AGT inhibitors and their uses

Abstract

The present application relates to an RNAi agent or a pharmaceutically acceptable salt thereof, wherein the structure of the RNAi agent contains a carrier structure and an interfering nucleic acid. The present application also relates to a method for using the RNAi agent to inhibit the expression of the AGT gene and a method for preventing and treating AGT-related diseases.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to an RNAi agent for inhibiting AGT gene expression and its application. Background Art

[0002] RNAi

[0003] RNAi (RNA interference) was discovered in 1998 by Andrew Z. Fire and others in an antisense RNA inhibition experiment in Caenorhabditis elegans, and this process was called RNAi. This discovery was rated as one of the top ten scientific advances in 2001 by Science magazine, and ranked first among the top ten scientific advances in 2002. Since then, siRNA with RNAi as its mechanism of action has attracted widespread attention as a potential gene therapy drug. In 2006, Andrew Z. Fire and Craig C. Mello won the Nobel Prize in Physiology or Medicine for their contributions to the study of RNAi mechanism. RNAi can be triggered by double-stranded RNA (dsRNA) in many organisms, including animals, plants and fungi. During the RNAi process, a nuclease called "Dicer" cuts or "dices" the long-chain dsRNA into small fragments of 21 to 25 nucleotides. These small fragments, called small interfering RNA (siRNA), have their antisense strands (Guide strands) loaded onto the Argonaute protein (AGO2). AGO2 loading occurs in the RISC-loading complex, a ternary complex consisting of the Argonaute protein, Dicer, and dsRNA binding protein (TRBP for short). During the loading process, the sense strand (Passenger strand) is cleaved and expelled by AGO2. AGO2 then uses the antisense strand to bind to mRNAs containing completely complementary sequences and then catalyzes the cleavage of these mRNAs, causing the mRNA split to lose its role as a translation template, thereby preventing the synthesis of related proteins. After cleavage, the cleaved mRNA is released, and the RISC-loading complex loaded with the antisense strand is recycled for another round of cleavage.

[0004] According to statistics, among the disease-related proteins in the human body, more than 80% of the proteins cannot be targeted by the current conventional small molecule drugs and biological macromolecule preparations, and are non-drugable proteins. Gene therapy, which aims to treat diseases through gene expression, silencing and other functions, is considered by the industry to be the third generation of therapeutic drugs after chemical small molecule drugs and biological macromolecule drugs. This therapy achieves the treatment of diseases at the gene level and is not restricted by non-drugable proteins. As the most mainstream type of RNAi technology in gene therapy, RNAi technology treats diseases at the mRNA level, which is more efficient than chemical small molecule drugs and biological macromolecule drugs at the protein level. Using RNAi technology, the sense and antisense chain sequences of siRNA with high specificity and good inhibitory effect can be designed according to specific gene sequences. These single-stranded sequences are synthesized through solid phase, and then the sense chain and antisense chain are paired into siRNA according to the base pairing principle in a specific annealing buffer, and finally delivered to the corresponding target in the body through a carrier system, degrading the target mRNA and destroying the function of the target mRNA as a translation template, thereby preventing the synthesis of related proteins.

[0005] siRNA delivery system

[0006] siRNA is unstable in blood and tissues and is easily degraded by nucleases. In order to improve the stability of siRNA, the sense strand and / or antisense strand of siRNA can be modified, but these chemical modifications only provide limited protection from nuclease degradation and may ultimately affect the activity of siRNA. Therefore, a corresponding delivery system is also needed to ensure that siRNA passes through the cell membrane safely and efficiently. Since siRNA has a large molecular weight, carries a large amount of negative charge, and has high water solubility, it cannot pass through the cell membrane smoothly and reach the cell.

[0007] The basic structure of liposomes is composed of a hydrophilic core and a phospholipid bilayer. It has a phospholipid bilayer similar to a biological membrane and has high biocompatibility. Therefore, liposomes once became the most popular and widely used siRNA carrier. Liposome-mediated siRNA delivery mainly encapsulates siRNA into liposomes to protect siRNA from degradation by nucleases, improve the efficiency of siRNA passing through cell membrane barriers, and thus promote cell absorption. For example, anionic liposomes, pH-sensitive liposomes, immunoliposomes, fusogenic liposomes, and cationic lipids, etc. Although certain progress has been made, liposomes themselves are prone to induce inflammatory reactions. Before administration, a variety of antihistamines and hormones such as cilitizine and dexamethasone must be used to reduce possible acute inflammatory reactions. Therefore, in actual clinical applications, it is not suitable for all treatment areas, especially for diseases such as chronic hepatitis B that have a long treatment cycle. The cumulative toxicity that may occur with long-term use is a potential safety hazard. Therefore, a safer and more effective carrier system is needed to deliver siRNA.

[0008] The asialoglycoprotein receptor (ASGPR) in the liver is a receptor specifically expressed by hepatocytes and is a highly efficient endocytic receptor. Since the secondary end of various glycoproteins exposed after enzyme or acid hydrolysis of sialic acid in the body under physiological conditions is a galactose residue, the sugar that ASGPR specifically binds is galactosyl, so it is also called a galactose-specific receptor. Monosaccharide and polysaccharide molecules such as galactose, galactosamine, and N-acetylgalactosamine have high affinity for ASGPR. The main physiological function of ASGPR is to mediate the clearance of substances such as asialoglycoproteins and lipoproteins in the blood, and it is closely related to the occurrence and development of liver diseases such as viral hepatitis, cirrhosis, and liver cancer. The discovery of this characteristic of ASGPR plays an important role in the diagnosis and treatment of hepatic diseases (Ashwell G, Harford J, Carbohydrate specific Receptors of the Liver, Ann Rev Biochem 1982 51:531-554). The therapeutic drugs for liver-derived diseases containing galactose or galactosamine and their derivatives in the structure can specifically bind to ASGPR, thereby having active liver targeting and requiring no other carrier system for delivery.

[0009] Angiotensinogen (AGT) and hypertension

[0010] Blood pressure refers to the pressure of blood against the walls of blood vessels in the circulatory system. Blood pressure is primarily due to the beating of an animal's heart. During each heartbeat, blood pressure varies between a maximum (systolic) blood pressure (SBP) and a minimum (diastolic) blood pressure (DBP). Mean arterial pressure (MAP) is the average arterial pressure during the heart cycle. Blood pressure can be measured by a sphygmomanometer (i.e., a blood pressure measuring device). Normal blood pressure at rest is in the range of 100-140 mmHg systolic and 60-90 mmHg diastolic, and is usually expressed as systolic (highest reading) / diastolic (lowest reading) mmHg.

[0011] Hypertension is defined as systolic blood pressure (SBP) ≥ 140 mmHg and / or diastolic blood pressure (DBP) ≥ 90 mmHg without the use of antihypertensive drugs. Hypertension is divided into grades 1, 2, and 3 according to the level of blood pressure increase. Cardiovascular risk stratification is performed based on blood pressure levels, cardiovascular risk factors, target organ damage, clinical complications, and diabetes, and is divided into four levels: low risk, moderate risk, high risk, and very high risk. The blood pressure classification and definition are as follows:

[0012]

[0013] According to the cause of the disease, hypertension can also be divided into primary hypertension and secondary hypertension. Primary hypertension is hypertension caused by multiple factors, or hypertension caused by unknown reasons, including genetics, region, sodium and water retention, sympathetic excitement, RAS activation and other various reasons, so primary hypertension can only be controlled, not cured. Secondary hypertension refers to increased blood pressure caused by a certain disease. Hypertension is one of the clinical symptoms of the primary disease, accounting for 95%. Generally, secondary hypertension is common in renal hypertension, renal artery stenosis, primary aldosteronism, pheochromocytoma, polyarteritis, etc.

[0014] There is a close causal relationship between blood pressure levels and the risk of cardiovascular and cerebrovascular disease and death. Studies have found that the baseline blood pressure ranges from 115 / 75mmHg to 185 / 115mmHg, with an average follow-up of 12 years. The results show that the SBP or DBP in the clinic is continuously, independently, and directly positively correlated with the risk of stroke, coronary heart disease events, and cardiovascular death. For every 20mmHg increase in SBP or 10mmHg increase in DBP, the risk of cardiovascular and cerebrovascular disease doubles, among which heart failure and stroke are the two complications most closely associated with blood pressure levels. It is also found that the incidence of end-stage renal disease (ESRD) is also significantly increased. In severe hypertension, the incidence of ESRD is more than 11 times that of people with normal blood pressure, and even at the high normal level, it is 1.9 times. Therefore, the fundamental goal of hypertension treatment is to effectively prevent or delay the occurrence of complications such as stroke, myocardial infarction, heart failure, and renal insufficiency by lowering blood pressure, effectively control the disease process of hypertension, and prevent the occurrence of severe hypertension such as hypertensive emergencies and sub-acute diseases.

[0015] The Renin-Angiotensin-Aldosterone System, abbreviated as RAAS or RAS (renin-angiotensin system), is a system in the human body that regulates cardiovascular function. It is led by the sympathetic nervous system and secretes angiotensin, which has the effect of constricting blood vessels. Excessive stimulation or activity of the RAS pathway is one of the causes of hypertension.

[0016] Angiotensinogen (AGT), a member of the serpin family, is also known as SERPINA8. It is encoded by the AGT gene and is the only precursor of all angiotensin peptides in the RAS. Human AGT has 485 amino acids, including a 33-amino acid signal peptide. It is mainly produced in the liver and released into the systemic circulation, during which renin converts it into angiotensin I. Angiotensin I is then converted into angiotensin II by angiotensin converting enzyme (ACE). Angiotensin I can stimulate the adrenal medulla to secrete adrenaline, but its direct effect on vasoconstriction is not obvious; angiotensin II can cause systemic arterioles to constrict and increase blood pressure. In addition, it can also promote the secretion of aldosterone by the adrenal cortex. Aldosterone acts on the renal tubules to retain sodium, water, and potassium, thereby causing an increase in blood volume and increased blood pressure. The mechanism of action is as follows: Fig.10 shown.

[0017] There are five main categories of antihypertensive drugs in clinical frontline. Commonly used antihypertensive drugs are divided into five categories, generally represented by the letters ABCD. A includes angiotensin-converting enzyme inhibitors ACEI (such as pril drugs) and angiotensin II antagonists ARB (such as sartan drugs), B refers to β1-receptor antagonists (such as loral drugs), C refers to dihydropyridine calcium channel antagonists (such as dihydropyridine drugs), and D refers to diuretics (such as thiazide drugs). The mechanism of action of these five types of antihypertensive drugs is not exactly the same. According to the different ages and blood pressure levels of patients, clinical blood pressure control is firstly controlled by one drug. If one drug has poor control effect on blood pressure, two or even three antihypertensive drugs are used in combination. If the three antihypertensive drugs cannot control blood pressure to the normal range, it is called refractory hypertension. Refractory hypertension is a common clinical problem in the treatment of hypertension and a difficult problem in treatment.

[0018] The five types of drugs currently commonly used in clinical practice require long-term, uninterrupted daily use. Some patients have poor compliance, and side effects are the main reason for poor compliance. ACEI and ARB can cause dry cough and edema, and severe cases can lead to renal insufficiency; CB drugs have side effects such as accelerated heartbeat, flushing, headache, and swollen feet; B drugs can also cause fatigue and affect blood sugar and blood lipid metabolism; D drugs can cause body weakness and cramps, and severe cases can lead to gout.

[0019] Therefore, there is a need in the art for more effective treatments with new mechanisms of action that are different from existing clinical drugs and with fewer side effects. Summary of the invention

[0020] The present application provides an AGT RNAi agent, which can prevent and / or treat diseases related to the RAS pathway, such as hypertension, including "non-refractory hypertension" and "refractory hypertension", by specifically interfering with the mRNA of the AGT gene, destroying its function as a translation template, and preventing the expression of angiotensinogen AGT protein.

[0021] The RNAi agent of the present application can be formed by base pairing of a sense strand and an antisense strand, wherein the sense strand and the antisense strand are at least 80% complementary to each other, and the 2' position of some or all nucleotide sugars can be fluorine or methoxy, and the phosphate esters between at least three consecutive nucleotides at the end can be thiolated.

[0022] The structure of the RNAi agent of the present application may also contain structures 5'MVIP and 3'MVIP that make the RNAi agent have liver targeting specificity, wherein 5'MVIP can be coupled to the 5' end of the sense strand and / or antisense strand of the RNAi agent, 3'MVIP can be coupled to the 3' end of the antisense strand and / or sense strand of the RNAi agent, 5'MVIP and 3'MVIP can both contain a (liver targeting specific) targeting unit X, a branch chain L, a linker B and a connecting chain D, and 5'MVIP can also contain a transfer point R connected to the 5' end of the sense strand or antisense strand of the RNAi agent 1 , 3'MVIP may also include a transition point R connected to the 3' end of the sense strand or antisense strand of the RNAi agent 2 The targeting unit X, the branch chain L, the linker B and the connecting chain D can be the same or different inside the 5'MVIP and the 3'MVIP or between the 5'MVIP and the 3'MVIP. In vitro and in vivo efficacy experiments have shown that this RNAi agent directly degrades AGT mRNA, continuously and efficiently inhibits AGT gene expression, and can be used to treat and / or prevent AGT gene-mediated diseases, such as hypertension.

[0023] In one aspect, the present application provides an RNAi agent or a pharmaceutically acceptable salt thereof, wherein the structure of the RNAi agent contains a carrier structure and an interfering nucleic acid, and the structure thereof is shown in Formula IIIa, IIIb or IIIc:

[0024]

[0025]

[0026] in,

[0027] The interfering nucleic acid targets the AGT gene, and includes an antisense strand and a sense strand;

[0028] The vector structure includes 5'MVIP (5'MultiValent Import Platform) and / or 3'MVIP (3'MultiValent Import Platform);

[0029] The 5'MVIP consists of a transition point R 1 , connecting chain D, linker B, branch chain L and liver-targeting specific ligand X, the 3'MVIP consists of a transfer point R 2 , connecting chain D, linker B, branch chain L and liver-targeting specific ligand X, the 5'MVIP is connected by a transfer point R 1 Connected to the 5' end of the sense strand or the 5' end of the antisense strand, the 3'MVIP is connected to the 5' end of the sense strand through the transition point R 2Connected to the 3' end of the sense strand or the 3' end of the antisense strand, n and m are each independently any integer of 0-4.

[0030] In certain embodiments, the interfering nucleic acid is used to inhibit the expression of the AGT gene.

[0031] In certain embodiments, the interfering nucleic acid comprises siRNA or miRNA.

[0032] In certain embodiments, wherein n+m=an integer of 2-6, preferably n+m=2, 3 or 4, more preferably 4.

[0033] In certain embodiments, the 5'MVIP is selected from any one of 5'MVIP01 to 5'MVIP22 in Table 10, and / or the 3'MVIP is selected from any one of 3'MVIP01 to 3'MVIP27 in Table 11.

[0034] In certain embodiments, the sense strand is substantially homologous to any one of SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:17 and SEQ ID NO:18, or a sequence that differs therefrom by no more than 3 nucleotides.

[0035] In certain embodiments, the antisense strand comprises the following nucleotide sequence: any one of SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:35 and SEQ ID NO:36, or a sequence that differs therefrom by no more than 3 nucleotides.

[0036] In certain embodiments, the sense strand comprises any one of SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO: 44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:53 and SEQ ID NO:54, or a sequence that differs therefrom by no more than 3 nucleotides, and the antisense strand comprises any one of SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:71 and SEQ ID NO:72, or a sequence that differs therefrom by no more than 3 nucleotides.

[0037] In certain embodiments, the interfering nucleic acid comprises any one or more of Kylo-09-DS01, Kylo-09-DS07, Kylo-09-DS08, Kylo-09-DS10, Kylo-09-DS11, Kylo-09-DS12, Kylo-09-DS17, Kylo-09-DS18, Kylo-09-DS37 to Kylo-09-DS54.

[0038] In certain embodiments, the RNAi agent or a pharmaceutically acceptable salt thereof includes any one or more of Kylo-09-DS122, Kylo-09-DS131 to Kylo-09-DS147 in Table 18.

[0039] In certain embodiments, the RNAi agent or a pharmaceutically acceptable salt thereof includes any one or more of Kylo-09-DS122, Kylo-09-DS131, Kylo-09-DS141, Kylo-09-DS142 and Kylo-09-DS147 in Table 19.

[0040] On the other hand, the present application provides an RNAi agent for inhibiting the expression of the AGT gene or a pharmaceutically acceptable salt thereof, which comprises an antisense strand, wherein the antisense strand comprises at least 12 consecutive nucleotides that are substantially complementary to the nucleotides at the corresponding positions selected from the following sequences or a sequence that differs from the nucleotides by no more than 3 nucleotides: AGT mRNA NM_001382817.3 starting at positions 1854-1874, 1907-1927, 1895-1915, 1352-1372, 1903-1923, 2019-2039, 1853-1873 and 1818-1838 or a sequence of at least 12 consecutive nucleotides or a sequence that differs by no more than 3 nucleotides from the starting positions 1822-1842, 1875-1895, 1863-1883, 1320-1340, 1871-1891, 1987-2007, 1821-1841 and 1786-1806 in NM_001384479.1.

[0041] In certain embodiments, the RNAi agent comprises a single-stranded or double-stranded nucleic acid molecule.

[0042] In certain embodiments, the RNAi agent comprises siRNA or miRNA.

[0043] In certain embodiments, the RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof further comprises a sense strand, wherein the antisense strand and the sense strand form a complementary region comprising at least 12 consecutive nucleotides.

[0044] In certain embodiments, one or more nucleotides on the sense strand and / or antisense strand are modified to form modified nucleotides.

[0045] In certain embodiments, the RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof further comprises a ligand, and the ligand is coupled to the sense strand and / or antisense strand via a carrier structure.

[0046] On the other hand, the present application provides a cell comprising the aforementioned RNAi agent or a pharmaceutically acceptable salt thereof, or the aforementioned RNAi agent that inhibits AGT gene expression or a pharmaceutically acceptable salt thereof.

[0047] On the other hand, the present application provides a pharmaceutical composition comprising the aforementioned RNAi agent or a pharmaceutically acceptable salt thereof, or the aforementioned RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient, carrier and / or diluent.

[0048] On the other hand, the present application provides a method for reducing the expression of AGT mRNA or protein in cells or tissues, which comprises contacting the cells or tissues with an effective amount of the aforementioned RNAi agent or a pharmaceutically acceptable salt thereof, the aforementioned RNAi agent that inhibits AGT gene expression or a pharmaceutically acceptable salt thereof, and / or the aforementioned pharmaceutical composition.

[0049] On the other hand, the present application provides the use of the aforementioned RNAi agent or a pharmaceutically acceptable salt thereof, the aforementioned RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition in the preparation of a drug for preventing and / or treating a disease or condition or reducing the risk of a disease or condition.

[0050] On the other hand, the present application provides a method for preventing and / or treating a disease or condition, the method comprising administering an effective amount of the aforementioned RNAi agent or a pharmaceutically acceptable salt thereof, the aforementioned RNAi agent that inhibits AGT gene expression or a pharmaceutically acceptable salt thereof, and / or the aforementioned pharmaceutical composition to a subject in need thereof.

[0051] On the other hand, the present application provides a drug kit comprising the aforementioned RNAi agent or a pharmaceutically acceptable salt thereof, the aforementioned RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition.

[0052] The AGT RNAi agent of the present application interferes with AGT mRNA, destroys its function as a translation template, adjusts the expression level of AGT protein (the only precursor of all angiotensin peptides of RAS) in the systemic circulation, regulates blood pressure at the genetic level and from the source of the RAS system, and provides a new treatment model to combat various types of hypertension, including primary and secondary hypertension, as well as refractory hypertension (TRH), so that patients with chronic heart failure who are resistant to refractory hypertension (TRH) or reduced ejection fraction have drugs available. The AGT RNAi agent of the present application also has a unique long-term effect, and it is possible to administer the drug once a quarter or half a year, which has incomparable advantages over current small molecule hypertension treatment drugs.

[0053] Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of the present application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the description in the drawings and specification of the present application is merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The specific features of the invention involved in this application are shown in the attached claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and drawings described in detail below. The drawings are briefly described as follows:

[0055] Figure 1A Shown is the AGT mRNA level in Hep 3B cells after RNAi intervention in Example 2 of the present application;

[0056] Figure 1B Shown is the AGT mRNA level in HepG2 cells after RNAi intervention in Example 2 of the present application;

[0057] Figures 2A-2D Shown is the HPLC graph of the stability test of the RNAi agent at different time periods in Example 3 of the present application;

[0058] Figure 3 Shown is a high-resolution mass spectrum of ERCd-01-c2 synthesized in Example 4.1.1.5 of the present application;

[0059] Figure 4 Shown is the high-resolution mass spectrum of 3'MVIP17-c1 synthesized in Example 4.1.2.6 of the present application;

[0060] Figure 5Shown is a high-resolution mass spectrum of 5'MVIP09-ERCd-PFP-c2 synthesized in Example 4.2.1.2 of the present application;

[0061] Figure 6 Shown is the average level of hAGT after administration of transgenic mice in Example 6 of the present application;

[0062] Figure 7 Shown is the average level of hAGT in serum of transgenic mice after administration in Example 7 of the present application.

[0063] Figure 8 Shown is the average level of AGT in serum of cynomolgus monkeys after administration in Example 8 of the present application.

[0064] Figures 9A-9E Shown are the structural formulas of Kylo-09-DS122, Kylo-09-DS131, Kylo-09-DS141, Kylo-09-DS142, and Kylo-09-DS147.

[0065] Fig.10 Shown is the mechanism of action of angiotensinogen (AGT). DETAILED DESCRIPTION

[0066] The following is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0067] Definition of terms

[0068] In this application, the term "angiotensinogen" can be used interchangeably with the term "AGT", and examples of AGT mRNA sequences are readily available using publicly available databases, such as GenBank, UniProt, OMIM, and the Macaca Genome Project website. The term "AGT" includes human AGT, whose mRNA sequence can be found, for example, in GenBank NM_001382817.3 or GenBank NM_001384479.1; cynomolgus monkey AGT, whose amino acid and complete coding sequence can be found, for example, in GenBank Accession No. GI:90075391 (AB170313.1); mouse (Mus musculus) AGT, whose amino acid and complete coding sequence can be found, for example, in GenBank Accession No. GI:113461997 (NM_007428.3); and rat AGT (Rattus norvegicus) AGT, whose amino acid and complete coding sequence can be found, for example, in GenBank Accession No. GI:51036672 (NM_134432). The term "AGT" as used herein also refers to naturally occurring DNA sequence variations of the AGT gene, such as single nucleotide polymorphisms (SNPs) in the AGT gene.

[0069] In this application, the terms "iRNA", "RNAi agent", "iRNA agent", "RNA interfering agent" are used interchangeably and generally refer to an agent comprising RNA as the term is defined herein, and which can mediate targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. iRNA directs sequence-specific degradation of mRNA via a process known as RNA interference (RNAi). iRNA regulates (e.g., inhibits) expression of the AGT gene in a cell (e.g., a cell in a subject such as a mammalian subject).

[0070] In certain embodiments, the RNAi agent may be a single-stranded siRNA (ssRNAi) introduced into a cell or organism to inhibit a target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which then cuts the target mRNA. Single-stranded siRNAs are generally 15 to 30 nucleotides and are chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Pat. No. 8,101,348 and Lima et al. (2012) Cell 150: 883-894, the entire contents of each of which are incorporated herein by reference. Any antisense nucleotide sequence described herein can be used as a single-stranded siRNA chemically modified as described herein or by the methods described in Lima et al. (2012) Cell 150: 883-894.

[0071] In certain embodiments, the "iRNA" used in the present application is a double-stranded RNA, and is referred to herein as a "double-stranded RNAi agent", "double-stranded RNA (dsRNA) molecule", "dsRNA agent" or "dsRNA". The term "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel and substantially complementary nucleic acid chains, referred to as having "sense" and "antisense" orientations relative to the target RNA (i.e., AGT gene). In some embodiments of the present application, double-stranded RNA (dsRNA) triggers degradation of target RNA (e.g., mRNA) through a post-transcriptional gene silencing mechanism (referred to herein as RNA interference or RNAi).

[0072] The duplex structure can be any length that allows the desired target RNA to be specifically degraded by the RISC pathway, and can be within the length range of about 19 to 36 base pairs, for example, a length of about 19-30 base pairs, for example, a length of about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 base pairs. The range and length in the middle of the above range and length are also included as part of the present application. In certain embodiments, the iRNA agent of the present application is a dsRNA with 15-23 nucleotides in each chain, which interacts with a target RNA sequence (e.g., an AGT gene) to guide the cutting of the target RNA. In certain embodiments, the iRNA of the present application is a dsRNA of 24-30 nucleotides, which interacts with a target RNA sequence (e.g., an AGT target mRNA sequence) to guide the cutting of the target RNA.

[0073] Typically, most of the nucleotides of each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each strand or both strands may also include one or more non-ribonucleotides, for example, deoxyribonucleotides or modified nucleotides. In addition, "iRNA" as used in this specification may include ribonucleotides with chemical modifications; iRNA may include substantial modifications at multiple nucleotides. The term "modified nucleotide" as used herein means a nucleotide independently having a modified sugar moiety, a modified internucleotide connection or a modified nucleobase, or any combination thereof. Therefore, the term "modified nucleotide" encompasses replacement, addition or removal of, for example, functional groups or atoms of internucleotide connections, sugar moieties or nucleobases. Modifications suitable for use in the present application's agents include all types of modifications disclosed herein or known in the art.

[0074] In this application, the terms "nucleic acid" and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides or analogs thereof) of any length. Polynucleotides can have any three-dimensional structure and can perform any function. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, ESTs or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, DNA separated from any sequence, RNA separated from any sequence, nucleic acid probes, siRNA, miRNA, shRNA, RNAi agents and primers. Polynucleotides can be modified or substituted at one or more bases, sugars and / or phosphates with any of the various modifications or substitutions described herein or known in the art. Polynucleotides can include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure can be modified before or after polymer assembly. The nucleotide sequence can be blocked by non-nucleotide components. Polynucleotides can be modified after polymerization, for example by coupling with a labeling component. The term can be double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the present application as a polynucleotide includes a double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.

[0075] In the present application, the term "target nucleic acid" or "target sequence" generally refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the AGT gene, including mRNA that is an RNA processing product of the primary transcription product. The target portion of the sequence should be at least long enough to serve as a substrate for iRNA-guided cleavage at or near the portion of the nucleotide sequence of the mRNA molecule formed during transcription of the AGT gene. In one embodiment, the target sequence is within the protein coding region of AGT. The target sequence may be about 19-36 nucleotides in length, for example, preferably about 19-30 nucleotides in length. Ranges and lengths intermediate to the above ranges and lengths are also included as part of the present application.

[0076] In this application, the term "nucleotide sequence" generally refers to a series or order of nucleobases, nucleotides and / or nucleosides, whether modified or unmodified, described by a series of letters using standard nucleotide nomenclature and the symbols for modified nucleotides described in this application.

[0077] In the present application, the term "oligonucleotide" generally refers to a polymer formed by connecting a plurality of nucleotide residues (deoxyribonucleotides or ribonucleotides, or its relevant structural variants or synthetic analogs) by a phosphodiester bond (or its relevant structural variants or synthetic analogs). Therefore, although the term "oligonucleotide" generally refers to a naturally occurring nucleotide polymer in which nucleotide residues and the connection therebetween are naturally occurring, it should be understood that the scope of the term also includes various analogs, including but not limited to: peptide nucleic acid (PNA), phosphoramidate, phosphorothioate, methylphosphonate, 2-O-methylribonucleic acid, etc. The exact size of the molecule can depend on specific applications. Oligonucleotides are generally shorter in length, usually approximately having 10-30 nucleotide residues, but the term can also refer to molecules of any length, although the terms "polynucleotide" or "nucleic acid" are generally used for larger oligonucleotides.

[0078] In certain embodiments, the oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and / or unmodified deoxyribonucleosides (DNA) and / or one or more modified nucleosides. The term "modified oligonucleotide" generally refers to an oligonucleotide comprising at least one modified nucleoside and / or at least one modified internucleoside linkage.

[0079] In the present application, the term "modified nucleoside" generally means a nucleoside comprising at least one chemical modification compared to a naturally occurring RNA or DNA nucleoside. The modified nucleoside comprises a modified sugar moiety and / or a modified nucleobase.

[0080] In the present application, the term "nucleobase" generally refers to a heterocyclic pyrimidine or purine compound, which is a component of all nucleic acids and includes adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U). Nucleotides may include modified nucleotides or nucleotide mimetics, abasic sites (Ab or X) or substitute substitutions. As used in the present application, "nucleobase sequence" generally refers to the order of consecutive nucleobases that are independent of any sugar, linkage or nucleobase modification. The term "unmodified nucleobase" or "naturally occurring nucleobase" generally refers to naturally occurring heterocyclic nucleobases of RNA or DNA: purine bases adenine (A) and guanine (G); and pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C) and uracil (U). "Modified nucleobase" generally refers to any nucleobase that is not a naturally occurring nucleobase.

[0081] In this application, the term "sugar moiety" generally refers to the naturally occurring sugar moiety or modified sugar moiety of a nucleoside. The term "naturally occurring sugar moiety" generally refers to the ribofuranosyl group as found in naturally occurring RNA or the deoxyribofuranosyl group as found in naturally occurring DNA. "Modified sugar moiety" means a substituted sugar moiety or sugar surrogate.

[0082] In this application, the term "internucleoside linkage" generally means a covalent linkage between adjacent nucleosides in an oligonucleotide. A "naturally occurring internucleoside linkage" means a 3' to 5' phosphodiester linkage. A "modified internucleoside linkage" means any internucleoside linkage other than a naturally occurring internucleoside linkage.

[0083] In the present application, the term "antisense oligonucleotide" refers to a single-stranded oligonucleotide molecule having a core base sequence complementary to the corresponding fragment of a target nucleic acid (e.g., a target genomic sequence, an mRNA precursor, or an mRNA molecule). In certain embodiments, the antisense oligonucleotide has a length of 12 to 30 core bases. In certain embodiments, the antisense oligonucleotide is an unmodified or modified nucleic acid having a nucleotide sequence complementary to a target nucleic acid (e.g., an AGT polynucleotide).

[0084] In the present application, the term "antisense strand" generally refers to a strand of an RNAi agent (e.g., dsRNA) that includes a region that is substantially complementary to a target sequence. When used herein, the term "complementarity region" generally refers to a region that is substantially complementary to a sequence (e.g., a target sequence) defined in the present application on the antisense strand. When the complementary region is not completely complementary to the target sequence, the mispairing may be in the interior or terminal region of the molecule. Typically, the most tolerated mispairing is in the terminal region, for example, within 5, 4, 3, or 2 nucleotides at the 5' end and / or 3' end.

[0085] In the present application, the term "sense strand" (S) generally refers to a strand of an RNAi agent, comprising a region substantially complementary to a region of the antisense strand as defined herein. A "sense" strand is sometimes referred to as a "sense" strand, a "passenger" strand or an "anti-guide" strand. With their sequence, the antisense strand targets the desired mRNA, while the sense strand targets different targets. Therefore, if the antisense strand is incorporated into RISC, the correct target is targeted. The incorporation of the sense strand can result in off-target effects. These off-target effects can be limited by using modifications or using 5' end caps on the sense strand.

[0086] In the present application, the term "complementary" refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence (such as an RNAi agent sense strand or AGT mRNA) to hybridize (form base pair hydrogen bonds) and form a duplex or double helix structure with an oligonucleotide or polynucleotide comprising a second nucleotide sequence under certain conditions when used to describe a first nucleotide sequence (such as an RNAi agent antisense strand). Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and include natural or modified nucleotides or nucleotide mimetics, as long as the above requirements for their hybridization ability are achieved. "Complementary" does not necessarily have core base complementarity on each nucleoside. On the contrary, some mispairings can be tolerated.

[0087] In the present application, the term "completely complementary" generally means that all (100%) bases in the continuous sequence of the first polynucleotide will hybridize with the same number of bases in the continuous sequence of the second polynucleotide. The continuous sequence may contain all or part of the first or second nucleotide sequence. As used in the present application, "partially complementary" generally means that in the hybridized nuclear base sequence pair, at least about 70% of the bases in the continuous sequence of the first polynucleotide will hybridize with the same number of bases in the continuous sequence of the second polynucleotide. As used in the present application, "substantially complementary" generally means that in the hybridized nuclear base sequence pair, at least about 90% of the bases in the continuous sequence of the first polynucleotide will hybridize with the same number of bases in the continuous sequence of the second polynucleotide. The terms "complementary", "completely complementary" and "substantially complementary" as used in the present application can be used in terms of base matching between the sense strand and the antisense strand of the RNAi agent or between the antisense strand of the RNAi agent and the sequence of the AGT mRNA. Sequence identity or complementarity does not depend on modification. For the purposes of determining identity or complementarity, for example, a and Af are complementary to U (or T) and identical to A.

[0088] In this application, the term "homologous" or "homology" generally refers to the number of nucleotides of a subject nucleic acid sequence that have been matched to the same nucleotides of a reference nucleic acid sequence, typically determined by sequence analysis programs (e.g., Karlin and Altschul, 1990, PNAS 87:2264-2268; Karlin and Altschul, 1993, PNAS 90:5873-5877), or by visual inspection. As used herein, the term "complete homology" or "complete homology" generally refers to complete (100%) homology or "identity" between a reference sequence and a subject nucleic acid sequence. As used herein, the terms "substantially homologous" or "substantial homology" generally refer to nucleotides that share at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) homology between the subject sequence and the nucleotides at the same nucleotide position in the reference sequence.

[0089] In this application, the term "ligand" generally refers to any compound or molecule that can be covalently or otherwise chemically bound to a biologically active substance (such as an oligonucleotide). In certain embodiments, a ligand can interact directly or indirectly with another compound such as a receptor, and the receptor that interacts with the ligand can be present on the cell surface, or alternatively can be an intracellular and / or intercellular receptor, and the interaction of the ligand with the receptor can result in a biochemical reaction, or can be simply a physical interaction or combination.

[0090] In this application, the terms "induce", "inhibit", "enhance", "elevate", "increase", "decrease", "lower" and the like generally refer to the quantitative difference between two states. For example, "an amount effective to inhibit the activity or expression of AGT" means that the level of activity or expression of AGT in the treated sample will be lower than the level of activity or expression of AGT in the untreated sample. The terms are applicable, for example, to expression levels and activity levels. The terms "reduce" and "reduce" are used interchangeably and generally refer to any change that is less than the original. "Reduce" and "reduce" are relative terms and require comparison between before and after measurement. "Reduce" and "reduce" include complete depletion.

[0091] In certain embodiments, the term "reduction" can be detected by standard methods known in the art (such as those described in the present application), and the expression level / amount of a gene, gene product, such as a protein or a biomarker in a first sample is compared with the expression level / amount of a corresponding gene, gene product, such as a protein or a biomarker in a second sample by about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% overall reduction. In certain embodiments, the term "reduction" refers to the reduction in the expression level / amount of a gene or a biomarker in a first sample, wherein the reduction is at least about 0.9 times, 0.8 times, 0.7 times, 0.6 times, 0.5 times, 0.4 times, 0.3 times, 0.2 times, 0.1 times, 0.05 times, or 0.01 times of the expression level / amount of a corresponding gene or a biomarker in a second sample. In certain embodiments, the first sample is a sample obtained from a subject, and the second sample is a reference sample.

[0092] In the present application, the term "expression" generally refers to the process by which a gene ultimately produces a protein. Expression includes, but is not limited to, transcription, post-transcriptional modification (eg, splicing, polyadenylation, addition of a 5'-cap), and translation.

[0093] In the present application, the term "pharmaceutically acceptable" generally refers to one or more non-toxic substances that do not interfere with the effectiveness of the biological activity of the active ingredient. Such preparations may generally contain salts, excipients, buffers, preservatives, compatible carriers and optional other therapeutic agents. Such pharmaceutically acceptable preparations may also generally contain compatible solid or liquid fillers, diluents or encapsulating materials suitable for administration to humans. When used in medicine, the salt should be a pharmaceutically acceptable salt, but non-pharmaceutically acceptable salts can be conveniently used to prepare pharmaceutically acceptable salts, and they cannot be excluded from the scope of this application. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, boric acid, formic acid, malonic acid, succinic acid, etc. Pharmaceutically acceptable salts may also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts or calcium salts.

[0094] In the present application, the term "lipid nanoparticle" or "LNP" generally refers to a vesicle comprising a lipid layer that encapsulates a pharmacologically active molecule (such as a nucleic acid molecule, e.g., a plasmid from which an iRNA or iRNA is transcribed). LNP is described in, e.g., Chinese Patent No. CN103189057B, the entire contents of which are incorporated herein by reference.

[0095] In the present application, the term "prevention and / or treatment" not only includes prevention and / or treatment of diseases, but also generally includes prevention of the onset of diseases, slowing down or reversing the progression of diseases, preventing or slowing down the onset of one or more symptoms associated with diseases, reducing and / or alleviating one or more symptoms associated with diseases, reducing the severity and / or duration of diseases and / or preventing further increase in the severity of diseases and / or any symptoms associated with them, preventing, reducing or reversing any physiological damage caused by diseases, and any pharmacological effects that are generally beneficial to patients being treated. The RNAi agent or pharmaceutical composition of the present application does not need to achieve complete cure or eradication of any symptoms or manifestations of diseases to form a feasible therapeutic agent. As recognized in the relevant field, drugs used as therapeutic agents can reduce the severity of a given disease state, but do not need to eliminate each manifestation of the disease to be considered as a useful therapeutic agent. Similarly, the treatment of prophylactic administration constitutes a feasible preventive agent that does not need to completely and effectively prevent the onset of symptoms. Simply reducing the effects of the disease in a subject (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or reducing the likelihood that the disease will occur or worsen, is sufficient.

[0096] In this application, the terms "disease" or "disorder" are used interchangeably and generally refer to any deviation of a subject from a normal state, such as any change in the state of the body or certain organs that prevents or disturbs the performance of functions, and / or causes symptoms such as discomfort, dysfunction, pain or even death in the person suffering from the disease or contacting it. Disease or disorder may also be referred to as distemper, ailment, ailment, malady, disorder, sickness, illness, complaint, inderdisposion or affectation.

[0097] In this application, the term "angiotensinogen-related disease" or "AGT-related disease" generally refers to a disease or disorder caused by or associated with activation of the renin-angiotensin-aldosterone system (RAAS), or a disease or disorder whose symptoms or progression respond to RAAS inactivation. The term "angiotensinogen-related disease" includes diseases, disorders or conditions that benefit from reduced AGT expression. Such diseases are generally associated with high blood pressure. Non-limiting examples of angiotensinogen-related diseases include hypertension, e.g., borderline hypertension (also known as prehypertension), essential hypertension (also known as essential hypertension or idiopathic hypertension), secondary hypertension (also known as non-essential hypertension), isolated systolic or diastolic hypertension, pregnancy-related hypertension (e.g., preeclampsia, eclampsia, and postpartum preeclampsia), diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension (also known as renal hypertension), Goldblatt's hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease (including peripheral vascular disease), Diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, sleep apnea, heart failure (e.g., left ventricular systolic dysfunction), myocardial infarction, angina pectoris, stroke, renal disease (e.g., chronic kidney disease or diabetic nephropathy, optionally in the case of pregnancy), renal failure (e.g., chronic renal failure) and systemic sclerosis (e.g., scleroderma renal crisis). In certain embodiments, AGT related diseases include intrauterine growth retardation (IUGR) or fetal growth restriction. In certain embodiments, AGT related disorders also include obesity, hepatic steatosis / fatty liver, for example, non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty liver disease (NAFLD), glucose intolerance, type 2 diabetes (non-insulin dependent diabetes) and metabolic syndrome. In certain embodiments, hypertension includes hypertension associated with low plasma renin activity or plasma renin concentration.

[0098] In the present application, the term "administering" generally refers to introducing the present application's pharmaceutical preparation into the body of a subject by any introduction or delivery route. Any method known to those skilled in the art for contacting cells, organs or tissues with the drug can be used. The administration may include, but is not limited to, intravenous, intraarterial, intranasal, intraperitoneal, intramuscular, subcutaneous transdermal or oral administration. The daily dose may be divided into one, two or more suitable forms of dosage to be administered at one, two or more times during a certain time period.

[0099] In the present application, the term "contact" generally refers to two or more different types of substances being contacted together in any order, in any manner, and for any duration. Contact may occur in vivo, ex vivo, or in vitro. In some embodiments, it may refer to making the RNAi agent or composition of the present application directly contact cells or tissues. In other embodiments, the term refers to making the RNAi agent or composition of the present application indirectly contact cells or tissues. For example, the method of the present application includes a method in which a subject contacts the RNAi agent or composition of the present application, and then the RNAi agent or composition contacts cells or tissues by diffusion or any other active transport or passive transport process known in the art (compounds circulate in the body through this process).

[0100] In the present application, the term "effective amount" or "effective dose" generally refers to an amount sufficient to achieve or at least partially achieve the desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is generally any amount of a drug that promotes disease regression (which is demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of the disease asymptomatic period, or the prevention of damage or disability caused by suffering from the disease) when used alone or in combination with another therapeutic agent. A "preventive effective amount" or "preventive effective dose" of a drug generally refers to an amount of a drug that inhibits the development or recurrence of a disease when administered alone or in combination with another therapeutic agent to a subject at risk of disease development or disease recurrence. The ability of a therapeutic agent or preventive agent to promote disease regression or inhibit disease development or recurrence can be evaluated using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems predicting efficacy in humans, or by measuring the activity of a pharmaceutical agent in an in vitro assay. In certain embodiments, an "effective amount" refers to the amount of an RNAi agent that produces an expected pharmacological, therapeutic or preventive result.

[0101] In this application, the term "subject" generally refers to a human or non-human animal (including mammals) that needs diagnosis, prognosis, improvement, prevention and / or treatment of a disease, such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), livestock (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cattle, goats, sheep, pigs) and experimental animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, newborns, infants, adolescents and adult subjects. Subjects include animal disease models.

[0102] In this application, the terms "include", "comprising", "having", "may", "containing" and their variations are generally intended to be open transitional phrases, terms or words that do not exclude the possibility of additional actions or structures. The term "consisting of..." generally means that no other components (or similarly, features, integers, steps, etc.) can be present. Unless the context clearly dictates otherwise, singular forms such as "a", "an", "the" in English, "a", "a kind" and "said / the" in Chinese generally include plural forms of the referred things.

[0103] In this application, the term "about" generally means approximately, in the region of, roughly, or around. When the term "about" is used to refer to a numerical range, a cutoff or a specific value is used to indicate that the stated value may differ from the recited value by up to 10%. Thus, the term "about" can be used to cover variations of ±10% or less, ±5% or less, ±1% or less, ±0.5% or less, or ±0.1% or less from a specific value.

[0104] It should be understood that the term "at least" preceding a number or a series of numbers includes the number adjacent to the term "at least", and all subsequent numbers or integers logically included, as clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 19 nucleotides in a nucleic acid molecule of 21 nucleotides" means that 19, 20 or 21 nucleotides have the indicated property. When "at least" appears before a series of numbers or ranges, it should be understood that "at least" can modify each number in the series or range.

[0105] It should be understood that "not more than" or "less than" as used herein refers to a value or integer that is adjacent to the phrase and logically lower, such as from the logic of the context, to zero. For example, a duplex having an overhang of "not more than 3 nucleotides" has an overhang of 3, 2, 1 or 0 nucleotides. When "not more than" appears before a series of numbers or ranges, it should be understood that "not more than" can modify each number in the series or range. As used herein, ranges include both upper and lower limits. DETAILED DESCRIPTION OF THE INVENTION

[0107] RNAi Agents

[0108] In one aspect, the present application provides RNAi agents that inhibit the expression of the AGT gene.

[0109] In certain embodiments, the RNAi agent comprises a single-stranded oligonucleotide or double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of an AGT gene in a cell, such as a cell of a subject (e.g., a mammal, such as a person susceptible to AGT-related disorders such as hypertension). Wherein, the dsRNA comprises an antisense strand having a complementary region complementary to at least a portion of the mRNA formed in the expression of the AGT gene. The complementary region is about 12-30 nucleotides in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13 or 12 nucleotides in length).

[0110] dsRNA comprises two RNA chains, which can complement and hybridize to form a duplex structure (complementary region) under the conditions used by dsRNA. One chain (antisense strand) of dsRNA comprises a complementary region that is substantially complementary to the target sequence and is usually completely complementary. The target sequence can be derived from the sequence of mRNA formed during the expression of the AGT gene. The other chain (sense strand) comprises a region complementary to the antisense strand, so that when combined under suitable conditions, the two chains can hybridize and form a duplex structure. Typically, the length of the duplex structure is 12 to 30 base pairs. Similarly, the length of the complementary region to the target sequence is 12 to 30 nucleotides.

[0111] In certain embodiments, the dsRNA is about 19 to about 23 nucleotides in length, or about 24 to about 30 nucleotides in length. Typically, the length of the dsRNA is sufficient to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that a dsRNA having a length greater than about 21-23 nucleotides can be used as a substrate for Dicer. It is also understood by those skilled in the art that the region of the RNA targeted for cutting is typically a portion of a larger RNA molecule (typically an mRNA molecule). A "portion" of a target is a continuous nucleotide of an mRNA target that is long enough to allow it to be a substrate for RNAi-guided cutting (i.e., cutting via a RISC pathway).

[0112] Those skilled in the art will also understand that the duplex region is the main functional part of the dsRNA, for example, a duplex region of about 19 to about 30 base pairs, for example, about 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20 base pairs. Therefore, in one embodiment, in order to achieve a functional duplex (e.g., 15-30 base pairs) that targets the desired RNA for cleavage, the RNA molecule or RNA molecule complex having a duplex region of more than 30 base pairs is a dsRNA.

[0113] In one aspect, the RNAi agent of the present application or a pharmaceutically acceptable salt thereof comprises an antisense strand, wherein the antisense strand comprises at least 12 consecutive nucleotides that are substantially complementary to the nucleotides at the corresponding positions selected from the following sequences, or a sequence that differs by no more than 3 nucleotides therefrom: at least 12 consecutive nucleotides starting at positions 1854-1874, 1907-1927, 1895-1915, 1352-1372, 1903-1923, 2019-2039, 1853-1873 and 1818-1838 in AGT mRNA NM_001384479.1, or a sequence that differs by no more than 3 nucleotides therefrom, or a sequence that differs by no more than 12 consecutive nucleotides starting at positions 1822-1842, at least 12 consecutive nucleotides of 1875-1895, 1863-1883, 1320-1340, 1871-1891, 1987-2007, 1821-1841 and 1786-1806, or a sequence that differs by no more than 3 nucleotides therefrom.

[0114] In certain embodiments, the duplex structure (complementary region) formed by the antisense strand and the sense strand comprises at least 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides.

[0115] In certain embodiments, the sense strand of the RNAi agent has substantial homology to a target sequence in Table 1.

[0116] Table 1 Target sequences

[0117]

[0118]

[0119] Among them, g = guanylate, a = adenylate, t = thymine, c = cytidine.

[0120] Table 2 Antisense strand sequence

[0121] SEQ ID NO. Single Chain Code Antisense strand sequence 5'→3' 19 AS1 uuuuguuucacaaacaagcun 20 AS2 uuuauuacuaacacaagggan 21 AS3 uauacuuuaauuuuaaaaccn 22 AS4 uacuuuaauuuuaaaacccan 23 AS5 uuguucaaaaaucacaagcan 24 AS6 uuaauuuuaaaacccaauuun 25 AS7 auacuuuaauuuuaaaacccn 26 AS8 uaaaacccaauuuuuuguucun 27 AS9 uuuugcagcgacuagcaccan 28 AS10 ucaagcucaaaaaaaaugcun 29 AS11 uuuaauuuuaaaacccaauun 30 AS12 uucaaaaaucacaagcaucun 31 AS13 uaagcuguuggguagacucun 32 AS14 uucacaaacaagcuggucggn 33 AS15 uuguccuggaugucacuccan 34 AS16 uauuacagacacuacacggan 35 AS17 uuuguuucacaaacaagcugn 36 AS18 uuuuggaacaguagucccgcn

[0122] Wherein n is a or u or g or c, g = guanylate, a = adenylate, u = uridylate, c = cytidine.

[0123] In some screening embodiments, the sense strand and antisense strand of the RNAi agent are selected from the sequences in Table 3 or differ from each sequence in Table 3 by one, two or three nucleotides.

[0124] Table 3 Screening RNAi agent sequences

[0125]

[0126] Among them, g = guanylate, a = adenylate, u = uridylate, c = cytidine.

[0127] It should be understood that, although the sequences in Table 3 are not described as modified or conjugated sequences, the RNA of the iRNA (e.g., dsRNA) of the present application may include any one of the sequences shown in Table 3, or a modified sequence of Table 4, 5 or 6, or a conjugated sequence of Table 13, 14, 16 or 17. In other words, the present application encompasses unmodified, unconjugated, modified or conjugated dsRNA as described herein.

[0128] In some embodiments, the RNAi agent can be added to the cell line in the form of liposome-nucleic acid nanoparticles for sequence screening. Patents US9233971B2, US9080186B2, CN102985548B and CN103189057B on lipid compounds and methods for preparing liposome-nucleic acid nanoparticles are fully introduced into this specification.

[0129] In some embodiments, the amphoteric lipids in the lipid compound are preferably macrocyclic lipid compounds D1C1, T1C1, T1C6, T4C4, B2C1, B2C6, B2C7 and M10C1.

[0130] It is well known to those skilled in the art that dsRNAs having a duplex structure of about 20 to 23 base pairs, for example, 21 base pairs, have been found to be particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20: 6877-6888). However, others have found that shorter or longer RNA duplex structures are also effective (Chu and Rana (2007) RNA 14: 1714-1719; Kim et al. (2005) Nat Biotech 23: 222-226). It is reasonable to expect that a duplex having a few nucleotides minus or added at one or both ends of a sequence in Tables 1, 2 and 3 may be similarly effective compared to the dsRNA. Therefore, inhibitory dsRNAs having a sequence of at least 18, 19, 20, 21 or more consecutive nucleotides derived from a sequence in Tables 1, 2 and 3 and differing by no more than about 5, 10, 15, 20, 25 or 30% in their ability to inhibit AGT gene expression from a dsRNA comprising the entire sequence are included within the scope of the present application.

[0131] In addition, the RNAs provided in Tables 1, 2 and 3 identify sites in the AGT transcript that are susceptible to RISC-mediated cleavage. Therefore, the present application further includes an iRNA that targets one of these sites. If the iRNA promotes cleavage of the transcript at any position within the specific site, then as used herein, an iRNA is referred to as targeting the specific site of the RNA transcript, and such an iRNA typically includes at least about 12, 13, 14, 15, 16, 17, 18 or 19 consecutive nucleotides from a sequence provided in Tables 1, 2 and 3.

[0132] The dsRNA described in the present application may further include one or more single-stranded nucleotide overhangs, for example, 1, 2, 3 or 4 nucleotides. The dsRNA with at least one nucleotide overhang may have an inhibitory property superior to its flat-ended counterpart. The nucleotide overhang may comprise nucleotide / nucleoside analogs or their composition, including deoxynucleotides / nucleosides. The overhang may be on a sense strand, an antisense strand or any combination thereof. In addition, the nucleotides of the overhang may be present in the 5'-end, 3'-end or both ends of the antisense or sense strand of the dsRNA. The overhang may be caused by one chain being longer than another chain, or by two chains of the same length being staggered. The overhang may form a mismatch with the target mRNA or it may be complementary to the targeted gene sequence or may be another sequence.

[0133] DsRNA can also contain only a single overhang, which can enhance the interference activity of RNAi agents without affecting their overall stability. For example, a single-stranded overhang can be located at the 3'-end of the sense strand, or alternatively, at the 3'-end of the antisense strand. RNAi agents can also have a flat end, located at the 5'-end of the antisense strand (or the 3'-end of the sense strand), and vice versa. Typically, the antisense strand of an RNAi agent has a nucleotide overhang at the 3'-end, and the 5'-end is a flat end. Although it is not desirable to be bound by theory, the asymmetric flat end at the 5'-end of the antisense strand and the 3'-end overhang of the antisense strand are conducive to the guide strand being loaded into the RISC process.

[0134] In certain embodiments, the overhang is present at the 3'-end of the sense strand, the antisense strand, or both strands. In certain embodiments, the 3'-overhang is present in the antisense strand. In certain embodiments, the 3'-overhang is present in the sense strand.

[0135] In certain embodiments, the dsRNA is 21 nucleotides long and blunt-ended.

[0136] In certain embodiments, the dsRNA is 21 nucleotides in length, and both the sense strand and the antisense strand have an overhang of 2 nucleotides at the 3' end.

[0137] Modified RNAi Agents

[0138] In order to enhance the stability of the RNAi agent described in the present application in vivo, the sense strand and antisense strand of the above-mentioned RNAi agent can be modified without affecting its activity or even enhancing its activity, wherein the nucleotides can have a modifying group, and the entire strand or part of the strand can be modified. In certain embodiments, one or more nucleotides on the sense strand and / or antisense strand are modified to form modified nucleotides.

[0139] In certain embodiments, the RNA of the RNAi agent (e.g., dsRNA) of the present application is unmodified, and does not include, for example, chemical modifications or couplings known in the art and described herein. In other embodiments, the RNA of the RNAi agent (e.g., dsRNA) of the present application is chemically modified to strengthen stability or other favorable properties. In other embodiments of the present application, all nucleotides or substantially all nucleotides of the RNAi agent of the present application are modified, i.e., the chain of the RNAi agent has no more than 5,4,3, 2 or 1 unmodified nucleotides.

[0140] Nucleic acids as described herein can be synthesized and / or modified using methods known in the art, such as those described in "Current protocols in nucleic acid chemistry", Beaucage, SL et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-terminal modifications (phosphorylation, coupling, reverse connection) or 3'-terminal modifications (coupling, DNA nucleotides, reverse connection, etc.); base modifications, such as the use of stabilized bases, destabilized bases, or bases that are paired with the expanded partner library, bases are replaced, bases are removed (abasic nucleotides) or coupled bases; sugar modifications (e.g., 2'-position or 4'-position) or sugar replacement; or backbone modifications, including modifications or replacements of phosphodiester linkages. In the RNAi agents provided herein, both the sense and antisense strands of the RNAi agents do not need to be uniformly modified, and one or more modifications can be incorporated into their individual nucleotides.

[0141] In certain embodiments, the modified nucleotides are selected from: deoxyribonucleotides, nucleotide mimetics, abasic nucleotides, 2'-modified nucleotides, 3' to 3' linked (inverted) nucleotides, nucleotides containing non-natural bases, bridged nucleotides, peptide nucleic acids (PNA), unlocked nucleobase analogs, locked nucleotides, 3'-O-methoxy (2' internucleoside linkage) nucleotides, 2'-F-arabino nucleotides, 5'-Me / 2'-fluorine-banded nucleotides, morpholino nucleotides, vinylphosphonate deoxyribonucleotides, vinylphosphonate-containing nucleotides and cyclopropylphosphonate-containing nucleotides.

[0142] In certain embodiments, the 2'-modified nucleotides include: 2'-O-methyl nucleotides, 2'-deoxy-2'-fluoro nucleotides, 2'-deoxy nucleotides, 2'-methoxyethyl nucleotides, 2'-amino nucleotides and / or 2'-alkyl nucleotides.

[0143] In certain embodiments, at least one of the 2' positions of the sugar moieties at positions 7, 12, and 14 from the 5' end of the antisense strand is fluorine. For example, at least one of the 2' positions of the sugar moieties at positions 7, 12, and 14 from the 5' end of the antisense strand is fluorine.

[0144] In certain embodiments, except for the 7th, 12th, and 14th nucleotides starting from the 5' end of the antisense strand, at least one of the 2' positions of the sugar moieties of the remaining nucleotides is a methoxy group.

[0145] In certain embodiments, at least one of the 5th, 7th, 8th, and 9th nucleotide sugars at the 5' end of the sense strand is fluorine. For example, the 5th, 7th, 8th, and 9th nucleotide sugars at the 5' end of the sense strand are all fluorine.

[0146] In certain embodiments, except for the 5th, 7th, 8th, and 9th nucleotides starting from the 5' end of the sense strand, at least one of the 2' positions of the sugar moieties of the remaining nucleotides is a methoxy group.

[0147] For example, the -OH at the 2' position of the sugar group of some or all nucleotides of the sense strand or antisense strand can be substituted, wherein the substituent group is fluorine or methoxy, preferably the 2' position of the nucleotides at positions 5, 7, 8, and 9 from the 5' end of the sense strand is fluorine, and the 2' position of the nucleotides at positions 7, 12, and 14 from the 5' end of the antisense strand is fluorine, and the 2' positions of the remaining nucleotides are all methoxy.

[0148] For example, the 2' positions of the 7th, 12th and 14th nucleotide sugar groups starting from the 5' end of the antisense strand are all fluorine, and the 2' positions of the remaining nucleotide sugar groups are all methoxy.

[0149] For example, the 2' positions of the 5th, 7th, 8th and 9th nucleotide sugar groups starting from the 5' end of the sense strand are all fluorine, and the 2' positions of the remaining nucleotide sugar groups are all methoxy.

[0150] In certain embodiments, there are at least two consecutive phosphorothioate bonds between the nucleotides in the sense strand and / or the antisense strand.

[0151] In certain embodiments, there are at least two consecutive phosphorothioate bonds between three consecutive nucleotides at the end of the sense strand and / or the end of the antisense strand.

[0152] For example, there are at least two consecutive phosphorothioate bonds between three consecutive nucleotides at the 5' end and the 3' end of the sense strand and the antisense strand.

[0153] For another example, the 2' position of the 5th, 7th, 8th, and 9th nucleotides from the 5' end of the sense strand is fluorine, and the 2' position of the 7th, 12th, and 14th nucleotides from the 5' end of the antisense strand is fluorine, and the 2' position of the remaining nucleotides is methoxy, and there are at least two consecutive phosphorothioate bonds between the three consecutive nucleotides at the 5' and 3' ends of the sense and antisense strands.

[0154] In some screening embodiments, the sense strand and antisense strand of the RNAi agent are selected from the sequences in Table 4 or differ from each sequence in Table 4 by one, two or three nucleotides.

[0155] Table 4 Screening RNA inhibitor sequences

[0156]

[0157]

[0158] Among them, G = 2'-methoxyguanylic acid, A = 2'-methoxyadenylic acid, U = 2'-methoxyuridylic acid, C = 2'-methoxycytidylic acid; u = uridylic acid, c = cytidylic acid.

[0159] In some embodiments, the antisense strand in the RNAi agent is selected from the sequences in Table 5. The 2' positions of the 7th, 12th, and 14th nucleotides from the 5' end of the antisense strand are fluorine, the 2' positions of the remaining nucleotides are methoxy, and the phosphate bonds between at least three adjacent nucleotides at the end of the antisense strand can be thiolated.

[0160] Table 5 Modified sequences of antisense strand

[0161]

[0162]

[0163]

[0164] Among them, G = 2'-O-methylguanylate, A = 2'-O-methyladenylate, U = 2'-O-methyluridylate, C = 2'-O-methylcytidine; fG = 2'-fluoroguanylate, fA = 2'-fluoroadenylate, fU = 2'-fluorouridylate, fC = 2'-fluorocytidine; Gs = 2'-O-methyl-3'-thioguanylate, As = 2'-O-methyl-3'-thioadenylate, Us = 2'-O-methyl-3'-thiouridine, Cs = 2'-O-methyl-3'-thiocytidine.

[0165] In some embodiments, the sense strand in the RNAi agent is selected from the sequences in Table 6. The 2' positions of the 5th, 7th, 8th, and 9th nucleotides from the 5' end of the sense strand are fluorine, the 2' positions of the remaining nucleotides are methoxy, and the phosphate bonds between at least 3 adjacent nucleotides at the end of the antisense strand can be thiolated.

[0166] Table 6 Sense chain modified sequence

[0167]

[0168]

[0169]

[0170] In certain embodiments, the sense strand of the RNAi agents described herein differs from each sequence in Table 6 by one, two, or three nucleotides.

[0171] In certain embodiments, the combination of the sense strand and the antisense strand in the RNAi agent is selected from Table 7.

[0172] Table 7 RNAi agents

[0173]

[0174]

[0175]

[0176] RNAi agent coupled to a carrier

[0177] Another aspect of the RNAi agent of the present application relates to the manner of coupling the interfering nucleic acid to the carrier to enhance the stability, activity, cellular distribution or cellular uptake of the RNAi agent.

[0178] In certain embodiments, the distribution, targeting or stability of the RNAi agent is altered by introducing a ligand for a target tissue receptor into the vector. For example, a specific ligand can provide enhanced affinity for a selected target (e.g., a molecule, a cell or cell type, a compartment (e.g., a cell or organ compartment, a body tissue, an organ or region)) compared to a species in which the ligand is not present.

[0179] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL) or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, for example synthetic polyamino acids.

[0180] Part can also include targeting group, for example, cell or tissue targeting agent combined with specified cell type such as nephrocyte, for example lectin, glycoprotein, lipid or protein, for example antibody.Targeting group can be thyrotropin, melanocyte stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acid, multivalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, vitamin A, biotin or RGD peptide or RGD peptide mimetic.In some embodiments, this part is multivalent galactose, for example, N-acetyl-galactosamine.

[0181] The sense strand and antisense strand included in the RNAi agent of the present application can be conveniently and routinely prepared by the known technology of solid phase synthesis. Any other method known in the art for this type of synthesis, such as liquid phase synthesis or fermentation, can be used additionally or alternatively. It is also known to use similar technology to prepare other oligonucleotides (such as thiophosphates and alkylated derivatives).

[0182] In certain embodiments, in addition to standard nucleoside phosphoramidite monomers and non-standard nucleoside phosphoramidite monomers that are commercially available and routinely used in oligonucleotide synthesis, the oligonucleotides or linked nucleotides of the present application can be synthesized by an automated synthesizer using a phosphoramidite method derived from a carrier-nucleoside phosphoramidite monomer.

[0183] In certain embodiments, the ligand conjugation method of the present invention is coupled to the 5' end and / or 3' end of the antisense strand, and / or the 5' end and / or 3' end of the sense strand through a carrier structure.

[0184] For example, the carrier structure can be coupled to the 5' end and / or the 3' end of the sense strand; or the carrier structure can be coupled to the 5' end of the antisense strand and the carrier structure is coupled to the 3' end of the sense strand; or the carrier structure can be coupled to the 3' end of the antisense strand and the ligand is coupled to the 5' end of the sense strand; or the carrier structure is coupled to both the 5' end and the 3' end of the sense strand.

[0185] In certain embodiments, the carrier structure includes 5'MVIP and 3'MVIP, wherein the 5'MVIP is coupled to the 5' end of the sense strand and / or antisense strand, and the 3'MVIP is coupled to the 3' end of the antisense strand and / or sense strand, the structure of the 5'MVIP is as shown in Formula I, and the structure of the 3'MVIP is as shown in Formula II.

[0186] (XL) n -BDR 1 -,

[0187] I

[0188] (XL) m -BDR 2 -,

[0189] II

[0190] in,

[0191] X is a targeting-specific ligand;

[0192] L is a branched chain;

[0193] B is a connector;

[0194] D is the connecting chain;

[0195] R 1 and R 2 is a transfer point;

[0196] The 5'MVIP is linked to the transition point R 1 Connected to the 5' end of the sense strand or the 5' end of the antisense strand, the 3'MVIP is connected to the 5' end of the sense strand through the transition point R 2 Connected to the 3' end of the sense strand or the 3' end of the antisense strand, n and m are each independently any integer of 0-4.

[0197] In certain embodiments, wherein X is a tissue-specific targeting ligand

[0198] In certain embodiments, the X-liver targeting specific ligand.

[0199] In certain embodiments, wherein said R 1 or R 2 The linkage to the sense strand or antisense strand is via phosphate or modified phosphate, preferably via phosphate or phosphorothioate linkage.

[0200] In certain embodiments, wherein n+m=an integer of 2-6, preferably n+m=2, 3 or 4, more preferably 4.

[0201] In certain embodiments, m or n may be 0, ie, there is no 3'MVIP or 5'MVIP.

[0202] In certain embodiments, when n=0 (ie, no 5'MVIP is present), the structure of the 3'MVIP may be:

[0203] In certain embodiments, when n=1, the structure of the 3'MVIP can be:

[0204]

[0205] In certain embodiments, when n=2, the structure of the 3'MVIP can be:

[0206]

[0207] In certain embodiments, when n=3, the structure of the 3'MVIP can be:

[0208]

[0209] In certain embodiments, when n=4, the structure of the 3'MVIP may be:

[0210]

[0211] In certain embodiments, the n refers to the sum of n placed in the 5'MVIP at the 5' end of both the sense and antisense strands of the RNAi agent, and the m refers to the sum of m placed in the 3'MVIP at the 3' end of both the sense and antisense strands of the RNAi agent.

[0212] In certain embodiments, the R 1 and R 2 The structure contains -NH-, -S- and / or -O-, R 1 and R 2 Through the -NH-, -S- or -O- in the structure, it is connected to the connecting strand D and the 5' end and 3' end of the sense strand and / or antisense strand, respectively. 1 and R 2 Same or different.

[0213] In certain embodiments, the R 1 and R 2 It is an optional straight chain, or a straight chain or cyclic structure with an amide group, a carboxyl group or an alkyl group as a branch, wherein the cyclic structure includes a saturated or unsaturated aliphatic carbocyclic group, or a five-membered or six-membered heterocyclic group or an aromatic hydrocarbon group containing sulfur, oxygen or nitrogen atoms.

[0214] In certain embodiments, the R 1 and / or R 2 -E 1 (CH 2 ) x CH 2 E 2 -, wherein x is any integer from 3 to 12, group E 1 and E 2 They can be -NH-, -S- or -O-, respectively.

[0215] In certain embodiments, the R 1 and / or R 2 -E 1 (CH 2 ) x1 CH(OH)(CH 2 ) x2 E 2 -, wherein x1 or x2 is independently any integer from 3 to 10, E 1 and E 2 They can be -NH-, -S- or -O-, respectively.

[0216] In certain embodiments, the R 1 It is a heterocyclic or carbocyclic structure containing N, S or O:

[0217]

[0218] In some embodiments, the transfer point R 1 -NH(CH 2 ) x CH 2 O-, wherein x is any integer from 3 to 12, preferably any integer from 4 to 6, can be introduced by the following two phosphoramidite monomers:

[0219] i. One of -O- or -S- is used for R 1 The synthesis of phosphoramidite monomers is connected to the 5' end of the sense strand or antisense strand of the RNAi agent by solid phase synthesis. In the structure, -NH-, -S- or -O- is used to connect with the connecting strand D in 5'MVIP, thereby introducing the liver-targeting specific ligand X at the 5' end of the sense strand or antisense strand of the RNAi agent. The exemplary structure of the monomer introduced into the 5' end of the sense strand or antisense strand of the RNAi agent is as follows:

[0220]

[0221] In certain embodiments, the following structures are preferred:

[0222]

[0223] ii.R 1 In the structure, -NH-, -S- or -O- is first connected to the connecting chain D, and the other -NH-, -S- or -O- is used to form an ester with the phosphoramidite in the synthesis of the 5'MVIP phosphoramidite monomer. The structure of the sense chain or antisense chain 5'MVIP phosphoramidite monomer is shown below:

[0224]

[0225] In certain embodiments, the sense strand or antisense strand 5'MVIP phosphoramidite monomer preferably has the following structure:

[0226]

[0227] When n in the general formula is 1-4, the linker B in the above monomer is branched 1 to 4 times to obtain the corresponding monomer compound. With the help of the above monomer compound, the liver-targeting specific ligand X is introduced into the 5' end of the sense chain or antisense chain through solid phase synthesis.

[0228] In certain embodiments, the transition point R 1 -NH(CH 2 ) x CH 2 O-, wherein x can be an integer from 3 to 12, preferably an integer from 4 to 6.

[0229] In certain embodiments, the 5'MVIP phosphoramidite monomer structure is selected from the following structures:

[0230]

[0231]

[0232]

[0233] In certain embodiments, the R 2 It is a heterocyclic or carbocyclic structure containing N, S or O:

[0234]

[0235] In certain embodiments, the R 2 -NH(CH 2 ) x1 CH(OH)(CH 2 ) x2 CH 2 O-, wherein x1 is any integer from 1 to 4, and x2 is any integer from 0 to 4.

[0236] The R 2 is through the reaction of succinic anhydride and R2 While -NH-, -S- or -O- in the structure forms esters or amides, it is also coupled with -NH- in the blank Solid Support to form 3'MVIP solid spport, and then 3'MVIP is introduced into the 3' end of the sense chain or antisense chain through the phosphoramidite solid phase synthesis method.

[0237] In certain embodiments, R 2 The heterocyclic ring in the structure is a pyrrole ring or a piperidine ring, which is connected to the connecting chain D of 3'MVIP through the nitrogen heteroatom in the ring. The exemplary structure of 3'MVIP solid spport is as follows:

[0238]

[0239] When m in the general formula is 1-4, the linker B part in the above monomer is branched 1 to 4 times to obtain the corresponding Solid Support.

[0240] In certain embodiments, R 2 For-B 4 (CH 2 ) x1 CH(OH)(CH 2 ) x2 CH 2 B 5 -, where x1 is an integer from 1 to 4, x2 is an integer from 0 to 4, B 4 and B 5 They are -NH-, -S- or -O- respectively.

[0241]

[0242] When m in the general formula is 1-4, the linker B part in the above monomer is branched 1 to 4 times to obtain the corresponding Solid Support.

[0243] In certain embodiments, R 2 -NHCH 2 CH(OH)CH 2 O-. The exemplary structure of the introduced 3'MVIP solid spport is as follows:

[0244]

[0245] When m in the general formula is 1-4, the linker B part in the above monomer is branched 1 to 4 times to obtain the corresponding Solid Support.

[0246] In certain embodiments, the 3'MVIP solid support structure is as follows:

[0247]

[0248]

[0249]

[0250] In certain embodiments, the liver-targeting specific ligand X is selected from structures for enhancing the uptake of RNAi agents by hepatocytes, and may be lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide-mimicking structures. In the RNAi agents provided in the present application, the liver-targeting specific ligand X introduced into the ends of the sense strand or antisense strand of the RNAi agent may be the same or different, for example, in terms of properties, some may be structures for enhancing liver targeting, some may be structures for regulating the pharmacokinetics of the RNAi agent in vivo, and some may be structures with in vivo dissolution activity. In certain embodiments, the liver-targeting specific ligand X is selected from one or more monosaccharides and their derivatives in the following structures.

[0251] In certain embodiments, the monosaccharide is selected from one or more of the following structures: mannose, galactose, D-arabinose, glucose, fructose, xylose, glucosamine, ribose. The monosaccharide derivative is selected from mannose derivatives, galactose derivatives, glucose derivatives, ribose derivatives and other derivatives.

[0252] In certain embodiments, the targeting unit X is selected from galactose, galactosamine, N-acetylgalactosamine and derivatives thereof, and its general structural formula is as follows:

[0253]

[0254] Among them, W 1 is a hydrogen or hydroxyl protecting group, which may be the same or different; W is -OH, -NHCOOH or -NHCO(CH 2 ) q CH 3 , where q is an integer from 0 to 4; W 2 is -NH-, O, S or C.

[0255] In certain embodiments, the targeting unit X is N-acetylgalactosamine and its derivatives.

[0256] In certain embodiments, the targeting unit X is selected from the following structures:

[0257]

[0258] Wherein, W is selected from -OH, -NHCOOH or -NHCO(CH 2 )q CH 3 One or two of the following, wherein q is an integer from 0 to 4.

[0259] In certain embodiments, the liver-targeting specific ligand X in the same 5'MVIP or 3'MVIP structure may be the same or different.

[0260] In certain embodiments, X between 5'MVIP and 3'MVIP may be the same or different.

[0261] In certain embodiments, L is -NH-, -C(=O)-, -O-, -S-, amide, phosphoryl, thiophosphoryl, C 4 -C 10 C is an aliphatic carbocyclic group, a phenyl group, or a combination of these groups 4 -C 18 Straight chain.

[0262] In certain embodiments, the L further has a hydroxyethyl group or a carboxylic acid side chain.

[0263] In certain embodiments, L is a C containing an amide group or a six-membered aliphatic carbocyclic group. 7 -C 18 Straight chain.

[0264] In certain embodiments, L is selected from one or more of the following structures:

[0265]

[0266] Wherein, r1 is any integer from 1 to 12, r2 is any integer from 0 to 20, and Z is H, an alkyl group or an amide group.

[0267] In certain embodiments, the structure of B is related to the number of Xs that can be introduced, and B contains -NH-, C, O, S, amide, phosphoryl, thiophosphoryl, and when n or m is 1, it is a straight chain, and when n or m is 2, 3 or 4, the number of forks is 2, 3 or 4, respectively.

[0268] In certain embodiments, B is selected from the following structures:

[0269]

[0270] Among them, A 1 and A 2 Each is independently C, O, S, -NH-, carbonyl, amide, phosphoryl or thiophosphoryl, and r is an integer of 0-4.

[0271] In certain embodiments, B is selected from the following structures:

[0272]

[0273]

[0274] Wherein, r is any integer from 0 to 4.

[0275] In certain embodiments, B is selected from the following structures:

[0276]

[0277]

[0278]

[0279] In certain embodiments, B is selected from the following structures:

[0280]

[0281] In certain embodiments, D is -NH-, C=O, O, S, amide, phosphoryl, thiophosphoryl, aromatic hydrocarbon, C 4 -C 10 C is an aliphatic carbocyclic group, a five-membered or six-membered heterocyclic group containing 1 to 3 nitrogen atoms, or a combination of these groups. 3 -C 18 Straight chain.

[0282] In certain embodiments, the D further contains a hydroxymethyl group, a methyl tert-butyl group, a methylphenol group, a C 5 -C 6 Side chain of an aliphatic ring group.

[0283] In certain embodiments, D is a C containing two C═O, a six-membered aliphatic carbocyclic group or a phenyl group. 3 -C 10 Straight chain.

[0284] In certain embodiments, D is a C containing two C=O 3 -C 10 Straight chain.

[0285] In certain embodiments, D is selected from the following structures:

[0286]

[0287]

[0288] Wherein, each p is independently any integer from 1 to 20; s is an integer from 2 to 13; Z 1 and Z 2are the same or different substituent groups.

[0289] In certain embodiments, D is selected from the following structures:

[0290]

[0291]

[0292]

[0293] In certain embodiments, D is selected from the following structures:

[0294]

[0295] In certain embodiments, (XL) in the 5'MVIP structure n -BD- and 3'MVIP structures (XL) m -BD- is selected from one or more of the following structures:

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308] In certain embodiments, the X, L, D, and B are the same or different within each of the 5'MVIP and the 3'MVIP or between the 5'MVIP and the 3'MVIP.

[0309] In certain embodiments, (XL) in the 5'MVIP structure n-BD- is selected from the structures shown in Table 8:

[0310] Table 8 5'MVIP (XL) n -BD-Structure

[0311]

[0312]

[0313]

[0314]

[0315] In certain embodiments, 5'MVIP may not be present, in which case m may be an integer of 2-4.

[0316] In certain embodiments, (XL) in the 3'MVIP structure m -BD- is selected from the structures shown in Table 9:

[0317] Table 9 3'MVIP (XL) m -BD-Structure

[0318]

[0319]

[0320]

[0321]

[0322] In certain embodiments, (XL) in the 5'MVIP ligand structure n -BD- and R 1 The combinations are shown in Table 10.

[0323] Table 10 5'MVIP Medium (XL) n -BD- and R 1 Combination of

[0324]

[0325]

[0326] In certain embodiments, 3'MVIP may not be present, in which case n may be 2-4.

[0327] In certain embodiments, (XL) in the 3'MVIP ligand structure m -BD- and R 2 The combinations are shown in Table 11.

[0328] Table 11 3'MVIP (XL) m -BD- and R 2 combination

[0329]

[0330]

[0331]

[0332] In certain embodiments, the 5'MVIP is selected from any one of 5'MVIP01 to 5'MVIP22 in Table 10.

[0333] In certain embodiments, the 5'MVIP is selected from:

[0334]

[0335] In certain embodiments, the 3'MVIP is selected from any one of 3'MVIP01 to 3'MVIP27 in Table 11.

[0336] In certain embodiments, the 3'MVIP is selected from:

[0337]

[0338]

[0339] In certain embodiments, different 5'MVIP and 3'MVIP combinations in Table 12 are selected to be inserted into different positions of the sense strand and / or antisense strand of the RNAi agent, including the ends and the middle of the sequence, to examine the effects on the expression level of AGT mRNA.

[0340] Table 12 Combination of 5'MVIP and 3'MVIP

[0341]

[0342]

[0343] For example, the 3' end of the antisense strand sequence in Table 5 can be coupled to the carrier structure 3'MVIP09. Exemplarily, the antisense strand in the RNAi agent can be selected from the sequences in Table 13 below.

[0344] Table 13 Antisense strand coupled to 3'MVIP

[0345]

[0346]

[0347]

[0348] In certain embodiments, the antisense strand of the RNAi agent described herein differs from each sequence in Table 13 by one, two, or three nucleotides.

[0349] For example, the 5' end of the sense strand sequence in Table 6 can be coupled to the carrier structure 5'MVIP09. Exemplarily, the sense strand in the RNAi agent can be selected from the sequences in Table 14 below.

[0350] Table 14 Sense strand coupled to 5'MVIP

[0351]

[0352]

[0353]

[0354] In certain embodiments, the sense strand of the RNAi agents described herein differs from each sequence in Table 14 by one, two, or three nucleotides.

[0355] AGT is mainly expressed in the liver, and its expression is limited to humans and non-primates. In some in vivo embodiments, monkeys are the preferred model for preclinical studies. In some in vivo test embodiments, the RNAi agent is selected from the sequences in Table 15.

[0356] Table 15 RNAi agents containing 5'MVIP09 / 3'MVIP09 combination

[0357]

[0358]

[0359] In certain embodiments, the sense and antisense strands of the RNAi agents described herein differ from each sequence in Table 15 by one, two, or three nucleotides.

[0360] In certain embodiments, the 5' end and / or 3' end of the antisense strand UsCsAAGCfUCAAAfAAfAAAUGCsUsG (SEQ ID NO: 118) of the RNAi agent is linked to 5'MVIP and / or 3'MVIP of different structures, and the antisense strand is selected from the following Table 16:

[0361] Table 16 5'MVIP and / or 3'MVIP coupled antisense strand

[0362]

[0363]

[0364]

[0365] In certain embodiments, the antisense strand of the RNAi agents described herein differs from each sequence in Table 16 by one, two, or three nucleotides.

[0366] In certain embodiments, the 5' end and / or 3' end of the sense strand GsCsAUfUUfUfUfUUUGAGCUUGAsAsG (SEQID NO: 194) of the RNAi agent is linked to 5'MVIP and / or 3'MVIP of different structures, and the sense strand is selected from Table 17 below.

[0367] Table 17 5'MVIP and / or 3'MVIP coupled positive strand

[0368]

[0369]

[0370] In certain embodiments, the sense strand of the RNAi agents described herein differs from each sequence in Table 17 by one, two, or three nucleotides.

[0371] In certain embodiments, the RNAi agents described herein are formed by random pairing of the sense strands in Table 17 or sequences differing from these sense strands by one, two or three nucleotides and the antisense strands in Table 16 or sequences differing from these antisense strands by one, two or three nucleotides.

[0372] In certain embodiments, the RNAi agents described herein are synthesized by annealing the antisense strand in Table 16 and the sense strand in Table 17, see Table 18. The n+m in these RNAi agents are 2, 3, 4, 5 and 6, respectively. The positions of 5'MVIP and / or 3'MVIP coupling include the 5' end and / or 3' end of the antisense strand, the 5' end and / or 3' end of the sense strand, the 5' end of the antisense strand and the 3' end of the sense strand, and the 5' end and 3' end of the sense strand. Where n+m=2, 3, 4, 5 and 6.

[0373] Table 18 RNAi agents containing a combination of 5'MVIP and 3'MVIP

[0374]

[0375]

[0376] In certain embodiments, the RNAi agent described herein or a pharmaceutically acceptable salt thereof is preferably prepared or synthesized in the form of a sodium salt and a triethylamine salt or other pharmaceutically acceptable salts.

[0377] In certain embodiments, the RNAi agent or a pharmaceutically acceptable salt thereof is more preferably a sodium salt or a triethylamine salt thereof.

[0378] Pharmaceutical composition

[0379] The present application also includes a pharmaceutical composition comprising the RNAi agent of the present application or a pharmaceutically acceptable salt thereof.

[0380] In one embodiment, provided herein is a pharmaceutical composition comprising an RNAi agent as described herein and a pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical composition comprising the RNAi agent can be used to prevent and / or treat AGT-related disorders, for example, hypertension. Such pharmaceutical compositions are formulated according to the mode of delivery. An example scheme is a composition formulated for systemic administration with parenteral delivery, for example, subcutaneous (SC), intramuscular (IM) or intravenous (IV) delivery. The pharmaceutical composition of the present application can be administered at a dose sufficient to inhibit AGT gene expression.

[0381] A pharmaceutically acceptable "excipient" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmaceutically inert vehicle for delivering one or more nucleic acids to an animal. Excipients can be liquid or solid and are selected taking into account the planned mode of administration to provide the desired volume, consistency, etc. when combined with the nucleic acid and other components in a given pharmaceutical composition. RNAi agents can be delivered in a manner that targets specific tissues (e.g., hepatocytes).

[0382] In certain embodiments, the pharmaceutical composition further comprises a delivery vehicle (such as nanoparticles, dendrimers, polymers, liposomes or cationic delivery systems),

[0383] In certain embodiments, the delivery vehicle comprises a liposome.

[0384] In certain embodiments, the delivery vehicle comprises nanolipids that are capable of forming liposome-nucleic acid nanoparticles with nucleic acid molecules.

[0385] In certain embodiments, the delivery vehicle comprises the amphiphilic lipid compound M10C1.

[0386] The pharmaceutical compositions of the present application include, but are not limited to, solutions, emulsions, and formulations containing liposomes. These compositions can be produced from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Formulations include those that target the liver. The pharmaceutical formulations of the present application that can be conveniently presented in unit dosage form can be prepared according to conventional techniques known to the pharmaceutical industry. Such techniques include the step of combining the active ingredient with a pharmaceutical excipient or excipient.

[0387] use

[0388] On the other hand, the present application provides a method for reducing AGT mRNA or protein expression in cells or tissues, comprising contacting the cells or tissues with an effective amount of the aforementioned RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof, and / or the aforementioned pharmaceutical composition.

[0389] Cells suitable for treatment using the method of the present application may be any cell expressing the AGT gene, for example, liver cells, brain cells, gallbladder cells, heart cells or kidney cells, but preferably liver cells. Cells suitable for use in the method of the present application may be mammalian cells, and when contacted with cells expressing the AGT gene, the RNAi agent inhibits the expression of the AGT gene (e.g., human, primate, non-primate or rat AGT gene) by at least about 50%, for example, as determined by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as immunofluorescence analysis, Western blotting or flow cytometry.

[0390] In certain embodiments, the tissue is liver tissue.

[0391] In certain embodiments, the cells and tissues are ex vivo.

[0392] In certain embodiments, the cells and tissues are in a subject.

[0393] As used herein, the term "inhibit" can be used interchangeably with "reduce," "lower," "silence," "down-regulate," "suppress," and other similar terms, and includes any level of inhibition. The expression of the AGT gene can be evaluated based on the level or level change of any variable related to the expression of the AGT gene, for example, the level of AGT mRNA or the level of AGT protein. This level can be analyzed in a single cell or in a cell population (including, for example, a sample derived from a subject). Inhibition can be evaluated by a decrease in the absolute or relative level of one or more variables related to AGT expression compared to a control level. The control level can be any type of control level used in the art, for example, a baseline level before administration or a level measured from a similar subject, cell or sample that has not been treated or treated with a control (such as, for example, a buffer-only control or an inactive agent control).

[0394] The inhibition of AGT gene expression can be shown by a reduction in the amount of mRNA expressed by a first cell or cell population (such cells may, for example, be present in a sample derived from a subject) in which the AGT gene is transcribed and treated (e.g., by contacting one or more cells with the RNAi agent of the present application, or by administering the RNAi agent of the present application to a subject in which the cell is present) so that the expression of the AGT gene is inhibited, compared to a second cell or cell population that is substantially the same as the first cell or cell population but not so treated (control cells that are not treated with the RNAi agent or are not treated with the RNAi agent targeting the gene of interest). In a preferred embodiment, inhibition is evaluated in a cell line that highly expresses AGT using an appropriate concentration of siRNA by the method provided in Example 2, and the mRNA level in the intervened cell is expressed as a percentage of the mRNA level in the non-intervention control cell.

[0395] In other embodiments, inhibition of AGT gene expression can be evaluated by a decrease in a parameter functionally associated with AGT gene expression, e.g., AGT protein levels in the blood or serum of a subject. AGT gene silencing can be measured in any cell expressing AGT (endogenous or exogenous from an expression construct) and by any assay known in the art.

[0396] The inhibition of AGT protein expression can be represented by a decrease in the level of AGT protein expressed by a cell or cell group or a subject sample (e.g., the level of protein in a blood sample derived from a subject). As described above, for the evaluation of mRNA inhibition, the inhibition of protein expression levels of treated cells or cell groups can be similarly expressed as a percentage of the protein level of a control cell or cell group, or a change in the protein level in a subject sample (e.g., blood or serum derived therefrom).

[0397] Control cells, cell groups or subject samples that can be used to evaluate AGT gene inhibition include cells, cell groups or subject samples that have not been contacted with the RNAi agent of the present application. For example, control cells, cell groups or subject samples can be derived from a single subject (e.g., a human or animal subject) or an appropriately matched population control before treatment with an RNAi agent.

[0398] The AGT mRNA level expressed by a cell or cell group can be measured by any method known in the art for evaluating mRNA expression. For example, qRT-PCR evaluates the reduction of gene expression. The reduction of protein production can be evaluated by any method known in the art, for example, ELISA. In certain embodiments, a puncture liver biopsy sample is used as a tissue material for monitoring the reduction of AGT gene or protein expression. In other embodiments, a blood sample is used as a subject sample for monitoring the reduction of AGT protein expression.

[0399] On the other hand, the present application provides the aforementioned RNAi agent for inhibiting AGT gene expression or its pharmaceutically acceptable salt, or the use of the aforementioned pharmaceutical composition in preparing a drug for preventing and / or treating a disease or condition or reducing the risk of a disease or condition.

[0400] In certain embodiments, the disease or condition comprises a disease or condition associated with AGT.

[0401] In certain embodiments, the disease or condition is selected from the group consisting of hypertension, hypertension, critical hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, hypertension associated with low plasma renin activity or plasma renin concentration, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, stroke, kidney disease, renal failure, systemic sclerosis, intrauterine growth retardation (IUGR), Fetal growth restriction, obesity, hepatic steatosis / fatty liver, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD); glucose intolerance, type 2 diabetes (non-insulin-dependent diabetes mellitus), and metabolic syndrome.

[0402] On the other hand, the present application provides a method for preventing and / or treating a disease or condition, which comprises administering an effective amount of the aforementioned RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof, and / or the aforementioned pharmaceutical composition to a subject in need thereof.

[0403] The in vivo method of the present application may include administering to a subject a composition comprising an RNAi agent, wherein the RNAi agent includes a nucleotide sequence complementary to at least a portion of an RNA transcript of an AGT gene of a mammal to which the RNAi agent is administered. The composition may be administered in any manner known in the art, including, but not limited to, oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the composition is administered by intravenous infusion or injection. In certain embodiments, the composition is administered by subcutaneous injection. In certain embodiments, the composition is administered by intramuscular injection.

[0404] The RNAi agent of the present application can also be administered as a "free RNAi agent". A free RNAi agent is administered in the absence of a pharmaceutical composition. Naked RNAi agents can be in a suitable buffer. The buffer may contain acetate, citrate, prolamin, carbonate or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate buffered saline (PBS). The pH and osmotic pressure of the buffer containing the RNAi agent can be adjusted so as to be suitable for administration to a subject.

[0405] Alternatively, the RNAi agents of the present application can be administered as a pharmaceutical composition, such as a liposomal formulation.

[0406] The pharmaceutical composition of the present application can be administered at a dosage sufficient to inhibit AGT gene expression. Typically, the suitable dosage of the RNAi agent of the present application is in the range of about 0.001 to about 200.0 mg per kilogram of recipient body weight per day, typically in the range of about 1 to 50 mg per kilogram of body weight per day. Typically, the suitable dosage of the RNAi agent of the present application is in the range of about 0.1 mg / kg to about 5.0 mg / kg, for example, in the range of about 0.3 mg / kg to about 3.0 mg / kg.

[0407] In one embodiment, the method comprises administering a composition as described herein such that target AGT gene expression is reduced, such as for about 1, 2, 3, 4, 5, 6, 1-6, 1-3 or 3-6 months per dose. In certain embodiments, the composition is administered once every 3-6 months.

[0408] In certain embodiments, after the initial treatment regimen, the treatment is administered less frequently. A repeated dosage regimen may include regularly administering a therapeutic amount of RNAi agent, such as once a month to once a year. In certain embodiments, the RNAi agent is administered about once a month to about once every three months, or about once every three months to about once every six months.

[0409] After the initial treatment regimen, treatment can be administered less frequently. The duration of treatment can be determined based on the severity of the disease.

[0410] In other embodiments, a single dose of the pharmaceutical composition can be long-acting, so that the dosage is applied at intervals of no more than 1, 2, 3 or 4 months. In some embodiments of the present application, a single dose of the pharmaceutical composition of the present application is applied about once a month. In other embodiments of the present application, a single dose of the pharmaceutical composition of the present application is applied quarterly (i.e., about every 3 months). In other embodiments of the present application, a single dose of the pharmaceutical composition of the present application is applied 2 times a year (i.e., about once every 6 months).

[0411] Those skilled in the art will appreciate that certain factors may affect the dosage and administration schedule required to effectively treat a subject, including, but not limited to: mutations present in the subject, previous treatments, the subject's general health or age, and other diseases present. In addition, treating a subject with a prophylactic and / or therapeutically effective amount of a composition may include a single treatment or a series of treatments as desired.

[0412] In certain embodiments, it further comprises determining the level of AGT in a sample from said subject.

[0413] For example, it further comprises determining the AGT protein level in a blood sample, a serum sample or a urine sample from the subject.

[0414] In certain embodiments, it further comprises administering to said subject an additional therapeutic agent for treating hypertension.

[0415] For example, the additional therapeutic agent can be selected from: diuretics, angiotensin converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, beta-blockers, vasodilators, calcium channel blockers, aldosterone antagonists, alpha2-agonists, renin inhibitors, alpha-blockers, peripherally acting adrenergic agents, selective D1 receptor partial agonists, non-selective alpha-adrenergic antagonists, synthetic steroidal anti-mineralocorticoids, angiotensin receptor-neprilysin inhibitors (ARNi), sacubitril / valsartan; or endothelin receptor antagonists (ERA), sitaxsentan, ambrisentan, atrasentan, BQ-123, zilpotentan, bosentan, macitentan and tezosentan; combinations of any of the above therapeutic agents; and hypertension therapeutic agents formulated as a combination of agents.

[0416] In certain embodiments, the additional therapeutic agent comprises an angiotensin II receptor antagonist.

[0417] For example, the angiotensin II receptor antagonist can be selected from the group consisting of losartan, valsartan, olmesartan, eprosartan and azilsartan.

[0418] In another aspect, the present application provides a cell comprising the aforementioned RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof.

[0419] On the other hand, the present application provides a drug kit comprising the aforementioned RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition.

[0420] Specifically, the present application also discloses the following implementation modes:

[0421] 1. A RNAi agent or a pharmaceutically acceptable salt thereof, wherein the structure of the RNAi agent contains a carrier structure and an interfering nucleic acid, and the structure thereof is shown in Formula IIIa, IIIb or IIIc:

[0422]

[0423]

[0424] in,

[0425] The interfering nucleic acid targets the AGT gene, and includes an antisense strand and a sense strand;

[0426] The vector structure includes 5'MVIP (5'MultiValent Import Platform) and 3'MVIP (3'MultiValent Import Platform);

[0427] The 5'MVIP consists of a transition point R 1 , connecting chain D, linker B, branch chain L and liver-targeting specific ligand X, the 3'MVIP consists of a transfer point R 2 , connecting chain D, linker B, branch chain L and liver-targeting specific ligand X, the 5'MVIP is connected by a transfer point R 1 Connected to the 5' end of the sense strand or the 5' end of the antisense strand, the 3'MVIP is connected to the 5' end of the sense strand through the transition point R 2 Connected to the 3' end of the sense strand or the 3' end of the antisense strand, n and m are each independently any integer of 0-4.

[0428] 2. The RNAi agent or a pharmaceutically acceptable salt thereof according to embodiment 1, wherein the interfering nucleic acid comprises siRNA or miRNA.

[0429] 3. The RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-2, wherein n+m=an integer of 2-6, preferably n+m=2, 3 or 4, and more preferably 4.

[0430] 4. The RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-3, wherein the R 1 is a heterocyclic or carbocyclic structure containing N, S or O:

[0431]

[0432] Alternatively, the R 1 -NH(CH 2 ) x CH 2 O-, wherein x is any integer from 3 to 12, preferably any integer from 4 to 6.

[0433] 5. The RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-4, wherein the R 2 is a heterocyclic or carbocyclic structure containing N, S or O:

[0434]

[0435] Alternatively, the R 2 -NH(CH 2 ) x1 CH(OH)(CH 2 ) x2 CH 2 O-, wherein x1 is any integer from 1 to 4, and x2 is any integer from 0 to 4.

[0436] 6. The RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1 to 5, wherein X is selected from a structure for enhancing uptake of the RNAi agent by hepatocytes.

[0437] 7. The RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-6, wherein X is selected from monosaccharides and their derivatives.

[0438] 8. The RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1 to 7, wherein X is N-acetylgalactosamine and its derivatives.

[0439] 9. The RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-8, wherein X is selected from the following structures:

[0440]

[0441] Wherein, W is selected from -OH, -NHCOOH or -NHCO(CH 2 ) q CH 3 One or two of the following, wherein q is an integer from 0 to 4.

[0442] 10. The RNAi agent or pharmaceutically acceptable salt thereof according to any one of embodiments 1-9, wherein L is selected from one or more of the following structures:

[0443]

[0444] Wherein, r1 is any integer from 1 to 12, r2 is any integer from 0 to 20, and Z is H, an alkyl group or an amide group.

[0445] 11. The RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-10, wherein B is selected from the following structures:

[0446]

[0447] Among them, A 1 and A 2 Each is independently C, O, S, -NH-, carbonyl, amide, phosphoryl or thiophosphoryl, and r is an integer of 0-4.

[0448] 12. The RNAi agent or pharmaceutically acceptable salt thereof according to any one of embodiments 1-11, wherein D is selected from the following structures:

[0449]

[0450]

[0451] Wherein, each p is independently any integer from 1 to 20; s is an integer from 2 to 13; Z 1 and Z 2 are the same or different substituent groups.

[0452] 13. The RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-12, wherein (XL) in the 5'MVIP structure n -BD- and 3'MVIP structures (XL) m -BD- is selected from one or more of the following structures:

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466] 14. The RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1 to 13, wherein X, L, D and B are the same or different within each of 5'MVIP and 3'MVIP or between 5'MVIP and 3'MVIP.

[0467] 15. The RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-14, wherein the 5'MVIP is selected from any one of 5'MVIP01 to 5'MVIP22 in Table 10.

[0468] 16. The RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-15, wherein the 3'MVIP is selected from any one of 3'MVIP01 to 3'MVIP27 in Table 11.

[0469] 17. The RNAi agent or pharmaceutically acceptable salt thereof according to any one of embodiments 1 to 16, wherein the combination of the sense strand 5'MVIP and the antisense strand 3'MVIP is 5'MVIP01 / 3'MVIP01, 5'MVIP01 / 3'MVIP17 or 5'MVIP09 / 3'MVIP09; or the combination of the sense strand 5'MVIP and the antisense strand 3'MVIP is 5'MVIP01 / 3'MVIP09 or 5'MVIP09 / 3'MVIP01.

[0470] 18. The RNAi agent or pharmaceutically acceptable salt thereof according to any one of embodiments 1-17, wherein the antisense strand and the sense strand form a complementary region comprising at least 12 consecutive nucleotides, wherein the sense strand comprises AGT mRNA A sequence of at least 12 consecutive nucleotides starting at positions 1854-1874, 1907-1927, 1895-1915, 1352-1372, 1903-1923, 2019-2039, 1853-1873 and 1818-1838 in NM_001382817.3, or a sequence of at least 12 consecutive nucleotides starting at positions 1822-1842, 1875-1895, 1863-1883, 1320-1340, 1871-1891, 1987-2007, 1821-1841 and 1786-1806, or a sequence of at least 12 consecutive nucleotides starting at positions 1822-1842, 1875-1895, 1863-1883, 1320-1340, 1871-1891, 1987-2007, 1821-1841 and 1786-1806, or a sequence of at least 12 consecutive nucleotides starting at positions 18

[0471] 19. The RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-18, wherein the sense strand and the antisense strand are at least about 80% base complementary.

[0472] 20. The RNAi agent according to any one of embodiments 1-19, or a pharmaceutically acceptable salt thereof, wherein the sense strand and the antisense strand are each independently 15-30 nucleotides, preferably 17-25 nucleotides, and more preferably 19-23 nucleotides.

[0473] 21. The RNAi agent or pharmaceutically acceptable salt thereof according to any one of embodiments 1-20, wherein the sense strand is substantially homologous to any one of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 17 and SEQ ID NO: 18, or a sequence that differs therefrom by no more than 3 nucleotides.

[0474] 22. The RNAi agent or pharmaceutically acceptable salt thereof according to any one of embodiments 1-21, wherein the antisense strand comprises any one of SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 35 and SEQ ID NO: 36, or a sequence that differs therefrom by no more than 3 nucleotides.

[0475] 23. The RNAi agent or pharmaceutically acceptable salt thereof according to any one of embodiments 1-22, wherein the sense strand comprises any one of SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:53 and SEQ ID NO:54, or a sequence that differs therefrom by no more than 3 nucleotides.

[0476] 24. The RNAi agent or pharmaceutically acceptable salt thereof according to any one of embodiments 1-23, wherein the antisense strand comprises any one of SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:71 and SEQ ID NO:72, or a sequence that differs therefrom by no more than 3 nucleotides.

[0477] 25. The RNAi agent according to any one of embodiments 1-24, or a pharmaceutically acceptable salt thereof, wherein one or more nucleotides on the sense strand and / or antisense strand are modified to form modified nucleotides.

[0478] 26. The RNAi agent of embodiment 25 or a pharmaceutically acceptable salt thereof, wherein the modified nucleotides are selected from deoxyribonucleotides, nucleotide mimetics, abasic nucleotides, 2'-modified nucleotides, 3' to 3' linked (inverted) nucleotides, nucleotides containing non-natural bases, bridged nucleotides, peptide nucleic acids (PNAs), unlocked nucleobase analogs, locked nucleotides, 3'-O-methoxy (2' internucleoside linkage) nucleotides, 2'-fluoro-arabino nucleotides, 5'-methyl / 2'-fluoro-banded nucleotides, morpholino nucleotides, vinylphosphonate deoxyribonucleotides, vinylphosphonate-containing nucleotides, and cyclopropylphosphonate-containing nucleotides.

[0479] 27. The RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-26, wherein the 2′ position of some or all of the nucleotide sugar groups of the sense strand and the antisense strand is fluorine or methoxy.

[0480] 28. The RNAi agent according to any one of embodiments 1-27, or a pharmaceutically acceptable salt thereof, wherein at least two consecutive phosphorothioate bonds exist between three consecutive nucleotides at the end of the sense strand and / or the end of the antisense strand.

[0481] 29. The RNAi agent or pharmaceutically acceptable salt thereof according to any one of embodiments 1-28, wherein the 2' position of the sugar group of the 7th, 12th and 14th nucleotides starting from the 5' end of the antisense chain is fluorine, the 2' position of the sugar group of the remaining nucleotides of the antisense chain is methoxy, and there are at least two consecutive thiophosphate bonds between the three consecutive nucleotides at the end of the antisense chain; the 2' position of the sugar group of the 5th, 7th, 8th and 9th nucleotides starting from the 5' end of the sense chain is fluorine, the 2' position of the sugar group of the remaining nucleotides of the sense chain is methoxy; and there are at least two consecutive thiophosphate bonds between the three consecutive nucleotides at the end of the sense chain.

[0482] 30. The RNAi agent according to any one of embodiments 1-29, or a pharmaceutically acceptable salt thereof, wherein the antisense strand comprises any one of SEQ ID NO: 109, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 125 and SEQ ID NO: 126, or a sequence that differs therefrom by no more than 3 nucleotides.

[0483] 31. The RNAi agent or pharmaceutically acceptable salt thereof according to any one of embodiments 1-30, wherein the sense strand comprises any one of SEQ ID NO: 185, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 201 and SEQ ID NO: 202, or a sequence that differs therefrom by no more than 3 nucleotides.

[0484] 32. The RNAi agent according to any one of embodiments 1-31, or a pharmaceutically acceptable salt thereof, wherein the interfering nucleic acid comprises any one of Kylo-09-DS01 to Kylo-09-DS112.

[0485] 33. An RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof, comprising an antisense strand, wherein the antisense strand comprises at least 12 consecutive nucleotides that are substantially complementary to the nucleotides at the corresponding positions selected from the following sequence or a sequence that differs from it by no more than 3 nucleotides: AGT mRNA At least 12 consecutive nucleotides starting at positions 1854-1874, 1907-1927, 1895-1915, 1352-1372, 1903-1923, 2019-2039, 1853-1873 and 1818-1838 in NM_001382817.3, or a sequence that differs by no more than 3 nucleotides therefrom, or at least 12 consecutive nucleotides starting at positions 1822-1842, 1875-1895, 1863-1883, 1320-1340, 1871-1891, 1987-2007, 1821-1841 and 1786-1806 in NM_001384479.1, or a sequence that differs by no more than 3 nucleotides therefrom.

[0486] 34. The RNAi agent for inhibiting AGT gene expression according to embodiment 33 or a pharmaceutically acceptable salt thereof, wherein the antisense chain length is 15-30 nucleotides, preferably 17-25 nucleotides, and more preferably 19-23 nucleotides.

[0487] 35. An RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-34, or a pharmaceutically acceptable salt thereof, wherein the antisense strand comprises the following nucleotide sequence: any one of SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO: 26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:35 and SEQ ID NO:36, or a sequence that differs therefrom by no more than 3 nucleotides.

[0488] 36. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-35, or a pharmaceutically acceptable salt thereof, wherein the RNAi agent comprises a single-stranded or double-stranded nucleic acid molecule.

[0489] 37. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-36, or a pharmaceutically acceptable salt thereof, wherein the RNAi agent comprises siRNA or miRNA.

[0490] 38. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33 to 37, or a pharmaceutically acceptable salt thereof, further comprising a sense strand, wherein the antisense strand and the sense strand form a complementary region comprising at least 12 consecutive nucleotides.

[0491] 39. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33 to 38, or a pharmaceutically acceptable salt thereof, wherein the complementary region comprises 12 to 25 consecutive nucleotide base pairs.

[0492] 40. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-39, or a pharmaceutically acceptable salt thereof, wherein the sense strand and the antisense strand have at least about 80% base complementarity.

[0493] 41. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-40 or a pharmaceutically acceptable salt thereof, wherein the positive strand comprises AGT mRNA At least 12 consecutive nucleotides starting at positions 1854-1874, 1907-1927, 1895-1915, 1352-1372, 1903-1923, 2019-2039, 1853-1873 and 1818-1838 in NM_001382817.3, or a sequence that differs by no more than 3 nucleotides therefrom, or at least 12 consecutive nucleotides starting at positions 1822-1842, 1875-1895, 1863-1883, 1320-1340, 1871-1891, 1987-2007, 1821-1841 and 1786-1806 in NM_001384479.1, or a sequence that differs by no more than 3 nucleotides therefrom.

[0494] 42. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-41, or a pharmaceutically acceptable salt thereof, wherein the sense chain length is 15-30 nucleotides, preferably 17-25 nucleotides, and more preferably 19-23 nucleotides.

[0495] 43. An RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-43, or a pharmaceutically acceptable salt thereof, wherein the positive strand has substantial homology to any one of SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:17 and SEQ ID NO:18, or a sequence that differs therefrom by no more than 3 nucleotides.

[0496] 44. An RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-43, or a pharmaceutically acceptable salt thereof, wherein the positive strand is substantially homologous to any one of SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:17 and SEQ ID NO:18, or a sequence that differs from it by no more than 3 nucleotides, and the antisense strand comprises any one of SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:35 and SEQ ID NO:36, or a sequence that differs from it by no more than 3 nucleotides.

[0497] 45. An RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-44, or a pharmaceutically acceptable salt thereof, wherein the antisense strand comprises any one of SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:71 and SEQ ID NO:72, or a sequence that differs therefrom by no more than 3 nucleotides.

[0498] 46. ​​An RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-45, or a pharmaceutically acceptable salt thereof, wherein the positive chain comprises any one of SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:53 and SEQ ID NO:54, or a sequence that differs therefrom by no more than 3 nucleotides.

[0499] 47. An RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-46, or a pharmaceutically acceptable salt thereof, wherein the antisense strand comprises any one of SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:71 and SEQ ID NO:72, or a sequence that differs therefrom by no more than 3 nucleotides, and the positive strand comprises any one of SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:53 and SEQ ID NO:54, or a sequence that differs therefrom by no more than 3 nucleotides.

[0500] 48. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-47, or a pharmaceutically acceptable salt thereof, wherein one or more nucleotides on the sense strand and / or antisense strand are modified to form modified nucleotides.

[0501] 49. An RNAi agent for inhibiting AGT gene expression according to embodiment 48, or a pharmaceutically acceptable salt thereof, wherein the modified nucleotides are selected from: deoxyribonucleotides, nucleotide mimetics, abasic nucleotides, 2'-modified nucleotides, 3' to 3' linked (inverted) nucleotides, nucleotides containing non-natural bases, bridging nucleotides, peptide nucleic acids (PNAs), unlocked nucleobase analogs, locked nucleotides, 3'-O-methoxy (2' internucleoside linkage) nucleotides, 2'-fluoro-arabino nucleotides, 5'-methyl / 2'-fluoro-ribonucleotides, morpholino nucleotides, vinylphosphonate deoxyribonucleotides, vinylphosphonate-containing nucleotides, and cyclopropylphosphonate-containing nucleotides.

[0502] 50. The RNAi agent for inhibiting AGT gene expression according to embodiment 49 or a pharmaceutically acceptable salt thereof, wherein the 2'-modified nucleotides include: 2'-O-methyl nucleotides, 2'-deoxy-2'-fluoro nucleotides, 2'-deoxy nucleotides, 2'-methoxyethyl nucleotides, 2'-amino nucleotides and / or 2'-alkyl nucleotides.

[0503] 51. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-50, or a pharmaceutically acceptable salt thereof, wherein the 2'-substituent group of part or all of the nucleotide sugar groups of the sense chain and the antisense chain is fluorine or methoxy.

[0504] 52. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-51 or a pharmaceutically acceptable salt thereof, wherein at least two consecutive phosphorothioate bonds exist between three consecutive nucleotides at the end of the sense strand and / or the end of the antisense strand.

[0505] 53. An RNAi agent or a pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 33-52, wherein the 2' position of the sugar group of the 7th, 12th and 14th nucleotides starting from the 5' end of the antisense chain is fluorine, the 2' position of the sugar group of the remaining nucleotides of the antisense chain is methoxy, and there are at least two consecutive thiophosphate bonds between the three consecutive nucleotides at the end of the antisense chain; the 2' position of the sugar group of the 5th, 7th, 8th and 9th nucleotides starting from the 5' end of the sense chain is fluorine, the 2' position of the sugar group of the remaining nucleotides of the sense chain is methoxy; and there are at least two consecutive thiophosphate bonds between the three consecutive nucleotides at the end of the sense chain.

[0506] 54. An RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-53, or a pharmaceutically acceptable salt thereof, wherein the antisense strand comprises any one of SEQ ID NO: 109, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 125 and SEQ ID NO: 126, or a sequence that differs therefrom by no more than 3 nucleotides.

[0507] 55. An RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-54, or a pharmaceutically acceptable salt thereof, wherein the positive chain comprises any one of SEQ ID NO: 185, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 201 and SEQ ID NO: 202, or a sequence that differs therefrom by no more than 3 nucleotides.

[0508] 56. An RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-55, or a pharmaceutically acceptable salt thereof, wherein the interfering nucleic acid comprises any one or more of Kylo-09-DS01, Kylo-09-DS07, Kylo-09-DS08, Kylo-09-DS10, Kylo-09-DS11, Kylo-09-DS12, Kylo-09-DS17, Kylo-09-DS18, Kylo-09-DS37 to Kylo-09-DS54.

[0509] 57. The RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof according to any one of embodiments 33-56, further comprising a ligand, wherein the ligand is coupled to the sense strand and / or antisense strand via a carrier structure.

[0510] 58. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-57 or a pharmaceutically acceptable salt thereof, wherein the ligand comprises a targeting ligand.

[0511] 59. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-58 or a pharmaceutically acceptable salt thereof, wherein the targeting ligand is coupled to the 5' end and / or 3' end of the antisense chain via a carrier structure.

[0512] 60. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-59 or a pharmaceutically acceptable salt thereof, wherein the carrier structure is attached to the 5' end and / or the 3' end of the sense strand.

[0513] 61. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 57-60, or a pharmaceutically acceptable salt thereof, wherein the carrier structure comprises 5'MVIP and 3'MVIP, wherein the 5'MVIP is coupled to the 5' end of the sense strand and / or antisense strand, and the 3'MVIP is coupled to the 3' end of the antisense strand and / or sense strand, the structure of the 5'MVIP is as shown in Formula I, and the structure of the 3'MVIP is as shown in Formula II,

[0514] (XL) n -BDR 1 -,

[0515] I

[0516] (XL) m -BDR 2 -,

[0517] II

[0518] in,

[0519] X is a targeting-specific ligand;

[0520] L is a branched chain;

[0521] B is a connector;

[0522] D is the connecting chain;

[0523] R 1 and R 2 is a transfer point;

[0524] The 5'MVIP is linked to the transition point R 1 Connected to the 5' end of the sense strand or the 5' end of the antisense strand, the 3'MVIP is connected to the 5' end of the sense strand through the transition point R 2 Connected to the 3' end of the sense strand or the 3' end of the antisense strand, n and m are each independently any integer of 0-4.

[0525] 62. The RNAi agent for inhibiting AGT gene expression according to embodiment 61 or a pharmaceutically acceptable salt thereof, wherein n+m=an integer of 2-6, preferably n+m=2, 3 or 4, and more preferably 4.

[0526] 63. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 61-62 or a pharmaceutically acceptable salt thereof, wherein the R 1 or R 2 The sense strand or antisense strand is linked via phosphate or modified phosphate, preferably via phosphate or phosphorothioate.

[0527] 64. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 61-63 or a pharmaceutically acceptable salt thereof, wherein the R 1 It is a heterocyclic or carbocyclic structure containing N, S or O:

[0528]

[0529] Alternatively, the R 1 -NH(CH 2 ) x CH 2 O-, wherein x is any integer from 3 to 12, preferably any integer from 4 to 6.

[0530] 65. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 61-64 or a pharmaceutically acceptable salt thereof, wherein the R 2 It is a heterocyclic or carbocyclic structure containing N, S or O:

[0531]

[0532] Alternatively, the R 2 -NH(CH 2 ) x1 CH(OH)(CH 2 ) x2 CH 2 O-, wherein x1 is any integer from 1 to 4, and x2 is any integer from 0 to 4.

[0533] 66. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 61-65 or a pharmaceutically acceptable salt thereof, wherein X is a targeting ligand selected from structures for enhancing the uptake of RNAi agents by hepatocytes.

[0534] 67. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 61-66 or a pharmaceutically acceptable salt thereof, wherein X is selected from monosaccharides and their derivatives.

[0535] 68. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 61-67 or a pharmaceutically acceptable salt thereof, wherein X is selected from galactose, galactosamine, N-acetylgalactosamine and derivatives thereof.

[0536] 69. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 61 to 68, or a pharmaceutically acceptable salt thereof, wherein X is N-acetylgalactosamine and its derivatives.

[0537] 70. According to the RNAi agent for inhibiting AGT gene expression according to any one of embodiments 61-69 or a pharmaceutically acceptable salt thereof, the X is selected from the following structures:

[0538]

[0539] Wherein, W is selected from -OH, -NHCOOH or -NHCO(CH 2 ) q CH 3 One or two of the following, wherein q is an integer from 0 to 4.

[0540] 71. According to the RNAi agent for inhibiting AGT gene expression according to any one of embodiments 61-70 or a pharmaceutically acceptable salt thereof, the L is selected from one or more of the following structures:

[0541]

[0542] Wherein, r1 is any integer from 1 to 12, r2 is any integer from 0 to 20, and Z is H, an alkyl group or an amide group.

[0543] 72. According to the RNAi agent for inhibiting AGT gene expression according to any one of embodiments 61-71 or a pharmaceutically acceptable salt thereof, the B is selected from the following structures:

[0544]

[0545]

[0546] Among them, A 1 and A2 Each is independently C, O, S, -NH-, carbonyl, amide, phosphoryl or thiophosphoryl, and r is an integer of 0-4.

[0547] 73. According to the RNAi agent for inhibiting AGT gene expression according to any one of embodiments 61-72 or a pharmaceutically acceptable salt thereof, the D is selected from the following structures:

[0548]

[0549]

[0550]

[0551] Wherein, each p is independently any integer from 1 to 20; s is an integer from 2 to 13; Z 1 and Z 2 are the same or different substituent groups.

[0552] 74. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 61-73 or a pharmaceutically acceptable salt thereof, wherein (XL) in the 5'MVIP structure n -BD- and 3'MVIP structures (XL) m -BD- is selected from one or more of the following structures:

[0553]

[0554]

[0555]

[0556]

[0557]

[0558]

[0559]

[0560]

[0561]

[0562]

[0563]

[0564]

[0565]

[0566] 75. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 61 to 74, or a pharmaceutically acceptable salt thereof, wherein X, L, D, and B are the same or different within each of 5'MVIP and 3'MVIP or between 5'MVIP and 3'MVIP.

[0567] 76. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 61 to 75, or a pharmaceutically acceptable salt thereof, wherein the 5'MVIP is selected from any one of 5'MVIP01 to 5'MVIP22 in Table 10.

[0568] 77. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 61 to 76, or a pharmaceutically acceptable salt thereof, wherein the 3'MVIP is selected from any one of 3'MVIP01 to 3'MVIP27 in Table 11.

[0569] 78. According to the RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof as described in any one of embodiments 61-77, the combination of the sense chain 5'MVIP and the antisense chain 3'MVIP is 5'MVIP01 / 3'MVIP01, 5'MVIP01 / 3'MVIP17 or 5'MVIP09 / 3'MVIP09; or the combination of the sense chain 5'MVIP and the sense chain 3'MVIP is 5'MVIP01 / 3'MVIP09 or 5'MVIP09 / 3'MVIP01.

[0570] 79. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33 to 78, or a pharmaceutically acceptable salt thereof, wherein the antisense strand comprises any one or more of AS131, AS137, AS138, AS140, AS141, AS142, AS147 and AS148.

[0571] 80. According to any one of embodiments 33-79, the RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof, the positive strand comprises any one or more of S131, S137, S138, S140, S141, S142, S147 and S148.

[0572] 81. The RNAi agent or a pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 33-80, wherein the RNAi agent or a pharmaceutically acceptable salt thereof is any one or more of Kylo-09-DS113, Kylo-09-DS119, Kylo-09-DS120, Kylo-09-DS122, Kylo-09-DS123, Kylo-09-DS124, Kylo-09-DS129 and Kylo-09-DS130 in Table 15.

[0573] 82. According to the RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof according to any one of embodiments 33-81, the antisense strand is selected from any one or more of AS140, AS207 to AS266.

[0574] 83. According to the RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof according to any one of embodiments 33-82, the positive strand is selected from any one or more of S140, S207 to S264.

[0575] 84. According to any one of embodiments 33-83, the RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof, the antisense strand is selected from any one or more of AS140, AS207 SEQ ID NO: 413 to AS266, and the positive strand is selected from any one or more of S140, S207 to S264.

[0576] 85. The RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-84, or a pharmaceutically acceptable salt thereof, comprises any one or more of Kylo-09-DS122, Kylo-09-DS131, Kylo-09-DS141, Kylo-09-DS142 and Kylo-09-DS147 in Table 18.

[0577] 86. A cell comprising the RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-32, or the RNAi agent or a pharmaceutically acceptable salt thereof that inhibits AGT gene expression according to any one of embodiments 33-85.

[0578] 87. A pharmaceutical composition comprising the RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-32, or the RNAi agent or a pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 33-85, and optionally a pharmaceutically acceptable excipient, carrier and / or diluent.

[0579] 88. The pharmaceutical composition of claim 87, further comprising a delivery vehicle.

[0580] 89. The pharmaceutical composition of claim 88, wherein the delivery vehicle comprises a liposome.

[0581] 90. The pharmaceutical composition of claim 89, wherein the delivery vehicle comprises nanolipids.

[0582] 91. A method for reducing AGT mRNA or protein expression in cells or tissues, comprising contacting the cells or tissues with an effective amount of the RNAi agent or a pharmaceutically acceptable salt thereof described in any one of embodiments 1-32, the RNAi agent or a pharmaceutically acceptable salt thereof that inhibits AGT gene expression described in any one of embodiments 33-85, and / or the pharmaceutical composition described in any one of embodiments 87-90.

[0583] 92. A method according to embodiment 91, wherein the cells are hepatocytes.

[0584] 93. The method of embodiment 91, wherein the tissue is liver tissue.

[0585] 94. A method according to embodiment 91, wherein the cells and tissues are ex vivo.

[0586] 95. A method according to embodiment 91, wherein the cells and tissues are in a subject.

[0587] 96. Use of the RNAi agent or a pharmaceutically acceptable salt thereof described in any one of embodiments 1-32, the RNAi agent or a pharmaceutically acceptable salt thereof for inhibiting AGT gene expression described in any one of embodiments 33-85, or the pharmaceutical composition described in any one of embodiments 87-90 in the preparation of a drug for preventing and / or treating a disease or condition or reducing the risk of a disease or condition.

[0588] 97. The use according to embodiment 96, wherein the disease or condition comprises a disease or condition associated with AGT.

[0589] 98. The use according to embodiment 96, wherein the disease or condition is selected from: hypertension, hypertension, critical hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, hypertension associated with low plasma renin activity or plasma renin concentration, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, Ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, stroke, renal disease, renal failure, systemic sclerosis, intrauterine growth retardation (IUGR), fetal growth restriction, obesity, hepatic steatosis / fatty liver, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD); glucose intolerance, type 2 diabetes mellitus (non-insulin-dependent diabetes mellitus), and metabolic syndrome.

[0590] 99. A method for preventing and / or treating a disease or condition, the method comprising administering to a subject in need thereof an effective amount of the RNAi agent or a pharmaceutically acceptable salt thereof described in any one of embodiments 1-32, the RNAi agent or a pharmaceutically acceptable salt thereof that inhibits AGT gene expression described in any one of embodiments 33-85, and / or the pharmaceutical composition described in any one of embodiments 87-90.

[0591] 100. The method of embodiment 99, wherein the administering comprises administering to the subject subcutaneously, intravenously, orally, rectally, or intraperitoneally.

[0592] 101. A method according to embodiment 99, wherein the RNAi agent, RNAi agent that inhibits AGT gene expression, or pharmaceutical composition is administered to the subject at a dose of about 0.01 mg / kg to about 50 mg / kg.

[0593] 102. The method of embodiment 99, further comprising determining the level of AGT in a sample from the subject.

[0594] 103. A method according to embodiment 99, wherein the AGT level in the subject's sample is the AGT protein level in a blood sample, a serum sample or a urine sample.

[0595] 104. The method of embodiment 99, further comprising administering to the subject an additional therapeutic agent for treating hypertension.

[0596] 105. The method of embodiment 104, wherein the additional therapeutic agent is selected from: a diuretic, angiotensin converting enzyme (ACE) inhibitor, angiotensin II receptor antagonist, a beta-blocker, a vasodilator, a calcium channel blocker, an aldosterone antagonist, an alpha2-agonist, a renin inhibitor, an alpha-blocker, a peripherally acting adrenergic agent, a selective D1 receptor partial agonist, a non-selective alpha-adrenergic antagonist, a synthetic steroidal antimineralocorticoid agent, angiotensin receptor-neprilysin inhibitor (ARNi), sacubitril / valsartan; or an endothelin receptor antagonist (ERA), sitaxsentan, ambrisentan, atrasentan, BQ-123, zilpotentan, bosentan, macitentan and tezosentan; a combination of any of the above therapeutic agents; and a hypertension therapeutic agent formulated as a combination of agents.

[0597] 106. The method of embodiment 104, wherein the additional therapeutic agent comprises an angiotensin II receptor antagonist.

[0598] 107. The method of embodiment 106, wherein the angiotensin II receptor antagonist is selected from the group consisting of: losartan, valsartan, olmesartan, eprosartan and azilsartan.

[0599] 108. A drug kit comprising the RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-32, the RNAi agent or a pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 33-85, or the pharmaceutical composition according to any one of embodiments 87-90.

[0600] Without intending to be bound by any theory, the following examples are merely intended to illustrate the RNAi agent, preparation method and use of the present application, and are not intended to limit the scope of the invention of the present application.

[0601] Example

[0602] illustrate:

[0603] The Chinese name of DMSO is dimethyl sulfoxide;

[0604] The Chinese name of DMF is N,N-dimethylformamide;

[0605] The Chinese name of HOBt is 1-hydroxybenzotriazole;

[0606] The Chinese name of HBTU is O-benzotriazole-tetramethyluronium hexafluorophosphate;

[0607] The Chinese name of DIPEA (DIEA) is N,N-diisopropylethylamine;

[0608] The Chinese name of DCM is dichloromethane;

[0609] The Chinese name of DMAP is 4-dimethylaminopyridine;

[0610] The Chinese name of DMT-CL is 4,4'-dimethoxytriphenylmethane;

[0611] The Chinese name of MEOH is methanol;

[0612] The Chinese name of TBTU is O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate;

[0613] The name of the solid phase carrier is macroporous aminomethyl resin (Resin).

[0614] Example 1 Solid Phase Phosphoramidite Method for Synthesizing siRNA

[0615] The sense strands in Table 1 and the antisense strands in Table 2 were synthesized according to a standard solid phase phosphoramidite method, and the sense strands were complementary annealed with the corresponding antisense strands to obtain siRNA.

[0616] The basic steps of the solid phase phosphoramidite method include:

[0617] 1) Deprotection: removing the hydroxyl protecting group (DMTr) of the starting monomer Solid Support;

[0618] 2) Coupling: Add the first phosphoramidite monomer and the coupling reaction occurs from 3' to 5' direction;

[0619] 3) Oxidation: oxidizing the obtained nucleoside phosphite to a more stable nucleoside phosphate (i.e., oxidizing trivalent phosphorus to pentavalent phosphorus);

[0620] 4) Blocking: Block the 5'-OH of the failed nucleotide sequence in the previous step to prevent it from further participating in the reaction; repeat the above steps until the last phosphoramidite monomer is connected; then use methylamine aqueous solution and ammonia water to cleave the ester bond between SolidSupport and the starting monomer, and remove the protecting groups on each base and phosphate on the resulting nucleotide sequence; after HPLC separation and purification, filter sterilize and lyophilize to obtain the corresponding sense chain or antisense chain.

[0621] Annealing process description:

[0622] Dissolve the sense chain and antisense chain freeze-dried powder separately, mix them in equal moles, add an appropriate amount of water for injection, and add an appropriate amount of TRIS buffer solution. Shake gently for about 1 to 2 minutes to mix the solution evenly. Heat the water bath to 92°C to 95°C. Heat the above reaction solution in a water bath for 3min to 5min, and shake gently to heat the solution evenly. Cool naturally to room temperature. Obtain a colorless or slightly yellow transparent liquid, take a sample for inspection, and measure the concentration. The RNAi agent described in this application is obtained by the solid phase phosphoramidite method to obtain the respective sense chain and antisense chain, and the sense chain and the corresponding antisense chain are complementary annealed to obtain the final product.

[0623] Example 2 In vitro RNAi agent inhibition of AGT gene expression test

[0624] Take the RNAi agent prepared according to the method described in Example 1 (Kylo-09-DS01 to Kylo-09-DS18 in Table 3). Mix the RNAi agent aqueous solution and the DOTMA organic solution to form a water-insoluble precipitate, separate and dry the precipitate, dissolve it in chloroform, and further mix it with other lipid chloroform solutions, the other lipids include M10C1 and PEG600-cholesterol. The mixture is vacuum centrifuged and evaporated and dried overnight to obtain a nanolipid-encapsulated RNAi agent, wherein the weight ratio of DOTMA, M10C1 and PEG600-cholesterol to the RNAi agent is 1 to 1.6, 1.5 to 2.5 and 2.5 to 3.5.

[0625] DMEM containing 10% fetal bovine serum was used to prepare sample solutions of RNAi agents (RNAi agents in Table 3: Kylo-09-DS01 to Kylo-09-DS18) with corresponding concentrations. 5Hep3B cells were inoculated with 10% fetal bovine serum DMEM medium at 37°C and 5% CO2 for 24 hours. Sample interventions of different concentrations (10nM, 1nM, 0.1nM) were added. After incubation for 72 hours, the cell samples were collected. 1ml of Ezol lysis buffer was added to the collected cell samples and vortexed to mix. 0.2ml of chloroform was added, and the mixture was shaken vigorously for 10s and placed at room temperature for 1 minute. Centrifuge at 12,000xg for 15min at 4°C. The supernatant aqueous phase was transferred to another new RNase-free centrifuge tube and an equal volume of 100% ethanol was added. All samples were aspirated and added to a mini-spin centrifuge column with a 2ml collection tube. Centrifuge at 8,000xg for 15s at room temperature and discard the flow-through. The remaining sample was transferred to the centrifuge column and the previous step was repeated. Add 700 μl WB to the centrifuge column, cover lightly, centrifuge at 8,000×g at room temperature for 15 seconds, discard the flow-through, repeat the previous step, and wash the centrifuge column twice with 500 μl WB. Determine the AGT mRNA level by QRT-PCR. Compare with the supernatant of Hep3B cells without intervention to calibrate the relative percentage of AGT mRNA in the sample intervention group. The test results are shown in Tables 19 and Figure 1A .

[0626] Table 19 AGT mRNA levels in Hep3B cells after RNAi intervention

[0627]

[0628] The experimental results showed that the RNAi agents in Table 3 formed by annealing the sense chain in Table 1 and the antisense chain in Table 2 showed different degrees of inhibitory effect on the expression level of AGT mRNA in Hep3B cells at different concentrations, among which Kylo-09-DS01, Kylo-09-DS07, Kylo-09-DS08, Kylo-09-DS10, Kylo-09-DS11, Kylo-09-DS12, Kylo-09-DS17 and Kylo-09-DS18 showed obvious dose-dependence in inhibiting the expression level of AGT mRNA in Hep3B cells, and the corresponding sense chain sequences were SEQ ID NO. 37, 43, 44, 46, 47, 48, 53 and 54 and the corresponding antisense chain sequences were SEQ ID NO. 55, 61, 62, 64, 65, 66, 71 and 72.

[0629] The selected Kylo-09-DS01, Kylo-09-DS07, Kylo-09-DS08, Kylo-09-DS10, Kylo-09-DS11, Kylo-09-DS12, Kylo-09-DS17 and Kylo-09-DS18 were operated with reference to the Hep 3B cell experiment to investigate the intervention effect of different concentrations of RNAi agents (10nM, 0.1nM) on HepG 2 cells, and the AGT mRNA level was determined by QRT-PCR. Compared with the supernatant of HepG 2 cells without intervention, the relative percentage of AGT mRNA in the sample intervention group was calibrated. The experimental results are shown in Tables 20 and 20 below. Figure 1B .

[0630] Table 20 AGT mRNA levels in HepG2 cells after RNAi intervention

[0631]

[0632] Example 3 Study on the stability of modified siRNA in plasma

[0633] The RNAi agent involved in this embodiment is selected from Table 7, and the parent chain is the sequence selected from Example 2 (the sense chain sequence is SEQ ID NO.37, 43, 44, 46, 47, 48, 53 and 54 and the corresponding antisense chain sequence is SEQ ID NO.55, 61, 62, 64, 65, 66, 71 and 72). The 2' position of the nucleotides at positions 7, 12 and 14 from the 5' end of the antisense chain is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between at least three adjacent nucleotides at the end of the antisense chain can be thiolated, the 2' position of the nucleotides at positions 5, 7, 8 and 9 from the 5' end of the sense chain is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between at least three adjacent nucleotides at the end of the antisense chain can be thiolated.

[0634] The purpose of this example is to verify that the above-mentioned modification method can enhance the stability of RNAi agents in human serum. The test results are shown in Table 21 and Figure 2.

[0635] Among them, Figure 2 shows the HPLC graphs of stability detection of RNAi Kylo-09-DS46 and its parent chain Kylo-09-DS10 at different time periods.

[0636] Table 21 Peak area ratio of full-length double-stranded RNAi agent to 0 h at different time periods in human serum.

[0637]

[0638] The above test results show that the 2' position of the nucleotides at the 7th, 12th and 14th positions starting from the 5' end of the antisense chain of the respective unmodified parent chain is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between at least 3 adjacent nucleotides at the end of the antisense chain can be thiolated, and the 2' position of the nucleotides at the 5th, 7th, 8th and 9th positions starting from the 5' end of the sense chain is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between at least 3 adjacent nucleotides at the end of the antisense chain can be thiolated. The modification method can effectively enhance the stability of RNAi agents in human serum. The enhancement effect is sequence-specific, and different sequences have different stability enhancement effects.

[0639] Example 4 Synthesis of 5'MVIP and 3'MVIP compounds

[0640] When the 3' end of the sense strand or antisense strand of the RNAi agent of the present invention is coupled with the carrier structure 3'MVIP, the solid support of 3'MVIP is used as the starting monomer of solid phase synthesis. When the 5' end of the sense strand or antisense strand of the RNAi agent of the present invention is coupled with the carrier structure 5'MVIP, the 5'MVIP phosphoramidite monomer is used as the last monomer of solid phase synthesis.

[0641] When the 3' end of the sense strand or antisense strand of the RNAi agent of the present application is coupled with 3'MVIP, the solid support of 3'MVIP is used as the starting monomer for solid phase synthesis, and the general formula of the solid support of 3'MVIP is as follows:

[0642]

[0643] When m is 1-4, the linker B part in the general formula is branched 1 to 4 times to obtain the corresponding SolidSupport of 3'MVIP.

[0644] For example, when m is 1, the obtained Solid Support is used as the RNAi agent Kylo-09-DS142 (structural formula see Fig.9D ) is used as the starting monomer for the solid phase synthesis of the sense chain of the RNAi agent Kylo-09-DS141 (structural formula see 9C) and the antisense chain of Kylo-09-DS122; when m is 2, the obtained SolidSupport is used as the starting monomer for the solid phase synthesis of the sense chain of the RNAi agent Kylo-09-DS141 (structural formula see 9C) and the antisense chain of Kylo-09-DS122;

[0645] When m is 3, the obtained Solid Support is used as the starting monomer for the solid phase synthesis of the antisense strand of the RNAi agent Kylo-09-DS147.

[0646] When there is 5'MVIP at the 5' end of the sense strand or antisense strand of the RNAi agent of the present application, the 5'MVIP phosphoramidite monomer is the last phosphoramidite monomer synthesized as the sense strand or antisense strand in the solid phase. The general formula of the 5'MVIP phosphoramidite monomer is as follows:

[0647]

[0648] When n is 1-4, the linker B in the general formula is branched 1 to 4 times to obtain the corresponding 5'MVIP phosphoramidite monomer.

[0649] For example, when n is 1, the resulting 5'MVIP phosphoramidite monomer is used as the last monomer in the solid phase synthesis of the positive chain of the RNAi agents Kylo-09-DS141, Kylo-09-DS131 (structural formula 9B) and Kylo-09-DS147 (structural formula see 9E); when n is equal to 2, the resulting 5'MVIP phosphoramidite monomer is used as the last monomer in the solid phase synthesis of the positive chain of Kylo-09-DS122 (structural formula see 9A) and Kylo-09-DS142.

[0650] When n is equal to 3, the obtained 5'MVIP phosphoramidite monomer with three ligands X can be used as the last monomer in the solid phase synthesis of the sense chain or the antisense chain.

[0651] Before the phosphoramidite solid phase synthesis of the sense strands and antisense strands of the RNAi agents described in the present application, the corresponding 3'MVIP Solid Support and 5'MVIP phosphoramidite monomers need to be chemically synthesized.

[0652] This embodiment exemplifies the chemical synthesis process of the 3'MVIP Solid Support and 5'MVIP phosphoramidite monomer of the following RNAi agent, as described below:

[0653] 4.1 Synthesis of Solid Support of 3'MVIP

[0654] 4.1.1 Synthesis of Solid Support of 3'MVIP09

[0655]

[0656] Description of the synthesis process:

[0657] 4.1.1.1 Synthesis of ERC-01-c1

[0658]

[0659] Weigh 2-amino-1,3-propanediol (5.0 g, 54.9 mmol), add 50 mL of DMSO, 5 mL of sodium hydroxide solution (1 g / mL), cool to 0°C, add tert-butyl acrylate (20 mL, 137.8 mol) dropwise over 2 hours, react at room temperature for 48 hours, add petroleum ether (100 mL), wash twice with saturated brine, dry the organic layer, pass through a chromatography column (eluent: ethyl acetate: petroleum ether = 25%-75%), add 0.05% triethylamine to the column, and obtain 6.2 g of colorless oil.

[0660] 4.1.1.2 Synthesis of ERC-01-c2

[0661]

[0662] Weigh ERC-01-c1 (6.2 g, 17.9 mmol), add 50 mL of dichloromethane and 23 mL of sodium carbonate solution (25%), add benzyl chloroformate (8.2 mL, 57.4 mmol) dropwise at room temperature for 2 hours, react at room temperature overnight, wash 3 times with saturated brine, dry over anhydrous sodium sulfate, evaporate the solvent, and pass through a chromatography column (ethyl acetate: petroleum ether = 5%-30%) to obtain 4.0 g of an oily substance.

[0663] 4.1.1.3 Synthesis of ERC-01-c3

[0664]

[0665] Take ERC-01-c2 (4.0 g, 8.3 mmol) and add 12 mL of formic acid, react at room temperature overnight, and evaporate the solvent under reduced pressure to obtain 2.8 g of the product.

[0666] 4.1.1.4 Synthesis of ERCd-01-c1

[0667]

[0668] Compound ERC-01-c3 (1.11 g, 3.0 mmol) and dlSANC-c4 (3.6 g, 8.04 mmol) were added to DMF (60 mL), followed by HOBt (2.24 g) and HBTU (3.36 g), and then DIEA (4.16 mL) was slowly added. The reaction solution was stirred at room temperature for 3 hours. Water was then added, and the aqueous layer was extracted with dichloromethane (2 x 10 mL). The organic layers were combined, and then washed with saturated sodium bicarbonate (80 mL), water (2 x 60 mL), and saturated brine (60 mL) in sequence. Dry with anhydrous sodium sulfate, evaporate to dryness under reduced pressure, and purify with silica gel column chromatography (eluent: 3-15% MeOH in DCM). 3.24 g of light yellow solid was obtained.

[0669] 4.1.1.5 Synthesis of ERCd-01-c2

[0670]

[0671] ERCd-01-c1 (3.24 g, 2.6 mmol) was dissolved in methanol (60 mL), and 10% palladium carbon (0.3 g) and acetic acid (2.0 mL) were added. Then hydrogenation was added under normal pressure and the reaction was allowed to proceed overnight. The reaction solution was filtered through diatomaceous earth, and the filtrate was evaporated to dryness under reduced pressure to obtain 2.9 g of an oily substance ERCd-01-c2, the high-resolution mass spectrum of which is shown in Figure 3 .

[0672] 4.1.1.6 Synthesis of 3'MVIP09-c1

[0673]

[0674] SANCd-01-c0 (0.824 g, 1.5 mmol) and ERCd-01-c2 (1.09 g, 1.0 mmol) were added to the reaction bottle in sequence, and then 10 mL of DCM was added, stirred to dissolve, and TBTU (0.963 g) and DIPEA (0.517 g) were added in sequence, and the reaction was allowed to proceed overnight. Water was added, and the mixture was extracted with DCM. The organic phase was then washed with saturated brine, dried, filtered, and concentrated, and finally purified by a silica gel column to obtain 1.3 g of the product.

[0675] 4.1.1.7 Synthesis of 3'MVIP09-c2

[0676]

[0677] To the reaction bottle, 3'MVIP09-c1 (1.62 g, 1 μmol) and 10 mL of DCM were added in sequence, and the mixture was dissolved by stirring at room temperature. DMAP (0.366 g) and succinic anhydride (0.2 g, 3 μmol) were added in sequence, and the mixture was reacted by stirring at room temperature. After TLC analysis, if the reaction was qualified, DCM was concentrated, water was added, and the mixture was extracted with DCM. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated, and finally purified by silica gel column to obtain 1.55 g of the product.

[0678] 4.1.1.8 Solid Support Synthesis of 3'MVIP09

[0679]

[0680] 3'MVIP09-c2 (0.86 g, 0.5 μmol) and 10 mL DMF were added to the reaction bottle in sequence to dissolve, and then HBTU (0.19 g), DIPEA (0.194 g) and macroporous aminomethyl resin (2.0 g) were added in sequence. The mixture was shaken for 24 h and filtered. The resin was washed with 10% methanol / DCM and capped with 25% acetic acid / pyridine. The degree of substitution was 150 μmol / g.

[0681] 4.1.2 Synthesis of Solid Support for 3'MVIP17

[0682]

[0683] 4.1.2.1 Synthesis of SANC-01-c1

[0684]

[0685] The synthesis steps refer to the synthesis of 4.1.1.1.ERC-01-c1.

[0686] 4.1.2.2 Synthesis of SANC-01-c2

[0687]

[0688] The synthesis steps refer to 4.1.1.2. Synthesis of ERC-01-c2.

[0689] 4.1.2.3 Synthesis of SANC-01-c3

[0690]

[0691] The synthesis steps refer to 4.1.1.3. Synthesis of ERC-01-c3.

[0692] 4.1.2.4 Synthesis of SANCd-01-c1

[0693]

[0694] The synthesis steps refer to the synthesis of 4.1.1.4.ERCd-01-c1.

[0695] 4.1.2.5 Synthesis of SANCd-01-c2

[0696]

[0697] The synthesis steps refer to 4.1.1.5. Synthesis of ERCd-01-c2.

[0698] 4.1.2.6 Synthesis of 3'MVIP17-c1

[0699]

[0700] The synthesis steps refer to the synthesis of 4.1.1.6.3'MVIP09-c1, and the high-resolution mass spectrum of the synthesized 3'MVIP17-c1 is shown in Figure 4.

[0701] 4.1.2.7 Synthesis of 3'MVIP17-c2

[0702]

[0703] The synthesis steps refer to 4.1.1.7.3'MVIP09-c2 synthesis.

[0704] 4.1.2.8 Solid Support Synthesis of 3'MVIP17

[0705]

[0706] The synthesis steps refer to 4.1.1.8 Solid Support Synthesis of 3'MVIP09.

[0707] 4.1.3 Synthesis of Solid Support of 3'MVIP01:

[0708]

[0709] Description of the synthesis process:

[0710] 4.1.3.1 Synthesis of 3'MVIP01-c1

[0711]

[0712] The synthesis steps refer to 4.1.1.6.3'MVIP09-c1 synthesis.

[0713] 4.1.3.2 Synthesis of 3'MVIP01-c2

[0714]

[0715] The synthesis steps refer to 5.1.1.7.3'MVIP09-c2 synthesis.

[0716] 4.1.3.3 Solid Support Synthesis of 3'MVIP01

[0717]

[0718] The synthesis steps refer to 4.1.1.8.3' Solid Support synthesis of MVIP09.

[0719] 4.2. Synthesis of 5'MVIP phosphoramidite monomer

[0720] 4.2.1 When n is 2, the obtained 5'MVIP phosphoramidite monomer is used as the last monomer 5'MVIP09 phosphoramidite monomer in the solid phase synthesis of the positive chain of Kylo-09-DS122 and Kylo-09-DS142:

[0721]

[0722] 4.2.1.1 Synthesis of 5'MVIP09-ERCd-PFP-c1

[0723]

[0724] Weigh ERCd-01-c2 (2.18 g, 2.0 mmol) and dissolve it in DMF (50 mL). Add benzyl glutarate (0.53 g, 2.4 mmol), DIPEA (0.78 g) and TBTU (0.84 g). Stir at room temperature overnight, quench with water (50 mL), extract with DCM (30 mL*3), wash with 10% citric acid (50 mL*3), saturated sodium bicarbonate 50 mL and pyridine 100 mL, dry over anhydrous sodium sulfate, filter, rotary evaporate, and purify by column to obtain the product 5'MVIP09-ERCd-PFP-c1 (2.15 g).

[0725] 4.2.1.2 Synthesis of 5'MVIP09-ERCd-PFP-c2

[0726]

[0727] Weigh 5'MVIP09-ERCd-PFP-c1 (2.15 g, 1.66 mmol) and 10% palladium carbon (0.21 g), add methanol (50 mL), stir and hydrogenate at room temperature overnight. After the reaction is completed, filter the palladium carbon through diatomaceous earth and rotary evaporate to obtain a crude product of 5'MVIP09-ERCd-PFP-c2 (1.9 g). Its high-resolution mass spectrum is shown below. Figure 5 shown.

[0728] 4.2.1.3 Synthesis of 5'MVIP09-ERCd-PFP

[0729]

[0730] Weigh the crude product of 5'MVIP09-ERCd-PFP-c2 (1.9 g, 1.58 mmol) and dissolve it in DCM (60 mL). Add DIPEA (1.33 g), cool it, add pentafluorophenol trifluoroacetate (2.21 g, 7.9 mmol), stir at room temperature for 2 h, then evaporate it by rotary evaporation, dissolve it in DCM (60 mL), wash it with saturated sodium bicarbonate (30 mL*3), 10% citric acid (30 mL*1), and saturated brine (50 mL*1), dry it with anhydrous sodium sulfate, filter it, and evaporate it by rotary evaporation to obtain the crude product of 5'MVIP09-ERCd-PFP (2.35 g). After drying, use it directly in the next reaction without purification.

[0731] 4.2.1.4 Synthesis of 5'MVIP09 phosphoramidite monomer-c1

[0732]

[0733] The crude product of 5'MVIP09-ERCd-PFP (2.35 g, 1.58 mmol) was dissolved in DCM (60 mL), and DIPEA (0.82 g, 6.32 mmol) and 6-amino-1-hexanol (0.37 g, 3.16 mmol) were added, and the mixture was stirred at room temperature overnight. 10% citric acid (30 mL) was added, and the mixture was extracted with DCM (30 mL*3), washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, evaporated, and purified by column to obtain the product 5'MVIP09 monomer-c1 (1.73 g).

[0734] 4.2.1.5 5'MVIP09 phosphoramidite monomer

[0735]

[0736] Weigh 5'MVIP09 phosphoramidite monomer-c1 (1.3 g, 1.0 mmol) and dissolve it in acetonitrile (30 mL), add diisopropylamine triazole (0.22 g), add bis-(diisopropylamino)(2-cyanoethoxy)phosphine (0.36 g, 1.2 mmol) dropwise under ice bath, react at room temperature for 4 h, control by HPLC, after the reaction is qualified, concentrate and purify by column to obtain the product 5'MVIP09 monomer (1.2 g).

[0737] When n is 1, the obtained 5'MVIP phosphoramidite monomer is used as the last monomer in the solid phase synthesis of the positive chain of Kylo-09-DS141, Kylo-09-DS131, and Kylo-09-DS147, and the code is 5'MVIP01:

[0738]

[0739] Phosphoramidite monomer of 5'MVIP01 Weigh YICd-01-c2 (1.12 g, 2.0 mmol), and refer to 4.2.1.1. to 4.2.1.5 for the remaining operations.

[0740] Example 5 Synthesis of RNAi Agents Conjugated with 5'MVIP09 / 3'MVIP 09 and Different siRNAs

[0741] Description of the synthesis of the antisense strand in Table 13: Purge the reagent bottle with argon for at least 2 minutes. Add phosphoramidite monomer and acetonitrile to the reagent bottle in sequence, tighten the bottle cap, and shake until the solid is visually dissolved. Then add 3A molecular sieves and let it stand for more than 8 hours for use. Purge the reagent bottle with argon for at least 2 minutes. Add hydrogenated xanthan and dry pyridine to the reagent bottle in sequence, tighten the bottle cap, and shake until the solid is visually dissolved, and store it for use. Confirm that the following operations are performed under room temperature 20-30oC environmental conditions: Weigh the 3'MVIP carrier, add it to the reagent bottle, then add acetonitrile, and shake to mix evenly. Transfer the carrier to the synthesis column, and use acetonitrile to elute the residual carrier in the reagent bottle and transfer it to the synthesis column. After elution, add acetonitrile to fill the synthesis column and record the amount of acetonitrile used. Install and fix the synthesis column according to the instrument operation.

[0742] Connect the above-prepared monomer solution, CAP A, CAP B, oxidant, thiolation agent, activator, decapping agent and acetonitrile to the corresponding pipelines of AKTA PILOT100, ensuring that the pipelines are inserted into the bottom of the reagent bottle.

[0743] After the synthesis method is set up, the instrument is ready to work. Click Run to start the synthesis. Observe and record the area of ​​each detritylation peak online. During the synthesis process, add the deprotection reagent according to the actual amount used.

[0744] After the synthesis is completed, argon is purged into the synthesis column for ≥2h, and the synthesis column is unloaded according to the operating procedures. The solid phase carrier in the synthesis column is transferred to the reaction bottle, methylamine aqueous solution and ammonia water are added, and the reaction bottle is placed in a shaker at 35°C for 2-3 hours. Filter the solution into a round-bottom flask, wash the residual solid phase with 50% ethanol aqueous solution, filter again and combine with the previous filtrate, connect the round-bottom flask to a rotary evaporator, set the water temperature to 50°C and steam until no distillation, add ethanol to the round-bottom flask, mix well, and steam again until no distillation, and repeat the operation until white powder appears at the bottom of the bottle. The obtained white powder is formulated into a solution, purified using a reverse chromatography column, and sampled to detect OD260 and purity. The purified antisense chain solution is divided into a syringe bottle and freeze-dried for standby use, and the product is sealed and stored in a -20°C refrigerator.

[0745] The synthesis operation of the sense strand in Table 14 is the same as that of the antisense strand, wherein the carrier used for the column is the Universal carrier. The intermediate is added with DIPEA to prepare a solution, and 5'MVIP phosphoramidite monomer is added and mixed, and the reaction bottle is placed in a shaker at 35°C for 2-3 hours.

[0746] Table 15 Synthesis annealing process description of RNAi agent:

[0747] Take the antisense strand in Table 13, take the antisense strand in Table 14 that is base-paired with the antisense strand in Table 13, mix them in a reaction bottle at 1:1 equimolar ratio, and after 5 minutes in a water bath at 95°C, turn off the power of the water bath to cool it naturally to below 40°C. Add 3M sodium acetate aqueous solution to the double-stranded solution, mix well, then add an appropriate volume of anhydrous ethanol, mix well, and put the reaction solution in a -20°C refrigerator for 45min. Set the refrigerated high-speed centrifuge to 4°C for pre-cooling. After the temperature is reached, put in the double-stranded solution and start the centrifuge. Take out the double-stranded solution after centrifugation, remove the supernatant, add ultrapure water to completely dissolve the solid, and take samples to detect OD260 and purity. The purified double-stranded solution is divided into cillin bottles and freeze-dried for standby use, and the product is sealed and stored in a -20°C refrigerator.

[0748] Example 6 Study on RNAi activity of 5'MVIP09 / 3'MVIP09 coupled with different siRNAs

[0749] Female AGT transgenic mice of appropriate age were used for experimental evaluation. The test samples were the RNAi agents Kylo-09-DS113 to Kylo-09-DS130 in Table 15. 3 mg / kg was administered by subcutaneous injection on Day 0; the administration volume was 100-200 μL. Blood was collected on days 0, 7, 14, 21, 28 and 35 after administration, and the serum was separated and stored at -80°C. The hAGT level in the serum was determined by the Elisa method, and the test results are shown in Tables 22 and Figure 6 .

[0750] Table 22 Average levels of hAGT in serum of transgenic mice after administration

[0751]

[0752]

[0753] The results show that the RNAi agents Kylo-09-DS113, 119, 120, 122, 123, 124, 129 and 130 obtained by 5'MVIP09 / 3'MVIP09 coupling with the preferred sequences of Example 2 (sense strand sequences are SEQ ID NO. 37, 43, 44, 46, 47, 48, 53 and 54 and the corresponding antisense strand sequences are SEQ ID NO. 55, 61, 62, 64, 65, 66, 71 and 72) are still significantly active and have good sustainability in vivo. This example verifies that the 5'MVIP09 / 3'MVIP09 vector structure can achieve safe delivery of siRNA with significant effects.

[0754] Example 7 Study on the effect of 5'MVIP and 3'MVIP with different structures coupled to the same siRNA on the activity of RNAi agents

[0755] The antisense strand in Table 16 and the sense strand in Table 17 were synthesized according to the method described in Example 5, and the RNAi agents Kylo-09-DS122, Kylo-09-DS147-160, Kylo-09-DS161 and Kylo-09-171 in Table 18 were synthesized by pairing and annealing. Female human AGT transgenic mice of appropriate age were used for experimental evaluation. The mice were given subcutaneous injections on Day 0: 3 mg / kg; the administration volume was 100-200 μL. Blood was collected 14 days after administration, serum was separated, and the hAGT level in serum was determined by Elisa method. The test results are shown in Table 23 and Figure 7 .

[0756] Table 23 Average levels of hAGT in serum of transgenic mice

[0757]

[0758] The above test results show that the positions where the vector structures 5'MVIP and / or 3'MVIP are coupled include the 5' end and / or 3' end of the antisense strand, the 5' end and / or 3' end of the sense strand, the 5' end of the antisense strand and the 3' end of the sense strand, and the 5' end and 3' end of the sense strand. Various vector structures are coupled to the same AGT siRNA at different positions, and the resulting RNAi agents have different inhibitory effects on the hAGT level of transgenic mice. Among them, 5'MVIP09, 20, 19 and 10, where n and m are 2 respectively, are coupled to the sense strand, while 3'MVIP09, 10, 15 and 12 are coupled to the antisense strand respectively. The RNAi agents Kylo-09-DS122, 151, 152 and 153 obtained by annealing and pairing have better inhibitory effects on the hAGT level of transgenic mice than other combinations; n and m Different, the inhibitory effect of RNAi agents Kylo-09-DS147, 148, 149 and 150 with n+m equal to 4 is second. Among the RNAi agents obtained by coupling the 5' end of the antisense chain with a carrier structure, the inhibitory effect of Kylo-09-DS160 obtained by combining 5'MVIP21 and 3'MVIP18 also reached 67.3%. Although the n+m of Kylo-09-DS161 and Kylo-09-DS171 is 5 and 6 respectively, they do not show the advantage of more branches in the inhibitory effect, which is speculated to be related to the specificity of the sequence or the steric hindrance of the introduced carrier structure. The test results show that the RNAi agents obtained by combining 5'MVIP selected from Table 10 and / or 3'MVIP selected from Table 11 into a carrier structure have a certain inhibitory effect on the hAGT expression level in transgenic mice.

[0759] Example 8 Exploration of the efficacy of the RNAi agent formed by the combination of 5'MVIP09 / 3'MVIP09 in cynomolgus monkeys

[0760] The purpose of this example is to study the effect of RNAi obtained by coupling the same AGT siRNA with different structures of 5'MVIP and 3'MVIP on the inhibitory activity in crab-eating monkeys. The corresponding RNAi agents Kylo-09-DS131, Kylo-09-DS141, Kylo-09-DS142, Kylo-09-DS147 or Kylo-09-DS122 were prepared according to the method described in Example 6. 18 male eluted monkeys aged 3 to 5 years were selected and randomly divided into a control group (n=3) and a drug administration group (n=3) according to body weight after adaptive feeding. The dosage was 3 mg / kg, and the administration volume was 3 ml / kg. The day of group administration was defined as Day 0. Blood was collected 7 days, 14 days, 21 days, 28 days, 35 days, 42 days and 49 days after the first administration. After separating the plasma, the AGT level in the serum was determined by the Elisa method. The test results are shown in Tables 24 and Figure 8 .

[0761] Table 24 Average AGT levels in cynomolgus monkey serum

[0762]

[0763] The experimental results showed that the combination of the sense chain 5'MVIP and the antisense chain 3'MVIP 5'MVIP01 / 3'MVIP01, 5'MVIP01 / 3'MVIP17 or 5'MVIP09 / 3'MVIP09 or the combination of the sense chain 5'MVIP and the sense chain 3'MVIP 5'MVIP01 / 3'MVIP09 or 5'MVIP09 / 3'MVIP01 all showed significant and sustained effects in inhibiting the AGT level in crab-eating monkeys. Sequence Listing <110> Xiamen Ganbaoli Biopharmaceutical Co., Ltd. <120> AGT inhibitors and their uses <130> 0249-PA-004 <160> 260 <170> PatentIn version 3.5 <210> 1 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S1 <400> 1 gcttgtttgt gaaacaaaaa a 21 <210> 2 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S2 <400> 2 cccttgtgtt agtaataaac g 21 <210> 3 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S3 <400> 3 gttttaaaat taaagtatac a <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S4 <400> 4 gggttttaaa attack <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S5 <400> 5 gcttgtgatt tttgaacaat a <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S6 <400> 6 aattgggttt seat g <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S7 <400> 7 ggttttaaaa ttaagtata c <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S8 <400> 8 gaacaaaaat tgggttttaa a 21 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S9 <400> 9 ggtgctagtc gctgcaaaac t 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S10 <400> 10 gcattttttt tgagcttgaa g 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S11 <400> 11 attgggtttt aaaattaaag t 21 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S12 <400> 12 gatgcttgtg atttttgaac a 21 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S13 <400> 13 gagtctaccc aacagcttaa c 21 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S14 <400> 14 cgaccagctt gtttgtgaaa c 21 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S15 <400> 15 ggagtgacat ccaggacaac t 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S16 <400> 16 ccgtgtagtg tctgtaatac c 21 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S17 <400> 17 agcttgtttg tgaaacaaaa a 21 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S18 <400> 18 cgggactact gttccaaaaa g 21 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS1 <220> <221> n represents a, u, g or c <222> (21)..(21) <220> <221> misc_feature <222> (21)..(21) <223> n is a, c, g, t or u <400> 19 uuuuguuuca caaacaagcu n 21 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS2 <220> <221> n represents a, u, g or c <222> (21)..(21) <220> <221> misc_feature <222> (21)..(21) <223> n is a, c, g, t or u <400> 20 uuuauuacua acacaaggga n 21 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS3 <220> <221> n represents a, u, g or c <222> (21)..(21) <220> <221> misc_feature <222> (21)..(21) <223> n is a, c, g, t or u <400> 21 uauacuuuaa uuuuaaaacc n 21 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS4 <220> <221> n represents a, u, g or c <222> (21)..(21) <220> <221> misc_feature <222> (21)..(21) <223> n is a, c, g, t or u <400> 22 uacuuuaauu uuaaaaccca n 21 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS5 <220> <221> n represents a, u, g or c <222> (21)..(21) <220> <221> misc_feature <222> (21)..(21) <223> n is a, c, g, t or u <400> 23 uuguucaaaa aucacaagca n 21 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS6 <220> <221> n represents a, u, g or c <222> (21)..(21) <220> <221> misc_feature <222> (21)..(21) <223> n is a, c, g, t or u <400> 24 uuaauuuuaa aacccaauuu n 21 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS7 <220> <221> n represents a, u, g or c <222> (21)..(21) <220> <221> misc_feature <222> (21)..(21) <223> n is a, c, g, t or u <400> 25 auacuuuaau uuuaaaaccc n 21 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS8 <220> <221> n represents a, u, g or c <222> (21)..(21) <220> <221> misc_feature <222> (21)..(21) <223> n is a, c, g, t or u <400> 26 uaaaacccaa uuuuuguucu n 21 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS9 <220> <221> n represents a, u, g or c <222> (21)..(21) <220> <221> misc_feature <222> (21)..(21) <223> n is a, c, g, t or u <400> 27 uuuugcagcg acuagcacca n 21 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS10 <220> <221> n represents a, u, g or c <222> (21)..(21) <220> <221> misc_feature <222> (21)..(21) <223> n is a, c, g, t or u <400> 28 ucaagcucaa aaaaaaugcu n 21 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS11 <220> <221> n represents a, u, g or c <222> (21)..(21) <220> <221> misc_feature <222> (21)..(21) <223> n is a, c, g, t or u <400> 29 uuuaauuuua aaacccaauu n 21 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS12 <220> <221> n represents a, u, g or c <222> (21)..(21) <220> <221> misc_feature <222> (21)..(21) <223> n is a, c, g, t or u <400> 30 uucaaaaauc acaagcaucu n 21 <210> 31 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS13 <220> <221> n represents a, u, g or c <222> (21)..(21) <220> <221> misc_feature <222> (21)..(21) <223> n is a, c, g, t or u <400> 31 uaagcuguug gguagacucu n 21 <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS14 <220> <221> n represents a, u, g or c <222> (21)..(21) <220> <221> misc_feature <222> (21)..(21) <223> n is a, c, g, t or u <400> 32 uucacaaaca agcuggucgg n 21 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS15 <220> <221> n represents a, u, g or c <222> (21)..(21) <220> <221> misc_feature <222> (21)..(21) <223> n is a, c, g, t or u <400> 33 uuguccugga ugucacucca n 21 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS16 <220> <221> n represents a, u, g or c <222> (21)..(21) <220> <221> misc_feature <222> (21)..(21) <223> n is a, c, g, t or u <400> 34 uauuacagac acuacacgga n 21 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS17 <220> <221> n represents a, u, g or c <222> (21)..(21) <220> <221> misc_feature <222> (21)..(21) <223> n is a, c, g, t or u <400> 35 uuuguuucac aaacaagcug n 21 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS18 <220> <221> n represents a, u, g or c <222> (21)..(21) <220> <221> misc_feature <222> (21)..(21) <223> n is a, c, g, t or u <400> 36 uuuuggaaca guagucccgc n 21 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S19 <400> 37 gcuuguuugu gaaacaaaaa a 21 <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S20 <400> 38 cccuuguguu aguaauaaac g 21 <210> 39 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S21 <400> 39 guuuuaaaau uaaaguauac a 21 <210> 40 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S22 <400> 40 ggguuuuaaa auuaaaguau a 21 <210> 41 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S23 <400> 41 gcuugugauu uuugaacaau a 21 <210> 42 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S24 <400> 42 aauuggguuu uaaaauuaaa g 21 <210> 43 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S25 <400> 43 gguuuuaaaa uuaaaguaua c 21 <210> 44 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S26 <400> 44 gaacaaaaau uggguuuuaa a 21 <210> 45 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S27 <400> 45 ggugcuaguc gcugcaaaac u 21 <210> 46 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S28 <400> 46 gcauuuuuuu ugagcuugaa g 21 <210> 47 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S29 <400> 47 auuggguuuu aaaauuaaag u 21 <210> 48 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S30 <400> 48 gaugcuugug auuuuugaac a 21 <210> 49 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S31 <400> 49 gagucuaccc aacagcuuaa c 21 <210> 50 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S32 <400> 50 cgaccagcuu guuugugaaa c 21 <210> 51 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S33 <400> 51 ggagugacau ccaggacaac u 21 <210> 52 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S34 <400> 52 ccguguagug ucuguaauac c 21 <210> 53 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S35 <400> 53 agcuuguuug ugaaacaaaa a 21 <210> 54 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> S36 <400> 54 cgggacuacu guuccaaaaa g 21 <210> 55 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS19 <400> 55 uuuuguuuca caaacaagcu g 21 <210> 56 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS20 <400> 56 uuuauuacua acacaaggga g 21 <210> 57 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS21 <400> 57 uauacuuuaa uuuuaaaacc c 21 <210> 58 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS22 <400> 58 uacuuuaauu uuaaaaccca a 21 <210> 59 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS23 <400> 59 uuguucaaaa aucacaagca u 21 <210> 60 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS24 <400> 60 uuaauuuuaa aacccaauuu u 21 <210> 61 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS25 <400> 61 auacuuuaau uuuaaaaccc a 21 <210> 62 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS26 <400> 62 uaaaacccaa uuuuuguucu c 21 <210> 63 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS27 <400> 63 uuuugcagcg acuagcacca g 21 <210> 64 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS28 <400> 64 ucaagcucaa aaaaaaugcu g 21 <210> 65 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS29 <400> 65 uuuaauuuua aaacccaauu u 21 <210> 66 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS30 <400> 66 uucaaaaauc acaagcaucu g 21 <210> 67 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS31 <400> 67 uaagcuguug gguagacucu g 21 <210> 68 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS32 <400> 68 uucacaaaca agcuggucgg u 21 <210> 69 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS33 <400> 69 uuguccugga ugucacucca g 21 <210> 70 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS34 <400> 70 uauuacagac acuacacgga g 21 <210> 71 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS35 <400> 71 uuuguuucac aaacaagcug g 21 <210> 72 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS36 <400> 72 uuuuggaaca guagucccgc g 21 <210> 73 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S37 <220> <221> misc_feature <222> (1)..(21) <223> gRepresentative 2'-Kiji-based Acid, aRepresentative 2'-Kei-based Adenanoic Acid,c Representative 2'-Kei-based Adenanoic Acid <400> 73 gcuuguugu gaaacaaaa a 21 <210> 74 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S38 <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, and a stands for 2'-methoxyadenylic acid <400> 74 cccuuguguu aguaauaaac g 21 <210> 75 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S39 <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, and a stands for 2'-methoxyadenylic acid <400> 75 guuuuaaaau uaaaguauac a 21 <210> 76 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S40 <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, and a stands for 2'-methoxyadenylic acid <400> 76 ggguuuuaaa auuaaaguau a 21 <210> 77 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S41 <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanylic acid, a stands for 2'-methoxyadenylic acid, and u stands for 2'-methoxyuridylic acid <400> 77 gcuugugauu uuugaacaau a 21 <210> 78 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S42 <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, a stands for 2'-methoxyadenylic acid <400> 78 aauuggguuu uaaaauuaaa g 21 <210> 79 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S43 <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, a stands for 2'-methoxyadenylic acid <400> 79 gguuuuaaaa uuaaaguaua c 21 <210> 80 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S44 <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, a stands for 2'-methoxyadenylic acid <400> 80 gaacaaaaau uggguuuuaa a 21 <210> 81 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S45 <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, a stands for 2'-methoxyadenylic acid <400> 81 ggugcuaguc gcugcaaaac u 21 <210> 82 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S46 <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, a stands for 2'-methoxyadenylic acid <400> 82 gcauuuuuuuu ugagcuugaa g 21 <210> 83 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S47 <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, a stands for 2'-methoxyadenylic acid <400> 83 auuggguuuu aaaauuaaag u 21 <210> 84 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S48 <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, a stands for 2'-methoxyadenylic acid <400> 84 gaugcuugug auuuuugaac a 21 <210> 85 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S49 <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, a stands for 2'-methoxyadenylic acid <400> 85 gagucuaccc aacagcuuaa c 21 <210> 86 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S50 <220> <221> misc_feature <222> (1)..(21) <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, a stands for 2'-methoxyadenylic acid <400> 86 cgaccagcuu guuugugaaa c 21 <210> 87 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S51 <220> <221> misc_feature <222> (1)..(21) <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, a stands for 2'-methoxyadenylic acid <400> 87 ggagugacau ccaggacaac u 21 <210> 88 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S52 <220> <221> misc_feature <222> (1)..(21) <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, a stands for 2'-methoxyadenylic acid <400> 88 ccguguagug ucuguaauac c 21 <210> 89 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S53 <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, a stands for 2'-methoxyadenylic acid <400> 89 agcuuguuug ugaaacaaaa a 21 <210> 90 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S54 <220> <221> misc_feature <222> (1)..(21) <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, a stands for 2'-methoxyadenylic acid <400> 90 cgggacuacu guuccaaaaa g 21 <210> 91 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS37 <220> <221> misc_feature <222> (1)..(3) <223> u represents 2'-methoxyuridylic acid <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, and a stands for 2'-methoxyadenylic acid <220> <221> misc_feature <222> (6)..(8) <223> u represents 2'-methoxyuridylic acid <220> <221> misc_feature <222> (19)..(19) <220> <221> misc_feature <222> (19)..(19) <223> c represents 2'-methoxycytidylic acid <400> 91 uuuuguuuca caaacaagcu g 21 <210> 92 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS38 <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, a stands for 2'-methoxyadenylic acid <220> <221> misc_feature <222> (1)..(2) <223> u represents 2'-methoxyuridylic acid <220> <221> misc_feature <222> (5) <223> u represents 2'-methoxyuridylic acid <220> <221> misc_feature <222> (8) <223> c represents 2'-methoxycytidylic acid <220> <221> misc_feature <222> (14)..(14) <223> c represents 2'-methoxycytidylic acid <400> 92 uuuauuacua acacaaggga g 21 <210> 93 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS39 <220> <221> misc_feature <222> (1)..(21) <223> a represents 2'-methoxyadenosine, c represents 2'-methoxycytidine <220> <221> misc_feature <222> (4)..(21) <223> u represents 2'-methoxyuridylic acid <400> 93 uauacuuuaa uuuuaaaacc c 21 <210> 94 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS40 <220> <221> misc_feature <222> (1)..(21) <223> a represents 2'-methoxyadenosine, c represents 2'-methoxycytidine <220> <221> misc_feature <222> (4)..(5) <223> u represents 2'-methoxyuridylic acid <220> <221> misc_feature <222> (9)..(11) <223> u represents 2'-methoxyuridylic acid <400> 94 uacuuuaauu uuaaaaccca a 21 <210> 95 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS41 <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, a stands for 2'-methoxyadenylic acid <220> <221> misc_feature <222> (1) <223> u represents 2'-methoxyuridylic acid <220> <221> misc_feature <222> (4)..(21) <223> u represents 2'-methoxyuridylic acid <400> 95 uuguucaaaa aucacaagca u 21 <210> 96 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS42 <220> <221> misc_feature <222> (1)..(21) <223> a represents 2'-methoxyadenosine <220> <221> misc_feature <222> (1) <223> u represents 2'-methoxyuridylic acid <220> <221> misc_feature <222> (5)..(7) <223> u represents 2'-methoxyuridylic acid <220> <221> misc_feature <222> (18)..(21) <223> u represents 2'-methoxyuridylic acid <400> 96 uuaauuuuaa aacccaauuu u 21 <210> 97 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS43 <220> <221> misc_feature <222> (1)..(21) <223> a represents 2'-methoxyadenosine, c represents 2'-methoxycytidine <220> <221> misc_feature <222> (10)..(12) <223> u represents 2'-methoxyuridylic acid <400> 97 auacuuuaau uuuaaaaccc a 21 <210> 98 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS44 <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, a stands for 2'-methoxyadenylic acid <220> <221> misc_feature <222> (6)..(7) <223> c represents 2'-methoxycytidylic acid <220> <221> misc_feature <222> (11)..(14) <223> u represents 2'-methoxyuridylic acid <220> <221> misc_feature <222> (17)..(21) <223> u represents 2'-methoxyuridylic acid <220> <221> misc_feature <222> (19)..(21) <223> c represents 2'-methoxycytidylic acid <400> 98 uaaaacccaa uuuuuguucu c 21 <210> 99 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS45 <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, a stands for 2'-methoxyadenylic acid <220> <221> misc_feature <222> (1)..(3) <223> u represents 2'-methoxyuridylic acid <220> <221> misc_feature <222> (12)..(12) <223> c represents 2'-methoxycytidylic acid <220> <221> misc_feature <222> (18) <223> c represents 2'-methoxycytidylic acid <400> 99 uuuugcagcg acuagcacca g 21 <210> 100 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS46 <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, a stands for 2'-methoxyadenylic acid <220> <221> misc_feature <222> (1) <223> u represents 2'-methoxyuridylic acid <220> <221> misc_feature <222> (6) <223> c represents 2'-methoxycytidylic acid <220> <221> misc_feature <222> (7) <223> u represents 2'-methoxyuridylic acid <220> <221> misc_feature <222> (19)..(19) <223> c represents 2'-methoxycytidylic acid <400> 100 ucaagcucaa aaaaaaugcu g 21 <210> 101 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS47 <220> <221> misc_feature <222> (1)..(21) <223> a represents 2'-methoxyadenosine <220> <221> misc_feature <222> (1)..(2) <223> u represents 2'-methoxyuridylic acid <220> <221> misc_feature <222> (6)..(8) <223> u represents 2'-methoxyuridylic acid <220> <221> misc_feature <222> (14)..(15) <223> c represents 2'-methoxycytidylic acid <220> <221> misc_feature <222> (19)..(21) <223> u represents 2'-methoxyuridylic acid <400> 101 uuuaauuuua aaacccaauu u 21 <210> 102 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS48 <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, a stands for 2'-methoxyadenylic acid <220> <221> misc_feature <222> (1)..(2) <223> u represents 2'-methoxyuridylic acid <220> <221> misc_feature <222> (10)..(10) <223> c represents 2'-methoxycytidylic acid <220> <221> misc_feature <222> (18) <223> u represents 2'-methoxyuridylic acid <400> 102 uucaaaaauc acaagcaucu g 21 <210> 103 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS49 <220> <221> misc_feature <222> (1)..(21) <223> g represents 2'-methoxyguanosine monophosphate, a represents 2'-methoxyadenosine, u represents 2'-methoxyuridylic acid, and c represents 2'-methoxycytidylic acid <400> 103 uaagcuguug gguagacucu g 21 <210> 104 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS50 <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, a stands for 2'-methoxyadenylic acid, and u stands for 2'-methoxyuridylic acid <400> 104 uucacaaaca agcuggucgg u 21 <210> 105 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS51 <220> <221> misc_feature <222> (1)..(21) <223> g represents 2'-methoxyguanosine monophosphate, a represents 2'-methoxyadenylic acid, and c represents 2'-methoxycytidylic acid <220> <221> misc_feature <222> (1) <223> u represents 2'-methoxyuridylic acid <220> <221> misc_feature <222> (4) <223> u represents 2'-methoxyuridylic acid <220> <221> misc_feature <222> (13)..(17) <223> u represents 2'-methoxyuridylic acid <400> 105 uuguccugga ugucacucca g 21 <210> 106 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS52 <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, a stands for 2'-methoxyadenylic acid <220> <221> misc_feature <222> (3) <223> u represents 2'-methoxyuridylic acid <220> <221> misc_feature <222> (10)..(12) <223> c represents 2'-methoxycytidylic acid <220> <221> misc_feature <222> (17) <223> c represents 2'-methoxycytidylic acid <400> 106 uauuacagac acuacacgga g 21 <210> 107 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS53 <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, a stands for 2'-methoxyadenylic acid <220> <221> misc_feature <222> (1)..(2) <223> u represents 2'-methoxyuridylic acid <220> <221> misc_feature <222> (5)..(7) <223> u represents 2'-methoxyuridylic acid <220> <221> misc_feature <222> (14)..(18) <223> c represents 2'-methoxycytidylic acid <400> 107 uuuguuucac aaacaagcug g 21 <210> 108 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS54 <220> <221> misc_feature <222> (1)..(21) <223> g stands for 2'-methoxyguanosine monophosphate, a stands for 2'-methoxyadenylic acid <220> <221> misc_feature <222> (1)..(3) <223> u represents 2'-methoxyuridylic acid <220> <221> misc_feature <222> (15) <223> u represents 2'-methoxyuridylic acid <220> <221> misc_feature <222> (16)..(20) <223> c represents 2'-methoxycytidylic acid <400> 108 uuuuggaaca guagucccgc g 21 <210> 109 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS55 <220> <221> misc_feature <222> (1)..(21) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 109 uuuuguuuca caaacaagcu g 21 <210> 110 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS56 <220> <221> misc_feature <222> (1)..(21) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 110 uuuauuacua acacaaggga g 21 <210> 111 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS57 <220> <221> misc_feature <222> (1)..(21) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 111 uauacuuuaa uuuuaaaacc c 21 <210> 112 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS58 <220> <221> misc_feature <222> (1)..(21) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 112 uacuuuaauu uuaaaaccca a 21 <210> 113 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS59 <220> <221> misc_feature <222> (1)..(21) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 113 uuguucaaaa aucacaagca u 21 <210> 114 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS60 <220> <221> misc_feature <222> (1)..(21) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 114 uuaauuuuaa aacccaauuu u 21 <210> 115 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS61 <220> <221> misc_feature <222> (1)..(21) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 115 auacuuuaau uuuaaaaccc a 21 <210> 116 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS62 <220> <221> misc_feature <222> (1)..(21) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 116 uaaaacccaa uuuuuguucu c 21 <210> 117 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS63 <220> <221> misc_feature <222> (1)..(21) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 117 uuuugcagcg acuagcacca g 21 <210> 118 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS64 <220> <221> misc_feature <222> (1)..(21) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 118 ucaagcucaa aaaaaaugcu g 21 <210> 119 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS65 <220> <221> misc_feature <222> (1)..(21) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 119 uuuaauuuua aaacccaauu u 21 <210> 120 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS66 <220> <221> misc_feature <222> (1)..(21) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 120 uucaaaaauc acaagcaucu g 21 <210> 121 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS67 <220> <221> misc_feature <222> (1)..(21) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 121 uaagcuguug gguagacucu g 21 <210> 122 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS68 <220> <221> misc_feature <222> (1)..(21) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 122 uucacaaaca agcuggucgg u 21 <210> 123 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS69 <220> <221> misc_feature <222> (1)..(21) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 123 uuguccugga ugucacucca g 21 <210> 124 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS70 <220> <221> misc_feature <222> (1)..(21) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 124 uauuacagac acuacacgga g 21 <210> 125 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS71 <220> <221> misc_feature <222> (1)..(21) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 125 uuuguuucac aaacaagcug g 21 <210> 126 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS72 <220> <221> misc_feature <222> (1)..(21) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 126 uuuuggaaca guagucccgc g 21 <210> 127 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS73 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 127 aagaaguugg ccagcaucc 19 <210> 128 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS74 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 128 uauacggaag cccaagaag 19 <210> 129 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS75 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 129 cugugcaugc cauauauac 19 <210> 130 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS76 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 130 cauggaccac gccccauag 19 <210> 131 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS77 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 131 aaagacagcc guuggggag 19 <210> 132 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS78 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 132 ucuuguccac ccagaacuc 19 <210> 133 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS79 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 133 agacccucca ccuugucca 19 <210> 134 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS80 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 134 agugagaccc uccaccuug 19 <210> 135 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS81 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 135 aaagugagac ccuccaccu 19 <210> 136 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS82 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 136 guugagggag uuuugcugg 19 <210> 137 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS83 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 137 aguugaggga guuuugcug 19 <210> 138 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS84 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 138 uccaguugag ggaguuuug 19 <210> 139 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS85 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 139 ucuucaucca guugaggga 19 <210> 140 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS86 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 140 uucuucaucc aguugaggg 19 <210> 141 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS87 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 141 aguuucuuca uccaguuga 19 <210> 142 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS88 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 142 uugcucaauu uuugcaggu 19 <210> 143 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS89 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 143 cauugcucaa uuuuugcag 19 <210> 144 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS90 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 144 ucauugcuca auuuuugca 19 <210> 145 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS91 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 145 gucauugcuc aauuuuugc 19 <210> 146 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS92 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 146 ugugggcucu cucucaucc 19 <210> 147 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS93 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 147 uugaucauac acagcaaac 19 <210> 148 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS94 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 148 uuugaucaua cacagcaaa 19 <210> 149 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS95 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 149 aaagguggga gacuggggg 19 <210> 150 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS96 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 150 cauuagaaga aaagguggg 19 <210> 151 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS97 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 151 ucauuagaag aaaaggugg 19 <210> 152 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS98 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 152 cucauuagaa gaaaaggug 19 <210> 153 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS99 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 153 ucgguuggaa uucuuuuug 19 <210> 154 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS100 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 154 aaacaagcug gucgguugg 19 <210> 155 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS101 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 155 ucacaaacaa gcuggucgg 19 <210> 156 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS102 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 156 uucacaaaca agcuggucg 19 <210> 157 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS103 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 157 uuucacaaac aagcugguc 19 <210> 158 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS104 <220> <221> misc_feature <222> (1)..(19) <223> From the 5' end, the 7th, 12th, and 14th nucleotides have a fluorine 2' position and the remaining nucleotides have a fluorine 2' position. The phosphate bonds between the three adjacent nucleotides at the 5' and 3' ends are thiolated. <400> 158 uuuuguuuca caaacaagc 19 <210> 159 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS105 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 159 uuuuuguuuc acaaacaag 19 <210> 160 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS106 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 160 uuuuuuuguuu cacaaacaa 19 <210> 161 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS107 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 161 acuuuuuuugu uucacaaac 19 <210> 162 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS108 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 162 acacuuuuuu guuucacaa 19 <210> 163 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS109 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 163 aaaagggaac acuuuuuug 19 <210> 164 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS110 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 164 ucucaacuug aaaagggaa 19 <210> 165 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS111 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 165 uguucucaac uugaaaagg 19 <210> 166 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS112 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 166 aacccaauuu uuguucuca 19 <210> 167 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS113 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 167 uaaaacccaa uuuuuguuc 19 <210> 168 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS114 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 168 uuuuaaaacc caauuuuug 19 <210> 169 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS115 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 169 auucaagaca cuaaauaca 19 <210> 170 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS116 <220> <221> misc_feature <222> (1)..(19) <223> From the 5' end, the 7th, 12th, and 14th nucleotides have a fluorine 2' position and the remaining nucleotides have a fluorine 2' position. The phosphate bonds between the three adjacent nucleotides at the 5' and 3' ends are thiolated. <400> 170 ucuuacauuc aagacacua 19 <210> 171 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS117 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 171 ucauguucuu acauucaag 19 <210> 172 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS118 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 172 gucauguucu uacauucaa 19 <210> 173 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS119 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 173 aucuguggaa aaaacuaag 19 <210> 174 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS120 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 174 aaaucacaag caucugugg 19 <210> 175 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AS121 <220> <221> misc_feature <222> (1)..(19) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 175 aaaaaucaca agcaucugu 19 <210> 176 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AS122 <220> <221> misc_feature <222> (1)..(20) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 176 cggacaaauc agcgaugugu 20 <210> 177 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AS123 <220> <221> misc_feature <222> (1)..(20) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 177 ccaaaaagaa uuccaauuga 20 <210> 178 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AS124 <220> <221> misc_feature <222> (1)..(20) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 178 accgaccagc uuguuuguga 20 <210> 179 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AS125 <220> <221> misc_feature <222> (1)..(20) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 179 agcgcgggac uacuguucca 20 <210> 180 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AS126 <220> <221> misc_feature <222> (1)..(20) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 180 gcgcgggacu acuguuccaa 20 <210> 181 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AS127 <220> <221> misc_feature <222> (1)..(20) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 181 aaccgaccag cuuguuugug 20 <210> 182 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AS128 <220> <221> misc_feature <222> (1)..(20) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 182 uguucccuuu ucaaguugag 20 <210> 183 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AS129 <220> <221> misc_feature <222> (1)..(20) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 183 ucccuuuuca aguugagaac 20 <210> 184 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> AS130 <220> <221> misc_feature <222> (1)..(21) <223> The 2' position of the 7th, 12th, and 14th nucleotides from the 5' end is fluorine and the 2' position of the remaining nucleotides is methoxy, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 184 uauacucuca uuguggauga c 21 <210> 185 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S55 <220> <221> misc_feature <222> (1)..(21) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 185 gcuuguuugu gaaacaaaaa a 21 <210> 186 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S56 <220> <221> misc_feature <222> (1)..(21) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 186 cccuuguguu aguaauaaac g 21 <210> 187 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S57 <220> <221> misc_feature <222> (1)..(21) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 187 guuuuaaaau uaaaguauac a 21 <210> 188 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> 58 <220> <221> misc_feature <222> (1)..(21) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 188 ggguuuuaaa auuaaaguau a 21 <210> 189 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S59 <220> <221> misc_feature <222> (1)..(21) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 189 gcuugugauu uuugaacaau a 21 <210> 190 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S60 <220> <221> misc_feature <222> (1)..(21) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 190 aauuggguuu uaaaauuaaa g 21 <210> 191 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S61 <220> <221> misc_feature <222> (1)..(21) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 191 gguuuuaaaa uuaaaguaua c 21 <210> 192 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S62 <220> <221> misc_feature <222> (1)..(21) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 192 gaacaaaaau uggguuuuaa a 21 <210> 193 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S63 <220> <221> misc_feature <222> (1)..(21) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 193 ggugcuaguc gcugcaaaac u 21 <210> 194 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S64 <220> <221> misc_feature <222> (1)..(21) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 194 gcauuuuuuuu ugagcuugaa g 21 <210> 195 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S65 <220> <221> misc_feature <222> (1)..(21) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 195 auuggguuuu aaaauuaaag u 21 <210> 196 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S66 <220> <221> misc_feature <222> (1)..(21) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 196 gaugcuugug auuuuugaac a 21 <210> 197 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S67 <220> <221> misc_feature <222> (1)..(21) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 197 gagucuaccc aacagcuuaa c 21 <210> 198 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S68 <220> <221> misc_feature <222> (1)..(21) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 198 cgaccagcuu guuugugaaa c 21 <210> 199 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S69 <220> <221> misc_feature <222> (1)..(21) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 199 ggagugacau ccaggacaac u 21 <210> 200 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S70 <220> <221> misc_feature <222> (1)..(21) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 200 ccguguagug ucuguaauac c 21 <210> 201 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S71 <220> <221> misc_feature <222> (1)..(21) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 201 agcuuguuug ugaaacaaaa a 21 <210> 202 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S72 <220> <221> misc_feature <222> (1)..(21) <223> From the 5' end, the 5th, 7th, 8th, and 9th nucleotides have a fluorine 2' position and the remaining nucleotides have a fluorine 2' position. The phosphate bonds between the three adjacent nucleotides at the 5' and 3' ends are thiolated. <400> 202 cgggacuacu guuccaaaaa g 21 <210> 203 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S73 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 203 ggaugcuggc caacuucuu 19 <210> 204 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S74 <220> <221> misc_feature <222> (1)..(19) <223> From the 5' end, the 5th, 7th, 8th, and 9th nucleotides have a fluorine 2' position and the remaining nucleotides have a fluorine 2' position. The phosphate bonds between the three adjacent nucleotides at the 5' and 3' ends are thiolated. <400> 204 cuucuugggc uuccguaua 19 <210> 205 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S75 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 205 guauauaugg caugcacag 19 <210> 206 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S76 <220> <221> misc_feature <222> (1)..(19) <223> From the 5' end, the 5th, 7th, 8th, and 9th nucleotides have a fluorine 2' position and the remaining nucleotides have a fluorine 2' position. The phosphate bonds between the three adjacent nucleotides at the 5' and 3' ends are thiolated. <400> 206 cuauggggcg ugguccaug 19 <210> 207 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S77 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 207 cuccccaacg gcugucuuu 19 <210> 208 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S78 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 208 gcugugacag gauggaaga 19 <210> 209 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S79 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 209 uggacaaggu ggagggucu 19 <210> 210 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S80 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 210 caagguggag ggucucacu 19 <210> 211 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S81 <220> <221> misc_feature <222> (1)..(19) <223> From the 5' end, the 5th, 7th, 8th, and 9th nucleotides have a fluorine 2' position and the remaining nucleotides have a fluorine 2' position. The phosphate bonds between the three adjacent nucleotides at the 5' and 3' ends are thiolated. <400> 211 agguggaggg ucucacuuu 19 <210> 212 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S82 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 212 ccagcaaaac ucccucaac 19 <210> 213 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S83 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 213 cagcaaaacu cccucaacu 19 <210> 214 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S84 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 214 caaaacuccc ucaacugga 19 <210> 215 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S85 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 215 ucccucaacu ggaugaaga 19 <210> 216 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S86 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 216 cccucaacug gaugaagaa 19 <210> 217 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S87 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 217 ucaacuggau gaagaaacu 19 <210> 218 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S88 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 218 accugcaaaa auugagcaa 19 <210> 219 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S89 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 219 cugcaaaaau ugagcaaug 19 <210> 220 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S90 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 220 ugcaaaaauu gagcaauga 19 <210> 221 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S91 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 221 gcaaaaauug agcaaugac 19 <210> 222 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S92 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 222 ggaugagaga gagcccaca 19 <210> 223 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S93 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 223 guuugcugug uaugaucaa 19 <210> 224 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S94 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 224 uuugcugugu augaucaaa 19 <210> 225 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S95 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 225 cccccagucu cccaccuuu 19 <210> 226 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S96 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 226 cccaccuuuu cuucuaaug 19 <210> 227 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S97 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 227 ccaccuuuuc uucuaauga 19 <210> 228 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S98 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 228 caccuuuucu ucuaaugag 19 <210> 229 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S99 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 229 caaaaagaau uccaaccga 19 <210> 230 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S100 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 230 ccaaccgacc agcuuguuu 19 <210> 231 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S101 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 231 ccgaccagcu uguuuguga 19 <210> 232 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S102 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 232 cgaccagcuu guuugugaa 19 <210> 233 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S103 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 233 gaccagcuug uuugugaaa 19 <210> 234 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S104 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 234 gcuuguuugu gaaacaaaa 19 <210> 235 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S105 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 235 cuuguuugug aaacaaaaa 19 <210> 236 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S106 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 236 uuguuuguga aacaaaaaa 19 <210> 237 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S107 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 237 guuugugaaa caaaaaagu 19 <210> 238 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S108 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 238 uugugaaaca aaaaagugu 19 <210> 239 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S109 <220> <221> misc_feature <222> (1)..(19) <223> From the 5' end, the 5th, 7th, 8th, and 9th nucleotides have a fluorine 2' position and the remaining nucleotides have a fluorine 2' position. The phosphate bonds between the three adjacent nucleotides at the 5' and 3' ends are thiolated. <400> 239 caaaaaagug uucccuuuu 19 <210> 240 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S110 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 240 uucccuuuuc aaguugaga 19 <210> 241 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S111 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 241 ccuuuucaag uugagaaca 19 <210> 242 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S112 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 242 ugagaacaaa aauuggguu 19 <210> 243 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S113 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 243 gaacaaaaau uggguuuua 19 <210> 244 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S114 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 244 caaaaauugg guuuuaaaa 19 <210> 245 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S115 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 245 uguauuuagu gucuugaau 19 <210> 246 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S116 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 246 uagugucuug aauguaaga 19 <210> 247 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S117 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 247 cuugaaugua agaacauga 19 <210> 248 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S118 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 248 uugaauguaa gaacaugac 19 <210> 249 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S119 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 249 cuuaguuuuu uccacagau 19 <210> 250 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S120 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 250 ccacagaugc uugugauuu 19 <210> 251 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> S121 <220> <221> misc_feature <222> (1)..(19) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 251 acagaugcuu gugauuuuu 19 <210> 252 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> S122 <220> <221> misc_feature <222> (1)..(20) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 252 acacaucgcu gauuuguccg 20 <210> 253 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> S123 <220> <221> misc_feature <222> (1)..(20) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 253 ucgguuggaa uucuuuuugg 20 <210> 254 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> S124 <220> <221> misc_feature <222> (1)..(20) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 254 ucacaaacaa gcuggucggu 20 <210> 255 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> S125 <220> <221> misc_feature <222> (1)..(20) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 255 uggaacagua gucccgcgcu 20 <210> 256 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> S126 <220> <221> misc_feature <222> (1)..(20) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 256 uuggaacagu agucccgcgc 20 <210> 257 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> S127 <220> <221> misc_feature <222> (1)..(20) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 257 cacaaacaag cuggucgguu 20 <210> 258 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> S128 <220> <221> misc_feature <222> (1)..(20) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine 2' position and the remaining nucleotides have a methoxy 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 258 cucaacuuga aaagggaaca 20 <210> 259 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> S129 <220> <221> misc_feature <222> (1)..(20) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 259 guucucaacu ugaaaaggga 20 <210> 260 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> S130 <220> <221> misc_feature <222> (1)..(21) <223> The 5th, 7th, 8th, and 9th nucleotides from the 5' end have a fluorine at the 2' position and the remaining nucleotides have a methoxy at the 2' position, and the phosphate bonds between the 3 adjacent nucleotides at the 5' end and the 3' end are thiolated. <400> 260 gucauccaca augagaguac a 21

Claims

1. An RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof, wherein: The RNAi agent is formed by base pairing of a sense strand and an antisense strand with a length of 21-23 nucleotides, and the antisense strand comprises SEQ ID NO.64, and the sense strand comprises SEQ ID NO.

46.

2. The RNAi agent for inhibiting AGT gene expression according to claim 1 or a pharmaceutically acceptable salt thereof, wherein: The RNAi agent is siRNA.

3. The RNAi agent for inhibiting AGT gene expression according to claim 2 or a pharmaceutically acceptable salt thereof, wherein: The antisense strand comprises SEQ ID NO.118, wherein SEQ ID NO.118 is UsCsAAGCfUCAAAfAAfAAAUGCsUsG, wherein G=2'-O-methylguanylate, A=2'-O-methyladenylate, U=2'-O-methyluridylate, C=2'-O-methylcytidine; fA=2'-fluoroadenylate, fU=2'-fluorouridylate; Us=2'-O-methyl-3'-thiouridine, Cs=2'-O-methyl-3'-thiocytidine.

4. The RNAi agent for inhibiting AGT gene expression according to claim 3 or a pharmaceutically acceptable salt thereof, wherein: The positive strand comprises SEQ ID NO.194, wherein SEQ ID NO.194 is GsCsAUfUUfUfUfUUUGAGCUUGAsAsG, wherein G=2'-O-methylguanylate, A=2'-O-methyladenylate, U=2'-O-methyluridylate, C=2'-O-methylcytidine; fU=2'-fluorouridylate; Gs=2'-O-methyl-3'-thioguanylate, As=2'-O-methyl-3'-thioadenosine, Cs=2'-O-methyl-3'-thiocytidine.

5. The RNAi agent for inhibiting AGT gene expression according to claim 4 or a pharmaceutically acceptable salt thereof, wherein: The RNAi agent is selected from the following sense strand and antisense strand combinations:

6. The RNAi agent for inhibiting AGT gene expression according to claim 5 or a pharmaceutically acceptable salt thereof, wherein: The RNAi agent further comprises a carrier structure, the carrier structure is coupled to the sense strand and / or the antisense strand, and the carrier structure comprises a ligand.

7. The RNAi agent for inhibiting AGT gene expression according to claim 6 or a pharmaceutically acceptable salt thereof, wherein: The carrier structure includes 5'MVIP and 3'MVIP, wherein the 5'MVIP is coupled to the 5' end of the sense strand and / or antisense strand, and the 3'MVIP is coupled to the 3' end of the antisense strand and / or sense strand. The structure of the 5'MVIP is shown in Formula I, and the structure of the 3'MVIP is shown in Formula II. (X-L) n -B-D-R1-, (X-L) m -B-D-R2-, II Wherein, the 5'MVIP is selected from any one of 5'MVIP01 to 5'MVIP07, 5'MVIP09 to 5'MVIP15, and 5'MVIP17 to 5'MVIP22 as shown below: Among them, 5'MVIP (XL) n The -BD- structure is as follows: The 3'MVIP is selected from any one of 3'MVIP01 to 3'MVIP07, 3'MVIP09 to 3'MVIP15, 3'MVIP17 to 3'MVIP27 as shown below: Wherein, the 3'MVIP (XL) m -BD- has the following structure:

8. The RNAi agent for inhibiting AGT gene expression according to claim 7 or a pharmaceutically acceptable salt thereof, wherein: The combination of the sense strand 5'MVIP and the antisense strand 3'MVIP is 5'MVIP01 / 3'MVIP01, 5'MVIP01 / 3'MVIP17 or 5'MVIP09 / 3'MVIP09; or the combination of the sense strand 5'MVIP and the antisense strand 3'MVIP is 5'MVIP01 / 3'MVIP09 or 5'MVIP09 / 3'MVIP01.

9. The RNAi agent for inhibiting AGT gene expression according to claim 7 or a pharmaceutically acceptable salt thereof, wherein: The antisense strand and its vector structure are AS140 as shown below: Single-strand code antisense strand sequence 5'→3' AS140 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP09.

10. The RNAi agent for inhibiting AGT gene expression according to claim 7 or a pharmaceutically acceptable salt thereof, wherein: The positive chain and its carrier structure are S140 as shown below: Single-strand code sense strand sequence 5'→3' S140 5'MVIP09-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG.

11. The RNAi agent for inhibiting AGT gene expression according to claim 8 or a pharmaceutically acceptable salt thereof, wherein: The RNAi agent is selected from the following combination:

12. The RNAi agent for inhibiting AGT gene expression according to claim 7 or a pharmaceutically acceptable salt thereof, wherein: The antisense strand and its carrier structure are selected from AS140, AS207-AS220, AS222-AS224, AS227-AS244, AS246-AS252, AS254-AS257 and AS259-AS266 as shown below:

13. The RNAi agent for inhibiting AGT gene expression according to claim 12 or a pharmaceutically acceptable salt thereof, wherein: The sense strand and its carrier structure are selected from S140, S207-S222, S224-S226, S229-S246, S248-S254, S256-S259 and S261-S264 as shown below:

14. The RNAi agent for inhibiting AGT gene expression according to claim 8 or a pharmaceutically acceptable salt thereof, wherein: The RNAi agent is selected from Kylo-09-DS122, Kylo-09-DS131, Kylo-09-DS141, Kylo-09-DS142 and Kylo-09-DS147 as shown below: 15 . A cell comprising the RNAi agent for inhibiting AGT gene expression according to any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof. 16 . A pharmaceutical composition comprising the RNAi agent for inhibiting AGT gene expression according to any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient.

17. The pharmaceutical composition of claim 16, further comprising a delivery vehicle.

18. The pharmaceutical composition according to claim 17, wherein The delivery vehicle includes liposomes.

19. The pharmaceutical composition according to claim 17, wherein The delivery vehicle comprises nanolipids.

20. Use of the RNAi agent for inhibiting AGT gene expression according to any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to any one of claims 16 to 19 in the preparation of a medicament for preventing and / or treating a disease or condition associated with AGT or reducing the risk of a disease or condition associated with AGT, wherein: The disease or condition associated with AGT is hypertension.

21. A medicine kit comprising the RNAi agent for inhibiting AGT gene expression according to any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof, or comprising the pharmaceutical composition according to any one of claims 16 to 19.

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