dsRNA molecules for regulating AGT expression

Abstract

The invention provides a double-stranded RNA for suppressing the expression of angiotensinogen (AGT) in cells, a vector containing its coding nucleotide, and cells, as well as a method for treating a disease or symptom mediated by or associated with AGT expression in a subject using the above dsRNA, vector, or cells.

Description

[Technical Field]

[0001] This disclosure relates to the field of RNA interference. [Background technology]

[0002] Angiotensinogen (AGT), a type of alpha-globulin, is located at the uppermost level of the renin-angiotensin-aldosterone system (RAAS) blood pressure control pathway and is the sole precursor of all angiotensin peptides. Inactive AGT is converted to active angiotensin II (Ang-II) by sequential cleavage by renin and angiotensin-converting enzyme. Ang-II then acts by binding to its receptors (AT1-AT4).

[0003] Angiotensinogen is primarily synthesized and secreted by the liver and is present in the plasma α-globulin fraction. In addition, angiotensinogen is also present in various tissues that express local RAAS. Elevated angiotensinogen levels are associated with essential hypertension. Transgenic mice expressing the rat angiotensinogen gene were hypertensive, while mice lacking the angiotensinogen gene were hypotensive.

[0004] The regulation of AGT production is multifactorial and occurs at both the transcriptional and post-transcriptional levels. AngII itself, glucocorticoids, estrogen, thyroxine, growth hormone, and various cytokines all influence AGT production. Physiological and pathological conditions such as pregnancy, oral contraceptive use, increased glucocorticoid secretion (due to hyperadrenaline or chronic stress), and chronic inflammation can lead to increased AGT production. Conversely, pituitary, thyroid, gonadal, or adrenal dysfunction is associated with low plasma AGT levels.

[0005] The primary source of circulating AGT is hepatocytes. These cells secrete AGT via a homeostatic pathway, resulting in relatively stable plasma levels unaffected by acute stress or cardiovascular changes. In addition to the liver, tissues such as adipocytes and epithelial cells of the proximal tubules of the kidney, as well as the brain, also express Agt, and its production in these tissues contributes to the activation of local ras. In the brain, AGT is mainly expressed in astrocytes (Milsted et al., 1990), while AT1 is present in brain endothelial cells (Wosik et al., 2007). Interestingly, mice lacking AGT exhibit rupture of the blood-brain barrier and impaired occlusion at tight junctions (Wosik et al., 2007), strongly suggesting that astrocytes maintain the properties of the blood-brain barrier via the AGT-Ang-II-AT1 pathway. Furthermore, evidence of a positive correlation between BMI and circulating AGT levels suggests that AGT production in adipocytes contributes to circulating AGT levels.

[0006] AGT was the first gene found to be associated with essential hypertension. Human genetic evidence and animal experimental evidence indicate that blocking or suppressing AGT expression can result in a significant antihypertensive effect. In this regard, one method of treating hypertension is to reduce AGT expression using small interfering RNAs (siRNAs) based on the RNA interference mechanism.

[0007] The technology requires compositions and methods for treating diseases, disorders, and conditions related to angiotensinogen.

[0008] Reducing AGT expression using double-stranded RNA (dsRNA), particularly small interfering RNA (siRNA), based on RNA interference mechanisms, represents a novel approach to treating angiotensinogen-related diseases, disorders, and conditions. [Overview of the Initiative] [Means for solving the problem]

[0009] This disclosure provides novel double-stranded RNA (dsRNA) for suppressing the expression of angiotensinogen (AGT) in cells, vectors, cells, kits, and pharmaceutical compositions containing the same. This disclosure also provides methods for suppressing or reducing the expression of the angiotensinogen (AGT) gene, or for treating diseases or disorders in which reducing angiotensinogen (AGT) expression is beneficial, using the dsRNA, vectors, cells, pharmaceutical compositions, and kits.

[0010] In a first embodiment, the present disclosure provides a double-stranded nucleotide (dsRNA) for repressing the expression of angiotensinogen (AGT) in cells, wherein the dsRNA comprises a sense strand and an antisense strand forming a double-stranded region, each independently having a length of 15 to 30 nucleotides, and the antisense strand comprises a nucleotide sequence of at least 15 adjacent nucleotides in the nucleotide sequence described in any one of SEQ ID NOs. 21 to 40.

[0011] In some embodiments, the sense strand comprises a nucleotide sequence of at least 15 adjacent nucleotides in the nucleotide sequence described in any one of Sequence IDs 1 to 20.

[0012] In some embodiments, the above dsRNA is siRNA.

[0013] In some embodiments, the double-stranded region has a length of 15 to 25 nucleotide pairs, preferably 16 to 23 nucleotide pairs, and more preferably 18 to 20 nucleotide pairs.

[0014] In some embodiments, the antisense strand comprises a nucleotide sequence of at least 16 adjacent nucleotides, a nucleotide sequence of at least 17 adjacent nucleotides, a nucleotide sequence of at least 18 adjacent nucleotides, a nucleotide sequence of at least 19 adjacent nucleotides, or a nucleotide sequence of at least 20 adjacent nucleotides in any one of the nucleotide sequences described in SEQ ID NOs: 21 to 40. In some preferred embodiments, the antisense strand comprises a nucleotide sequence described in any one of the nucleotide sequences described in SEQ ID NOs: 21 to 40.

[0015] In some embodiments, the sense strand comprises a nucleotide sequence of at least 16 adjacent nucleotides, a nucleotide sequence of at least 17 adjacent nucleotides, or a nucleotide sequence of at least 18 adjacent nucleotides in the nucleotide sequence described in any one of SEQ ID NOs: 1 to 20. In some preferred embodiments, the sense strand comprises a nucleotide sequence described in any one of SEQ ID NOs: 1 to 20.

[0016] In one preferred embodiment, the siRNA comprises one of the following sense strand sequence and antisense strand sequence pairs: (1) Sense chain: Sequence ID 1, Antisense chain: Sequence ID 21; (2) Sense chain: Sequence ID 2, Antisense chain: Sequence ID 22; (3) Sense chain: Sequence ID 3, Antisense chain: Sequence ID 23; (4) Sense chain: Sequence ID 4, Antisense chain: Sequence ID 24; (5) Sense chain: Sequence ID 5, Antisense chain: Sequence ID 25; (6) Sense chain: Sequence ID 6, Antisense chain: Sequence ID 26; (7) Sense chain: Sequence ID 7, Antisense chain: Sequence ID 27; (8) Sense chain: Sequence ID 8, Antisense chain: Sequence ID 28; (9) Sense chain: Sequence ID 9, Antisense chain: Sequence ID 29; (10) Sense chain: Sequence ID 10, Antisense chain: Sequence ID 30; (11) Sense chain: Sequence ID 11, Antisense chain: Sequence ID 31; (12) Sense chain: Sequence ID 12, Antisense chain: Sequence ID 32; (13) Sense chain: Sequence ID 13, Antisense chain: Sequence ID 33; (14) Sense chain: Sequence ID 14, Antisense chain: Sequence ID 34; (15) Sense chain: Sequence ID 15, Antisense chain: Sequence ID 35; (16) Sense chain: Sequence ID 16, Antisense chain: Sequence ID 36; (17) Sense chain: Sequence ID 17, Antisense chain: Sequence ID 37; (18) Sense chain: Sequence ID 18, Antisense chain: Sequence ID 38; (19) Sense chain: Sequence ID 19, Antisense chain: Sequence ID 39; and (20) Sense chain: Sequence ID 20, Antisense chain: Sequence ID 40.

[0017] In some embodiments, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides. In some other embodiments, all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

[0018] In some other embodiments, the sense strand and the antisense strand each independently include one or more nucleotide modifications selected from the group consisting of 2'-O-methyl modifications, 2'-fluoro modifications, SCP modifications, glycol modifications, and phosphorothioate internucleotide linkage modifications.

[0019] In some embodiments, the antisense strand is 21 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1, 3, 5, 9, 11, 13, 15, 17, 19, and 21, and 2'-fluoro modified nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counted from the 5' end); and / or (ii) Internucleotide links of phosphorothioate between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counted from the 5' end) It has.

[0020] In some embodiments, the antisense strand is 21 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1, 3, 5, 9, 11, 13, 15, 17, 19, 20, and 21, and 2'-fluoro modified nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, and 18 (counted from the 5' end); and / or (ii) Internucleotide links of phosphorothioate between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counted from the 5' end) It has.

[0021] In some embodiments, the antisense strand is 21 nucleotides long, and (i) 2'-O-methyl-modified nucleotides at positions 3, 4, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, and 21 (counted from the 5' end); (ii) The 2'-fluoromodified nucleotide at position 14 (counted from the 5' end); (iii) 2'-deoxy modifications at positions 2, 5, 7, and 12 (counted from the 5' end); (iv) SCP modification at position 1 (counted from the 5' end); and / or (v) Internucleotide links of phosphorothioate between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counted from the 5' end) It has.

[0022] In some embodiments, the antisense strand is 21 nucleotides long, and (i) 2'-O-methyl-modified nucleotides at positions 3, 5, 9, 11, 13, 15, 17, 19, 20, and 21 (counted from the 5' end); (ii) 2'-fluoromodified nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, and 18 (counted from the 5' end); (iii) GNA modification at position 7 (counted from the 5' end); (iv) SCP modification at position 1 (counted from the 5' end); and / or (v) Internucleotide links of phosphorothioate between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counted from the 5' end) It has.

[0023] In some embodiments, the sense strand is 19 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1-6 and 10-19 and 2'-fluoro modified nucleotides at positions 7-9 (counted from the 5' end); and / or (ii) Phosphothioate internucleotide bonds (counted from the 5' end) between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between nucleotide positions 18 and 19 It has.

[0024] In some embodiments, the antisense strand includes a sequence selected from any one of the sequences described in Sequence IDs 81 to 105.

[0025] In some embodiments, the sense strand includes an array selected from any one of the arrays described in Sequence IDs 41 to 59.

[0026] In some preferred embodiments, the dsRNA comprises one of the following pairs of sense strand sequence and antisense strand sequence: Sense chain: Sequence ID 41, Antisense chain: Sequence ID 81; Sense chain: Sequence ID 42, Antisense chain: Sequence ID 82; Sense chain: SEQ ID NO: 43, Antisense chain: SEQ ID NO: 83; Sense chain: Sequence ID 44, Antisense chain: Sequence ID 84; Sense chain: SEQ ID NO: 45, Antisense chain: SEQ ID NO: 85; Sense chain: Sequence ID 46, Antisense chain: Sequence ID 86; Sense chain: Sequence ID 47, Antisense chain: Sequence ID 87; Sense chain: SEQ ID NO: 48, Antisense chain: SEQ ID NO: 88; Sense chain: Sequence ID 49, Antisense chain: Sequence ID 89; Sense chain: SEQ ID NO: 50, Antisense chain: SEQ ID NO: 90; Sense chain: Sequence ID 51, Antisense chain: Sequence ID 91; Sense chain: Sequence ID 52, Antisense chain: Sequence ID 92; Sense chain: SEQ ID NO: 53, Antisense chain: SEQ ID NO: 93; Sense chain: Sequence ID 54, Antisense chain: Sequence ID 94; Sense chain: SEQ ID NO: 55, Antisense chain: SEQ ID NO: 95; Sense chain: SEQ ID NO: 56, Antisense chain: SEQ ID NO: 96; Sense chain: Sequence ID 57, Antisense chain: Sequence ID 97; Sense strand: SEQ ID NO: 58, Antisense strand: SEQ ID NO: 98; and Sense chain: Sequence ID 59, Antisense chain: Sequence ID 99.

[0027] In some embodiments, the sense strand includes an array selected from any one of the arrays described in sequence numbers 113 to 131.

[0028] In some embodiments, the dsRNA includes one of the following pairs of sense strand sequence and antisense strand sequence: Sense chain: Sequence ID 113, Antisense chain: Sequence ID 81; Sense chain: Sequence ID 114, Antisense chain: Sequence ID 82; Sense chain: Sequence ID 115, Antisense chain: Sequence ID 83; Sense chain: Sequence ID 116, Antisense chain: Sequence ID 84; Sense chain: Sequence ID 117, Antisense chain: Sequence ID 85; Sense chain: Sequence ID 118, Antisense chain: Sequence ID 86; Sense chain: Sequence ID 119, Antisense chain: Sequence ID 87; Sense chain: Sequence ID 120, Antisense chain: Sequence ID 88; Sense chain: Sequence ID 121, Antisense chain: Sequence ID 89; Sense chain: Sequence ID 122, Antisense chain: Sequence ID 90; Sense chain: Sequence ID 123, Antisense chain: Sequence ID 91; Sense chain: Sequence ID 124, Antisense chain: Sequence ID 92; Sense chain: Sequence ID 125, Antisense chain: Sequence ID 93; Sense chain: Sequence ID 126, Antisense chain: Sequence ID 94; Sense chain: Sequence ID 127, Antisense chain: Sequence ID 95; Sense chain: Sequence ID 128, Antisense chain: Sequence ID 96; Sense chain: Sequence ID 129, Antisense chain: Sequence ID 97; Sense strand: SEQ ID NO: 130, Antisense strand: SEQ ID NO: 98; and Sense chain: Sequence ID 131, Antisense chain: Sequence ID 99.

[0029] In some embodiments, the dsRNA is further conjugated to a ligand moiety containing N-acetylgalactosamine, preferably the 3' end of the sense strand is conjugated to the ligand moiety.

[0030] In some embodiments, the ligand has the following structure. [ka] During the ceremony, [ka] This indicates a point that connects to the sense strand of dsRNA via a phosphate group or phosphorothioate group.

[0031] In some preferred embodiments, the dsRNA comprises one of the following pairs of sense strand sequence and antisense strand sequence: Sense chain: SEQ ID NO: 60, Antisense chain: SEQ ID NO: 81; Sense chain: Sequence ID 61, Antisense chain: Sequence ID 82; Sense chain: Sequence ID 62, Antisense chain: Sequence ID 83; Sense chain: Sequence ID 63, Antisense chain: Sequence ID 84; Sense chain: Sequence ID 64, Antisense chain: Sequence ID 85; Sense chain: Sequence ID 65, Antisense chain: Sequence ID 86; Sense chain: Sequence ID 66, Antisense chain: Sequence ID 87; Sense chain: Sequence ID 67, Antisense chain: Sequence ID 88; Sense chain: SEQ ID NO: 68, Antisense chain: SEQ ID NO: 89; Sense chain: SEQ ID NO: 69, Antisense chain: SEQ ID NO: 90; Sense chain: Sequence ID 70, Antisense chain: Sequence ID 91; Sense chain: Sequence ID 71, Antisense chain: Sequence ID 92; Sense chain: Sequence ID 72, Antisense chain: Sequence ID 93; Sense chain: Sequence ID 73, Antisense chain: Sequence ID 94; Sense chain: Sequence ID 74, Antisense chain: Sequence ID 95; Sense chain: Sequence ID 75, Antisense chain: Sequence ID 96; Sense chain: Sequence ID 76, Antisense chain: Sequence ID 97; Sense chain: Sequence ID 77, Antisense chain: Sequence ID 98; Sense chain: SEQ ID NO: 78, Antisense chain: SEQ ID NO: 99; Sense chain: Sequence ID 72, Antisense chain: Sequence ID 100; Sense chain: Sequence ID 72, Antisense chain: Sequence ID 101; Sense chain: Sequence ID 72, Antisense chain: Sequence ID 102; Sense chain: Sequence ID 72, Antisense chain: Sequence ID 103; Sense strand: Sequence ID 79, Antisense strand: Sequence ID 104; and Sense chain: Sequence ID 79, Antisense chain: Sequence ID 105.

[0032] In a second embodiment, the Disclosure provides a vector comprising a nucleotide sequence encoding the dsRNA of the Disclosure.

[0033] In a third embodiment, the Disclosure provides cells comprising the dsRNA or vector of the Disclosure.

[0034] In a fourth aspect, the Disclosure provides a pharmaceutical composition comprising the dsRNA, vector, or cell of the Disclosure and, optionally, a pharmaceutically acceptable carrier or excipient.

[0035] In a fifth embodiment, the Disclosure provides a kit comprising the dsRNA, vector, or cells of the Disclosure.

[0036] In a sixth aspect, the Disclosure provides a method for reducing angiotensinogen (AGT) in a subject, comprising the step of administering the dsRNA, vector, cells, or pharmaceutical composition of the Disclosure to the subject. The Disclosure also provides a method for treating a disease or disorder in which reducing the expression of angiotensinogen (AGT) in a subject is beneficial, comprising the step of administering the dsRNA, vector, cells, or pharmaceutical composition of the Disclosure to the subject. The Disclosure also provides a method for preventing at least one symptom in a subject suffering from a disease or disorder in which reducing the expression of angiotensinogen (AGT) is beneficial, comprising the step of administering the dsRNA, vector, cells, or pharmaceutical composition of the Disclosure to the subject.

[0037] In some embodiments, diseases or disorders for which reducing the expression of the above-mentioned angiotensinogen (AGT) is beneficial are AGT-mediated diseases or AGT-related diseases.

[0038] In some embodiments, the AGT-mediated disease or AGT-related disease is selected from the group consisting of hypertension, intraocular hypertension, glaucoma, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, stroke, nephropathy, renal failure, systemic sclerosis, intrauterine growth restriction (IUGR), fetal growth restriction, obesity, fatty liver / fatty liver disease, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD); glucose intolerance, type 2 diabetes mellitus (non-insulin-dependent diabetes mellitus), and metabolic syndrome. In some embodiments, the hypertension is selected from the group consisting of borderline hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, treatment-resistant hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, and unstable hypertension.

[0039] In some embodiments, the methods provided in this disclosure for treating a disease or disorder in which reducing the expression of angiotensinogen (AGT) in a subject is beneficial, for preventing at least one symptom in a patient suffering from a disease or disorder in which reducing the expression of angiotensinogen (AGT) is beneficial, or for reducing angiotensinogen (AGT) in a subject, include subcutaneous, topical, or intravenous administration of the dsRNA, vector, cells, or pharmaceutical composition to the subject.

[0040] In some embodiments, the subject is a human patient. [Brief explanation of the drawing]

[0041] [Figure 1] This figure shows the effect of the test compound on reducing serum AGT levels in cynomolgus monkeys.

[0042] [Figure 2] This figure shows the effect of test compounds on reducing systolic blood pressure in cynomolgus monkeys.

[0043] [Figure 3] This figure shows the effect of the test compound on reducing diastolic blood pressure in cynomolgus monkeys.

[0044] [Figure 4] This figure shows the effect of test compounds on reducing mean arterial pressure in cynomolgus monkeys. [Modes for carrying out the invention]

[0045] Embodiments of this disclosure will be described below through specific examples. Those skilled in the art will readily understand other advantages and effects of this disclosure from this specification. Furthermore, this disclosure can also be implemented or applied through other specific embodiments. Various details in this specification can be modified or changed in various ways on other viewpoints and uses without departing from the spirit of this disclosure.

[0046] Please understand that the scope of protection of this disclosure is not limited to the following specific embodiments. Furthermore, please understand that the terminology used in the embodiments of this disclosure is intended to describe the specific embodiments, not to limit the scope of protection of this disclosure.

[0047] In the specification and claims of this disclosure, the singular forms "a," "an," and "the" include the plural forms unless explicitly stated otherwise in the context.

[0048] Where numerical ranges are given in embodiments, it should be understood that, unless otherwise specifically mentioned in this disclosure, any values ​​at both endpoints of each numerical range and any values ​​between those endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. In addition to the specific methods, apparatus, and materials used in embodiments, those skilled in the art can also implement this disclosure using similar or equivalent prior art methods, apparatus, and materials, based on their understanding of the prior art and the specifications of this disclosure, and all of these are within the scope of protection of this disclosure. Embodiments of this disclosure are described in more detail below.

[0049] definition

[0050] In this specification, “double-stranded region” refers to a region comprising two nucleic acid strands that are antiparallel and complementary or substantially complementary to each other.

[0051] In this specification, "double-stranded RNA" or "dsRNA" refers to a ribonucleic acid molecule or a complex of ribonucleic acid molecules containing the double-stranded region as defined above. The two parts forming the double-stranded region may be two different parts of a larger RNA molecule, or they may be separate RNA molecules.

[0052] When the two parts are separate RNA molecules, dsRNA as used herein refers to small interfering RNA or short interfering RNA, and is abbreviated as siRNA.

[0053] When two parts are two distinct parts of a larger molecule, that is, when a double-stranded region is formed by the 3' end of one part joining the 5' end of another part via one or more uninterrupted nucleotides, the uninterrupted nucleotides used in this joining are called a "hairpin loop." When two parts are covalently joined by means other than a hairpin loop to form a double-stranded region, the joining structure is called a "linker." When such dsRNA is introduced into a cell, it can be cleaved by an endribonuclease called a dicer enzyme within the cell to become siRNA.

[0054] In this specification, “siRNA” refers to a class of dsRNA molecules comprising a sense strand and an antisense strand that can mediate the silencing of a target RNA (e.g., mRNA such as a protein-coding gene transcript) that is complementary or substantially complementary to the antisense strand. siRNA is generally double-stranded and comprises an antisense strand complementary to its target RNA and a sense strand complementary or substantially complementary to the antisense strand. For convenience, in this specification, such mRNA is also referred to as “silencing target mRNA,” and such a gene is also referred to as “target gene.” Typically, silencing target RNA is an endogenous gene or a pathogen gene. In addition, RNA other than mRNA (e.g., tRNA) and viral RNA can also be targeted.

[0055] In this specification, “antisense strand” refers to a strand in dsRNA, particularly in siRNA, which includes a region that is completely or substantially complementary to its target sequence.

[0056] In this specification, the “complementary region” refers to a region on the antisense strand that is completely or substantially complementary to its target mRNA sequence. If the complementary region is not completely complementary to its target sequence, mismatches may be located in the internal or terminal regions of the molecule. Typically, the most acceptable mismatches are located in terminal regions of, for example, 5' and / or 3' ends, within 5, 4, 3, 2, or 1 nucleotide. The region of the antisense strand that is most sensitive to mismatches is called the “seed region.” For example, in an siRNA containing a 19nt strand, position 19 (5' to 3') may tolerate several mismatches.

[0057] In this specification, “complementary” refers to the ability of a first polynucleotide to hybridize with a second polynucleotide under certain conditions, such as stringent conditions. For example, stringent conditions include 400 mM NaCl, 40 mM PIPES, pH 6.4, 1 mM EDTA, and 12–16 hours at 50°C or 70°C.

[0058] In this specification, a “complementary” sequence for satisfying the above requirements regarding its hybridizing ability may include, and may consist solely of, non-Watson-Crick base pairs and / or base pairs formed with non-natural and modified nucleotides. Examples of such non-Watson-Crick base pairs include, but are not limited to, G:U fluctuation base pairs or Hoogsteen base pairs.

[0059] In this specification, a polynucleotide that is "at least partially complementary" or "substantially complementary" to messenger RNA (mRNA) means a polynucleotide that is substantially complementary to the continuous portion of the mRNA of interest (such as the mRNA encoding AGT). For example, a polynucleotide is at least partially complementary to the mRNA encoding AGT if its sequence is substantially complementary to the uninterrupted portion of the AGT mRNA.

[0060] In this specification, “complementary,” “fully complementary,” and “substantially complementary” can be used with respect to base pairs between the sense strand and antisense strand of dsRNA, particularly siRNA, or between the antisense strand and target sequence of dsRNA, particularly siRNA.

[0061] In this specification, “sense strand” refers to one strand of siRNA that contains a region substantially complementary to the antisense strand region as defined herein.

[0062] A "nucleoside" is a compound containing two components: one is a purine or pyrimidine base, and the other is ribose or deoxyribose. A "nucleotide" is a compound containing three components: one is a purine or pyrimidine base, another is ribose or deoxyribose, and the third is phosphate. An "oligonucleotide" refers to a nucleic acid molecule (RNA or DNA) that is less than 100, 200, 300, or 400 nucleotides in length, for example.

[0063] A "base" is the fundamental building block of nucleosides, nucleotides, and nucleic acids, and is also called a "nitrogen base" because it always contains nitrogen. Unless otherwise specified, capital letters in this specification, namely A, U, T, G, and C, indicate the bases of nucleotides, representing adenine, uracil, thymine, guanine, and cytosine, respectively.

[0064] In this specification, “nucleotide overhang” refers to at least one unpaired nucleotide protruding from the double-stranded region of an siRNA. A nucleotide overhang exists, for example, when the 3' end of one strand of an siRNA is longer than the 5' end of the other strand, or vice versa. An siRNA may contain overhangs having at least one nucleotide, at least two nucleotides, at least three nucleotides, at least four nucleotides, or at least five or more nucleotides. A nucleotide overhang may contain or be composed of nucleotides / modified nucleotides (including deoxyribonucleotides / nucleosides). One or more overhangs may be on the sense strand or the antisense strand, or a combination thereof. An overhang having one or more nucleotides may be located at the 5' end, 3' end, or both ends of the antisense strand or sense strand of an siRNA.

[0065] "Blunt-ended" means that there are no unpaired nucleotides, or nucleotide overhangs, at the end of a double-stranded siRNA molecule. "Blunt-ended siRNA" is siRNA that is double-stranded throughout its entire length, meaning that there are no nucleotide overhangs at any end of the molecule.

[0066] The dsRNAs, particularly siRNAs, of this disclosure are substantially all modified nucleotides. For example, substantially all nucleotides of the sense strand are modified nucleotides, substantially all nucleotides of the antisense strand are modified nucleotides, or substantially all nucleotides of both the sense and antisense strands are modified nucleotides. In other embodiments of this disclosure, all nucleotides of the dsRNAs, particularly siRNAs, of this disclosure are modified nucleotides. For example, all nucleotides of the sense strand are modified nucleotides, all nucleotides of the antisense strand are modified nucleotides, or all nucleotides of both the sense and antisense strands are modified nucleotides. In this specification, “substantially all nucleotides are modified” means that the majority (but not necessarily all) of the nucleotides of the dsRNAs, particularly siRNAs, of this disclosure are modified, and that there may be at most 5, 4, 3, 2, or 1 unmodified nucleotide.

[0067] In this specification, "modified nucleotide" is not particularly limited to, but includes 2'-O-alkyl-modified nucleotides (such as 2'-O-methyl-modified nucleotides or 2'-methoxyethyl-modified nucleotides), 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, SCP-modified nucleotides, inosine ribonucleotides, debased nucleotides, reverse debased deoxyribonucleotides, nucleotides containing a phosphorothioate group, vinyl phosphate-modified nucleotides, locked nucleotides, unlocked nucleotides, 2'-amino-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, and morpholino This includes nucleotides, phosphoramidates, nucleotides containing non-natural bases, cholesteryl derivatives or terminal nucleotides bonded to a bisdecylamide dodecanoate group, deoxyribonucleotides, 3'-terminal deoxythymine (dT) nucleotides, sterically restricted nucleotides, restricted ethyl nucleotides, 2'-hydroxy-modified nucleotides, nucleotides containing a methylphosphonate group, nucleotides containing 5'-phosphate, nucleotides containing a 5'-phosphate mimetic, glycol-modified nucleotides (GNAs), phosphorothioate internucleotide bond modifications, and 2-O-(N-methylacetamide)-modified nucleotides, etc.

[0068] For example, a "2'-fluoromodified nucleotide" refers to a nucleotide in which the hydroxyl atom at the 2' position of the ribosyl group is replaced with a fluorine atom. A "2'-O-methyl-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl atom of the ribosyl group is replaced with a methoxyl atom.

[0069] A "phosphorothioate-containing nucleotide" refers to a nucleotide in which one or more oxygen atoms on the phosphate group of the nucleotide are replaced with sulfur atoms. A "phosphorothioate internucleotide bond modification" refers to a modification in which two adjacent nucleotides are joined by a phosphorothioate.

[0070] In some embodiments, the sense strand of the dsRNA, particularly siRNA, of the Disclosure has one or two phosphorothioate nucleotide linkage modifications at positions 1–5 (counted from the 5' end) and / or one or two phosphorothioate nucleotide linkage modifications at positions 1–5 (counted from the 3' end), and / or the antisense strand of the dsRNA, particularly siRNA, of the Disclosure has one or two phosphorothioate nucleotide linkage modifications at positions 1–5 (counted from the 5' end) and / or one or two phosphorothioate nucleotide linkage modifications at positions 1–5 (counted from the 3' end).

[0071] In some embodiments, the sense strand of the dsRNA, particularly siRNA, of the Disclosure has one or two phosphorothioate nucleotide linkage modifications at positions 1-4 (counted from the 5' end) and / or one or two phosphorothioate nucleotide linkage modifications at positions 1-4 (counted from the 3' end), and / or the antisense strand of the dsRNA, particularly siRNA, of the Disclosure has one or two phosphorothioate nucleotide linkage modifications at positions 1-4 (counted from the 5' end) and / or one or two phosphorothioate nucleotide linkage modifications at positions 1-4 (counted from the 3' end).

[0072] In some embodiments, the sense strand of the dsRNA, particularly siRNA, of the Disclosure has one or two phosphorothioate nucleotide linkage modifications at positions 1-3 (counted from the 5' end) and / or one or two phosphorothioate nucleotide linkage modifications at positions 1-3 (counted from the 3' end), and / or the antisense strand of the dsRNA, particularly siRNA, of the Disclosure has one or two phosphorothioate nucleotide linkage modifications at positions 1-3 (counted from the 5' end) and / or one or two phosphorothioate nucleotide linkage modifications at positions 1-3 (counted from the 3' end).

[0073] In some embodiments, the sense strand of the dsRNA, particularly siRNA, of the Disclosure has one or two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counted from the 5' end) and / or one or two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counted from the 3' end) and / or the antisense strand of the dsRNA, particularly siRNA, of the Disclosure has one or two phosphorothioate nucleotide linkage modifications at positions 1-2 (counted from the 5' end) and / or one or two phosphorothioate nucleotide linkage modifications at positions 1-2 (counted from the 3' end).

[0074] In some embodiments, the sense strand of the dsRNA, particularly siRNA, of the Disclosure has one or two phosphorothioate nucleotide binding modifications at positions 1 and 2 (counted from the 5' end) and / or one or two phosphorothioate nucleotide binding modifications at positions 1 and 2 (counted from the 3' end), and / or the antisense strand of the dsRNA, particularly siRNA, of the Disclosure has one or two phosphorothioate nucleotide binding modifications at positions 1-2 (counted from the 5' end) and / or one or two phosphorothioate nucleotide binding modifications at positions 1-2 (counted from the 3' end), the 3' end of the sense strand is bound to the ligand moiety, and the nucleotide at the 3' end of the sense strand is bound to the ligand moiety by phosphate binding.

[0075] In some embodiments, the sense strand of the dsRNA, particularly siRNA, of the Disclosure has one or two phosphorothioate internucleotide binding modifications at positions 1 and 2 (counted from the 5' end) and / or one or two phosphorothioate internucleotide binding modifications at positions 1 and 2 (counted from the 3' end), and / or the antisense strand of the dsRNA, particularly siRNA, of the Disclosure has one or two phosphorothioate internucleotide binding modifications at positions 1-2 (counted from the 5' end) and / or one or two phosphorothioate internucleotide binding modifications at positions 1-2 (counted from the 3' end), the 3' end of the sense strand is bound to the ligand moiety, and the nucleotide at the 3' end of the sense strand is bound to the ligand moiety by a phosphorothioate bond.

[0076] In this specification, “ligand moiety” refers to a chemical component conjugated with dsRNA, particularly siRNA, which can alter the distribution, target orientation, or lifetime of the dsRNA, particularly siRNA. In some embodiments, such ligand moieties improve affinity to selected targets, such as molecules, cells or cell types, and compartments (e.g., compartments of cells or organs, tissues, organs, or regions of the body), compared to siRNA without a ligand moiety. In some embodiments, the ligand moiety targets the asialocrycoprotein receptor (ASGPR) on hepatocytes. When the ligand moiety binds to ASGPR, internalization by clathrin-coated vesicles is mediated. As the endosome matures, the lysosomal pH decreases, promoting dissociation of the ligand-receptor complex and releasing the dsRNA, particularly siRNA. Conjugating a ligand moiety that targets the asialocrycoprotein receptor (ASGPR) on hepatocytes provides efficacy and stability of dsRNA, particularly siRNA, in vivo or intracellularly. This facilitates subcutaneous administration of dsRNA, particularly siRNA.

[0077] In this specification, “suppression” is used interchangeably with similar terms such as “reduction,” “silencing,” and “downward control,” and encompasses any level of suppression.

[0078] "Suppressing angiotensinogen (AGT) expression" refers to suppressing the expression of any AGT gene and its variants or mutants. Therefore, in the case of genetically modified cells, cell populations, or organisms, the AGT gene may be a wild-type AGT gene, a mutant AGT gene, or a transgenic AGT gene.

[0079] "Suppression of AGT gene expression" encompasses any level of suppression of the AGT gene, including at least partial suppression of AGT gene expression. AGT gene expression can be assessed based on the level of any variable related to AGT gene expression, such as AGT mRNA levels or AGT protein levels, or a change in such levels. This level may be assessed in a single cell or in a population of cells (e.g., including a sample derived from the subject).

[0080] Suppression can be assessed by reducing the absolute or relative level of one or more variables related to AGT expression compared to the control level. The control level can be any type of control level used in the field, such as the baseline level before administration or the level determined for subjects, cells, or samples treated with similar untreated or controlled (such as a buffer-only control or an inactivator control).

[0081] A "hydroxyl protecting group" refers to a group that prevents hydroxyl from chemically reacting and can be removed under certain conditions to regenerate hydroxyl. Examples of hydroxyl protecting groups mainly include silane, acyl, or ether-based protecting groups, preferably trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, and 9-fluorenyl methoxycarboxymethylcellulose. Examples include carbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), di-p-methoxytrityl (DMTr), methoxymethyl (MOM), benzyloxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), and p-methoxybenzyloxymethyl (PMBM).

[0082] "Halo" or "halogen" refers to substitutions by fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0083] "C 1-6 "Haloalkyl" is the same as the above "C 1-6 This refers to a case where "alkyl" is substituted with one or more halogen groups. In some embodiments, C 1-4 Haloalkyl is particularly preferred, C 1-2Haloalkyl is even more preferred. Examples of haloalkyl include, but are not particularly limited to, -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, and 2,2,2-trifluoro-1,1-dimethyl-ethyl, etc. The haloalkyl may be substituted at any substitutable bonding point with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, etc.

[0084] “C 1-6 alkylene” refers to a divalent group formed by removing another hydrogen of C 1-6 alkyl, and may or may not be substituted. In some embodiments, C 1-4 alkylene, C 2-4 alkylene, and C 1-2 alkylene are preferred. Examples of unsubstituted alkylene include, but are not particularly limited to, methylene group (-CH2-), ethylene group (-CH2CH2-), propylene group (-CH2CH2CH2-), butylene group (-CH2CH2CH2CH2-), pentylene group (-CH2CH2CH2CH2CH2-), and hexylene group (-CH2CH2CH2CH2CH2CH2-), etc. Examples of the above-mentioned substituted alkylene, for example, alkylene substituted with one or more alkyl (methyl) groups include, but are not particularly limited to, substituted methylene (-CH(CH3)- and -C(CH3)2-), substituted ethylene (-CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, and -CH2C(CH3)2-), substituted propylene (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2C(CH)2-), etc.

[0085] In this specification, “vector” refers to a nucleic acid molecule capable of amplifying or expressing another nucleic acid to which it is bound. In general, “vector” in this specification is a vector capable of self-replication in a host cell, preferably a multicopy vector. In addition, vectors typically have markers, such as antibiotic resistance genes, for selecting transformants. Furthermore, vectors may have promoters and / or terminators for expressing the introduced gene. Vectors may be, for example, bacterial plasmid-derived vectors, viral vectors, yeast plasmid-derived vectors, bacteriophage-derived vectors, cosmids, phagemids, and the like.

[0086] In this specification, “to treat” and “treatment,” etc., refer to administering a drug or performing a procedure to obtain an effect. The effect may be preventive in that it completely or partially prevents a disease or its symptoms, and / or therapeutic in that it affects the partial or complete cure of the disease and / or its symptoms. In this specification, “to treat” may include treating a disease or disorder (such as cancer) in a mammal, in particular a human, and includes (a) preventing the onset of the disease or its symptoms (such as a disease that may be related to or caused by the underlying disease) in a subject susceptible to the disease but not diagnosed with the disease; (b) suppressing the disease, i.e., preventing its progression; and (c) alleviating the disease, i.e., causing the disease to regress. Treatment may also refer to the success of treating, improving or preventing cancer, and may include objective or subjective parameters such as disappearance; remission; reduction of symptoms or a disease state that is tolerable to the patient; delay of exacerbation or decline; or reduction of weakness in the exacerbation endpoint. Treatment or improvement of symptoms is based on one or more objective or subjective parameters, including the results of a physician’s examination. Accordingly, “treatment” includes administering the antibodies, compositions, or conjugates disclosed herein to prevent, delay, alleviate, halt, or suppress the onset of symptoms or conditions associated with a disease (such as cancer). “Therapeutic effect” means reducing, eliminating, or preventing a disease, its symptoms, or its side effects in a subject.

[0087] In this specification, “effective dose” means the amount that, when administered to a subject for the treatment of a disease, produces a therapeutic effect on that disease.

[0088] In this specification, “subject” refers to a mammal subject for which diagnosis, cure, or treatment is desired. “Mammal” for therapeutic purposes refers to any animal classified as a mammal, including humans, dogs, horses, cattle, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, and other domestic animals such as monkeys, laboratory animals, zoo animals, sports animals, or pet animals.

[0089] I.dsRNA

[0090] This disclosure provides a double-stranded RNA (dsRNA) for suppressing the expression of angiotensinogen (AGT) in cells, wherein the dsRNA comprises a sense strand and an antisense strand forming a double-stranded region, each independently having a length of 15 to 30 nucleotides, and the antisense strand contains the nucleotide sequence of at least 15 adjacent nucleotides in the nucleotide sequence described in any one of Sequence IDs 21 to 40.

[0091] In some embodiments, the double-stranded regions formed by the sense strand and the antisense strand are completely complementary. In other embodiments, the double-stranded regions formed by the sense strand and the antisense strand are substantially complementary and may include one, two, three, four, or five non-complementary sites.

[0092] In some specific embodiments, the sense strand comprises a nucleotide sequence of at least 15 adjacent nucleotides in the nucleotide sequence described in any one of Sequence IDs 1 to 20.

[0093] In some embodiments, the sense strand and the antisense strand are each independently 15 to 27 nucleotides long, preferably 18 to 25 nucleotides long, and more preferably 19 to 21 nucleotides long.

[0094] In some embodiments, the double-stranded region has a length of 15 to 25 nucleotide pairs, preferably 16 to 23 nucleotide pairs, and more preferably 18 to 20 nucleotide pairs.

[0095] In some embodiments, the dsRNA of the Disclosure is siRNA. In other embodiments, a hairpin loop is formed between the sense strand and the antisense strand of the dsRNA of the Disclosure.

[0096] In some embodiments, one or both of the sense strand and the antisense strand include a 3' overhang and / or 5' overhang having at least one nucleotide. In some specific embodiments, one or both of the sense strand and the antisense strand include a 3' overhang and / or 5' overhang having at least one nucleotide. In some specific embodiments, one or both of the sense strand and the antisense strand include a 3' overhang and / or 5' overhang having one nucleotide. In some specific embodiments, one or both of the sense strand and the antisense strand include a 3' overhang and / or 5' overhang having two nucleotides. In some specific embodiments, one or both of the sense strand and the antisense strand include a 3' overhang and / or 5' overhang having three nucleotides. In some specific embodiments, one or both of the sense strand and the antisense strand include a 3' overhang and / or 5' overhang having four nucleotides.

[0097] In some specific embodiments, the antisense strand includes a 3' overhang and / or a 5' overhang having one nucleotide. In some embodiments, the antisense strand includes a 3' overhang and / or a 5' overhang having two nucleotides. In some embodiments, the antisense strand includes a 3' overhang and / or a 5' overhang having three nucleotides. In some embodiments, the antisense strand includes a 3' overhang and / or a 5' overhang having four nucleotides. In some preferred embodiments, the antisense strand includes a 3' overhang and / or a 5' overhang having at least two nucleotides.

[0098] The antisense strand preferably includes a 3' overhang and / or a 5' overhang having two nucleotides.

[0099] In some embodiments, the sense chain and the antisense chain are of the same length.

[0100] In some embodiments, the total length of the sense strand is complementary to the total length of the antisense strand, forming a double helix, i.e., having blunt ends.

[0101] In some other embodiments, the sense strand and the antisense strand are of the same length, and a portion of the sense strand is complementary to a portion of the antisense strand, i.e., both the sense strand and the antisense strand have a 5' overhang. In some embodiments, the sense strand and the antisense strand are of different lengths. In a preferred embodiment, the 5' end of the antisense strand has an overhang of at least one nucleotide, more preferably two or three nucleotides.

[0102] The dsRNAs of this disclosure include dsRNAs having a nucleotide overhang at one end (i.e., a substance having one overhang and one blunt end), or dsRNAs having nucleotide overhangs at both ends. For example, the 5' end of the sense strand of a dsRNA includes an overhang having one or more nucleotides, and the 3' end of the sense strand includes an overhang having one or more nucleotides. For example, the 5' end of the antisense strand of a dsRNA includes an overhang having one or more nucleotides, and the 3' end of the antisense strand includes an overhang having one or more nucleotides. For example, the 5' end of the sense strand of a dsRNA includes an overhang having one or more nucleotides, and the 5' end of the antisense strand includes an overhang having one or more nucleotides. For example, the 3' end of the sense strand of a dsRNA includes an overhang having one or more nucleotides, and the 3' end of the antisense strand includes an overhang having one or more nucleotides. For example, the 5' end of the sense strand of dsRNA contains an overhang with one or more nucleotides, and the 3' end of the sense strand contains a blunt end. For example, the 3' end of the sense strand of dsRNA contains an overhang with one or more nucleotides, and the 3' end of the sense strand contains a blunt end. For example, the 5' end of the antisense strand of dsRNA contains an overhang with one or more nucleotides, and the 3' end of the antisense strand contains a blunt end. For example, the 3' end of the antisense strand of dsRNA contains an overhang with one or more nucleotides, and the 5' end of the antisense strand contains a blunt end.

[0103] In some preferred embodiments, the 3' end of the antisense strand of the dsRNA of the Disclosure includes an overhang having one or more nucleotides, and the 5' end of the antisense strand includes a blunt end. In some more preferred embodiments, the 3' end of the antisense strand of the dsRNA of the Disclosure includes an overhang having one, two, three, or four nucleotides, and the 5' end of the antisense strand includes a blunt end. In some more preferred embodiments, the 3' end of the antisense strand of the dsRNA of the Disclosure includes an overhang having two nucleotides, and the 5' end of the antisense strand includes a blunt end.

[0104] In some embodiments, the antisense strand comprises a nucleotide sequence of at least 16 adjacent nucleotides, a nucleotide sequence of at least 17 adjacent nucleotides, a nucleotide sequence of at least 18 adjacent nucleotides, a nucleotide sequence of at least 19 adjacent nucleotides, or a nucleotide sequence of at least 20 adjacent nucleotides in the nucleotide sequence described in any one of SEQ ID NOs: 21 to 40, and preferably, the antisense strand comprises a nucleotide sequence described in any one of SEQ ID NOs: 21 to 40.

[0105] In some embodiments, the sense strand comprises a nucleotide sequence of at least 16 adjacent nucleotides, a nucleotide sequence of at least 17 adjacent nucleotides, or a nucleotide sequence of at least 18 adjacent nucleotides from any one of the nucleotide sequences described in SEQ ID NOs: 1 to 20, and preferably, the sense strand comprises a nucleotide sequence described in any one of SEQ ID NOs: 1 to 20.

[0106] In some embodiments, the dsRNA includes any sense-antisense sequence pair from the sense-antisense sequence pairs listed in Table 3.

[0107] II. Modification of Nucleotides

[0108] In some embodiments, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides. In some embodiments, at least 80% of the nucleotides of the sense strand are modified nucleotides, and / or at least 80%, at least 85%, at least 90%, at least 92%, and at least 95% of the nucleotides of the antisense strand are modified nucleotides. In some embodiments, at least 80% of the nucleotides of the antisense strand are modified nucleotides, and / or at least 80%, at least 85%, at least 90%, at least 92%, and at least 95% of the nucleotides of the sense strand are modified nucleotides.

[0109] In some embodiments, all nucleotides of the sense strand are modified nucleotides and / or all nucleotides of the antisense strand are modified nucleotides.

[0110] The nucleotide modifications described herein may be modifications to the phosphate group, ribose group, and / or base of the nucleotide.

[0111] In some specific embodiments, the sense chain and the antisense chain are independently 2'-O-alkyl-modified nucleotides (such as 2'-O-methyl-modified nucleotides), 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, debased nucleotides, reverse debased deoxyribonucleotides, nucleotides containing a phosphorothioate group, vinyl phosphate-modified nucleotides, locked nucleotides, unlocked nucleotides, 2'-amino-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, morpholino nucleotides, phosphoramidates, non-phosphorine nucleotides. The nucleotide modification comprises one or more nucleotide modifications selected from the group consisting of nucleotides containing a natural base, terminal nucleotides bonded to a cholesteryl derivative or bisdecylamide dodecanoate group, deoxyribonucleotides, 3'-terminal deoxythymine (dT) nucleotides, sterically restricted nucleotides, restricted ethyl nucleotides, 2'-hydroxy-modified nucleotides, nucleotides containing a methylphosphonate group, nucleotides containing 5'-phosphate, nucleotides containing a 5'-phosphate mimetic, SCP modifications, glycol-modified nucleotides (GNAs), phosphorothioate internucleotide bond modifications, and 2-O-(N-methylacetamide)-modified nucleotides.

[0112] In some preferred embodiments, the sense strand and the antisense strand each independently include one or more nucleotide modifications selected from the group consisting of 2'-O-methyl modifications, 2'-fluoro modifications, SCP modifications, and phosphorothioate internucleotide bond modifications.

[0113] In some preferred embodiments, the sense strand and / or antisense strand comprises at least two 2'-fluoromodified nucleotides. In some preferred embodiments, the sense strand and / or antisense strand comprises at least eight 2'-O-methylmodified nucleotides. In some preferred embodiments, the 3' and / or 5' ends of the sense strand and / or antisense strand comprise 1 to 5 phosphorothioate nucleotide interbonds, preferably 2 to 3 phosphorothioate nucleotide interbonds. In some embodiments, the sense strand and / or antisense strand comprises adenine deoxyribonucleotide, thymine deoxyribonucleotide, guanine deoxyribonucleotide, and / or cytosine deoxyribonucleotide. In a more preferred embodiment, the sense strand and / or antisense strand comprises thymine deoxyribonucleotide. In the most preferred embodiment, the sense strand comprises thymine deoxyribonucleotide.

[0114] In some embodiments, the antisense strand is 21 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1, 3, 5, 9, 11, 13, 15, 17, 19, and 21, and 2'-fluoro modified nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counted from the 5' end); and / or (ii) Internucleotide links of phosphorothioate between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counted from the 5' end) It has.

[0115] In some other embodiments, the antisense strand is 21 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1, 3, 5, 9, 11, 13, 15, 17, 19, and 21, and 2'-fluoro modified nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counted from the 5' end); and / or (ii) Internucleotide links of phosphorothioate between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counted from the 5' end) It has.

[0116] In some other embodiments, the antisense strand is 21 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1, 3, 5, 9, 11, 13, 15, 17, 19, 20, and 21, and 2'-fluoro modified nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, and 18 (counted from the 5' end); and / or (ii) Internucleotide links of phosphorothioate between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counted from the 5' end) It has.

[0117] In some embodiments, the antisense strand is 21 nucleotides long, and (i) 2'-O-methyl-modified nucleotides at positions 3, 4, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, and 21 (counted from the 5' end); (ii) The 2'-fluoromodified nucleotide at position 14 (counted from the 5' end); (iii) 2'-deoxy modifications at positions 2, 5, 7, and 12 (counted from the 5' end); (iv) SCP modification at position 1 (counted from the 5' end); and / or (v) Internucleotide links of phosphorothioate between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counted from the 5' end) It has.

[0118] In some embodiments, the antisense strand is 21 nucleotides long, and (i) 2'-O-methyl-modified nucleotides at positions 3, 5, 9, 11, 13, 15, 17, 19, 20, and 21 (counted from the 5' end); (ii) 2'-fluoromodified nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, and 18 (counted from the 5' end); (iii) GNA modification at position 7 (counted from the 5' end); (iv) SCP modification at position 1 (counted from the 5' end); and / or (v) Internucleotide links of phosphorothioate between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counted from the 5' end) It has.

[0119] In some embodiments, the sense strand is 19 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1-6 and 10-19 and 2'-fluoro modified nucleotides at positions 7-9 (counted from the 5' end); and / or (ii) Phosphothioate internucleotide bonds (counted from the 5' end) between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between nucleotide positions 18 and 19 It has.

[0120] In some embodiments, the antisense strand includes a sequence selected from any one of the sequences described in Sequence IDs 81 to 105.

[0121] In some embodiments, the sense strand comprises a sequence selected from any one of the sequences described in Sequence IDs 41 to 59. In some preferred embodiments, the dsRNA comprises any of the modified sense strand sequence and modified antisense strand sequence pairs described in Table 6.

[0122] In some embodiments, the 3' end of the sense strand is modified and conjugated to a ligand. In some specific embodiments, the sense strand includes a sequence selected from any one of the sequences described in SEQ ID NOs: 113-131.

[0123] In some preferred embodiments, the dsRNA comprises one of the following pairs of modified sense strand sequences and modified antisense strand sequences: Sense chain: Sequence ID 113, Antisense chain: Sequence ID 81; Sense chain: Sequence ID 114, Antisense chain: Sequence ID 82; Sense chain: Sequence ID 115, Antisense chain: Sequence ID 83; Sense chain: Sequence ID 116, Antisense chain: Sequence ID 84; Sense chain: Sequence ID 117, Antisense chain: Sequence ID 85; Sense chain: Sequence ID 118, Antisense chain: Sequence ID 86; Sense chain: Sequence ID 119, Antisense chain: Sequence ID 87; Sense chain: Sequence ID 120, Antisense chain: Sequence ID 88; Sense chain: Sequence ID 121, Antisense chain: Sequence ID 89; Sense chain: Sequence ID 122, Antisense chain: Sequence ID 90; Sense chain: Sequence ID 123, Antisense chain: Sequence ID 91; Sense chain: Sequence ID 124, Antisense chain: Sequence ID 92; Sense chain: Sequence ID 125, Antisense chain: Sequence ID 93; Sense chain: Sequence ID 126, Antisense chain: Sequence ID 94; Sense chain: Sequence ID 127, Antisense chain: Sequence ID 95; Sense chain: Sequence ID 128, Antisense chain: Sequence ID 96; Sense chain: Sequence ID 129, Antisense chain: Sequence ID 97; Sense strand: SEQ ID NO: 130, Antisense strand: SEQ ID NO: 98; and Sense chain: Sequence ID 131, Antisense chain: Sequence ID 99

[0124] III. Ligand part

[0125] The dsRNAs of this disclosure are further conjugated to a ligand moiety containing N-acetylgalactosamine. In preferred embodiments, the sense strand of the dsRNA is conjugated to the ligand moiety. In some embodiments, the 3' end of the sense strand is conjugated to the ligand moiety. In other embodiments, the 5' end of the sense strand is conjugated to the ligand moiety.

[0126] In some embodiments, the ligand moiety includes a conjugate group represented by formula (X').

[0127] [ka]

[0128] During the ceremony, [ka] The symbol represents a point that connects to dsRNA;

[0129] Q is independent of H, [ka] is;

[0130] In the formula, L1 represents a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)). a -is;

[0131] L2 is a chemical bond or -CH2CH2C(O)-;

[0132] L3 is a chemical bond, -(NHCH2CH2) b -,-(NHCH2CH2CH2) b-, or -C(O)CH2-;

[0133] L4 is -(OCH2CH2) c -,-(OCH2CH2CH2) c -,-(OCH2CH2CH2CH2) c -,-(OCH2CH2CH2CH2CH2) c -, or -NHC(O)-(CH2) d -is;

[0134] During the ceremony, a = 0, 1, 2, or 3;

[0135] b = 1, 2, 3, 4, or 5;

[0136] c = 1, 2, 3, 4, or 5;

[0137] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0138] L is a chemical bond, -CH2O-, or -NHC(O)-;

[0139] L' represents a chemical bond, -C(O)NH-, -NHC(O)-, or -O(CH2CH2O). e -is;

[0140] In the formula, e is 1, 2, 3, 4, or 5;

[0141] T is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M-, or -C(O)-M-;

[0142] In the formula, M is [ka] is;

[0143] Both R1 and R2 form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0144] Alternatively, both R1 and R3 are set to -C 1-2 It forms an alkylene group, and R2 is H;

[0145] In the formula, R is -OR', -CH2OR', or -CH2CH2OR'; in the formula, R' is H, a hydroxyl protecting group, or a solid support, the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0146] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0147] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0148] In some embodiments, the conjugate group is represented by formula (I').

[0149] [ka]

[0150] During the ceremony, [ka] The symbol represents a point that connects to dsRNA;

[0151] Q is independent of H, [ka] is;

[0152] In the formula, L1 represents a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)). a -is;

[0153] L2 is a chemical bond or -CH2CH2C(O)-;

[0154] L3 is a chemical bond, -(NHCH2CH2) b -,-(NHCH2CH2CH2) b -, or -C(O)CH2-;

[0155] L4 is -(OCH2CH2) c -,-(OCH2CH2CH2) c -,-(OCH2CH2CH2CH2) c -,-(OCH2CH2CH2CH2CH2) c -, or -NHC(O)-(CH2) d -is;

[0156] During the ceremony, a = 0, 1, 2, or 3;

[0157] b = 1, 2, 3, 4, or 5;

[0158] c = 1, 2, 3, 4, or 5;

[0159] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0160] L is either -CH2O- or -NHC(O)-;

[0161] L' is a chemical bond, -C(O)NH-, or -NHC(O)-;

[0162] Both R1 and R2 form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0163] Alternatively, both R1 and R3 are set to -C 1-2 It forms an alkylene group, and R2 is H;

[0164] In the formula, R is -OR', -CH2OR', or -CH2CH2OR'; in the formula, R' is H, a hydroxyl protecting group, or a solid support, the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0165] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0166] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0167] In some specific embodiments, in the formula,

[0168] Q is independent of H or [ka] is;

[0169] In the formula, L1 is -CH2O- or -NHC(O)-(CH2NHC(O)) a -is;

[0170] L2 is -CH2CH2C(O)-;

[0171] L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -is;

[0172] L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -is;

[0173] During the ceremony, a = 0, 1, 2, or 3;

[0174] b = 1, 2, 3, 4, or 5;

[0175] c = 1, 2, 3, 4, or 5;

[0176] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0177] L is -CH2O-;

[0178] L’ is a chemical bond;

[0179] Both R1 and R2 form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0180] Or, both R1 and R3 form -C 1-2 [[ID=I0]]alkylene-, and R2 is H;

[0181] In the formula, R is -OR’, -CH2OR’, or -CH2CH2OR’; in the formula, R’ is H, a hydroxyl protecting group, or a solid support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4’-dimethoxytrityl;

[0182] [[ID=1S]]m = 0, I, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0183] n = 0, I, 2, I, 4, 5, 6, 7, 8, 9, or 10;

[0184] In some embodiments, the conjugate group is represented by formula (I’-1), formula (I’-2), or formula (I’-3).

[0185] [[ID=I9]]

Chemical formula

[0186] In the formula,

Chemical formula

[0187] Q is

Chemical formula

[0188] In the formula, L1 is -CH2O- or -NHC(O)-;

[0189] L2 is -CH2CH2C(O)-;

[0190] L3 is -(NHCH2CH2) b - or -(NHCH2CH2CH2) b -;

[0191] L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -;

[0192] wherein, b = 1, 2, 3, 4, or 5;

[0193] c = 1, 2, 3, 4, or 5;

[0194] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0195] L is -CH2O-;

[0196] R' is H, a hydroxyl protecting group, or a solid support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0197] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0198] [[ID=4�]]In some specific embodiments, wherein,

[0199] Q is independently H,

Chemical formula

[0202] L3 is -(NHCH2CH2) b -,-(NHCH2CH2CH2) b -, or -C(O)CH2-;

[0203] L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -is;

[0204] During the ceremony, a = 0, 1, 2, or 3;

[0205] b = 1, 2, 3, 4, or 5;

[0206] c = 1, 2, 3, 4, or 5;

[0207] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0208] L is either -CH2O- or -NHC(O)-;

[0209] L' represents a chemical bond or -C(O)NH-;

[0210] Both R1 and R2 form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0211] Alternatively, both R1 and R3 are set to -C 1-2 It forms an alkylene group, and R2 is H;

[0212] In the formula, R is -OR', -CH2OR', or -CH2CH2OR'; in the formula, R' is H, a hydroxyl protecting group, or a solid support, the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0213] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0214] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0215] In some embodiments, the conjugate group is represented by formula (II'-1) or formula (II'-2).

[0216] [ka]

[0217] During the ceremony, [ka] The symbol represents a point that connects to dsRNA;

[0218] Q is independent, [ka] is;

[0219] In the formula, L1 is -CH2O- or -CH2O-CH2CH2O-;

[0220] L3 is -(NHCH2CH2) b -,-(NHCH2CH2CH2) b -, or -C(O)CH2-;

[0221] L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -is;

[0222] During the ceremony, b = 1, 2, 3, 4, or 5;

[0223] c = 1, 2, 3, 4, or 5;

[0224] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0225] L is -NHC(O)-;

[0226] L' represents a chemical bond or -C(O)NH-;

[0227] R' is H, a hydroxyl protecting group, or a solid support, where the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0228] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0229] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0230] In some specific embodiments, in the formula,

[0231] Q is independent of H, [ka] is;

[0232] In the formula, L1 is -CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -is;

[0233] L2 is a chemical bond;

[0234] L3 is -(NHCH2CH2) b -,-(NHCH2CH2CH2) b -, or -C(O)CH2-;

[0235] L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -is;

[0236] During the ceremony, a = 0, 1, 2, or 3;

[0237] b = 1, 2, 3, 4, or 5;

[0238] c = 1, 2, 3, 4, or 5;

[0239] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0240] L is either -CH2O- or -NHC(O)-;

[0241] L' represents a chemical bond or -C(O)NH-;

[0242] Both R1 and R2 form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0243] Alternatively, both R1 and R3 are set to -C 1-2 It forms an alkylene group, and R2 is H;

[0244] In the formula, R is -OR', -CH2OR', or -CH2CH2OR'; in the formula, R' is H, a hydroxyl protecting group, or a solid support, the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0245] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0246] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0247] In some embodiments, the conjugate group in the formula is as shown in formula (II'-2).

[0248] [ka]

[0249] During the ceremony, [ka] The symbol represents a point that connects to dsRNA;

[0250] Q is independent, [ka] is;

[0251] In the formula, L1 is either -CH2- or -C(O)-;

[0252] L3 is -(NHCH2CH2) b -is;

[0253] L4 is -(OCH2CH2) c -is;

[0254] During the ceremony, b = 1, 2, 3, 4, or 5;

[0255] c = 1, 2, 3, 4, or 5;

[0256] L is either -CH2O- or -NHC(O)-;

[0257] R' is H, a hydroxyl protecting group, or a solid support, where the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0258] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0259] In some specific embodiments, the formula is:

[0260] Q is independent of H, [ka] is;

[0261] In the formula, L1 represents a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)). a -is;

[0262] L2 is a chemical bond or -CH2CH2C(O)-;

[0263] L3 is a chemical bond, -(NHCH2CH2) b -,-(NHCH2CH2CH2) b -, or -C(O)CH2-;

[0264] L4 is -(OCH2CH2) c -,-(OCH2CH2CH2) c -,-(OCH2CH2CH2CH2) c -,-(OCH2CH2CH2CH2CH2) c -, or -NHC(O)-(CH2) d -is;

[0265] During the ceremony, a = 0, 1, 2, or 3;

[0266] b = 1, 2, 3, 4, or 5;

[0267] c = 1, 2, 3, 4, or 5;

[0268] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0269] L is a chemical bond, -CH2O-, or -NHC(O)-;

[0270] L' represents a chemical bond, -C(O)NH-, -NHC(O)-, or -O(CH2CH2O). e -is;

[0271] In the formula, e is 1, 2, 3, 4, or 5;

[0272] T is a chemical bond, -CH2-, -M-, -CH2-M-, or -C(O)-M-;

[0273] In the formula, M is [ka] is;

[0274] Both R1 and R2 form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0275] Alternatively, both R1 and R3 are set to -C 1-2 It forms an alkylene group, and R2 is H;

[0276] In the formula, R is -OR', -CH2OR', or -CH2CH2OR'; in the formula, R' is H, a hydroxyl protecting group, or a solid support, the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0277] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0278] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0279] In some specific embodiments, in the formula,

[0280] T is -M-, -CH2-M-, or -C(O)-M-, where M is [ka] That is the case.

[0281] In some specific embodiments, in the formula,

[0282] Q is independent of H or [ka] is;

[0283] In the formula, L1 is -CH2O- or -NHC(O)-(CH2NHC(O)) a -is;

[0284] L2 is -CH2CH2C(O)-;

[0285] L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -is;

[0286] L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -is;

[0287] During the ceremony, a = 0, 1, 2, or 3;

[0288] b = 1, 2, 3, 4, or 5;

[0289] c = 1, 2, 3, 4, or 5;

[0290] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0291] L is a chemical bond or -CH2O-;

[0292] L' represents a chemical bond or -O(CH2CH2O) e -is;

[0293] In the formula, e is 1, 2, 3, 4, or 5;

[0294] Both R1 and R2 form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0295] Alternatively, both R1 and R3 are set to -C 1-2 It forms an alkylene group, and R2 is H;

[0296] In the formula, R is -OR', -CH2OR', or -CH2CH2OR'; in the formula, R' is H, a hydroxyl protecting group, or a solid support, the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0297] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0298] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0299] In the formula, T is as defined in the above embodiment.

[0300] In some embodiments, the conjugate group is represented by formula (III'-1), formula (III'-2), or formula (III'-3).

[0301] [ka]

[0302] In the formula, Q is [ka] is;

[0303] In the formula, L1 is either -CH2O- or -NHC(O)-;

[0304] L2 is -CH2CH2C(O)-;

[0305] L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -is;

[0306] L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -is;

[0307] During the ceremony, b = 1, 2, 3, 4, or 5;

[0308] c = 1, 2, 3, 4, or 5;

[0309] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0310] L is a chemical bond or -CH2O-;

[0311] In the formula, R' is H, a hydroxyl protecting group, or a solid support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0312] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0313] In the formula, T is as defined in the above embodiment.

[0314] In some specific embodiments, in the formula,

[0315] Q is independent of H, [ka] is;

[0316] In the formula, L1 is -CH2-, -CH2O-, or -C(O)-;

[0317] L2 is a chemical bond;

[0318] L3 is -(NHCH2CH2) b -,-(NHCH2CH2CH2) b -, or -C(O)CH2-;

[0319] L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -is;

[0320] During the ceremony, b = 1, 2, 3, 4, or 5;

[0321] c = 1, 2, 3, 4, or 5;

[0322] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0323] L is a chemical bond or -NHC(O)-;

[0324] L' is a chemical bond;

[0325] Both R1 and R2 form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0326] Alternatively, both R1 and R3 are set to -C 1-2 It forms an alkylene group, and R2 is H;

[0327] In the formula, R is -OR', -CH2OR', or -CH2CH2OR'; In the formula, R' is H, a hydroxyl protecting group, or a solid support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0328] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0329] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0330] In the formula, T is as defined in the above embodiment.

[0331] In some embodiments, the conjugate group is as shown in formula (IV-1) or formula (IV-2).

[0332] [ka]

[0333] During the ceremony,

[0334] Q is independent, [ka] is;

[0335] In the formula, L1 is -CH2-, -CH2O-, or -C(O)-;

[0336] L3 is -(NHCH2CH2) b -,-(NHCH2CH2CH2) b -, or -C(O)CH2-;

[0337] L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -is;

[0338] During the ceremony, b = 1, 2, 3, 4, or 5;

[0339] c = 1, 2, 3, 4, or 5;

[0340] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0341] L is a chemical bond or -NHC(O)-;

[0342] L' is a chemical bond;

[0343] In the formula, R' is H, a hydroxyl protecting group, or a solid support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0344] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0345] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0346] Where T is as defined in the above embodiments.

[0347] In some specific embodiments, in the formula:

[0348] Q is independently H,

Chemical formula

[0349] Where L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0350] L2 is a chemical bond or -CH2CH2C(O);

[0351] L3 is a chemical bond, -(NHCH2CH2) b -, -(NHCH2CH2CH2) b -, or -C(O)CH2;

[0352] L4 is -(OCH2CH2) c -, -(OCH2CH2CH2) c -, -(OCH2CH2CH2CH2) c -, -(OCH2CH2CH2CH2CH2) c -, or -NHC(O)-(CH2) d -;

[0353] Where a = 0, 1, 2, or 3;

[0354] b = 1, 2, 3, 4, or 5;

[0355] c = 1, 2, 3, 4, or 5;

[0356] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0357] L is a chemical bond, -CH2O-, or -NHC(O)-;

[0358] L' is -O(CH2CH2O) e -is;

[0359] In the formula, e is 1, 2, 3, 4, or 5;

[0360] T is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M-, or -C(O)-M-;

[0361] In the formula, M is [ka] is;

[0362] Both R1 and R2 form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0363] Alternatively, both R1 and R3 are set to -C 1-2 It forms an alkylene group, and R2 is H;

[0364] In the formula, R is -OR', -CH2OR', or -CH2CH2OR'; in the formula, R' is H, a hydroxyl protecting group, or a solid support, the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0365] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0366] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0367] In some preferred embodiments, the conjugate group in the formula is selected from Table 1 below.

[0368] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0369] In some preferred embodiments, the conjugate group in the formula is selected from Table 2 below.

[0370] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0371] In some embodiments, the ligand targets the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand targets the asialoglycoprotein receptor (ASGPR) on hepatocytes.

[0372] In some embodiments, the ligand has the following structure. [ka]

[0373] During the ceremony, [ka] This indicates a point that connects to dsRNA via a phosphate group or phosphorothioate group.

[0374] In some embodiments, the ligand has the following structure. [ka]

[0375] During the ceremony, [ka] This indicates a point that connects to dsRNA via a phosphate group or phosphorothioate group.

[0376] In some embodiments, the ligand has the following structure. [ka]

[0377] During the ceremony, [ka] This indicates a point that connects to the sense strand of dsRNA via a phosphate group or phosphorothioate group.

[0378] In a preferred embodiment, the ligand has the following structure. [ka]

[0379] During the ceremony, [ka] This indicates a point that connects to the sense strand of dsRNA via a phosphate group or phosphorothioate group.

[0380] In some embodiments, the sense strand comprises a ligand-conjugated sense strand sequence selected from any one of SEQ ID NOs.60 to 79. In some preferred embodiments, the dsRNA comprises one of the sense strand sequence-antisense strand sequence pairs selected from those listed in Table 7.

[0381] IV. Suppression of AGT gene expression

[0382] The dsRNAs of this disclosure can suppress AGT gene expression by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0383] Suppression of AGT gene expression can be manifested by a reduction in the level of mRNA expressed by a first cell or population of cells (such cells may be present, for example, in a sample derived from a subject). In this case, the AGT gene is transcribed, and the cells are treated (for example, by contacting the cells with the dsRNA of the Disclosure, or by administering the dsRNA of the Disclosure to a subject in which the cells are present or previously present) thereby suppressing AGT gene expression compared to a second cell or population of cells that is substantially the same as the first cell or population of cells but has not been treated.

[0384] In a preferred embodiment, the inhibition is evaluated by the level of mRNA in the treated cells as a percentage relative to the level of mRNA in the control cells, using the following formula. In some specific embodiments, 2 -△△Ct The values are calculated to compare the differences between the experimental group and the control group, where ΔΔCt = [(Ct 標的遺伝子,実験群 - Ct 参照遺伝子,実験群 ) - (Ct 標的遺伝子,コントロール群 - Ct 参照遺伝子,コントロール群 )].

[0385] Alternatively, the suppression of AGT gene expression, such as in terms of a decrease in parameters functionally related to AGT gene expression, for example, lipid levels and cholesterol levels (such as LDLc levels), may be evaluated. The silencing of the AGT gene can be determined in any cell that constitutively expresses AGT or any cell that expresses AGT by genomic manipulation, by any assay known in the art. The liver and kidneys are the major sites of AGT expression, and other important expression sites include the small intestine, white adipose tissue, and colon.

[0386] The suppression of AGT protein expression can be demonstrated by a reduction in the level of AGT protein expressed by the cell or population of cells (such as the level of protein expressed in a sample derived from the subject). Similar to the evaluation of mRNA suppression described above, the suppression of protein expression levels in the treated cells or population of cells can also be expressed as a percentage relative to the protein level in the control cells or population of cells.

[0387] Control cells or populations of cells that can be used to evaluate the suppression of AGT gene expression include cells or populations of cells not exposed to the dsRNA of the present disclosure. For example, the control cells or population of cells may be derived from an individual subject (such as a human or animal subject) before treatment with dsRNA. V. Vector

[0388] This disclosure provides a vector comprising a nucleotide sequence encoding the dsRNA of the Disclosure. The vector of the Disclosure can amplify and / or express the nucleotide encoding the dsRNA of the Disclosure ligated thereto. The vector of the Disclosure may be a virus, plasmid, or cosmid.

[0389] dsRNAs targeting the AGT gene can be expressed from transcription units inserted into DNA or RNA vectors. Expression can be transient (within a few hours to a few weeks) or persistent (a few weeks to several months or longer), depending on the specific construct used and the type of target tissue or cell. Nucleotides encoding dsRNAs targeting AGT mRNA can be introduced into linear constructs, circular plasmids, or viral vectors. Nucleotides encoding dsRNAs targeting AGT mRNA can be stably expressed by integration into the cell genome or by stable extrachromosomal inheritance. Generally, dsRNA expression vectors are typically DNA plasmids or viral vectors. Viral vector systems containing a coding sequence of dsRNA targeting AGT mRNA include, but are not limited to, (a) adenovirus vectors, (b) retrovirus vectors, (c) adeno-associated virus vectors, (d) herpes simplex virus vectors, (e) SV40 vectors, (f) polyomavirus vectors, (g) papillomavirus vectors, (h) picornavirus vectors, or (i) poxvirus vectors.

[0390] VI.Cells This disclosure provides cells containing the dsRNA or vector of this disclosure.

[0391] VII. Pharmaceutical Compositions This disclosure provides pharmaceutical compositions comprising the dsRNA, vector, or cells of this disclosure and, optionally, a pharmaceutically acceptable carrier or excipient.

[0392] In this specification, “pharmaceutically acceptable” means a compound, material, composition, and / or dosage form that is suitable for contact with human and animal tissues, within the bounds of appropriate medical judgment, without causing excessive toxicity, irritation, allergic reactions, or other problems or complications, and that is balanced by a reasonable benefit-risk ratio.

[0393] In this specification, pharmaceutically acceptable carriers refer to pharmaceutical carriers that facilitate the administration of vectors or cells containing dsRNA or sequences encoding it into the human body and / or promote their absorption or action. Examples include diluents, excipients such as water, fillers such as starch and sucrose, binders such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone, wetting agents such as glycerin, disintegrants such as agar, calcium carbonate, and sodium bicarbonate, absorption enhancers such as quaternary ammonium compounds, surfactants such as cetyl alcohol, adsorbents such as kaolin and bentonite, and lubricants such as talc, calcium / magnesium stearate, and polyethylene glycol. Other adjuvants such as fragrances and sweeteners may also be added to the composition.

[0394] The pharmaceutical compositions of this disclosure may include a pharmaceutically acceptable diluent or a sustained-release matrix into which the dsRNA or vector of this disclosure is embedded.

[0395] The pharmaceutical compositions of this disclosure may include a drug delivery system for delivering dsRNA. The drug delivery systems of this disclosure are not limited to, but include, nanoparticles (lipid nanoparticles, polymer nanoparticles, etc.), polymers, PEG, or cation delivery systems, polylactic acid (PLA) microspheres, poly(lactic acid-co-glycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid helices (cochleates), lipid microtubules, cholesterol, PEG lipids, such as PEG-2000-C-DMG and PEG-2000-DMG(Moderna), ALC-0159, and DSPC.

[0396] In some embodiments, the siRNA or vector in the pharmaceutical composition of this disclosure may be contained in polymers and polymer-based nanoparticles.

[0397] In some specific embodiments, the polymer is a polymer based on poly(lactic acid-coglycolic acid) (PLGA). In some specific embodiments, the PLGA-based polymer is modified to contain individual cationic groups.

[0398] In some specific embodiments, the polymer contains amine groups that can become cationic, such as polyethyleneimine (PEI) and poly(L-lysine) (PLL), and can form a complex with siRNA through electrostatic interactions to deliver siRNA into cells. In some embodiments, chemical modification of PEG and PLL improves in vivo efficacy and tolerability.

[0399] In some embodiments, siRNA, vectors, or cells in the pharmaceutical compositions of the present disclosure can be delivered by a cationic polymer, poly(β-aminoester) (PBAE).

[0400] VIII. Kit

[0401] This disclosure provides a kit comprising the dsRNA, vector, or cells described herein.

[0402] This disclosure also provides a kit for using the dsRNAs of this disclosure and / or for carrying out the methods of this disclosure. The kit may include one or more dsRNAs of this disclosure, vectors, or cells, and may further include instructions for use. These instructions may contain instructions for suppressing AGT expression in cells by contacting the cells with an amount of the dsRNAs or vectors of this disclosure that is effective in suppressing AGT expression.

[0403] When the dsRNA of the Disclosure is brought into contact with cells in vitro, the Kit of the Disclosure may further include, as necessary, means for bringing cells into contact with the dsRNA or vector of the Disclosure (e.g., an injection device), or means for measuring the inhibitory effect on AGT (e.g., means for measuring the inhibition of AGT mRNA or AGT protein). Such means for measuring the inhibition of AGT may include means for obtaining a sample (e.g., a plasma sample) from the subject.

[0404] When administering the dsRNA or vector of this disclosure, or cells into which such dsRNA or vector has been introduced in vitro, into the body, the kit of this disclosure may further include, as necessary, a device for administering the dsRNA, vector, or cells of this disclosure, or a device for determining a therapeutic or prophylactic dose.

[0405] IX. Treatment methods and pharmaceutical uses

[0406] This disclosure provides a method for reducing angiotensinogen (AGT) in a subject, comprising the step of administering the dsRNA, vector, cells, or pharmaceutical composition of this disclosure to the subject.

[0407] This disclosure provides a method for treating, preventing, suppressing or alleviating a disease or disorder in which reducing the expression of angiotensinogen (AGT) in a subject is beneficial, the method comprising the step of administering the dsRNA, vector, cells, or pharmaceutical composition of this disclosure to the subject. This disclosure also provides a method for treating, preventing, suppressing, or alleviating at least one symptom in a patient suffering from a disease or disorder in which reducing the expression of angiotensinogen (AGT) is beneficial.

[0408] In some embodiments, diseases or disorders for which reducing angiotensinogen (AGT) expression is beneficial are AGT-related diseases.

[0409] In some embodiments, the methods provided herein for reducing angiotensinogen (AGT) in a subject, for treating, preventing, suppressing or alleviating a disease or disorder in which reducing the expression of angiotensinogen (AGT) in a subject is beneficial, or for treating, preventing, suppressing or alleviating at least one symptom in a patient suffering from a disease or disorder in which reducing the expression of angiotensinogen (AGT) is beneficial, include subcutaneous, topical, or intravenous administration of the dsRNA, vector, cells, or pharmaceutical composition to the subject. In some embodiments, the subject is a human patient.

[0410] This disclosure also relates to the dsRNA, vector, cells, or pharmaceutical composition of this disclosure used to treat diseases or conditions associated with AGT expression in subjects.

[0411] This disclosure also relates to the use of the dsRNA, vectors, cells, or pharmaceutical compositions of this disclosure in the manufacture of a pharmaceutical product for treating, preventing, suppressing, or alleviating a disease or disorder in which reducing angiotensinogen (AGT) expression in a subject is beneficial. This disclosure also relates to the use of the dsRNA, vectors, cells, or pharmaceutical compositions of this disclosure in the manufacture of a pharmaceutical product for reducing angiotensinogen (AGT) in a subject. This disclosure also relates to the use of the dsRNA, vectors, cells, or pharmaceutical compositions of this disclosure in the manufacture of a pharmaceutical product for treating, preventing, suppressing, or alleviating at least one symptom in a patient suffering from a disease or disorder in which reducing angiotensinogen (AGT) expression is beneficial.

[0412] array

[0413] The RNA sequences provided in this disclosure target the human AGT gene (or target gene, target mRNA sequence, or target sequence). The target AGT mRNA sequence relates to the gene indicated by Genbank accession number NM_001393338.1. [Table 3]

[0414] Tables 4 and 5 represent the modified RNA sequences used in this disclosure, respectively.

[0415] The meanings of the abbreviations used in this specification are as follows:

[0416] "A," "U," "G," and "C" represent natural adenine ribonucleotide, uracil ribonucleotide, guanine ribonucleotide, and cytosine ribonucleotide, respectively.

[0417] The letter "d" indicates that the nucleotide adjacent to its right is a deoxyribonucleotide. For example, "dA", "dT", "dG", and "dC" represent adenine deoxyribonucleotide, thymine deoxyribonucleotide, guanine deoxyribonucleotide, and cytosine deoxyribonucleotide, respectively.

[0418] The letter "m" indicates that the nucleotide adjacent to it on the left is a 2'-OCH3 modified nucleotide. For example, "Am", "Um", "Gm", and "Cm" represent A, U, G, and C modified with 2'-OCH3, respectively.

[0419] The letter "f" indicates that the nucleotide adjacent to it on the left is a 2'-fluoro modified nucleotide. For example, "Af", "Uf", "Gf", and "Cf" represent A, U, G, and C modified with 2'-fluoro, respectively.

[0420] The letter "s" indicates that two adjacent nucleotides are linked by a phosphorothioate bond.

[0421] The "s-" symbol indicates that the nucleotide adjacent to the left and the delivery ligand adjacent to the right are linked by a phosphorothioate bond.

[0422] "VP" indicates that the nucleotide adjacent to the right is a vinyl phosphate-modified nucleotide, which is well known in this field. For example, see PCT publication numbers WO2011139702, WO2013033230, and WO2019105419.

[0423] Tgn and Agn are the adenosine-ethylene glycol nucleic acid (GNA)S isomer and the thymidine-ethylene glycol nucleic acid (GNA)S isomer, respectively, and are well known in the field. See, for example, PCT publication numbers WO2019222166A1 and WO2020132227A2. This has the following structure: [ka] The formula comprises, where B is thymine (in the case of the thymidine-ethylene glycol nucleic acid (GNA) S isomer) or adenine (in the case of the adenosine-ethylene glycol nucleic acid (GNA) S isomer).

[0424] "IB" represents a reverse debasic deoxyribonucleotide, which can include the following three structures depending on its position / conjugate mode in dsRNA. IB is well known in this field. See, for example, Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16, and PCT publication numbers WO2016011123 and WO2019051402. [ka]

[0425] [ka]

[0426] "L96" represents a GalNAc delivery ligand with the following structure, which is well known in this field, and in the formula, [ka] The symbol indicates a point that connects to dsRNA via a phosphate group or phosphorothioate group. See, for example, PCT publication numbers WO2009073809 and WO2009082607. [ka]

[0427] "GL6" represents a GalNAc delivery ligand with the following structure, where, [ka] This indicates a point that connects to dsRNA via a phosphate group or phosphorothioate group. [ka]

[0428] The structure of an "SCP-modified nucleotide" is as follows: [ka] (In the formula, Base is independently selected from H, a modified or unmodified base, or a leaving group) a modified nucleotide. The Base is preferably an unmodified base, including an adenine base, a guanine base, a uracil base, and a cytosine base. For example, SCP-U means that the base is a uracil base. [Table 4-1] [Table 4-2] [Table 5] [Table 6] [Table 7]

[0429] The contents of this disclosure will be further explained below with reference to examples. Please understand that the following examples are illustrative and should not be construed as limiting the scope of this disclosure. [Examples]

[0430] Unless otherwise specified, the sources of the commercially available materials used in the examples are as follows:

[0431] Huh7 cell line: Purchased from Cobioer Biosciences, Cat#CBP60202

[0432] Hep3B cell line: Purchased from Cobioer Biosciences, Cat#CBP60197

[0433] PHH cells: Purchased from Shanghai Xuanyi Biotechnology, Cat#QYLF-HPMC

[0434] HEK293A cell line: Purchased from Cobioer Biosciences, Cat#CBP60436

[0435] Balb / c mice: Purchased from Vital River Laboratory Animal Technology, Cat#Balb / c

[0436] Example 1: Preparation of Compound E7

[0437] 1. Preparation of intermediates 3-4

[0438] 1.1 Preparation of Compound 2 [ka]

[0439] At 15°C, compound 1 (300g, 2.01mol) was added to DCM (1.80L), to which benzyl(2,5-dioxopyrrolidine-1-yl) carbonate (600g, 2.40mol) was slowly added, and TEA (203g, 2.01mol, 280mL) was added dropwise. The mixture was then stirred at 25°C for 16 hours. TLC (dichloromethane:methanol = 10:1) confirmed that reactant 1 was retained (R f =0.32), and prominent spots (R f Compound 2 (=0.52) was newly detected. The reaction mixture was washed with saturated sodium bicarbonate solution (1.00 L x 2). The organic phase was washed with brine (1.00 L), dried over anhydrous Na2SO4, and concentrated under vacuum. Compound 2 (approximately 385 g) was obtained as yellow oil without further purification.

[0440] 1.2 Preparation of Compound 2A [ka]

[0441] At 0-15°C, DMAP (19.8g, 162 mmol) was added all at once to a pyridine (1.75L) solution of compound 4 (350g, 1.62mol, HCl) and Ac2O (994g, 9.74mol, 912mL), and TEA (164g, 1.62mol, 226mL) was added dropwise. The mixture was stirred at 25°C for 16 hours. LC-MS (product: RT=0.687 min) confirmed that the starting reactants had been completely consumed. At 25°C, HCl (1.40L) was added to the mixture and stirred for 30 minutes. The resulting mixture was then filtered, and the filtered cake was washed with HCl (300mL). The filtered cake was ground with water (1.45L) at 25°C for 30 minutes. The mixture was filtered, and the filtered cake was washed with water (175mL x 3). The filtered cake was recovered, and compound 2A (approximately 580g) was obtained as a white solid.

[0442] 1.3 Preparation of Compound 2B [ka]

[0443] Three reactions were carried out in parallel.

[0444] At 10-15°C, TMSOTf (137g, 616mmol, 111mL) was added dropwise over 0.5 hours to a solution of compound 2A (200g, 514mmol) in DCM (800mL). The mixture was then stirred at 25°C for 3 hours. Compound 2A (R) was analyzed by TLC (dichloromethane:methanol = 20:1). f =0.54) has been completely consumed, and a new spot (R f It was confirmed that compound 2B (=0.24) was formed. The three reactions were combined. The mixture was cooled to 0-15°C and slowly poured into NaHCO3 (300g dissolved in 3.00L of water) at 0-5°C. The organic phase was separated and the aqueous phase was extracted with DCM (1.00L x 3). The combined organic phase was dried with Na2SO4, filtered, and vacuum concentrated. Compound 2B (approximately 507g) for use in the next step was obtained as yellow oil without further purification.

[0445] 1.4 Preparation of Compound 3 [ka]

[0446] At 0-10°C, TMSOTf (84.4g, 380mmol, 69.0mL) was added dropwise to a mixture of compound 2B (250g, 759mmol) and compound 2 (151g, 531mmol) in DCM (1.00L). The mixture was stirred at 20°C for 12 hours. Compound 2(R) was analyzed by TLC (dichloromethane:methanol = 20:1). f =0.33) has been completely consumed, and a new spot (R f It was confirmed that compound 3 (=0.03) was formed. The combined reaction mixture was cooled to 0-5°C, then poured into NaHCO3 (100g in 1L of water) and stirred at 5-10°C for 10 minutes to separate the phases. The aqueous phase was extracted with DCM (500mL x 2). The combined organic phase was dried with Na2SO4, filtered, and vacuum concentrated. Compound 3 (approximately 360g) was obtained as yellow oil without further purification.

[0447] 1 1H NMR: (400 MHz, DMSO)

[0448] δ=7.79-7.37 (m, 1H), 7.35-7.26 (m, 5H), 5.21-5.20 (m, 1H), 5.00-4.95 (m, 3H), 4.55-4.53 (m, 1H), 4.03-3.86 (m, 3H), 3.61-3.59 (m, 1H), 3.59-3.57 (m, 1H), 3.48-3.40 (m, 6H), 3.39-3.31 (m, 2H), 3.14-3.13 (m, 2H), 2.09 (s, 3H), 1.99 (s, 3H), 1.88 (s, 3H), 1.76-1.74 (m, 3H)

[0449] 1.5 Preparation of intermediates 3-4 (TFA salts) [ka]

[0450] Three reactions were carried out in parallel.

[0451] Compound 3 (180 g, 293 mmol) and TFA (33.5 g, 293 mmol, 21.8 mL) were added to a mixture of Pd / C (18.0 g, 16.3 mmol, 10% content) in THF (1.80 L) under an argon atmosphere. The suspension was degassed and purged three times with hydrogen. The mixture was stirred under H2 (50 Psi) at 30°C for 2 hours. LC-MS (product: RT = 0.697 min) confirmed that compound 3 had been consumed and that a product peak was detected. The three reactions were combined. The mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to remove the solvent. Without further purification, intermediate 3-4 (TFA salt) (393 g, 660 mmol, yield 74.8%, purity 99.6%, TFA) was obtained as a yellow solid.

[0452] 1 1H NMR: (400 MHz, DMSO-d6)

[0453] δ = 7.92 (d, J = 9.1 Hz, 4H), 5.27-5.17 (m, 1H), 5.03-4.91 (m, 1H), 4.60-4.50 (m, 1H), 4.09-3.97 (m, 4H), 3.85 (s, 2H), 3.65-3.46 (m, 10H), 3.04-2.92 (m, 2H), 2.10 (s, 3H), 2.00 (s, 3H), 1.94-1.86 (m, 3H), 1.82-1.71 (m, 4H)

[0454] 2. Modulation of intermediate 3-3

[0455] 2.1 Modulation of compound 5

change

[0456] At 25°C, DIEA (30.3g, 234mmol, 40.8mL, 6.60eq) was added in one step to a 1.00L solution of compound 4B (10.0g, 35.5mmol, 1.00eq) and compounds 3-4 (46.3g, 78.2mmol, 2.20eq, TFA) prepared above in DCM (1.00L). The mixture was stirred at 25°C for 30 minutes. HBTU (30.3g, 234mmol, 40.8mL, 6.60eq) was added to the mixture. The mixture was stirred at 25°C for 16 hours. The reaction was confirmed to be complete by LCMS (product: RT=0.681 min). The mixture was concentrated under vacuum. At 20°C, the mixture was added to 0.50N HCl (200mL x 2) and then extracted with DCM (3 x 500mL). The combined organic phase was washed with saturated NaHCO3 (3 × 800 mL) to pH=8, then washed with brine (3 × 500 mL), dried over Na2SO4, and vacuum concentrated. The residue was purified by column chromatography (SiO2, DCM:MeOH = 50:1~15:1). The residue was vacuum concentrated at 40°C and purified by preparative MPLC (column: 800 g Agela C18; mobile phase: [water-ACN]; 15-45%, 25 min; 45%, 10 min). Compound 5 (approximately 180 g + 75.0 g + 87.0 g + 40.0 g + 38.0 g) was obtained as a yellow solid by vacuum drying.

[0457] 417.0g of compound 3-4 was divided into 9 batches and converted to compound 5.

[0458] 2.2 Preparation of Intermediate 3-3 [ka]

[0459] Under an argon atmosphere, Pd / C (3.00 g, 10% content) was added to THF (300 mL), to which compound 5 (73.0 g, 61.7 mmol, 1.00 eq) and TFA (7.04 g, 61.7 mmol, 4.57 mL, 1.00 eq) were added. The suspension was degassed and purged three times with hydrogen. The mixture was stirred under H2 (20 Psi) at 20°C for 16 hours. TLC (dichloromethane:methanol = 8:1, R) fThe reaction was confirmed to be complete by a coefficient of 0.0. The mixture was filtered through celite, and the filtrate was concentrated under pressure to remove the solvent, yielding compound 3-3 (approximately 33.4 g + 129 g + 75.0 g) as a white solid.

[0460] 1 1H NMR: (400 MHz, DMSO)

[0461] δ=8.53 (t, J = 5.2 Hz, 1H), 8.18 (d, J = 2.4 Hz, 3H), 8.03 (t, J = 5.2 Hz, 1H), 7.84 (dd, J = 3.6 Hz, 2H), 5.22 (d, J = 3.2 Hz, 2H), 4.96 (dd, J = 3.2 Hz, 2H), 4.55 (d, J =8.4 Hz, 2H), 4.02 (t, J =8.8 Hz, 6H), 3.77-3.59 (m, 5H), 3.58-3.45 (m, 21H), 3.40-3.20 (m, 4H), 2.18 (t, J = 7.6 Hz, 2H), 2.17 (d, J =8.0 Hz, 6H), 2.10 (s, 6H), 1.99 (s, 6H), 1.90-1.80 (m, 8H), 1.77 (s, 6H)

[0462] 3. Preparation of compound E7

[0463] 3.1 Preparation of Compound 3 [ka]

[0464] Compound 1 (2.00 g, 1.87 mmol, prepared according to the method of intermediate 3-3 described above) was dissolved in DCM (20.0 mL) at room temperature. DIEA (0.135 mL, 0.814 mmol) and Compound 2 (0.550 g, 0.814 mmol) were sequentially added to this solution, and the mixture was purged with nitrogen three times. The reaction mixture was stirred at 25°C for 16 hours. The MS response of the product was detected by liquid chromatography / tandem mass spectrometry (LC-MS / MS). Thin-layer chromatography (dichloromethane / methanol = 5 / 1) confirmed the disappearance of the starting material and the formation of new spots. The reaction mixture was concentrated under reduced pressure. The resulting crude product was purified by column chromatography (dichloromethane / methanol = 5 / 1) to obtain Compound 3 (approximately 780 mg) as a white solid.

[0465] 1 1H NMR (400 MHz, CD3OD)

[0466] δ=7.28-7.42 (m, 5H), 5.30-5.34 (m, 4H), 5.04-5.14 (m, 6H), 4.63-4.67 (m, 4H), 4.36-4.44 (m, 2H), 4.00-4.20 (m, 23H), 3.91-3.95 (m, 4H), 3.69-3.77 (m, 9H), 3.52-3.67 (m, 32H), 3.34-3.43 (m, 9H), 2.29-2.31 (m, 4H), 2.14 (s, 12H), 2.03 (s, 12H), 1.92-1.96 (m, 24H). LCMS: m / z = 1221.6 (M / 2+H) +

[0467] 3.2 Preparation of Compound 4 [ka]

[0468] Compound 3 (1.10 g, 0.451 mmol) was dissolved in MeOH (10.0 mL) at room temperature, and 10% mass fraction wet Pd / C (0.050 g, 0.451 mmol) was added to this solution. The reaction mixture was purged with hydrogen three times and then stirred at 25°C for 18 hours under a hydrogen atmosphere (14.696 psi). The MS response of the product was detected by LC-MS / MS. Furthermore, TLC (dichloromethane / methanol = 10 / 1, color: phosphomolybdic acid) confirmed that the starting material had been completely consumed and that new spots had been formed. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain compound 4 (approximately 840 mg) as a white solid.

[0469] 1 1H NMR (400 MHz, CD3OD)

[0470] δ=5.32-5.34 (m 4H), 5.06-5.10 (m, 4H), 4.63-4.65 (m, 4H), 4.38-4.40 (m, 2H), 3.99-4.20 (m, 20H), 3.90-3.97 (m, 4H), 3.69-3.76 (m, 6H), 3.50-3.68 (m, 36H), 3.35-3.44 (m, 11H), 2.28-2.38 (m, 4H), 2.15 (s, 12H), 2.03 (s, 12H), 1.90-1.94 (m, 24H). LCMS: m / z = 1154.7 (M / 2+H) +

[0471] 3.3 Preparation of Compound 6 [ka]

[0472] Compound 5 (232 mg, 0.364 mmol) was dissolved in DCM (10.0 mL) at room temperature. HBTU (207 mg, 0.546 mmol), DIEA (0.181 mL, 1.09 mmol), and Compound 4 (840 mg, 0.364 mmol) were added sequentially to the solution, and the mixture was purged with nitrogen three times. The reaction mixture was stirred at 25°C for 1 hour. Disappearance of the starting material was detected by LC-MS / MS. Furthermore, TLC (dichloromethane / methanol = 5 / 1) confirmed the disappearance of the starting material and the formation of new spots. The reaction mixture was concentrated under reduced pressure. The resulting crude product was purified by column chromatography (dichloromethane / methanol = 8 / 1 to 5 / 1) to obtain Compound 6 (approximately 620 mg) as a white solid.

[0473] 1 1H NMR (400 MHz, CD3OD)

[0474] δ=7.41-7.43 (m, 2H), 7.23-7.34 (m, 7H), 6.83-6.90 (m, 4H), 5.31-5.35 (m, 4H), 5.01-5.12 (m, 4H), 4.63-4.65 (m, 4H), 4.41-4.45 (m, 2H), 4.31-4.33 (m, 1H), 3.99-4.22 (m, 22H), 3.87-3.97 (m, 6H), 3.58-3.81 (m, 45H), 3.34-3.43 (m, 10H), 2.19-2.40 (m, 10H), 2.14 (s, 12H), 2.02 (s, 12H), 1.92-1.96 (mz, 24H), 1.48-1.63 (m, 4H), 1.28-1.38 (m, 8H). LCMS: m / z = 1460.0 (M / 2+H) +

[0475] 4. Preparation of compound E7 [ka]

[0476] Compound 6 (300 mg, 0.103 mmol) was dissolved in DCM (10.0 mL) at room temperature. DIEA (0.102 mL, 0.618 mmol), compound 7 (10.3 mg, 0.103 mmol), and DMAP (12.6 mg, 0.103 mmol) were sequentially added to this solution, and the mixture was purged with nitrogen three times. The reaction mixture was stirred at 25°C for 2 hours. Disappearance of the starting materials was detected by LC-MS / MS. The reaction mixture was concentrated under reduced pressure. The resulting crude product was separated by preparative MPLC (prep-HPLC, column: Waters Xbridge BEH C18 100*30 mm*10 μm; mobile phase: water-ACN; B%: 17%-57%, 5 min) to obtain compound E7 (53.0 mg, yield 17.08%, purity 78.94%) as a white solid.

[0477] 1 1H NMR (400 MHz, CD3OD) δ=7.41-7.45 (m, 2H), 7.17-7.34 (m, 7H), 6.85-6.89 (m, 4H), 5.32-5.36 (m, 4H), 5.03-5.13 (m, 4H), 4.63-4.67 (m, 4H), 4.38-4.47 (m, 2H), 4.32-4.34 (m, 1H), 4.01-4.26 (m, 22H), 3.88-4.00 (m, 6H), 3.77-3.81 (m, 7H), 3.49-3.76 (m, 45H), 3.33-3.47 (m, 10H), 2.56-2.62 (m, 2H), 2.45-2.55 (m, 3H), 2.21-2.38 (m, 7H), 2.14 (s, 12H), 2.05-2.11 (m, 2H), 2.02 (s, 12H), 1.92-1.96 (m, 24H), 1.47-1.68 (m, 4H), 1.28-1.34 (m, 8H)

[0478] MS: m / z = 3022.36 (M+H) +

[0479] Example 2 Preparation of siRNA

[0480] The siRNAs of this disclosure were prepared using the solid-phase phosphoramidite method, which is well known in this field. For specific details, please refer to, for example, PCT application numbers WO2016081444 and WO2019105419, but will also be briefly described below.

[0481] 1. Preparation of siRNA not conjugated to a ligand 1.1 Synthesis of sense strands (SS strands)

[0482] A solid-phase phosphoramidite synthesis method was employed, using a blank CPG solid support as the starting cycle. Nucleoside monomers were attached one by one from 3' to 5' according to the nucleotide sequence of the sense strand. Each bond of nucleoside monomer involves four step reactions: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for oligonucleotide synthesis on a 5 μmol scale are as follows.

[0483] Commercially available phosphoramidites modified with 2'-F and 2'-O-methyl were used. Nucleoside monomers were supplied as a 0.05 mol / L acetonitrile solution. The reaction conditions were the same for each step: temperature 25°C; deprotection was performed three times using a 3% trichloroacetic acid-dichloromethane solution; coupling was performed twice using a 0.25 mol / L ETT-acetonitrile solution as an activator; capping was performed twice using 10% anhydrous acetobiliary and pyridine / N-methylimidazole / acetonitrile (10:14:76, v / v / v); oxidation was performed twice using a 0.05 mol / L iodine / tetrahydrofuran / pyridine / water (70:20:10, v / v / v) solution; and thiolation was performed twice using 0.2 mol / L PADS added to acetonitrile / 3-methylpyridine (1:1, v / v). 1.2 Synthesis of antisense chains (AS chains)

[0484] A solid-phase phosphoramidite synthesis method was employed, using a blank CPG solid support as the starting cycle. Nucleoside monomers were attached one by one from 3' to 5' according to the nucleotide sequence of the antisense strand. Each nucleoside monomer bond involved four step reactions: deprotection, coupling, capping, and oxidation or thiolation. The conditions for synthesizing the antisense strand oligonucleotides on a 5 μmol scale were the same as those for the sense strand. 1.3 Purification and annealing of oligonucleotides

[0485] 1.3.1 Ammonolithesis

[0486] The synthesized solid support (sense chain or antisense chain) was added to a 5 mL centrifuge tube. A 3% diethylamine / ammonia solution (v / v) was added, and the mixture was reacted in a constant temperature water bath at 35°C for 16 hours (or at 55°C for 8 hours). The mixture was filtered, and the solid support was washed three times with 1 mL of ethanol / water each time. The filtrate was centrifuged and concentrated, and the crude product was purified.

[0487] 1.3.2 Purification

[0488] Methods for purification and desalting are well known to those skilled in the art. For example, a column was packed with a strong anionic filler, and after elution and purification using a sodium chloride-sodium hydroxide system, the product was recovered and pooled. Desalting was possible using a gel-packed purification column with pure water as the elution system.

[0489] 1.3.3 Annealing

[0490] According to Table 6, sense chains (SS chains) were mixed with antisense chains (AS chains) in a molar ratio (SS chain / AS chain = 1 / 1.05), heated in a water bath to 70-95°C for 3-5 minutes, allowed to cool naturally to room temperature, and the system was freeze-dried to obtain the product.

[0491] Ultimately, the siRNAs shown in Table 6 were obtained.

[0492] 2. Preparation of siRNA using a sense strand conjugated with a ligand

[0493] 2.1 Conjugation of Ligands to CPG Supports

[0494] Conjugation of compound E7 to CPG support

[0495] Compound E7 (53 mg, 0.018 mmol) and HBTU (13.3 mg, 0.035 mmol) were mixed and shaken to dissolve in acetonitrile (5 mL). Next, DIEA (9.0 mg, 0.07 mmol) and DMAP (2.1 mg, 0.018 mmol) were added and shaken until dissolved. Blank carrier resin (550 mg, CPG pore size 1000 Å) was weighed and added to the reaction mixture, and the mixture was left in a shaker at 20°C overnight. Samples were taken and monitored. Thin-layer chromatography (TLC) (developing solvent: DCM / methanol = 4 / 1; color: phosphomolybdic acid) was performed to confirm that the reaction was complete. The reaction mixture was filtered through a sand-core funnel. The filtration cake was washed with anhydrous acetonitrile (20 mL * 5) and collected. 530 mg of an off-white solid was obtained by suction filtration under reduced pressure using an oil pump for 6 hours.

[0496] The concentrated product (530 mg) was transferred to a 50 mL round-bottom flask. CapC (DMAP / acetonitrile), CapB (N-methylimidazole / pyridine / acetonitrile), and CapA (acetic anhydride / acetonitrile) were added sequentially. The mixture was then shaken overnight at room temperature. After filtration, the filtration cake was washed with acetonitrile (20 mL x 4) and collected. 200 mg of an off-white solid was obtained by suction filtration under reduced pressure using an oil pump for 8 hours. This was used for solid-phase synthesis.

[0497] 2.2 Synthesis of Sense Strands (SS Strands)

[0498] The solid-phase phosphoramidite method was employed, and the DL0043 solid support prepared above was used as the starting cycle. Nucleoside monomers were attached one by one from 3' to 5' according to the composition of the sense chain nucleotide. Each nucleoside monomer attachment involved four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for synthesizing 5 μmol of oligonucleotides were as follows.

[0499] Nucleoside monomers were supplied as a 0.05 mol / L acetonitrile solution. The conditions for each step were the same: temperature 25°C; deprotection was performed three times using a 3% trichloroacetic acid-dichloromethane solution; coupling was performed twice using a 0.25 mol / L ETT-acetonitrile solution as an activator; capping was performed twice using 10% anhydrous acetonenitrile and pyridine / N-methylimidazole / acetonitrile (10:14:76, v / v / v); oxidation was performed twice using a 0.05 mol / L iodine tetrahydrofuran / pyridine / water (70:20:10, v / v / v) solution; and thiolation was performed twice using a 0.2 mol / L PADS acetonitrile / 3-methylpyridine (1:1, v / v) solution.

[0500] 2.3 Synthesis of antisense chains (AS chains)

[0501] A solid-phase phosphoramidite method was employed, using a blank CPG solid support as the starting cycle. Nucleoside monomers were attached one by one from 3' to 5' according to the composition of the antisense chain nucleotide. Each nucleoside monomer attachment involved four steps: deprotection, coupling, capping, and oxidation or thiolation. The conditions for synthesizing 5 μmol of oligonucleotide as the antisense chain were the same as those for the sense chain.

[0502] 2.4 Purification and annealing of oligonucleotides

[0503] 2.4.1 Ammonolithesis

[0504] The synthesized solid support (sense chain or antisense chain) was transferred to a 5 mL centrifuge tube, and 3% diethylamine / ammonia (v / v) was added. The mixture was reacted in a constant temperature water bath at 35°C for 16 hours (or at 55°C for 8 hours), and then filtered. The solid support was washed three times with 1 mL of ethanol / water each time. The filtrate was concentrated by centrifugation, and the crude product was purified.

[0505] 2.4.2 Purification

[0506] Methods for purification and desalting are well known to those skilled in the art. For example, elution and purification may be performed using a strongly anion-packed column and a sodium chloride-sodium hydroxide system. The product can be recovered in a tube and desalted using a gel-packed purification column with pure water as the eluent.

[0507] 2.4.3 Annealing

[0508] According to Table 7, the sense strand (SS strand) was mixed with the antisense strand (AS strand) in a molar ratio (SS strand / AS strand = 1 / 1.05). The mixture was heated in a water bath to 70–95°C, held for 3–5 minutes, and then allowed to cool naturally to room temperature. The system was freeze-dried to obtain the product. Finally, the siRNAs shown in Table 7 were obtained. DR005677 was used as a control.

[0509] Example 3: Screening of activity in Huh7 cell line Cell transfection

[0510] On day 1, the Huh7 cell line was digested, resuspended, and counted. The resuspended cells were placed in a 96-well plate at a rate of 100 μL / well (1 × 10⁶ cells). 4 Cells were seeded in wells and transfection was performed 18 hours later.

[0511] On the second day, the 20 μM siRNA stock solution was diluted with Opti-MEM. 198 μL of Opti-MEM was added to 2 μL of the siRNA stock solution. The final concentrations of siRNA were as shown below. The solutions were thoroughly mixed by pipetting and allowed to stand until use.

[0512] On day 2, 9 μL of RNAiMAX (Thermo, 13778150) was diluted with 14.1 μL of Opti-MEM, gently mixed by pipetting, and allowed to stand at room temperature for 5 minutes. Then, 15 μL of the prepared RNAi-MAX mixture and 15 μL of diluted siRNA were gently mixed by pipetting (avoiding the formation of air bubbles) and allowed to stand at room temperature for 10 minutes. The mixture was transferred to a 96-well plate at a rate of 10 μL / well. After incubation in a 5% CO2 incubator at 37°C for 24 hours, the plates were extracted (siRNA was not added to the control group).

[0513] RNA extraction

[0514] Cellular RNA was extracted using a nucleic acid extractor (Hangzhou Allsheng, Auto-pure96) according to the protocol of the high-throughput cell RNA extraction kit (FireGen, FG0417-L).

[0515] RNA reverse transcription

[0516] Denaturation reaction mixtures were prepared according to the PrimeScript® II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Preparation volume per well: 1 μL of oligo-dT primer, 1 μL of dNTP mixture, and 12.5 μL of template RNA. The reaction mixtures were incubated at 65°C for 5 minutes using a conventional PCR instrument, followed by rapid cooling on ice for 2 minutes.

[0517] Reverse transcription reaction mixtures were prepared according to the PrimeScript® II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contained 4 μL of 5× Prime Script II Buffer, 0.5 μL of RNase Inhibitor, and 1 μL of PrimeScript II RTase.

[0518] The denaturation reaction mixture (14.5 μL) was gently mixed with the reverse transcription reaction mixture. Reverse transcription was performed in a conventional PCR instrument by incubation at 42°C for 45 minutes, and the enzyme was inactivated by further incubation at 95°C for 5 minutes. Subsequently, the reverse transcript (cDNA) was cooled at 4°C.

[0519] After reverse transcription, 30 μL of DNase / RNase-free distilled water was added to the cDNA sample in each well.

[0520] Fluorescence Quantitative PCR

[0521] A fluorescence quantitative PCR reaction was performed in a 20 μL system (ABI, QuantStudio3) referring to the procedure for TaqMan® Fast Advanced Master Mix (ABI, 4444965). The reaction program was as follows: (50°C, 2 min) × 1 cycle; (95°C, 20 sec) × 1 cycle; (95°C, 1 sec; 60°C, 24 sec) × 40 cycles. [Table 8]

[0522] Data statistics

[0523] 2 -△△Ct The value was calculated and converted to a percentage to obtain the residue suppression rate. △△Ct=[(Ct 標的遺伝子,実験群 -Ct 参照遺伝子,実験群 )-(Ct 標的遺伝子,コントロール群 -Ct 参照遺伝子,コントロール群 )]

[0524] The target gene was hAGT, and the reference gene was hACTB.

[0525] The final concentration of siRNA was set to 10 nM, and cell line activity screening of siRNA compounds was performed. The results of the experimental screening are shown in Table 9. [Table 9]

[0526] Example 4: Screening of activity in human primary hepatocytes (PHH cells) Cell transfection

[0527] 1.4 mL of rat tail collagen solution (Sigma, C3867) was added to 40.6 mL of DNase / RNase-free distilled water and thoroughly mixed. The mixture was added to 96-well culture plates at a rate of 40 μL / well and coated overnight at 4°C. The coating medium was removed the following day.

[0528] On the second day, prior to use, the coated cell plates were rinsed with DPBS and aspirated. PHH (Shanghai Xuanyi Biotechnology, Cat#: QYLF-HPMC) was recovered at 37°C, transferred to recovery medium, centrifuged, resuspended, and counted. PHH was then added to 96-well plates at a rate of 90 μL / well (2 × 10⁶). 4 Cells were seeded in wells. The culture medium was replaced with complete medium after 4 hours, and transfected after 18 hours.

[0529] On the third day, the 20 μM siRNA stock solution was diluted with Opti-MEM. 198 μL of Opti-MEM was added to 2 μL of the 20 μM siRNA stock solution and thoroughly mixed by pipetting to obtain the first concentration point. Serial dilutions were then performed as determined experimentally.

[0530] On day 3, 0.9 μL of RNAiMAX (Thermo, 13778150) was diluted with 14.1 μL of Opti-MEM, gently mixed by pipetting, and allowed to stand at room temperature for 5 minutes. Then, 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted compound were gently mixed by pipetting (avoiding the formation of air bubbles) and allowed to stand at room temperature for 10 minutes. The mixture was transferred to a 96-well plate at a rate of 10 μL / well. After incubation in a 5% CO2 incubator at 37°C for 24 hours, RNA was extracted.

[0531] RNA extraction

[0532] Cellular RNA was extracted using a nucleic acid extractor (Hangzhou Allsheng, Auto-pure96) according to the protocol of a high-throughput cell RNA extraction kit (FireGen, FG0412).

[0533] RNA reverse transcription

[0534] A denaturation reaction mixture was prepared according to the PrimeScript® II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contained 1 μL of Oligo dT Primer, 1 μL of dNTP Mixture, and 12.5 μL of template RNA. The denaturation reaction was carried out by incubation at 65°C for 5 minutes in a conventional PCR instrument. The mixture was rapidly cooled on ice for 2 minutes.

[0535] Reverse transcription reaction mixtures were prepared according to the PrimeScript® II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contained 4 μL of 5× Prime Script II Buffer, 0.5 μL of RNase Inhibitor, and 1 μL of PrimeScript II RTase.

[0536] The denaturation reaction mixture (14.5 μL) was gently mixed with the reverse transcription reaction mixture. Reverse transcription was performed in a conventional PCR instrument by incubation at 42°C for 45 minutes, and the enzyme was inactivated by further incubation at 95°C for 5 minutes. Subsequently, the reverse transcript (cDNA) was cooled at 4°C.

[0537] After reverse transcription, 30 μL of DNase / RNase-free distilled water was added to the cDNA sample in each well.

[0538] Fluorescence Quantitative PCR

[0539] A fluorescence quantitative PCR reaction was performed in a 20 μL system (ABI, QuantStudio3) referring to the procedure for TaqMan® Fast Advanced Master Mix (ABI, 4444965). The reaction program was as follows: (50°C, 2 min) × 1 cycle; (95°C, 20 sec) × 1 cycle; (95°C, 1 sec; 60°C, 24 sec) × 40 cycles. [Table 10]

[0540] Data statistics

[0541] 2 -△△Ct The value was calculated and converted to a percentage to obtain the residue suppression rate. △△Ct=[(Ct 標的遺伝子,実験群 -Ct 参照遺伝子,実験群 )-(Ct 標的遺伝子,コントロール群 -Ct 参照遺伝子,コントロール群 )]

[0542] The initial concentration of siRNA was set to 10 nM, and five concentration points (10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM) were obtained by serial dilution fivefold. The activity of siRNA in primary human liver cells was screened. The screening results are shown in Table 10. In this table, the residual inhibition rate is listed in columns 2-6, and the IC50 value is listed in column 7. [Table 11]

[0543] Example 5: Screening for off-target activity in psiCHECK2 GSSM-5Hits Plasmid preparation

[0544] Corresponding antisense strand off-target plasmids were designed according to the siRNA sequence. The psiCHECK2 GSSM-5Hits recombinant plasmid was prepared by Sangon Biotech (Shanghai). This recombinant plasmid was used diluted to 1000 ng / μL.

[0545] Cell transfection

[0546] 100 μL of HEK293A cell suspension (Cobioer Biosciences, Cat#CBP60436) was seeded per well in a 96-well plate (8 × 10⁶). 3 Cells / wells).

[0547] The following day, the complete culture medium in the wells was aspirated and then replaced with 80 μL / well of Opti-MEM medium, and the cells were starved for approximately 1.5 hours.

[0548] siRNA preparation: siRNA was diluted threefold from a final concentration of 40 nM to obtain 11 concentration points (10 nM, 3.3333 nM, 1.1111 nM, 0.37037 nM, 0.12346 nM, 0.04115 nM, 0.01372 nM, 0.00457 nM, 0.00152 nM, 0.00051 nM, and 0.00017 nM).

[0549] Plasmid mixture preparation: The preparation volume per well was 0.01 μL / well of plasmid and 8.99 μL / well of Opti-MEM.

[0550] Preparation of Lipo mixture: Lipo 2000 (Lipofectamine® 2000 Transfection Reagent, Thermo, 11668019) was diluted with Opti-MEM by adding 0.2 μL of Lipo 2000 and 9.8 μL of Opti-MEM to each well, and the resulting Lipo mixture was allowed to stand at room temperature for 5 minutes.

[0551] 22 μL of the prepared Lipo mixture, 2.2 μL of siRNA, and 19.8 μL of the plasmid mixture were individually dispensed into the corresponding wells to form the "Well A" mixture. After mixing by pipetting, the mixtures were incubated at room temperature for 20 minutes before co-transfection. 20 μL / well of the "Well A" mixture was added to each cell well. Combined with the original 80 μL of Opti-MEM, the final volume was 100 μL per well. After incubation at 37°C for 4 hours in a 5% CO2 incubator, 100 μL of DMEM medium containing 20% ​​fetal bovine serum was added to each well. The cells were incubated at 37°C for 24 hours in a 5% CO2 incubator before detection.

[0552] Result detection

[0553] Before the experiment, the mixed Dual-Glo® luciferase (Dual-Glo® Luciferase Assay System, Promega, E2940) was thawed. After equilibration to room temperature, substrate I was prepared by adding DMEM in a 1:1 ratio to each tube and used immediately after preparation. Dual-Glo® Stop&Glo® Buffer was thawed. After equilibration to room temperature, substrate II was prepared by mixing it with Dual-Glo® Stop&Glo® Substrate in a 100:1 ratio and used immediately after preparation. The old culture medium in the 96-well culture plate was aspirated using a vacuum pump. 150 μL of substrate I / well was added and incubated on a shaker at room temperature for 10 minutes. 120 μL of substrate I was transferred to a 96-well microplate and the firefly chemiluminescence value was read using a microplate reader (Tecan, Infinite 200). Next, 60 μL of substrate II / well was added, and the mixture was incubated on a shaker at room temperature for 10 minutes. Then, the chemiluminescence values ​​of Renilla were read using a microplate reader.

[0554] Data analysis and processing

[0555] The fluorescence activity is measured using a microplate reader, the collected Renilla signals are normalized using a Firefly signal standard, and the inhibitory effect of siRNA is compared to the untreated results (residual inhibitory activity). The calculation process is as follows:

[0556] Normalized Ren / Fir ratio: Ratio = Renilla (sea lichen luciferase) / Firefly (firefly luciferase).

[0557] Residual inhibition rate = (RatiosiRNA / Ratiocontrol) * 100% (calculated as the average of two replication wells, where Ratiocontrol is the Ratio value of the control well (without siRNA) (calculated as the average of two replication wells)).

[0558] Drawing: Drawing using Graphpad Prism

[0559] 50% inhibitory concentration (IC50): In this experiment, the top and bottom are plotted, and the IC50 value is obtained according to the following equation: Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * HillSlope)) (where Y = 50, X = log(concentration)).

[0560] Table 12 shows the results of screening for off-target activity of siRNA in psiCHECK2 GSSM-5Hits. In this table, the residual inhibition rate is listed in columns 2-12, and the IC50 value is listed in column 13. [Table 12]

[0561] Example 6: Activity detection in AAV humanized mouse model AAV8 virus (purchased from OBiO Tech) containing a human-derived AGT gene fragment was used in a 1 x 10⁻¹⁶ experiment. 11 The virus was injected into mice via the tail vein at a dose of vg. The day of virus injection was defined as day -14. Serum was collected from the mice on day -1, and AGT protein was detected by ELISA as a baseline (detection kit purchased from Abcam Company, Cat#ab267592). The mice were divided into groups, with 3 mice per group. On day 0, the siRNA drug was administered via tail vein infusion at a dose of 1 mg / kg. Serum was collected on days 7, 14, 28, and 42 (D7, D14, D28, D42) after administration, and AGT protein levels were detected using the above kit. The survival rate was calculated with the baseline level set at 100%. [Table 13]

[0562] Example 7: In vivo activity evaluation in a transgenic mouse model After the adaptation period, blood was collected from the orbital vein of each male C57BL / 6J transgenic mouse on day 4 prior to administration (D-4), and serum samples were collected after centrifugation. The level of human AGT protein in the serum of each mouse was detected by ELISA assay as the experimental baseline, and the mice were divided into administration groups (4 mice per group) according to the baseline level. On the day of administration (D0), each group of mice was administered either physiological saline or the siRNA disclosed herein by subcutaneous injection at a dose of 1 or 3 mg / kg (mpk) and a dosing volume of 5 ml / kg, respectively. Mouse serum was collected on days 7, 14, 21, 28, 35, 42, 56, and 70 after administration, and the human AGT protein level was measured by ELISA assay (assay kit purchased from Abcam, Cat#ab267592). The siRNA knockdown efficiency was calculated by comparing the human AGT protein level in the serum of each mouse before and after administration. The experimental results are shown in Table 14. [Table 14]

[0563] Example 8: In vivo pharmacodynamic evaluation of siRNA in diet-induced hypertensive cynomolgus monkeys Sixteen male hypertensive cynomolgus monkeys were divided into four groups according to serum AGT levels, body weight, and blood pressure. They were administered either saline (i.e., vehicle group) or a single subcutaneous injection of the siRNA of this disclosure at a dose of 10 mg / kg and a dosing volume of 1 ml / kg. Serum was collected on day 6 before administration (D-6), on the day of administration (D0), and on days 3, 7, 14, 21, 28, 35, 42, 55, 70, 77, 84, 91, 98, and 105 after administration. Serum AGT protein levels were detected by ELISA assay (IBL-Japan, Cat#:27412). Blood pressure was measured weekly using a high-resolution oscilloscope (HDO) at the left wrist or base of the tail (tail cuff device). The siRNA knockdown efficiency was calculated by comparing serum AGT protein levels in each monkey on the day of administration and at various time points after administration. The magnitude of the reduction in serum AGT protein, systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) by each compound is shown in Figures 1, 2, 3, and 4.

[0564] The results showed that each compound achieved a very high rate of suppression of serum AGT protein (maximum suppression > 98%). On day 105 after administration, DR009219 and DR009221 still showed suppression rates of over 90%. From day 14 onward, all compounds in each group significantly reduced systolic blood pressure, with the maximum reduction ranging from 40 mmHg to 50 mmHg. On day 105 after administration, the systolic blood pressure of monkeys in each treatment group was still significantly lower than that of the control group. Each compound also significantly reduced diastolic blood pressure, with the maximum reduction ranging from 20 mmHg to 30 mmHg. On day 105 after administration, the diastolic blood pressure of monkeys in each treatment group was still significantly lower than that of the control group.

Claims

1. A double-stranded nucleotide (dsRNA) for suppressing the expression of angiotensinogen (AGT) in cells, wherein the dsRNA comprises a sense strand and an antisense strand forming a double-stranded region, each of which is independently 15 to 30 nucleotides long, and the antisense strand contains the nucleotide sequence of at least 15 adjacent nucleotides in the nucleotide sequence described in any one of Sequence ID No. 21 to 40.

2. The dsRNA according to claim 1, wherein the sense strand comprises the nucleotide sequence of at least 15 adjacent nucleotides in the nucleotide sequence described in any one of SEQ ID NOs: 1 to 20.

3. The dsRNA according to claim 1 or 2, wherein the dsRNA is siRNA.

4. The dsRNA according to any one of claims 1 to 3, wherein the double-stranded region has a length of 15 to 25 nucleotide pairs, preferably 16 to 23 nucleotide pairs, and more preferably 18 to 20 nucleotide pairs.

5. The antisense strand comprises a nucleotide sequence of at least 16 adjacent nucleotides, a nucleotide sequence of at least 17 adjacent nucleotides, a nucleotide sequence of at least 18 adjacent nucleotides, a nucleotide sequence of at least 19 adjacent nucleotides, or a nucleotide sequence of at least 20 adjacent nucleotides in the nucleotide sequence described in any one of SEQ ID NOs: 21 to 40, and preferably comprises a nucleotide sequence described in any one of SEQ ID NOs: 21 to 40, the dsRNA according to any one of claims 1 to 4.

6. The sense strand comprises a nucleotide sequence of at least 16 adjacent nucleotides, a nucleotide sequence of at least 17 adjacent nucleotides, or a nucleotide sequence of at least 18 adjacent nucleotides in the nucleotide sequence described in any one of SEQ ID NOs: 1 to 20, and preferably comprises a nucleotide sequence described in any one of SEQ ID NOs: 1 to 20, the dsRNA according to any one of claims 1 to 5.

7. The above siRNA is a dsRNA according to any one of the following pairs of sense strand sequence and antisense strand sequence: (1) Sense chain: Sequence ID 1, Antisense chain: Sequence ID 21; (2) Sense chain: Sequence ID 2, Antisense chain: Sequence ID 22; (3) Sense chain: Sequence ID 3, Antisense chain: Sequence ID 23; (4) Sense chain: Sequence ID 4, Antisense chain: Sequence ID 24; (5) Sense chain: Sequence ID 5, Antisense chain: Sequence ID 25; (6) Sense strand: Sequence ID 6, Antisense strand: Sequence ID 26; (7) Sense strand: Sequence ID 7, Antisense strand: Sequence ID 27; (8) Sense strand: Sequence ID 8, Antisense strand: Sequence ID 28; (9) Sense chain: Sequence ID 9, Antisense chain: Sequence ID 29; (10) Sense chain: Sequence ID 10, Antisense chain: Sequence ID 30; (11) Sense chain: Sequence ID 11, Antisense chain: Sequence ID 31; (12) Sense chain: Sequence ID 12, Antisense chain: Sequence ID 32; (13) Sense chain: Sequence ID 13, Antisense chain: Sequence ID 33; (14) Sense chain: Sequence ID 14, Antisense chain: Sequence ID 34; (15) Sense chain: Sequence ID 15, Antisense chain: Sequence ID 35; (16) Sense chain: Sequence ID 16, Antisense chain: Sequence ID 36; (17) Sense chain: Sequence ID 17, Antisense chain: Sequence ID 37; (18) Sense chain: Sequence ID 18, Antisense chain: Sequence ID 38; (19) Sense strand: Sequence ID 19, Antisense strand: Sequence ID 39; and (20) Sense strand: Sequence ID 20, Antisense strand: Sequence ID 40.

8. The dsRNA according to any one of claims 1 to 7, wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, or all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

9. The dsRNA according to any one of claims 1 to 8, wherein the sense strand and the antisense strand each independently include one or more nucleotide modifications selected from the group consisting of 2'-O-methyl modification, 2'-fluoro modification, SCP modification, glycol modification, and phosphorothioate internucleotide bond modification.

10. The above antisense strand is 21 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1, 3, 5, 9, 11, 13, 15, 17, 19, and 21, and 2'-fluoro modified nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counted from the 5' end); and / or (ii) Inter-phosphorothioate nucleotide bonds (counted from the 5' end) between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21. A dsRNA according to any one of claims 1 to 9, having the following characteristics.

11. The above antisense strand is 21 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 3, 4, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, and 21 (counted from the 5' end); (ii) The 2'-fluoromodified nucleotide at position 14 (counted from the 5' end); (iii) 2'-deoxy modifications at positions 2, 5, 7, and 12 (counted from the 5' end); (iv) SCP modification at position 1 (counted from the 5' end); and / or (v) Phosphothioate internucleotide bonds (counted from the 5' end) between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 A dsRNA according to any one of claims 1 to 9, having the following characteristics.

12. The above antisense strand is 21 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 3, 5, 9, 11, 13, 15, 17, 19, 20, and 21 (counted from the 5' end); (ii) 2'-fluoromodified nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, and 18 (counted from the 5' end); (iii) GNA modification at position 7 (counted from the 5' end); (iv) SCP modification at position 1 (counted from the 5' end); and / or (v) Phosphothioate internucleotide bonds (counted from the 5' end) between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 A dsRNA according to any one of claims 1 to 9, having the following characteristics.

13. The sense strand described above is 19 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1-6 and 10-19 and 2'-fluoro modified nucleotides at positions 7-9 (counted from the 5' end); and / or (ii) Phosphothioate internucleotide bonds between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between nucleotide positions 18 and 19 (counted from the 5' end) A dsRNA according to any one of claims 1 to 9, having the following characteristics.

14. The dsRNA according to any one of claims 8 to 12, wherein the antisense strand comprises a sequence selected from any one of the sequences described in Sequence ID No. 81 to 105.

15. The dsRNA according to any one of claims 8-9 and 13-14, wherein the sense strand comprises a sequence selected from any one of the sequences described in Sequence ID No. 41-59.

16. The dsRNA according to claim 15, wherein the above dsRNA comprises any pair of sense strand sequence and antisense strand sequence selected from the following: Sense chain: Sequence ID 41, Antisense chain: Sequence ID 81; Sense chain: Sequence ID 42, Antisense chain: Sequence ID 82; Sense chain: SEQ ID NO: 43, Antisense chain: SEQ ID NO: 83; Sense chain: Sequence ID 44, Antisense chain: Sequence ID 84; Sense chain: Sequence ID 45, Antisense chain: Sequence ID 85; Sense chain: Sequence ID 46, Antisense chain: Sequence ID 86; Sense strand: Sequence ID 47, Antisense strand: Sequence ID 87; Sense strand: Sequence ID 48, Antisense strand: Sequence ID 88; Sense strand: Sequence ID 49, Antisense strand: Sequence ID 89; Sense chain: Sequence ID 50, Antisense chain: Sequence ID 90; Sense chain: SEQ ID NO: 51, Antisense chain: SEQ ID NO: 91; Sense chain: Sequence ID 52, Antisense chain: Sequence ID 92; Sense chain: SEQ ID NO: 53, Antisense chain: SEQ ID NO: 93; Sense chain: Sequence ID 54, Antisense chain: Sequence ID 94; Sense chain: Sequence ID 55, Antisense chain: Sequence ID 95; Sense strand: Sequence ID 56, Antisense strand: Sequence ID 96; Sense strand: Sequence ID 57, Antisense strand: Sequence ID 97; Sense strand: SEQ ID NO: 58, Antisense strand: SEQ ID NO: 98; and Sense strand: Sequence ID 59, Antisense strand: Sequence ID 99.

17. The dsRNA according to any one of claims 8-9 and 13-14, wherein the sense strand includes a sequence selected from any one of the sequences described in SEQ ID NOs: 113-131.

18. The dsRNA according to claim 17, wherein the above dsRNA comprises any pair of sense strand sequence and antisense strand sequence selected from the following: Sense chain: SEQ ID NO: 113, Antisense chain: SEQ ID NO: 81; Sense chain: Sequence ID 114, Antisense chain: Sequence ID 82; Sense chain: SEQ ID NO: 115, Antisense chain: SEQ ID NO: 83; Sense chain: Sequence ID 116, Antisense chain: Sequence ID 84; Sense chain: Sequence ID 117, Antisense chain: Sequence ID 85; Sense chain: Sequence ID 118, Antisense chain: Sequence ID 86; Sense chain: SEQ ID NO: 119, Antisense chain: SEQ ID NO: 87; Sense strand: Sequence ID 120, Antisense strand: Sequence ID 88; Sense chain: SEQ ID NO: 121, Antisense chain: SEQ ID NO: 89; Sense chain: Sequence ID 122, Antisense chain: Sequence ID 90; Sense chain: SEQ ID NO: 123, Antisense chain: SEQ ID NO: 91; Sense chain: Sequence ID 124, Antisense chain: Sequence ID 92; Sense chain: SEQ ID NO: 125, Antisense chain: SEQ ID NO: 93; Sense chain: SEQ ID NO: 126, Antisense chain: SEQ ID NO: 94; Sense strand: Sequence ID 127, Antisense strand: Sequence ID 95; Sense strand: Sequence ID 128, Antisense strand: Sequence ID 96; Sense strand: Sequence ID 129, Antisense strand: Sequence ID 97; Sense strand: SEQ ID NO: 130, Antisense strand: SEQ ID NO: 98; and Sense chain: Sequence ID 131, Antisense chain: Sequence ID 99.

19. The dsRNA according to any one of claims 1 to 18, wherein the above dsRNA is further conjugated to a ligand portion containing N-acetylgalactosamine, preferably the 3' end of the sense strand is conjugated to the ligand portion.

20. The dsRNA according to any one of claims 19, wherein the ligand described above has the following structure: 【Chemistry 1】 (In the formula, 【Chemistry 2】 (This indicates a point where the sense strand of the dsRNA is connected via a phosphate group or a phosphorothioate group.)

21. The dsRNA according to claim 19 or claim 20, wherein the sense strand comprises a sequence selected from any one of the sequences described in SEQ ID NOs. 60 to 79.

22. The dsRNA according to claim 21, comprising any pair of sense strand sequence and antisense strand sequence selected from the following: Sense strand: Sequence ID 60, Antisense strand: Sequence ID 81; Sense chain: Sequence ID 61, Antisense chain: Sequence ID 82; Sense chain: Sequence ID 62, Antisense chain: Sequence ID 83; Sense chain: Sequence ID 63, Antisense chain: Sequence ID 84; Sense chain: Sequence ID 64, Antisense chain: Sequence ID 85; Sense strand: Sequence ID 65, Antisense strand: Sequence ID 86; Sense strand: Sequence ID 66, Antisense strand: Sequence ID 87; Sense strand: Sequence ID 67, Antisense strand: Sequence ID 88; Sense strand: Sequence ID 68, Antisense strand: Sequence ID 89; Sense strand: Sequence ID 69, Antisense strand: Sequence ID 90; Sense strand: SEQ ID NO: 70, Antisense strand: SEQ ID NO: 91; Sense chain: SEQ ID NO: 71, Antisense chain: SEQ ID NO: 92; Sense chain: Sequence ID 72, Antisense chain: Sequence ID 93; Sense chain: Sequence ID 73, Antisense chain: Sequence ID 94; Sense strand: Sequence ID 74, Antisense strand: Sequence ID 95; Sense strand: Sequence ID 75, Antisense strand: Sequence ID 96; Sense strand: SEQ ID NO: 76, Antisense strand: SEQ ID NO: 97; Sense strand: Sequence ID 77, Antisense strand: Sequence ID 98; Sense strand: Sequence ID 78, Antisense strand: Sequence ID 99; Sense chain: Sequence ID 72, Antisense chain: Sequence ID 100; Sense chain: SEQ ID NO: 72, Antisense chain: SEQ ID NO: 101; Sense chain: SEQ ID NO: 72, Antisense chain: SEQ ID NO: 102; Sense chain: SEQ ID NO: 72, Antisense chain: SEQ ID NO: 103; Sense strand: Sequence ID 79, Antisense strand: Sequence ID 104; and Sense strand: Sequence ID 79, Antisense strand: Sequence ID 105.

23. A vector comprising a nucleotide sequence encoding the dsRNA according to any one of claims 1 to 22.

24. A cell comprising the dsRNA described in any one of claims 1 to 22 or the vector described in claim 23.

25. A pharmaceutical composition comprising a dsRNA according to any one of claims 1 to 22, a vector according to claim 23, or a cell according to claim 24, and optionally a pharmaceutically acceptable carrier or excipient.

26. A kit comprising the dsRNA according to any one of claims 1 to 22, the vector according to claim 23, or the cells according to claim 24.

27. A method for treating a disease or disorder in which reducing the expression of angiotensinogen (AGT) in a subject is beneficial, comprising the step of administering to the subject a dsRNA according to any one of claims 1 to 22, a vector according to claim 23, cells according to claim 24, or a pharmaceutical composition according to claim 25.

28. A method for preventing at least one symptom in a subject having a disease or disorder for which reducing the expression of angiotensinogen (AGT) is beneficial, comprising the step of administering to the subject a dsRNA according to any one of claims 1 to 22, a vector according to claim 23, cells according to claim 24, or a pharmaceutical composition according to claim 25.

29. The method according to claim 27 or 28, wherein the disease or disorder for which reducing the expression of the above-mentioned angiotensinogen (AGT) is beneficial is an AGT-mediated disease or an AGT-related disease.

30. The method according to claim 29, wherein the above-mentioned AGT-mediated disease or AGT-related disease is selected from the group consisting of hypertension, intraocular hypertension, glaucoma, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, stroke, nephropathy, renal failure, systemic sclerosis, intrauterine growth restriction (IUGR), fetal growth restriction, obesity, fatty liver / fatty liver disease, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD); glucose intolerance, type 2 diabetes mellitus (non-insulin-dependent diabetes mellitus), and metabolic syndrome.

31. The method according to claim 30, wherein the hypertension described above is selected from the group consisting of borderline hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, treatment-resistant hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, and unstable hypertension.

32. A method for reducing the level of angiotensinogen (AGT) in a subject, comprising the step of administering to the subject a dsRNA according to any one of claims 1 to 22, a vector according to claim 23, cells according to claim 24, or a pharmaceutical composition according to claim 25.

33. The method according to any one of claims 27 to 32, wherein the dsRNA, vector, cells, or pharmaceutical composition is administered by subcutaneous, topical, or intravenous administration.

34. The method according to any one of claims 27 to 33, wherein the subject is a human.

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