand comprises the sequence shown in SEQ ID NO: 471, and the antisense strand comprises the sequence shown in SEQ ID NO: 678;

[0143] (8) the sense strand comprises the sequence shown in SEQ ID NO: 478, and the antisense strand comprises the sequence shown in SEQ ID NO: 679;

[0144] (9) the sense strand comprises the sequence shown in SEQ ID NO: 483, and the antisense strand comprises the sequence shown in SEQ ID NO: 680;

[0145] (10) the sense strand comprises the sequence shown in SEQ ID NO: 594, and the antisense strand comprises the sequence shown in SEQ ID NO: 764;

[0146] (11) the sense strand comprises the sequence shown in SEQ ID NO: 593, and the antisense strand comprises the sequence shown in SEQ ID NO: 610;

[0147] (12) the sense strand comprises the sequence shown in SEQ ID NO: 592, and the antisense strand comprises the sequence shown in SEQ ID NO: 773;

[0148] (13) the sense strand comprises the sequence shown in SEQ ID NO: 592, and the antisense strand comprises the sequence shown in SEQ ID NO: 774;

[0149] (14) the sense strand comprises the sequence shown in SEQ ID NO: 592, and the antisense strand comprises the sequence shown in SEQ ID NO: 775;

[0150] (15) the sense strand comprises the sequence shown in SEQ ID NO: 595, and the antisense strand comprises the sequence shown in SEQ ID NO: 776;

[0151] (16) the sense strand comprises the sequence shown in SEQ ID NO: 596, and the antisense strand comprises the sequence shown in SEQ ID NO: 777;

[0152] (17) the sense strand comprises the sequence shown in SEQ ID NO: 597, and the antisense strand comprises the sequence shown in SEQ ID NO: 778;

[0153] (18) the sense strand comprises the sequence shown in SEQ ID NO: 598, and the antisense strand comprises the sequence shown in SEQ ID NO: 779;

[0154] (19) the sense strand comprises the sequence shown in SEQ ID NO: 518, and the antisense strand comprises the sequence shown in SEQ ID NO: 780;

[0155] (20) the sense strand comprises the sequence shown in SEQ ID NO: 536, and the antisense strand comprises the sequence shown in SEQ ID NO: 782;

[0156] (21) the sense strand comprises the sequence shown in SEQ ID NO: 548, and the antisense strand comprises the sequence shown in SEQ ID NO: 783;

[0157] (22) the sense strand comprises the sequence shown in SEQ ID NO: 554, and the antisense strand comprises the sequence shown in SEQ ID NO: 785;

[0158] (23) the sense strand comprises the sequence shown in SEQ ID NO: 592, and the antisense strand comprises the sequence shown in SEQ ID NO: 854;

[0159] (24) the sense strand comprises the sequence shown in SEQ ID NO: 592, and the antisense strand comprises the sequence shown in SEQ ID NO: 857;

[0160] (25) the sense strand comprises the sequence shown in SEQ ID NO: 592, and the antisense strand comprises the sequence shown in SEQ ID NO: 864;

[0161] (26) the sense strand comprises the sequence shown in SEQ ID NO: 822, and the antisense strand comprises the sequence shown in SEQ ID NO: 839;

[0162] (27) the sense strand comprises the sequence shown in SEQ ID NO: 827, and the antisense strand comprises the sequence shown in SEQ ID NO: 839;

[0163] (28) the sense strand comprises the sequence shown in SEQ ID NO: 831, and the antisense strand comprises the sequence shown in SEQ ID NO: 839;

[0164] (29) the sense strand comprises the sequence shown in SEQ ID NO: 598, and the antisense strand comprises the sequence shown in SEQ ID NO: 840;

[0165] (30) the sense strand comprises the sequence shown in SEQ ID NO: 832, and the antisense strand comprises the sequence shown in SEQ ID NO: 841;

[0166] (31) the sense strand comprises the sequence shown in SEQ ID NO: 832, and the antisense strand comprises the sequence shown in SEQ ID NO: 842;

[0167] (32) the sense strand comprises the sequence shown in SEQ ID NO: 832, and the antisense strand comprises the sequence shown in SEQ ID NO: 848;

[0168] (33) the sense strand comprises the sequence shown in SEQ ID NO: 832, and the antisense strand comprises the sequence shown in SEQ ID NO: 849;

[0169] (34) the sense strand comprises the sequence shown in SEQ ID NO: 832, and the antisense strand comprises the sequence shown in SEQ ID NO: 850;

[0170] (35) the sense strand comprises the sequence shown in SEQ ID NO: 833, and the antisense strand comprises the sequence shown in SEQ ID NO: 858;

[0171] (36) the sense strand comprises the sequence shown in SEQ ID NO: 834, and the antisense strand comprises the sequence shown in SEQ ID NO: 859;

[0172] (37) the sense strand comprises the sequence shown in SEQ ID NO: 835, and the antisense strand comprises the sequence shown in SEQ ID NO: 860;

[0173] (38) the sense strand comprises the sequence shown in SEQ ID NO: 836, and the antisense strand comprises the sequence shown in SEQ ID NO: 861;

[0174] (39) the sense strand comprises the sequence shown in SEQ ID NO: 837, and the antisense strand comprises the sequence shown in SEQ ID NO: 862;

[0175] (40) the sense strand comprises the sequence shown in SEQ ID NO: 838, and the antisense strand comprises the sequence shown in SEQ ID NO: 863;

[0176] (41) the sense strand comprises the sequence shown in SEQ ID NO: 882, and the antisense strand comprises the sequence shown in SEQ ID NO: 894;

[0177] (42) the sense strand comprises the sequence shown in SEQ ID NO: 882, and the antisense strand comprises the sequence shown in SEQ ID NO: 902;

[0178] (43) the sense strand comprises the sequence shown in SEQ ID NO: 884, and the antisense strand comprises the sequence shown in SEQ ID NO: 896;

[0179] (44) the sense strand comprises the sequence shown in SEQ ID NO: 884, and the antisense strand comprises the sequence shown in SEQ ID NO: 903;

[0180] (45) the sense strand comprises the sequence shown in SEQ ID NO: 592, and the antisense strand comprises the sequence shown in SEQ ID NO: 905;

[0181] (46) the sense strand comprises the sequence shown in SEQ ID NO: 592, and the antisense strand comprises the sequence shown in SEQ ID NO: 907;

[0182] (47) the sense strand comprises the sequence shown in SEQ ID NO: 882, and the antisense strand comprises the sequence shown in SEQ ID NO: 910; and

[0183] (48) the sense strand comprises the sequence shown in SEQ ID NO: 882, and the antisense strand comprises the sequence shown in SEQ ID NO: 911;

[0184] wherein each strand is independently 19 to 25 nucleotides in length.

[0185] In some preferred embodiments, the aforementioned oligonucleotide comprises any one selected from the following sense strand and antisense strand combinations:

[0186] (1) the sense strand represented by SEQ ID NO: 594, and the antisense strand represented by SEQ ID NO: 764;

[0187] (2) the sense strand represented by SEQ ID NO: 593, and the antisense strand represented by SEQ ID NO: 610;

[0188] (3) the sense strand represented by SEQ ID NO: 592, and the antisense strand represented by SEQ ID NO: 773;

[0189] (4) the sense strand represented by SEQ ID NO: 592, and the antisense strand represented by SEQ ID NO: 774;

[0190] (5) the sense strand represented by SEQ ID NO: 592, and the antisense strand represented by SEQ ID NO: 775;

[0191] (6) the sense strand represented by SEQ ID NO: 595, and the antisense strand represented by SEQ ID NO: 776;

[0192] (7) the sense strand represented by SEQ ID NO: 596, and the antisense strand represented by SEQ ID NO: 777;

[0193] (8) the sense strand represented by SEQ ID NO: 597, and the antisense strand represented by SEQ ID NO: 778;

[0194] (9) the sense strand represented by SEQ ID NO: 598, and the antisense strand represented by SEQ ID NO: 779;

[0195] (10) the sense strand represented by SEQ ID NO: 518, and the antisense strand represented by SEQ ID NO: 780;

[0196] (11) the sense strand represented by SEQ ID NO: 536, and the antisense strand represented by SEQ ID NO: 782;

[0197] (12) the sense strand represented by SEQ ID NO: 548, and the antisense strand represented by SEQ ID NO: 783;

[0198] (13) the sense strand represented by SEQ ID NO: 554, and the antisense strand represented by SEQ ID NO: 785;

[0199] (14) the sense strand represented by SEQ ID NO: 592, and the antisense strand represented by SEQ ID NO: 854;

[0200] (15) the sense strand represented by SEQ ID NO: 592, and the antisense strand represented by SEQ ID NO: 857;

[0201] (16) the sense strand represented by SEQ ID NO: 592, and the antisense strand represented by SEQ ID NO: 864;

[0202] (17) the sense strand represented by SEQ ID NO: 822, and the antisense strand represented by SEQ ID NO: 839;

[0203] (18) the sense strand represented by SEQ ID NO: 827, and the antisense strand represented by SEQ ID NO: 839;

[0204] (19) the sense strand represented by SEQ ID NO: 831, and the antisense strand represented by SEQ ID NO: 839;

[0205] (20) the sense strand represented by SEQ ID NO: 598, and the antisense strand represented by SEQ ID NO: 840;

[0206] (21) the sense strand represented by SEQ ID NO: 832, and the antisense strand represented by SEQ ID NO: 841;

[0207] (22) the sense strand represented by SEQ ID NO: 832, and the antisense strand represented by SEQ ID NO: 842;

[0208] (23) the sense strand represented by SEQ ID NO: 832, and the antisense strand represented by SEQ ID NO: 848;

[0209] (24) the sense strand represented by SEQ ID NO: 832, and the antisense strand represented by SEQ ID NO: 849;

[0210] (25) the sense strand represented by SEQ ID NO: 832, and the antisense strand represented by SEQ ID NO: 850;

[0211] (26) the sense strand represented by SEQ ID NO: 833, and the antisense strand represented by SEQ ID NO: 858;

[0212] (27) the sense strand represented by SEQ ID NO: 834, and the antisense strand represented by SEQ ID NO: 859;

[0213] (28) the sense strand represented by SEQ ID NO: 835, and the antisense strand represented by SEQ ID NO: 860;

[0214] (29) the sense strand represented by SEQ ID NO: 836, and the antisense strand represented by SEQ ID NO: 861;

[0215] (30) the sense strand represented by SEQ ID NO: 837, and the antisense strand represented by SEQ ID NO: 862;

[0216] (31) the sense strand represented by SEQ ID NO: 838, and the antisense strand represented by SEQ ID NO: 863;

[0217] (32) the sense strand represented by SEQ ID NO: 882, and the antisense strand represented by SEQ ID NO: 894;

[0218] (33) the sense strand represented by SEQ ID NO: 882, and the antisense strand represented by SEQ ID NO: 902;

[0219] (34) the sense strand represented by SEQ ID NO: 884, and the antisense strand represented by SEQ ID NO: 896;

[0220] (35) the sense strand represented by SEQ ID NO: 884, and the antisense strand represented by SEQ ID NO: 903;

[0221] (36) the sense strand represented by SEQ ID NO: 592, and the antisense strand represented by SEQ ID NO: 905;

[0222] (37) the sense strand represented by SEQ ID NO: 592, and the antisense strand represented by SEQ ID NO: 907;

[0223] (38) the sense strand shown in SEQ ID NO: 882, and the antisense strand shown in SEQ ID NO: 910; and

[0224] (39) The sense strand represented by SEQ ID NO: 882, and the antisense strand represented by SEQ ID NO: 911.

[0225] In some preferred embodiments, the aforementioned oligonucleotide comprises any one selected from the following sense strand and antisense strand combinations:

[0226] (1) the sense strand represented by SEQ ID NO: 594, and the antisense strand represented by SEQ ID NO: 764;

[0227] (2) the sense strand represented by SEQ ID NO: 592, and the antisense strand represented by SEQ ID NO: 773;

[0228] (3) the sense strand represented by SEQ ID NO: 592, and the antisense strand represented by SEQ ID NO: 774;

[0229] (4) the sense strand represented by SEQ ID NO: 592, and the antisense strand represented by SEQ ID NO: 775;

[0230] (5) the sense strand represented by SEQ ID NO: 595, and the antisense strand represented by SEQ ID NO: 776;

[0231] (6) the sense strand represented by SEQ ID NO: 598, and the antisense strand represented by SEQ ID NO: 779;

[0232] (7) the sense strand shown in SEQ ID NO: 518, and the antisense strand shown in SEQ ID NO: 780

[0233] (8) the sense strand represented by SEQ ID NO: 592, and the antisense strand represented by SEQ ID NO: 854;

[0234] (9) the sense strand represented by SEQ ID NO: 592, and the antisense strand represented by SEQ ID NO: 857;

[0235] (10) the sense strand represented by SEQ ID NO: 592, and the antisense strand represented by SEQ ID NO: 864;

[0236] (11) the sense strand represented by SEQ ID NO: 822, and the antisense strand represented by SEQ ID NO: 839;

[0237] (12) the sense strand represented by SEQ ID NO: 827, and the antisense strand represented by SEQ ID NO: 839;

[0238] (13) the sense strand represented by SEQ ID NO: 831, and the antisense strand represented by SEQ ID NO: 839;

[0239] (14) the sense strand represented by SEQ ID NO: 598, and the antisense strand represented by SEQ ID NO: 840;

[0240] (15) the sense strand represented by SEQ ID NO: 832, and the antisense strand represented by SEQ ID NO: 841;

[0241] (16) the sense strand represented by SEQ ID NO: 832, and the antisense strand represented by SEQ ID NO: 842;

[0242] (17) the sense strand represented by SEQ ID NO: 832, and the antisense strand represented by SEQ ID NO: 848;

[0243] (18) the sense strand represented by SEQ ID NO: 832, and the antisense strand represented by SEQ ID NO: 849;

[0244] (19) the sense strand represented by SEQ ID NO: 832, and the antisense strand represented by SEQ ID NO: 850;

[0245] (20) the sense strand represented by SEQ ID NO: 833, and the antisense strand represented by SEQ ID NO: 858;

[0246] (21) the sense strand represented by SEQ ID NO: 834, and the antisense strand represented by SEQ ID NO: 859;

[0247] (22) the sense strand represented by SEQ ID NO: 835, and the antisense strand represented by SEQ ID NO: 860;

[0248] (23) the sense strand represented by SEQ ID NO: 836, and the antisense strand represented by SEQ ID NO: 861;

[0249] (24) the sense strand represented by SEQ ID NO: 837, and the antisense strand represented by SEQ ID NO: 862;

[0250] (25) the sense strand represented by SEQ ID NO: 838, and the antisense strand represented by SEQ ID NO: 863;

[0251] (26) the sense strand represented by SEQ ID NO: 882, and the antisense strand represented by SEQ ID NO: 894;

[0252] (27) the sense strand represented by SEQ ID NO: 882, and the antisense strand represented by SEQ ID NO: 902;

[0253] (28) the sense strand represented by SEQ ID NO: 884, and the antisense strand represented by SEQ ID NO: 896;

[0254] (29) the sense strand represented by SEQ ID NO: 884, and the antisense strand represented by SEQ ID NO: 903;

[0255] (30) the sense strand represented by SEQ ID NO: 592, and the antisense strand represented by SEQ ID NO: 905;

[0256] (31) the sense strand represented by SEQ ID NO: 592, and the antisense strand represented by SEQ ID NO: 907;

[0257] (32) the sense strand shown in SEQ ID NO: 882, and the antisense strand shown in SEQ ID NO: 910; and

[0258] (33) The sense strand shown in SEQ ID NO: 882, and the antisense strand shown in SEQ ID NO: 911.

[0259] In some embodiments, at least one nucleotide of the aforementioned oligonucleotide or its salt is conjugated to one or more targeting ligands to form a siRNA conjugate. The aforementioned siRNA conjugate contains the above-mentioned siRNA and a conjugated group that is conjugated to the siRNA. The term "oligonucleotide salt" refers to an oligonucleotide compound in the form of a salt. Oligonucleotide salts include salts of oligonucleotide conjugated compounds and salts of unconjugated oligonucleotide compounds. Oligonucleotide salts are advantageously present in the form of solid powders.

[0260] In general, the aforementioned conjugated group includes at least one pharmaceutically acceptable targeting ligand and an optional linker, and the aforementioned siRNA, the aforementioned linker and the aforementioned targeting ligand are connected in sequence. The targeting group can be a ligand conventionally used in the field of siRNA administration, such as the various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference. In some embodiments, the aforementioned targeting ligand is 2-4. The aforementioned siRNA molecule can be non-covalently or covalently conjugated to the aforementioned conjugated group, for example, it can be covalently conjugated to the aforementioned conjugated group. The conjugation site of siRNA with the conjugated group can be at the 3' end or 5' end of the siRNA sense strand or antisense strand, or can be in the internal sequence of the siRNA. In some embodiments, the conjugation site of the aforementioned siRNA with the conjugated group is at the 3' end or 5' end of the siRNA sense strand. In some embodiments, the conjugation site of the aforementioned siRNA with the conjugated group is at the 3' end or 5' end of the siRNA antisense strand. In some preferred embodiments, the conjugation site between the aforementioned siRNA and the conjugation group is at the 3' end of the siRNA sense strand.

[0261] In some embodiments, the targeting ligand comprises an asialoglycoprotein receptor ligand. In some embodiments, the asialoglycoprotein receptor ligand comprises or consists of one or more galactose derivatives. As used herein, the term "galactose derivative" includes galactose and lactose derivatives having an affinity for the asialoglycoprotein receptor equal to or greater than that of galactose. Galactose derivatives include, but are not limited to, galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, N-n-butyryl-galactosamine, and N-isobutyrylgalactosamine. Galactose derivatives and clusters of galactose derivatives that can be used to target oligonucleotides and other molecules to the liver in vivo are known in the art. Galactose derivatives have been used to target molecules to hepatocytes in vivo by binding to the asialoglycoprotein receptor (ASGPr) expressed on the surface of hepatocytes. Binding of ASGPr ligands to ASGPr(s) facilitates cell-specific targeting to hepatocytes and endocytosis of molecules into hepatocytes. The ASGPr ligand can be a monomer (e.g., having a single galactose derivative) or a polymer (e.g., having multiple galactose derivatives). The galactose derivative or galactose derivative cluster can be linked to the 3' end or 5' end of the siRNA using methods known in the art.

[0262] In some embodiments, the pharmaceutically acceptable targeting ligand in the aforementioned siRNA conjugate can be galactose or N-acetylgalactosamine (GalNAc), wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent. It should be understood that the monovalent, divalent, trivalent, and tetravalent respectively refer to the siRNA molecule and the conjugated group containing galactose or N-acetylgalactosamine as the targeting ligand to form the siRNA conjugate, and the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule in the siRNA conjugate is 1: 1, 1: 2, 1: 3, or 1: 4. In some embodiments, the pharmaceutically acceptable targeting ligand is N-acetylgalactosamine. In some embodiments, when the siRNA of the present invention is conjugated to a conjugated group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA described herein is conjugated to a conjugation group comprising N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0263] In some embodiments, the targeting ligand is selected from a carbohydrate, an amino sugar, cholesterol, a polypeptide, or a lipid.

[0264] In some embodiments, the targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

[0265] In some embodiments, the aforementioned GalNAc moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.

[0266] In some embodiments, the targeting ligand is A1 or L96. A1 is a GalNAc targeting ligand represented by formula (XII); L96 is N-[tris(GalNAc-alkyl)amidodecanoyl]-4-hydroxyprolinol Hyp-(GalNAc-alkyl)3 represented by formula (V) (XIII).

[0267] In another aspect, the present disclosure provides a composition comprising the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

[0268] In some embodiments, the composition is in the form of an oral dosage, an intravenous injection, a subcutaneous injection, or an intramuscular injection.

[0269] In some preferred embodiments of the present disclosure, the composition is in the form of a subcutaneous injection.

[0270] In some embodiments, the aforementioned combination further comprises other drugs for treating and / or preventing AGT-related diseases.

[0271] In another aspect, the present disclosure provides a use of the aforementioned oligonucleotide or a pharmaceutically acceptable salt or composition thereof in the preparation of a medicament for treating and / or preventing AGT-related diseases.

[0272] Various preparations have been developed to promote the use of oligonucleotides. For example, oligonucleotides can be delivered to a subject or a cellular environment using a preparation that minimizes degradation, promotes delivery and / or intake, or provides another beneficial property for the oligonucleotides in the preparation. In some embodiments, compositions comprising oligonucleotides (for example, single-stranded or double-stranded oligonucleotides) for reducing the expression of AGT are provided herein. Such compositions can be suitably prepared so that when applied to a subject (in the direct environment of the target cell or systemically), enough oligonucleotides of a portion enter the cell to reduce AGT expression. Any of the various suitable oligonucleotide preparations can be used to deliver oligonucleotides for reducing AGT, as disclosed herein. In some embodiments, oligonucleotides or their conjugates are formulated in a buffer solution, such as a phosphate buffered saline solution, a liposome, a micellar structure, and a shell. The buffer solution can be selected from the solution of acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In some embodiments, the oligonucleotides or their conjugates exposed (that is, without a delivery agent) are formulated in water or an aqueous solution (for example, the water of pH regulation). In some embodiments, the naked oligonucleotide or its conjugate is formulated in an alkaline buffered aqueous solution (eg, PBS).

[0273] The preparation of oligonucleotides with cationic lipids can be used to facilitate transfection of oligonucleotides into cells. For example, cationic lipids such as lipofectin, cationic glycerol derivatives and polycationic molecules (e.g., polylysine) can be used.

[0274] Thus, in some embodiments, the formulation comprises lipid nanoparticles. In some embodiments, the excipient comprises a liposome, lipid, lipid complex, microsphere, microparticle, nanosphere, or nanoparticle, or can be otherwise formulated for administration to a cell, tissue, organ, or body of a subject in need thereof.

[0275] In some embodiments, preparations as disclosed herein include excipients. In some embodiments, excipients give stability of the improvement of active ingredient, absorption of improvement, solubility and / or therapeutic enhancement to the composition. In some embodiments, excipients are buffers (for example, sodium citrate, sodium phosphate, tris alkali or sodium hydroxide) or vehicles (for example, buffered solution, petrolatum, dimethyl sulfoxide or mineral oil). In some embodiments, oligonucleotides are freeze-dried for extending their shelf life, and then solution is made before use (for example, being applied to a subject). Therefore, the excipient in the composition comprising any one of the oligonucleotides described herein can be a lyoprotectant (for example, mannitol, lactose, polyethylene glycol or polyvinyl pyrrolidone) or a collapse temperature modifier (for example, dextran, ficoll or gelatin).

[0276] In some embodiments, the pharmaceutical composition is formulated to be compatible with the intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Typically, the route of administration is intravenous or subcutaneous.

[0277] Pharmaceutical compositions suitable for injectable applications include sterile aqueous solutions (when water-soluble) or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. For intravenous or subcutaneous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL.TM. (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The aforementioned carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. In many cases, it is preferred to include isotonic agents, for example sugars, polyols such as mannitol, sorbitol and sodium chloride in the composition. Sterile injectable solutions can be prepared by being incorporated into the solvent of selection with the desired amount of oligonucleotide and the desired above-listed composition or in combination, followed by filtration sterilization.

[0278] In some embodiments, the composition may contain at least about 0.1% or more of the therapeutic agent (e.g., an oligonucleotide for reducing AGT expression), although the percentage of the active ingredient(s) may be between about 1% and about 80% or more of the weight or volume of the total composition. One skilled in the art of preparing such pharmaceutical formulations will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and therefore various dosages and treatment regimens may be desired.

[0279] Even though many of the embodiments relate to liver-targeted delivery of any of the oligonucleotides disclosed herein, targeting other tissues is also contemplated.

[0280] In some embodiments, administration of an oligonucleotide as described herein results in a decrease in the level of AGT expression in a cell. In some embodiments, the decrease in the level of AGT expression can be a decrease to 1% or less, 5% or less, 10% or less, 15% or less, 20% or less, 25% or less, 30% or less, 35% or less, 40% or less, 45% or less, 50% or less, 55% or less, 60% or less, 70% or less, 80% or less, or 90% or less compared to an appropriate control level of AGT. An appropriate control level can be the level of AGT expression in a cell or cell population that has not been contacted with an oligonucleotide as described herein. In some embodiments, the effect of delivering an oligonucleotide to a cell according to the methods disclosed herein is evaluated after a limited period of time. For example, the level of AGT in the blood can be analyzed at least 8 hours, 12 hours, 18 hours, 24 hours after the aforementioned oligonucleotide is introduced into the cell; or at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks.

[0281] In some embodiments, the oligonucleotide is delivered in the form of a transgenic engineered to express an oligonucleotide disclosed herein in a cell (e.g., in the form of an shRNA). In some embodiments, the oligonucleotide is delivered using a transgenic engineered to express any oligonucleotide disclosed herein. Transgenics can be delivered using viral vectors (e.g., adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, or herpes simplex virus) or non-viral vectors (e.g., plasmid or synthetic mRNA). In some embodiments, the transgenic can be injected directly into the subject.

[0282] In another aspect, the present disclosure provides a method of preventing at least one symptom in a subject having a disorder that would benefit from decreased AGT expression, e.g., an AGT-related disease, e.g., hypertension, e.g., critical hypertension (also known as prehypertension), essential hypertension (also known as primary hypertension or idiopathic hypertension), secondary hypertension (also known as non-essential hypertension), hypertensive crisis (also known as malignant hypertension), hypertensive urgency, isolated systolic or diastolic hypertension, pregnancy-related hypertension (e.g., preeclampsia, eclampsia, and postpartum preeclampsia), diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension (also known as renal hypertension), Goldblatt's hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension, Hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease (including peripheral vascular disease), diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other glucocorticoid excess states (including chronic steroid treatment), pheochromocytoma, nephrinoma, secondary aldosteronism and other mineralocorticoid excess states, sleep apnea, thyroid / parathyroid disease, heart failure (e.g., left ventricular systolic dysfunction), myocardial infarction, angina pectoris, stroke, diabetes (e.g., diabetic nephropathy), kidney disease (e.g., chronic kidney disease or diabetic nephropathy, optionally in a pregnancy environment), renal failure (e.g., chronic renal failure), cognitive impairment (such as Alzheimer's disease) and systemic sclerosis (e.g., scleroderma renal crisis). In certain embodiments, AGT-related diseases include intrauterine growth retardation (IUGR) and fetal growth restriction. The method comprises administering to a subject a therapeutically effective amount of an iRNA agent (eg, dsRNA) or vector of the invention, thereby preventing at least one symptom in a subject suffering from a disorder that would benefit from reduced AGT expression.

[0283] In certain aspects, the present disclosure provides methods for preventing a disease, disorder, symptom or condition as described herein in a subject by administering a therapeutic agent (e.g., an oligonucleotide or a vector encoding the same or a transgene) to the subject. In some embodiments, the subject to be treated is one who would benefit therapeutically from a reduction in the amount of, for example, AGT protein.

[0284] The methods described herein generally involve administering to a subject an effective amount (i.e., an amount capable of producing the desired therapeutic result) of an oligonucleotide. A therapeutically acceptable amount can be an amount capable of treating a disease or condition. The appropriate dosage for any one subject will depend on certain factors, including the subject's size, body surface area, age, the specific composition to be administered, one or more active ingredients in the composition, the time and route of administration, overall health, and other drugs administered concurrently.

[0285] In some embodiments, any of the compositions disclosed herein is administered to a subject enterally (e.g., orally, through a gastric feeding tube, through a duodenal feeding tube, via gastrostomy, or rectally), parenterally (e.g., subcutaneously, intravenously, intraarterially, intramuscularly), topically (e.g., epidermally, by inhalation, via eye drops, or through a mucous membrane), or by direct injection into a target organ (e.g., the subject's liver). Typically, the oligonucleotides disclosed herein are administered intravenously or subcutaneously.

[0286] In some embodiments, the oligonucleotide is administered at a dosage in the range of about 0.001 mg / kg to about 200 mg / kg (e.g., about 0.1 mg / kg to about 100 mg / kg). In some embodiments, the oligonucleotide is administered to a subject at a dosage in the range of about 0.1 mg / kg to about 50 mg / kg, preferably about 0.1 mg / kg to about 20 mg / kg, 0.3 mg / kg to about 18 mg / kg, 0.5 mg / kg to about 15 mg / kg, or 0.5 mg / kg to about 12 mg / kg, more preferably about 1 mg / kg to about 10 mg / kg.

[0287] In some embodiments, the oligonucleotide is administered at a fixed dose of about 50 mg to about 800 mg. In some embodiments, the oligonucleotide is administered to a subject at a fixed dose of about 50 mg to about 200 mg, about 200 mg to about 400 mg, or about 400 mg to about 800 mg. In some embodiments, the oligonucleotide is administered to a subject at a fixed dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, 500 mg, about 600 mg, about 700 mg, or about 800 mg.

[0288] As one set of non-limiting examples, the oligonucleotides of the present disclosure will typically be administered once a year, twice a year, quarterly (once every three months), every two months (once every two months), monthly, or weekly. In some embodiments, a fixed dose is administered to the subject at monthly intervals. In some embodiments, a fixed dose is administered to the subject at six-month intervals.

[0289] In some embodiments, the subject is administered a fixed dose of about 150 mg approximately once every six months. In some embodiments, the subject is administered a fixed dose of about 300 mg approximately once every six months. In some embodiments, the subject is administered a fixed dose of about 300 mg approximately once every six months. In some embodiments, the subject is administered a fixed dose of about 600 mg approximately once every six months. In some embodiments, the subject is administered a fixed dose of about 800 mg approximately once every six months. In some embodiments, the subject is administered a fixed dose of about 800 mg approximately once every six months.

[0290] In some embodiments, the subject to be treated is a human (e.g., a human patient) or a non-human primate or other mammalian subject. Other exemplary subjects include domestic animals such as dogs and cats; livestock such as horses, cows, pigs, sheep, goats, and chickens; and animals such as mice, rats, guinea pigs, and hamsters.

[0291] In another aspect, the present disclosure provides a method for inhibiting angiotensinogen (AGT) gene expression in a subject, comprising administering to the subject a fixed dose of about 50 mg to about 800 mg of an oligonucleotide or a salt thereof.

[0292] In another aspect, the present disclosure provides a method for preventing or treating a subject suffering from angiotensinogen (AGT)-related disorder, the method comprising administering to the subject a fixed dose of about 50 mg to about 800 mg of an oligonucleotide or a salt thereof.

[0293] In another aspect, the present disclosure provides a method for lowering blood pressure levels in a subject, the method comprising administering to the subject a fixed dose of about 50 mg to about 800 mg of an oligonucleotide or a salt thereof.

[0294] In some embodiments, the method further comprises administering to the subject other therapeutic agents for treating hypertension. In some embodiments, the other therapeutic agents are selected from the following: diuretics, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, beta-blockers, vasodilators, calcium channel blockers, aldosterone antagonists, alpha2-agonists, renin inhibitors, alpha-blockers, peripherally acting adrenergic agents, selective D1 receptor partial agonists, non-selective alpha-adrenergic antagonists, synthetic steroidal antimineralocorticoids; any combination of the above; and hypertension therapeutic agents formulated as a combination of agents. In some embodiments, the other therapeutic agents include angiotensin II receptor antagonists. In other embodiments, angiotensin II receptor antagonists are selected from the following: losartan, valsartan, olmesartan, eprosartan, and azilsartan.

[0295] For purposes of clarity and conciseness, features are described herein as part of the same or separate embodiments; however, it will be understood that the scope of the present disclosure may include embodiments having a combination of all or some of the described features.

[0296] Hereinafter, the present disclosure will be described in more detail with reference to specific examples. However, the examples are for illustrative purposes only and have no limiting effect on the present disclosure.

[0297] Example

[0298] Unless the source of a reagent is specifically given herein, such reagent can be obtained from any molecular biology reagent supplier at quality / purity standards appropriate for molecular biology.

[0299] The abbreviation of the nucleotide monomer used in the nucleic acid sequence representation.It will be understood that these monomers, when present in an oligonucleotide, are interconnected by 5'-3' phosphodiester bonds unless otherwise indicated.And it will be understood that when nucleotide contains 2'-fluorine modification, fluorine replaces the hydroxyl group (that is, it is 2'-deoxy-2'-fluorine nucleotide) at this position in the parent nucleotide.

[0300] Table A. Nucleotide monomer abbreviations used in nucleic acid sequence representation

[0301] Preparation of targeting ligands

[0302] (i) L96 was prepared according to the method described in patent CN104717982B.

[0303] (ii) The preparation steps of A1 are as follows:

[0304] (1) Synthesis of intermediate E1

[0305] F1 (199.46 mg, 0.41 mmol) was dissolved in DMF and activated with HBTU (155.39 mg, 0.41 mmol). DIEA (67.81 L, 0.41 mmol) was then added and stirred for 30 min. Intermediate G1 (100.0 mg, 0.14 mmol) was added to the reaction system. After stirring overnight, the reaction was complete by TLC. After concentration under reduced pressure, column chromatography (PE:EA = 10:1) was performed to obtain intermediate I-F1 (174.98 mg, 58% yield). Intermediate I-F1 was debenzylated with palladium on carbon under hydrogen catalysis to obtain intermediate II-F1 (156.34, 95%). Intermediate II-F1 activated with HBTU was coupled with (3R,5S)-5-[[bis(4-methoxyphenyl)phenylmethoxy]methyl]-3-pyrrolidinol in DMF to obtain intermediate E1. 1 HNMR (400MHz, DMSO-D6): δ7.91-7.86(m,8H),7.51-7.45(m,6H),7.40-7.32(m,10H),7.02-6.95(m,3H),6.03-5.89( m,4H),5.31(d,3H),5.07-4.99(m,4H),4.43-4.37(m,3H),4.23-4.19(m,4H),4.17-4.13(m,6H),4.05-4.00(m,6H),3 .88-3.81(m,6H),3.77(s,6H),3.69-3.66(m,9H),3.50-3.47(m,12H),3.40-3.37(m,6H),3.20-3.16(m,16H),2.60- 2.57(m,2H),2.46(s,9H),2.30(s,9H),2.10(m,6H),2.07(s,9H),1.98(s,9H),1.56-1.45(m,8H),1.32-1.27(m,4H). HRMS[M+H] + m / z 2467 (calcd for C 115 H 168 N 14 O 45 ,2466).

[0306] (2) Synthesis of solid phase support A1 with protective groups

[0307] Intermediate E1 was treated with succinic anhydride in the presence of DMAP and Et3N to obtain the hemisuccinate intermediate E1-1 in quantitative yield. The hemisuccinate intermediate E1-1 was coupled with the amino groups of a 60% divinylbenzene (DVB) cross-linked (aminomethyl) polystyrene resin with an amine content of 250 mol / g to obtain a solid support A1 with a loading of 100 mol / g and a protecting group.

[0308] Preparation of oligonucleotides

[0309] (1) Preparation of siRNA

[0310] The siRNA sequence is synthesized separately on a solid support via a sense strand (SS) and an antisense strand (AS), and is obtained after deprotection, cleavage, purification, annealing, purification, and lyophilization.

[0311] Solid-phase synthesis (Figure 2): Sense and antisense strands are synthesized separately on a solid support using phosphoramidite technology using an automated oligonucleotide synthesizer. Examples of such synthesizers include the AKTA Oligopilot (Cytiva) and the Dr. Oligo 192XLc (Kunshan Berleke Precision Instrument Co., Ltd.). Solid-phase synthesis begins at the 3' end of the sequence and sequentially couples monomers into the sequence. Each coupling of a phosphoramidite monomer involves four chemical steps: 1) unblocking or deprotection (removal of the hydroxyl protecting group); 2) coupling; 3) oxidation; and 4) capping. All phosphoramidite monomers, reagents, and purification consumables used were commercially available, including various phosphoramidite monomers (e.g., 5'-O-(4,4'-Dimethoxytrityl)-2'-O-methyl-Uridine-3'-CE-Phosphoramidite) purchased from Shanghai Zhaowei Technology Development Co., Ltd., and reaction reagents (e.g., 40 wt% aqueous methylamine solution, 28 wt% aqueous ammonium hydroxide solution) purchased from Sigma-Aldrich LLC. The siRNA synthesis and purification methods used herein are described in US20130178612A1 and US2015100197A1, among others; the synthesis methods for sequences containing VPUm and APU structures are described in J. Med. Chem. 2018, 61, 734-744.

[0312] (2) Preparation of double-stranded RNA (dsRNA) agents

[0313] (a) Synthesis of the positive chain

[0314] The solid-phase phosphoramidite method is a well-established method for oligonucleotide synthesis. It utilizes a computer-controlled synthesizer and is carried out within a stainless steel synthesis column. Sense strand synthesis begins with a solid support loaded with a targeting ligand (e.g., L96 and A1), or directly from the solid support. The solid-phase synthesizer controls different pipelines, injecting various raw materials, reagents, and solvents in a 3' to 5' sequence order, ligating phosphoramidite nucleoside monomers one by one. The reaction involves four cycles: DMT protection group removal, condensation, oxidation or thiolation, and capping. Each cycle adds a single nucleotide unit to the oligonucleotide, yielding sequences of 19 or 21 nucleotide units. Following synthesis, the protecting group (2-cyanoethyl) is removed on the solid-phase synthesis column, and the synthesized sequence is cleaved from the solid support via aminolysis. The resulting product is filtered, the filter cake washed with ethanol, and the filtrate and washings are collected and concentrated to yield the crude sense strand. The crude product is purified by chromatography (SOURCE 15Q) and lyophilized to yield the desired sense strand. Among them, in the synthesizer, the siRNA sense chain conjugate is synthesized starting from the solid support loaded with the targeting ligand (such as L96); and the siRNA is synthesized directly starting from the solid support.

[0315] (b) Synthesis of antisense strand

[0316] The synthesis of the antisense strand is similar to that of the sense strand. A solid-phase synthesizer controls different pipelines to inject different raw materials, reagents, and solvents in the order of 3' to 5' of the sequence, connecting the phosphoramidite nucleoside monomers one by one. The reaction process includes four cycles: DMT protection group removal, condensation reaction, oxidation or thiolation reaction, and end-capping reaction. Each cycle connects a nucleotide unit to obtain an oligonucleotide sequence of 21 or 23 nucleotide units. After synthesis, the protecting group (2-cyanoethyl) is removed on a solid-phase synthesis column, and the synthesized sequence is cleaved from the solid phase support by aminolysis. The residue is filtered, the filter cake is washed with ethanol, and the filtrate and washing liquid are collected and concentrated to obtain the crude antisense strand. The crude product is purified by chromatography (SOURCE 15Q), ultrafiltration, and lyophilization to obtain the target antisense strand siRNA.

[0317] (c) Preparation of double-stranded siRNA

[0318] Dissolve the AS and SS strands separately in injection water, mix at a defined ratio (1.01:1.0-1.2:1.0), incubate at 30-50°C for 30-90 minutes, cool to room temperature, and freeze-dry to obtain double-stranded siRNA.

[0319] According to the same method, the double-stranded siRNA agents shown in Tables 2, 3 and 4 below were prepared.

[0320] Example 1. AGT-siRNA activity screening in liver cell lines in vitro

[0321] First, a computer-based algorithm was used to generate candidate oligonucleotide sequences complementary to human AGT mRNA (NM_000029.3, Table 1). Some of these sequences were also complementary to or had no more than two mismatches with cynomolgus macaque AGT mRNA (XM_005443695.2, Table 1). Some of these sequences were designed as double-stranded siRNAs with a 19 / 21 sense / antisense pairing, with the antisense strand having two overhangs complementary to the mRNA sequence; in some cases, the antisense strand had non-complementary UU overhangs. Some of these sequences were designed as double-stranded siRNAs with a 21 / 23 sense / antisense pairing, with the antisense strand having two overhangs complementary to the mRNA sequence. Some of these sequences were designed as double-stranded siRNAs with 21 / 21 or 23 / 23 pairings. In some of these complementary pairs, the first base at the 5' end of the antisense strand (the last base at the 3' end of the sense strand) was replaced with a base that did not match the AGT mRNA.

[0322] Table 1 Human and cynomolgus monkey AGT mRNA sequences

[0323] Certain oligonucleotides may include a diol nucleic acid (GNA) modification. In certain oligonucleotides, individual ribonucleic acids are replaced with (Tgn), which is a thymidine-diol nucleic acid (GNA) S-isomer of formula (I). In certain oligonucleotides, individual ribonucleic acids are replaced with (Cgn), which is a cytidine-diol nucleic acid (GNA) S-isomer of formula (II). In certain oligonucleotides, individual ribonucleic acids are replaced with (Agn), which is an adenosine-diol nucleic acid (GNA) S-isomer of formula (III). In certain oligonucleotides, individual ribonucleic acids are replaced with (Ggn), which is a guanosine-diol nucleic acid (GNA) S-isomer of formula (IV). Represents the connection with the rest of the oligonucleotide. The structural formulas of Tgn, Cgn, Agn and Ggn are as follows:

[0324] Certain oligonucleotide sequences may comprise 2'-5'-phosphodiester bonds, wherein adjacent pairs of nucleoside units are linked 2'-5' to 5'-2'. In certain oligonucleotides, individual ribonucleic acids are replaced by (U-2'5'), which is a uridine-2'-phosphate of formula (V). In certain oligonucleotides, individual ribonucleic acids are replaced by (G-2'5'), which is a guanosine-2'-phosphate of formula (VI). In certain oligonucleotides, individual ribonucleic acids are replaced by (C-2'5'), which is a cytidine-2'-phosphate of formula (VII). In certain oligonucleotides, individual ribonucleic acids are replaced by (A-2'5'), which is an adenosine-2'-phosphate of formula (VIII). In certain oligonucleotides, individual ribonucleic acids are replaced by (T-2'5'), which is a thymidine-2'-phosphate of formula (IX).

[0325] In Tables 2, 3, and 4, "G," "C," "A," "U," "T," and "I" generally represent nucleotides based on guanine, cytosine, adenine, uracil, thymine, and hypoxanthine, respectively. The naked sequences in Tables 2, 3, and 4 refer to unmodified oligonucleotide sequences.

[0326] For nucleotide modifications: m represents 2'-methoxy; f represents 2'-deoxy-2'-fluoro; s represents phosphorothioate; APU is a 5'-phosphate analog-modified uridylic acid (2'-acetylamino-5'-vinylphosphonate-uridylic acid) represented by formula (X); VPUm is a 5'-phosphate analog-modified uridylic acid (2'-methoxy-5'-vinylphosphonate-uridylic acid) represented by formula (XI):

[0327] A1 is a GalNAc targeting ligand represented by formula (XII); L96 is N-[tris(GalNAc-alkyl)amidodecanoyl]-4-hydroxyprolinol (Hyp-(GalNAc-alkyl)3) represented by formula (XIII):

[0328] Table 2 Oligonucleotide naked sequences

[0329] Table 3 Modified oligonucleotides

[0330] Table 4 siRNA sequences with targeting ligands

[0331] (1) Hep3B cell culture and transfection:

[0332] Human hepatoma cell line Hep3B (Shanghai Fushen Biotechnology Co., Ltd.) was used and cultured in a 37°C, 5% CO2 incubator using Dulbecco's Modified Eagle's Medium (Hyclone, SH30022.01, 2 g / L Glucose) supplemented with 10% FBS (aqlabteech, AQ-MV-06600) and 1% penicillin-streptomycin (Keygen Biotechnology, KGY0023). Cells were digested with trypsin (Amresco, 0458-250G) when the confluence reached 90%, and counted using a Nexcelom cellometer Mini. 150 μl of cell suspension was plated per well in a 96-well plate, and the cell number was 2*10 4 cells / well and wait for them to adhere to the wall for transfection the next day.

[0333] Using Lipofectamine TM For transfection, 5 μl (50 nM) of the diluted compound was dispersed in 20 μl Opti-MEM (thermofisher, 1105821), and 0.2 μl of RNAiMAX was dispersed in 25 μl Opti-MEM. The final concentration of the siRNA was 5 nM. After incubation for 5 minutes, the cells were mixed with the compound dispersion and incubated for 10 minutes. The cells were then added to the transfection complex (n = 2) and cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0334] (2) Cell lysis

[0335] One day after transfection, the culture medium was removed and washed with PBS. 50 μl of lysis buffer was added to each well and stored at -80°C.

[0336] (3) RT-qPCR

[0337] A mixture of 2.5 μl buffer, 0.2 μl Enzyme Mix (Foregene, DRT-02011), 0.4 μl lysis buffer, and 0.4 μl 5 μM primer was prepared in an RNase-free centrifuge tube. The mixture was made up to 5 μl with RNase-free ddH2O. Each sample was repeated three times. The 96-well plate was placed in a qPCR instrument (ROCGENE, Archimed) and the program was executed: stage 1, 42°C, 5 min; 95°C, 10 sec; amplification, 95°C, 5 sec; 59°C, 20 sec; 72°C, 10 sec; 40 cycles; melting curve, 95°C, 15 sec, 59°C, 60 sec, 95°C, 15 sec.

[0338] (4) Data statistical analysis:

[0339] Export the data to EXCEL format and use CT AGT -CT hTBP The control group was normalized. In order to calculate the fold change of relative silencing efficiency, the data were analyzed using the △△CT method. The results are shown in Tables 5 and 6. The data in each table are obtained from a separate experiment. Due to different cell batches, the silencing efficiency of the target gene will vary.

[0340] When the dosage was 0.05 nM, the inhibitory effects of naked sequence AL0181001-AL0061069 and AL0181070-AL0181223 groups on the AGT gene expression level of Hep3B cell lines are shown in Tables 5 and 6. The inhibition rate was between 10-90%, among which AL0181001, AL0181002, AL0181003, AL0181004, AL0181005, AL0181006, AL0181012, AL0181014, AL0181019, AL0181020, AL0181021, AL0181033, AL0181038, AL0181040, AL0181042, AL0181043, AL0181045, AL0181046, and AL0181047 were the most common cytokines. 181050, AL0181051, AL0181053, AL0181056, AL0181059, AL0181060, AL0181061, AL0181062, AL0181063, AL0181065, AL 0181066, AL0181067, AL0181069, AL0181153, AL0181074, AL0181154, AL0181156, AL0181077, AL0181078, AL0181160, L0181084, AL0181167, AL0181089, AL0181169, AL0181170, AL0181171, AL0181090, AL0181091, AL0181092, AL0181097, AL0181098, AL0181099, AL0181178, AL0181102, AL0181103, AL0181104, AL0181105, AL0181181, AL0181183, AL0181185 , AL0181108, AL0181110, AL0181111, AL0181112, AL0181187, AL0181189, AL0181113, AL0181115, AL0181195, AL0181128, AL0181135, AL0181136, AL0181215, AL0181217, AL0181218, AL0181147 and AL0181222 groups had a knockdown rate of AGT mRNA greater than 75%, or even greater than 90%.

[0341] Table 2 Naked sequence AGT siRNA knockdown level

[0342] Table 6 Naked sequence AGT siRNA knockdown level

[0343] After sorting the mRNA inhibition rate from high to low, the top 15 were selected and siRNAs were designed based on the sequence position. All sequences were chemically modified. When the dosage was 0.5 nM and 0.05 nM, the knockdown of AGT mRNA in the modified sequence AL0185001-AL0185180 group was shown in Table 7.

[0344] Table 7 Knockdown levels of modified sequence AGT siRNA

[0345] Example 2. In vitro activity screening of chemically modified AGT-siRNA in primary monkey hepatocytes

[0346] (1) Cynomolgus monkey hepatocyte cell culture and transfection:

[0347] Cynomolgus macaque hepatocytes (cmTCSC, Beijing Red Biotech Co., Ltd.) were used. Culture medium was prewarmed. Thawing medium (HEPO24, Beijing Red Biotech Co., Ltd.) was removed from the biosafety cabinet and 4 mL of FBS was added to 36 mL of thawing medium (HEPO24). This was heated in a 37°C water bath for 10 minutes. The cells were then treated with coating medium (HEPO44, Beijing Red Biotech Co., Ltd.) in a CO2 incubator at 37°C for 0.5 h. The cells were removed from liquid nitrogen and lysed in a 37°C water bath. After approximately 2 minutes, the cells were removed and transferred to 40 mL of prewarmed thawing medium. The cryovial was rinsed with 2 mL of complete thawing medium. The cell suspension was centrifuged at 180 × g for 1 minute, the supernatant discarded, and 2 mL of prewarmed CM seeding medium (CMHEP054, Beijing Red Biotech Co., Ltd.) was added. The cell suspension was gently dispersed by pipetting, and 20 μl of the cell suspension was collected for counting. According to the counting results, 12-well plates were seeded with 3*10^5 / well and cultured in a 37°C, 5% CO2 incubator. After 4-5 hours of adherence, the CM seeding medium was aspirated and replaced with pre-warmed culture medium (Beijing Red Biotech Co., Ltd., CMHEP064). Transfection was performed 6 hours after adherence. Lipofectamine was used. TMTransfection was performed using 3000 Transfection Reagent (thermofisher, L3000150). 50 nM modified siRNA (Suzhou Beixin Biotechnology Co., Ltd.) was diluted with 50 μl Opti-MEM (thermofisher, 1105821) for system ①, and 3 μl Lip3000 was diluted with 50 μl Opti-MEM for system ②. After standing for 5 min, systems ① and ② were mixed and allowed to stand for another 15 min. The cells were then added dropwise to a 12-well plate. DMEM / F12 complete medium was replaced 4 h after transfection, and the 12-well plate was placed in an incubator and incubated for 48 h.

[0348] (2) Total RNA was extracted using RNA-Quick Purification Kit (RN001, Yishan Biotechnology):

[0349] Remove the 12-well plate from the incubator, aspirate the medium, wash once with an appropriate amount of PBS, add 500 μl of lysis buffer to each well, and transfer the supernatant to a fresh 1.5 ml centrifuge tube. Add 500 μl of absolute ethanol to the lysed cells and mix thoroughly (if precipitation occurs, this is normal; continue with the procedure). Invert the centrifuge tube several times or pipette vigorously 10 times to disperse the precipitate. Then add the liquid to the spin column, place the tubes symmetrically in a centrifuge (Eppendorf, 5430), and centrifuge at 4000 × g for 1 min. Remove the centrifuge tube and add 500 μl of wash buffer to the column. Centrifuge at 12000 × g for 1 min. When removing the column after centrifugation, be careful not to allow the waste liquid in the collection tube to come into contact with the RNA column to prevent contamination. Discard the waste liquid and return the RNA column to the collection tube. Centrifuge the empty tube once to completely remove any residual wash buffer. Place the column in a clean, RNase-free 1.5 ml centrifuge tube and air dry it with the lid open for 2 minutes. Add 30 μl of elution buffer to the center of the RNA column membrane. Let stand at room temperature for 2 minutes. Centrifuge at 2000 × g for 1 minute to elute the RNA, then place on ice. Measure the eluted RNA concentration for subsequent experiments. The extracted RNA can be used immediately for subsequent experiments or stored at -80°C for later use.

[0350] (3) Use cDNA was synthesized using IIQ RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (Novozyme, R223-01):

[0351] In an RNase-free centrifuge tube, prepare a mixture of 4 μl of 4×g DNA wiper mix, 1 μg of template RNA, and 16 μl of RNase-free ddH₂O to remove genomic DNA. Gently pipette to mix thoroughly and incubate at 42°C for 2 minutes. Then, add 4 μl of 5× HiScript II qRT SuperMix II directly to the reaction tube and gently pipette to mix thoroughly. Place the tube in a PCR instrument (Applied Biosystems, 9700) and cycle at 50°C for 15 minutes, 85°C for 5 seconds, and hold at 4°C. The product can be used immediately for qPCR reactions or stored at -20°C and used within six months. For long-term storage, aliquot and store at -80°C. Avoid repeated freeze-thaw cycles for cDNA.

[0352] (4) qPCR quantification using ChamQ SYBR qPCR Master Mix (Novozymes, Q311-02):

[0353] A mixture of 10 μl 2× ChamQ SYBR qPCR Master Mix, 0.5 μl Forword primer (Ruiboxing), 0.5 μl Reverse primer (Ruiboxing), 1 μl Template cDNA, and 8 μl ddH2O was prepared to 20 μl system. Each sample was replicated three times. The 96-well plate was placed in a qPCR instrument (ROCGENE, Archimed) and the following program was performed: initial denaturation, 95°C, 30 sec; amplification, 95°C, 10 sec, 60°C, 30 sec, 40 cycles; melting curve, 95°C, 15 sec, 60°C, 60 sec, 95°C, 15 sec.

[0354] (5) Data statistical analysis:

[0355] Export the data to EXCEL format and use CT AGT -CT GAPDH The control group was normalized. To calculate the fold change of relative silencing efficiency, the data were analyzed using the △△CT method. The mean and standard deviation of the three replicate data were calculated. The screening results of seven monkey primary cells are shown in Tables 8 to 14. The data in each table are obtained from a separate experiment. Due to different cell batches, the silencing efficiency of the target gene will vary. In some experiments, low concentrations may occur due to the cell state. For example, at a dosage of 0.01 nM in Table 14, the deviation between the biological replicates is large. Reference should be made to other concentrations and data with smaller errors at the same concentration.

[0356] As shown in Table 8, using the AL0185001 group as the quality control standard, at a dosage of 0.5 nM, the inhibitory effects of the siRNAs in the AL0185055-AL0185057, AL0185061-64, and AL0185091-AL0185094 groups were all greater than 90%. At a low dosage of 0.05 nM, except for the AL0185062 and AL01850663 groups, the inhibition rates of the other siRNAs were all greater than 80%.

[0357] As shown in Table 9, using the AL0185001 group as the quality control standard, at a dose of 0.5 nM, all siRNAs except AL0185081 and AL0185188 demonstrated superior inhibitory efficacy compared to the AL0185001 group. At a low dose of 0.05 nM, all siRNAs except AL0185089 exhibited superior inhibition rates compared to the AL0185001 group. The AL0185188-AL0185195 groups, based on the AL0185081, AL0185085, AL0185087, and AL0185089 groups, were modified with thermally labile bases (VPU and APU). These results demonstrate that VPU or APU modifications can further enhance the efficacy of siRNAs.

[0358] As shown in Table 10, using the AL0185001 group as the quality control standard, at a dosage of 0.5 nM, except for the AL0185104 group, the inhibitory effects of other siRNAs were all around 70%; at a low dosage of 0.05 nM, the inhibition rates of other siRNAs decreased.

[0359] As can be seen from Table 11, using the AL0185001 group as the quality control standard, when the dosage was 0.5 nM, the inhibitory effects of siRNA in the AL0185210, AL0185215, AL0185219, AL0185220, AL0185221, AL0185222, AL0185228, AL0185229, and AL0185230 groups were all higher than 98%; when the dosage was a low concentration of 0.05 nM, the inhibitory effects were all higher than 94%. All of the above siRNAs were modified with thermolabile bases (VPU and APU). The AL0185222 group also had a (Tgn) modification; the AL0185228 group also had a (C-2'5') modification; the AL0185229 group also had an (A-2'5') modification; and the AL0185230 group also had a (U-2'5') modification. The results showed that VPU modification further enhanced the efficacy of siRNA. GNA and 2'5' modifications reduced miRNA-like off-target activity in the seed region (positions 2-8 of the siRNA antisense strand), while still achieving an inhibitory effect of greater than 98% on AGT mRNA. The results showed that VPU, APU, GNA, and 2'5' modifications further enhanced the efficacy of the siRNA.

[0360] As shown in Table 12, using the AL0185001 group as the quality control standard, at a dosage of 0.01 nM or 0.05 nM, the efficacy can be further improved by introducing Im modifications at certain positions, such as the AL0185234, AL0185237, and AL0185244 groups.

[0361] As shown in Table 13, the efficacy of the drug can be further improved by replacing the VPUm or base modification with DNA modification. For example, compared with the AL0185258 group, the efficacy can be increased by 2-3 times after adding VPUm or replacing the DNA modification, especially at lower dosage concentrations. For example, the silencing efficiency of the target protein mRNA in the AL0185255, AL0185259, AL0185257, AL0185268, AL0185269 and AL0185270 groups can reach more than 80%.

[0362] As shown in Table 14, the addition of A-2'5', U-2'5', C-2'5', or G-2'5' modifications at positions 6-7 of the antisense strand stabilizes its efficacy because these modifications reduce the thermal stability of the seed region and thus the risk of off-target effects. At a dose of 0.05 nM, the AL0185259 group exhibited the best efficacy. At a concentration of 0.01 nM, the silencing activity of the siRNA was enhanced compared to that of the siRNAs without A-2'5', U-2'5', C-2'5', or G-2'5' modifications, as shown in the AL0185272, AL0185274, AL0185277, and AL0185278 sequences. This suggests, to some extent, that at relatively low doses, the A-2'5', U-2'5', C-2'5', or G-2'5' modifications can sometimes achieve better target gene silencing effects.

[0363] Table 8 Inhibitory results of AGT siRNA in primary hepatocytes of cynomolgus monkeys

[0364] Table 9 Inhibition results of AGT siRNA in primary hepatocytes of cynomolgus monkey II

[0365] Table 10 Inhibitory results of AGT siRNA in primary hepatocytes of cynomolgus monkeys III

[0366] Table 11 Inhibitory results of AGT siRNA in primary hepatocytes of cynomolgus monkeys IV

[0367] Table 12 Inhibition results of AGT siRNA in primary hepatocytes of cynomolgus monkeys V

[0368] Table 13 Inhibitory results of AGT siRNA in primary hepatocytes of cynomolgus monkeys VI

[0369] Table 14 Inhibitory results of AGT siRNA in primary hepatocytes of cynomolgus monkeys VII

[0370] Example 3. Screening of AGT siRNA activity in hepatic cell lines

[0371] (1) Cell culture and transfection:

[0372] Human hepatoma cell lines Hep3B, Huh7, and HepG2 (Shanghai Fushen Biotechnology Co., Ltd.) were cultured in a 37°C, 5% CO2 incubator using Dulbecco's Modified Eagle's Medium (Hyclone, SH30022.01, 2 g / L Glucose) supplemented with 10% FBS (aqlabteech, AQ-MV-06600) and 1% penicillin-streptomycin (Keygen Biotechnology, KGY0023). Cells were digested with trypsin (Amresco, 0458-250G) when the confluence reached 90%, and counted using a Nexcelom cellometer Mini. 150 μl of cell suspension was plated per well in a 96-well plate, with a cell number of 2 × 10 4 cells / well and wait for them to adhere to the wall for transfection the next day.

[0373] Using Lipofectamine TM For transfection, 5 μl (5 nM, 0.5 nM, and 0.1 nM) of the diluted compound was dispersed in 20 μL Opti-MEM (thermofisher, 1105821), and 0.2 μL of RNAiMAX was dispersed in 25 μL Opti-MEM. The final siRNA concentrations were 0.5 nM, 0.05 nM, and 0.01 nM, respectively. After incubation for 5 minutes, the cells were mixed with the compound dispersion and incubated for 10 minutes. The cells were then added to the transfection complex (n = 2) and cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0374] (2) Cell lysis

[0375] One day after transfection, the culture medium was removed and washed with PBS. 50 μl of lysis buffer was added to each well and stored at -80°C.

[0376] (3) RT-qPCR

[0377] A mixture of 2.5 μl buffer, 0.2 μl Enzyme Mix (Foregene, DRT-02011), 0.4 μl lysis buffer, and 0.4 μl 5 μM primer was prepared in an RNase-free centrifuge tube. The mixture was made up to 5 μl with RNase-free ddH2O. Each sample was repeated three times. The 96-well plate was placed in a qPCR instrument (ROCGENE, Archimed) and the program was executed: stage 1, 42°C, 5 min; 95°C, 10 sec; amplification, 95°C, 5 sec; 59°C, 20 sec; 72°C, 10 sec; 40 cycles; melting curve, 95°C, 15 sec, 59°C, 60 sec, 95°C, 15 sec.

[0378] (4) Data statistical analysis:

[0379] Export the data to EXCEL format and use CT AGT -CT hTBP The control group was normalized, and the data were analyzed using the ΔΔCT method to calculate the fold change in relative silencing efficiency. The results are shown in Tables 15 and 16. The data in each table are obtained from a separate experiment. Due to different cell batches, the silencing efficiency of the target gene may vary.

[0380] As shown in Table 15, when the dosage was 0.5 nM and 0.01 nM, the average inhibitory effect of the naked sequence AL0181240 group on the AGT gene expression level in Hep3B, HepG2 and Huh7 cell lines was better than that of other sequences in most cases, especially at the low dose of 0.01 nM.

[0381] As shown in Table 16, when the dosage was 0.5 nM, 0.05 nM and 0.01 nM, the silencing efficiency of AGT mRNA in the modified sequence groups AL0185199, AL0185234 and AL0185237 was significantly better than that in the AL0185300 group at the three dosages (P < 0.05).

[0382] Table 15 Inhibitory effect of AGT siRNA in hepatocyte cell lines

[0383] Table 16 Inhibitory effect of AGT siRNA in hepatocyte cell lines

[0384] Note: Paired t test with AL018300

[0385] Example 4. In vivo testing of AGT RNAi agents in mice I

[0386] The experiment used SPF male B6.Cg-Tg(hAGT)2041Sig / J mice (sourced from Jackson laboratory) aged 8-9 weeks. Pre-dose serum samples were obtained on day 0 of administration, and the mice were randomly divided into groups according to hAGT levels. B6.Cg-Tg(hAGT)2041Sig / J mice were given a single subcutaneous dose of 3 mg / kg of AGT RNAi agent. Blood was collected from the mice (blood was collected from the eyeball and sent for testing within 1 hour after blood collection) at 1, 2, 3, 4, 5, 6, 7, and 8 weeks after administration, and the hAGT expression level was measured. The level of hAGT knockdown was detected using the pre-dose control as the control. During the experiment, no animals showed signs of death or dying. Clinical observation showed no obvious abnormalities in all animals.

[0387] The experimental groups are shown in Table 17; the changes in hAGT levels are shown in Figure 3.

[0388] Table 17 Dosage Grouping

[0389] As can be seen from Figure 3 , compared with pre-drug administration, at the third week after drug intervention, the knockdown effects of all AGT siRNAs except AL0187004 reached the lowest level (more than 90%), and then gradually recovered. After the sixth week, the hAGT levels in the blood of the AL0187002, AL0187004, and AL0187009 groups had returned to the pre-drug administration level. Drug intervention can significantly reduce hAGT levels in the blood of mice. The most significant and longest-lasting reduction was in the AL0187003, AL0187006, AL0187007, AL0187008, AL0187010, and AL0187011 groups. Moreover, after 8 weeks of administration, the drug's hAGT inhibition was still about 50%, which was significantly different from the AL0187002 group in the AL0187005, AL0187006, AL0187007, AL0187008, AL0187010, and AL0187011 groups (P < 0.05).

[0390] Example 4. In vivo testing of AGT RNAi agents in mice II

[0391] siRNA sequences with good in vitro and in vivo efficacy were modified to investigate their effects on efficacy. SPF male, 8- to 9-week-old B6.Cg-Tg(hAGT)2041Sig / J mice (from Jackson Laboratory) were used. Pre-dose serum samples were obtained on day 0, and the mice were randomly divided into groups based on hAGT levels. B6.Cg-Tg(hAGT)2041Sig / J mice were subcutaneously administered a single dose of 3 mg / kg of the AGT RNAi agent. Blood samples (from the eyeballs and sent for analysis within 1 hour) were collected at 1, 2, 3, 4, and 5 weeks after administration to assess hAGT expression levels and to determine hAGT knockdown levels, using the pre-dose control as the control. No animals died or showed signs of dying during the experiment. Clinical observations revealed no significant abnormalities in any of the animals.

[0392] The experimental groups are shown in Table 18; the changes in hAGT levels are shown in Figure 4.

[0393] Table 18 Dosage grouping table

[0394] As shown in Figure 4, compared with pre-drug administration, at week 2 after drug intervention, the knockdown effect of all AGT siRNAs, except AL0187002, AL0187013, and AL0187015, reached its lowest level (over 90%), followed by a gradual and slow recovery. After week 5, hAGT levels in the blood recovered faster in the AL0187002 and AL0187013 groups. Drug intervention significantly reduced hAGT levels in the blood of mice, with the most significant reductions in the AL0187006, AL0187012, AL0187014, AL0187015, AL0187016, AL0187017, AL0187018, and AL0187019 groups. Even after 5 weeks of drug administration, hAGT inhibition by the drugs remained approximately 75%, significantly different from that in the AL0187002 group (P < 0.05).

[0395] Example 5. In vivo testing of AGT RNAi agents in mice III

[0396] siRNA sequences with good in vitro and in vivo efficacy were modified to investigate their effects on efficacy. SPF male, 8- to 9-week-old B6.Cg-Tg(hAGT)2041Sig / J mice (from Jackson Laboratory) were used. Pre-dose serum samples were obtained on day 0, and the mice were randomly divided into groups based on hAGT levels. B6.Cg-Tg(hAGT)2041Sig / J mice were subcutaneously administered a single dose of 3 mg / kg of the AGT RNAi agent. Blood samples (from the eyeballs and sent for analysis within 1 hour) were collected at 1, 2, 3, 4, and 5 weeks after administration to assess hAGT expression levels and to determine hAGT knockdown levels, using the pre-dose control as the control. No animals died or showed signs of dying during the experiment. Clinical observations revealed no significant abnormalities in any of the animals.

[0397] The experimental groups are shown in Table 19; the changes in hAGT levels are shown in Figures 5 and 6.

[0398] Table 19 Dosage grouping table

[0399] As shown in Figures 5 and 6 , compared to pre-drug administration, at week 2 after drug intervention, except for the AL0187002, AL0187021, AL0187030, and AL0187033 groups, the knockdown effect of AGT siRNAs reached its lowest level (over 90%), then gradually recovered. After week 5, hAGT levels in the blood of the AL0187030, AL0187033, and AL0187002 groups recovered rapidly. Drug intervention significantly reduced hAGT levels in the blood of mice, with the most significant reduction observed in the AL0187020, AL0187023, AL0187024, AL0187025, and AL0187028 groups. Even after 5 weeks of drug administration, hAGT inhibition by the drug still reached 80%, significantly different from that of the AL0187002 group (P < 0.05).

[0400] Example 6. In vivo testing of AGT RNAi agents in mice IV

[0401] The experiment was the same as in Example 5, with grouping shown in Tables 20 and 21. The two experiments were conducted independently. During the experiments, no animals showed signs of death or dying. Clinical observations revealed no significant abnormalities in any of the animals. Changes in hAGT levels are shown in Figures 7 and 8.

[0402] Table 20 Dosage grouping table

[0403] Table 21 Dosage grouping table

[0404] As shown in Figure 7, compared to pre-drug administration, the knockdown effect of AGT siRNAs reached its lowest level (over 80%) in the second week after drug intervention, with the exception of the AL0187041 group, and then gradually recovered. Drug intervention significantly reduced hAGT levels in the mouse blood, with the most significant reductions seen in the AL0187024, AL0187037, AL0187040, AL0187043, AL0187045, AL0187047, AL0187048, and AL0187049 groups. Even after five weeks of drug administration, hAGT inhibition still reached 80%.

[0405] As shown in Figure 8, at a 1 mg / kg dose, AGT siRNA knockdown reached its lowest level two weeks after drug intervention, then gradually recovered. All drug interventions significantly reduced hAGT levels in the mouse blood, with the most significant reductions seen in the AL0187024, AL0187040, AL0187047, and AL0187051 groups. Even after four weeks of drug administration, hAGT inhibition remained at 80%.

[0406] Example 7. Efficacy testing of AGT RNAi agents in non-human primates (NHPs)

[0407] siRNA sequences that demonstrated promising efficacy in hAGT transgenic mice were evaluated in cynomolgus monkeys. Male cynomolgus monkeys aged 10-22 years (sourced from Kunming Keling Biotechnology Co., Ltd. (KBI)) were used. Pre-dose serum samples were obtained on day 0 of dosing, and the monkeys were randomly divided into groups based on AGT levels. All experimental groups were administered 1 mg / kg of the AGT RNAi agent. The experiment was divided into the following three groups:

[0408] Group 1: Blood samples were collected from cynomolgus monkeys at 1, 2, 3, 4, and 5 weeks after dosing to measure AGT expression levels, with pre-dose control used as the control. Clinical observations during the experiment revealed no significant abnormalities in any of the animals. Experimental groupings are shown in Table 22; changes in AGT levels are shown in Figure 9.

[0409] Group 2: Blood samples were collected from cynomolgus monkeys at 1, 2, 3, 4, 5, and 6 weeks after dosing to measure AGT expression levels, with pre-dose control used as the control. Clinical observations during the experiment revealed no significant abnormalities in any of the animals. Experimental groupings are shown in Table 23; changes in AGT levels are shown in Figure 10.

[0410] Group 3: Blood samples were collected from cynomolgus monkeys at 1, 2, 3, 4, 5, 6, 7, 8, and 10 weeks after dosing to measure AGT expression levels. The pre-dose control was used as the control to determine the level of AGT knockdown. Clinical observations during the experiment revealed no significant abnormalities in any of the animals. The experimental groups are shown in Table 24; changes in AGT levels are shown in Figure 11.

[0411] Table 22 Dosage grouping table

[0412] Table 23 Dosage grouping table

[0413] Table 24 Dosage grouping table

[0414] As shown in Figures 9, 10, and 11, the knockdown effect of all AGT siRNAs reached its lowest level three weeks after drug intervention, then gradually recovered. All drug interventions significantly reduced AGT levels in the blood of cynomolgus monkeys. In the first group of experiments, the most significant effects were seen in the AL0187020, AL0187022, and AL0187024 groups. Even after five weeks of drug administration, AGT inhibition by the drugs still reached 75%, significantly different from that of the AL0187002 group (P < 0.05). In the second and third groups of experiments, the AL0187047, AL0187040, and AL0187051 groups showed significant efficacy, with AGT inhibition still reaching 70% and even exceeding 80% after seven weeks of drug administration.

Claims

1. An oligonucleotide for reducing AGT expression or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand has a sequence having at least 80% sequence identity with a sequence shown in any one of SEQ ID NOs: 2-219, 789-804, 868-871 or a fragment thereof, or a modified sequence thereof, and preferably has a sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity; and the antisense strand has a sequence having at least 80% sequence identity with a sequence shown in any one of SEQ ID NOs: 221-424, 805-821, 872-875 or a fragment thereof, or a modified sequence thereof, and preferably has a sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity; And / or, the modified sequence of the sense strand comprises a sequence selected from any one of SEQ ID NOs: 426-598, 822-838, 876-886; And / or, the modified sequence of the antisense strand comprises a sequence selected from any one of SEQ ID NOs: 600-676, 678-788, 839-867, 887-911.

2. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1, wherein The oligonucleotide or a pharmaceutically acceptable salt thereof is selected from carboxylate salt, sodium salt, triethylamine salt and other pharmaceutically acceptable salts.

3. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein: The sense strand comprises an unmodified oligonucleotide selected from any one of SEQ ID NOs: 74, 101, 107, 108, 111-113, 129, 133, 135, 137, 148, 166, 178, 184, 789, 794, 798, 799, 800, 801, 802, 803, 804, 870, or a modified oligonucleotide selected from any one of SEQ ID NOs: 471, 477, 478, 481-483, 518, 536, 548, 554, 592-598, 822, 827, 831, 832, 833, 834, 835, 836, 837, 838; the antisense strand comprises an unmodified oligonucleotide selected from any one of SEQ ID NOs: 74, 101, 107, 108, 111-113, 129, 133, 135, 137, 148, 166, 178, 184, 789, 794, 798, 799, 800, 801, 802, 803, 804, 870, or a modified oligonucleotide selected from any one of SEQ ID NOs: 471, 477, 478, 481-483, 518 NO: 259, 281, 286, 311, 317, 318, 319, 334, 338, 340, 342, 353, 371, 383, 389, 805, 808, 811, 812, 813, 814, 815, 816, 817, 818, 874, or an unmodified oligonucleotide of any one of SEQ ID The modified oligonucleotides described in any one of NO:643, 644, 645, 647, 648, 681, 678, 679, 680, 764, 610, 773, 774, 775, 776, 777, 778, 779, 780, 782, 783, 785, 839, 854, 857, 858, 859, 860, 861, 862, 863, 864.

4. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein: The oligonucleotide comprises any one selected from the following sense strand and antisense strand combinations: (1) the sense strand comprises the sequence shown in SEQ ID NO: 101, and the antisense strand comprises the sequence shown in SEQ ID NO: 311; (2) the sense strand comprises the sequence shown in SEQ ID NO: 107, and the antisense strand comprises the sequence shown in SEQ ID NO: 281; (3) the sense strand comprises the sequence shown in SEQ ID NO: 108, and the antisense strand comprises the sequence shown in SEQ ID NO: 286; (4) the sense strand comprises the sequence shown in SEQ ID NO:111, and the antisense strand comprises the sequence shown in SEQ ID NO:317; (5) the sense strand comprises the sequence shown in SEQ ID NO: 112, and the antisense strand comprises the sequence shown in SEQ ID NO: 318; (6) the sense strand comprises the sequence shown in SEQ ID NO:113, and the antisense strand comprises the sequence shown in SEQ ID NO:319; (7) the sense strand comprises the sequence shown in SEQ ID NO:129, and the antisense strand comprises the sequence shown in SEQ ID NO:334; (8) the sense strand comprises the sequence shown in SEQ ID NO: 133, and the antisense strand comprises the sequence shown in SEQ ID NO: 338; (9) the sense strand comprises the sequence shown in SEQ ID NO: 135, and the antisense strand comprises the sequence shown in SEQ ID NO: 340; (10) the sense strand comprises the sequence shown in SEQ ID NO: 137, and the antisense strand comprises the sequence shown in SEQ ID NO: 342; (11) the sense strand comprises the sequence shown in SEQ ID NO: 148, and the antisense strand comprises the sequence shown in SEQ ID NO: 353; (12) the sense strand comprises the sequence shown in SEQ ID NO: 166, and the antisense strand comprises the sequence shown in SEQ ID NO: 371; (13) the sense strand comprises the sequence shown in SEQ ID NO: 178, and the antisense strand comprises the sequence shown in SEQ ID NO: 383; (14) the sense strand comprises the sequence shown in SEQ ID NO: 184, and the antisense strand comprises the sequence shown in SEQ ID NO: 389; (15) the sense strand comprises the sequence shown in SEQ ID NO:113, and the antisense strand comprises the sequence shown in SEQ ID NO:808; (16) the sense strand comprises the sequence shown in SEQ ID NO:113, and the antisense strand comprises the sequence shown in SEQ ID NO:811; (17) the sense strand comprises the sequence shown in SEQ ID NO:113, and the antisense strand comprises the sequence shown in SEQ ID NO:818; (18) the sense strand comprises the sequence shown in SEQ ID NO:789, and the antisense strand comprises the sequence shown in SEQ ID NO:805; (19) the sense strand comprises the sequence shown in SEQ ID NO:794, and the antisense strand comprises the sequence shown in SEQ ID NO:805; (20) the sense strand comprises the sequence shown in SEQ ID NO:798, and the antisense strand comprises the sequence shown in SEQ ID NO:805; (21) the sense strand comprises the sequence shown in SEQ ID NO:799, and the antisense strand comprises the sequence shown in SEQ ID NO:812; (22) the sense strand comprises the sequence shown in SEQ ID NO: 800, and the antisense strand comprises the sequence shown in SEQ ID NO: 813; (23) the sense strand comprises the sequence shown in SEQ ID NO:801, and the antisense strand comprises the sequence shown in SEQ ID NO:814; (24) the sense strand comprises the sequence shown in SEQ ID NO:802, and the antisense strand comprises the sequence shown in SEQ ID NO:815; (25) the sense strand comprises the sequence shown in SEQ ID NO:803, and the antisense strand comprises the sequence shown in SEQ ID NO:816; (26) the sense strand comprises the sequence shown in SEQ ID NO:804, and the antisense strand comprises the sequence shown in SEQ ID NO:817; (27) the sense strand comprises the sequence shown in SEQ ID NO:868, and the antisense strand comprises the sequence shown in SEQ ID NO:872; (28) the sense strand comprises the sequence shown in SEQ ID NO: 869, and the antisense strand comprises the sequence shown in SEQ ID NO: 873; (29) the sense strand comprises the sequence shown in SEQ ID NO: 870, and the antisense strand comprises the sequence shown in SEQ ID NO: 874; (30) the sense strand comprises the sequence shown in SEQ ID NO: 871, and the antisense strand comprises the sequence shown in SEQ ID NO: 875; and (31) the sense strand comprises the sequence shown in SEQ ID NO:74, and the antisense strand comprises the sequence shown in SEQ ID NO:259; wherein each strand is independently 19 to 25 nucleotides in length.

5. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein: The oligonucleotide comprises at least one modified nucleotide; And / or, at least one of the modified nucleotides is selected from the group consisting of deoxynucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, 2'-5'-linked ribonucleotides (3'-RNA), unlocked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-hydroxy modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides including non-natural bases, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides including thiophosphate groups, nucleotides including methylphosphonate groups, nucleotides including 5'-phosphates, nucleotides including 5'-phosphate mimetics, vinyl-phosphonate nucleotides, thermolabile nucleotides, glycol modified nucleotides, nucleotides including 2' phosphates, and 2-O-(N-methylacetamide) modified nucleotides; and combinations thereof; and / or, at least one of the modified nucleotides is selected from the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl and ethylene glycol; and combinations thereof; and / or, at least one of the modified nucleotides is selected from the group consisting of deoxynucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, glycol modified nucleotides (GNA), nucleotides comprising 2' phosphates, and nucleotides comprising thiophosphate groups; and combinations thereof; and / or, the oligonucleotide comprises at least one 2'-modified nucleotide; and / or, the 2'-modified nucleotides are selected from one or more of 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-acylamino modified nucleotides, 2'-deoxy modified nucleotides, 2'-O-allyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-hydroxy modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, 2'-deoxy nucleotides, nucleotides including 2' phosphates, and 2'-O-(N-methylacetamide) modified nucleotides; and / or, the 2'-modification is a modification selected from the group consisting of 2'-methoxy, 2'-acetylamino, 2'-aminoethyl, 2'-fluoro, 2'-O-methoxyethyl and 2'-fluoro-β-d-arabinonucleotide; And / or, the oligonucleotide may comprise a diol nucleic acid (GNA) modification; preferably, the diol nucleic acid (GNA) modification is selected from adenosine-diol nucleic acid, cytidine-diol nucleic acid, thymidine-diol nucleic acid and guanosine-diol nucleic acid; preferably, the diol nucleic acid (GNA) modification is selected from thymidine-diol nucleic acid S-isomer (Tgn) shown in formula (I), cytidine-diol nucleic acid S-isomer (Cgn) shown in formula (II), adenosine-diol nucleic acid S-isomer (Agn) shown in formula (III) and guanosine-diol nucleic acid S-isomer (Ggn) shown in formula (IV); And / or, the oligonucleotide may contain a 2'-5'-phosphodiester bond, preferably, the oligonucleotide contains a uridine-2'-phosphate (U-2'5') shown in formula (V), a guanosine-2'-phosphate (G-2'5') shown in formula (VI); a cytidine-2'-phosphate (C-2'5') shown in formula (VII); an adenosine-2'-phosphate (A-2'5') shown in formula (VIII) and a thymidine-2'-phosphate (T-2'5') shown in formula (IX); and / or, the oligonucleotide further comprises a 5'-phosphate analog modified nucleotide; And / or, the 5'-phosphate analogue modified nucleotide is APU shown in formula (X); preferably, the 5'-phosphate analogue modified nucleotide is VPUm shown in formula (XI):

6. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein: The oligonucleotide comprises at least one modified internucleotide linkage; And / or, the at least one modified internucleotide bond is a phosphorothioate bond.

7. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein: The oligonucleotide comprises any one selected from the following sense strand and antisense strand combinations: (1) the sense strand comprises the sequence shown in SEQ ID NO:477, and the antisense strand comprises the sequence shown in SEQ ID NO:643; (2) the sense strand comprises the sequence shown in SEQ ID NO:471, and the antisense strand comprises the sequence shown in SEQ ID NO:644; (3) the sense strand comprises the sequence shown in SEQ ID NO:478, and the antisense strand comprises the sequence shown in SEQ ID NO:645; (4) the sense strand comprises the sequence shown in SEQ ID NO:482, and the antisense strand comprises the sequence shown in SEQ ID NO:647; (5) the sense strand comprises the sequence shown in SEQ ID NO:483, and the antisense strand comprises the sequence shown in SEQ ID NO:648; (6) the sense strand comprises the sequence shown in SEQ ID NO:481, and the antisense strand comprises the sequence shown in SEQ ID NO:681; (7) the sense strand comprises the sequence shown in SEQ ID NO:471, and the antisense strand comprises the sequence shown in SEQ ID NO:678; (8) the sense strand comprises the sequence shown in SEQ ID NO:478, and the antisense strand comprises the sequence shown in SEQ ID NO:679; (9) the sense strand comprises the sequence shown in SEQ ID NO:483, and the antisense strand comprises the sequence shown in SEQ ID NO:680; (10) the sense strand comprises the sequence shown in SEQ ID NO:594, and the antisense strand comprises the sequence shown in SEQ ID NO:764; (11) the sense strand comprises the sequence shown in SEQ ID NO:593, and the antisense strand comprises the sequence shown in SEQ ID NO:610; (12) the sense strand comprises the sequence shown in SEQ ID NO:592, and the antisense strand comprises the sequence shown in SEQ ID NO:773; (13) the sense strand comprises the sequence shown in SEQ ID NO:592, and the antisense strand comprises the sequence shown in SEQ ID NO:774; (14) the sense strand comprises the sequence shown in SEQ ID NO:592, and the antisense strand comprises the sequence shown in SEQ ID NO:775; (15) the sense strand comprises the sequence shown in SEQ ID NO:595, and the antisense strand comprises the sequence shown in SEQ ID NO:776; (16) the sense strand comprises the sequence shown in SEQ ID NO:596, and the antisense strand comprises the sequence shown in SEQ ID NO:777; (17) the sense strand comprises the sequence shown in SEQ ID NO:597, and the antisense strand comprises the sequence shown in SEQ ID NO:778; (18) the sense strand comprises the sequence shown in SEQ ID NO:598, and the antisense strand comprises the sequence shown in SEQ ID NO:779; (19) the sense strand comprises the sequence shown in SEQ ID NO:518, and the antisense strand comprises the sequence shown in SEQ ID NO:780; (20) the sense strand comprises the sequence shown in SEQ ID NO:536, and the antisense strand comprises the sequence shown in SEQ ID NO:782; (21) the sense strand comprises the sequence shown in SEQ ID NO:548, and the antisense strand comprises the sequence shown in SEQ ID NO:783; (22) the sense strand comprises the sequence shown in SEQ ID NO:554, and the antisense strand comprises the sequence shown in SEQ ID NO:785; (23) the sense strand comprises the sequence shown in SEQ ID NO:592, and the antisense strand comprises the sequence shown in SEQ ID NO:854; (24) the sense strand comprises the sequence shown in SEQ ID NO:592, and the antisense strand comprises the sequence shown in SEQ ID NO:857; (25) the sense strand comprises the sequence shown in SEQ ID NO:592, and the antisense strand comprises the sequence shown in SEQ ID NO:864; (26) the sense strand comprises the sequence shown in SEQ ID NO:822, and the antisense strand comprises the sequence shown in SEQ ID NO:839; (27) the sense strand comprises the sequence shown in SEQ ID NO:827, and the antisense strand comprises the sequence shown in SEQ ID NO:839; (28) the sense strand comprises the sequence shown in SEQ ID NO:831, and the antisense strand comprises the sequence shown in SEQ ID NO:839; (29) the sense strand comprises the sequence shown in SEQ ID NO:598, and the antisense strand comprises the sequence shown in SEQ ID NO:840; (30) the sense strand comprises the sequence shown in SEQ ID NO:832, and the antisense strand comprises the sequence shown in SEQ ID NO:841; (31) the sense strand comprises the sequence shown in SEQ ID NO: 832, and the antisense strand comprises the sequence shown in SEQ ID NO: 842; (32) the sense strand comprises the sequence shown in SEQ ID NO:832, and the antisense strand comprises the sequence shown in SEQ ID NO:848; (33) the sense strand comprises the sequence shown in SEQ ID NO:832, and the antisense strand comprises the sequence shown in SEQ ID NO:849; (34) the sense strand comprises the sequence shown in SEQ ID NO:832, and the antisense strand comprises the sequence shown in SEQ ID NO:850; (35) the sense strand comprises the sequence shown in SEQ ID NO:833, and the antisense strand comprises the sequence shown in SEQ ID NO:858; (36) the sense strand comprises the sequence shown in SEQ ID NO:834, and the antisense strand comprises the sequence shown in SEQ ID NO:859; (37) the sense strand comprises the sequence shown in SEQ ID NO:835, and the antisense strand comprises the sequence shown in SEQ ID NO:860; (38) the sense strand comprises the sequence shown in SEQ ID NO:836, and the antisense strand comprises the sequence shown in SEQ ID NO:861; (39) the sense strand comprises the sequence shown in SEQ ID NO:837, and the antisense strand comprises the sequence shown in SEQ ID NO:862; (40) the sense strand comprises the sequence shown in SEQ ID NO:838, and the antisense strand comprises the sequence shown in SEQ ID NO:863; (41) the sense strand comprises the sequence shown in SEQ ID NO: 882, and the antisense strand comprises the sequence shown in SEQ ID NO: 894; (42) the sense strand comprises the sequence shown in SEQ ID NO:882, and the antisense strand comprises the sequence shown in SEQ ID NO:902; (43) the sense strand comprises the sequence shown in SEQ ID NO: 884, and the antisense strand comprises the sequence shown in SEQ ID NO: 896; (44) the sense strand comprises the sequence shown in SEQ ID NO:884, and the antisense strand comprises the sequence shown in SEQ ID NO:903; (45) the sense strand comprises the sequence shown in SEQ ID NO:592, and the antisense strand comprises the sequence shown in SEQ ID NO:905; (46) the sense strand comprises the sequence shown in SEQ ID NO:592, and the antisense strand comprises the sequence shown in SEQ ID NO:907; (47) the sense strand comprises the sequence shown in SEQ ID NO: 882, and the antisense strand comprises the sequence shown in SEQ ID NO: 910; and (48) the sense strand comprises the sequence shown in SEQ ID NO:882, and the antisense strand comprises the sequence shown in SEQ ID NO:911; wherein each strand is independently 19 to 25 nucleotides in length.

8. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, wherein: At least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands; and / or, the targeting ligand comprises a carbohydrate, an amino sugar, cholesterol, a polypeptide or a lipid; and / or, the targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety; and / or, the GalNac moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety or a tetravalent GalNAc moiety; And / or, the targeting ligand is A1 represented by formula (XII): And / or, the targeting ligand is L96 represented by formula (XIII):

9. A composition comprising the oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, and optionally a pharmaceutically acceptable carrier; And / or, the dosage form of the composition is oral preparation or injection; the injection is selected from intravenous injection, subcutaneous injection or intramuscular injection; And / or, the combination further comprises other drugs for treating and / or preventing AGT-related diseases.

10. Use of the oligonucleotide according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof or the composition according to claim 9 in the preparation of a medicament for treating and / or preventing AGT-related diseases; Alternatively, the AGT-related disease is selected from hypertension, e.g., borderline hypertension (also known as prehypertension), essential hypertension (also known as primary hypertension or idiopathic hypertension), secondary hypertension (also known as non-essential hypertension), hypertensive crisis (also known as malignant hypertension), hypertensive urgency, isolated systolic or diastolic hypertension, pregnancy-related hypertension (e.g., preeclampsia, eclampsia and postpartum preeclampsia), diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension (also known as renal 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 sclerosis, vascular disease (including peripheral vascular disease), diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, Cushing's syndrome and other glucocorticoid excess states (including chronic steroid therapy), pheochromocytoma, nephrinoma, secondary aldosteronism and other mineralocorticoid excess states, sleep apnea, thyroid / parathyroid disease, heart failure (e.g., left ventricular systolic dysfunction), myocardial infarction, angina pectoris, stroke, diabetes (e.g., diabetic nephropathy), kidney disease (e.g., chronic kidney disease or diabetic nephropathy, optionally in the setting of pregnancy), renal failure (e.g., chronic renal failure), cognitive impairment (such as Alzheimer's disease), and systemic sclerosis (e.g., scleroderma renal crisis); Alternatively, the AGT-related disorder is selected from intrauterine growth retardation (IUGR) and fetal growth restriction.

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