Double-stranded oligonucleotide containing terminal nucleotide modification and use thereof for inhibiting AGT gene expression

Abstract

The present invention belongs to the technical field of nucleic acid drugs. Provided are a double-stranded oligonucleotide containing a terminal nucleotide modification and the use thereof for inhibiting angiotensinogen (AGT) gene expression. The provided double-stranded oligonucleotide can induce the silencing complex (RISC)-mediated cleavage of an RNA transcript of the AGT gene and inhibit the expression of the AGT gene, thereby helping to treat or prevent diseases or conditions mediated by the AGT gene.

Description

Double-stranded oligonucleotides containing terminal nucleotide modifications and their application in suppressing AGT gene expression

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. 202411487769.0, filed on October 24, 2024, entitled "Double-stranded oligonucleotides containing terminal nucleotide modifications and their application in inhibiting AGT gene expression", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of nucleic acid drug technology, specifically to a double-stranded oligonucleotide containing terminal nucleotide modification and its application in inhibiting AGT gene expression. Background Technology

[0004] Angiotensinogen (AGT), also known as SERPINA8 or ANHU, is a member of the serpin family and a component of the renin-angiotensin-aldosterone system (RAAS), which plays a crucial role in blood pressure regulation. Renin is secreted into the circulation by glomerular cells in the kidneys. It is primarily produced in the liver and released into the circulation, where renin converts it into angiotensin I. Angiotensin I is then converted to angiotensin II by angiotensin-converting enzyme (ACE). Angiotensin II is a peptide hormone that causes vasoconstriction, which in turn increases blood pressure. Angiotensin II also stimulates the secretion of aldosterone, a hormone from the adrenal cortex. Aldosterone causes the kidneys to increase the reabsorption of sodium and water, leading to an increase in body fluid volume, which in turn increases blood pressure. Overstimulation or activity of the RAAS pathway can lead to high blood pressure. Chronic high blood pressure is called hypertension. In hypertensive patients, high blood pressure requires the heart to work harder to circulate blood through the blood vessels.

[0005] The World Health Organization (WHO) has identified hypertension as a leading cause of cardiovascular disease. Hypertension is a major risk factor for a wide range of diseases, conditions, and symptoms, including shortened life expectancy, chronic kidney disease, stroke, myocardial infarction, heart failure, vascular aneurysms (e.g., aortic aneurysm), peripheral artery disease, cardiac injury (e.g., cardiomegaly or hypertrophy), and other cardiovascular-related diseases, conditions, and / or symptoms.

[0006] Although a large number of antihypertensive drugs are available for treating hypertension, more than two-thirds of subjects cannot achieve control with a single antihypertensive drug and require two or more antihypertensive drugs from different drug classes. This further reduces the number of subjects with controlled blood pressure due to increased adherence and side effects with increased medication use. Therefore, alternative and combination therapies for subjects with angiotensinogen-related diseases remain to be investigated.

[0007] In view of this, this disclosure is hereby made. Summary of the Invention

[0008] This disclosure provides a double-stranded oligonucleotide that induces cleavage of the RNA transcript of the angiotensinogen (AGT) gene mediated by the silencing complex (RISC), thereby inhibiting AGT gene expression and can be used to treat or prevent diseases or symptoms mediated by the angiotensinogen (AGT) gene. This disclosure also provides conjugates comprising the double-stranded oligonucleotide, pharmaceutical compositions, kits, and methods and uses thereof for inhibiting or reducing AGT gene expression or treating AGT gene-mediated diseases or symptoms.

[0009] This disclosure includes the following technical solutions:

[0010] In a first aspect, this disclosure provides a double-stranded oligonucleotide for inhibiting AGT gene expression, the double-stranded oligonucleotide comprising a sense strand and an antisense strand, wherein the antisense strand comprises or is selected from a sequence of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides as shown in Formula (A), or comprises a nucleotide sequence having a difference of 1, 2, or 3 nucleotides between said consecutive nucleotides; the sense strand comprises at least 17 nucleotides, and the sense strand is complementary or substantially complementary to the antisense strand to form a double-stranded region, wherein substantially complementary means that the mismatch between the sense strand and the antisense strand in the double-stranded region does not exceed 3 nucleotides;

[0011] Formula (A)5'-X1GUUUCUUCAUCCAGUUGA(X2) m (X3) n -3';

[0012] Where X1 is selected from A or U;

[0013] (X2) m (X3) n The bases X2 and X3 are each independently selected from A, U, G, C or T, and m and n are independently selected from 0 or 1, and m+n≥1.

[0014] Furthermore, at least one of the nucleotides X2 and X3 is a [2'-R1-2'-R2] disubstituted modified nucleotide, wherein the disubstituted modified nucleotide refers to a nucleotide in which both the hydroxyl and hydrogen at the 2' position of the ribose are substituted, wherein R1 and R2 are each independently selected from halogen (preferably F), optionally substituted C1-C6 alkyl (preferably methyl or ethyl) or optionally substituted C1-C6 alkoxy (preferably methoxy).

[0015] In some embodiments of this disclosure, each nucleotide in the double-stranded oligonucleotide is independently selected from modified or unmodified nucleotides.

[0016] According to embodiments of this disclosure, all nucleotides in the double-stranded oligonucleotide are substantially selected from modified nucleotides. Here, "all nucleotides in the double-stranded oligonucleotide are substantially selected from modified nucleotides" means that most, but not all, of the nucleotides in the double-stranded oligonucleotide are modified nucleotides, and may contain no more than 5, 4, 3, 2, or 1 unmodified nucleotide.

[0017] In an optional embodiment of this disclosure, each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides:

[0018] 2'-Fluorinated nucleotides, 2'-deoxynucleotides, 2'-O-methylnucleotides, 2'-O-(CH2) x Nucleotides modified with -O-R3, 2'-O-Si(R4)3, 2'-amino, debased, nucleotide-like, or [2'-F-2'-methyl] disubstituted.

[0019] The nucleotides described in this disclosure are selected from one or more of peptide nucleic acid (PNA), morpholino nucleic acid (MNA), bridged nucleic acid (BNA), locked nucleic acid (LNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), or unlocked nucleic acid (UNA).

[0020] According to embodiments of this disclosure, the sense chain and / or the antisense chain independently comprise one or more thiophosphate bonds.

[0021] In a second aspect of this disclosure, a conjugate is provided comprising the double-stranded oligonucleotide described in the first aspect of this disclosure, and one or more ligands conjugated to the double-stranded oligonucleotide capable of binding to a cell receptor.

[0022] In some alternative embodiments of this disclosure, the ligand comprises at least one N-acetyl galactosamine (GalNAc).

[0023] In some alternative embodiments of this disclosure, the ligand is selected from structures shown in formula (I), or stereoisomers thereof, tautomers thereof, or pharmaceutically acceptable salts thereof:

[0024] In formula (Ⅰ), * represents the conjugation site between the ligand and the double-stranded oligonucleotide, and j is selected from 1, 2, 3 or 4;

[0025] In some alternative embodiments of this disclosure, each Z is independently selected from hydroxyl or thiol groups. In some specific embodiments of this disclosure, each Z is a hydroxyl group.

[0026] In some embodiments of this disclosure, each p is independently 1 or 2.

[0027] In some specific embodiments of this disclosure, each p is 1.

[0028] In some embodiments of this disclosure, each q is independently 1 or 2.

[0029] In some specific implementations of this disclosure, each q is 1.

[0030] In some specific embodiments of this disclosure, each p is 1 and each q is 1.

[0031] Each L is independently selected from C1-C 30 alkylene or Among them, each R L2a Each is independently selected from C1-C 10 Alkylene, each R L2b Each is independently selected from O, S, NH or -NH-C(O)-, and k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0032] In a third aspect, this disclosure provides a pharmaceutical composition comprising any of the following and pharmaceutically acceptable excipients:

[0033] (I) The double-stranded oligonucleotides described in the first aspect of this disclosure; and / or

[0034] (II) The conjugate described in the second aspect of this disclosure.

[0035] In a fourth aspect of this disclosure, the following are provided for use in the preparation of medicaments for alleviating, preventing, and / or treating AGT gene-mediated diseases or conditions:

[0036] (I) The double-stranded oligonucleotides described in the first aspect of this disclosure; and / or

[0037] (II) The conjugates described in the second aspect of this disclosure; and / or

[0038] (III) The pharmaceutical composition described in the third aspect of this disclosure.

[0039] According to embodiments of this disclosure, the diseases or conditions include hypertension, borderline hypertension, primary hypertension, secondary hypertension, hypertensive crisis, hypertensive urgency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, intractable hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, and ventricular fibrosis.

[0040] In a fifth aspect, this disclosure provides a method for inhibiting intracellular AGT gene expression, the method comprising contacting the cell with any of the following:

[0041] (I) The double-stranded oligonucleotides described in the first aspect of this disclosure; and / or

[0042] (II) The conjugates described in the second aspect of this disclosure; and / or

[0043] (III) The pharmaceutical composition described in the third aspect of this disclosure.

[0044] The methods disclosed herein can be used to inhibit angiotensinogen expression. For example, they can inhibit angiotensinogen expression in animals; or for non-disease treatment purposes, such as inhibiting angiotensinogen expression in vitro for further research.

[0045] In a sixth aspect, this disclosure provides a method for alleviating, treating, and / or preventing AGT gene-mediated diseases or conditions, said method comprising administering any of the following to a subject:

[0046] (I) The double-stranded oligonucleotides described in the first aspect of this disclosure; and / or

[0047] (II) The conjugates described in the second aspect of this disclosure; and / or

[0048] (III) The pharmaceutical composition described in the third aspect of this disclosure.

[0049] In a seventh aspect of this disclosure, a kit is provided, the kit comprising any of the following:

[0050] (I) The double-stranded oligonucleotides described in the first aspect of this disclosure; and / or

[0051] (II) The conjugates described in the second aspect of this disclosure; and / or

[0052] (III) The pharmaceutical composition described in the third aspect of this disclosure.

[0053] The double-stranded oligonucleotides, conjugates, and pharmaceutical compositions disclosed herein can induce cleavage of the RNA transcript of the angiotensinogen (AGT) gene mediated by the silencing complex (RISC), inhibit the expression of the AGT gene, and help treat or prevent diseases or conditions mediated by the angiotensinogen (AGT) gene. Attached Figure Description

[0054] Figure 1 shows the changes in AGT protein levels in cynomolgus monkey serum after administration of the siRNA conjugate in Example 1. Detailed Implementation

[0055] The technical solutions of this disclosure will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. Based on the embodiments of this disclosure, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this disclosure.

[0056] Terminology Explanation

[0057] In this disclosure, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this disclosure but do not exclude other contents.

[0058] In this disclosure, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0059] In this disclosure, "2'-position monosubstituted modification" refers to the substitution of the 2'-hydroxyl group of the ribose in a nucleotide.

[0060] In this disclosure, "2'-position double substitution modification" refers to the simultaneous substitution of both the 2'-hydroxyl group and the 2'-hydrogen group of the ribose in a nucleotide. For example, a nucleotide with [2'-F-2'-methyl] substitution modification has the following structural formula:

[0061] In this disclosure, the term "small interfering RNA (siRNA)" is a double-stranded RNA of 17 to 25 nucleotides in length, comprising a sense strand and an antisense strand. siRNA mediates targeted cleavage of RNA transcripts via the RISC pathway by forming an RNA-induced silencing complex (RISC). Specifically, siRNA directs the specific degradation of mRNA sequences through known RNA interference (RNAi) processes, inhibiting the translation of mRNA into amino acids and its conversion into proteins. For example, siRNA can regulate (e.g., inhibit) the expression of AGT in cells.

[0062] In this disclosure, the term "antisense strand (or guide strand)" includes a region substantially complementary to a target sequence, such as the mRNA of AGT. "Sense strand (or lagging strand)" refers to an iRNA strand containing an iRNA strand substantially complementary to the antisense strand. The term "substantially complementary" means fully complementary or at least partially complementary, for example, the antisense strand being fully complementary or at least partially complementary to the target sequence. In the case of partial complementarity, mismatches can be present within the molecule or in terminal regions, wherein the most tolerant mismatches are present in terminal regions, for example, within 5, 4, 3, or 2 nucleotides at the 5'- and / or 3'-terminus of the iRNA.

[0063] It should be noted that "at least partially substantially complementary" to mRNA means that the antisense strand has a polynucleotide substantially complementary to a continuous portion of the mRNA of interest (e.g., the mRNA encoding angiotensinogen). Alternatively, if a polynucleotide is substantially non-discontinuously complementary to a portion of the mRNA encoding angiotensinogen, then the antisense strand is complementary to at least a portion of the angiotensinogen mRNA.

[0064] In this disclosure, the term "complementary" refers to the ability of an oligonucleotide of the first sequence to hybridize with an oligonucleotide of the second sequence under certain conditions and form a double-stranded structure.

[0065] In this disclosure, the terms "nucleotide difference," "nucleotide base difference," and "nucleotide sequence difference" are used interchangeably. A nucleotide difference refers to a change in the type of bases of nucleotides at the same or corresponding positions compared to the original nucleotide sequence. For example, if a nucleotide base in the original nucleotide sequence is A, and the nucleotide base at the same or corresponding position is changed to U, C, G, or dT, dC, dG, etc., then a nucleotide sequence difference is considered to exist at that position. It should be noted that if, compared to the original nucleotide sequence, the nucleotides at the same or corresponding positions differ only in the presence or type of modification, then a nucleotide sequence difference is not considered to exist at that position.

[0066] In this disclosure, the term "protrusion" refers to at least one unpaired nucleotide protruding from the double-stranded oligonucleotide double helix structure, which is also a nucleotide sequence in the siRNA structure other than the double-stranded region. For example, a nucleotide protrusion exists when the 3' end of one strand of the sense strand and / or antisense strand extends beyond the 5' end of the other strand, or when the 5' end of one strand of the sense strand and / or antisense strand extends beyond the 3' end of the other strand. The protrusion may contain at least one nucleotide, at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more nucleotides. The nucleotide protrusion may contain or be composed of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The protrusion may be located on the sense strand, antisense strand, or any combination thereof. Furthermore, the nucleotide of the protrusion may appear at the 5' end, 3' end, or both ends of the antisense or sense strand.

[0067] In this disclosure, the term "inhibition of AGT gene expression" includes inhibition of the AGT gene at any level, such as at least partial inhibition of AGT gene expression, such as inhibition of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%. AGT gene expression can be evaluated based on the level of any variable associated with AGT gene expression, such as the mRNA or protein level of AGT. Inhibition can be evaluated by a reduction in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level utilized in the art, such as baseline levels before administration, or levels measured in similar subjects, cells, or samples that have never been treated or have been treated with a control (e.g., a buffer-only control or an active agent-free control).

[0068] In this disclosure, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0069] In this disclosure, the term "pharmaceutically acceptable acid addition salt" refers to a salt formed with an inorganic or organic acid that retains the bioavailability of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromic acid sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetate, trifluoroacetate, propionate, hexanoate, octanoate, undecenoate, glycolate, gluconate, lactate, sebate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, and 4-aminosalicylic acid. These salts can be prepared by methods known in the art.

[0070] In this disclosure, the term "pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic or organic base that retains the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, thiocyanate, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts, with sodium salts being the most preferred. Salts derived from organic bases include, but are not limited to, the following: primary amines, secondary amines, and tertiary amines; substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. These salts can be prepared by methods known in the art.

[0071] In this disclosure, the term "alkyl" refers to a saturated aliphatic hydrocarbon group, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, and their various branched isomers. In some embodiments, alkyl groups containing 1 to 6 carbon atoms are selected. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. By way of example only, "C1-4 alkyl" means having one to four carbon atoms in an alkyl chain, i.e., the C1-4 alkyl is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl. The alkyl group can be substituted or unsubstituted.

[0072] In this disclosure, the term "alkoxy" refers to -O-alkyl, wherein the definition of alkyl is as shown above. Non-limiting examples of said alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexyloxy. By way of example only, "C..." 1-10 "alkylene" indicates that the alkyl chain has one to ten carbon atoms, i.e., the C atoms. 1-10 The alkylene group is selected from methylene (-CH2-), ethylene (=CH2CH3), 1,2-ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), isopropylene (-CH2CH(CH3)-), etc.

[0073] In this disclosure, the term "hydroxyl group" refers to the -OH group.

[0074] In this disclosure, the term "halogen" or "halogenated" refers to any radioactive-stable atom in Group VII of the periodic table, such as fluorine, chlorine, bromine, or iodine, wherein fluorine and chlorine are preferred, and fluorine is more preferred.

[0075] In this disclosure, the term "link," when referring to a connection between two molecules, means that the two molecules are linked by a covalent bond or by a non-covalent bond (e.g., a hydrogen bond or an ionic bond), including direct and indirect links. The term "direct link" refers to the connection between a first compound or group and a second compound or group without any intercalating atoms or groups. The term "indirect link" refers to the connection between a first compound or group and a second compound or group via an intermediate group, compound, or molecule (e.g., a linking group).

[0076] In this disclosure, the term "optionally substituted" is used to define a variable that may be unsubstituted or substituted.

[0077] In this disclosure, the term "unsubstituted" means that the specified group does not contain substituents.

[0078] In this disclosure, the terms “substituted,” “replaced,” and “substituted” are used interchangeably to indicate that any one or more hydrogen atoms in the given structure are specifically substituented (e.g., C). 1-3 Alkyl, C 1-3 The substituted group may be replaced by an alkoxy or halogen group, provided that the normal valence of the specified atom does not exceed the valence of the substituted atom and the substitution produces a stable compound. Unless otherwise indicated, a substituted group may have one substituent at each substituted position of the group. When more than one position in the given structural formula can be substituted by one or more substituents selected from a particular group, then the substituents may be substituted at each substituted position in the same or different manner.

[0079] In this disclosure, the structural formulas of "compound", "ligand" and "support" contain bonds. This indicates that the configuration is not specified. If chiral isomers exist in the chemical structure, the bond... It can be Or simultaneously include Two configurations. Although all the above structural formulas are shown in some isomer forms for simplicity, this disclosure can include all isomers, such as tautomers, rotational isomers, geometric isomers, diastereomers, racemates, and enantiomers.

[0080] In this disclosure, "conjugation" refers to the connection between two or more chemical parts through covalent linkage; "conjugated compound" refers to a compound formed by the covalent linkage between the chemical parts; and "conjugated molecule" is understood as a specific compound that can be reactively conjugated to an oligonucleotide to ultimately form the oligonucleotide conjugated compound of this disclosure.

[0081] In this disclosure, "pharmaceutical composition" can refer to a drug for the treatment of a disease or for use in in vitro cell culture experiments. When used for the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with excipients constituting one or more adjunct components. Typically, the composition is prepared by uniformly and adequately combining active siRNA with liquid excipients, finely pulverized solid excipients, or both.

[0082] In this disclosure, the term "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated. Preferably, "pharmaceutical acceptable" as used in this disclosure means approved by a federal regulatory agency or national government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, particularly in humans.

[0083] In this disclosure, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a specific target dosage form. The use of any conventional excipients that are incompatible with the siRNA of this disclosure, such as those that produce any adverse biological effects or interactions with any other component of the pharmaceutically acceptable composition in a harmful manner, is also within the scope of this disclosure.

[0084] In addition to any conventional excipients, the use of any siRNA incompatible with the present disclosure, such as any adverse biological effects produced or interactions with any other component of a pharmaceutically acceptable composition in a harmful manner, is also within the scope of this disclosure.

[0085] In this disclosure, the terms “prevention” and “avoidance” are used interchangeably to refer to methods for obtaining beneficial or desired results, including but not limited to preventive benefits. To obtain a “preventive benefit,” the conjugate, RNAi reagent, or composition may be given to a subject at risk of developing a specific disease, or to a subject who reports one or more physiological symptoms of a disease, even if a diagnosis of the disease may not have been made.

[0086] In this disclosure, the term "treatment" means the use of a drug to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" covers diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in individuals susceptible to the disease but not yet diagnosed with it; (b) suppression of a disease, such as inhibiting disease progression; or (c) alleviating a disease, such as reducing symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or siRNA to an individual to treat, cure, alleviate, improve, reduce, or suppress a disease in that individual, including but not limited to administration of a drug containing the siRNA or siRNA conjugate described herein to an individual in need.

[0087] In this disclosure, the term "subject" refers to any animal being examined, studied, or treated, and is not intended to limit this disclosure to any particular type of subject. In some embodiments of this disclosure, humans are preferred subjects, while in other embodiments, non-human animals are preferred subjects, including but not limited to mice, monkeys, ferrets, cattle, sheep, goats, pigs, chickens, turkeys, dogs, cats, horses, and reptiles.

[0088] The present disclosure is further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present disclosure in any way.

[0089] Double-stranded oligonucleotides

[0090] In a first aspect, this disclosure provides a double-stranded oligonucleotide for inhibiting AGT gene expression, the double-stranded oligonucleotide comprising a sense strand and an antisense strand, wherein the antisense strand comprises or is selected from a sequence of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides as shown in Formula (A), or comprises a nucleotide sequence having a difference of 1, 2, or 3 nucleotides between said consecutive nucleotides; the sense strand comprises at least 17 nucleotides, and the sense strand is complementary or substantially complementary to the antisense strand to form a double-stranded region, wherein substantially complementary means that the mismatch between the sense strand and the antisense strand in the double-stranded region does not exceed 3 nucleotides;

[0091] Formula (A)5'-X1GUUUCUUCAUCCAGUUGA(X2) m (X3) n -3';

[0092] Where X1 is selected from A or U;

[0093] (X2) m (X3) n The bases X2 and X3 are each independently selected from A, U, G, C or T, and m and n are independently selected from 0 or 1, and m+n≥1.

[0094] Furthermore, at least one of the nucleotides X2 and X3 is a [2'-R1-2'-R2] disubstituted modified nucleotide, wherein the disubstituted modified nucleotide refers to a nucleotide in which both the hydroxyl and hydrogen at the 2' position of the ribose are substituted, wherein R1 and R2 are each independently selected from halogen (preferably F), optionally substituted C1-C6 alkyl (preferably methyl or ethyl) or optionally substituted C1-C6 alkoxy (preferably methoxy).

[0095] In some embodiments of this disclosure, X1 is selected from A. In other embodiments of this disclosure, X1 is selected from U.

[0096] In some alternative embodiments of this disclosure, (X2) m (X3) n The value is selected from GG, UU, TT, CC, AA, G, U, T, C, or A. In some other alternative embodiments of this disclosure, (X2) m (X3) n Selected from UU or U. In some specific embodiments of this disclosure, (X2) m (X3) n Selected from UU. In some other embodiments of this disclosure, (X2) m (X3) n Selected from U.

[0097] According to embodiments of this disclosure, the antisense strand comprises or is selected from any one of the sequences shown in A1)-A3), or a nucleotide sequence that differs from any of the above sequences by one or two nucleotides:

[0098] A1)5'-AGUUUCUUCCAUCCAGUUGAUU-3';

[0099] A2)5'-AGUUUCUUCAUCCAGUUGAU-3';

[0100] A3)5'-UGUUUCUUCCAUCCAGUUGAUU-3'.

[0101] According to embodiments of this disclosure, the positive chain comprises or is selected from any of the sequences shown in B1)-B2) a nucleotide sequence of at least 15, at least 16, at least 17, at least 18 or at least 19 consecutive nucleotides, or comprises a nucleotide sequence having a difference of 1, 2 or 3 nucleotides between the consecutive nucleotides.

[0102] B1)5'-UCAACUGGAUGAAGAAACU-3';

[0103] B2)5'-UCAACUGGAUGAAGAAACA-3'.

[0104] In specific implementations of this disclosure, the antisense chain includes or is selected from any of the sequences shown in A1)-A3), and the justice chain includes or is selected from any of the sequences shown in B1)-B2).

[0105] In some specific embodiments of this disclosure, the double-stranded oligonucleotide is selected from one or more of the following groups:

[0106] 1) The antisense strand has the nucleotide sequence shown in A1 (SEQ ID NO.3), and the sense strand has the nucleotide sequence shown in B1 (SEQ ID NO.1);

[0107] 2) The antisense strand has the nucleotide sequence shown in A2 (SEQ ID NO.4), and the sense strand has the nucleotide sequence shown in B1 (SEQ ID NO.1);

[0108] 3) The antisense strand has the nucleotide sequence shown in A3 (SEQ ID NO.5), and the sense strand has the nucleotide sequence shown in B2 (SEQ ID NO.2).

[0109] Table 1. Naked sequence nucleotide information of double-stranded oligonucleotides.

[0110] In some embodiments of this disclosure, each nucleotide in the double-stranded oligonucleotide is independently selected from modified or unmodified nucleotides.

[0111] In some embodiments of this disclosure, at least one nucleotide in the sense or antisense strand of the double-stranded oligonucleotide is a modified nucleotide, for example, a ribose group and optionally a phosphate group modified nucleotide group, but not limited thereto.

[0112] According to embodiments of this disclosure, all nucleotides in the double-stranded oligonucleotide are substantially selected from modified nucleotides. Here, "all nucleotides in the double-stranded oligonucleotide are substantially selected from modified nucleotides" means that most, but not all, of the nucleotides in the double-stranded oligonucleotide are modified nucleotides, and may contain no more than 5, 4, 3, 2, or 1 unmodified nucleotide.

[0113] According to embodiments of this disclosure, all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides, and the modification is selected from one or both of the following: mono- or di-substitution modification at the 2' position of the ribosyl group of the nucleotide.

[0114] The structural formula of the unmodified nucleotide is as follows: Base represents nucleoside bases, and the nucleoside bases on each nucleotide are independently selected from uracil (U), thymine (T), cytosine (C), adenine (A), or guanine (G).

[0115] In an optional embodiment of this disclosure, each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides:

[0116] 2'-Fluorinated nucleotides, 2'-deoxynucleotides, 2'-O-methylnucleotides, 2'-O-(CH2) xNucleotides modified with -O-R3, 2'-O-Si(R4)3, 2'-amino, debased, nucleotide-like, or [2'-F-2'-methyl] disubstituted.

[0117] The nucleotides described in this disclosure are selected from one or more of peptide nucleic acid (PNA), morpholino nucleic acid (MNA), bridged nucleic acid (BNA), locked nucleic acid (LNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), or unlocked nucleic acid (UNA).

[0118] Where x is selected from 1 or 2, and R3 is selected from C with optional substitution. 1-6 Alkyl or optionally substituted C 1-6 alkoxy group, if R3 contains a substituent, the substituent is selected from halogens, C 1-6 Alkoxy, hydroxy, or amino.

[0119] R4 is independently selected from the arbitrarily substituted C. 1-6 Alkyl group, wherein if R4 contains a substituent, the substituent is selected from halogen, C1-C3 alkyl or C1-C3 alkoxy.

[0120] In this disclosure, 2'-O-(CH2) x -R3 modified nucleotides refer to nucleotides in which the 2' hydroxyl group of the ribosome is modified with -O-(CH2). x -R3 substitution. Where x is selected as 1, the 2'-O-(CH2) is... x The -R3 modified nucleotide is selected from nucleotides modified with 2'-O-ethoxymethyl or 2'-O-2,2,2-trifluoroethoxymethyl. When x is selected as 2, the 2'-O-(CH2) x -R3 modified nucleotides are selected from nucleotides modified with 2'-O-methoxyethyl (also known as nucleotides modified with 2'-O-moe).

[0121] In some specific embodiments of this disclosure, the 2'-O-(CH2) x The -O-R3 modified nucleotide is selected from nucleotides modified with 2'-O-methoxyethyl or nucleotides modified with 2'-O-ethoxymethyl.

[0122] In this disclosure, "2'-O-Si(R4)3 modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosome with -O-Si(R4)3. Exemplary examples include nucleotides modified with 2'-O-TBDMS, nucleotides modified with 2'-O-TIPS, or nucleotides modified with 2'-O-TOM; wherein, the structural formula of TBDMS is... The structural formula of TIPS is: The structural formula of TOM is:

[0123] In some specific embodiments of this disclosure, the 2'-O-Si(R4)3 modified nucleotide is selected from nucleotides modified with 2'-O-TBDMS, nucleotides modified with 2'-O-TIPS, or nucleotides modified with 2'-O-TOM.

[0124] In some specific embodiments of this disclosure, the structural formula of the [2'-F-2'-methyl] disubstituted modified nucleotide is as follows: Base represents the nucleobases A, U, G, C, or T.

[0125] According to embodiments of this disclosure, all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides, and each nucleotide in the double-stranded oligonucleotide is independently selected from at least three of the following modified nucleotides: 2'-fluorine modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, and [2'-F-2'-methyl] disubstituted modified nucleotides.

[0126] In some specific embodiments of this disclosure, the antisense strand of the double-stranded oligonucleotide contains at least one nucleotide modified with 2'-O-methoxyethyl.

[0127] In some specific embodiments of this disclosure, the nucleotide at position 20 and / or position 21 of the antisense strand is a [2'-F-2'-methyl] disubstituted nucleotide, directed from the 5' end to the 3' end.

[0128] In some specific embodiments of this disclosure, the nucleotide at position 19 of the positive strand is a [2'-F-2'-methyl] disubstituted nucleotide, oriented from the 5' end to the 3' end.

[0129] In some embodiments of this disclosure, at least three nucleotides at positions 7-10 of the nucleotide sequence in the positive strand, in the direction from the 5' end to the 3' end, are selected from 2'-fluorinated nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methylated nucleotides.

[0130] In some embodiments of this disclosure, at least three nucleotides at positions 7-10 of the nucleotide sequence in the positive strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 19 is selected from [2'-F-2'-methyl] disubstituted nucleotides, and the nucleotides at the remaining positions are independently selected from 2'-O-methyl-modified nucleotides.

[0131] According to embodiments of this disclosure, in the direction from the 5' end to the 3' end, at least four nucleotides at positions 2, 6, 9-12, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at positions 2, 6, 14, and 16 are selected from 2'-fluoro-modified nucleotides, the nucleotides at positions 20 and / or 21 are selected from [2'-F-2'-methyl] disubstituted nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0132] According to embodiments of this disclosure, at least four nucleotides at positions 2, 6, 9-12, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at positions 2, 6, 14, and 16 are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 8 and / or 15 is selected from 2'-O-methoxyethyl-modified nucleotides, the nucleotide at position 20 and / or 21 is selected from [2'-F-2'-methyl]disubstituted nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0133] According to embodiments of this disclosure, each nucleotide of the double-stranded oligonucleotide is independently selected from modified nucleotides, the modification being selected from any one of the following (1)-(2):

[0134] 1) In the direction from the 5' end to the 3' end, the nucleotides at positions 7-10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; and in the antisense strand, the nucleotides at positions 2, 6, 9, 14 and 16 of the nucleotide sequence are selected from 2'-fluoro-modified nucleotides, the nucleotides at positions 20 and / or 21 are selected from [2'-F-2'-methyl] disubstituted nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0135] 2) In the direction from the 5' end to the 3' end, the nucleotides at positions 7-10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; and in the antisense strand, the nucleotides at positions 2, 6, 9, 14 and 16 of the nucleotide sequence are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, the nucleotide at position 20 and / or 21 is selected from [2'-F-2'-methyl]disubstituted nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0136] According to embodiments of this disclosure, the sense chain and / or the antisense chain independently comprise one or more thiophosphate bonds.

[0137] According to embodiments of this disclosure, at least one or at least two of the following nucleotides in the positive strand are linked by phosphate thioester bonds:

[0138] The connection between the first and second nucleotides at the 5' end of the positive strand;

[0139] The connection between the second and third nucleotides at the 5' end of the positive strand;

[0140] The connection between the first and second nucleotides at the 3' end of the positive strand;

[0141] The connection between the second and third nucleotides at the 3' end of the positive strand;

[0142] as well as,

[0143] The following nucleotides in the antisense strand are linked by phosphate thioester bonds:

[0144] The link between the first and second nucleotides at the 5' end of the antisense strand, and the link between the second and third nucleotides;

[0145] The connection between the first and second nucleotides at the 3' end of the antisense strand, and the connection between the second and third nucleotides.

[0146] In some embodiments of this disclosure, the internucleotide bond between the 10th and 11th nucleotides of the antisense strand is selected from a phosphate thioester bond, with the direction from the 5' end to the 3' end.

[0147] According to embodiments of this disclosure, the positive strand of the double-stranded oligonucleotide comprises or is selected from any of the modified nucleotide sequences shown in MB1)-MB4) below, in the 5' to 3' end direction:

[0148] MB1)UmsCmsAmAmCmUmGfGfAfUfGmAmAmGmAmAmAmCmsUm;

[0149] MB2)UmsCmAmAmCmUmGfGfAfUfGmAmAmGmAmAmAmsCmsUm;

[0150] MB3)UmsCmAmAmCmUmGfGfAfUfGmAmAmGmAmAmAmsCms(NM);

[0151] MB4)UmsCmsAmAmCmUmGfGfAfUfGmAmAmGmAmAmAmCmsAm.

[0152] According to embodiments of this disclosure, the antisense strand of the double-stranded oligonucleotide, in the 5' to 3' end direction, comprises or is selected from any of the modified nucleotide sequences shown below (MA1)-MA5):

[0153] MA1)AmsGfsUmUmUmCfUmUmCfAmsUmCmCmAfGmUfUmGmAms(NM)s(NM);

[0154] MA2)AmsGfsUmUmUmCfUmUmCfAmsUmCmCmAfG(moe)UfUmGmAms(NM)s(NM);

[0155] MA3)AmsGfsUmUmUmCfUmUmCfAmUmCmCmAfG(moe)UfUmGmsAms(NM);

[0156] MA4)AmsGfsUmUmUmCfUmUmCfAmUmCmCmAfG(moe)UfUmGmAms(NM)s(NM);

[0157] MA5)UmsGfsUmUmUmCfUmUmCfAmUmCmCmAfG(moe)UfUmGmAms(NM)s(NM).

[0158] In some specific embodiments of this disclosure, the double-stranded oligonucleotides include one or more of groups 1) to 5) shown in Table 2.

[0159] Table 2 Nucleotide information for double-stranded oligonucleotide modification sequences

[0160] Wherein, C, G, U, A, and T represent cytidine-3'-phosphate, guanosine-3'-phosphate, uridine-3'-phosphate, adenosine-3'-phosphate, and thymidine-3'-phosphate, respectively; m indicates that the nucleotide represented by the uppercase letter to the left of the letter m is a 2'-O-methyl modified nucleotide; f indicates that the nucleotide represented by the uppercase letter to the left of the letter f is a 2'-fluorine modified nucleotide; (moe) indicates that the nucleotide represented by the uppercase letter to the left of the combined identifier (moe) is a 2'-O-methoxyethyl modified nucleotide; s indicates that the nucleotide bond between the nucleotides adjacent to its left and right is a phosphate thiophosphate bond;

[0161] (NM) represents a nucleotide in which the hydroxyl and hydrogen at the 2' position of the ribose are replaced by [2'-F-2'-methyl], with the structural formula as follows:

[0162] In an optional embodiment, the diolithonucleotide is selected from siRNA.

[0163] Conjugate

[0164] In a second aspect of this disclosure, a conjugate is provided comprising the double-stranded oligonucleotide described in the first aspect of this disclosure, and one or more ligands conjugated to the double-stranded oligonucleotide capable of binding to a cell receptor.

[0165] The ligand can be covalently or chemically conjugated to the double-stranded oligonucleotide, and the binding of the ligand and the cell receptor can promote the specific targeting of the conjugate to the target cell and internalize the conjugate into the target cell to inhibit the translation of AGT mRNA into amino acids and conversion into proteins in the target cell. This can effectively alleviate, prevent and / or treat diseases or symptoms mediated by the angiotensinogen (AGT) gene.

[0166] In some alternative embodiments of this disclosure, the ligand is selected from desialyl glycoprotein receptor ligand (ASGPR ligand).

[0167] In some alternative embodiments of this disclosure, the ligand comprises at least one N-acetyl galactosamine (GalNAc).

[0168] In some alternative embodiments of this disclosure, the ligand is selected from structures shown in formula (I), or stereoisomers thereof, tautomers thereof, or pharmaceutically acceptable salts thereof:

[0169] In formula (Ⅰ), * represents the conjugation site between the ligand and the double-stranded oligonucleotide, and j is selected from 1, 2, 3 or 4;

[0170] In some alternative embodiments of this disclosure, each Z is independently selected from hydroxyl or thiol groups. In some specific embodiments of this disclosure, each Z is a hydroxyl group.

[0171] In some embodiments of this disclosure, each p is independently 1 or 2.

[0172] In some specific embodiments of this disclosure, each p is 1.

[0173] In some embodiments of this disclosure, each q is independently 1 or 2.

[0174] In some specific implementations of this disclosure, each q is 1.

[0175] In some specific embodiments of this disclosure, each p is 1 and each q is 1.

[0176] Each L is independently selected from C1-C 30 alkylene or Among them, each R L2a Each is independently selected from C1-C 10 Alkylene, each R L2b Each is independently selected from O, S, NH or -NH-C(O)-, and k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0177] Furthermore, in some specific embodiments of this disclosure, the ligand has a structure as shown in Formula (II), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0178] In formula (II), j is selected from 1, 2, 3, or 4; L is independently selected from -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9-, -(CH2) 10 -, -CH2-NH-CO-(CH2)5-, -(CH2)2-NH-CO-(CH2)4-, -(CH2)3-NH-CO-(CH2)3-, -(CH2)4-NH-CO-(CH2)2-, or -(CH2)5-NH-CO-CH2-.

[0179] In some alternative embodiments of this disclosure, each L is independently selected from -CH2-CH2- or -(CH2)2-NH-CO-(CH2)4-.

[0180] In some specific embodiments of this disclosure, L is selected from -CH2-CH2-.

[0181] In some specific embodiments of this disclosure, L is selected from -(CH2)2-NH-CO-(CH2)4-.

[0182] In some specific embodiments of this disclosure, the ligand has a structure shown in formula (III), formula (IV), formula (V) or formula (VI), or a stereoisomer thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0183] According to embodiments of this disclosure, the number of ligands is selected from one, and one of the ligands is conjugated to the 3' end of the positive strand of the double-stranded oligonucleotide.

[0184] According to embodiments of this disclosure, the number of ligands is selected from two, and the two ligands are respectively conjugated to the 5' end of the sense strand and the 3' end of the antisense strand of the double-stranded oligonucleotide.

[0185] In some specific embodiments of this disclosure, the conjugate includes one or more of R303079, R303082, R303086, R303087, R303088, and R303089 shown in Table 3.

[0186] Table 3 Nucleotide information of the conjugates

[0187] In this context, (CR01008×2), (CR01008×3), and (CR01008×4) all represent ligands; if the ligand is located at the 5' end of the sense chain, it indicates that the ligand is conjugated to the 5' end of the sense chain; if the ligand is located at the 3' end of the sense chain, it indicates that the ligand is conjugated to the 3' end of the sense chain; if the ligand is located at the 3' end of the antisense chain, it indicates that the ligand is conjugated to the 3' end of the antisense chain.

[0188] The structural formula for (CR01008×2) is:

[0189] The structural formula of (CR01008×3) is:

[0190] The structural formula of (CR01008×4) is:

[0191] Pharmaceutical Composition

[0192] In a third aspect, this disclosure provides a pharmaceutical composition comprising any of the following and pharmaceutically acceptable excipients:

[0193] (I) The double-stranded oligonucleotides described in the first aspect of this disclosure; and / or

[0194] (II) The conjugate described in the second aspect of this disclosure.

[0195] Both the double-stranded oligonucleotides and conjugates can degrade AGT mRNA and inhibit AGT expression. Therefore, the pharmaceutical compositions disclosed herein can effectively prevent and / or treat diseases or conditions mediated by the angiotensinogen (AGT) gene.

[0196] The pharmaceutical compositions disclosed herein include formulations suitable for parenteral administration. The formulations can be conveniently available in unit dosage forms and can be prepared by any method known in the pharmaceutical field. The amount of active ingredient in a single-dose form, typically the amount of siRNA producing the therapeutic effect, can be prepared in combination with excipients.

[0197] use

[0198] In a fourth aspect of this disclosure, the following are provided for use in the preparation of medicaments for alleviating, preventing, and / or treating AGT gene-mediated diseases or conditions:

[0199] (I) The double-stranded oligonucleotides described in the first aspect of this disclosure; and / or

[0200] (II) The conjugates described in the second aspect of this disclosure; and / or

[0201] (III) The pharmaceutical composition described in the third aspect of this disclosure.

[0202] According to embodiments of this disclosure, the diseases or conditions mediated by the AGT gene include hypertension, borderline hypertension, primary hypertension, secondary hypertension, hypertensive crisis, hypertensive urgency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, intractable hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, or ventricular fibrosis.

[0203] Methods to inhibit intracellular AGT gene expression

[0204] In a fifth aspect, this disclosure provides a method for inhibiting intracellular AGT gene expression, the method comprising contacting the cell with any of the following:

[0205] (I) The double-stranded oligonucleotides described in the first aspect of this disclosure; and / or

[0206] (II) The conjugates described in the second aspect of this disclosure; and / or

[0207] (III) The pharmaceutical composition described in the third aspect of this disclosure.

[0208] The methods disclosed herein can be used to inhibit angiotensinogen expression. For example, they can inhibit angiotensinogen expression in animals; or for non-disease treatment purposes, such as inhibiting angiotensinogen expression in vitro for further research.

[0209] Disease prevention and / or treatment

[0210] In a sixth aspect, this disclosure provides a method for alleviating, treating, and / or preventing AGT gene-mediated diseases or conditions, said method comprising administering any of the following to a subject:

[0211] (I) The double-stranded oligonucleotides described in the first aspect of this disclosure; and / or

[0212] (II) The conjugates described in the second aspect of this disclosure; and / or

[0213] (III) The pharmaceutical composition described in the third aspect of this disclosure.

[0214] According to embodiments of this disclosure, the AGT-mediated diseases or conditions include those related to the mRNA levels of AGT gene expression.

[0215] According to embodiments of this disclosure, the diseases or conditions mediated by the AGT gene include hypertension, borderline hypertension, primary hypertension, secondary hypertension, hypertensive crisis, hypertensive urgency, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, intractable hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, or ventricular fibrosis.

[0216] The effective amount of the double-stranded oligonucleotides, conjugates, or pharmaceutical compositions described in this disclosure may vary depending on the mode of administration and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, such as four times a day, three times a day, twice a day, once a day, or every other day, or the number of daily doses may be proportionally reduced.

[0217] The medication may be administered to the subject via any suitable route known in the art, including but not limited to: oral or parenteral routes, including intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and local administration (including oral and sublingual administration), preferably intravenous administration.

[0218] Reagent test kit

[0219] In a seventh aspect of this disclosure, a kit is provided, the kit comprising any of the following:

[0220] (I) The double-stranded oligonucleotides described in the first aspect of this disclosure; and / or

[0221] (II) The conjugates described in the second aspect of this disclosure; and / or

[0222] (III) The pharmaceutical composition described in the third aspect of this disclosure.

[0223] The double-stranded oligonucleotides and conjugates disclosed herein can degrade AGT mRNA and inhibit AGT expression, which is helpful for AGT-related scientific research and clinical treatment and / or prevention of diseases or conditions mediated by the angiotensinogen gene.

[0224] Unless otherwise stated, all siRNA sequences used in this disclosure were synthesized by Kunshan Aotai Biotechnology Co., Ltd.; all PCR primers used in this disclosure were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0225] The data on AGT protein detection at different time points in the in vivo activity assay disclosed in this disclosure are summarized below:

[0226] In the context of this disclosure, unless otherwise stated, in vivo activity data are presented in hard copy. It is noted that all experimental data were plotted and analyzed using GraphPadprism 8.0 software.

[0227] In the context of this disclosure, unless otherwise stated, all reagent ratios provided below are calculated on a volume ratio (v / v).

[0228] Preparation of compounds

[0229] Unless otherwise stated, all reagents used in the preparation of the compounds in this disclosure were purchased from Beijing Coupling Technology Co., Ltd. Information on the main reagents is shown in Table 4.

[0230] Table 4

[0231] CPG stands for Controlled Pore Glass carrier.

[0232] Preparation of compound CR01008 (Example 1):

[0233] (1-1) The synthetic structure of compound CR01008 is shown below:

[0234] The synthetic route for compound CR01008 is shown below:

[0235] (1-1-1) Synthesis of Compound 2:

[0236] Compound 1 (trans-4-(Boc-amino)cyclohexylformaldehyde, 10.0 g, 1.0 eq) and an aqueous formaldehyde solution (8.9 g, 37 wt%, 2.4 eq) were dissolved in 33 mL of methanol. 13 mL of a 45.3 wt% KOH aqueous solution was added dropwise. After the addition was complete, the mixture was stirred at 25 °C for 30 minutes, then heated to 60 °C and refluxed at 60 °C for 2 hours. After the reaction was complete, the reaction solution was cooled to room temperature and then evaporated under reduced pressure to obtain a crude white solid. A small amount of water was added to the crude product, and the mixture was slurried and filtered to obtain compound 2 (9 g, yield 78.9%), a white solid. MS-ESI (m / z) = 260 [M+H] + .

[0237] (1-1-2) Synthesis of compound 3:

[0238] Compound 2 (9 g, 1 eq) was dissolved in 70 mL of 1,4-dioxane, and a solution of 1,4-dioxane in hydrogen chloride (45 mL, 4 M) was added. The mixture was stirred at 25 °C for 1 hour. After the reaction was completed, the reaction solution was evaporated to dryness under reduced pressure to give compound 3 (6.8 g, 100% yield) as a white solid.

[0239] (1-1-3) Synthesis of compound 5:

[0240] Compound 3 (1.8 g, 2.0 eq), compound 4 (5-[[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)-2-tetrahydropyranyl]oxy]valeric acid, 2.1 g, 1.0 eq), and DIEA (N,N-diisopropylethylamine, 3.5 g, 6.0 eq) were dissolved in 15 mL of DMF. HBTU (1.9 g, 1.1 eq) was added, and the mixture was stirred at 25 °C for 3 hours under a nitrogen atmosphere. After the reaction was complete, the reaction mixture was evaporated to dryness under reduced pressure and purified by reversed-phase column chromatography (22 v / v acetonitrile aqueous solution) to give compound 5 (1.78 g, yield 64.4%) as a white solid. MS-ESI (m / z) = 589 [M+H] + .

[0241] (1-1-4) Synthesis of compound 6:

[0242] Compound 5 (1.54 g, 1.0 eq) was dissolved in 15 mL of pyridine. The reaction system was cooled to 0 °C using an ice-water bath, and DMTrCl (4,4'-dimethoxytriphenylchloromethane, 1.32 g, 1.5 eq) was added at 0 °C. The reaction was carried out at 25 °C for 3 hours, and the reaction was quenched by adding 15 mL of methanol. After the reaction was completed, the reaction solution was evaporated to dryness under reduced pressure, and purified by reversed-phase column chromatography (60 v / v aqueous solution of acetonitrile) to give compound 6 (1 g, yield 42.7%) as a yellow solid. MS-ESI (m / z) = 891 [M+H] + .

[0243] (1-1-5) Synthesis of compound CR01008:

[0244] Compound 6 (1.08 g, 1.0 eq) was dissolved in 20 mL of anhydrous dichloromethane. DCI (115 mg, 0.8 eq) and compound 7 (bis(diisopropylamino)(2-cyanoethoxy)phosphine, 732 mg, 2.1 eq) were added separately. The mixture was purged with nitrogen three times and stirred at 25 °C for 2 hours. After the reaction was complete, 20 mL of saturated sodium bicarbonate aqueous solution was added to the reaction solution, and the mixture was extracted three times with 20 mL of dichloromethane (3 × 20 mL). The organic phases were combined, evaporated to dryness under reduced pressure, purified by reversed-phase chromatography (72 vol% acetonitrile aqueous solution), and dried under vacuum for 12 hours to obtain a white powder, compound CR01008 (1 g, yield 76.0%). MS-ESI (m / z) = 1091 [M + Na]. + .

[0245] H NMR(400MHz, DMSO-d6)δ1.05(d,J=6.7Hz,6H).1.14(d,J=6.7Hz,6H),1.37–1.17(m,5H),1.60–1.40(m,6H),1.68–1.62(m,1H),1.80(s,3H), 1.80(s,3H),1.92(s,3H),2.02(s,5H),2.13(s,3H),2.71(t,J=5.9Hz,2H),2.79(d,J=8.4Hz,1H),2.87(d,J=8.4Hz,1H),3.36(s,1H),3.58– 3.39(m,3H),3.69–3.60(m,2H),3.75(s,7H),3.90(dt,J=11.2,8.8Hz,1H),4.05(s,3H),4.51(d,J=8.4Hz,1H),4.99(dd,J=11.3,3.4Hz,1H) ,5.24(d,J=3.4Hz,1H),5.78(s,1H),6.93–6.87(m,4H),7.35–7.21(m,7H),7.44–7.37(m,2H),7.66(d,J=7.8Hz,1H),7.84(d,J=9.2Hz,1H).

[0246] (1-2) Synthesis of compound CR01008Z:

[0247] The synthetic route for compound CR01008Z is shown below:

[0248] (1-2-1) Synthesis of Compound 9:

[0249] Compound 6 (500 mg) was dissolved in 10 mL of dichloromethane. Compound 8 (succinic anhydride, 112 mg), DMAP (6.8 mg), and TEA (226.2 mg) were added. The mixture was purged with nitrogen three times, and the reaction was stirred at 25 °C for 16 hours. The mixture was then flash purified to give compound 9 (300 mg, yield 53.6%). MS-ESI (m / z) = 10¹³ [M + Na] + .

[0250] (1-2-2) Synthesis of compound CR01008Z:

[0251] Compound 9 (50 mg), aminoCPG (1.25 g, 80 μmol / g, 0.1 mmol), HBTU (27 mg), and DIEA (12 mg) were added to a 20 ml sample vial, and the mixture was reacted on a shaker for 16 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was first washed once with 10 ml of acetonitrile (1 × 10 ml) and then dried under vacuum. The dried filter cake, DMAP (3 mg), Cap1 (10 ml, 200 V), and Cap2 (1 ml, 20 V) were added to a 20 ml sample vial, and the mixture was reacted on a shaker for 6 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was first washed once with 10 ml of acetonitrile (1 × 10 ml) and then dried under vacuum to obtain compound CR01008Z (1.03 g, loading 20-30 μmol / g).

[0252] Cap1 and Cap2 are capping reagents. Cap1 is a pyridine / acetonitrile mixed solution of N-methylimidazolium with a concentration of 20% by volume, and the volume ratio of pyridine to acetonitrile is 3:5. Cap2 is an acetonitrile solution of acetic anhydride with a concentration of 20% by volume.

[0253] Preparation Example 2: Synthesis of compound NM054:

[0254] In this preparation example, the synthetic route of compound NM054 is shown below:

[0255] (2-1) Synthesis of compound NM054-2:

[0256] Compound NM054-1 (3 g, 11.54 mmol, 1.0 eq, (2'R)-2'-deoxy-2'-fluoro-2'-methylurea, CAS No. 863329-66-2) and pyridine (30 ml) were added to a 500 ml reaction vessel. The mixture was cooled to 0 °C, and 4,4'-bismethoxytriphenylmethylchloro (4.29 g, 12.7 mmol, 1.1 eq) was added in portions. The mixture was purged with nitrogen three times, and the reaction system was stirred at 25 °C for 3 hours under a nitrogen atmosphere. HPLC showed no starting material. After the reaction was complete, the reaction solution was concentrated, and purified with purified water (50 ml) and ethyl acetate (50 ml) to separate the organic phase. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give compound NM054-2 (2.7 g, yield 41.7%). MS ESI (m / z) = 563.0 [M+H] + .

[0257] (2-2) Synthesis of compound NM054:

[0258] Compound NM054-2 (2.7 g, 4.8 mmol, 1.0 eq) was added to a 100 mL reaction vessel. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.74 g, 5.76 mmol, 1.2 eq) was added in portions, followed by 4,5-dicyanimidazole (0.45 g, 3.8 mmol, 0.8 eq, abbreviated as DCI, CAS number 1122-28-7) and dichloromethane (27 mL). The mixture was purged with nitrogen three times, and the reaction system was stirred at 25 °C for 3 hours under a nitrogen atmosphere. After the reaction was complete, sodium bicarbonate aqueous solution (20 mL) was added to the reaction solution. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reversed-phase column chromatography (elution: acetonitrile / water = 90 / 10, v / v) to obtain compound NM054 (3.0 g). MS ESI(m / z) = 763[M+H] + .

[0259] Preparation of siRNA (Example 3):

[0260] (3-1) Synthesis of the Justice Chain (SS):

[0261] The phosphoramidite solid-phase synthesis method for nucleic acids involves starting with a compound on a solid support and sequentially linking nucleoside monomers one by one along the nucleotide sequence in the 3'-5' direction. In this synthesis, compound NM054 is considered as a nucleoside monomer.

[0262] Each connection of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfidation. The synthetic conditions are given below:

[0263] The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0264] The deprotection reaction conditions were the same for each step. The deprotection reaction conditions were: temperature 25℃, reaction time 70 seconds, deprotection reagent was a dichloromethane solution of dichloroacetic acid (3% by volume), and the molar ratio of dichloroacetic acid to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid support was 5:1.

[0265] The conditions for each coupling reaction were identical. The coupling reaction conditions were as follows: temperature 25℃, molar ratio of nucleic acid sequence to nucleoside monomer on the solid-phase support 1:10, molar ratio of nucleic acid sequence to coupling reagent on the solid-phase support 1:65, reaction time 600 seconds, coupling reagent 0.5M acetonitrile solution of 5-ethylthio-1H-tetrazole, and thioreagent 0.2M acetonitrile / pyridine mixed solution of hydrogenated xanthanin (acetonitrile and pyridine volume ratio 1:1).

[0266] The conditions for each capping reaction were identical. The conditions for the capping reaction were: temperature 25℃; reaction time 2 minutes; the capping reagent solution was a 1:1 molar ratio of Cap1 and Cap2, where Cap1 was a 20% (v / v) N-methylimidazole pyridine / acetonitrile mixture with a pyridine to acetonitrile volume ratio of 3:5, and Cap2 was a 20% (v / v) acetic anhydride acetonitrile solution; the molar ratio of N-methylimidazole in Cap1 and acetic anhydride in Cap2 to the nucleic acid sequence linked on the solid-phase support was 1:1:1.

[0267] The conditions for each oxidation reaction were identical. The oxidation reaction conditions were: temperature 25°C; reaction time 3 seconds; oxidizing agent concentration of 0.05M iodine solution, with a molar ratio of iodine to the nucleic acid sequence linked on the solid support in the coupling reaction of 30:1; the oxidation reaction was carried out in a water / pyridine mixed solvent (water to pyridine volume ratio 1:9). The sulfidation reaction conditions were: temperature 25°C; reaction time 360 ​​seconds; thioreagent concentration of 0.2M hydroflavin in pyridine solution, with a molar ratio of thioreagent to the nucleic acid sequence linked on the solid support in the coupling reaction of 4:1; the thioreagent reaction was carried out in a water / pyridine mixed solvent (water to pyridine volume ratio 1:9).

[0268] After the last nucleoside monomer was ligated, the nucleic acid sequence ligated on the solid-phase support was sequentially cut, deprotected, purified, and desalted, and then freeze-dried to obtain the positive strand, wherein:

[0269] The cleavage and deprotection conditions were as follows: The synthesized nucleotide sequence linked to a solid-phase support was added to 25% (w / w) ammonia solution at a concentration of 0.5 mL / μmol. The reaction was carried out at 55 °C for 16 hours. The solvent was removed, and the solution was concentrated to dryness under vacuum. After ammonia treatment, the product was dissolved in 0.4 mL / μmol N-methylpyrrolidone relative to the amount of single-stranded nucleic acid. Subsequently, 0.3 mL / μmol triethylamine and 0.6 mL / μmol triethylamine trifluoride were added to deprotect the 2'-O-TBDMS protection on the ribose.

[0270] Purification and desalting conditions: Nucleic acid purification was performed using a preparative ion chromatography column (Source 15Q) with a NaCl gradient elution. Specifically: eluent 1 was 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixture (water to acetonitrile volume ratio 9:1); eluent 2 was 1.5 M sodium chloride, 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixture (water to acetonitrile volume ratio 9:1); the elution gradient was eluent 1: eluent 2 = (100:0) - (50:50). The product eluates were collected and combined, and desalting was performed using a reversed-phase chromatography column. Desalting conditions included using a dextran gel column (g25 packing material) and elution with deionized water.

[0271] Detection: Purity was determined using ion exchange chromatography (IEX-HPLC); molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value. If the measured value and the theoretical value were consistent, it indicated that the positive strand of siRNA had been obtained.

[0272] (3-2) Synthesis of antisense strand (AS):

[0273] The antisense chain was synthesized using a general solid-phase support. The deprotection, coupling, capping, oxidation or sulfidation reaction conditions, cleavage and deprotection conditions, purification and desalting conditions of the solid-phase synthesis method of the antisense chain are the same as those of the synthesis of the sense chain in step (3-1).

[0274] Detection: Purity was determined using ion exchange chromatography (IEX-HPLC); molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value. If the measured value and the theoretical value were consistent, it indicated that the antisense strand of siRNA had been obtained.

[0275] (3-3) Synthesis of siRNA:

[0276] The sense strand synthesized in step (3-1) and the antisense strand synthesized in step (3-2) were mixed in an equimolar ratio, dissolved in water for injection, and heated to 95°C. The mixture was then slowly cooled to room temperature and kept at room temperature for 10 minutes to allow the sense and antisense strands to form a double-stranded structure through hydrogen bonds, thereby obtaining siRNA with the sense and antisense strands shown in Table 2.

[0277] Preparation Example 4: Synthesis of siRNA conjugates

[0278] (4-1) The synthesis of the chain of justice

[0279] The phosphoramide solid-phase synthesis method for nucleic acids involves starting with a carrier compound (e.g., Universal CPG carrier, Universal PS carrier, or compound CR01008Z) and sequentially linking nucleoside monomers one by one along the nucleotide sequence from the 3' end to the 5' end. During the synthesis, compounds CR01008 and NM054 are each considered as a nucleoside monomer.

[0280] Each connection of a nucleoside monomer involves four steps: deprotection, coupling, capping, oxidation, or sulfidation. In this preparation example, the conditions for deprotection, coupling, capping, oxidation, or sulfidation, as well as the conditions for cleavage and deprotection, purification, and desalting, are the same as those for the synthesis of the positive chain in step (3-1).

[0281] During the synthesis of the justice chain, two-cluster CR01008 carriers (denoted as (CR01008×2) or (CR01008)×2), three-cluster CR01008 carriers (denoted as (CR01008×3) or (CR01008)×3), and four-cluster CR01008 carriers (denoted as (CR01008×4) or (CR01008)×4) were obtained.

[0282] The structural formula of the two clusters CR01008 is:

[0283] The structural formula of the three-cluster CR01008 is:

[0284] The structural formula of the four-cluster CR01008 is:

[0285] (4-2) Synthesis of antisense strands

[0286] The antisense strand of this preparation example was synthesized according to the antisense strand synthesis method shown in step (3-2) of preparation example 3.

[0287] (4-3) Synthesize siRNA conjugates according to the method shown in step (3-3) of Preparation Example 3.

[0288] The structural formula of the siRNA conjugate (CR01008)×3 vector conjugated to the 3' end of the positive strand is shown below:

[0289] in, This indicates siRNA, where SS represents the sense strand of siRNA and AS represents the antisense strand of siRNA. (CR01008×3) is conjugated to the 3' end of the sense strand of siRNA via a phosphodiester bond.

[0290] The structural formula of the siRNA conjugate (CR01008)×4 vector conjugated to the 3' end of the positive strand is shown below:

[0291] in, This indicates siRNA, where SS represents the sense strand of siRNA and AS represents the antisense strand of siRNA. (CR01008×4) is conjugated to the 3' end of the sense strand of siRNA via a phosphodiester bond.

[0292] The structural formulas of the siRNA conjugates with two (CR01008)×2 vectors conjugated to the 5' end of the sense strand and the 3' end of the antisense strand, respectively, are shown below:

[0293] in, This indicates siRNA, where SS represents the sense strand of the siRNA and AS represents the antisense strand of the siRNA. Two (CR01008×2) are conjugated to the 5' end of the sense strand and the 3' end of the antisense strand of the siRNA via phosphodiester bonds, respectively.

[0294] Biological detection experiments:

[0295] Unless otherwise stated, all reagents, consumables and instruments used in biological testing experiments in this disclosure are commercially available products.

[0296] Example 1: Comparison of the efficacy of siRNA conjugates in normal cynomolgus monkeys:

[0297] In this embodiment, the expression of AGT protein in the serum of cynomolgus monkeys at different time points after a single dose of RZ003089, R303079, R303082, R303086, R303087, R303088, and R303089 was determined by ELISA.

[0298] The nucleotide information for conjugate RZ003089 is as follows:

[0299] Chain of Justice (5'-3'): UmsCmsAmAmCmUmGfGfAfUfGmAmAmGmAmAmAmCmUm_(CR01008×3);

[0300] Antonym chain (5'-3'): AmsGfsUmUmUmCfUmUmCfAmUmCmCmAfG(moe)UfUmGmAmsGmsGm.

[0301] Animal grouping, drug administration, and tissue sample collection:

[0302] Healthy cynomolgus monkeys weighing 3-5 kg ​​were divided into groups of three males each, based on their serum AGT protein levels. Each test group received a predetermined dose of the drug conjugate, with a solvent control group also included. Dosage was calculated based on body weight for all animals, and administration was a single subcutaneous injection into the interscapular region of the back. Each drug conjugate was administered at a dose of 6 mg (calculated as siRNA) / mL in 0.9% sodium chloride injection, with an administration volume of 1 mL / kg (cynomolgus monkey body weight), meaning the dose of each drug conjugate was 6 mg (calculated as siRNA) / kg (cynomolgus monkey body weight). The solvent control group received 1 mL / kg (cynomolgus monkey body weight) of 0.9% sodium chloride injection without the siRNA conjugate. Serum samples were collected from cynomolgus monkeys on the day of drug administration (Day 1, D0, before drug administration), Day 7 (D7), Day 14 (D14), Day 21 (D21), Day 28 (D28), Day 35 (D35), and Day 42 (D42). AGT protein expression was measured using a human angiotensinogen (AGT) assay kit (IBL, 27412). The experiment is ongoing.

[0303] Table 5 shows the changes in AGT protein levels in cynomolgus monkey serum after administration of the siRNA conjugate.

[0304] The results of Example 1 are shown in Table 5 and Figure 1. The results show that RZ003089, R303079, R303082, R303086, R303087, R303088, and R303089 can significantly reduce the serum AGT protein level in cynomolgus monkeys when administered as a single dose of 6 mg / kg. With the extension of observation time, R303082 and R303089 are slightly more effective than RZ003089 in reducing serum AGT protein.

[0305] The above specific embodiments are merely illustrative of the present invention and do not represent a limitation thereof. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and substance of this disclosure, and these modifications and improvements are also considered to be within the scope of protection of this disclosure.

Claims

1. A double-stranded oligonucleotide that inhibits AGT gene expression, characterized in that, The double-stranded oligonucleotide comprises a sense strand and an antisense strand. The antisense strand comprises or is selected from a sequence of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides as shown in Formula (A), or comprises a nucleotide sequence that differs from the consecutive nucleotides by 1, 2, or 3 nucleotides. The sense strand comprises at least 17 nucleotides, and the sense strand is complementary or substantially complementary to the antisense strand to form a double-stranded region. The substantially complementary nature means that the mismatch between the sense strand and the antisense strand in the double-stranded region does not exceed 3 nucleotides. Formula (A)5'-X1GUUUCUUCAUCCAGUGA(X2) m (X3) n -3'; Where X1 is selected from A or U; (X2) m (X3) n The bases that protrude at the 3' end are represented by X2 and X3, which are each independently selected from A, U, G, C or T, and m and n are independently selected from 0 or 1, and m+n≥1; Furthermore, at least one of the nucleotides X2 and X3 is a [2'-R1-2'-R2] disubstituted modified nucleotide, wherein the disubstituted modified nucleotide refers to a nucleotide in which both the hydroxyl and hydrogen at the 2' position of the ribose are substituted, wherein R1 and R2 are each independently selected from halogens, optionally substituted C1-C6 alkyl groups, or optionally substituted C1-C6 alkoxy groups.

2. The double-stranded oligonucleotide according to claim 1, characterized in that, The antisense strand comprises or is selected from any of the sequences shown in A1)-A3), or a nucleotide sequence that differs from any of the above sequences by one or two nucleotides: A1)5'-AGUUUCUUCCAUCCAGUUGAUU-3'; A2)5'-AGUUUCUUCAUCCAGUUGAU-3'; A3)5'-UGUUUCUUCAUCCAGUUGAUU-3'; The positive chain comprises or is selected from any of the sequences shown in B1)-B2) a nucleotide sequence of at least 15, at least 16, at least 17, at least 18 or at least 19 consecutive nucleotides, or comprises a nucleotide sequence that differs from the consecutive nucleotides by 1, 2 or 3 nucleotides. B1)5'-UCAACUGGAUGAAGAAACU-3'; B2)5'-UCAACUGGAUGAAGAAACA-3'.

3. The double-stranded oligonucleotide according to claim 1, characterized in that, The antisense chain contains or is selected from any of the sequences shown in A1)-A3), and the justice chain contains or is selected from any of the sequences shown in B1)-B2).

4. The double-stranded oligonucleotide according to claim 3, characterized in that, The double-stranded oligonucleotide is selected from one or more of the following groups: 1) The antisense strand has the nucleotide sequence shown in A1, and the sense strand has the nucleotide sequence shown in B1; 2) The antisense strand has the nucleotide sequence shown in A2, and the sense strand has the nucleotide sequence shown in B1; 3) The antisense strand has the nucleotide sequence shown in A3, and the sense strand has the nucleotide sequence shown in B2.

5. The double-stranded oligonucleotide according to claim 1, characterized in that, All nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides, and the modification is selected from one or both of the following: a mono-substitution at the 2' position or a double-substitution at the 2' position of the ribosyl group of the nucleotide. Each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides: 2'-Fluorinated nucleotides, 2'-deoxynucleotides, 2'-O-methylnucleotides, 2'-O-(CH2) x -O-R3 modified nucleotides, 2'-amino modified nucleotides, debased nucleotides, nucleotide-like nucleotides, or [2'-F-2'-methyl] disubstituted nucleotides; Where x is selected from 1 or 2, and R3 is selected from C with optional substitution. 1-6 Alkyl or optionally substituted C 1-6 alkoxy group, if R3 contains a substituent, the substituent is selected from halogens, C 1-6 alkoxy, hydroxy, or amino groups; The structural formula of the [2'-F-2'-methyl] disubstituted nucleotide is as follows: Base represents the nucleobases A, U, G, C, or T.

6. The double-stranded oligonucleotide according to claim 5, characterized in that, All nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides, and each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides: 2'-fluorine modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, and [2'-F-2'-methyl] disubstituted modified nucleotides.

7. The double-stranded oligonucleotide according to claim 6, characterized in that, Each nucleotide of the double-stranded oligonucleotide is independently selected from modified nucleotides, the modifications being selected from any one of the following (1)-(2): 1) In the direction from the 5' end to the 3' end, the nucleotides at positions 7-10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; and in the antisense strand, the nucleotides at positions 2, 6, 9, 14 and 16 are selected from 2'-fluoro-modified nucleotides, the nucleotides at positions 20 and / or 21 are selected from [2'-F-2'-methyl] disubstituted nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; 2) In the direction from the 5' end to the 3' end, the nucleotides at positions 7-10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; and in the antisense strand, the nucleotides at positions 2, 6, 9, 14, and 16 are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, the nucleotide at position 20 and / or 21 is selected from [2'-F-2'-methyl]disubstituted nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; The sense chain and / or the antisense chain independently contain one or more thiophosphate bonds.

8. The double-stranded oligonucleotide according to claim 7, characterized in that, At least one or at least two of the following nucleotides in the positive strand are linked by phosphate thioester bonds: The connection between the first and second nucleotides at the 5' end of the positive strand; The connection between the second and third nucleotides at the 5' end of the positive strand; The connection between the first and second nucleotides at the 3' end of the positive strand; The connection between the second and third nucleotides at the 3' end of the positive strand; as well as, The following nucleotides of the antisense strand are linked by phosphate thioester bonds: the link between the first and second nucleotides at the 5' end of the antisense strand, and the link between the second and third nucleotides; the link between the first and second nucleotides at the 3' end of the antisense strand, and the link between the second and third nucleotides.

9. The double-stranded oligonucleotide according to claim 8, characterized in that, The positive strand of the double-stranded oligonucleotide, in the 5' to 3' direction, comprises or is selected from any one of the modified nucleotide sequences shown in MB1)-MB4) below: MB1)UmsCmsAmAmCmUmGfGfAfUfGmAmAmGmAmAmAmCmsUm; MB2)UmsCmAmAmCmUmGfGfAfUfGmAmAmGmAmAmAmsCmsUm; MB3)UmsCmAmAmCmUmGfGfAfUfGmAmAmGmAmAmAmsCms(NM); MB4)UmsCmsAmAmCmUmGfGfAfUfGmAmAmGmAmAmAmCmsAm; The antisense strand of the double-stranded oligonucleotide, in the 5' to 3' end direction, comprises or is selected from any of the modified nucleotide sequences shown below (MA1)-MA5): MA1)AmsGfsUmUmUmCfUmUmCfAmsUmCmCmAfGmUfUmGmAms(NM)s(NM); MA2)AmsGfsUmUmUmCfUmUmCfAmsUmCmCmAfG(moe)UfUmGmAms(NM)s(NM); MA3)AmsGfsUmUmUmCfUmUmCfAmUmCmCmAfG(moe)UfUmGmsAms(NM); MA4)AmsGfsUmUmUmCfUmUmCfAmUmCmCmAfG(moe)UfUmGmAms(NM)s(NM); MA5)UmsGfsUmUmUmCfUmUmCfAmUmCmCmAfG(moe)UfUmGmAms(NM)s(NM); Wherein, m indicates that the nucleotide represented by the uppercase letter to the left of the letter m is a 2'-O-methyl modified nucleotide; f indicates that the nucleotide represented by the uppercase letter to the left of the letter f is a 2'-fluorine modified nucleotide; (moe) indicates that the nucleotide represented by the uppercase letter to the left of the combined identifier (moe) is a 2'-O-methoxyethyl modified nucleotide; s indicates that the nucleotide bond between the nucleotides to the left and right of the letter s is selected from a phosphate thioester bond; (NM) represents a nucleotide in which the hydroxyl and hydrogen at the 2' position of the ribose are replaced by [2'-F-2'-methyl], with the structural formula as follows:

10. The double-stranded oligonucleotide according to claim 9, characterized in that, The double-stranded oligonucleotide is selected from one or more groups (1) to (5): Group 1: A double-stranded oligonucleotide consisting of SEQ ID NO. 6 and SEQ ID NO. 7; Group 2: A double-stranded oligonucleotide consisting of SEQ ID NO. 8 and SEQ ID NO. 9; Group 3: A double-stranded oligonucleotide consisting of SEQ ID NO.10 and SEQ ID NO.11; Group 4: A double-stranded oligonucleotide consisting of SEQ ID NO.12 and SEQ ID NO.13; Group 5: Double-stranded oligonucleotides consisting of SEQ ID NO.14 and SEQ ID NO.

15.

11. A conjugate, characterized in that, The conjugate comprises the double-stranded oligonucleotide according to any one of claims 1-10, and one or more ligands conjugated to the double-stranded oligonucleotide capable of binding to cell surface receptors; The ligand has the structure shown in Formula (I), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof: In formula (Ⅰ), * represents the conjugation site between the ligand and the double-stranded oligonucleotide; j is selected from 1, 2, 3, or 4; Each Z is independently selected from either a hydroxyl or a thiol group; Each p is independently either 1 or 2; Each q is independently either 1 or 2; Each L is independently selected from C1-C 30 alkylene or Among them, each R L2a Each is independently selected from C1-C 10 Alkylene, each R L2b Each is independently selected from O, S, NH or -NH-C(O)-, and k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

12. The conjugate according to claim 11, characterized in that, The ligand has a structure as shown in Formula (II), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof: In formula (II), j is selected from 1, 2, 3, or 4; L is independently selected from -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9-, -(CH2) 10 -, -CH2-NH-CO-(CH2)5-, -(CH2)2-NH-CO-(CH2)4-, -(CH2)3-NH-CO-(CH2)3-, -(CH2)4-NH-CO-(CH2)2-, or -(CH2)5-NH-CO-CH2-.

13. The conjugate according to claim 12, characterized in that, The ligand has the structure shown in formula (III), formula (IV), formula (V) or formula (VI), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:

14. The conjugate according to claim 13, characterized in that, The number of ligands is selected from one, and one ligand is conjugated to the 3' end of the sense strand of the double-stranded oligonucleotide; or, The number of ligands is selected from two, and the two ligands are respectively conjugated to the 5' end of the sense strand and the 3' end of the antisense strand of the double-stranded oligonucleotide.

15. The conjugate according to claim 14, characterized in that, The conjugate is selected from one or more of R303079, R303082, R303086, R303087, R303088, and R303089.

16. A pharmaceutical composition comprising any of the following and pharmaceutically acceptable excipients: (I) the double-stranded oligonucleotide according to any one of claims 1-10; and / or (II) The conjugate according to any one of claims 11-15.

17. A method for inhibiting intracellular AGT gene expression, the method comprising contacting the cell with any of the following: (I) the double-stranded oligonucleotide according to any one of claims 1-10; and / or (II) The conjugate according to any one of claims 11-15; and / or (III) The pharmaceutical composition according to claim 16.

18. A method for alleviating, treating, and / or preventing AGT gene-mediated diseases or conditions, said method comprising administering to a subject any of the following: (I) the double-stranded oligonucleotide according to any one of claims 1-10; and / or (II) The conjugate according to any one of claims 11-15; and / or (III) The pharmaceutical composition according to claim 16.

19. The use of any of the following in the preparation of a medicament for the relief, prevention, and / or treatment of AGT gene-mediated diseases or conditions: (I) the double-stranded oligonucleotide according to any one of claims 1-10; and / or (II) The conjugate according to any one of claims 11-15; and / or (III) The pharmaceutical composition according to claim 16.

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