WO2023066236A1 - Agt抑制剂及其用途 - Google Patents

Agt抑制剂及其用途 Download PDF

Info

Publication number
WO2023066236A1
WO2023066236A1 PCT/CN2022/125877 CN2022125877W WO2023066236A1 WO 2023066236 A1 WO2023066236 A1 WO 2023066236A1 CN 2022125877 W CN2022125877 W CN 2022125877W WO 2023066236 A1 WO2023066236 A1 WO 2023066236A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
rnai agent
kylo
nucleotides
pharmaceutically acceptable
Prior art date
Application number
PCT/CN2022/125877
Other languages
English (en)
French (fr)
Inventor
崔坤元
王圣军
陈清燕
Original Assignee
厦门甘宝利生物医药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 厦门甘宝利生物医药有限公司 filed Critical 厦门甘宝利生物医药有限公司
Publication of WO2023066236A1 publication Critical patent/WO2023066236A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present application relates to the field of biomedicine, in particular to an RNAi agent for inhibiting AGT gene expression and its application.
  • RNAi RNA interference
  • Andrew Z. Fire et al. when performing antisense RNA inhibition experiments in Caenorhabditis elegans, and called this process RNAi.
  • This discovery was rated as one of the top ten scientific advances in 2001 by the "Science” magazine, and ranked first in the top ten scientific advances in 2002. Since then, siRNA with RNAi as the mechanism of action has attracted widespread attention as a potential gene therapy drug.
  • Andrew Fahe and Craig C. Mello Craig C. Contribution to the Nobel Prize in Physiology or Medicine.
  • RNAi is triggered by double-stranded RNA (dsRNA) in many organisms, including animals, plants, and fungi.
  • dsRNA double-stranded RNA
  • RNAi an endonuclease called "Dicer” cuts or “cuts” the long-chain dsRNA. Ding" into small fragments of 21-25 nucleotides in length. These small fragments are called small interfering RNA (siRNA), and the antisense strand (Guide strand) is loaded onto the Argonaute protein (AGO2).
  • siRNA small interfering RNA
  • AGO2 loading occurs in the RISC-loading complex, a ternary complex consisting of Argonaute protein, Dicer, and dsRNA-binding protein (TRBP for short).
  • the sense strand Passenger strand
  • Passenger strand is cleaved by AGO2 and expelled.
  • AGO2 then uses the antisense strand to bind to mRNAs containing perfectly complementary sequences, and then catalyzes the cleavage of these mRNAs, causing mRNA cleavage to lose its role as a translation template, thereby preventing the synthesis of related proteins. After cleavage, the cleaved mRNA is released and the antisense-loaded RISC-loading complex is recycled for another round of cleavage.
  • RNAi technology treats diseases at the mRNA level, which is more efficient than chemical small molecule drugs and biomacromolecular drugs at the protein level.
  • the sense strand and antisense strand sequence of siRNA with high specificity and good inhibitory effect can be designed, these single-strand sequences are synthesized by solid phase, and then the sense strand and antisense strand are annealed in a specific
  • the buffer is paired into siRNA according to the principle of base pairing, and finally transported to the corresponding target in the body through the carrier system to degrade the target mRNA and destroy the function of the target mRNA as a translation template, thereby preventing the synthesis of related proteins.
  • siRNA is unstable in blood and tissue and is easily degraded by nucleases.
  • the sense strand and/or antisense strand of siRNA can be modified, but these chemical modifications only provide limited resistance to nuclease degradation. protective effect and may ultimately affect the activity of siRNA. Therefore, a corresponding delivery system is also needed to ensure that siRNA can safely and efficiently pass through the cell membrane. Due to the large molecular weight of siRNA, a large number of negative charges, and high water solubility, it cannot pass through the cell membrane to reach the cell.
  • liposomes 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 vector.
  • Liposome-mediated siRNA delivery mainly encapsulates siRNA into liposomes to protect siRNA from nuclease degradation and improve the efficiency of siRNA passing through cell membrane barriers, thereby promoting cell absorption.
  • liposomes themselves are prone to inflammatory reactions , a variety of antihistamines and hormones such as cetizine and dexamethasone must be used before administration to reduce possible acute inflammatory reactions, so it is not suitable for all therapeutic areas in actual clinical applications, especially like For diseases such as chronic hepatitis B with a long treatment cycle, the accumulated toxicity that may be produced by long-term use is a potential safety hazard. Therefore, a safer and more effective carrier system is needed to deliver siRNA.
  • the asialoglycoprotein receptor (ASGPR) in the liver is a receptor specifically expressed in hepatocytes and is a highly efficient endocytic receptor.
  • ASGPR asialoglycoprotein receptor
  • the secondary end of various glycoproteins exposed after enzymatic or acid hydrolysis of sialic acid is galactose residues, so the sugar specifically bound by ASGPR is galactosyl, so it is also called galactose-specific receptor .
  • Monosaccharide and polysaccharide molecules such as galactose, galactosamine, and N-acetylgalactosamine all have high affinity for ASGPR.
  • ASGPR The main physiological function of ASGPR is to mediate the clearance of asialoglycoprotein, lipoprotein and other substances in the blood, and it is closely related to the occurrence and development of viral hepatitis, cirrhosis, liver cancer and other liver diseases.
  • the discovery of this characteristic of ASGPR plays an important role in the diagnosis and treatment of liver-derived diseases (Ashwell G, Harford J, Carbohydrate specific Receptors of the Liver, Ann Rev Biochem1982 51:531-554).
  • Drugs for the treatment of hepatic diseases containing galactose or galactosamine and their derivatives in their structure can specifically bind to ASGPR, thereby having active liver targeting, and do not need other carrier systems for delivery.
  • Angiotensinogen AGT
  • hypertension ACT
  • Blood pressure is the pressure of blood on the walls of blood vessels in the circulatory system. Blood pressure is mainly due to the beating of the animal's heart. During each heartbeat, blood pressure varies between a maximum (systolic) blood pressure (SBP) and a minimum (diastolic) blood pressure (DBP).
  • SBP maximum blood pressure
  • DBP minimum blood pressure
  • MAP Mean arterial pressure
  • Blood pressure can be measured with a sphygmomanometer (ie, a blood pressure monitor).
  • 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.
  • Hypertension was defined as systolic blood pressure (SBP) ⁇ 140 mmHg and/or diastolic blood pressure (DBP) ⁇ 90 mmHg without the use of antihypertensive drugs. According to the level of elevated blood pressure, hypertension is divided into grade 1, grade 2 and grade 3. Cardiovascular risk was stratified according to blood pressure level, cardiovascular risk factors, target organ damage, clinical complications and diabetes, and divided into 4 levels of low risk, intermediate risk, high risk and very high risk. Blood pressure is classified and defined as follows:
  • hypertension can also be divided into primary hypertension and secondary hypertension.
  • Essential hypertension is hypertension caused by a variety of factors, or hypertension caused by unknown reasons. Sexual hypertension can only be controlled, not cured.
  • Secondary hypertension refers to an increase in 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, Takayasu arteritis and so on.
  • 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 severe hypertension such as hypertensive emergencies and subemergencies. Blood pressure occurs.
  • RAS Renin-angiotensin-aldosterone system
  • RAAS renin-angiotensin system
  • RAS renin-angiotensin system
  • Angiotensinogen (AGT), a member of the serpin family, also known as SERPINA8, is encoded by the AGT gene and is the only precursor of all angiotensin peptides in RAS.
  • Human AGT has 485 amino acids, including a 33 amino acid signal peptide, is mainly produced in the liver, and released into the systemic circulation, during which renin converts it to angiotensin I.
  • Angiotensin I is subsequently converted to angiotensin II by angiotensin converting enzyme (ACE).
  • ACE angiotensin converting enzyme
  • Angiotensin I can stimulate the adrenal medulla to secrete adrenaline, but the effect of direct contraction of blood vessels is not obvious; angiotensin II can cause the contraction of small arteries throughout the body to increase blood pressure.
  • aldosterone acts on Kidney tubules play the roles of sodium retention, water retention, and potassium excretion, which cause blood volume to increase and blood pressure to increase. The mechanism of action is shown in Figure 10.
  • ARB Antagonist ARB
  • B ⁇ 1-receptor antagonists
  • C dihydropyridine calcium channel antagonists
  • D refers to Diuretics (such as thiazides).
  • the mechanism of action of these five types of antihypertensive drugs is not exactly the same.
  • one drug is used first. If the effect of one drug on blood pressure control is not good, two drugs are used in combination. Even three blood pressure medications. If none of the three antihypertensive drugs can control blood pressure to the normal range, it is called refractory hypertension.
  • Refractory hypertension is a relatively common clinical problem in the treatment of hypertension, and it is also a difficult problem in treatment.
  • ACEI and ARB can cause dry cough and edema, which can seriously cause renal failure. Insufficient function; side effects of CB drugs can be rapid heartbeat, blush, headache, and swollen feet; B drugs can also cause fatigue and affect blood sugar and blood lipid metabolism; D drugs can cause body weakness, cramps, and severe gout.
  • This application provides an AGT RNAi agent, which specifically interferes with the mRNA of the AGT gene, destroys its function as a translation template, and prevents the expression of the angiotensinogen AGT protein, thereby preventing and/or treating the RAS pathway-related Diseases, such as hypertension, include “non-refractory hypertension” and “refractory hypertension”.
  • the RNAi agent of the present application can be formed by base pairing of the sense strand and the antisense strand, the sense strand and the antisense strand are at least 80% base complementary to each other, and part or all of the 2' positions of the nucleotide sugar groups can be fluorine Or methoxy, the terminal can have at least 3 consecutive phosphates between nucleotides that are thiolated.
  • the structure of the RNAi agent of the present application may also contain the structures 5'MVIP and 3'MVIP that make the RNAi agent have liver targeting specificity, wherein the 5'MVIP can be coupled to the sense strand and/or the reverse strand of the RNAi agent.
  • the 5' end of the sense strand, the 3'MVIP can be coupled to the 3' end of the antisense strand and/or the sense strand of the RNAi agent, and both the 5'MVIP and the 3'MVIP can contain a (liver targeting specific) targeting unit X, branch chain L, linker B and connecting chain D, 5'MVIP can also comprise the transfer point R 1 that is connected with the sense strand or antisense strand 5' end of the RNAi agent, and 3'MVIP can also comprise the transition point R 1 connected with the said RNAi agent sense strand or antisense strand.
  • the transition point R 2 connected to the 3' end of the sense strand or the antisense strand of the RNAi agent, the targeting unit X, the branch chain L, the linker B and the connecting strand D are inside or 5' of the 5'MVIP and the 3'MVIP respectively MVIP and 3'MVIP may be the same or different.
  • the present application provides an RNAi agent or a pharmaceutically acceptable salt thereof, the structure of the RNAi agent contains a carrier structure and an interfering nucleic acid, and its structure is shown in formula IIIa, IIIb or IIIc:
  • the interfering nucleic acid targets the AGT gene, which includes an antisense strand and a sense strand;
  • the carrier structure includes 5'MVIP (5'MultiValent Import Platform) and/or 3'MVIP (3'MultiValent Import Platform);
  • the 5'MVIP consists of a transfer point R 1 , a connecting chain D, a linker B, a branched chain L and a liver-targeting specific ligand X
  • the 3'MVIP consists of a transfer point R 2 , a connecting chain D, a linker B.
  • the 5'MVIP is connected to the 5' end of the sense chain or the 5' end of the antisense chain through the transfer point R1
  • the 3'MVIP is connected to the 5' end of the antisense chain through the transfer point R1.
  • Point R 2 is 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 from 0 to 4.
  • interfering nucleic acid is used to inhibit AGT gene expression
  • interfering nucleic acid comprises siRNA or miRNA.
  • the 5'MVIP is selected from any one of 5'MVIP01 to 5'MVIP22 in Table 10
  • the 3'MVIP is selected from any of 3'MVIP01 to 3'MVIP27 in Table 11 either one.
  • said sense strand is identical to 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:12, Any one of ID NO: 17 and SEQ ID NO: 18, or a sequence that differs therefrom by no more than 3 nucleotides, has substantial homology.
  • the antisense strand comprises the following nucleotide sequences: SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, Any one of 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.
  • said sense strand comprises 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: 48, SEQ ID NO: Any one of ID NO:53 and SEQ ID NO:54 or a sequence with a difference of no more than 3 nucleotides
  • the antisense strand includes 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.
  • said interfering nucleic acid comprises Kylo-09-DS01, Kylo-09-DS07, Kylo-09-DS08, Kylo-09-DS10, Kylo-09-DS11, Kylo-09-DS12, Kylo - Any one or more of 09-DS17, Kylo-09-DS18, Kylo-09-DS37 to Kylo-09-DS54.
  • 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.
  • the RNAi agent or a pharmaceutically acceptable salt thereof includes Kylo-09-DS122, Kylo-09-DS131, Kylo-09-DS141, Kylo-09-DS142 and Kylo- Any one or more of 09-DS147.
  • RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof which comprises an antisense strand
  • the antisense strand comprises at least 12 corresponding positions selected from the following sequences: Contiguous nucleotides with substantially complementary nucleotides or a sequence differing therefrom by no more than 3 nucleotides: AGT mRNA NM_001382817.3 starts at 1854-1874, 1907-1927, 1895-1915, 1352-1372, At least 12 consecutive nucleotides of 1903-1923, 2019-2039, 1853-1873 and 1818-1838 or a sequence with a difference of no more than 3 nucleotides or the starting position in NM_001384479.1 is 1822-1842, 1875- 1895, 1863-1883, 1320-1340, 1871-1891, 1987-2007, 1821-1841 and 1786-1806 sequences of at least 12 consecutive nucleotides or no more than 3 nucleotides different from
  • the RNAi agent comprises a single- or double-stranded nucleic acid molecule.
  • the RNAi agent comprises siRNA or miRNA.
  • 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, and the complementary region includes at least 12 consecutive nucleotides.
  • one or more nucleotides on the sense strand and/or the antisense strand are modified to form modified nucleotides.
  • 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 the antisense strand through a carrier structure.
  • the present application provides a cell 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.
  • the present application provides a pharmaceutical composition, which comprises 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 any Optionally pharmaceutically acceptable excipients, carriers and/or diluents.
  • the present application provides a method for reducing AGT mRNA or protein expression in cells or tissues, which includes making cells or tissues react with an effective amount of the aforementioned RNAi agent or a pharmaceutically acceptable salt thereof, the aforementioned AGT inhibiting RNAi agent for gene expression or a pharmaceutically acceptable salt thereof, and/or the aforementioned pharmaceutical composition.
  • the present application provides the aforementioned RNAi agent or its pharmaceutically acceptable salt, the aforementioned RNAi agent for inhibiting AGT gene expression or its pharmaceutically acceptable salt, or the aforementioned pharmaceutical composition in the preparation of medicine Use, the medicine is used for preventing and/or treating a disease or a disease or reducing the risk of a disease or a disease.
  • the present application provides a method for preventing and/or treating a disease or a disease, the method comprising administering an effective amount of the aforementioned RNAi agent or a pharmaceutically acceptable salt thereof, to a subject in need thereof,
  • the aforementioned RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof, and/or the aforementioned pharmaceutical composition are administered to a subject in need thereof.
  • the present application provides a 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 drug combination.
  • the AGT RNAi agent of the present application interferes with AGT mRNA, destroys its function as a translation template, and adjusts the expression level of AGT protein (the only precursor of all RAS angiotensin peptides) in the systemic circulation, from the gene level and from the source of the RAS system
  • the regulation of blood pressure provides a brand-new treatment model to fight against various types of hypertension, including anti-indigenous and secondary, including resistant hypertension (TRH), so that those resistant to resistant hypertension ( TRH) or patients with chronic heart failure with reduced ejection fraction are available.
  • TRH resistant hypertension
  • the AGT RNAi agent of the present application also has a unique long-acting property, and it may be administered once a quarter or half a year, which has incomparable advantages over current small-molecule hypertension therapeutic drugs.
  • FIG. 1A shows is the AGT mRNA level in the Hep 3B cell after the RNAi intervention in the embodiment 2 of the present application;
  • FIG. 1 B shows is the AGT mRNA level in the HepG 2 cell after the RNAi intervention in the embodiment 2 of the present application;
  • Figures 2A-2D show the HPLC graphs of the stability detection of the RNAi agent in different periods of time in Example 3 of the present application;
  • Figure 3 shows the high-resolution mass spectrum of ERCd-01-c2 synthesized in Example 4.1.1.5 of the present application
  • Figure 4 shows the high-resolution mass spectrum of 3'MVIP17-c1 synthesized in Example 4.1.2.6 of the present application
  • Figure 5 shows the high-resolution mass spectrum of 5'MVIP09-ERCd-PFP-c2 synthesized in Example 4.2.1.2 of the present application;
  • Figure 6 shows the average level of hAGT after administration of transgenic mice in Example 6 of the present application
  • Figure 7 shows the average level of hAGT in the serum of the transgenic mice in Example 7 of the present application after administration.
  • Figure 8 shows the average level of AGT in the serum of cynomolgus monkeys in Example 8 of the present application after administration.
  • Figures 9A-9E show the structural formulas of Kylo-09-DS122, Kylo-09-DS131, Kylo-09-DS141, Kylo-09-DS142 and Kylo-09-DS147.
  • FIG 10 shows the mechanism of action of angiotensinogen (AGT).
  • angiotensinogen is used interchangeably with the term “AGT”, and examples of AGT mRNA sequences are readily available using published databases, e.g., GenBank, UniProt, OMIM, and the Macaque (Macaca) Genome Project website.
  • 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 in, for example, GenBank Accession No. GI: 90075391 (AB170313.
  • mouse (Mus musculus) AGT its amino acid and complete coding sequence can be found in for example, GenBank accession number GI: 113461997 (NM_007428.3); and rat AGT (Rattus norvegicus) AGT, its amino acid and complete coding sequence can be See, eg, GenBank Accession No. GI:51036672 (NM_134432).
  • 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.
  • RNAi agent RNA agent
  • RNA interfering agent an agent comprising RNA as defined by the term herein, and which can be complexed by RNA-induced silencing
  • the RISC pathway mediates targeted cleavage of RNA transcripts.
  • iRNAs direct the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).
  • RNAi RNA interference
  • the iRNA modulates (eg, suppresses) expression of the AGT gene in a cell (eg, a cell in a subject, such as a mammalian subject).
  • an RNAi agent can be a single-stranded siRNA (ssRNAi) introduced into a cell or organism to inhibit a target mRNA.
  • the single-stranded RNAi agent binds the RISC endonuclease Argonaute 2, which then cleaves the target mRNA.
  • Single-stranded siRNAs are typically 15 to 30 nucleotides in length and are chemically modified. Design and testing of single-stranded siRNAs are described in US Patent 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 of the antisense nucleotide sequences described herein can be used as the single-stranded siRNA described herein or chemically modified by the methods described in Lima et al. (2012) Cell 150:883-894.
  • an "iRNA” as used herein 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” .
  • dsRNA refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel and substantially complementary nucleic acid strands, referred to as having a “sense” and "Anti-sense” orientation.
  • double-stranded RNA triggers the degradation of target RNA (eg, mRNA) through a post-transcriptional gene silencing mechanism (referred to herein as RNA interference or RNAi).
  • target RNA eg, mRNA
  • RNA interference post-transcriptional gene silencing mechanism
  • the duplex structure can be of any length that permits specific degradation of the desired target RNA by the RISC pathway, and can be in the range of about 19 to 36 base pairs in length, e.g., about 19-30 base pairs in length, For example, about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length. Ranges and lengths intermediate to the above ranges and lengths are also included as part of this application.
  • an iRNA agent of the present application is a dsRNA comprising 15-23 nucleotides per strand that interacts with a target RNA sequence (eg, the AGT gene) to direct cleavage of the target RNA.
  • an iRNA of the present application is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence (eg, an AGT target mRNA sequence) to direct cleavage of the target RNA.
  • each or both strands may also include one or more non-ribonucleotides, e.g., deoxyribose nucleotides or modified nucleotides.
  • iRNA as used in this specification may include ribonucleotides with chemical modifications; iRNA may include substantial modifications at multiple nucleotides.
  • modified nucleotide as used herein means a nucleotide independently having a modified sugar moiety, a modified internucleotide linkage, or a modified nucleobase, or any combination thereof.
  • modified nucleotide encompasses the substitution, addition or removal of eg functional groups or atoms to internucleotide linkages, sugar moieties or nucleobases. Modifications applicable to the agents of the present application include all types of modifications disclosed herein or known in the art.
  • nucleic acid and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides or analogs thereof) of any length.
  • a polynucleotide can have any three-dimensional structure and can perform any function.
  • genes or gene fragments such as probes, primers, EST or SAGE tags
  • exons introns
  • messenger RNA mRNA
  • transfer RNA transfer RNA
  • ribosomal RNA nuclear Enzymes
  • cDNA recombinant polynucleotides
  • branched polynucleotides vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, siRNA, miRNA, shRNA, RNAi reagents and primers.
  • a polynucleotide may 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.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be made either before or after polymer assembly. Nucleotide sequences can be blocked by non-nucleotide components.
  • Polynucleotides can be modified after polymerization, for example by conjugation with labeling components. The term can refer to both double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of the present application as a polynucleotide includes the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.
  • target nucleic acid or "target sequence” generally refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during AGT gene transcription, including mRNA that is the RNA processing product of the main transcription product.
  • the target portion of the sequence should be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of the mRNA molecule formed during AGT gene transcription.
  • the target sequence is within the protein coding region of AGT.
  • the target sequence may be about 19-36 nucleotides in length, eg, preferably about 19-30 nucleotides in length. Ranges and lengths intermediate to the above ranges and lengths are also included as part of this application.
  • nucleotide sequence generally refers to a series or sequence of nucleobases, nucleotides and/or nucleosides, whether modified or unmodified, using standard nucleotide nomenclature and the present application The symbols for the modified nucleotides are described by a series of letters.
  • oligonucleotide generally refers to a oligonucleotide composed of multiple nucleotide residues (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogs thereof) via a phosphate diphosphate Ester linkages (or related structural variants or synthetic analogues thereof) link the constituent polymers.
  • oligonucleotide generally refers to polymers of nucleotides in which nucleotide residues and linkages between them are naturally occurring, it is to be understood that the term also includes within the scope of the term various analogs, including But not limited to: peptide nucleic acid (PNA), phosphoramidate, phosphorothioate, methylphosphonate, 2-O-methyl ribonucleic acid, etc. The exact size of the molecule can depend on the particular application.
  • PNA peptide nucleic acid
  • phosphoramidate phosphoramidate
  • phosphorothioate phosphorothioate
  • methylphosphonate 2-O-methyl ribonucleic acid
  • Oligonucleotides are generally shorter in length, usually about 10-30 nucleotide residues, but the term can refer to molecules of any length, although the terms "polynucleotide” or “nucleic acid” are generally used for larger Oligonucleotides.
  • an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNA) and/or one or more modified nucleosides.
  • modified oligonucleotide generally means an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage.
  • modified nucleoside generally means a nucleoside comprising at least one chemical modification compared to a naturally occurring RNA or DNA nucleoside.
  • Modified nucleosides comprise modified sugar moieties and/or modified nucleobases.
  • nucleobase generally means 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 surrogate replacement moieties. As used herein, “nucleobase sequence” generally means the sequence of contiguous nucleobases independent of any sugar, linkage, or nucleobase modification.
  • unmodified nucleobase or “naturally occurring nucleobase” generally means the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G); and The pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C) and uracil (U).
  • Modified nucleobase generally means any nucleobase that is not a naturally occurring nucleobase.
  • sugar moiety generally means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.
  • naturally occurring sugar moiety generally means a ribofuranosyl group as found in naturally occurring RNA or a deoxyribofuranosyl group as found in naturally occurring DNA.
  • Modified sugar moiety means a substituted sugar moiety or sugar substitute.
  • internucleoside linkage generally means a covalent linkage between adjacent nucleosides in an oligonucleotide.
  • Naturalally occurring internucleoside linkage means a 3' to 5' phosphodiester linkage.
  • Modified internucleoside linkage means any internucleoside linkage other than a naturally occurring internucleoside linkage.
  • antisense oligonucleotide refers to a single-stranded oligonucleotide molecule having a core complementary to a corresponding fragment of a target nucleic acid (for example, a target genomic sequence, mRNA precursor, or mRNA molecule). base sequence.
  • the antisense oligonucleotides are 12 to 30 nucleobases in length.
  • an antisense oligonucleotide is an unmodified or modified nucleic acid having a nucleotide sequence that is complementary to the sequence of a target nucleic acid (eg, an AGT polynucleotide).
  • the term "antisense strand” generally refers to the strand of an RNAi agent (eg, dsRNA) that includes a region that is substantially complementary to a target sequence.
  • region of complementarity generally refers to the region of the antisense strand that is substantially complementary to a sequence as defined herein (eg, a target sequence).
  • the mismatch can be in an internal or terminal region of the molecule.
  • the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3 or 2 nucleotides of the 5' end and/or the 3' end.
  • the term "sense strand” generally refers to a strand of an RNAi agent that includes a region that is substantially complementary to a region that is the term antisense strand as defined herein.
  • the "justice” strand is sometimes called the “sense” strand, the “passenger” strand, or the “anti-guidance” strand.
  • the antisense strand targets the desired mRNA, while the sense strand targets a different target. Therefore, if the antisense strand is incorporated into RISC, the correct target is targeted. Incorporation of the sense strand can lead to off-target effects. These off-target effects can be limited by the use of modifications on the sense strand or by the use of 5' caps.
  • the term "complementary" when used to describe a first nucleotide sequence (such as the sense strand of an RNAi agent or AGT mRNA) with respect to a second nucleotide sequence (such as the antisense strand of an RNAi agent) means An oligonucleotide or polynucleotide comprising a first nucleotide sequence hybridizes (forms base pair hydrogen bonds) to an oligonucleotide or polynucleotide comprising a second nucleotide sequence under certain conditions and forms a double strand ability to form bulk or double helix structures.
  • Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and include natural or modified nucleotides or nucleotide mimetics, provided the above The need was fulfilled with regard to their ability to hybridize. "Complementary” does not necessarily have nucleobase complementarity at every nucleoside. Instead, some mismatches can be tolerated.
  • the term “fully complementary” generally means that all (100%) of the bases in the contiguous sequence of the first polynucleotide will hybridize to the same number of bases in the contiguous sequence of the second polynucleotide .
  • the contiguous sequence may comprise all or part of the first or second nucleotide sequence.
  • “partially complementary” generally means that in hybridized nucleobase sequence pairs, at least about 70% of the bases in the contiguous sequence of the first polynucleotide will be identical to the contiguous sequence of the second polynucleotide. Hybridization of the same number of bases.
  • substantially complementary generally means that in hybridized nucleobase sequence pairs, at least about 90% of the bases in the contiguous sequence of the first polynucleotide will be contiguous with those of the second polynucleotide. The same number of bases in the sequence hybridize.
  • the terms “complementary”, “fully complementary” and “substantially complementary” as used in this application may be 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 Used for base matching. Sequence identity or complementarity is independent of modification. For purposes of determining identity or complementarity, for example, a and Af are complementary to U (or T) and identical to A.
  • 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, usually by sequence analysis procedures (e.g., Karlin and Altschul, 1990, PNAS 87:2264-2268; Karlin and Altschul, 1993, PNAS 90:5873-5877), or by visual inspection.
  • sequence analysis procedures e.g., Karlin and Altschul, 1990, PNAS 87:2264-2268; Karlin and Altschul, 1993, PNAS 90:5873-5877
  • the terms “perfect homology” or “perfect homology” generally refer to complete (100%) homology or "identity" between a reference sequence and a subject nucleic acid sequence.
  • the term "substantially homologous” or “substantially homologous” generally means that the subject sequence shares at least 50% (e.g., at least 55%) of the nucleotides at the same nucleotide position in the reference sequence. , 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) homologous nucleotides.
  • the term "ligand” generally refers to any compound or molecule capable of covalently or otherwise chemically binding to a biologically active substance such as an oligonucleotide.
  • the ligand is capable of interacting directly or indirectly with another compound, such as a receptor, which may be present on the cell surface, or alternatively may be intracellular and/or Or intercellular receptors, the interaction of the ligand with the receptor can result in a biochemical reaction, or can simply be a physical interaction or binding.
  • the terms “induce”, “inhibit”, “enhance”, “elevate”, “increase”, “decrease”, “decrease”, etc. generally denote a quantitative difference between two states.
  • an amount effective to inhibit the activity or expression of AGT means that the level of AGT activity or expression in a treated sample will be lower than the level of AGT activity or expression in an untreated sample.
  • the terms apply, for example, to expression levels and activity levels.
  • the terms “reduce” and “decrease” are used interchangeably and generally mean any change from the original.
  • “Reduce” and “decrease” are relative terms that require a comparison between before and after measurements.
  • “Reduce” and “decrease” include complete depletion.
  • the term "reduces" the level of expression of a gene, gene product such as a protein or a biomarker in a first sample, detectable by standard methods known in the art (such as those described in this application) /Amount is about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60% compared to the expression level/amount of the corresponding gene, gene product, such as protein or biomarker in the second sample , 70%, 80%, 85%, 90%, 95%, or 100% overall reduction.
  • the term "decrease” refers to a reduction in the expression level/amount of a gene or biomarker in a first sample, wherein the reduction is the expression level/amount of the corresponding gene or biomarker in a second sample 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.
  • the first sample is a sample obtained from a subject and the second sample is a reference sample.
  • expression generally means the process by which a gene ultimately produces a protein.
  • Expression includes, but is not limited to, transcription, post-transcriptional modifications (eg, splicing, polyadenylation, addition of a 5'-cap), and translation.
  • 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 formulations will generally contain salts, excipients, buffers, preservatives, compatible carriers and, optionally, other therapeutic agents.
  • Such pharmaceutically acceptable formulations may also generally contain compatible solid or liquid fillers, diluents or encapsulating materials suitable for human administration.
  • 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 the present application.
  • Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, salts prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, boric , formic acid, malonic acid, succinic acid, etc.
  • Pharmaceutically acceptable salts can also be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium or calcium salts.
  • lipid nanoparticle generally refers to a vesicle comprising a lipid layer encapsulating a pharmacologically active molecule, such as a nucleic acid molecule, eg, iRNA or a plasmid from which the iRNA is transcribed.
  • a pharmacologically active molecule such as a nucleic acid molecule, eg, iRNA or a plasmid from which the iRNA is transcribed.
  • LNPs are described, for example, in Chinese Patent No. CN103189057B, the entire contents of which are incorporated herein by reference.
  • prevention and/or treatment includes not only preventing and/or treating a disease, but also generally preventing the onset of a disease, slowing or reversing the progression of a disease, preventing or slowing down one or more symptoms associated with a disease onset, reduction and/or alleviation of one or more symptoms associated with the disease, reduction of the severity and/or duration of the disease and/or any symptoms associated therewith and/or prevention of the disease and/or any symptoms associated therewith prevent, reduce or reverse any physiological impairment caused by the disease, and generally any pharmacological effect that is beneficial to the patient being treated.
  • An RNAi agent or pharmaceutical composition of the present application need not achieve a complete cure or eradicate any symptom or manifestation of a disease to be a viable therapeutic agent.
  • drugs used as therapeutic agents may reduce the severity of a given disease state, but need not eliminate every manifestation of the disease to be considered a useful therapeutic agent.
  • a treatment administered prophylactically need not be fully effective in preventing the onset of the disorder to constitute a viable prophylactic. Simply reducing the effects of disease in a subject (for example, 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 disease occurrence or The possibility of deterioration is enough.
  • disease or “condition” are used interchangeably and generally refer to any deviation from the normal state of a subject, such as any change in the state of the body or certain organs that prevents or disrupts the performance of function , and/or cause symptoms such as malaise, dysfunction, suffering or even death in those who are sick or come into contact with it.
  • a disease or condition may also be called a disorder, ailing, ailment, malady, disorder, sickness, illness, complaint, inderdisposion or affectation.
  • angiotensinogen-associated disease or “AGT-associated disease” generally refers to a disease or disorder caused by or associated with activation of the renin-angiotensin-aldosterone system (RAAS), or its symptoms or A disease or disorder whose progression is in response to RAAS inactivation.
  • RAAS renin-angiotensin-aldosterone system
  • angiotensinogen-related disease includes diseases, disorders or conditions that benefit from reduced expression of AGT. These disorders are often associated with high blood pressure.
  • angiotensinogen-related diseases include hypertension, e.g., borderline hypertension (also known as prehypertension), essential hypertension (also known as native hypertension or essential hypertension) , secondary hypertension (also known as non-native hypertension), isolated systolic or diastolic hypertension, pregnancy-associated hypertension (eg, preeclampsia, eclampsia, and postpartum preeclampsia), diabetic Hypertension, resistant hypertension, resistant hypertension, paroxysmal hypertension, renovascular hypertension (also known as renal hypertension), Goldblatt's hypertension, ocular hypertension, glaucoma, pulmonary hypertension Blood pressure, 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
  • the AGT-associated disorder comprises intrauterine growth retardation (IUGR) or fetal growth restriction.
  • AGT-related disorders also include obesity, hepatic steatosis/fatty liver, e.g., nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD), glucose intolerance, type 2 diabetes (non-insulin-dependent diabetes mellitus) and metabolic syndrome.
  • hypertension includes hypertension associated with low plasma renin activity or plasma renin concentration.
  • the term "administration" generally refers to introducing the pharmaceutical formulation of the present application into the body of a subject by any route of introduction or delivery. Any method known to those skilled in the art for contacting cells, organs or tissues with the drug may be used. Such administration may include, without limitation, intravenous, intraarterial, intranasal, intraperitoneal, intramuscular, subcutaneous transdermal or oral.
  • the daily dose may be divided into one, two or more doses of suitable form to be administered at one, two or more times during a certain period of time.
  • the term "contacting" generally means that two or more substances of different types are brought into contact together in any order, in any manner, and for any length of time. Exposure can occur in vivo, ex vivo or in vitro. In some embodiments, it may refer to directly contacting cells or tissues with the RNAi agent or composition of the present application. In other embodiments, the term refers to indirect contact of an RNAi agent or composition of the present application with a cell or tissue.
  • the methods of the present application include wherein the subject is exposed to the RNAi agent or composition of the present application, and then the RNAi agent or composition passes through diffusion or any other active or passive transport process known in the art (the process by which the compound is transported in vivo). Circulation) A method of contacting cells or tissues.
  • 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 typically one that, when used alone or in combination with another therapeutic agent, promotes regression of disease (by reducing the severity of disease symptoms, frequency of asymptomatic periods of disease), any amount of drug that is evidenced by an increase in the degree and duration of the disease, or by the prevention of impairment or disability due to the presence of a disease.
  • a “prophylactically effective amount” or “prophylactically effective dose” of a drug generally refers to the amount of the drug that, alone or in combination with another therapeutic agent, inhibits the development or recurrence of the disease when administered to a subject at risk of disease development or disease recurrence .
  • the ability of a therapeutic or prophylactic agent to promote disease regression or inhibit disease progression or recurrence can be assessed using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems Efficacy in humans is predicted, or by assaying the activity of the agent in an in vitro assay.
  • an "effective amount” refers to the amount of an RNAi agent that produces a desired pharmacological, therapeutic, or prophylactic result.
  • the term "subject” generally refers to a human or non-human animal (including mammals), such as a human, a non-human primate (ape, or , gibbons, gorillas, chimpanzees, orangutans, macaques), domestic animals (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs) and laboratory animals (mice, rats, rabbits , guinea pig).
  • Human subjects include fetal, neonatal, infant, adolescent and adult subjects.
  • Subjects include animal disease models.
  • the term “about” generally means approximately, in the vicinity of, roughly, or around.
  • a cut-off or a specific value is used to indicate that the stated value may vary by as much as 10% from the recited value.
  • the term “about” may be used to encompass a variation of ⁇ 10% or less, ⁇ 5% or less, ⁇ 1% or less, ⁇ 0.5% or less, or ⁇ 0.5% or less from the specified value. 0.1% or less variation.
  • the term "at least" preceding a number or series of numbers includes the number adjacent to that term “at least”, and all subsequent numbers or integers logically included, as clear from the context.
  • the number of nucleotides in a nucleic acid molecule must be an integer.
  • “at least 19 nucleotides in a nucleic acid molecule of 21 nucleotides” means that 19, 20, or 21 nucleotides have the indicated property.
  • not more than or “below” as used herein refers to the logically lower value or integer adjacent to the phrase, such as, from the logic of the context, to zero.
  • a duplex having an overhang of "no more than 3 nucleotides” has an overhang of 3, 2, 1 or 0 nucleotides.
  • not exceeding precedes a series or range of numbers, it is understood that “not exceeding” modifies each number in the series or range.
  • ranges include both upper and lower limits.
  • the present application provides an RNAi agent that inhibits the expression of the AGT gene.
  • the RNAi agent comprises a monoclonal agent for inhibiting expression of the AGT gene in a cell, such as a cell of a subject (e.g., a mammal, such as a human predisposed to developing an AGT-related disorder such as hypertension).
  • Stranded oligonucleotides or double-stranded ribonucleic acid (dsRNA) molecules are included in the dsRNA molecules.
  • the dsRNA includes an antisense strand having a complementary region complementary to at least a part of mRNA formed during expression of the AGT gene.
  • the region of complementarity 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).
  • a dsRNA comprises two RNA strands that can be complementary and hybridize to form a duplex structure (complementary region) under the conditions used for the dsRNA.
  • One strand of the dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and often perfectly complementary, to the target sequence.
  • the target sequence may be derived from a sequence that forms mRNA during expression of the AGT gene.
  • the other strand includes a region that is complementary to the antisense strand such that when combined under appropriate conditions, the two strands can hybridize and form a duplex structure.
  • the duplex structure is 12 to 30 base pairs in length.
  • the region of complementarity to the target sequence is 12 to 30 nucleotides in length.
  • the dsRNA is about 19 to about 23 nucleotides in length, or about 24 to about 30 nucleotides in length.
  • the dsRNA is long enough to serve as a substrate for the Dicer enzyme.
  • dsRNAs greater than about 21-23 nucleotides in length are known in the art to serve as substrates for Dicer.
  • the region of RNA that is targeted for cleavage is typically part of a larger RNA molecule, typically an mRNA molecule.
  • a "portion" of a target is contiguous nucleotides of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage (ie, cleavage via the RISC pathway).
  • 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 bp.
  • RNA molecules having a duplex region of more than 30 base pairs is dsRNA.
  • 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 nucleotides substantially complementary to the corresponding positions selected from the following sequences: Consecutive nucleotides or a sequence with a difference of no more than 3 nucleotides: AGT mRNA NM_001382817.3 starts at 1854-1874, 1907-1927, 1895-1915, 1352-1372, 1903-1923, 2019-2039 , 1853-1873 and 1818-1838 of at least 12 consecutive nucleotides or a sequence with a difference of no more than 3 nucleotides or the starting position in NM_001384479.1 is 1822-1842, 1875-1895, 1863-1883, 1320 - 1340, 1871-1891, 1987-2007, 1821-1841 and 1786-1806 sequences of at least 12 consecutive nucleotides or no more than 3 nucleotides difference therefrom.
  • said complementary region comprises at least 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides.
  • the sense strand of the RNAi agent has substantial homology to the target sequences in Table 1.
  • g guanylic acid
  • a adenylic acid
  • t thymidylic acid
  • c cytidylic acid
  • n is a or u or g or c
  • g guanylic acid
  • a adenylic acid
  • u uridylic acid
  • c cytidylic acid
  • 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.
  • g guanylic acid
  • a adenylic acid
  • u uridylic acid
  • c cytidylic acid
  • RNA of the iRNA for example, dsRNA
  • the present application may comprise any one of the sequences shown in Table 3, or modified Tables 4, 5 or the sequence of 6, or the sequence of Table 13, 14, 16 or 17 coupled.
  • the present application encompasses unmodified, unconjugated, modified or conjugated dsRNA as described herein.
  • the RNAi agent can be added to the cell line in the form of liposome-nucleic acid nanoparticles for sequence screening.
  • the methods for preparing lipid compounds and liposome-nucleic acid nanoparticles in patents US9233971B2, US9080186B2, CN102985548B and CN103189057B are fully incorporated into this specification.
  • amphoteric lipids in the lipid compounds are preferably macrocyclic lipid compounds D1C1, T1C1, T1C6, T4C4, B2C1, B2C6, B2C7 and M10C1.
  • dsRNAs having a duplex structure of about 20 to 23 base pairs, for example, 21 base pairs have been considered to be particularly effective at inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877- 6888).
  • RNA duplex structures also work (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). It is reasonable to expect that duplexes with a few nucleotides subtracted or added at one or both ends from a sequence in Tables 1, 2 and 3 would be similarly efficient compared to the dsRNA.
  • a sequence having at least 18, 19, 20, 21 or more contiguous nucleotides derived from one of the sequences in Tables 1, 2 and 3 and differing from a dsRNA comprising the full sequence in its ability to inhibit expression of the AGT gene dsRNAs that are suppressed by no more than about 5, 10, 15, 20, 25 or 30% are encompassed within the scope of this application.
  • RNAs provided in Tables 1, 2 and 3 identify sites in AGT transcripts that are susceptible to RISC-mediated cleavage. Accordingly, further encompassed herein are iRNAs targeted within one of such sites. As used herein, an iRNA is said to target within a specific site of an RNA transcript if the iRNA promotes cleavage of the transcript at any position within that specific site, and such iRNAs typically include those provided in Tables 1, 2, and 3. At least about 12, 13, 14, 15, 16, 17, 18 or 19 contiguous nucleotides of a sequence of .
  • the dsRNA described herein may further comprise one or more single-stranded nucleotide overhangs, eg, 1, 2, 3 or 4 nucleotides.
  • a dsRNA having at least one nucleotide overhang can have superior inhibitory properties than its blunt-ended counterpart.
  • Nucleotide overhangs may comprise nucleotide/nucleoside analogs or compositions thereof, including deoxynucleotides/nucleosides. The overhang can be on the sense strand, the antisense strand, or any combination thereof.
  • overhang nucleotides may be present at the 5'-end, 3'-end, or both ends of the antisense or sense strand of the dsRNA.
  • the overhang can be caused by one strand being longer than the other, or by two strands of the same length being interleaved.
  • the overhang may form a mismatch with the target mRNA or it may be complementary to the sequence of the gene being targeted or it may be another sequence.
  • the dsRNA can also contain only a single overhang, which can enhance the interference activity of the RNAi agent without affecting its overall stability.
  • 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 blunt ends, located at the 5'-end of the antisense strand (or 3'-end of the sense strand), or vice versa.
  • the antisense strand of an RNAi agent has a nucleotide overhang at the 3'-end and a blunt 5'-end.
  • the asymmetric blunt end of the 5'-end of the antisense strand and the overhang at the 3'-end of the antisense strand facilitate the guide strand loading process in RISC.
  • the overhang is present at the 3'-end of the sense strand, the antisense strand, or both strands. In certain embodiments, this 3'-overhang is present in the antisense strand. In certain embodiments, this 3'-overhang is present in the sense strand.
  • the dsRNA is a double-ended blunt 21 nucleotides in length.
  • the dsRNA is 21 nucleotides in length, and both the sense and antisense strands have a 2 nucleotide overhang at the 3' end.
  • the sense strand and antisense strand of the above RNAi agent can be modified, and the nucleotides therein can have The modifying group can modify the whole chain or part of it.
  • one or more nucleotides on the sense strand and/or the antisense strand are modified to form modified nucleotides.
  • the RNA of the RNAi agent (eg, dsRNA) of the present application is unmodified and does not comprise, for example, chemical modifications or conjugations known in the art and described herein.
  • the RNA of an RNAi agent (eg, dsRNA) of the present application is chemically modified to enhance stability or other favorable properties.
  • all or substantially all nucleotides of the RNAi agent of the present application are modified, that is, there are no more than 5, 4, 3, 2 or 1 unmodified cores in the chain of the RNAi agent glycosides.
  • Nucleic acids as described herein can be synthesized and/or modified using methods well known in the art, such as those described in "Current protocols in nucleic acid chemistry", Beaucage, S.L. et al. (eds.), John Wiley & Sons, Inc., New York , NY, USA, which is incorporated herein by reference.
  • Modifications include, for example, terminal modifications, for example, 5'-end modifications (phosphorylation, coupling, reverse linkage) or 3'-end modifications (coupling, DNA nucleotides, reverse linkage, etc.); base modifications, For example, use of stabilizing bases, destabilizing bases, or substitution, removal of bases (abasic nucleotides), or coupling of bases with bases that base-pair with expanded partner pools; sugar modifications (e.g., 2'-position or 4'-position) or sugar substitution; or backbone modification, including modification or substitution of a phosphodiester linkage.
  • RNAi agent provided in the present application, neither the sense strand nor the antisense strand of the RNAi agent needs to be uniformly modified, and one or more than one modification can be incorporated in a single nucleotide.
  • the modified nucleotides are selected from the group consisting of: deoxyribonucleotides, nucleotide mimetics, abasic nucleotides, 2'-modified nucleotides, 3' to 3' Linked (inverted) nucleotides, nucleotides with unnatural bases, bridging nucleotides, peptide nucleic acid (PNA), unlocked nucleobase analogs, locked nucleotides, 3'-O-methoxy (2' internucleoside linkage) nucleotides, 2'-F-arabinonucleotides, 5'-Me/2'-fluoronucleotides, morpholino nucleotides, vinylphosphonate deoxy Ribonucleotides, vinylphosphonate-containing nucleotides, and cyclopropylphosphonate-containing nucleotides.
  • 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.
  • At least one of the 2' positions of the 7th, 12th, and 14th nucleotide sugar groups starting from the 5' end of the antisense strand is fluorine.
  • the 2' position of the nucleotide sugar group at the 7th, 12th and 14th nucleotides starting from the 5' end of the antisense strand is all fluorine.
  • At least one of the 2' positions of the remaining nucleotide sugar groups is a methoxyl group.
  • At least one of the 2' positions of the 5', 7, 8, and 9 nucleotide sugar groups starting from the 5' end of the sense strand is fluorine.
  • the 2' positions of the 5', 7, 8, and 9 nucleotide sugar groups starting from the 5' end of the sense strand are all fluorine.
  • At least one of the 2' positions of the remaining nucleotide sugar groups is methoxy base.
  • part or all of the -OH at the 2' position of the nucleotide sugar group of the sense strand or the antisense strand can be substituted, wherein the substituting group is fluorine or methoxy, preferably from the 5' end of the sense strand
  • the substituting group is fluorine or methoxy, preferably from the 5' end of the sense strand
  • the first 5, 7, 8, and 9 nucleotide 2' positions are fluorine and the 7, 12, and 14 nucleotide 2' positions from the 5' end of the antisense strand are fluorine, and the rest of the core
  • the 2' position of the nucleotide is methoxy.
  • the 2' positions of the 7th, 12th and 14th nucleotide sugar groups starting from the 5' end of the antisense chain are all fluorine, and the 2' positions of the remaining nucleotide sugar groups are all methoxy groups.
  • the 2' positions of the 5', 7, 8, and 9 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.
  • the 5', 7, 8, and 9 nucleotide 2' positions from the 5' end of the sense strand are fluorine and the 7, 12, and 14 nucleotides from the 5' end of the antisense strand
  • the 2' position is fluorine
  • the 2' positions of the remaining nucleotides are all methoxy, and there are at least two consecutive nucleotides between the 5' end and the 3' end of the sense strand and the antisense strand. a phosphorothioate linkage.
  • the sense and antisense strands 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.
  • G 2'-methoxyguanylic acid
  • A 2'-methoxyadenylic acid
  • U 2'-methoxyuridylic acid
  • C 2'-methoxycytidylic acid
  • u uridylic acid
  • c cytidylic acid
  • the antisense strand in the RNAi agent is selected from the sequences in Table 5 below.
  • the 2' position of the 7th, 12th and 14th nucleotides starting from the 5' end of the antisense strand is fluorine, and the 2' position of the remaining nucleotides is a methoxyl group, and the end of the antisense strand has at least 3 Phosphate bonds between adjacent nucleotides can be thiolated.
  • G 2'-O-methylguanylic acid
  • A 2'-O-methyladenylic acid
  • U 2'-O-methyluridine
  • C 2'-O-methyl Cytidylic acid
  • fG 2'-fluoroguanylic acid
  • fA 2'-fluoroadenylic acid
  • fU 2'-fluorouridine acid
  • fC 2'-fluorocytidylic acid
  • the sense strand in the RNAi agent is selected from the sequences in Table 6 below.
  • the 2' position of the 5', 7, 8, and 9 nucleotides starting from the 5' end of the sense strand is fluorine, and the 2' position of the remaining nucleotides is methoxyl, and the end of the antisense strand has at least Phosphate bonds between 3 adjacent nucleotides can be thiolated.
  • the sense strand of an RNAi agent described herein differs from each sequence in Table 6 by one, two, or three nucleotides.
  • the combination of sense and antisense strands in the RNAi agent is selected from Table 7.
  • RNAi agent of the present application relates to the way of coupling the interfering nucleic acid to the carrier to enhance the stability, activity, cellular distribution or cellular uptake of the RNAi agent.
  • the distribution, targeting or stability of the RNAi agent is altered by introducing a ligand of a target tissue receptor into the carrier.
  • a specific ligand may provide specificity for a selected target (e.g., molecule, cell or cell type, compartment (e.g., cell or organ compartment, body tissue, organ or region)) compared to a species in which the ligand is not present. enhanced affinity.
  • Ligands can include naturally occurring substances such as proteins (e.g. human serum albumin (HSA), low density lipoprotein (LDL) or globulin); carbohydrates (e.g. dextran, pullulan, chitin, chitosan sugar, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid); or a lipid.
  • a ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, for example a synthetic polyamino acid.
  • Ligands may also include targeting moieties, such as cell or tissue targeting agents, such as lectins, glycoproteins, lipids or proteins, such as antibodies, that bind to a given cell type, such as kidney cells.
  • Targeting groups can be thyrotropin, melanin, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N- Acetyl-glucosamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acid, polyvalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipid , cholesterol, steroids, cholic acid, folic acid, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptidomimetic.
  • the ligand is a multivalent galactose, eg, N-ace
  • the sense strand and antisense strand contained in the RNAi agent of the present application can be conveniently and routinely prepared by the known technique of solid-phase synthesis. Any other method known in the art for such synthesis, such as solution phase synthesis or fermentation, may additionally or alternatively be used. It is also known to use similar techniques to prepare other oligonucleotides such as phosphorothioate and alkylated derivatives.
  • the oligonucleotide or Linked nucleotides can be synthesized by an automated synthesizer using the phosphoramidite method derived from carrier-nucleoside phosphoramidite monomers.
  • 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. ' end.
  • 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 coupled to the sense strand. 3' end; 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 the 5' end of the sense strand and 3' ends.
  • 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 the antisense strand, and the 3'MVIP 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 is a target-specific ligand
  • L is a branched chain
  • R 1 and R 2 are transfer points
  • the 5'MVIP is connected to the 5' end of the sense strand or the 5' end of the antisense strand through the transfer point R1
  • the 3'MVIP is connected to the 3 ' end of the sense strand or the 3' end of the antisense strand through the transfer point R2 connection
  • n and m are each independently any integer of 0-4.
  • tissue-specific targeting ligand is a tissue-specific targeting ligand
  • the liver X targets a specific ligand.
  • R 1 or R 2 is linked to said sense strand or antisense strand through phosphate or modified phosphate, preferably through phosphate or phosphorothioate.
  • m or n can be 0, i.e. no 3'MVIP or 5'MVIP is present.
  • the structure of the 3'MVIP may be:
  • the structure of the 3'MVIP may be:
  • the structure of the 3'MVIP may be:
  • the structure of the 3'MVIP can be:
  • the structure of the 3'MVIP may be:
  • said n refers to the sum of n placed in the sense strand and antisense strand 5' end 5'MVIP of said RNAi agent at the same time
  • said m refers to the sum of n placed on said RNAi agent at the same time The sum of m in the 3'MVIP at the 3' end of the sense and antisense strands of the agent.
  • the R 1 and R 2 have -NH-, -S- and/or -O- in the structure, and R 1 and R 2 have -NH-, -S- or -O in the structure - Linked to the linking strand D and the 5' and 3' ends of the sense strand and/or the antisense strand, respectively, R1 and R2 are the same or different.
  • the R 1 and R 2 are optionally straight chains, or straight chains or cyclic structures with amide, carboxyl or alkyl branched chains, and the cyclic structures include saturated or unsaturated A saturated aliphatic carbocyclic group, or a five-membered or six-membered heterocyclic group or an aromatic hydrocarbon group containing sulfur, oxygen or nitrogen atoms.
  • the R 1 and/or R 2 are -E 1 (CH 2 ) x CH 2 E 2 -, wherein x is any integer from 3 to 12, and the groups E 1 and E 2 can be respectively is -NH-, -S- or -O-.
  • the R 1 and/or R 2 are -E 1 (CH 2 ) x1 CH(OH)(CH 2 ) x2 E 2 -, wherein each of x1 or x2 is independently 3-10 Any integer, E 1 and E 2 can be -NH-, -S- or -O-, respectively.
  • the R is a heterocyclic or carbocyclic structure containing N, S or O:
  • the transition point R 1 is -NH(CH 2 ) x CH 2 O-, wherein x is any integer of 3-12, preferably any integer of 4-6, which can be obtained by the following two methods: A phosphoramidite monomer is introduced:
  • One of the -O- or -S- is used for the synthesis of the R 1 phosphoramidite monomer, which is connected to the 5' end of the sense or antisense strand of the RNAi agent by solid-phase synthesis.
  • -NH-, -S- or -O- is used to connect with the connecting strand D in the 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.
  • An exemplary structure of a monomer introduced into the 5' end of the sense or antisense strand of an RNAi agent is as follows:
  • the sense strand or antisense strand 5' MVIP phosphoramidite monomer preferably has the following structure:
  • the linker B in the above-mentioned monomer is branched 1 to 4 times to obtain the corresponding monomeric compound.
  • the liver-targeting specific ligand X It is introduced at the 5' end of the sense or antisense strand by solid phase synthesis.
  • the transition point R 1 is -NH(CH 2 ) x CH 2 O-, wherein x can be an integer of 3-12, preferably an integer of 4-6.
  • the 5'MVIP phosphoramidite monomer structure is selected from the following structures:
  • the R2 is a heterocyclic or carbocyclic structure containing N, S or O:
  • the R 2 is -NH(CH 2 ) x1 CH(OH)(CH 2 ) x2 CH 2 O-, wherein x1 is any integer from 1-4, and x2 is any integer from 0-4 .
  • R 2 described in this application is formed by succinic anhydride and -NH-, -S- or -O- in the structure of R 2 forming an ester or amide, and at the same time coupling with -NH- in the blank Solid Support to form 3 'MVIP solid spport, and then through the phosphoramidite solid-phase synthesis method, the 3'MVIP is introduced to the 3' end of the sense strand or the antisense strand.
  • the heterocyclic ring in the R2 structure is a pyrrole ring or a piperidine ring, which is connected to the connecting chain D of the 3'MVIP through a nitrogen heteroatom in the ring, and the exemplary structure of the 3'MVIP solid spport is as follows:
  • R 2 is -B 4 (CH 2 ) x1 CH(OH)(CH 2 ) x2 CH 2 B 5 -, wherein x1 is an integer of 1-4, x2 is an integer of 0-4, B 4 and B 5 are -NH-, -S- or -O-, respectively.
  • R2 is -NHCH2CH (OH) CH2O- .
  • the exemplary structure of introducing 3'MVIP solid spport is as follows:
  • the 3'MVIP solid support structure is as follows:
  • the liver-targeting specific ligand X is selected from structures used to enhance the uptake of RNAi agents by liver cells, which can be lipids, steroids, vitamins, sugars, proteins, peptides, polyamines and Peptide mimetic structure.
  • RNAi agents lipids, steroids, vitamins, sugars, proteins, peptides, polyamines and Peptide mimetic structure.
  • the liver-targeting specific ligands X introduced at the ends of the sense strand or antisense strand of the RNAi agent can be the same or different, for example, in terms of characteristics, some can enhance liver targeting , some can be the regulatory structure of the RNAi agent's pharmacokinetics in vivo, and some can be structures with in vivo dissolution activity.
  • the specific liver-targeting ligand X is selected from one or more monosaccharides and derivatives thereof in the following structures.
  • the monosaccharide is selected from one or more of the following structures: mannose, galactose, D-arabinose, glucose, fructose, xylose, glucosamine, ribose.
  • the monosaccharide derivatives are selected from mannose derivatives, galactose derivatives, glucose derivatives, ribose derivatives and other derivatives.
  • the targeting unit X is selected from galactose, galactosamine, N-acetylgalactosamine and derivatives thereof, and its general structural formula is as follows:
  • W 1 is hydrogen or a hydroxyl protecting group, which can be the same or different;
  • W is -OH, -NHCOOH or -NHCO(CH 2 ) q CH 3 , where q is an integer of 0-4;
  • W 2 is -NH- , O, S or C.
  • the targeting unit X is N-acetylgalactosamine and its derivatives.
  • the targeting unit X is selected from the following structures:
  • W is selected from one or both of -OH, -NHCOOH or -NHCO(CH 2 ) q CH 3 , wherein q is an integer of 0-4.
  • the specific liver-targeting ligands X can be the same or different in the same 5'MVIP or 3'MVIP structure.
  • X between 5'MVIP and 3'MVIP can be the same or different from each other.
  • the L also has a side chain of hydroxyethyl or carboxylic acid.
  • the L is a C 7 -C 18 straight chain containing an amide group or a six-membered aliphatic carbocyclic group.
  • the L is selected from one or more of the following structures:
  • r1 is any integer of 1-12
  • r2 is any integer of 0-20
  • Z is H, an alkyl group or an amido group.
  • the structure of B is related to the number of X that can be introduced, and the B contains -NH-, C, O, S, amido, phosphoryl, thiophosphoryl, when n or When m is 1, it is a straight chain; when n or m is 2, 3 or 4, the number of forks is 2, 3 or 4, respectively.
  • said B is selected from the following structures:
  • A1 and A2 are each independently C, O, S, -NH-, carbonyl, amido, phosphoryl or thiophosphoryl, and r is an integer of 0-4.
  • said B is selected from the following structures:
  • r is any integer of 0-4.
  • said B is selected from the following structures:
  • said B is selected from the following structures:
  • the D further has a side chain of a hydroxymethyl group, a methyl tert-butyl group, a methyl phenol group, or a C 5 -C 6 alicyclic group.
  • the D is a C 3 -C 10 straight chain containing two C ⁇ O, six-membered aliphatic carbocyclyl or phenyl.
  • the D is selected from the following structures:
  • the D is selected from the following structures:
  • the D is selected from the following structures:
  • (XL) n -BD- in the 5'MVIP structure and (XL) m -BD- in the 3'MVIP structure are selected from one or more of the following structures:
  • 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.
  • the (XL) n -BD- in the 5'MVIP structure is selected from the structures shown in Table 8:
  • 5'MVIP may also be absent, and m may be an integer of 2-4 at this time.
  • the (XL) m -BD- in the 3'MVIP structure is selected from the structures shown in Table 9:
  • the combination of (XL) n -BD- and R 1 in the 5′MVIP ligand structure is as shown in Table 10.
  • 3'MVIP may be absent, in which case n may be 2-4.
  • the (XL)mBD- and R2 combinations in the 3'MVIP ligand structure are as shown in Table 11.
  • the 5'MVIP is selected from any one of 5'MVIP01 to 5'MVIP22 in Table 10. In certain embodiments, the 5'MVIP is selected from:
  • the 3'MVIP is selected from any one of 3'MVIP01 to 3'MVIP27 in Table 11. In certain embodiments, the 3'MVIP is selected from:
  • 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 end and the middle position of the sequence, to investigate the effect on AGT mRNA expression level Impact.
  • the 3' end of the antisense strand sequence in Table 5 can be coupled to the vector construct 3'MVIP09.
  • the antisense strand in the RNAi agent can be selected from the sequences in Table 13 below.
  • the antisense strand of an RNAi agent described herein differs from each sequence in Table 13 by one, two, or three nucleotides.
  • the 5' end of the sense strand sequence in Table 6 can be coupled to the vector construct 5'MVIP09.
  • the sense strand in the RNAi agent can be selected from the sequences in Table 14 below.
  • the sense strand of an RNAi agent described herein differs from each sequence in Table 14 by one, two, or three nucleotides.
  • AGT is mainly expressed in the liver, and its expression is limited to humans and non-primates.
  • monkeys are the model of choice for preclinical studies.
  • the RNAi agent is selected from the sequences in Table 15.
  • Table 15 contains the RNAi agent of 5'MVIP09/3'MVIP09 combination
  • the sense and antisense strands of the RNAi agents described herein differ from each sequence in Table 15 by one, two, or three nucleotides.
  • 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, the The antisense strand is selected from Table 16 below:
  • the antisense strand of an RNAi agent described herein differs from each sequence in Table 16 by one, two, or three nucleotides.
  • the 5' end and/or 3' end of the sense strand GsCsAUfUUfUfUfUUUUGAGCUUGAsAsG (SEQ ID NO: 194) of the RNAi agent is linked to 5'MVIP and/or 3'MVIP of different structures, the sense Chains were selected from Table 17 below.
  • the sense strand of an RNAi agent described herein differs from each sequence in Table 17 by one, two, or three nucleotides.
  • the RNAi agent described in the application is composed of the sense strand of Table 17 or a sequence that differs from these sense strands by one, two or three nucleotides and the antisense strand in Table 16 or with these
  • the antisense strand is formed by random pairing of one, two or three nucleotide sequences.
  • the RNAi agent described in the present application is 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.
  • Table 18 contains RNAi agents combining 5'MVIP and 3'MVIP
  • RNAi agent or pharmaceutically acceptable salt thereof described herein is preferably prepared or synthesized in the form of sodium salt and triethylamine salt or other pharmaceutically acceptable salts.
  • the RNAi agent or its pharmaceutically acceptable salt is more preferably its sodium salt or triethylamine salt.
  • the present application also includes a pharmaceutical composition comprising the RNAi agent of the present application or a pharmaceutically acceptable salt thereof.
  • RNAi agent described herein and a pharmaceutically acceptable excipient.
  • Pharmaceutical compositions comprising RNAi agents are useful for the prevention and/or treatment of AGT-related disorders, eg, hypertension.
  • Such pharmaceutical compositions are formulated according to the mode of delivery.
  • One example protocol is formulation of compositions for systemic administration with parenteral delivery, eg, subcutaneous (SC), intramuscular (IM) or intravenous (IV) delivery.
  • the pharmaceutical composition of the present application can be administered at a dose sufficient to inhibit the expression of the AGT gene.
  • a pharmaceutically acceptable "adjuvant” or “excipient” is a pharmaceutically acceptable solvent, suspending agent, or any other pharmaceutically inert vehicle used to deliver one or more nucleic acids to an animal. Excipients can be liquid or solid and are selected with regard to the intended mode of administration to provide the desired volume, consistency, etc. when combined with the nucleic acid and other components of a given pharmaceutical composition. RNAi agents can be delivered in a manner that targets specific tissues (eg, liver cells).
  • the pharmaceutical composition further comprises a delivery vehicle (such as nanoparticles, dendrimers, polymers, liposomes or cationic delivery systems),
  • a delivery vehicle such as nanoparticles, dendrimers, polymers, liposomes or cationic delivery systems
  • the delivery vehicle comprises liposomes.
  • the delivery vehicle includes nanolipids capable of forming liposome-nucleic acid nanoparticles with nucleic acid molecules.
  • the delivery vehicle includes the amphoteric lipid compound M10C1.
  • compositions of the present application include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be produced from a variety of components including, but not limited to, pre-formed liquids, self-emulsifying solids, and self-emulsifying semi-solids. Formulations include those targeted to the liver.
  • the pharmaceutical formulations of the present application which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredient with pharmaceutical adjuvants or excipients.
  • the present application provides a method for reducing AGT mRNA or protein expression in cells or tissues, which includes making cells or tissues with an effective amount of the aforementioned RNAi agent that inhibits AGT gene expression or a pharmaceutically acceptable salt thereof, And/or contact with the aforementioned pharmaceutical composition.
  • Cells suitable for treatment using the methods of the present application may be any cells expressing the AGT gene, for example, liver cells, brain cells, gallbladder cells, heart cells or kidney cells, but are preferably liver cells.
  • Cells suitable for use in the methods of the present application may be mammalian cells, and the RNAi agent inhibits expression of the AGT gene (e.g., human, primate, non-primate, or rat AGT gene) when contacted with a cell expressing the AGT gene
  • At least about 50% for example, can be determined by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as immunofluorescence assays, Western blots or flow cytometric analysis techniques.
  • tissue is liver tissue.
  • the term “inhibit” is used interchangeably with “reduce”, “decrease”, “silence”, “downregulate”, “suppress” and other similar terms and include any level of inhibition.
  • Expression of the AGT gene can be assessed in terms of the level or change in level of any variable associated with AGT gene expression, for example, AGT mRNA levels or AGT protein levels. This level can be assayed in individual cells or in populations of cells including, for example, a sample derived from a subject. Inhibition can be assessed by a reduction in the absolute or relative level of one or more variables associated with AGT expression compared to control levels.
  • the control level can be any type of control level used in the art, for example, a pre-dose baseline level or a similar subject that is untreated or treated with a control (such as, for example, a buffer only control or no active agent control), The level measured in a cell or sample.
  • Inhibition of AGT gene expression can be by wherein the AGT gene is transcribed and has been processed (e.g., by contacting one or more cells with an RNAi agent of the present application, or by administering an RNAi agent of the present application to a subject in which the cells are present)
  • a first cell or cell population that inhibits AGT gene expression (such cells may, for example, be present in a sample derived from a subject) expresses an amount of mRNA that is substantially the same as that of the first cell or cell population but not so treated
  • a reduction compared to a second cell or population of cells control cells not treated with an RNAi agent or not treated with an RNAi agent targeting a gene of interest) is expressed.
  • inhibition is evaluated in a cell line with high AGT expression using the method provided in Example 2 using an appropriate concentration of siRNA, and the mRNA level in the intervened cells is expressed as the percentage of the mRNA level in the non-intervened control cells .
  • inhibition of AGT gene expression can be assessed by a reduction in a parameter functionally related to AGT gene expression, eg, AGT protein levels in the subject's blood or serum.
  • AGT gene silencing can be determined in any cell expressing AGT (endogenous or exogenous from an expression construct) and by any assay known in the art.
  • Inhibition of AGT protein expression can be manifested by a decrease in the level of AGT protein expressed by a cell or population of cells or a sample from a subject (eg, the level of protein in a blood sample derived from a subject).
  • a cell or population of cells or a sample from a subject e.g, the level of protein in a blood sample derived from a subject.
  • inhibition of protein expression levels in treated cells or cell populations can similarly be expressed as a percentage of protein levels in control cells or cell populations, or in a subject sample (e.g., blood or a derivative thereof) Changes in protein levels in serum).
  • 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.
  • a control cell, population of cells, or subject sample can be derived from a single subject (eg, a human or animal subject) or an appropriately matched population control prior to treatment with an RNAi agent.
  • the level of AGT mRNA expressed by a cell or population of cells can be determined using any method known in the art for assessing mRNA expression. For example, qRT-PCR, assessing reduction in gene expression. Reduction of protein production can be assessed by any method known in the art, eg, ELISA.
  • a needle liver biopsy sample is used as tissue material to monitor for decreased expression of the AGT gene or protein.
  • a blood sample is used as a sample of a subject to monitor for decreased expression of AGT protein.
  • the present application provides the aforementioned RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof, or the use of the aforementioned pharmaceutical composition in the preparation of medicines, which are used to prevent and/or treat diseases or condition or reduce the risk of a disease or condition.
  • the disease or disorder comprises a disease or disorder associated with AGT.
  • the disease or condition is selected from the group consisting of hypertension, hypertension, borderline hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, Pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, low plasma renin activity, or plasma renal Hypertension related to hormone concentration, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, arterial hypertension Atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, coarctation of the aorta, aortic aneurysm, ventricular fibrosis
  • the present application provides a method for preventing and/or treating a disease or a disease, the method comprising administering an effective amount of the aforementioned RNAi agent for inhibiting AGT gene expression or its pharmaceutically acceptable amount to a subject in need thereof. Acceptable salts, and/or the aforementioned pharmaceutical compositions.
  • compositions may be administered by any means known in the art, including but not limited to: oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intracerebroventricular, intraparenchymal, and intrathecal), intravenous, Intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration.
  • intracranial e.g., intracerebroventricular, intraparenchymal, and intrathecal
  • intravenous Intramuscular
  • subcutaneous e.g., transdermal
  • airway aerosol
  • nasal rectal
  • topical including buccal and sublingual
  • the compositions are administered by intravenous infusion or injection.
  • the composition is administered by subcutaneous injection.
  • the composition is administered by intramuscular injection.
  • RNAi agents of the present application can also be administered as "free RNAi agents.” Free RNAi agents are administered in the absence of the pharmaceutical composition. Naked RNAi agents can be in a suitable buffer.
  • the buffer may comprise acetate, citrate, prolamins, carbonate or phosphate, or any combination thereof.
  • the buffer is phosphate buffered saline (PBS). The pH and osmolarity of the buffer containing the RNAi agent can be adjusted for suitable administration to the subject.
  • RNAi agents of the present application can be administered as pharmaceutical compositions, such as liposomal formulations.
  • RNAi agents of the present application can be administered at a dose sufficient to inhibit the expression of the AGT gene.
  • suitable dosages of RNAi agents of the present application are in the range of about 0.001 to about 200.0 mg per kilogram of body weight of the recipient per day, usually in the range of about 1 to 50 mg per kilogram of body weight per day.
  • a suitable dose of the RNAi agent of the present application is in the range of about 0.1 mg/kg to about 5.0 mg/kg, such as in the range of about 0.3 mg/kg to about 3.0 mg/kg.
  • the method comprises administering a composition described herein such that target AGT gene expression is reduced, such as about 1, 2, 3, 4, 5, 6, 1-6, 1-3, or 3- 6 months.
  • the composition is administered every 3-6 months.
  • Treatment is administered less frequently.
  • Repeated dose regimens can include administering a therapeutic amount of an RNAi agent on a regular basis, such as monthly to yearly.
  • the RNAi agent is administered about once a month to about every three months, or about every three months to about every six months.
  • treatment may be administered less frequently.
  • duration of treatment may be determined by the severity of the disease.
  • a single dose of a pharmaceutical composition may be depoted such that doses are administered at intervals of no more than 1, 2, 3, or 4 months.
  • a single dose of the pharmaceutical composition of the present application is administered about once a month.
  • a single dose of the pharmaceutical composition of the present application is administered quarterly (ie, about every 3 months).
  • a single dose of the pharmaceutical composition of the present application is administered twice a year (ie, about once every 6 months).
  • treatment of a subject with a prophylactically and/or therapeutically effective amount of the composition can include a single treatment or a series of treatments as needed.
  • it further comprises determining the level of AGT in a sample from said subject.
  • it further comprises determining the level of AGT protein in a blood sample, serum sample or urine sample from said subject.
  • it further comprises administering to said subject an additional therapeutic agent for treating hypertension.
  • the additional therapeutic agent may be selected from: diuretics, angiotensin converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, beta-blockers, vasodilators, calcium channel blockers , aldosterone antagonists, ⁇ 2-agonists, renin inhibitors, ⁇ -blockers, peripherally acting adrenergic agents, selective D1 receptor partial agonists, nonselective ⁇ -adrenergic antagonists, synthetic Steroidal antimineralocorticoids, angiotensin receptor-neprilysin inhibitors (ARNi), sacubitril/valsartan; or endothelin receptor antagonists (ERA), sitaxsentan, Bisentan, atrasentan, BQ-123, zipotetan, bosentan, macitentan, and tizosentan; combinations of any of the foregoing; and therapeutic agents for hypertension formulated as a combination of agents.
  • ACE angiotensin converting enzyme
  • the additional therapeutic agent comprises an angiotensin II receptor antagonist.
  • angiotensin II receptor antagonist can be selected from: losartan, valsartan, olmesartan, eprosartan and azilsartan.
  • the present application provides a cell comprising the aforementioned RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof.
  • the present application provides a kit comprising the aforementioned RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition.
  • RNAi agent or a pharmaceutically acceptable salt thereof, the structure of the RNAi agent contains a carrier structure and an interfering nucleic acid, and its structure is as shown in formula IIIa, IIIb or IIIc:
  • the interfering nucleic acid targets the AGT gene, which includes an antisense strand and a sense strand;
  • the carrier structure includes 5'MVIP (5'MultiValent Import Platform) and 3'MVIP (3'MultiValent Import Platform);
  • the 5'MVIP consists of a transfer point R 1 , a connecting chain D, a linker B, a branched chain L and a liver-targeting specific ligand X
  • the 3'MVIP consists of a transfer point R 2 , a connecting chain D, a linker B.
  • the 5'MVIP is connected to the 5' end of the sense chain or the 5' end of the antisense chain through the transfer point R1
  • the 3'MVIP is connected to the 5' end of the antisense chain through the transfer point R1.
  • Point R 2 is 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 from 0 to 4.
  • RNAi agent or the pharmaceutically acceptable salt thereof according to embodiment 1, wherein the interfering nucleic acid comprises siRNA or miRNA.
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-3, wherein R is a heterocyclic or carbocyclic structure containing N, S or O:
  • the R 1 is -NH(CH 2 ) x CH 2 O-, wherein x is any integer of 3-12, preferably any integer of 4-6.
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-4, said R is a heterocyclic or carbocyclic structure containing N, S or O:
  • the R 2 is -NH(CH 2 ) x1 CH(OH)(CH 2 ) x2 CH 2 O-, wherein x1 is any integer from 1-4, and x2 is any integer from 0-4.
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-5, wherein X is selected from structures for enhancing the uptake of the RNAi agent by hepatocytes.
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-6, wherein X is selected from monosaccharides and derivatives thereof.
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-7, wherein X is N-acetylgalactosamine and its derivatives.
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-8, wherein X is selected from the following structures:
  • W is selected from one or both of -OH, -NHCOOH or -NHCO(CH 2 ) q CH 3 , wherein q is an integer of 0-4.
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-9, wherein L is selected from one or more of the following structures:
  • r1 is any integer of 1-12
  • r2 is any integer of 0-20
  • Z is H, an alkyl group or an amido group.
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-10, said B is selected from the following structures:
  • A1 and A2 are each independently C, O, S, -NH-, carbonyl, amido, phosphoryl or thiophosphoryl, and r is an integer of 0-4.
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-11, wherein D is selected from the following structures:
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-12, (XL)n-BD- in the 5'MVIP structure and (XL) n -BD- in the 3'MVIP structure ) m -BD- is selected from one or more of the following structures:
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-13, said X, L, D and B are inside each of 5'MVIP and 3'MVIP or 5'MVIP Same or different from 3'MVIP.
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-14 is selected from any one of 5'MVIP01 to 5'MVIP22 in Table 10.
  • the 3'MVIP is selected from any one of 3'MVIP01 to 3'MVIP27 in Table 11.
  • RNAi agent or pharmaceutically acceptable salt thereof according to any one of embodiments 1-16, wherein the combination of sense strand 5'MVIP and antisense strand 3'MVIP is 5'MVIP01/3'MVIP01, 5'MVIP01/3'MVIP17 or 5'MVIP09/3'MVIP09; or the combination of sense strand 5'MVIP and sense strand 3'MVIP is 5'MVIP01/3'MVIP09 or 5'MVIP09/3'MVIP01.
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-17, wherein the complementary region formed by the antisense strand and the sense strand comprises at least 12 consecutive cores nucleotides, wherein the sense strand comprises AGT mRNA NM_001382817.3 starting positions are 1854-1874, 1907-1927, 1895-1915, 1352-1372, 1903-1923, 2019-2039, 1853-1873 and 1818-1838
  • the sequence of at least 12 consecutive nucleotides or the sequence with a difference of no more than 3 nucleotides or the starting position in NM_001384479.1 is 1822-1842, 1875-1895, 1863-1883, 1320-1340, 1871-1891, 1987 -
  • RNAi agent according to any one of embodiments 1-18, or a pharmaceutically acceptable salt thereof, wherein there is at least about 80% base complementarity between the sense strand and the antisense strand.
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-19, wherein the sense strand and the antisense strand are each independently 15-30 nucleotides, preferably 17- 25 nucleotides, more preferably 19-23 nucleotides.
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-20, wherein the sense strand is identical to SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:8, Any one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 17 and SEQ ID NO: 18 or a sequence with a difference of no more than 3 nucleotides has substantial homology .
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-21, wherein the antisense strand comprises 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.
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-22, wherein the sense strand comprises SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:44, Any one of 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.
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-23, wherein the antisense strand comprises 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.
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-24, wherein one or more nucleotides on the sense strand and/or the antisense strand are modified to form Modified nucleotides.
  • RNAi agent according to embodiment 25 or a pharmaceutically acceptable salt thereof, wherein the modified nucleotide is selected from the group consisting of: deoxyribonucleotides, nucleotide mimics, abasic nucleotides, 2'-modified nucleotides, 3' to 3' linked (inverted) nucleotides, nucleotides containing unnatural bases, bridging nucleotides, peptide nucleic acid (PNA), unlocked nucleobase analogs, Locked nucleotides, 3'-O-methoxy (2' internucleoside linkage) nucleotides, 2'-fluoro-arabino nucleotides, 5'-methyl/2'-fluoro nucleotides , morpholino nucleotides, vinylphosphonate deoxyribonucleotides, vinylphosphonate-containing nucleotides, and cyclopropylphosphonate-containing nucleotides.
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-26, wherein part or all of the 2' positions of the nucleotide sugar groups of the sense strand and the antisense strand are fluorine or Methoxy.
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-27, wherein there is an At least two consecutive phosphorothioate linkages.
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-28, wherein the 7th, 12th, and 14th nucleotide sugar groups starting from the 5' end of the antisense strand are 2 The ' position is fluorine, the 2' position of the remaining nucleotide sugars of the antisense strand is methoxyl, and there are at least two consecutive phosphorothioates between the three consecutive nucleotides at the end of the antisense strand bond; the 5', 7, 8, and 9 nucleotide sugars at the 5' end of the sense strand are fluorine at the 2' position, and the 2' positions of the remaining nucleotide sugars of the sense strand are methoxy; and There are at least two consecutive phosphorothioate bonds between the three consecutive nucleotides at the end of the sense strand.
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-29, wherein the antisense strand comprises 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.
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-30, wherein the sense strand comprises SEQ ID NO: 185, SEQ ID NO: 191, SEQ ID NO: 192, Any one of 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.
  • RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-31, wherein the interfering nucleic acid comprises any one of Kylo-09-DS01 to Kylo-09-DS112.
  • RNAi agent or a pharmaceutically acceptable salt thereof that inhibits AGT gene expression comprising an antisense strand, wherein the antisense strand comprises at least 12 nucleotides that are substantially complementary to the corresponding positions selected from the following sequences Consecutive nucleotides or a sequence with a difference of no more than 3 nucleotides:
  • AGT mRNA NM_001382817.3 starts at 1854-1874, 1907-1927, 1895-1915, 1352-1372, 1903-1923, 2019- At least 12 consecutive nucleotides of 2039, 1853-1873 and 1818-1838 or the sequence with a difference of no more than 3 nucleotides or the starting position in NM_001384479.1 is 1822-1842, 1875-1895, 1863-1883, 1320-1340, 1871-1891, 1987-2007, 1821-1841 and 1786-1806 sequences of at least 12 consecutive nucleotides or a difference of no more than 3 nucleotides.
  • RNAi agent for inhibiting AGT gene expression according to embodiment 33 or a pharmaceutically acceptable salt thereof, wherein the length of the antisense chain is 15-30 nucleotides, preferably 17-25 nucleotides, More preferably 19-23 nucleotides.
  • RNAi agent or pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 33-34, wherein the antisense strand comprises the following nucleotide sequence: 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 not differing therefrom by more than A sequence of 3 nucleotides.
  • RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-35, or a pharmaceutically acceptable salt thereof, said RNAi agent comprising a single-stranded or double-stranded nucleic acid molecule.
  • RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof according to any one of embodiments 33-36, the RNAi agent comprising siRNA or miRNA.
  • RNAi agent or a pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 33-37, which further comprises a sense strand, wherein the complementary region formed by the antisense strand and the sense strand , the complementary region includes at least 12 consecutive nucleotides.
  • RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof according to any one of embodiments 33-38, wherein the complementary region comprises 12-25 consecutive nucleotide base pairs.
  • RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-39, or a pharmaceutically acceptable salt thereof, wherein there are at least about 80% of bases between the sense strand and the antisense strand complementary.
  • RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-40, or a pharmaceutically acceptable salt thereof, wherein the positive-sense strand comprises AGT mRNA NM_001382817.3 whose starting position is 1854-1874 , 1907-1927, 1895-1915, 1352-1372, 1903-1923, 2019-2039, 1853-1873, and 1818-1838 are sequences of at least 12 consecutive nucleotides or differ therefrom by no more than 3 nucleotides or NM_001384479 .1 At least 12 consecutive nucleotides starting from 1822-1842, 1875-1895, 1863-1883, 1320-1340, 1871-1891, 1987-2007, 1821-1841 and 1786-1806 or not different from them Sequences of more than 3 nucleotides.
  • RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-41 or a pharmaceutically acceptable salt thereof, wherein the length of the positive-sense strand is 15-30 nucleotides, preferably 17-25 nucleotides nucleotides, more preferably 19-23 nucleotides.
  • RNAi agent or pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 33-43, wherein the sense strand is identical to SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 7, SEQ ID NO: Any one of 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 differing therefrom by no more than 3 nucleotides has Essentially homologous.
  • RNAi agent or pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of the embodiments 33-43, wherein the sense strand is identical to SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 7, SEQ ID NO: Any one of 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 differing therefrom by no more than 3 nucleotides has Substantially homologous, and said antisense strand comprises 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:29, SEQ ID NO:30, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ Any one of ID NO: 35 and SEQ ID NO: 36 or a sequence that differs therefrom by no more than 3 nucleo
  • RNAi agent or pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 33-44, wherein the antisense strand comprises SEQ ID NO:55, SEQ ID NO:61, Any one of 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 from it by no more than 3 nucleotides .
  • RNAi agent or pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 33-45, wherein the sense strand comprises SEQ ID NO: 37, SEQ ID NO: 43, SEQ ID NO: 43, SEQ ID NO: Any one of 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.
  • RNAi agent or pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 33-46, wherein the antisense strand comprises SEQ ID NO:55, SEQ ID NO:61, Any one of 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 from it by no more than 3 nucleotides
  • the sense strand comprises 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: 48, SEQ ID NO: 53 and SEQ Any one of ID NO:54 or a sequence that differs from it by no more than 3 nucleotides.
  • RNAi agent or pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 33-47, wherein one or more nucleosides on the sense strand and/or the antisense strand Acids are modified to form modified nucleotides.
  • RNAi agent for inhibiting AGT gene expression according to embodiment 48 or a pharmaceutically acceptable salt thereof, wherein the modified nucleotides are selected from the group consisting of: deoxyribonucleotides, nucleotide mimics, alkalization base nucleotides, 2'-modified nucleotides, 3' to 3' linked (inverted) nucleotides, nucleotides containing unnatural bases, bridging nucleotides, peptide nucleic acid (PNA), unlocked nuclei Base analogs, locked nucleotides, 3'-O-methoxy (2' internucleoside linkage) nucleotides, 2'-fluoro-arabinonucleotides, 5'-methyl/2'- Fluorinated nucleotides, morpholino nucleotides, vinylphosphonate deoxyribonucleotides, vinylphosphonate-containing nucleotides, and cyclopropylphosphonate-containing nucleo
  • 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.
  • RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-50 or a pharmaceutically acceptable salt thereof, wherein part or all of the nucleotide sugars of the sense strand and the antisense strand are 2
  • the 'position substituting group is fluorine or methoxy.
  • RNAi agent or a pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 33-51, wherein three consecutive cores at the end of the sense strand and/or the end of the antisense strand There are at least two consecutive phosphorothioate linkages between nucleotides.
  • RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-52 or a pharmaceutically acceptable salt thereof, wherein the 7th, 12th, and 14th cores from the 5' end of the antisense strand are The 2' position of the nucleotide sugar group is fluorine, the 2' position of the remaining nucleotide sugar groups of the antisense chain is a methoxy group, and there are at least two consecutive nucleotides between the 3 consecutive nucleotides at the end of the antisense chain a phosphorothioate bond; the 5', 7, 8, and 9 nucleotide sugars at the 5' end of the sense strand are fluorine at the 2' position, and the 2' positions of the remaining nucleotide sugars of the sense chain are methoxy; and there are at least two consecutive phosphorothioate bonds between the three consecutive nucleotides at the end of the sense strand.
  • RNAi agent or pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 33-53, wherein the antisense strand comprises SEQ ID NO: 109, SEQ ID NO: 115, Any one of 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 from it by no more than 3 nucleotides .
  • RNAi agent or pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 33-54, wherein the sense strand comprises SEQ ID NO: 185, SEQ ID NO: 191, SEQ ID NO: 191, SEQ ID NO: Any one of 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.
  • RNAi agent or pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 33-55, wherein the interfering nucleic acid comprises Kylo-09-DS01, Kylo-09-DS07, Kylo Any one of -09-DS08, Kylo-09-DS10, Kylo-09-DS11, Kylo-09-DS12, Kylo-09-DS17, Kylo-09-DS18, Kylo-09-DS37 ⁇ Kylo-09-DS54 one or more species.
  • RNAi agent or pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 33-56, further comprising a ligand coupled to the sense strand and/or via a carrier structure or antisense strand.
  • RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof according to any one of embodiments 33-57, wherein the ligand comprises a targeting ligand.
  • 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' of the antisense strand through a carrier structure end and/or 3' end.
  • RNAi agent or a pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 33-59, wherein the carrier structure fits at the 5' end and/or 3' end of the sense strand .
  • 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 includes 5'MVIP and 3'MVIP, wherein the 5'MVIP 'MVIP is coupled to the 5' end of the sense strand and/or the antisense strand, and the 3'MVIP is coupled to the 3' end of the antisense strand and/or the sense strand, and the structure of the 5'MVIP is as follows: As shown in I, the structure of the 3'MVIP is shown in formula II,
  • X is a target-specific ligand
  • L is a branched chain
  • R 1 and R 2 are transfer points
  • the 5'MVIP is connected to the 5' end of the sense strand or the 5' end of the antisense strand through the transfer point R1
  • the 3'MVIP is connected to the 3 ' end of the sense strand or the 3' end of the antisense strand through the transfer point R2 connection
  • n and m are each independently any integer of 0-4.
  • RNAi agent or a pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 61-62, wherein said R 1 or R 2 and said sense strand or antisense strand pass through a phosphate An ester or a modified phosphate, preferably linked via a phosphate or phosphorothioate.
  • RNAi agent or a pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 61-63, wherein R is a heterocyclic or carbocyclic structure containing N, S or O:
  • the R 1 is -NH(CH 2 ) x CH 2 O-, wherein x is any integer of 3-12, preferably any integer of 4-6.
  • RNAi agent or a pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 61-64, wherein R is a heterocyclic or carbocyclic structure containing N, S or O:
  • the R 2 is -NH(CH 2 ) x1 CH(OH)(CH 2 ) x2 CH 2 O-, wherein x1 is any integer from 1-4, and x2 is any integer from 0-4.
  • RNAi agent or a pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 61-65, wherein X is a targeting ligand selected from the group used to enhance liver cells to RNAi agents ingested structure.
  • RNAi agent for inhibiting AGT gene expression or a pharmaceutically acceptable salt thereof according to any one of embodiments 61-66, wherein X is selected from monosaccharides and derivatives thereof.
  • RNAi agent or pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 61-67, wherein X is selected from the group consisting of galactose, galactosamine, N-acetylgalactosamine and its derivatives.
  • RNAi agent for inhibiting AGT gene expression according to any one of embodiments 61-68 or a pharmaceutically acceptable salt thereof, wherein X is N-acetylgalactosamine and its derivatives.
  • RNAi agent or a pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 61-69, wherein X is selected from the following structures:
  • W is selected from one or both of -OH, -NHCOOH or -NHCO(CH 2 ) q CH 3 , wherein q is an integer of 0-4.
  • RNAi agent for inhibiting AGT gene expression according to any one of embodiments 61-70 or a pharmaceutically acceptable salt thereof, wherein L is selected from one or more of the following structures:
  • r1 is any integer of 1-12
  • r2 is any integer of 0-20
  • Z is H, an alkyl group or an amido group.
  • RNAi agent or a pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 61-71, wherein B is selected from the following structures:
  • A1 and A2 are each independently C, O, S, -NH-, carbonyl, amido, phosphoryl or thiophosphoryl, and r is an integer of 0-4.
  • RNAi agent or a pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 61-72, wherein D is selected from the following structures:
  • RNAi agent for inhibiting AGT gene expression according to any one of embodiments 61-73 or a pharmaceutically acceptable salt thereof, the (XL) n -BD- and 3'MVIP in the 5'MVIP structure (XL) m -BD- in the structure is selected from one or more of the following structures:
  • RNAi agent for inhibiting AGT gene expression according to any one of embodiments 61-74 or a pharmaceutically acceptable salt thereof, wherein X, L, D, and B are respectively in 5'MVIP and 3'MVIP The same or different within or between 5'MVIP and 3'MVIP.
  • RNAi agent for inhibiting AGT gene expression according to any one of embodiments 61-75 or a pharmaceutically acceptable salt thereof, wherein the 5'MVIP is selected from 5'MVIP01 to 5'MVIP22 in Table 10 either one.
  • RNAi agent for inhibiting AGT gene expression according to any one of embodiments 61-76 or a pharmaceutically acceptable salt thereof, wherein the 3'MVIP is selected from any of 3'MVIP01 to 3'MVIP27 in Table 11 one.
  • RNAi agent for inhibiting AGT gene expression according to any one of embodiments 61-77 or a pharmaceutically acceptable salt thereof, 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 sense strand 3'MVIP is 5'MVIP01/3'MVIP09 or 5'MVIP09 /3' MVIP01.
  • RNAi agent or pharmaceutically acceptable salt thereof for inhibiting AGT gene expression includes AS131, AS137, AS138, AS140, AS141, AS142, AS147 and any one or more of AS148.
  • RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-79 or a pharmaceutically acceptable salt thereof, the sense strand includes S131, S137, S138, S140, S141, S142, S147 and Any one or more of S148.
  • 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 Kylo-09 in Table 15 - any of DS113, Kylo-09-DS119, Kylo-09-DS120, Kylo-09-DS122, Kylo-09-DS123, Kylo-09-DS124, Kylo-09-DS129 and Kylo-09-DS130 or Various.
  • RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-81 or a pharmaceutically acceptable salt thereof, wherein the antisense strand is selected from any one or more of AS140, AS207-AS266 kind.
  • RNAi agent for inhibiting AGT gene expression according to any one of embodiments 33-82 or a pharmaceutically acceptable salt thereof, wherein the positive-sense strand is selected from any one or more of S140, S207-S264 .
  • RNAi agent or pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 33-83, wherein the antisense strand is selected from any of AS140, AS207, SEQ ID NO: 413-AS266 One or more, and the sense strand is selected from any one or more of S140, S207-S264.
  • RNAi agent or pharmaceutically acceptable salt thereof for inhibiting AGT gene expression according to any one of embodiments 33-84, including Kylo-09-DS122, Kylo-09-DS131, and Kylo-09 in Table 18 - any one or more of DS141, Kylo-09-DS142 and Kylo-09-DS147.
  • a cell comprising the RNAi agent of any one of embodiments 1-32 or a pharmaceutically acceptable salt thereof, or the RNAi agent for inhibiting AGT gene expression of any one of embodiments 33-85 or its pharmaceutically acceptable salt.
  • a pharmaceutical composition comprising the RNAi agent or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-32, or the RNAi that inhibits AGT gene expression according to any one of embodiments 33-85 agent or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient, carrier and/or diluent.
  • composition of claim 87 further comprising a delivery vehicle.
  • composition of claim 88, wherein the delivery vehicle comprises liposomes.
  • composition of claim 89 wherein the delivery vehicle comprises nanolipids.
  • a method for reducing AGT mRNA or protein expression in a cell or tissue comprising making the cell or tissue an effective amount of the RNAi agent described in any one of embodiments 1-32 or a pharmaceutically acceptable salt thereof, The RNAi agent or pharmaceutically acceptable salt thereof for inhibiting AGT gene expression described in any one of Embodiments 33-85, and/or the pharmaceutical composition described in any one of Embodiments 87-90.
  • embodiment 96 wherein the disease or condition comprises an AGT-associated disease or condition.
  • the disease or condition is selected from the group consisting of: hypertension, hypertension, borderline hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt's hypertension, low plasma renin Hypertension related to active or plasma renin concentration, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertension Renal disease, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, coarctation of the aorta, aortic aneurysm, Ventricular fibrosis,
  • a method for preventing and/or treating a disease or disorder comprising administering an effective amount of the RNAi agent described in any one of embodiments 1-32 or a pharmaceutically acceptable amount thereof to a subject in need thereof Acceptable salts, the RNAi agent for inhibiting AGT gene expression described in any one of embodiments 33-85 or a pharmaceutically acceptable salt thereof, and/or the pharmaceutical composition described in any one of embodiments 87-90 .
  • administering comprises administering to the subject by subcutaneous, intravenous, oral, rectal or intraperitoneal administration routes.
  • RNAi agent RNAi agent that inhibits AGT gene expression, or pharmaceutical composition
  • the additional therapeutic agent is selected from the group consisting of diuretics, angiotensin converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, beta-blockers, vascular Dilators, calcium channel blockers, aldosterone antagonists, ⁇ 2-agonists, renin inhibitors, ⁇ -blockers, peripherally acting adrenergic agents, selective D1 receptor partial agonists, nonselective ⁇ - Adrenergic antagonists, synthetic steroidal antimineralocorticoids, angiotensin receptor-neprilysin inhibitors (ARNi), sacubitril/valsartan; or endothelin receptor antagonists (ERA ), sitaxsentan, ambrisentan, atrasentan, BQ-123, zippotentan, bosentan, macitentan, and tezosentan; combinations of any of the foregoing therapeutic agents; and formulations as pharmaceutical combinations therapeutic agent for hypertension.
  • ACE angiotensin converting enzyme
  • angiotensin II receptor antagonist is selected from the group consisting of: losartan, valsartan, olmesartan, eprosartan, and azilsartan.
  • kits comprising the RNAi agent of any one of embodiments 1-32 or a pharmaceutically acceptable salt thereof, the RNAi agent for inhibiting AGT gene expression of any one of embodiments 33-85, or A pharmaceutically acceptable salt thereof, or the pharmaceutical composition described in any one of embodiments 87-90.
  • DMSO dimethyl sulfoxide
  • the Chinese name of DMF is N,N-dimethylformamide
  • HOBt 1-hydroxybenzotriazole
  • HBTU O-benzotriazole-tetramethyluronium hexafluorophosphate
  • DIPEA N,N-Diisopropylethylamine
  • the Chinese name of DCM is dichloromethane
  • the Chinese name of DMAP is 4-dimethylaminopyridine
  • DMT-CL 4,4'-dimethoxytriphenylchloromethane
  • the Chinese name of MEOH is Methanol
  • the Chinese name of TBTU is O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroboric acid;
  • solid phase carrier such as macroporous aminomethyl resin (Resin).
  • Embodiment 1 The solid-phase phosphoramidite method of synthesizing siRNA
  • the sense strand in Table 1 and the antisense strand in Table 2 were synthesized according to the standard solid-phase phosphoramidite method, and the sense strand and the corresponding antisense strand were complementary annealed to obtain siRNA.
  • the basic steps of the solid-phase phosphoramidite method include:
  • nucleoside phosphite is oxidized into a more stable nucleoside phosphate (i.e. trivalent phosphorus is oxidized to pentavalent phosphorus);
  • the sense strand and the antisense strand lyophilized powder were redissolved and mixed equimolarly, an appropriate amount of water for injection was added, and an appropriate amount of TRIS buffer solution was added. Shake gently for about 1-2 minutes to mix the solution evenly. Raise the temperature of the water bath to 92°C to 95°C. Heat the above reaction solution in a water bath for 3-5 minutes, and shake gently to make the solution evenly heated. Naturally cool to room temperature. A colorless or light yellow transparent liquid is obtained, and a sample is sent for inspection to measure the concentration.
  • RNAi agent described in the present application obtains respective sense strands and antisense strands by the solid-phase phosphoramidite method, and the sense strand and the corresponding antisense strand are complementarily annealed to obtain the final product.
  • Example 2 RNAi agent inhibits AGT gene expression test in vitro
  • RNAi agents (Kylo-09-DS01-Kylo-09-DS18 in Table 3) prepared by the method described in Example 1.
  • the RNAi agent aqueous solution and the organic solution of DOTMA are mixed to form a water-insoluble precipitate, which is dissolved in chloroform after separation and drying, and further mixed with other lipid chloroform solutions, and other lipids include M10C1 and PEG600-cholesterol.
  • the mixture is vacuum-centrifuged, evaporated and dried overnight to obtain the RNAi agent coated with nano-lipid, wherein the weight ratio of DOTMA, M10C1 and PEG600-cholesterol to the RNAi agent is 1-1.6, 1.5-2.5 and 2.5-3.5.
  • DMEM containing 10% fetal bovine serum was used to prepare sample solutions of nano-lipid-encapsulated RNAi agents (RNAi agents in Table 3: Kylo-09-DS01 ⁇ Kylo-09-DS18) of corresponding concentrations.
  • RNAi agents in Table 3 Kylo-09-DS01 ⁇ Kylo-09-DS18
  • Hep3B cells were inoculated at a cell density of 10 5 in DMEM medium with 10% fetal bovine serum, 37°C, 5% CO2, cultured for 24 hours, then intervened with samples of different concentrations (10nM, 1nM, 0.1nM), incubated for 72 hours, and the cells were collected For samples, add 1ml of Ezol lysate to the collected cell samples, and vortex to mix.
  • RNAi agents in Table 3 formed by the annealing of the sense strand in Table 1 and the antisense strand in Table 2 exhibit different degrees of inhibitory effects 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 on AGTmRNA expression level in Hep3B cells Inhibition showed obvious dose-dependent tolerance, and the corresponding sense strand sequences were SEQ ID NO.37, 43, 44, 46, 47, 48, 53 and 54 and the corresponding antisense strand sequences were SEQ ID NO.55, 61, 62, 64, 65, 66, 71 and 72.
  • 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 refers to the experimental operation of Hep 3B cells to investigate the intervention effect of different concentrations of RNAi agents (10nM, 0.1nM) on HepG 2 cells, and measure the AGT mRNA level by QRT-PCR. Compared with the supernatant of unintervened HepG 2 cells, the relative percentage of AGT mRNA in the intervention group of the calibration sample was compared. The test results obtained are shown in Table 20 below and in Figure 1B.
  • RNAi agent involved in this embodiment is selected from Table 7, and the parent strand is the sequence preferred in Example 2 (the sense strand sequence is SEQ ID NO.37, 43, 44, 46, 47, 48, 53 and 54 and the corresponding Antisense strand sequence SEQ ID NO.55, 61, 62, 64, 65, 66, 71 and 72).
  • the 2' position of the 7th, 12th, and 14th nucleotides from the 5' end of the antisense strand is fluorine and the 2' position from the remaining nucleotides is a methoxyl group, and the end of the antisense strand is at least There are 3 phosphate bonds between adjacent nucleotides that can be thiolated, and the 2' positions of the 5th, 7th, 8th, and 9th nucleotides starting from the 5' end of the sense strand are fluorine and from the rest of the nucleosides
  • the 2' position of the acid is a methoxyl group, and at least three phosphate bonds between adjacent nucleotides at the end of the antisense chain can be thiolated.
  • the purpose of this example is to verify that the above-mentioned modification method can enhance the stability of the RNAi agent in human serum.
  • the test results are shown in Table 21 and FIG. 2 .
  • Figure 2 shows the HPLC graphs of the stability detection of RNAi Kylo-09-DS46 and its parent chain Kylo-09-DS10 at different time periods.
  • RNAi agent in different time lengths in human serum the peak area ratio % of the full-length duplex relative to 0h.
  • the solid support of the 3'MVIP serves as the starting monomer for solid-phase synthesis.
  • the sense strand or antisense strand of the RNAi agent of the present application is coupled with a carrier structure 5'MVIP, the 5'MVIP phosphoramidite monomer is used as the last monomer of solid-phase synthesis.
  • the solid support of 3'MVIP is used as the starting monomer for solid-phase synthesis, and the solid spport general formula of 3'MVIP is as follows :
  • the linker B in the general formula is branched 1 to 4 times to obtain the corresponding Solid Support of 3'MVIP.
  • the resulting Solid Support is used as the starting monomer for the solid-phase synthesis of the sense strand and the antisense strand of Kylo-09-DS133 of the RNAi agent Kylo-09-DS142 (see Figure 9D for the structural formula); when m is At 2 o'clock, the obtained Solid Support was used as the starting monomer for the solid-phase synthesis of the sense strand of the RNAi agent Kylo-09-DS141 (see structural formula 9C) and the antisense strand of Kylo-09-DS122;
  • 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.
  • the 5'MVIP phosphoramidite monomer is the last phosphoramidite monomer synthesized as the sense strand or the antisense strand in solid phase .
  • the general formula of 5'MVIP phosphoramidite monomer is as follows:
  • the linker B in the general formula is branched 1 to 4 times to obtain the corresponding 5'MVIP phosphoramidite monomer.
  • the resulting 5'MVIP phosphoramidite monomer is used as the sense agent of RNAi agents Kylo-09-DS141, Kylo-09-DS131 (structural formula 9B) and Kylo-09-DS147 (structural formula 9E).
  • the last monomer of chain solid-phase synthesis when n is equal to 2, the resulting 5'MVIP phosphoramidite monomer is solid-phase synthesized as the sense chain of Kylo-09-DS122 (see 9A for structural formula) and Kylo-09-DS142 The last single.
  • the resulting 5'MVIP phosphoramidite monomer with 3 ligands X can be used as the last monomer in the solid-phase synthesis of the sense strand or the antisense strand.
  • RNAi agents described in this application need to chemically synthesize the corresponding 3'MVIP Solid Support and 5'MVIP phosphoramidite monomers before performing the solid-phase synthesis of phosphoramidites.
  • This implementation exemplarily describes the chemical synthesis process of 3'MVIP Solid Support and 5'MVIP phosphoramidite monomers of the following RNAi agents, as follows:
  • ERCd-01-c1 (3.24g, 2.6mmol) was dissolved in methanol (60mL), and 10% palladium on carbon (0.3g), acetic acid (2.0mL) were added. Hydrogenation was then carried out under normal pressure, and the reaction was carried out overnight. The reaction solution was filtered with diatomaceous earth, and the filtrate was evaporated to dryness under reduced pressure to obtain 2.9 g of oily substance ERCd-01-c2, whose high-resolution mass spectrum is shown in Figure 3.
  • Synthesis steps refer to 4.1.1.1. Synthesis of ERC-01-c1.
  • Synthesis steps refer to 4.1.1.3. Synthesis of ERC-01-c3.
  • 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.
  • the synthesis steps refer to the synthesis of 4.1.1.7.3'MVIP09-c2.
  • the synthesis steps refer to the synthesis of 4.1.1.6.3'MVIP09-c1.
  • the synthesis steps refer to the synthesis of 5.1.1.7.3'MVIP09-c2.
  • the synthesis steps refer to the Solid Support synthesis of 4.1.1.8.3'MVIP09.
  • ERCd-01-c2 (2.18g, 2.0mmol) and dissolve it in DMF (50mL), add monobenzyl glutarate (0.53g, 2.4mmol), DIPEA (0.78g) and TBTU (0.84g), stir at room temperature Overnight, quenched with water (50mL), extracted with DCM (30mL*3), washed with 10% citric acid (50mL*3), saturated sodium bicarbonate 50mL and pyridine 100mL, dried over anhydrous sodium sulfate, filtered, rotary evaporated, passed through the column The purified product 5'MVIP09-ERCd-PFP-c1 (2.15g) was obtained.
  • 5'MVIP09-ERCd-PFP crude product (2.35g, 1.58mmol) was dissolved in DCM (60mL), added DIPEA (0.82g, 6.32mmol), 6-amino-1-hexanol (0.37g, 3.16mmol), stirred at room temperature React overnight. Add 10% citric acid (30mL), extract with DCM (30mL*3), wash with saturated brine (50mL), dry over anhydrous sodium sulfate, filter, rotary evaporate, and column-purify to obtain the product 5'MVIP09 monomer-c1 (1.73 g).
  • Antisense strand synthesis description in Table 13 The reagent bottle was purged with argon for at least 2 minutes. Add the phosphoramidite monomer and acetonitrile in turn to the reagent bottle, tighten the bottle cap, and shake until the solid is completely dissolved by visual inspection. Then add 3A molecular sieves and let it stand for more than 8 hours. Purge the reagent bottle with argon for at least 2 min. Add hydrogenated flavin and dry pyridine to the reagent bottle in turn, tighten the bottle cap, shake until the solid is completely dissolved by visual inspection, and temporarily store it for later use.
  • the argon gas purged the synthesis column for more than 2 hours, and unloaded the synthesis column according to the operating procedures.
  • the solid phase carrier in the synthesis column is transferred to the reaction bottle, and methylamine aqueous solution and ammonia water are added, and the reaction bottle is placed in a shaker at 35° C. for 2-3 hours.
  • the synthesis operation of the sense strand in Table 14 is the same as that of the antisense strand, and the column-packed carrier is a Universal carrier.
  • the purified double-strand solution was divided into vials for freeze-drying, and the product was sealed and stored in a -20°C refrigerator.
  • Age-appropriate female AGT transgenic mice were used for experimental evaluation.
  • the test products are RNAi agents Kylo-09-DS113 ⁇ Kylo-09-DS130 in Table 15.
  • 3 mg/kg was administered by subcutaneous injection; the administration volume was 100-200 ⁇ L.
  • Blood was collected on days 0, 7, 14, 21, 28 and 35 after administration, serum was separated and stored at -80°C.
  • the hAGT level in serum was measured by Elisa method, and the test results are shown in Table 22 and FIG. 6 .
  • Table 22 The average level of hAGT in the serum of transgenic mice after administration
  • Example 2 of 5'MVIP09/3'MVIP09 coupling (sense strand sequence is SEQ ID NO.37,43,44,46,47,48,53 and 54 and corresponding antisense strand sequence SEQ ID NO.55, 61, 62, 64, 65, 66, 71 and 72) obtained RNAi agent Kylo-09-DS113, 119, 120, 122, 123, 124, 129 and 130 are still significantly active in vivo And good durability.
  • This example verifies that the 5'MVIP09/3'MVIP09 carrier structure can realize the safe delivery of siRNA, and the effect is remarkable.
  • the carrier structure 5'MVIP and/or 3'MVIP coupling position includes the 5' end and/or 3' end of the antisense strand, the 5' end and/or 3' end of the sense strand, the antisense The 5' end of the sense strand and the 3' end of the sense strand, the 5' end and the 3' end of the sense strand, and various carrier structures coupled with the same AGT siRNA at different positions, the inhibitory effect of the obtained RNAi agent on the hAGT level of transgenic mice Showing differences, where 5'MVIP09, 20, 19 and 10 with n and m of 2 are coupled to the sense strand, while 3'MVIP09, 10, 15 and 12 are respectively coupled to the antisense strand, and the annealing pair is obtained RNAi agents Kylo-09-DS122, 151, 152, and 153 are superior to other combinations in inhibiting hAGT in transgenic mice; n and m are different, and the RNAi agents Kylo-09
  • the inhibitory effect of Kylo-09-DS160 obtained in the combination of 5'MVIP21 and 3'MVIP18 also reached 67.3%.
  • the n+m of Kylo-09-DS161 and Kylo-09-DS171 are 5 and 6, respectively, they do not show the advantage of more branching in the inhibitory effect, which is speculated to be related to the specificity of the sequence or the space of the introduced carrier structure related to steric hindrance.
  • the test results show that the RNAi agent selected from the 5'MVIP in Table 10 and/or the 3'MVIP in Table 11 combined into a vector structure has a certain inhibitory effect on the expression level of hAGT in the transgenic mice.
  • RNAi agent Kylo-09-DS131, Kylo-09-DS141, Kylo-09-DS142, Kylo-09-DS147 or Kylo-09-DS122 according to the method described in Example 6, select 18 3-5 years old
  • the administration dose is 3mg/kg, and the administration volume is 3ml/kg.
  • the day of group administration was defined as Day0, and blood was collected 7 days, 14 days, 21 days, 28 days, 35 days, 42 days and 49 days after the first administration, and after the plasma was separated, the AGT level in the serum was determined by Elisa method. The results are shown in Table 24 and Figure 8.
  • Table 24 The average level of AGT in serum of cynomolgus monkeys

Abstract

提供一种RNAi剂或其药学上可接受的盐，所述RNAi剂的结构中含有载体结构和干扰核酸。还提供使用所述RNAi剂以抑制AGT基因表达的方法及预防与治疗AGT相关疾病的方法。

Description

AGT抑制剂及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种抑制AGT基因表达的RNAi剂及其应用。
背景技术
RNAi
RNAi(RNA干扰)于1998年，由安德鲁·法厄(Andrew Z.Fire)等在秀丽隐杆线虫中进行反义RNA抑制实验时发现，并将这一过程称为RNAi。这一发现被《Science》杂志评为2001年的十大科学进展之一，并名列2002年十大科学进展之首。自此以后，以RNAi为作用机理的siRNA作为潜在的基因治疗药物得到人们广泛的关注，2006年，安德鲁·法厄与克雷格·梅洛(Craig C.Mello)由于在RNAi机制研究中的贡献获得诺贝尔生理或医学奖。RNAi是在许多生物中，包括动物、植物和真菌，都可由双链RNA(dsRNA)触发的，在RNAi过程中，一种称为“Dicer”的核酸内切酶将长链dsRNA切割或“切丁”成21～25个核苷酸长的小片段。这些小片段，被称为小干扰RNA(siRNA)，其中的反义链(Guide strand)被加载到Argonaute蛋白(AGO2)上。AGO2加载发生在RISC-loading复合物中，这是一个三元复合物，由Argonaute蛋白、Dicer和dsRNA结合蛋白(简称为TRBP)组成。在装载过程中，正义链(Passenger strand)链被AGO2裂解并排出。然后，AGO2使用反义链与包含完全互补序列的mRNA结合，然后催化这些mRNA的切割，致使mRNA分裂丧失翻译模板的作用，进而阻止相关蛋白质的合成。切割后，被切割的mRNA被释放，加载着反义链的RISC-loading复合物被循环用于另一轮的切割。
据统计，在人体内的疾病相关蛋白中，大约超过80％的蛋白质不能被目前常规的小分子药物以及生物大分子制剂所靶向，属于不可成药蛋白。旨在通过基因的表达、沉默等功能治疗疾病的基因治疗被业界认为是继化学小分子药物、生物大分子药物之后的第三代治疗药物，这种疗法在基因水平上实现对疾病的治疗，不受不可成药蛋白的制约。作为基因治疗中RNAi技术最主流的类型，RNAi技术是从mRNA的水平对疾病进行治疗，相比化学小分子药物及生物大分子药物在蛋白质水平的治疗具有更高的效率。利用RNAi技术，可以根据特定基因序列，设计出特异性高、抑制效果好的siRNA的正义链和反义链序列，通过固相合成这些单链序列，然后正义链与反义链在特定的退火缓冲液中按照碱基配对原则配对成siRNA，最后通过载体系统输送到体内相应靶点，降解目标mRNA，破坏目标mRNA作为翻译模板的功能，从而阻止相关蛋白的合成。
siRNA的递送系统
siRNA在血液和组织中不稳定，容易被核酸酶降解，为了提高siRNA的稳定性，可以通过对siRNA的正义链和/或反义链修饰，但这些化学修饰只提供有限的免受核酸酶降解的保护作用并且可能最终影响siRNA的活性。因此，还需要相应的传递系统来保障siRNA安全高效的穿过细胞膜。由于siRNA分子质量较大，且带有大量负电荷，而且具有高水溶解性，所以自身无法顺利穿越细胞膜到达细胞内。
脂质体基本结构是由亲水核和磷脂双分子层构成，具备类似生物膜的磷脂双分子层，拥有很高的生物相容性，所以脂质体一度成为最受欢迎、应用最广泛的siRNA载体。脂质体介导的siRNA递送主要将siRNA包裹到脂质体内，保护siRNA不被核酸酶降解，提高siRNA的通过细胞膜障碍的效率，从而促进细胞的吸收。例如阴离子脂质体、pH敏感性脂质体、免疫脂质体、膜融合脂质体(fusogenic liposome)和阳离子脂质等等，尽管取得了一定的进展，但脂质体本身容易引发炎症反应，给药前必须使用多种抗组胺和激素类如西利替嗪和地塞米松类等药物，以减少可能发生的急性炎症反应，因此在实际临床应用中并不适合所有治疗领域， 尤其像慢性乙肝这一类治疗周期长的疾病，长期使用可能产生的积蓄毒性是潜在的安全隐患，因此需要一种更安全有效的载体系统来递送siRNA。
肝脏中去唾液酸糖蛋白受体(ASGPR)，是肝细胞特异性表达的受体，是一种高效的内吞型受体。由于体内生理情况下各种糖蛋白在酶或酸水解唾液酸后，暴露出的次末端是半乳糖残基，所以ASGPR特异性结合的糖为半乳糖基，故又称半乳糖特异性受体。半乳糖、半乳糖胺、N-乙酰半乳糖胺等单糖和多糖分子都对ASGPR有高亲和性。ASGPR主要生理功能是介导血液中去唾液酸糖蛋白、脂蛋白等物质的清除，且与病毒性肝炎、肝硬化、肝癌等肝脏疾病的发生发展有着密切联系。ASGPR这一特性的发现，对肝源性疾病的诊断及治疗起着重要作用(Ashwell G、Harford J，Carbohydrate specific Receptors of the Liver，Ann Rev Biochem1982 51:531-554)。结构中含有半乳糖或半乳糖胺及其衍生物的肝源性疾病治疗药物可以特异性地与ASGPR亲和，从而具有主动肝靶向性，不需要其它的载体系统来输送。
血管紧张素原(AGT)与高血压
血压是指循环系统中血液对血管壁的压力。血压主要是由于动物心脏的跳动。在每次心跳期间，血压在最大(收缩期)血压(SBP)和最小(舒张期)血压(DBP)之间变化。平均动脉压(MAP)是心跳周期期间的平均动脉压。血压可以通过血压计(即血压测量计)测量。静息时的正常血压在100-140mmHg收缩期和60-90mmHg舒张期的范围内，并且通常表示为收缩压(最高读数)/舒张压(最低读数)mmHg。
在未使用降压药物的情况下,收缩压(SBP)≥140mmHg和(或)舒张压(DBP)≥90mmHg，定义为高血压。根据血压升高水平，将高血压分为1级、2级和3级。根据血压水平、心血管危险因素、靶器官损害、临床并发症和糖尿病进行心血管风险分层,分为低危、中危、高危和很高危4个层次。血压分类和定义如下：
Figure PCTCN2022125877-appb-000001
根据病症起因，高血压还可分为原发性高血压以及继发性高血压。原发性高血压是多种因素导致的高血压，或者是由不明原因导致的高血压，有遗传、地域或者是钠水潴留、交感兴奋、RAS激活等各种各样的原因，所以原发性高血压只能够控制，不能够根治。继发性高血压是指由某种疾病所导致血压增高，高血压是原发疾病的临床症状之一，占95％。一般继发性高血压常见于肾性高血压、肾动脉狭窄，原发性醛固酮增多症、嗜铬细胞瘤、多发性大动脉炎等等。
血压水平与心脑血管病发病和死亡风险之间存在密切的因果关系。有研究发现，基线血压从115/75mmHg到185/115mmHg，平均随访12年,结果发现诊室SBP或DBP与脑卒中、冠心病事件、心血管病死亡的风险呈连续、独立、直接的正相关关系。SBP每升高20mmHg或DBP每升高10mmHg，心、脑血管病发生的风险倍增，其中心力衰竭和脑卒中是与血压水平关联最密切的两种并发症，同时也发现终末期肾病(ESRD)的发生率也明显增加。在重度高血压，ESRD发生率是正常血压者的11倍以上,即使血压在正常高值水平也达1.9倍。因此高血压治疗的根本目标是通过降低血压，有效预防或延迟脑卒中、心肌梗死、心力衰竭、肾功能不全等并发症发生有效控制高血压的疾病进程，预防高血压急症、亚急症等重症高血压发生。
“肾素-血管紧张素-醛固酮系统”，英文缩写为RAAS或者RAS(肾素-血管紧张素系统)是人体内对心血管功能有调节作用的系统，受交感神经系统领导，分泌产生的血管紧张素具有收缩血管的作用。RAS途径的过度刺激或活动是高血压形成的原因之一。
血管紧张素原(AGT)，是serpin家族的成员，也被称为SERPINA8，由AGT基因编码，是RAS所有血管紧张素肽的唯一前体。人AGT有485个氨基酸，其中包括一个33个氨基酸的信号肽，主要在肝脏中产生，并且释放到体循环中，期间肾素将其转化为血管紧张素I。随后通过血管紧张素转化酶(ACE)将血管紧张素I转化成血管紧张素II。血管紧张素Ⅰ能刺激肾上腺髓质分泌肾上腺素，但直接收缩血管的作用不明显；血管紧张素Ⅱ能使全身小动脉收缩而升高血压，此外，还可促进肾上腺皮质分泌醛固酮，醛固酮作用于肾小管，起保钠、保水、排钾作用，从而引起血量增多，血压升高。作用机制如图10所示。
临床一线的降压药主要有五大类，通常使用的降压药分为五大类，一般用字母ABCD来表示，A包括血管紧张素转换酶抑制剂ACEI(如普利类药物)和血管紧张素II拮抗剂ARB(如沙坦类药物)、B是是指β1-受体拮抗剂(如洛尔类药物)、C是指二氢吡啶钙通道拮抗剂(如地平类药物)、D是指利尿剂(如噻嗪类药物)。这五类降压药的作用机制不完全一样，根据患者的不同年龄不同血压水平临床上对于血压的控制，首先采用一个药物，如果一个药物对血压的控制效果欠佳，则联合使用两个，甚至三个降压药。如果三个降压药都无法将血压控制到正常范围，则称之为难治型高血压。难治性高血压是高血压治疗中一个比较常见的临床问题，也是治疗方面的棘手的问题。
当前临床常用的这五类药物都需要长期每天不间断的用药，有些患者的依从性差，其中的副作用是导致依从性差的主要原因，ACEI类和ARB类都可引起干咳和水肿，严重可导致肾功能不全；CB类药副作用可见为心跳加速、脸红、头疼、脚肿；B类还可导致乏力，对血糖、血脂代谢影响；D类药可导致身体发软、抽筋，严重可导致痛风。
因此，本领域中存在不同于现有临床药物的新的作用机理的、副作用少的有更有效治疗的需要。
发明内容
本申请提供了一种AGT RNAi剂，其通过特异性的干扰AGT基因的mRNA，破坏其作为翻译模板的功能，阻止血管紧张素原AGT蛋白的表达，从而可以预防和/或治疗与RAS途径相关的疾病，如高血压，包括“非顽固性高血压”和“顽固性高血压”。
本申请的RNAi剂可以由正义链和反义链通过碱基配对形成，正义链和反义链彼此之间至少80％碱基互补，且部分或全部核苷酸糖基2’位可以是氟或甲氧基，末端可以至少有连续3个核苷酸之间的磷酸酯被硫代。
本申请的RNAi剂的结构中还可以含有使所述RNAi剂具有肝靶向特异性的结构5’MVIP和3'MVIP，其中5’MVIP可以偶联在所述RNAi剂正义链和/或反义链5’末端，3’MVIP可以偶联在所述RNAi剂反义链和/或正义链3’末端，5’MVIP和3’MVIP都可以包含有(肝靶向特异性)靶向单元X、支链L、接头B和连接链D，5’MVIP还可以包含与所述RNAi剂正义链或反义链5’末端连接的转接点R 1，3’MVIP还可以包含与所述RNAi剂正义链或反义链3’末端连接的转接点R 2，所述靶向单元X、支链L、接头B和连接链D在5’MVIP与3’MVIP各自的内部或5’MVIP与3’MVIP之间可以相同，也可以不同。通过体内外药效实验证明，这种RNAi剂直接降解AGT mRNA，持续高效地抑制AGT基因表达，可用于治疗或/和预防AGT基因介导的疾病，如高血压。
一方面，本申请提供一种RNAi剂或其药学上可接受的盐，所述RNAi剂的结构中含有载体结构和干扰核酸，其结构如式IIIa、IIIb或IIIc所示：
Figure PCTCN2022125877-appb-000002
其中，
所述干扰核酸靶向AGT基因，其包括反义链和正义链；
所述载体结构包括5’MVIP(5’MultiValent Import Platform)和/或3’MVIP(3’MultiValent Import Platform)；
所述5’MVIP由转接点R 1、连接链D、接头B、支链L和肝靶向特异性配体X组成，所述3’MVIP由转接点R 2、连接链D、接头B、支链L和肝靶向特异性配体X组成，所述5’MVIP通过转接点R 1与正义链5’端或反义链5’端连接，所述3’MVIP通过转接点R 2与正义链3’端或反义链3’端连接，n和m各自独立地为0-4的任意整数。
在某些实施方式中，其中所述干扰核酸用于抑制AGT基因表达，
在某些实施方式中，其中所述干扰核酸包括siRNA或miRNA。
在某些实施方式中，其中所述且n+m＝2-6的整数，优选n+m＝2、3或4，更优选为4。
在某些实施方式中，所述5’MVIP选自表10中5’MVIP01至5’MVIP22中的任一个，和/或所述3’MVIP选自表11中3’MVIP01至3’MVIP27的任一个。
在某些实施方式中，其中所述正义链与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和SEQ ID NO:18中任意一个或与其相差不超过3个核苷酸的序列具有基本上同源性。
在某些实施方式中，所述反义链包含以下的核苷酸序列：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和SEQ ID NO:36中任意一个或与其相差不超过3个核苷酸的序列。
在某些实施方式中，其中所述正义链包含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和SEQ ID NO:54中任意一个或与其相差不超过3个核苷酸的序列，且所述反义链包含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和SEQ ID NO:72中任意一个或与其相差不超过3个核苷酸的序列。
在某些实施方式中，其中所述干扰核酸包括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～Kylo-09-DS54中任意任一种或多种。
在某些实施方式中，所述的RNAi剂或其药学上可接受的盐包括表18中Kylo-09-DS122，Kylo-09-DS131至Kylo-09-DS147中的任一种或多种。
在某些实施方式中，所述的RNAi剂或其药学上可接受的盐包括表19中Kylo-09-DS122，Kylo-09-DS131，Kylo-09-DS141，Kylo-09-DS142和Kylo-09-DS147中的任一种或多种。
另一方面，本申请提供了一种抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其包含反义链，其中所述反义链包含至少12个与选自以下序列相应位置的核苷酸基本上互补的连续核苷酸或与其相差不超过3个核苷酸的序列：AGT mRNA NM_001382817.3中起始位置为1854-1874、1907-1927、1895-1915、1352-1372、1903-1923、2019-2039、1853-1873和1818-1838的至少连续12个核苷酸或与其相差不超过3个核苷酸的序列或NM_001384479.1中起始位置为1822-1842、1875-1895、1863-1883、1320-1340、1871-1891、1987-2007、1821-1841和1786-1806的至少连续12个核苷酸或与其相差不超过3个核苷酸的序列。
在某些实施方式中，所述RNAi剂包括单链或双链核酸分子。
在某些实施方式中，所述RNAi剂包括siRNA或miRNA。
在某些实施方式中，所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐还包含正义链，其中所述反义链和正义链形成的互补区，所述互补区包括至少12个连续核苷酸。
在某些实施方式中，其中所述正义链和/或反义链上的一个或多个核苷酸被修饰以形成修饰的核苷酸。
在某些实施方式中，所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐还包含配体，所述配体通过载体结构偶联于正义链和/或反义链。
另一方面，本申请提供了一种细胞，其包含前述的RNAi剂或其药学上可接受的盐，或者前述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐。
另一方面，本申请提供了一种药物组合物，其包含前述的RNAi剂或其药学上可接受的盐，或者前述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，以及任选地药学上可接受的赋形剂、载体和/或稀释剂。
另一方面，本申请提供了一种减少细胞或组织中AGT mRNA或蛋白质表达的方法，其包括使细胞或组织与有效量的前述的RNAi剂或其药学上可接受的盐、前述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，和/或前述的药物组合物接触。
另一方面，本申请提供了前述的RNAi剂或其药学上可接受的盐、前述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，或前述的药物组合物在制备药物中的用途，所述药物用于预防和/或治疗疾病或病症或者降低疾病或病症的风险。
另一方面，本申请提供了一种预防和/或治疗疾病或病症的方法，所述方法包括向有此需要的受试者施用有效量的前述的RNAi剂或其药学上可接受的盐、前述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，和/或前述的药物组合物。
另一方面，本申请提供了一种药盒，其包含前述的RNAi剂或其药学上可接受的盐、前述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，或前述的药物组合物。
本申请的AGT RNAi剂通过干扰AGT mRNA，破坏其作为翻译模板的功能，调整体循环中AGT蛋白(RAS所有血管紧张素肽的唯一前体)的表达水平，从基因层面和从RAS系统的源头上进行血压的调控，供了一种全新的治疗模式来对抗各类高血压，包括抗原发性和继发性的，也包括其中的顽固性高血压(TRH)，使那些抗顽固性高血压(TRH)或射血分数降低的慢性心力衰竭患者有药可用。本申请的AGT RNAi剂还具有独特的长效性，存在一季度或半年给药一次的可能，具有当前小分子高血压治疗药无可比拟的优势。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1A显示的是本申请实施例2中的RNAi干预后Hep 3B细胞中AGT mRNA水平；
图1B显示的是本申请实施例2中的RNAi干预后HepG 2细胞中AGT mRNA水平；
图2A-2D显示的是本申请实施例3中RNAi剂不同时段的稳定性检测的HPLC图；
图3显示的是本申请实施例4.1.1.5中合成的ERCd-01-c2的高分辨质谱图；
图4显示的是本申请实施例4.1.2.6中合成的3’MVIP17-c1的高分辨质谱图；
图5显示的是本申请实施例4.2.1.2中合成的5’MVIP09-ERCd-PFP-c2的高分辨质谱图；
图6显示的是本申请实施例6中的转基因小鼠给药后hAGT平均水平；
图7显示的是本申请实施例7中的转基因小鼠给药后血清中hAGT平均水平。
图8显示的是本申请实施例8中的食蟹猴给药后血清中AGT平均水平。
图9A-9E显示的是Kylo-09-DS122，Kylo-09-DS131，Kylo-09-DS141，Kylo-09-DS142和Kylo-09-DS147的结构式。
图10显示的是血管紧张素原(AGT)的作用机制。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“血管紧张素原”可以与术语“AGT”交换使用，AGT mRNA序列的实例容易利用已公开的数据库取得，例如，GenBank、UniProt、OMIM和猕猴(Macaca)基因组计划网站。术语“AGT”包括人类AGT，其mRNA序列可见于例如，GenBank NM_001382817.3或GenBank NM_001384479.1；食蟹猴AGT，其氨基酸与完整编码序列可见于，例如，GenBank登录号GI：90075391(AB170313.1)；小鼠(Mus musculus)AGT，其氨基 酸与完整编码序列可见于例如，GenBank登录号GI：113461997(NM_007428.3)；及大鼠AGT(Rattus norvegicus)AGT，其氨基酸与完整编码序列可见于例如，GenBank登录号GI：51036672(NM_134432)。本文所用术语“AGT”亦指AGT基因的天然存在的DNA序列变异，如AGT基因中的单核苷酸多态性(SNP)。
在本申请中，术语“iRNA”、“RNAi剂”、“iRNA剂”、“RNA干扰剂”可交换使用，通常指包含如本文术语所定义的RNA的药剂，且其可通过RNA诱导沉默复合物(RISC)途径介导RNA转录物的靶向切割。iRNA经由称为RNA干扰(RNAi)的过程指导mRNA的序列特异性降解。iRNA调控(例如，抑制)AGT基因在细胞(例如，受试者如哺乳动物受试者中的细胞)中的表达。
在某些实施方式中，RNAi剂可为引入细胞或生物体中来抑制靶mRNA的单链siRNA(ssRNAi)。单链RNAi剂结合RISC内切核酸酶Argonaute 2，其然后切割靶mRNA。单链siRNA一般为15至30个核苷酸且经化学修饰。单链siRNA的设计和测试说明于美国专利号8,101,348及Lima等人(2012)Cell 150：883-894中，其各自的完整内容以引用的方式并入本文中。本文所述的任何反义核苷酸序列均可用作本文所述的或通过Lima等人(2012)Cell 150：883-894中说明的方法进行化学修饰的单链siRNA。
在某些实施方式中，本申请所使用的“iRNA”是双链RNA，且本文中称为“双链RNAi剂”、“双链RNA(dsRNA)分子”、“dsRNA剂”或“dsRNA”。术语“dsRNA”指核糖核酸分子的复合物，其具有包含两个反向平行且基本上互补的核酸链的双链体结构，称为具有相对于靶RNA(即AGT基因)的“正义”和“反义”取向。在本申请的一些实施方式中，双链RNA(dsRNA)通过转录后基因沉默机制(本文中称为RNA干扰或RNAi)触发靶RNA(例如，mRNA)的降解。
双链体结构可为容许所需的靶RNA通过RISC途径的特异性降解的任何长度，且可在约19至36碱基对的长度范围内，例如，约19-30碱基对的长度，例如，约19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35或36碱基对的长度。上述范围与长度中间的范围与长度也包括为本申请的部分。在某些实施方式中，本申请的iRNA剂为各链包含15-23个核苷酸的dsRNA，其与靶RNA序列(例如，AGT基因)相互作用以指导靶RNA的切割。在某些实施方式中，本申请的iRNA为24-30个核苷酸的dsRNA，其与靶RNA序列(例如，AGT靶mRNA序列)相互作用以指导靶RNA的切割。
通常，dsRNA分子的各链的大多数核苷酸为核糖核苷酸，但如本文详细说明的，各链或两条链也可包括一个或多个非核糖核苷酸，例如，脱氧核糖核苷酸或修饰的核苷酸。此外，本说明书所用“iRNA”可包括具有化学修饰的核糖核苷酸；iRNA可在多个核苷酸处包括实质修饰。本文所用术语“修饰的核苷酸”意指独立地具有修饰的糖部分、修饰的核苷酸间连接或修饰的核碱基，或其任何组合的核苷酸。因此，术语“修饰的核苷酸”涵盖对核苷酸间连接、糖部分或核碱基的例如官能团或原子的置换、添加或移除。适用于本申请药剂的修饰包括本文所公开或本领域已知的所有类型的修饰。
在本申请中，术语“核酸”和“多核苷酸”可互换使用，并指任何长度的核苷酸(脱氧核糖核苷酸或者核糖核苷酸或者其类似物)的聚合形式。多核苷酸可以具有任何三维结构并且可以执行任何功能。以下是多核苷酸的非限制性实例：基因或基因片段(例如探针、引物、EST或SAGE标签)、外显子、内含子、信使RNA(mRNA)、转移RNA、核糖体RNA、核酶、cDNA、重组多核苷酸、分支的多核苷酸、质粒、载体、任何序列的分离的DNA、任何序列的分离的RNA、核酸探针、siRNA、miRNA、shRNA、RNAi试剂和引物。多核苷酸可以在一个或多个碱基、糖和/或磷酸酯处以本申请所述或本领域已知的任何各种修饰或取代进行修饰或取代。多核苷酸可以包含修饰的核苷酸，例如甲基化的核苷酸和核苷酸类似物。如果存在，可以在聚合物组装之前或之后对核苷酸结构进行修饰。核苷酸序列可以被非核苷酸组分阻断。多聚 核苷酸可以在聚合后修饰，例如通过与标记组分偶联。该术语可以是双链和单链分子。除非另有说明或要求，否则本申请作为多核苷酸的任何实施方式包括双链形式和已知或据预测构成双链形式的两种互补单链形式中的每一种。
在本申请中，术语“靶核酸”或“靶序列”通常指在AGT基因转录期间所形成mRNA分子的核苷酸序列的连续部分，包括作为主要转录产物的RNA加工产物的mRNA。序列的靶部分应至少足够长以作为在AGT基因转录期间形成的mRNA分子的核苷酸序列的该部分位置处或附近iRNA指导的切割的底物。在一个实施方式中，该靶序列在AGT的蛋白质编码区内。靶序列可为约19-36个核苷酸的长度，例如，优选约19-30个核苷酸的长度。上述范围和长度中间的范围和长度也包括为本申请的部分。
在本申请中，术语“核苷酸序列”通常是指一连串或一定顺序的核碱基、核苷酸和/或核苷，无论是经修饰还是未修饰，使用标准核苷酸命名和本申请所述的经修饰的核苷酸的符号表用一连串字母描述。
在本申请中，术语“寡核苷酸”通常是指由多个核苷酸残基(脱氧核糖核苷酸或核糖核苷酸，或其相关的结构变体或合成类似物)通过磷酸二酯键(或其相关的结构变体或合成类似物)连接组成的聚合物。因此，虽然术语“寡核苷酸”一般指其中核苷酸残基和它们之间的连接是天然产生的核苷酸聚合物，但应理解，该术语的范围也包括各种类似物，包括但不限于：肽核酸(PNA)、氨基磷酸酯、硫代磷酸酯、甲基膦酸酯、2-O-甲基核糖核酸等。该分子的确切大小可取决于具体应用。寡核苷酸一般长度较短，通常约有10-30个核苷酸残基，但该术语也可指任何长度的分子，尽管术语“多核苷酸”或“核酸”一般用于较大的寡核苷酸。
在某些实施方式中，寡核苷酸包含一个或多个未修饰的核糖核苷(RNA)和/或未修饰的脱氧核糖核苷(DNA)和/或一个或多个修饰核苷。术语“修饰寡核苷酸”通常意指包含至少一个修饰核苷和/或至少一个修饰的核苷间键联的寡核苷酸。
在本申请中，术语“修饰的核苷”通常意指与天然存在的RNA或DNA核苷相比包含至少一个化学修饰的核苷。修饰的核苷包含修饰的糖部分和/或修饰的核碱基。
在本申请中，术语“核碱基”通常意指杂环嘧啶或嘌呤化合物，它是所有核酸的组分且包括腺嘌呤(A)、鸟嘌呤(G)、胞嘧啶(C)、胸腺嘧啶(T)和尿嘧啶(U)。核苷酸可包括经修饰的核苷酸或核苷酸模拟物、无碱基位点(Ab或X)或替代物替代部分。如本申请所使用，“核碱基序列”通常意指不依赖于任何糖、键联或核碱基修饰的连续核碱基的顺序。术语“未修饰的核碱基”或“天然存在的核碱基”通常意指RNA或DNA的天然存在的杂环核碱基：嘌呤碱基腺嘌呤(A)和鸟嘌呤(G)；以及嘧啶碱基胸腺嘧啶(T)、胞嘧啶(C)(包括5-甲基C)和尿嘧啶(U)。“修饰的核碱基”通常意指并非天然存在的核碱基的任何核碱基。
在本申请中，术语“糖部分”通常意指核苷的天然存在的糖部分或修饰的糖部分。术语“天然存在的糖部分”通常意指如在天然存在的RNA中发现的呋喃核糖基或如在天然存在的DNA中发现的脱氧呋喃核糖基。“修饰的糖部分”意指取代的糖部分或糖替代物。
在本申请中，术语“核苷间键联”通常意指寡核苷酸中相邻核苷之间的共价键联。“天然存在的核苷间键联”意指3’至5’磷酸二酯键联。“修饰的核苷间键联”意指除了天然存在的核苷间键联之外的任何核苷间键联。
在本申请中，术语“反义寡核苷酸”是指单链寡核苷酸分子，其具有与靶核酸(例如，目标基因组序列，mRNA前体，或mRNA分子)的相应片段互补的核碱基序列。在某些实施方案中，反义寡核苷酸的长度为12至30个核碱基。在某些实施方案中，反义寡核苷酸是具有与靶核酸(如AGT多核苷酸)序列互补的核苷酸序列的未经修饰或经修饰的核酸。
在本申请中，术语“反义链”通常是指RNAi剂(例如dsRNA)的包括与靶序列实质上互补的区域的链。在本文中使用时，术语“互补性区域”通常指反义链上与本申请定义的序列(例如靶序列)实质上互补的区域。当互补性区域与靶序列不完全互补时，错配可以在分子的内部 或末端区域。通常，最被容许的错配在末端区域，例如，在5’末端和/或3’末端的5、4、3或2个核苷酸内。
在本申请中，术语“正义链”(S)通常是指RNAi剂的这样一条链，所述链包括与作为在此定义的术语反义链的区域基本互补的区域。“正义”链有时被称为“有义”链，“过客”链或“反引导”链。借助它们的序列，反义链靶向所希望的mRNA，同时正义链靶向不同靶标。因此，如果反义链被掺入RISC中，则正确的靶标被靶向。正义链的掺入可以导致脱靶效应。这些脱靶效应可以通过在正义链上使用修饰或使用5’端帽加以限制。
在本申请中，术语“互补”当用于描述就第二核苷酸序列(如RNAi剂反义链)而言的第一核苷酸序列(如RNAi剂正义链或AGT mRNA)时是指包含第一核苷酸序列的寡核苷酸或多核苷酸在一定条件下与包含第二核苷酸序列的寡核苷酸或多核苷酸杂交(形成碱基对氢键)并形成双链体或双螺旋结构的能力。互补序列包括沃森-克里克碱基对(Watson-Crick base pairs)或非沃森-克里克碱基对，并且包括天然或经修饰的核苷酸或核苷酸模拟物，只要以上关于它们的杂交能力而言的需求得以实现。“互补”不必需在每个核苷上均具有核碱基互补性。相反，可以容忍一些错配。
在本申请中，术语“完全互补”通常意指第一多核苷酸的连续序列中的全部(100％)碱基将与第二多核苷酸的连续序列中的相同数目的碱基杂交。连续序列可以包含第一或第二核苷酸序列的全部或一部分。如本申请所用，“部分互补”通常意指在杂交的核碱基序列对中，第一多核苷酸的连续序列中至少约70％的碱基将与第二多核苷酸的连续序列中相同数目的碱基杂交。如本申请所用，“基本上互补”通常意指在杂交的核碱基序列对中，第一多核苷酸的连续序列中至少约90％的碱基将与第二多核苷酸的连续序列中相同数目的碱基杂交。如本申请所用的术语“互补”、“完全互补”和“基本上互补”可以就在RNAi剂的正义链与反义链之间或者在RNAi剂的反义链与AGT mRNA的序列之间的碱基匹配而言使用。序列同一性或互补性不依赖于修饰。为了测定同一性或互补性的目的，例如，a和Af与U(或T)互补并且与A同一。
在本申请中，术语“同源的”或“同源性”通常是指已经与参考核酸序列的相同核苷酸匹配的主题核酸序列的核苷酸的数量，通常通过序列分析程序(例如，Karlin和Altschul,1990,PNAS 87:2264-2268；Karlin和Altschul,1993,PNAS 90:5873-5877)，或通过目视检查确定。如本文所使用的，术语“完全同源性”或“完全同源的”一般是指在参考序列和主题核酸序列之间的完全(100％)同源性或“同一性”。如本文所使用的，术语“基本上同源的”或“基本上同源性”一般是指主题序列与参考序列中相同核苷酸位置的核苷酸共有至少50％(例如，至少55％、60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％或99％)的同源的核苷酸。
在本申请中，术语“配体”通常是指能够共价地或以其它化学方式与生物活性物质(如寡核苷酸)结合的任何化合物或分子。在某些实施方式中，配体能够与另一种化合物例如受体直接或间接地相互作用，与配体相互作用的受体可以存在于细胞表面上，或可替代地可以是细胞内和/或细胞间受体，配体与受体的相互作用可以导致生化反应，或可以仅仅是物理相互作用或结合。
在本申请中，术语“诱导”、“抑制”、“加强”、“升高”、“增加”、“减少”、“降低”等通常表示两个状态之间的定量差异。例如，“有效抑制AGT的活性或表达的量”意指处理样品中AGT的活性或表达的水平将低于未处理样品中AGT活性或表达的水平。所述术语例如适用于表达水平和活性水平。术语“减少”和“降低”可互换使用并且通常表示小于原来的任何变化。“减少”和“降低”是相对的术语，需要在测量前和测量后间进行比较。“减少”和“降低”包括完全耗竭。
在某些实施方式中，术语“降低”可以通过本领域已知标准方法(诸如本申请中描述的那 些)检测的，基因、基因产物例如蛋白质或生物标志物在第一样品中的表达水平/量与相应基因、基因产品例如蛋白质或生物标志物在第二样品中的表达水平/量相比约5％、10％、20％、25％、30％、40％、50％、60％、70％、80％、85％、90％、95％、或100％的总体降低。在某些实施方式中，术语“降低”指第一样品中基因或生物标志物的表达水平/量的降低，其中该降低是第二样品中相应基因或生物标志物的表达水平/量的至少约0.9倍、0.8倍、0.7倍、0.6倍、0.5倍、0.4倍、0.3倍、0.2倍、0.1倍、0.05倍、或0.01倍。在某些实施方式中，第一样品是自受试者获得的样品，而第二样品是参照样品。
在本申请中，术语“表达”通常意指基因最终产生蛋白质的过程。表达包括但不限于转录、转录后修饰(例如，剪接、聚腺苷酸化、添加5’-帽)以及翻译。
在本申请中，术语“药学上可接受的”通常是指不干扰活性成分生物学活性的有效性的一种或多种无毒物质。这类制剂通常可含有盐、赋形剂、缓冲剂、防腐剂、相容性载体和任选的其它治疗剂。这类药学上可接受的制剂通常也可包含适合给予人的相容性固体或液体填料、稀释剂或包囊材料。用于医药时，盐应该是药学上可接受的盐，但可方便地使用非药学上可接受的盐来制备药学上可接受的盐，不能将它们排除在本申请范围以外。这类药理学和药学上可接受的盐包括但不限于由以下酸制备的盐：氢氯酸、氢溴酸、硫酸、硝酸、磷酸、马来酸、乙酸、水杨酸、柠檬酸、硼酸、甲酸、丙二酸、琥珀酸等。药学上可接受的盐也可制备成碱金属盐或碱土金属盐，如钠盐、钾盐或钙盐。
在本申请中，术语“脂质纳米颗粒”或“LNP”通常指包含包封药理活性分子(如核酸分子，例如，iRNA或iRNA从其转录的质粒)的脂质层的囊泡。LNP描述于例如，中国专利号CN103189057B中，其完整内容以引用方式并入本文中。
在本申请中，术语“预防和/或治疗”不仅包括预防和/或治疗疾病，还通常包括预防疾病的发作，减缓或逆转疾病的进展，预防或减缓与疾病相关的一种或多种症状的发作，减少和/或减轻与疾病相关的一种或多种症状，降低疾病和/或与其相关的任何症状的严重程度和/或持续时间和/或预防疾病和/或与其相关的任何症状的严重程度的进一步增加，预防、减少或逆转由疾病引起的任何生理损伤，以及通常对正在治疗的患者有益的任何药理学作用。本申请的RNAi剂或药物组合物形成可行的治疗剂不需要实现完全治愈或根除疾病的任何症状或表现。如在相关领域中所认识到的，用作治疗剂的药物可降低给定疾病状态的严重程度，但不需要消除疾病的每种表现才能被认为是有用治疗剂。类似地，预防性施用的治疗构成可行的预防剂不需要完全有效地预防病症的发作。简单地在受试者中减少疾病的影响(例如，通过减少其症状的数量或严重程度，或通过提高另一种治疗的有效性，或通过产生另一种有益效果)，或减少疾病发生或恶化的可能性就足够了。
在本申请中，术语“疾病”或“病症”可以互换使用，通常是指受试者与正常状态的任意偏离，例如身体或某些器官的状态的任何变化，妨碍或扰乱了功能的履行，和/或在患病或与其接触的人中引起症状例如不适、机能障碍、痛苦或甚至死亡。疾病或病症还可以称为失调(distemper)、不适(ailing)、小病(ailment)、疾病(malady)、紊乱(disorder)、疾病(sickness)、生病(illness)、身体不适(complaint)、inderdisposion或affectation。
在本申请中，术语“血管紧张素原相关疾病”或“AGT相关疾病”通常是指因肾素-血管紧张素-醛固酮系统(RAAS)激活导致或与其相关的疾病或障碍，或其症状或进展响应于RAAS失活的疾病或障碍。术语“血管紧张素原相关疾病”包括因AGT表达降低而受益的疾病、障碍或病症。这类疾病通常与高的血压相关。血管紧张素原相关疾病的非限制实例包括高血压，例如，临界性高血压(也称为高血压前期)、原发性高血压(也称为本态性高血压或特发性高血压)、继发性高血压(也称为非本态性高血压)、孤立性收缩期或舒张期高血压、妊娠相关高血压(例如，先兆子痫、子痫和产后先兆子痫)、糖尿病性高血压、顽固性高血压、难治性高血压、阵发性高血压、肾血管性高血压(也称为肾高血压)、戈德布拉特氏高血压、眼高压、青 光眼、肺动脉高血压、门静脉高血压、系统性静脉高血压、收缩期高血压、不稳定性高血压；高血压性心脏病、高血压性肾病、动脉粥样硬化、动脉硬化、血管病变(包括外周血管疾病)、糖尿病性肾病、糖尿病性视网膜病、慢性心力衰竭、心肌病、糖尿病性心肌病、肾小球硬化症、主动脉缩窄、主动脉瘤、心室纤维化、睡眠呼吸暂停、心力衰竭(例如，左心室收缩功能障碍)、心肌梗塞、心绞痛、中风、肾疾病(例如，慢性肾脏病或糖尿病性肾病变，任选地在妊娠的情况中)、肾衰竭(例如，慢性肾衰竭)和全身性硬化(例如，硬皮病肾危象)。在某些实施方式中，AGT相关疾病包括宫内发育迟缓(IUGR)或胎儿生长受限。在某些实施方式中，AGT相关障碍也包括肥胖、肝脂肪变性/脂肪肝，例如，非酒精性脂肪性肝炎(NASH)及非酒精性脂肪肝病(NAFLD)、葡萄糖耐受不良、2型糖尿病(非胰岛素依赖型糖尿病)和代谢综合征。在某些实施方式中，高血压包括与低的血浆肾素活性或血浆肾素浓度相关的高血压。
本申请中，术语“施用”通常是指通过任意引入或递送途径将本申请药物制剂引入受试者的身体中。可以采用本领域技术人员已知的用于使细胞、器官或组织与所述药物接触的任何方法。所述施用可以包括而不限于静脉内、动脉内、鼻内、腹内、肌内、皮下透皮或口服。每日剂量可以划分成一个、两个或更多个合适形式的剂量以在某个时间段期间的一个、两个或更多个时间施用。
在本申请中，术语“接触”通常是指两种两个或更多个不同类型的物质以任何顺序、任何方式以及任何时长接触在一起。接触可以发生在体内(in vivo)、间接体内(ex vivo)或体外(in vitro)。在某些实施方式中，可以是指使本申请的RNAi剂或组合物直接接触细胞或组织。在另一些实施方式中，该术语是指使本申请的RNAi剂或组合物间接接触细胞或组织。例如，本申请的方法包括其中受试者接触本申请的RNAi剂或组合物，然后RNAi剂或组合物通过扩散或本领域已知的任何其他主动运输或被动运输过程(化合物通过该过程在体内循环)接触细胞或组织的方法。
在本申请中，术语“有效量”或“有效剂量”通常是指足以实现或至少部分实现所需效果的量。药物或治疗剂的“治疗有效量”或“治疗有效剂量”通常是当单独使用或与另一种治疗剂组合使用时促进疾病消退(这通过疾病症状严重程度的降低、疾病无症状期的频度和持续时间的增加、或者由于罹患疾病而引起的损害或残疾的预防来证明)的任何药物量。药物的“预防有效量”或“预防有效剂量”通常是指当单独或与另一种治疗剂组合给有疾病发展或疾病复发的风险的受试者施用时抑制疾病的发展或复发的药物量。可以使用本领域技术人员已知的多种方法对治疗剂或预防剂促进疾病消退或抑制疾病发展或复发的能力进行评估，比如在处于临床试验期间的人类受试者中、在动物模型系统中预测对人类的功效、或者通过在体外测定中测定药剂的活性。在某些实施方式中，“有效量”是指产生预期药理学、治疗性或预防性结果的RNAi剂的量。
在本申请中，术语“受试者”通常是指需要诊断、预后、改善、预防和/或治疗疾病的人或非人动物(包括哺乳动物)，诸如人、非人灵长类动物(猿、长臂猿、大猩猩、黑猩猩、猩猩、猕猴)、家畜(狗和猫)、农场动物(家禽如鸡和鸭、马、牛、山羊、绵羊、猪)和实验动物(小鼠、大鼠、兔、豚鼠)。人受试者包括胎儿、新生儿、婴儿、青少年和成人受试者。受试者包括动物疾病模型。
在本申请中，术语“包括”、“包含”、“具有”、“可以”、“含有”及其变体通常旨在是开放式过渡性短语、术语或词语，其不排除额外行为或结构的可能性。术语“由……组成”通常表示不能存在别的组分(或同样地，特征、整数、步骤、等)。除非上下文另有明确规定，单数形式如英文的“a”，“an”，“the”，中文的“一个”、“一种”和“所述/该”一般包括所指代事物的复数形式。
在本申请中，术语“约”通常意指大约(approximately)、在......的附近(intheregionof)、粗略地(roughly)、或左右(around)。当术语“约”当用于指涉数值范围时，截值或特定数值用于指 示所载明的数值可与该列举数值有多达10％的差异。因此，术语“约”可用于涵盖自特定值±10％或更少的变异、±5％或更少的变异、±1％或更少的变异、±0.5％或更少的变异、或±0.1％或更少的变异。
应理解，数字或一系列数字之前的术语“至少”包括与该术语“至少”相邻的数字，及逻辑上包括在内的所有后续数字或整数，如从上下文中明确的。例如，核酸分子中的核苷酸数目必需为整数。例如，“21个核苷酸的核酸分子中的至少19个核苷酸”意指19、20或21个核苷酸具有所指示的性质。当“至少”出现在一系列数字或范围之前时，应理解“至少”可修饰该系列或范围中每一个数字。
应理解本文中采用的“不超过”或“低于”指与该短语相邻且逻辑上较低的值或整数，如从上下文逻辑而言，至零。例如，具有“不超过3个核苷酸”的突出端的双链体具有3、2、1或0个核苷酸的突出端。当“不超过”出现在一系列数字或范围之前时，应理解，“不超过”可修饰该系列或范围内每个数字。本文所采用范围同时包括上限与下限。
发明详述
RNAi剂
一方面，本申请提供抑制AGT基因表达的RNAi剂。
在某些的实施方式中，RNAi剂包括用于抑制在细胞中，如受试者(例如，哺乳动物，如易于发生AGT相关障碍例如高血压的人)的细胞中，AGT基因的表达的单链寡核苷酸或双链核糖核酸(dsRNA)分子。其中，该dsRNA包括反义链，其具有与在AGT基因的表达中所形成mRNA的至少一部分互补的互补区。该互补区为约12-30个核苷酸长度(例如，约30、29、28、27、26、25、24、23、22、21、20、19、18、17、16、15、14、13或12个核苷酸长度)。
dsRNA包括两个RNA链，其可在dsRNA使用的条件下互补并杂交形成双链体结构(互补区)。dsRNA的一条链(反义链)包括与靶序列基本上互补且通常完全互补的互补区。靶序列可源自AGT基因表达期间形成mRNA的序列。另一条链(正义链)包括与反义链互补的区域，使得在合适条件下组合时，两条链可杂交并形成双链体结构。通常，双链体结构的长度为12至30个碱基对。类似地，与靶序列的互补区的长度为12至30个核苷酸。
在某些实施方式中，dsRNA为约19至约23个核苷酸长度，或约24至约30个核苷酸长度。通常，dsRNA的长度足以用作Dicer酶的底物。例如，本领域公知，长度大于约21-23个核苷酸的dsRNA可用作Dicer的底物。本领域技术人员也了解，被靶向切割的RNA的区域通常为较大RNA分子(通常mRNA分子)的部分。靶的“一部分”为mRNA靶的连续核苷酸，其长度足以允许其为RNAi指导切割(即经由RISC途径的切割)的底物。
本领域技术人员也应理解，双链体区为dsRNA的主要功能部分，例如，约19至约30碱基对的双链体区，例如，约19-30、19-29、19-28、19-27、19-26、19-25、19-24、19-23、19-22、19-21、19-20碱基对。因此在一个实施方式中，为了达到成为靶向所需RNA进行切割的功能性双链体(例如，15-30碱基对)的程度，具有超过30碱基对的双链体区的RNA分子或RNA分子的复合物为dsRNA。
在一个方面中，本申请的RNAi剂或其药学上可接受的盐，其包含反义链，其中所述反义链包含至少12个与选自以下序列相应位置的核苷酸基本上互补的连续核苷酸或与其相差不超过3个核苷酸的序列：AGT mRNA NM_001382817.3中起始位置为1854-1874、1907-1927、1895-1915、1352-1372、1903-1923、2019-2039、1853-1873和1818-1838的至少连续12个核苷酸或与其相差不超过3个核苷酸的序列或NM_001384479.1中起始位置为1822-1842、1875-1895、1863-1883、1320-1340、1871-1891、1987-2007、1821-1841和1786-1806的至少连续12个核苷酸或与其相差不超过3个核苷酸的序列。
在某些实施方式中，其中所述反义链和正义链形成的双链体结构(互补区)，所述互补区包括至少12、13、14、15、16、17、18、19、20或21个连续核苷酸。
在某些实施方式中，所述RNAi剂的正义链与表1中的靶序列具有基本上同源性。
表1靶序列
Figure PCTCN2022125877-appb-000003
其中，g＝鸟苷酸，a＝腺苷酸，t＝胸腺苷酸，c＝胞苷酸。
表2反义链序列
SEQ ID NO. 单链代码 反义链序列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 uaaaacccaauuuuuguucun
27 AS9 uuuugcagcgacuagcaccan
28 AS10 ucaagcucaaaaaaaaugcun
SEQ ID NO. 单链代码 反义链序列5'→3'
29 AS11 uuuaauuuuaaaacccaauun
30 AS12 uucaaaaaucacaagcaucun
31 AS13 uaagcuguuggguagacucun
32 AS14 uucacaaacaagcuggucggn
33 AS15 uuguccuggaugucacuccan
34 AS16 uauuacagacacuacacggan
35 AS17 uuuguuucacaaacaagcugn
36 AS18 uuuuggaacaguagucccgcn
其中n为a或u或g或c，g＝鸟苷酸，a＝腺苷酸，u＝尿苷酸，c＝胞苷酸。
在一些筛选实施方案中，所述的RNAi剂的正义链和反义链选自表3中序列或与表3中的各序列相差一个、两个或三个核苷酸。
表3筛选RNAi剂序列
Figure PCTCN2022125877-appb-000004
其中，g＝鸟苷酸，a＝腺苷酸，u＝尿苷酸，c＝胞苷酸。
应理解，虽然表3中的序列未说明为修饰或偶联的序列，但本申请的iRNA(例如，dsRNA)的RNA可包含表3所示的任何一个序列、或经修饰的表4、5或6的序列、或偶联的表13、14、16或17的序列。换言之，本申请涵盖如本文所述的未修饰的、未偶联的、修饰的或偶联的dsRNA。
在一些实施方案中，所述的RNAi剂可以通过脂质体-核酸纳米颗粒的方式加药到细胞系 中进行序列筛选。专利US9233971B2，US9080186B2，CN102985548B和CN103189057B有关脂质化合物及脂质体-核酸纳米颗粒制备的方法全文引入本说明书。
在一些实施方案中，其中所述的脂质化合物中的两性脂质优选大环脂类化合物D1C1，T1C1，T1C6，T4C4，B2C1，B2C6，B2C7和M10C1。
本领域技术人员公知，具有约20至23碱基对，例如，21碱基对的双链体结构的dsRNA已经被认为能特别有效地诱导RNA干扰(Elbashir等人，EMBO 2001，20：6877-6888)。然而，其他人已发现更短或更长的RNA双链体结构也有效(Chu和Rana(2007)RNA14：1714-1719；Kim等人(2005)Nat Biotech 23：222-226)。可以合理地预期，由表1、2和3中一个序列在一端或两端减去或增加几个核苷酸的双链体与所述dsRNA相比可以类似地有效。因此，具有源自表1、2和3中一个序列的至少18、19、20、21或更多个连续核苷酸的序列且在抑制AGT基因表达的能力方面与包含全序列的dsRNA的差异不超过约5、10、15、20、25或30％的抑制的dsRNA均包括在本申请范围内。
此外，表1、2和3提供的RNA识别了在AGT转录物中易发生RISC介导的切割的位点。因此，本申请的进一步包含靶向这类位点之一内的iRNA。如果iRNA促进转录物在该特定位点内任何位置的切割，则如本文所用iRNA被称为靶向RNA转录物的该特定位点内，这种iRNA通常包括来自表1、2和3中提供的一个序列的至少约12、13、14、15、16、17、18或19个连续核苷酸。
本申请所述dsRNA可进一步包括一个或多个单链核苷酸突出端，例如，1、2、3或4个核苷酸。具有至少一个核苷酸突出端的dsRNA可以具有比其平端的对应物优异的抑制性质。核苷酸突出端可包含核苷酸/核苷类似物或其组成，包括脱氧核苷酸/核苷。该突出端可在正义链、反义链或其任何组合上。此外，突出端的核苷酸可存在于dsRNA的反义或正义链的5’-端、3’-端或两端。该突出端可由一条链长于另一条链所造成，或由相同长度的两条链交错造成。该突出端可与靶mRNA形成错配或其可与所靶向的基因序列互补或可为另一个序列。
dsRNA也可以只含有单一突出端，其可以增强RNAi剂的干扰活性，而不影响其整体稳定性。例如，单链突出端可以位于正义链的3’-端，或可选地，在反义链的3’-端。RNAi剂还可以具有平端，位于反义链的5’-端(或正义链的3’-端)，反之亦然。通常，RNAi剂的反义链在3’-端具有核苷酸突出端，而5’-端是平端。尽管不希望受到理论的束缚，反义链的5’-端的不对称平端和反义链的3’-端突出端有利于引导链载入RISC过程中。
在某些实施方式中，突出端存在于正义链、反义链或两条链的3’-端。在某些实施方式中，此3’-突出端存在于反义链中。在某些实施方式中，此3’-突出端存在于正义链中。
在某些实施方式中，所述dsRNA是21个核苷酸长的双端平端体。
在某些实施方式中，所述dsRNA具有21个核苷酸长度，且正义链和反义链在3’端均具有2个核苷酸的突出端。
修饰的RNAi剂
为了增强本申请所述的RNAi剂在体内的稳定性，在不影响其活性甚至增强其活性的情况下，可以对上述RNAi剂的正义链和反义链进行修饰，其中的核苷酸可以有修饰基团，可以整条链或者部分修饰。在某些实施方式中，其中所述正义链和/或反义链上的一个或多个核苷酸被修饰以形成修饰的核苷酸。
在某些实施方式中，本申请的RNAi剂(例如，dsRNA)的RNA未修饰，且不包含例如，本领域已知及本文所述的化学修饰或偶联。在其他实施方式中，本申请的RNAi剂(例如，dsRNA)的RNA经化学修饰以加强稳定性或其他有利特性。本申请的其他实施方式中，本申请的RNAi剂的所有核苷酸或基本上所有核苷酸被修饰，即RNAi剂的链存在不超过5、4、3、2或1个未修饰的核苷酸。
如本申请所述的核酸可采用本领域上公知的方法合成和/或修饰，如那些描述于“Current  protocols in nucleic acid chemistry”，Beaucage，S.L.等人(编辑)，JohnWiley&Sons，Inc.，New York，NY，USA中的，其以引用方式并入本文中。修饰包括例如，末端修饰，例如，5’-端修饰(磷酸化、偶联、反向连接)或3’-端修饰(偶联、DNA核苷酸、反向连接等)；碱基修饰，例如，使用稳定化碱基、失稳碱基或用与扩异展的伴体库碱基配对的碱基替代、移除碱基(无碱基核苷酸)或偶联碱基；糖修饰(例如，2’-位或4’-位)或糖的替代；或主链修饰，包括磷酸二酯连接的修饰或替换。在本申请提供的RNAi剂中，所述RNAi剂的正义链和反义链均不需要均匀修饰，可在其单个核苷酸中掺入一种或一种以上的修饰。
在某些实施方式中，其中所述修饰的核苷酸选自：脱氧核糖核苷酸、核苷酸模拟物、脱碱基核苷酸、2’-修饰核苷酸、3’至3’连接(倒置)核苷酸、含非天然碱基的核苷酸、桥接核苷酸、肽核酸(PNA)、解锁的核碱基类似物、锁定核苷酸、3’-O-甲氧基(2’核苷间连接)核苷酸、2’-F-阿拉伯糖核苷酸、5’-Me/2’-氟带核苷酸、吗啉代核苷酸、乙烯基膦酸酯脱氧核糖核苷酸、含乙烯基膦酸酯的核苷酸和含环丙基膦酸酯的核苷酸。
在某些实施方式中，其中所述2’-修饰核苷酸包括：2’-O-甲基核苷酸、2’-脱氧-2’-氟核苷酸、2’-脱氧核苷酸、2’-甲氧基乙基核苷酸、2’-氨基核苷酸和/或2’-烷基核苷酸。
在某些实施方式中，其中所述反义链5’末端开始的第7、12、14位核苷酸糖基2’位至少一个是氟。例如，其中所述反义链5’末端开始的第7、12、14位核苷酸糖基2’位均是氟。
在某些实施方式中，其中所述反义链除了5’末端开始的第7、12、14位核苷酸之外，其余的核苷酸糖基的2’位至少一个是甲氧基。
在某些实施方式中，其中所述正义链5’末端开始的第5、7、8、9位核苷酸糖基2’位至少一个是氟。例如，所述正义链5’末端开始的第5、7、8、9位核苷酸糖基2’位均是氟。
在某些实施方式中，其中所述正义链除了5’末端开始的第5、7、8、9位核苷酸之外，其余的核苷酸糖基的2’位的至少一个是甲氧基。
例如，所述正义链或反义链的部分或全部核苷酸糖基2’位的-OH可以被取代，其中，所述取代基团为氟或甲氧基，优选从正义链5’末端开始的第5、7、8、9位的核苷酸2’位是氟和从反义链的5’末端开始的第7、12、14位核苷酸2’位是氟，其余的核苷酸2’位均是甲氧基。
例如，所述反义链5’末端开始的第7、12、14位核苷酸糖基2’位均是氟，其余的核苷酸糖基的2’位均是甲氧基。
例如，所述正义链5’末端开始的第5、7、8、9位核苷酸糖基2’位均是氟，其余的核苷酸糖基的2’位均是甲氧基。
在某些实施方式中，其中所述正义链和/或反义链的核苷酸之间存在至少连续两个硫代磷酸酯键。
在某些实施方式中，其中所述正义链末端和/或反义链末端的3个连续的核苷酸之间存在至少连续两个硫代磷酸酯键。
例如，其中所述正义链和反义链的5’端和3’端的3个连续的核苷酸之间存在至少连续两个硫代磷酸酯键。
又例如，从正义链5’末端开始的第5、7、8、9位的核苷酸2’位是氟和从反义链的5’末端开始的第7、12、14位核苷酸2’位是氟，其余的核苷酸2’位均是甲氧基，且所述正义链和反义链的5’端和3’端的3个连续的核苷酸之间存在至少连续两个硫代磷酸酯键。
在一些筛选实施方案中，所述的RNAi剂的正义链和反义链选自表4中序列或与表4中的各序列相差一个、两个或三个核苷酸。
表4筛选RNA抑制剂序列
Figure PCTCN2022125877-appb-000005
其中，G＝2’-甲氧基鸟苷酸，A＝2’-甲氧基腺苷酸，U＝2’-甲氧基尿苷酸，C＝2’-甲氧基胞苷酸；u＝尿苷酸，c＝胞苷酸。
在一些实施例方案中，在所述RNAi剂中的反义链选自下列表5中的序列。从反义链5’末端开始的第7、12、14位的核苷酸2’位是氟，其余的核苷酸2’位是甲氧基，且所述反义链的末端至少有3个相邻核苷酸之间的磷酸酯键可以硫代。
表5反义链的修饰序列
SEQ ID NO. 单链代码 反义链序列5'→3'
109 AS55 UsUsUUGUfUUCACfAAfACAAGCsUsG
110 AS56 UsUsUAUUfACUAAfCAfCAAGGGsAsG
111 AS57 UsAsUACUfUUAAUfUUfUAAAACsCsC
112 AS58 UsAsCUUUfAAUUUfUAfAAACCCsAsA
113 AS59 UsUsGUUCfAAAAAfUCfACAAGCsAsU
114 AS60 UsUsAAUUfUUAAAfACfCCAAUUsUsU
115 AS61 AsUsACUUfUAAUUfUUfAAAACCsCsA
116 AS62 UsAsAAACfCCAAUfUUfUUGUUCsUsC
117 AS63 UsUsUUGCfAGCGAfCUfAGCACCsAsG
118 AS64 UsCsAAGCfUCAAAfAAfAAAUGCsUsG
119 AS65 UsUsUAAUfUUUAAfAAfCCCAAUsUsU
120 AS66 UsUsCAAAfAAUCAfCAfAGCAUCsUsG
SEQ ID NO. 单链代码 反义链序列5'→3'
121 AS67 UsAsAGCUfGUUGGfGUfAGACUCsUsG
122 AS68 UsUsCACAfAACAAfGCfUGGUCGsGsU
123 AS69 UsUsGUCCfUGGAUfGUfCACUCCsAsG
124 AS70 UsAsUUACfAGACAfCUfACACGGsAsG
125 AS71 UsUsUGUUfUCACAfAAfCAAGCUsGsG
126 AS72 UsUsUUGGfAACAGfUAfGUCCCGsCsG
127 AS73 AsAsGAAGfUUGGCfCAfGCAUsCsC
128 AS74 UsAsUACGfGAAGCfCCfAAGAsAsG
129 AS75 CsUsGUGCfAUGCCfAUfAUAUsAsC
130 AS76 CsAsUGGAfCCACGfCCfCCAUsAsG
131 AS77 AsAsAGACfAGCCGfUUfGGGGsAsG
132 AS78 UsCsUUGUfCCACCfCAfGAACsUsC
133 AS79 AsGsACCCfUCCACfCUfUGUCsCsA
134 AS80 AsGsUGAGfACCCUfCCfACCUsUsG
135 AS81 AsAsAGUGfAGACCfCUfCCACsCsU
136 AS82 GsUsUGAGfGGAGUfUUfUGCUsGsG
137 AS83 AsGsUUGAfGGGAGfUUfUUGCsUsG
138 AS84 UsCsCAGUfUGAGGfGAfGUUUsUsG
139 AS85 UsCsUUCAfUCCAGfUUfGAGGsGsA
140 AS86 UsUsCUUCfAUCCAfGUfUGAGsGsG
141 AS87 AsGsUUUCfUUCAUfCCfAGUUsGsA
142 AS88 UsUsGCUCfAAUUUfUUfGCAGsGsU
143 AS89 CsAsUUGCfUCAAUfUUfUUGCsAsG
144 AS90 UsCsAUUGfCUCAAfUUfUUUGsCsA
145 AS91 GsUsCAUUfGCUCAfAUfUUUUsGsC
146 AS92 UsGsUGGGfCUCUCfUCfUCAUsCsC
147 AS93 UsUsGAUCfAUACAfCAfGCAAsAsC
148 AS94 UsUsUGAUfCAUACfACfAGCAsAsA
149 AS95 AsAsAGGUfGGGAGfACfUGGGsGsG
150 AS96 CsAsUUAGfAAGAAfAAfGGUGsGsG
151 AS97 UsCsAUUAfGAAGAfAAfAGGUsGsG
152 AS98 CsUsCAUUfAGAAGfAAfAAGGsUsG
153 AS99 UsCsGGUUfGGAAUfUCfUUUUsUsG
154 AS100 AsAsACAAfGCUGGfUCfGGUUsGsG
155 AS101 UsCsACAAfACAAGfCUfGGUCsGsG
156 AS102 UsUsCACAfAACAAfGCfUGGUsCsG
157 AS103 UsUsUCACfAAACAfAGfCUGGsUsC
158 AS104 UsUsUUGUfUUCACfAAfACAAsGsC
159 AS105 UsUsUUUGfUUUCAfCAfAACAsAsG
SEQ ID NO. 单链代码 反义链序列5'→3'
160 AS106 UsUsUUUUfGUUUCfACfAAACsAsA
161 AS107 AsCsUUUUfUUGUUfUCfACAAsAsC
162 AS108 AsCsACUUfUUUUGfUUfUCACsAsA
163 AS109 AsAsAAGGfGAACAfCUfUUUUsUsG
164 AS110 UsCsUCAAfCUUGAfAAfAGGGsAsA
165 AS111 UsGsUUCUfCAACUfUGfAAAAsGsG
166 AS112 AsAsCCCAfAUUUUfUGfUUCUsCsA
167 AS113 UsAsAAACfCCAAUfUUfUUGUsUsC
168 AS114 UsUsUUAAfAACCCfAAfUUUUsUsG
169 AS115 AsUsUCAAfGACACfUAfAAUAsCsA
170 AS116 UsCsUUACfAUUCAfAGfACACsUsA
171 AS117 UsCsAUGUfUCUUAfCAfUUCAsAsG
172 AS118 GsUsCAUGfUUCUUfACfAUUCsAsA
173 AS119 AsUsCUGUfGGAAAfAAfACUAsAsG
174 AS120 AsAsAUCAfCAAGCfAUfCUGUsGsG
175 AS121 AsAsAAAUfCACAAfGCfAUCUsGsU
176 AS122 CsGsGACAfAAUCAfGCfGAUGUsGsU
177 AS123 CsCsAAAAfAGAAUfUCfCAAUUsGsA
178 AS124 AsCsCGACfCAGCUfUGfUUUGUsGsA
179 AS125 AsGsCGCGfGGACUfACfUGUUCsCsA
180 AS126 GsCsGCGGfGACUAfCUfGUUCCsAsA
181 AS127 AsAsCCGAfCCAGCfUUfGUUUGsUsG
182 AS128 UsGsUUCCfCUUUUfCAfAGUUGsAsG
183 AS129 UsCsCCUUfUUCAAfGUfUGAGAsAsC
184 AS130 UsAsUACUfCUCAUfUGfUGGAUGsAsC
其中，G＝2’-O-甲基鸟苷酸，A＝2’-O-甲基腺苷酸，U＝2’-O-甲基尿苷酸，C＝2’-O-甲基胞苷酸；fG＝2’-氟鸟苷酸，fA＝2’-氟腺苷酸，fU＝2’-氟尿苷酸，fC＝2’-氟胞苷酸；Gs＝2'-O-甲基-3’-硫代鸟苷酸、As＝2'-O-甲基-3'-硫代腺甘酸、Us＝2'-O-甲基-3'-硫代尿甘酸、Cs＝2'-O-甲基-3'-硫代胞苷酸。
在一些实施例方案中，在所述RNAi剂中的正义链选自下列表6中的序列。从正义链5’末端开始的第5、7、8、9位的核苷酸2’位是氟，其余的核苷酸2’位是甲氧基，且所述反义链的末端至少有3个相邻核苷酸之间的磷酸酯键可以硫代。
表6正义链修饰序列
SEQ ID NO. 单链代码 正义链序列5'→3'
185 S55 GsCsUUfGUfUfUfGUGAAACAAAAsAsA
186 S56 CsCsCUfUGfUfGfUUAGUAAUAAAsCsG
187 S57 GsUsUUfUAfAfAfAUUAAAGUAUAsCsA
188 S58 GsGsGUfUUfUfAfAAAUUAAAGUAsUsA
189 S59 GsCsUUfGUfGfAfUUUUUGAACAAsUsA
190 S60 AsAsUUfGGfGfUfUUUAAAAUUAAsAsG
SEQ ID NO. 单链代码 正义链序列5'→3'
191 S61 GsGsUUfUUfAfAfAAUUAAAGUAUsAsC
192 S62 GsAsACfAAfAfAfAUUGGGUUUUAsAsA
193 S63 GsGsUGfCUfAfGfUCGCUGCAAAAsCsU
194 S64 GsCsAUfUUfUfUfUUUGAGCUUGAsAsG
195 S65 AsUsUGfGGfUfUfUUAAAAUUAAAsGsU
196 S66 GsAsUGfCUfUfGfUGAUUUUUGAAsCsA
197 S67 GsAsGUfCUfAfCfCCAACAGCUUAsAsC
198 S68 CsGsACfCAfGfCfUUGUUUGUGAAsAsC
199 S69 GsGsAGfUGfAfCfAUCCAGGACAAsCsU
200 S70 CsCsGUfGUfAfGfUGUCUGUAAUAsCsC
201 S71 AsGsCUfUGfUfUfUGUGAAACAAAsAsA
202 S72 CsGsGGfACfUfAfCUGUUCCAAAAsAsG
203 S73 GsGsAUfGCfUfGfGCCAACUUCsUsU
204 S74 CsUsUCfUUfGfGfGCUUCCGUAsUsA
205 S75 GsUsAUfAUfAfUfGGCAUGCACsAsG
206 S76 CsUsAUfGGfGfGfCGUGGUCCAsUsG
207 S77 CsUsCCfCCfAfAfCGGCUGUCUsUsU
208 S78 GsCsUGfUGfAfCfAGGAUGGAAsGsA
209 S79 UsGsGAfCAfAfGfGUGGAGGGUsCsU
210 S80 CsAsAGfGUfGfGfAGGGUCUCAsCsU
211 S81 AsGsGUfGGfAfGfGGUCUCACUsUsU
212 S82 CsCsAGfCAfAfAfACUCCCUCAsAsC
213 S83 CsAsGCfAAfAfAfCUCCCUCAAsCsU
214 S84 CsAsAAfACfUfCfCCUCAACUGsGsA
215 S85 UsCsCCfUCfAfAfCUGGAUGAAsGsA
216 S86 CsCsCUfCAfAfCfUGGAUGAAGsAsA
217 S87 UsCsAAfCUfGfGfAUGAAGAAAsCsU
218 S88 AsCsCUfGCfAfAfAAAUUGAGCsAsA
219 S89 CsUsGCfAAfAfAfAUUGAGCAAsUsG
220 S90 UsGsCAfAAfAfAfUUGAGCAAUsGsA
221 S91 GsCsAAfAAfAfUfUGAGCAAUGsAsC
222 S92 GsGsAUfGAfGfAfGAGAGCCCAsCsA
223 S93 GsUsUUfGCfUfGfUGUAUGAUCsAsA
224 S94 UsUsUGfCUfGfUfGUAUGAUCAsAsA
225 S95 CsCsCCfCAfGfUfCUCCCACCUsUsU
226 S96 CsCsCAfCCfUfUfUUCUUCUAAsUsG
227 S97 CsCsACfCUfUfUfUCUUCUAAUsGsA
228 S98 CsAsCCfUUfUfUfCUUCUAAUGsAsG
229 S99 CsAsAAfAAfGfAfAUUCCAACCsGsA
230 S100 CsCsAAfCCfGfAfCCAGCUUGUsUsU
231 S101 CsCsGAfCCfAfGfCUUGUUUGUsGsA
SEQ ID NO. 单链代码 正义链序列5'→3'
232 S102 CsGsACfCAfGfCfUUGUUUGUGsAsA
233 S103 GsAsCCfAGfCfUfUGUUUGUGAsAsA
234 S104 GsCsUUfGUfUfUfGUGAAACAAsAsA
235 S105 CsUsUGfUUfUfGfUGAAACAAAsAsA
236 S106 UsUsGUfUUfGfUfGAAACAAAAsAsA
237 S107 GsUsUUfGUfGfAfAACAAAAAAsGsU
238 S108 UsUsGUfGAfAfAfCAAAAAAGUsGsU
239 S109 CsAsAAfAAfAfGfUGUUCCCUUsUsU
240 S110 UsUsCCfCUfUfUfUCAAGUUGAsGsA
241 S111 CsCsUUfUUfCfAfAGUUGAGAAsCsA
242 S112 UsGsAGfAAfCfAfAAAAUUGGGsUsU
243 S113 GsAsACfAAfAfAfAUUGGGUUUsUsA
244 S114 CsAsAAfAAfUfUfGGGUUUUAAsAsA
245 S115 UsGsUAfUUfUfAfGUGUCUUGAsAsU
246 S116 UsAsGUfGUfCfUfUGAAUGUAAsGsA
247 S117 CsUsUGfAAfUfGfUAAGAACAUsGsA
248 S118 UsUsGAfAUfGfUfAAGAACAUGsAsC
249 S119 CsUsUAfGUfUfUfUUUCCACAGsAsU
250 S120 CsCsACfAGfAfUfGCUUGUGAUsUsU
251 S121 AsCsAGfAUfGfCfUUGUGAUUUsUsU
252 S122 AsCsACfAUfCfGfCUGAUUUGUCsCsG
253 S123 UsCsGGfUUfGfGfAAUUCUUUUUsGsG
254 S124 UsCsACfAAfAfCfAAGCUGGUCGsGsU
255 S125 UsGsGAfACfAfGfUAGUCCCGCGsCsU
256 S126 UsUsGGfAAfCfAfGUAGUCCCGCsGsC
257 S127 CsAsCAfAAfCfAfAGCUGGUCGGsUsU
258 S128 CsUsCAfACfUfUfGAAAAGGGAAsCsA
259 S129 GsUsUCfUCfAfAfCUUGAAAAGGsGsA
260 S130 GsUsCAfUCfCfAfCAAUGAGAGUAsCsA
在某些实施方案中，本申请所述的RNAi剂的正义链与表6中的各序列相差一个、两个或三个核苷酸。
在某些实施方案中，所述RNAi剂中正义链与反义链的组合选自表7。
表7 RNAi剂
正义链代码 反义链代码 双链代码
S55 AS55 Kylo-09-DS37
S56 AS56 Kylo-09-DS38
S57 AS57 Kylo-09-DS39
S58 AS58 Kylo-09-DS40
S59 AS59 Kylo-09-DS41
S60 AS60 Kylo-09-DS42
S61 AS61 Kylo-09-DS43
正义链代码 反义链代码 双链代码
S62 AS62 Kylo-09-DS44
S63 AS63 Kylo-09-DS45
S64 AS64 Kylo-09-DS46
S65 AS65 Kylo-09-DS47
S66 AS66 Kylo-09-DS48
S67 AS67 Kylo-09-DS49
S68 AS68 Kylo-09-DS50
S69 AS69 Kylo-09-DS51
S70 AS70 Kylo-09-DS52
S71 AS71 Kylo-09-DS53
S72 AS72 Kylo-09-DS54
S73 AS73 Kylo-09-DS55
S74 AS74 Kylo-09-DS56
S75 AS75 Kylo-09-DS57
S76 AS76 Kylo-09-DS58
S77 AS77 Kylo-09-DS59
S78 AS78 Kylo-09-DS60
S79 AS79 Kylo-09-DS61
S80 AS80 Kylo-09-DS62
S81 AS81 Kylo-09-DS63
S82 AS82 Kylo-09-DS64
S83 AS83 Kylo-09-DS65
S84 AS84 Kylo-09-DS66
S85 AS85 Kylo-09-DS67
S86 AS86 Kylo-09-DS68
S87 AS87 Kylo-09-DS69
S88 AS88 Kylo-09-DS70
S89 AS89 Kylo-09-DS71
S90 AS90 Kylo-09-DS72
S91 AS91 Kylo-09-DS73
S92 AS92 Kylo-09-DS74
S93 AS93 Kylo-09-DS75
S94 AS94 Kylo-09-DS76
S95 AS95 Kylo-09-DS77
S96 AS96 Kylo-09-DS78
S97 AS97 Kylo-09-DS79
S98 AS98 Kylo-09-DS80
S99 AS99 Kylo-09-DS81
S100 AS100 Kylo-09-DS82
S101 AS101 Kylo-09-DS83
S102 AS102 Kylo-09-DS84
S103 AS103 Kylo-09-DS85
正义链代码 反义链代码 双链代码
S104 AS104 Kylo-09-DS86
S105 AS105 Kylo-09-DS87
S106 AS106 Kylo-09-DS88
S107 AS107 Kylo-09-DS89
S108 AS108 Kylo-09-DS90
S109 AS109 Kylo-09-DS91
S110 AS110 Kylo-09-DS92
S111 AS111 Kylo-09-DS93
S112 AS112 Kylo-09-DS94
S113 AS113 Kylo-09-DS95
S114 AS114 Kylo-09-DS96
S115 AS115 Kylo-09-DS97
S116 AS116 Kylo-09-DS98
S117 AS117 Kylo-09-DS99
S118 AS118 Kylo-09-DS100
S119 AS119 Kylo-09-DS101
S120 AS120 Kylo-09-DS102
S121 AS121 Kylo-09-DS103
S122 AS122 Kylo-09-DS104
S123 AS123 Kylo-09-DS105
S124 AS124 Kylo-09-DS106
S125 AS125 Kylo-09-DS107
S126 AS126 Kylo-09-DS108
S127 AS127 Kylo-09-DS109
S128 AS128 Kylo-09-DS110
S129 AS129 Kylo-09-DS111
S130 AS130 Kylo-09-DS112
与载体偶联的RNAi剂
本申请RNAi剂的另方面涉及将干扰核酸与载体偶联的方式，以增强RNAi剂的稳定性、活性、细胞分布或细胞摄取。
在某些实施方式中，通过在载体中引入靶组织受体的配体，以改变RNAi剂的分布、靶向或稳定性。例如，与不存在配体的物种相比，专属性的配体可以提供针对所选靶(例如分子、细胞或细胞类型、区室(例如细胞或器官区室、身体组织、器官或区域))的增强的亲和力。
配体可以包括天然存在的物质，如蛋白质(例如人血清白蛋白(HSA)、低密度脂蛋白(LDL)或球蛋白)；碳水化合物(例如葡聚糖、茁霉多糖、壳多糖、壳聚糖、菊糖、环糊精、N-乙酰葡糖胺、N-乙酰半乳糖胺或透明质酸)；或脂质。配体也可以是重组或合成分子，如合成聚合物，例如合成的聚氨基酸。
配体也可以包括靶向基团，例如与指定的细胞类型如肾细胞结合的细胞或组织靶向剂，例如凝集素、糖蛋白、脂质或蛋白质，例如抗体。靶向基团可以是促甲状腺激素、促黑素、凝集素、糖蛋白、表面活性蛋白质A、黏蛋白碳水化合物、多价乳糖、多价半乳糖、N-乙酰基-半乳糖胺、N-乙酰基-葡糖胺多价甘露糖、多价岩藻糖、糖基化聚氨基酸、多价半乳糖、 转铁蛋白、双膦酸盐、聚谷氨酸、聚天冬氨酸、脂质、胆固醇、类固醇、胆酸、叶酸、维生素B12、维生素A、生物素、或RGD肽或RGD肽模拟物。在某些实施方式中，该配体为多价半乳糖，例如，N-乙酰基-半乳糖胺。
本申请RNAi剂所包含的正义链和反义链可以通过固相合成的公知技术方便且常规地制备。可另外地或替代地使用本领域中已知的用于这类合成的任何其他方法，如液相合成或发酵。使用相似的技术来制备其他寡核苷酸(如硫代磷酸酯和烷基化衍生物)也是已知的。
在某些实施方式中，除了可商购以及寡核苷酸合成中常规使用的标准核苷亚磷酰胺单体以及非标准核苷亚磷酰胺单体之外，本申请的寡核苷酸或连接的核苷酸可以通过自动合成仪使用衍生自载体-核苷亚磷酰胺单体的亚磷酰胺法合成。
在某些实施方式中，本发明所述配体缀合的方式通过载体结构偶联于反义链的5’末端和/或3’末端，和/或正义链的5’末端和/或3’末端。
例如，所述载体结构可以偶联于正义链的5’末端和/或3’末端；或所述载体结构可以偶联于反义链的5’末端且所述载体结构偶联于正义链的3’末端；或所述载体结构可以偶联于反义链的3’末端，且所述配体偶联于正义链的5’末端；或所述载体结构偶联于正义链的5’末端和3’末端。
在某些实施方式中，其中所述载体结构包括5’MVIP和3’MVIP，其中，所述5’MVIP偶联在所述正义链和/或反义链5’末端，所述3’MVIP偶联在所述反义链和/或正义链3’末端，所述5’MVIP的结构如式I所示，所述3’MVIP结构如式II所示，
(X-L) n-B-D-R 1-，
       I
(X-L) m-B-D-R 2-，
        II
其中，
X为靶向特异性配体；
L为支链；
B为接头；
D为连接链；
R 1和R 2为转接点；
所述5’MVIP通过转接点R 1与正义链5’端或反义链5’端连接，所述3’MVIP通过转接点R 2与正义链3’端或反义链3’端连接，n和m各自独立地为0-4的任意整数。
在某些实施方式中，其中所述X为组织的特异性靶向配体
在某些实施方式中，其中所述的X肝靶向特异性配体。
在某些实施方式中，其中所述R 1或R 2与所述正义链或反义链的连接通过磷酸酯或经修饰的磷酸酯，优选地通过磷酸酯或硫代磷酸酯相连接。
在某些实施方式中，其中所述且n+m＝2-6的整数，优选n+m＝2、3或4，更优选为4。
在某些实施方式中，m或n可以为0，即不存在3’MVIP或5’MVIP。
在某些实施方案中，当n＝0(即不存在5’MVIP)时，所述3’MVIP的结构可以为：
Figure PCTCN2022125877-appb-000006
在某些实施方案中，当n＝1时，所述3’MVIP的结构可以为：
Figure PCTCN2022125877-appb-000007
在某些实施方案中，当n＝2时，所述3’MVIP的结构可以为：
Figure PCTCN2022125877-appb-000008
在某些实施方案中，当n＝3时，所述3’MVIP的结构可以为：
Figure PCTCN2022125877-appb-000009
在某些实施方案中，当n＝4时，所述3’MVIP的结构可以为：
Figure PCTCN2022125877-appb-000010
在某些实施方案中，所述的n是指同时放在所述RNAi剂的正义链和反义链5’末端5'MVIP中n之和，所述的m是指同时放在所述RNAi剂的正义链和反义链3’末端3'MVIP中m之和。
在某些实施方案中，所述R 1和R 2结构中带有-NH-、-S-和/或-O-，R 1和R 2通过结构中-NH-、-S-或-O-分别与连接链D以及正义链和/或反义链5’末端和3’末端相连，R 1和R 2相同或不相同。
在某些实施方案中，所述R 1和R 2是任选直链，或带有酰胺基、羧基或烷基类支链的直链或者环状结构，所述环状结构包括饱和或不饱和的脂肪族碳环基，或者含有硫、氧或氮原子的五元或六元杂环基或芳香烃基。
在某些实施方案中，所述R 1和/或R 2为-E 1(CH 2) xCH 2E 2-，其中x为3-12的任意整数，基团E 1和E 2可以分别为-NH-、-S-或-O-。
在某些实施方案中，所述R 1和/或R 2为-E 1(CH 2) x1CH(OH)(CH 2) x2E 2-，其中x1或x2各自独立地为3-10的任意整数，E 1和E 2可以分别为-NH-、-S-或-O-。
在某些实施方案中，所述R 1为含有N、S或O的杂环或碳环结构：
Figure PCTCN2022125877-appb-000011
在某些实施方式中，所述转接点R 1为-NH(CH 2) xCH 2O-，其中x为3-12的任意整数，优 选为4-6的任意整数，可以通过以下两种亚磷酰胺单体引入：
i.其中的一个-O-或-S-用于R 1亚磷酰胺单体的合成，通过固相合成的方法接入RNAi剂正义链或反义链的5’端。结构中-NH-、-S-或-O-用于与5'MVIP中的连接链D连接，从而在RNAi剂正义链或反义链的5’端引入肝靶向特异性配体X。引入到RNAi剂正义链或反义链5’端的单体示例性结构如下：
Figure PCTCN2022125877-appb-000012
在某些实施方案中，优选以下结构：
Figure PCTCN2022125877-appb-000013
ii.R 1结构中-NH-、-S-或-O-先与连接链D连接，另外一个-NH-、-S-或-O-用于5'MVIP亚磷酰胺单体的合成中与亚磷酰胺成酯，正义链或反义链5’MVIP亚磷酰胺单体结构示例如下：
Figure PCTCN2022125877-appb-000014
在某些实施方案中，正义链或反义链5’MVIP亚磷酰胺单体优选以下结构：
Figure PCTCN2022125877-appb-000015
当通式中n为1-4时，上述的单体中接头B部分分别支化1至4次，以获得对应的单体化合物，借助上述的单体化合物，肝靶向特异性配体X通过固相合成被引入到正义链或反义链5’末端。
在某些实施方案中，转接点R 1为-NH(CH 2) xCH 2O-，其中x可以是3-12的整数，优选为4-6的整数。
在某些实施方案中，5'MVIP亚磷酰胺单体结构选自如下结构中：
Figure PCTCN2022125877-appb-000016
Figure PCTCN2022125877-appb-000017
Figure PCTCN2022125877-appb-000018
在某些实施方式中，所述R 2为含有N、S或O的杂环或碳环结构：
Figure PCTCN2022125877-appb-000019
在某些实施方式，所述R 2为-NH(CH 2) x1CH(OH)(CH 2) x2CH 2O-，其中x1为1-4的任意整数，x2为0-4的任意整数。
本申请所述的R 2是通过丁二酸酐与R 2结构中-NH-、-S-或-O-成酯或酰胺的同时，又与空白Solid Support中-NH-进行偶联，形成3'MVIP solid spport，再通过亚磷酰胺固相合成法，将3’MVIP引入到正义链或反义链的3’末端。
在某些实施方案中，R 2结构中的杂环为吡咯环或哌啶环，其通过环中的氮杂原子与3'MVIP的连接链D连接，3'MVIP solid spport示例性结构如下：
Figure PCTCN2022125877-appb-000020
当通式中m为1-4时，上述的单体中接头B部分分别支化1至4次，以获得对应的Solid Support。
在某些实施方案中，R 2为-B 4(CH 2) x1CH(OH)(CH 2) x2CH 2B 5-，其中x1为1-4的整数，x2为0-4的整数，B 4和B 5分别为-NH-、-S-或-O-。
Figure PCTCN2022125877-appb-000021
当通式中m为1-4时，上述的单体中接头B部分分别支化1至4次，以获得对应的Solid Support。
在某些实施方案中，R 2为-NHCH 2CH(OH)CH 2O-。引入3’MVIP solid spport示例性结构如下：
Figure PCTCN2022125877-appb-000022
当通式中m为1-4时，上述的单体中接头B部分分别支化1至4次，以获得对应的Solid Support。
在某些实施方案中，3’MVIP solid support结构如下：
Figure PCTCN2022125877-appb-000023
Figure PCTCN2022125877-appb-000024
Figure PCTCN2022125877-appb-000025
在某些实施方式中，所述肝靶向特异性配体X选自用于增强肝细胞对RNAi剂的摄取的结构，可以是脂质、类固醇、维生素、糖、蛋白质、肽、多胺及肽模拟结构。在本申请提供 的RNAi剂中，引入所述RNAi剂正义链或反义链末端的肝靶向特异性配体X可以相同，也可以不同，例如在特性上，有些可以是增强肝靶向性，有些可以是所述RNAi剂在体内药代动力学的调节结构，有些可以是具有体内溶解活性的结构。在某些实施方案中，所述肝靶向特异性配体X选自以下结构中的一种或多种单糖及其衍生物。
在某些实施方式中，所述单糖选自以下结构中的一种或多种：甘露糖、半乳糖、D-阿拉伯糖、葡萄糖、果糖、木糖、葡糖胺、核糖。所述单糖衍生物选自甘露糖衍生物、半乳糖衍生物、葡萄糖衍生物、核糖衍生物以及其他衍生物。
在某些实施方式中，所述靶向单元X选自半乳糖、半乳糖胺、N-乙酰半乳糖胺及其衍生物，其结构通式如下：
Figure PCTCN2022125877-appb-000026
其中，W 1为氢或羟基保护基，可以相同也可以不同；W为-OH、-NHCOOH或-NHCO(CH 2) qCH 3，其中q为0-4的整数；W 2为-NH-、O、S或C。
在某些实施方式中，所述靶向单元X为N-乙酰半乳糖胺及其衍生物。
在某些实施方式中，所述靶向单元X选自以下结构：
Figure PCTCN2022125877-appb-000027
其中，W选自-OH、-NHCOOH或-NHCO(CH 2) qCH 3中的一种或两种，其中q为0-4的整数。
在某些实施方案中，所述肝靶向特异性配体X在同一个5'MVIP或3'MVIP结构中可以相同，也可以不同。
在某些实施方案中，5'MVIP与3'MVIP彼此之间的X可以相同，也可以不同。
在某些实施方式中，所述L是含有-NH-、-C(＝O)-、-O-、-S-、酰胺基、磷酰基、硫代磷酰基、C 4-C 10脂肪族碳环基、苯基或者这些基团的组合的C 4-C 18直链。
在某些实施方式中，所述L还带有羟乙基或羧酸类的侧链。
在某些实施方式中，所述L为含酰胺基或六元脂肪族碳环基的C 7-C 18直链。
在某些实施方式中，所述L选自如下结构中的一种或多种：
Figure PCTCN2022125877-appb-000028
其中，r1是1-12的任意整数，r2为0-20的任意整数，Z为H、烷基或酰胺基。
在某些实施方式中，所述B的结构与能引入的X的数量有关，所述B中含-NH-、C、O、S、酰胺基、磷酰基、硫代磷酰基，当n或m为1时，其为一条直链，当n或m为2、3或4时，其分叉的次数分别为2、3或4。
在某些实施方式中，所述B选自以下结构：
Figure PCTCN2022125877-appb-000029
其中，A 1和A 2各自独立地是C、O、S、-NH-、羰基、酰胺基、磷酰基或硫代磷酰基，r为0-4的整数。
在某些实施方式中，所述B选自以下结构：
Figure PCTCN2022125877-appb-000030
Figure PCTCN2022125877-appb-000031
其中，r为0-4的任意整数。
在某些实施方式中，所述B选自以下结构：
Figure PCTCN2022125877-appb-000032
Figure PCTCN2022125877-appb-000033
Figure PCTCN2022125877-appb-000034
在某些实施方式中，所述B选自以下结构：
Figure PCTCN2022125877-appb-000035
在某些实施方式中，所述D是含有-NH-、C＝O、O、S、酰胺基、磷酰基、硫代磷酰基、芳香烃基、C 4-C 10脂肪族碳环基、含1-3个氮的五元或六元杂环基或者这些基团的组合的C 3-C 18直链。
在某些实施方式中，所述D还带有羟甲基、甲基叔丁基、甲基苯酚基、C 5-C 6脂肪环基的侧链。
在某些实施方式中，，所述D为含两个C＝O、六元脂肪族碳环基或苯基的C 3-C 10直链。
在某些实施方式中，所述D为含两个C＝O的C 3-C 10直链。
在某些实施方式中，所述D选自以下结构：
Figure PCTCN2022125877-appb-000036
Figure PCTCN2022125877-appb-000037
其中，每个p各自独立地为1-20的任意整数；s为2-13的整数；Z 1和Z 2为相同或者不同的取代基团。
在某些实施方式中，所述D选自以下结构：
Figure PCTCN2022125877-appb-000038
Figure PCTCN2022125877-appb-000039
Figure PCTCN2022125877-appb-000040
在某些实施方式中，所述D选自以下结构：
Figure PCTCN2022125877-appb-000041
在某些实施方式中，所述5’MVIP结构中的(X-L) n-B-D-和3’MVIP结构中的(X-L) m-B-D-选自以下结构中的一种或多种：
Figure PCTCN2022125877-appb-000042
Figure PCTCN2022125877-appb-000043
Figure PCTCN2022125877-appb-000044
Figure PCTCN2022125877-appb-000045
Figure PCTCN2022125877-appb-000046
Figure PCTCN2022125877-appb-000047
Figure PCTCN2022125877-appb-000048
Figure PCTCN2022125877-appb-000049
Figure PCTCN2022125877-appb-000050
Figure PCTCN2022125877-appb-000051
Figure PCTCN2022125877-appb-000052
Figure PCTCN2022125877-appb-000053
在某些实施方式中，所述X、L、D、及B在5’MVIP与3’MVIP各自的内部或5’MVIP与3’MVIP之间相同或不同。
在某些实施方案中，所述5’MVIP结构中的(X-L) n-B-D-选自表8中所示的结构：
表8 5’MVIP的(X-L) n-B-D-结构
Figure PCTCN2022125877-appb-000054
Figure PCTCN2022125877-appb-000055
Figure PCTCN2022125877-appb-000056
Figure PCTCN2022125877-appb-000057
在某些实施方案中，5’MVIP也可以不存在，这时候m可以为2-4的整数。
在某些实施方案中，所述3’MVIP结构中的(X-L) m-B-D-选自表9中所示的结构：
表9 3’MVIP的(X-L) m-B-D-结构
Figure PCTCN2022125877-appb-000058
Figure PCTCN2022125877-appb-000059
Figure PCTCN2022125877-appb-000060
Figure PCTCN2022125877-appb-000061
在某些实施方案中，5’MVIP配体结构中的(X-L) n-B-D-与R 1的组合如表10所示。
表10 5’MVIP中(X-L) n-B-D-与R 1的组合
Figure PCTCN2022125877-appb-000062
Figure PCTCN2022125877-appb-000063
在某些实施方案中，3’MVIP可以不存在，这时候n可以是2-4。
在某些实施方案中，3’MVIP配体结构中的(X-L)m-B-D-与R 2组合如表11所示。
表11 3’MVIP的(X-L) m-B-D-与R 2组合
Figure PCTCN2022125877-appb-000064
Figure PCTCN2022125877-appb-000065
Figure PCTCN2022125877-appb-000066
在某些实施方式中，所述5’MVIP选自表10中5’MVIP01至5’MVIP22中的任一个。在某些实施方式中，所述5’MVIP选自：
Figure PCTCN2022125877-appb-000067
在某些实施方式中，所述3’MVIP选自表11中3’MVIP01至3’MVIP27的任一个。在某些实施方式中，所述3’MVIP选自：
Figure PCTCN2022125877-appb-000068
Figure PCTCN2022125877-appb-000069
在某些实施方案中，选择表12中不同5'MVIP和3'MVIP组合接入RNAi剂的正义链和或反义链的不同位置，包括末端及序列的中间位置，考察对AGT mRNA表达水平的影响。
表12 5'MVIP与3'MVIP的组合
Figure PCTCN2022125877-appb-000070
Figure PCTCN2022125877-appb-000071
例如，可以将表5中的反义链序列的3’末端与载体结构3'MVIP09偶联。示例性的，所述RNAi剂中的反义链可以选自下列表13中的序列。
表13与3’MVIP偶联的反义链
单链代码 反义链序列5'→3'
AS131 UsUsUUGUfUUCACfAAfACAAGCsUsG-3'MVIP09
AS132 UsUsUAUUfACUAAfCAfCAAGGGsAsG-3'MVIP09
AS133 UsAsUACUfUUAAUfUUfUAAAACsCsC-3'MVIP09
AS134 UsAsCUUUfAAUUUfUAfAAACCCsAsA-3'MVIP09
AS135 UsUsGUUCfAAAAAfUCfACAAGCsAsU-3'MVIP09
AS136 UsUsAAUUfUUAAAfACfCCAAUUsUsU-3'MVIP09
AS137 AsUsACUUfUAAUUfUUfAAAACCsCsA-3'MVIP09
AS138 UsAsAAACfCCAAUfUUfUUGUUCsUsC-3'MVIP09
AS139 UsUsUUGCfAGCGAfCUfAGCACCsAsG-3'MVIP09
AS140 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP09
AS141 UsUsUAAUfUUUAAfAAfCCCAAUsUsU-3'MVIP09
AS142 UsUsCAAAfAAUCAfCAfAGCAUCsUsG-3'MVIP09
AS143 UsAsAGCUfGUUGGfGUfAGACUCsUsG-3'MVIP09
AS144 UsUsCACAfAACAAfGCfUGGUCGsGsU-3'MVIP09
AS145 UsUsGUCCfUGGAUfGUfCACUCCsAsG-3'MVIP09
AS146 UsAsUUACfAGACAfCUfACACGGsAsG-3'MVIP09
AS147 UsUsUGUUfUCACAfAAfCAAGCUsGsG-3'MVIP09
AS148 UsUsUUGGfAACAGfUAfGUCCCGsCsG-3'MVIP09
单链代码 反义链序列5'→3'
AS149 AsAsGAAGfUUGGCfCAfGCAUsCsC-3'MVIP09
AS150 UsAsUACGfGAAGCfCCfAAGAsAsG-3'MVIP09
AS151 CsUsGUGCfAUGCCfAUfAUAUsAsC-3'MVIP09
AS152 CsAsUGGAfCCACGfCCfCCAUsAsG-3'MVIP09
AS153 AsAsAGACfAGCCGfUUfGGGGsAsG-3'MVIP09
AS154 UsCsUUGUfCCACCfCAfGAACsUsC-3'MVIP09
AS155 AsGsACCCfUCCACfCUfUGUCsCsA-3'MVIP09
AS156 AsGsUGAGfACCCUfCCfACCUsUsG-3'MVIP09
AS157 AsAsAGUGfAGACCfCUfCCACsCsU-3'MVIP09
AS158 GsUsUGAGfGGAGUfUUfUGCUsGsG-3'MVIP09
AS159 AsGsUUGAfGGGAGfUUfUUGCsUsG-3'MVIP09
AS160 UsCsCAGUfUGAGGfGAfGUUUsUsG-3'MVIP09
AS161 UsCsUUCAfUCCAGfUUfGAGGsGsA-3'MVIP09
AS162 UsUsCUUCfAUCCAfGUfUGAGsGsG-3'MVIP09
AS163 AsGsUUUCfUUCAUfCCfAGUUsGsA-3'MVIP09
AS164 UsUsGCUCfAAUUUfUUfGCAGsGsU-3'MVIP09
AS165 CsAsUUGCfUCAAUfUUfUUGCsAsG-3'MVIP09
AS166 UsCsAUUGfCUCAAfUUfUUUGsCsA-3'MVIP09
AS167 GsUsCAUUfGCUCAfAUfUUUUsGsC-3'MVIP09
AS168 UsGsUGGGfCUCUCfUCfUCAUsCsC-3'MVIP09
AS169 UsUsGAUCfAUACAfCAfGCAAsAsC-3'MVIP09
AS170 UsUsUGAUfCAUACfACfAGCAsAsA-3'MVIP09
AS171 AsAsAGGUfGGGAGfACfUGGGsGsG-3'MVIP09
AS172 CsAsUUAGfAAGAAfAAfGGUGsGsG-3'MVIP09
AS173 UsCsAUUAfGAAGAfAAfAGGUsGsG-3'MVIP09
AS174 CsUsCAUUfAGAAGfAAfAAGGsUsG-3'MVIP09
AS175 UsCsGGUUfGGAAUfUCfUUUUsUsG-3'MVIP09
AS176 AsAsACAAfGCUGGfUCfGGUUsGsG-3'MVIP09
AS177 UsCsACAAfACAAGfCUfGGUCsGsG-3'MVIP09
AS178 UsUsCACAfAACAAfGCfUGGUsCsG-3'MVIP09
AS179 UsUsUCACfAAACAfAGfCUGGsUsC-3'MVIP09
AS180 UsUsUUGUfUUCACfAAfACAAsGsC-3'MVIP09
AS181 UsUsUUUGfUUUCAfCAfAACAsAsG-3'MVIP09
AS182 UsUsUUUUfGUUUCfACfAAACsAsA-3'MVIP09
AS183 AsCsUUUUfUUGUUfUCfACAAsAsC-3'MVIP09
AS184 AsCsACUUfUUUUGfUUfUCACsAsA-3'MVIP09
AS185 AsAsAAGGfGAACAfCUfUUUUsUsG-3'MVIP09
AS186 UsCsUCAAfCUUGAfAAfAGGGsAsA-3'MVIP09
AS187 UsGsUUCUfCAACUfUGfAAAAsGsG-3'MVIP09
单链代码 反义链序列5'→3'
AS188 AsAsCCCAfAUUUUfUGfUUCUsCsA-3'MVIP09
AS189 UsAsAAACfCCAAUfUUfUUGUsUsC-3'MVIP09
AS190 UsUsUUAAfAACCCfAAfUUUUsUsG-3'MVIP09
AS191 AsUsUCAAfGACACfUAfAAUAsCsA-3'MVIP09
AS192 UsCsUUACfAUUCAfAGfACACsUsA-3'MVIP09
AS193 UsCsAUGUfUCUUAfCAfUUCAsAsG-3'MVIP09
AS194 GsUsCAUGfUUCUUfACfAUUCsAsA-3'MVIP09
AS195 AsUsCUGUfGGAAAfAAfACUAsAsG-3'MVIP09
AS196 AsAsAUCAfCAAGCfAUfCUGUsGsG-3'MVIP09
AS197 AsAsAAAUfCACAAfGCfAUCUsGsU-3'MVIP09
AS198 CsGsGACAfAAUCAfGCfGAUGUsGsU-3'MVIP09
AS199 CsCsAAAAfAGAAUfUCfCAAUUsGsA-3'MVIP09
AS200 AsCsCGACfCAGCUfUGfUUUGUsGsA-3'MVIP09
AS201 AsGsCGCGfGGACUfACfUGUUCsCsA-3'MVIP09
AS202 GsCsGCGGfGACUAfCUfGUUCCsAsA-3'MVIP09
AS203 AsAsCCGAfCCAGCfUUfGUUUGsUsG-3'MVIP09
AS204 UsGsUUCCfCUUUUfCAfAGUUGsAsG-3'MVIP09
AS205 UsCsCCUUfUUCAAfGUfUGAGAsAsC-3'MVIP09
AS206 UsAsUACUfCUCAUfUGfUGGAUGsAsC-3'MVIP09
在某些实施方案中，本申请所述的RNAi剂的反义链与表13中的各序列相差一个、两个或三个核苷酸。
例如，可以将表6中的正义链序列的5’末端与载体结构5'MVIP09偶联。示例性的，所述RNAi剂中的正义链可以选自下列表14中的序列。
表14与5’MVIP偶联的正义链
单链代码 正义链序列5'→3'
S131 5'MVIP09-GsCsUUfGUfUfUfGUGAAACAAAAsAsA
S132 5'MVIP09-CsCsCUfUGfUfGfUUAGUAAUAAAsCsG
S133 5'MVIP09-GsUsUUfUAfAfAfAUUAAAGUAUAsCsA
S134 5'MVIP09-GsGsGUfUUfUfAfAAAUUAAAGUAsUsA
S135 5'MVIP09-GsCsUUfGUfGfAfUUUUUGAACAAsUsA
S136 5'MVIP09-AsAsUUfGGfGfUfUUUAAAAUUAAsAsG
S137 5'MVIP09-GsGsUUfUUfAfAfAAUUAAAGUAUsAsC
S138 5'MVIP09-GsAsACfAAfAfAfAUUGGGUUUUAsAsA
S139 5'MVIP09-GsGsUGfCUfAfGfUCGCUGCAAAAsCsU
S140 5'MVIP09-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG
S141 5'MVIP09-AsUsUGfGGfUfUfUUAAAAUUAAAsGsU
S142 5'MVIP09-GsAsUGfCUfUfGfUGAUUUUUGAAsCsA
S143 5'MVIP09-GsAsGUfCUfAfCfCCAACAGCUUAsAsC
S144 5'MVIP09-CsGsACfCAfGfCfUUGUUUGUGAAsAsC
S145 5'MVIP09-GsGsAGfUGfAfCfAUCCAGGACAAsCsU
单链代码 正义链序列5'→3'
S146 5'MVIP09-CsCsGUfGUfAfGfUGUCUGUAAUAsCsC
S147 5'MVIP09-AsGsCUfUGfUfUfUGUGAAACAAAsAsA
S148 5'MVIP09-CsGsGGfACfUfAfCUGUUCCAAAAsAsG
S149 5'MVIP09-GsGsAUfGCfUfGfGCCAACUUCsUsU
S150 5'MVIP09-CsUsUCfUUfGfGfGCUUCCGUAsUsA
S151 5'MVIP09-GsUsAUfAUfAfUfGGCAUGCACsAsG
S152 5'MVIP09-CsUsAUfGGfGfGfCGUGGUCCAsUsG
S153 5'MVIP09-CsUsCCfCCfAfAfCGGCUGUCUsUsU
S154 5'MVIP09-GsCsUGfUGfAfCfAGGAUGGAAsGsA
S155 5'MVIP09-UsGsGAfCAfAfGfGUGGAGGGUsCsU
S156 5'MVIP09-CsAsAGfGUfGfGfAGGGUCUCAsCsU
S157 5'MVIP09-AsGsGUfGGfAfGfGGUCUCACUsUsU
S158 5'MVIP09-CsCsAGfCAfAfAfACUCCCUCAsAsC
S159 5'MVIP09-CsAsGCfAAfAfAfCUCCCUCAAsCsU
S160 5'MVIP09-CsAsAAfACfUfCfCCUCAACUGsGsA
S161 5'MVIP09-UsCsCCfUCfAfAfCUGGAUGAAsGsA
S162 5'MVIP09-CsCsCUfCAfAfCfUGGAUGAAGsAsA
S163 5'MVIP09-UsCsAAfCUfGfGfAUGAAGAAAsCsU
S164 5'MVIP09-AsCsCUfGCfAfAfAAAUUGAGCsAsA
S165 5'MVIP09-CsUsGCfAAfAfAfAUUGAGCAAsUsG
S166 5'MVIP09-UsGsCAfAAfAfAfUUGAGCAAUsGsA
S167 5'MVIP09-GsCsAAfAAfAfUfUGAGCAAUGsAsC
S168 5'MVIP09-GsGsAUfGAfGfAfGAGAGCCCAsCsA
S169 5'MVIP09-GsUsUUfGCfUfGfUGUAUGAUCsAsA
S170 5'MVIP09-UsUsUGfCUfGfUfGUAUGAUCAsAsA
S171 5'MVIP09-CsCsCCfCAfGfUfCUCCCACCUsUsU
S172 5'MVIP09-CsCsCAfCCfUfUfUUCUUCUAAsUsG
S173 5'MVIP09-CsCsACfCUfUfUfUCUUCUAAUsGsA
S174 5'MVIP09-CsAsCCfUUfUfUfCUUCUAAUGsAsG
S175 5'MVIP09-CsAsAAfAAfGfAfAUUCCAACCsGsA
S176 5'MVIP09-CsCsAAfCCfGfAfCCAGCUUGUsUsU
S177 5'MVIP09-CsCsGAfCCfAfGfCUUGUUUGUsGsA
S178 5'MVIP09-CsGsACfCAfGfCfUUGUUUGUGsAsA
S179 5'MVIP09-GsAsCCfAGfCfUfUGUUUGUGAsAsA
S180 5'MVIP09-GsCsUUfGUfUfUfGUGAAACAAsAsA
S181 5'MVIP09-CsUsUGfUUfUfGfUGAAACAAAsAsA
S182 5'MVIP09-UsUsGUfUUfGfUfGAAACAAAAsAsA
S183 5'MVIP09-GsUsUUfGUfGfAfAACAAAAAAsGsU
S184 5'MVIP09-UsUsGUfGAfAfAfCAAAAAAGUsGsU
S185 5'MVIP09-CsAsAAfAAfAfGfUGUUCCCUUsUsU
S186 5'MVIP09-UsUsCCfCUfUfUfUCAAGUUGAsGsA
单链代码 正义链序列5'→3'
S187 5'MVIP09-CsCsUUfUUfCfAfAGUUGAGAAsCsA
S188 5'MVIP09-UsGsAGfAAfCfAfAAAAUUGGGsUsU
S189 5'MVIP09-GsAsACfAAfAfAfAUUGGGUUUsUsA
S190 5'MVIP09-CsAsAAfAAfUfUfGGGUUUUAAsAsA
S191 5'MVIP09-UsGsUAfUUfUfAfGUGUCUUGAsAsU
S192 5'MVIP09-UsAsGUfGUfCfUfUGAAUGUAAsGsA
S193 5'MVIP09-CsUsUGfAAfUfGfUAAGAACAUsGsA
S194 5'MVIP09-UsUsGAfAUfGfUfAAGAACAUGsAsC
S195 5'MVIP09-CsUsUAfGUfUfUfUUUCCACAGsAsU
S196 5'MVIP09-CsCsACfAGfAfUfGCUUGUGAUsUsU
S197 5'MVIP09-AsCsAGfAUfGfCfUUGUGAUUUsUsU
S198 5'MVIP09-AsCsACfAUfCfGfCUGAUUUGUCsCsG
S199 5'MVIP09-UsCsGGfUUfGfGfAAUUCUUUUUsGsG
S200 5'MVIP09-UsCsACfAAfAfCfAAGCUGGUCGsGsU
S201 5'MVIP09-UsGsGAfACfAfGfUAGUCCCGCGsCsU
S202 5'MVIP09-UsUsGGfAAfCfAfGUAGUCCCGCsGsC
S203 5'MVIP09-CsAsCAfAAfCfAfAGCUGGUCGGsUsU
S204 5'MVIP09-CsUsCAfACfUfUfGAAAAGGGAAsCsA
S205 5'MVIP09-GsUsUCfUCfAfAfCUUGAAAAGGsGsA
S206 5'MVIP09-GsUsCAfUCfCfAfCAAUGAGAGUAsCsA
在某些实施方案中，本申请所述的RNAi剂的正义链与表14中的各序列相差一个、两个或三个核苷酸。
AGT主要表达在肝脏，其表达仅限于人类和非灵长类动物。在一些体内实施方案中，猴是临床前研究首选模型。在一些体内试验实施方案中，所述的RNAi剂选自表15中的序列。
表15含5’MVIP09/3’MVIP09组合的RNAi剂
Figure PCTCN2022125877-appb-000072
Figure PCTCN2022125877-appb-000073
在某些实施方案中，本申请所述的RNAi剂的正义链和反义链与表15中的各序列相差一个、两个或三个核苷酸。
在某些实施方案中，所述RNAi剂的反义链UsCsAAGCfUCAAAfAAfAAAUGCsUsG(SEQ ID NO:118)的5'末端和/或3'端与不同结构的5'MVIP和/或3'MVIP连接，所述反义链选自下表16：
表16 5'MVIP和/或3'MVIP偶联的反义链
单链代码 反义链序列5'→3'
AS140 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP09
AS207 5'MVIP17-UsCsAAGCfUCAAAfAAfAAAUGCsUsG
AS208 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP01
AS209 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP17
AS210 5'MVIP01-UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP01
AS211 5'MVIP09-UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP09
AS212 5'MVIP17-UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP17
AS213 5'MVIP01-UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP17
AS214 5'MVIP17-UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP01
AS215 5'MVIP01-UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP09
AS216 5'MVIP09-UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP01
AS217 5'MVIP09-UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP17
单链代码 反义链序列5'→3'
AS218 5'MVIP17-UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP09
AS219 5'MVIP12-UsCsAAGCfUCAAAfAAfAAAUGCsUsG
AS220 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP19
AS221 5'MVIP16-UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP16
AS222 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP17
AS223 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP18
AS224 5'MVIP03-UsCsAAGCfUCAAAfAAfAAAUGCsUsG
AS225 5'MVIP08-UsCsAAGCfUCAAAfAAfAAAUGCsUsG
AS226 5'MVIP16-UsCsAAGCfUCAAAfAAfAAAUGCsUsG
AS227 5'MVIP13-UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP06
AS228 5'MVIP04-UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP06
AS229 5'MVIP11-UsCsAAGCfUCAAAfAAfAAAUGCsUsG
AS230 5'MVIP11-UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP14
AS231 5'MVIP15-UsCsAAGCfUCAAAfAAfAAAUGCsUsG
AS232 5'MVIP02-UsCsAAGCfUCAAAfAAfAAAUGCsUsG
AS233 5'MVIP05-UsCsAAGCfUCAAAfAAfAAAUGCsUsG
AS234 5'MVIP06-UsCsAAGCfUCAAAfAAfAAAUGCsUsG
AS235 5'MVIP07-UsCsAAGCfUCAAAfAAfAAAUGCsUsG
AS236 5'MVIP10-UsCsAAGCfUCAAAfAAfAAAUGCsUsG
AS237 5'MVIP14-UsCsAAGCfUCAAAfAAfAAAUGCsUsG
AS238 5'MVIP18-UsCsAAGCfUCAAAfAAfAAAUGCsUsG
AS239 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP02
AS240 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP03
AS241 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP04
AS242 5'MVIP04-UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP04
AS243 5'MVIP03-UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP19
AS244 5'MVIP18-UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP18
AS245 5'MVIP08-UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP18
AS246 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP05
AS247 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP07
AS248 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP10
AS249 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP11
AS250 5'MVIP11-UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP11
AS251 5'MVIP15-UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP15
AS252 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP06
AS253 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP08
AS254 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP12
AS255 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP13
AS256 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP14
AS257 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP15
AS258 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP16
单链代码 反义链序列5'→3'
AS259 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP24
AS260 UsCsAAGCfUCAAAfAAfAAAUGCsUsG-3'MVIP27
AS261 5'MVIP19-UsCsAAGCfUCAAAfAAfAAAUGCsUsG
AS262 5'MVIP20-UsCsAAGCfUCAAAfAAfAAAUGCsUsG
AS263 5'MVIP21-UsCsAAGCfUCAAAfAAfAAAUGCsUsG
AS264 5'MVIP22-UsCsAAGCfUCAAAfAAfAAAUGCsUsG
AS265 5'MVIP01-UsCsAAGCfUCAAAfAAfAAAUGCsUsG
AS266 5'MVIP09-UsCsAAGCfUCAAAfAAfAAAUGCsUsG
在某些实施方案中，本申请所述的RNAi剂的反义链与表16中的各序列相差一个、两个或三个核苷酸。
在某些实施方案中，所述RNAi剂的正义链GsCsAUfUUfUfUfUUUGAGCUUGAsAsG(SEQ ID NO:194)的5'末端和/或3'端与不同结构的5'MVIP和/或3'MVIP连接，所述正义链选自下表17。
表17 5'MVIP和/或3'MVIP偶联的正义链
单链代码 正义链序列5'→3'
S140 5'MVIP09-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG
S207 GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP17
S208 GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP09
S209 GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP01
S210 5'MVIP17-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG
S211 5'MVIP01-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG
S212 5'MVIP01-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP01
S213 5'MVIP09-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP09
S214 5'MVIP17-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP17
S215 5'MVIP01-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP17
S216 5'MVIP17-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP01
S217 5'MVIP01-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP09
S218 5'MVIP09-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP01
S219 5'MVIP09-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP17
S220 5'MVIP17-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP09
S221 5'MVIP12-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG
S222 GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP19
S223 5'MVIP16-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP16
S224 GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP17
S225 GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP18
S226 5'MVIP03-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG
S227 5'MVIP08-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG
S228 5'MVIP16-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG
S229 5'MVIP13-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP06
S230 5'MVIP04-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP06
S231 5'MVIP11-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG
S232 5'MVIP11-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP14
单链代码 正义链序列5'→3'
S233 5'MVIP15-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG
S234 5'MVIP02-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG
S235 5'MVIP05-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG
S236 5'MVIP06-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG
S237 5'MVIP07-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG
S238 5'MVIP10-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG
S239 5'MVIP14-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG
S240 5'MVIP18-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG
S241 GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP02
S242 GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP03
S243 GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP04
S244 5'MVIP04-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP04
S245 5'MVIP03-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP19
S246 5'MVIP18-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP18
S247 5'MVIP08-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP18
S248 GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP05
S249 GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP07
S250 GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP10
S251 GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP11
S252 5'MVIP11-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP11
S253 5'MVIP15-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP15
S254 GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP06
S255 GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP08
S256 GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP12
S257 GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP13
S258 GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP14
S259 GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP15
S260 GsCsAUfUUfUfUfUUUGAGCUUGAsAsG-3'MVIP16
S261 5'MVIP19-GsCsAUfUUfUfUfUUUGAGCUUGAsAsG
S262 5'MVIP20--GsCsAUfUUfUfUfUUUGAGCUUGAsAsG
S263 5'MVIP21--GsCsAUfUUfUfUfUUUGAGCUUGAsAsG
S264 5'MVIP22--GsCsAUfUUfUfUfUUUGAGCUUGAsAsG
在某些实施方案中，本申请所述的RNAi剂的正义链与表17中的各序列相差一个、两个或三个核苷酸。
在某些实施方案中，本申请所述的RNAi剂是由表17的正义链或与这些正义链相差一个、两个或三个核苷酸的序列和表16中的反义链或与这些反义链一个、两个或三个核苷酸的序列随机配对形成的。
在某些实施方案中，本申请所述的RNAi剂是由表16中反义链和表17中正义链配对退火合成，见表18。这些RNAi剂中的n+m分别为2，3，4，5和6。5’MVIP和/或3’MVIP偶联的位置包括反义链的5’末端和/或3’末端、正义链的5’末端和/或3’末端、反义链的5’末端和正义链的3’末端、正义链的5’末端和3’末端。其中n+m＝2,3,4,5和6。
表18含有5’MVIP和3’MVIP组合的RNAi剂
Figure PCTCN2022125877-appb-000074
Figure PCTCN2022125877-appb-000075
在某些实施方案中，本申请所述的RNAi剂或其药学上可接受的盐优选以钠盐和三乙胺盐或其它可药用盐的形式制备或合成。
在某些实施方案中，所述RNAi剂或其药学上可接受的盐更优选为其钠盐或三乙胺盐。
药物组合物
本申请也包括包含本申请的RNAi剂或其药学上可接受的盐的药物组合物。
在一个实施方式中，本文提供包含本文所述的RNAi剂和药学上可接受的药用辅料的药物组合物。包含RNAi剂的药物组合物可用于预防和/或治疗AGT相关障碍，例如，高血压。这类药物组合物依据递送模式配制。一个实例方案为配制用于以肠胃外递送全身性施用的组合物，例如，皮下(SC)、肌内(IM)或静脉内(IV)递送。本申请的药物组合物可以足以抑制AGT基因表达的剂量施用。
药学上可接受的“辅料”或“赋形剂”是用于递送一种或多种核酸至动物的药学上可接受的溶剂、悬浮剂或任何其他药学上惰性的媒介物。赋形剂可为液体或固体，并考虑计划的 施用方式进行选择，以在与核酸及给定药物组合物中的其他组分组合时提供所需的体积、稠度等。RNAi剂可以靶向特定组织(例如，肝细胞)的方式递送。
在某些实施方式中，所述的药物组合物，其还包含递送媒介物(如纳米颗粒、树状聚合物、聚合物、脂质体或阳离子递送系统)，
在某些实施方式中，其中所述递送媒介物包括脂质体。
在某些实施方式中，其中所述递送媒介物包括纳米脂质，其能够与核酸分子形成脂质体-核酸纳米颗粒。
在某些实施方式中，所述递送媒介物包括两性脂质化合物M10C1。
本申请的药物组合物包括(但不限于)溶液、乳液和包含脂质体的制剂。这些组合物可由多种组分产生，包括(但不限于)预形成液体、自乳化固体和自乳化半固体。制剂包括靶向肝脏的那些。可以单位剂型方便地存在的本申请药物制剂可依据制药业公知的常规技术制备。这类技术包括将活性成分与药用辅料或赋形剂结合的步骤。
用途
另一方面，本申请提供一种减少细胞或组织中AGT mRNA或蛋白质表达的方法，其包括使细胞或组织与有效量的前述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，和/或前述的药物组合物接触。
适合使用本申请方法处理的细胞可为任何表达AGT基因的细胞，例如，肝脏细胞、脑细胞、胆囊细胞、心脏细胞或肾脏细胞，但优选为肝脏细胞。适合用于本申请方法的细胞可为哺乳动物细胞，当与表达AGT基因的细胞接触时，RNAi剂抑制AGT基因(例如，人类、灵长类、非灵长类或大鼠AGT基因)的表达至少约50％，例如可通过PCR或基于分支DNA(bDNA)的方法，或由基于蛋白质的方法，如免疫荧光分析法，蛋白质印迹法或流式细胞分析技术测定的。
在某些实施方式中，其中所述组织是肝脏组织。
在某些实施方式中，其中所述细胞和组织是离体的。
在某些实施方式中，其中所述细胞和组织在受试者体内。
本文所用术语“抑制”可与“减少”、“降低”、“沉默”、“下调”、“压制”及其他类似术语交换使用，且包括任何抑制水平。AGT基因的表达可依据与AGT基因表达相关的任何变量的水平或水平变化来评价，例如，AGT mRNA水平或AGT蛋白水平。这一水平可在单个细胞中或细胞群中(包括例如，源自受试者的样品)中分析。可通过与AGT表达相关的一种或多种变量与对照水平比较的绝对或相对水平的下降来评价抑制。对照水平可为本领域上采用的任何类型的对照水平，例如，给药前基线水平或从未处理或接受对照(如例如，仅缓冲剂对照或无活性剂对照)处理的类似受试者、细胞或样本测得的水平。
AGT基因表达的抑制可通过其中AGT基因被转录且已处理(例如，通过一个或多个细胞与本申请的RNAi剂接触，或通过施用本申请的RNAi剂于其中存在该细胞的受试者)使得抑制AGT基因表达的第一细胞或细胞群(这类细胞可例如存在于源自受试者的样品中)表达的mRNA量与基本上与该第一细胞或细胞群相同但未如此处理的第二细胞或细胞群(未用RNAi剂处理或未用靶向目的基因的RNAi剂处理的对照细胞)相比的降低来表现。在优选的实施方式中，抑制通过实施例2提供的方法使用siRNA合适的浓度在高表达AGT的细胞系中评价，并将被干预细胞中的mRNA水平表示为非干预对照细胞中mRNA水平的百分比。
在其他实施方式中，AGT基因表达的抑制可通过功能上与AGT基因表达相关的参数的降低来评价，例如，受试者血液或血清中的AGT蛋白水平。AGT基因沉默可在任何表达AGT的细胞(内源性或来自表达构建体的外源性)中且通过本领域已知的任何分析法测定。
AGT蛋白质表达的抑制可由细胞或细胞群或受试者样品表达的AGT蛋白水平(例如，源自受试者的血液样品中的蛋白质水平)的降低来表现。如上所述，对于mRNA抑制的评价， 处理细胞或细胞群的蛋白质表达水平的抑制可类似地表示为对照细胞或细胞群的蛋白质水平的百分比，或受试者样品(例如，血液或其衍生的血清)中的蛋白质水平的变化。
可用于评价AGT基因抑制的对照组细胞、细胞群或受试者样品包括未与本申请RNAi剂接触的细胞、细胞群或受试者样品。例如，对照细胞、细胞群或受试者样品可源自于用RNAi剂治疗前的单个受试者(例如，人类或动物受试者)或适当匹配的群体对照。
细胞或细胞群表达的AGT mRNA水平可采用本领域已知用于评价mRNA表达的任何方法测定。例如，qRT-PCR，评价基因表达的降低。可通过本领域中已知的任何方法，例如，ELISA，评价蛋白质产生的降低。在某些实施方式中，穿刺肝脏活检样品用作监测AGT基因或蛋白质表达降低的组织材料。其他实施方式中，血液样品用作监测AGT蛋白质表达降低的受试者样品。
另一方面，本申请提供前述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，或前述的药物组合物在制备药物中的用途，所述药物用于预防和/或治疗疾病或病症或者降低疾病或病症的风险。
在某些实施方式中，其中所述疾病或病症包括与AGT相关的疾病或病症。
在某些实施方式中，其中所述疾病或病症选自：高血压病、高血压、临界性高血压、原发性高血压、继发性高血压、孤立性收缩期或舒张期高血压、妊娠相关高血压、糖尿病性高血压、顽固性高血压、难治性高血压、阵发性高血压、肾血管性高血压、戈德布拉特氏高血压、低血浆肾素活性或血浆肾素浓度相关的高血压、眼高血压、青光眼、肺动脉高血压、门静脉高血压、系统性静脉高血压、收缩期高血压、不稳定性高血压；高血压性心脏病、高血压性肾病、动脉粥样硬化、动脉硬化、血管病变、糖尿病性肾病、糖尿病性视网膜病、慢性心力衰竭、心肌病、糖尿病性心肌病、肾小球硬化症、主动脉缩窄、主动脉瘤、心室纤维化、心力衰竭、心肌梗塞、心绞痛、中风、肾疾病、肾衰竭、系统性硬化症、宫内发育迟缓(IUGR)、胎儿生长受限、肥胖、肝脂肪变性/脂肪肝、非酒精性脂肪性肝炎(NASH)、非酒精性脂肪肝病(NAFLD)；葡萄糖耐受不良、2型糖尿病(非胰岛素依赖型糖尿病)和代谢综合征。
另一方面，本申请提供一种预防和/或治疗疾病或病症的方法，所述方法包括向有此需要的受试者施用有效量的前述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，和/或前述的药物组合物。
本申请的体内方法可包括对受试者施用包含RNAi剂的组合物，其中该RNAi剂包括与接受施用RNAi剂的哺乳动物的AGT基因的RNA转录物的至少一部分互补的核苷酸序列。组合物可采用本领域已知的任何方式施用，包括(但不限于)：经口、腹膜内或肠胃外途径，包括颅内(例如，脑室内、脑实质内和鞘内)、静脉内、肌内、皮下、透皮、气道(气雾剂)、经鼻、直肠和局部(包括颊内及舌下)施用。在某些实施方式中，组合物通过静脉内输注或注射施用。在某些实施方式中，组合物通过皮下注射施用。在某些实施方式中，该组合物通过肌内注射施用。
本申请的RNAi剂还可作为“游离RNAi剂”施用。游离RNAi剂是在没有药物组合物的存在下施用。裸RNAi剂可在合适缓冲液中。缓冲液可包含乙酸盐、柠檬酸盐、醇溶谷蛋白、碳酸盐或磷酸盐，或其任何组合。在一个实施方式中，缓冲液为磷酸盐缓冲盐水(PBS)。可以调整包含RNAi剂的缓冲液的pH和渗透压，以便适合施用于受试者。
或者，本申请的RNAi剂可作为药物组合物施用，如脂质体制剂。
本申请的药物组合物可以足以抑制AGT基因表达的剂量施用。通常，本申请的RNAi剂的合适剂量在每天每千克接受者体重约0.001至约200.0mg的范围内，通常在每天每千克体重约1至50mg的范围内。通常，本申请的RNAi剂的合适剂量在约0.1mg/kg至约5.0mg/kg的范围内，例如约0.3mg/kg至约3.0mg/kg的范围内。
在一个实施方式中，该方法包括施用本文所述的组合物，使得降低靶AGT基因表达，如 每剂约1、2、3、4、5、6、1-6、1-3或3-6个月。在某些实施方式中，该组合物每3-6个月施用一次。
在某些实施方式中，在初始治疗方案后，以较少的频率施用治疗。重复剂量方案可包括规律地施用治疗量的RNAi剂，如每月一次至一年一次。在某些实施方式中，RNAi剂约每月一次至约每三个月一次施用，或约每三个月一次至约每六个月一次施用。
在初始治疗方案后，可以较低的频率施用治疗。可依据疾病严重性确定治疗持续时间。
在其他实施方式中，单一剂量的药物组合物可以为长效的，使得剂量以不超过1、2、3或4个月的间隔施用。在本申请的一些实施方式中，本申请的药物组合物的单一剂量约每月施用一次。在本申请的其他实施方式中，本申请药物组合物的单一剂量按季(即约每3个月)施用。在本申请的其他实施方式中，本申请药物组合物的单一剂量每年施用2次(即约每6个月一次)。
本领域技术人员应理解，某些因素可影响有效治疗受试者所需的剂量和施用时间，包括(但不限于)：受试者中存在的突变、之前的治疗、受试者的一般健康或年龄和存在的其他疾病。此外，按照需要以预防和/或治疗有效量的组合物治疗受试者可包括单次治疗或一系列治疗。
在某些实施方式中，其进一步包括测定来自所述受试者的样品中的AGT水平。
例如，其进一步包括测定来自所述受试者血液样品、血清样品或尿液样品中的AGT蛋白质水平。
在某些实施方式中，其进一步包括对所述受试者施用用于治疗高血压的另外的治疗剂。
例如，其中该另外的治疗剂可以选自：利尿剂、血管紧张素转化酶(ACE)抑制剂、血管紧张素II受体拮抗剂、β-阻滞剂、血管扩张剂、钙通道阻滞剂、醛固酮拮抗剂、α2-激动剂、肾素抑制剂、α-阻滞剂、外周作用肾上腺素能剂、选择性D1受体部分激动剂、非选择性α-肾上腺素能拮抗剂、合成的甾体抗盐皮质激素剂、血管紧张素受体-脑啡肽酶抑制剂(ARNi)、沙库比曲/缬沙坦；或内皮素受体拮抗剂(ERA)、西他生坦、安贝生坦、阿曲生坦、BQ-123、齐泊腾坦、波生坦、马昔腾坦和替唑生坦；上述任何治疗剂的组合；及配制成药剂组合的高血压治疗剂。
在某些实施方式中，其中该另外的治疗剂包括血管紧张素II受体拮抗剂。
例如，其中该血管紧张素II受体拮抗剂可以选自：氯沙坦、缬沙坦、奥美沙坦、依普沙坦和阿齐沙坦。
另一方面，本申请提供一种细胞其包含前述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐。
另一方面，本申请提供一种药盒，其包含前述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，或前所述的药物组合物。
具体地，本申请还公开了以下实施方式：
1.一种RNAi剂或其药学上可接受的盐，所述RNAi剂的结构中含有载体结构和干扰核酸，其结构如式IIIa、IIIb或IIIc所示：
Figure PCTCN2022125877-appb-000076
其中，
所述干扰核酸靶向AGT基因，其包括反义链和正义链；
所述载体结构包括5’MVIP(5’MultiValent Import Platform)和3’MVIP(3’MultiValent Import Platform)；
所述5’MVIP由转接点R 1、连接链D、接头B、支链L和肝靶向特异性配体X组成，所述3’MVIP由转接点R 2、连接链D、接头B、支链L和肝靶向特异性配体X组成，所述5’MVIP通过转接点R 1与正义链5’端或反义链5’端连接，所述3’MVIP通过转接点R 2与正义链3’端或反义链3’端连接，n和m各自独立地为0-4的任意整数。
2.根据实施方式1所述的RNAi剂或其药学上可接受的盐，其中所述干扰核酸包括siRNA或miRNA。
3.根据实施方式1-2中任一项所述的RNAi剂或其药学上可接受的盐，其中所述且n+m＝2-6的整数，优选n+m＝2、3或4，更优选为4。
4.根据实施方式1-3中任一项所述的RNAi剂或其药学上可接受的盐，所述R 1为含有N、S或O的杂环或碳环结构：
Figure PCTCN2022125877-appb-000077
或者，所述R 1为-NH(CH 2) xCH 2O-，其中x为3-12的任意整数，优选为4-6的任意整数。
5.根据实施方式1-4中任一项所述的RNAi剂或其药学上可接受的盐，所述R 2为含有N、S或O的杂环或碳环结构：
Figure PCTCN2022125877-appb-000078
或者，所述R 2为-NH(CH 2) x1CH(OH)(CH 2) x2CH 2O-，其中x1为1-4的任意整数，x2为0-4的任意整数。
6.根据实施方式1-5中任一项所述的RNAi剂或其药学上可接受的盐，所述X选自用于增强肝细胞对RNAi剂的摄取的结构。
7.根据实施方式1-6中任一项所述的RNAi剂或其药学上可接受的盐，所述X选自单糖及其衍生物。
8.根据实施方式1-7中任一项所述的RNAi剂或其药学上可接受的盐，所述X为N-乙酰半乳糖胺及其衍生物。
9.根据实施方式1-8中任一项所述的RNAi剂或其药学上可接受的盐，所述X选自以下结构：
Figure PCTCN2022125877-appb-000079
其中，W选自-OH、-NHCOOH或-NHCO(CH 2) qCH 3中的一种或两种，其中q为0-4的整数。
10.根据实施方式1-9中任一项所述的RNAi剂或其药学上可接受的盐，所述L选自如下结构中的一种或多种：
Figure PCTCN2022125877-appb-000080
其中，r1是1-12的任意整数，r2为0-20的任意整数，Z为H、烷基或酰胺基。
11.根据实施方式1-10中任一项所述的RNAi剂或其药学上可接受的盐，所述B选自以下结构：
Figure PCTCN2022125877-appb-000081
其中，A 1和A 2各自独立地是C、O、S、-NH-、羰基、酰胺基、磷酰基或硫代磷酰基，r为0-4的整数。
12.根据实施方式1-11中任一项所述的RNAi剂或其药学上可接受的盐，所述D选自以下结构：
Figure PCTCN2022125877-appb-000082
Figure PCTCN2022125877-appb-000083
Figure PCTCN2022125877-appb-000084
其中，每个p各自独立地为1-20的任意整数；s为2-13的整数；Z 1和Z 2为相同或者不同的取代基团。
13.根据实施方式1-12中任一项所述的RNAi剂或其药学上可接受的盐，所述5’MVIP结构中的(X-L) n-B-D-和3’MVIP结构中的(X-L) m-B-D-选自以下结构中的一种或多种：
Figure PCTCN2022125877-appb-000085
Figure PCTCN2022125877-appb-000086
Figure PCTCN2022125877-appb-000087
Figure PCTCN2022125877-appb-000088
Figure PCTCN2022125877-appb-000089
Figure PCTCN2022125877-appb-000090
Figure PCTCN2022125877-appb-000091
Figure PCTCN2022125877-appb-000092
Figure PCTCN2022125877-appb-000093
Figure PCTCN2022125877-appb-000094
Figure PCTCN2022125877-appb-000095
Figure PCTCN2022125877-appb-000096
Figure PCTCN2022125877-appb-000097
14.根据实施方式1-13中任一项所述的RNAi剂或其药学上可接受的盐，所述X、L、D及B在5’MVIP与3’MVIP各自的内部或5’MVIP与3’MVIP之间相同或不同。
15.根据实施方式1-14中任一项所述的RNAi剂或其药学上可接受的盐，所述5’MVIP选自表10中5’MVIP01至5’MVIP22中的任一个。
16.根据实施方式1-15中任一项所述的RNAi剂或其药学上可接受的盐，所述3’MVIP选自表11中3’MVIP01至3’MVIP27的任一个。
17.根据实施方式1-16中任一项所述的RNAi剂或其药学上可接受的盐，其中正义链5’MVIP和反义链3’MVIP的组合为5’MVIP01/3’MVIP01、5’MVIP01/3’MVIP17或5’MVIP09/3’MVIP09；或者正义链5’MVIP和正义链3’MVIP的组合为5’MVIP01/3’MVIP09或5’MVIP09/3’MVIP01。
18.根据实施方式1-17中任一项所述的RNAi剂或其药学上可接受的盐，其中所述反义链和正义链形成的互补区，所述互补区包括至少12个连续核苷酸，其中所述正义链包含AGT mRNA NM_001382817.3中起始位置为1854-1874、1907-1927、1895-1915、1352-1372、1903-1923、2019-2039、1853-1873和1818-1838的至少连续12个核苷酸或与其相差不超过3个核苷酸的序列或NM_001384479.1中起始位置为1822-1842、1875-1895、1863-1883、1320-1340、1871-1891、1987-2007、1821-1841和1786-1806的至少连续12个核苷酸或与其相差不超过3个核苷酸的序列。
19.根据实施方式1-18中任一项所述的RNAi剂或其药学上可接受的盐，其中所述正义链和反义链之间至少有约80％的碱基互补。
20.根据实施方式1-19中任一项所述的RNAi剂或其药学上可接受的盐，其中所述正义链和反义链各自独立地为15-30个核苷酸，优选17-25个核苷酸，更优选19-23个核苷酸。
21.根据实施方式1-20中任一项所述的RNAi剂或其药学上可接受的盐，其中所述正义链与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和SEQ ID NO:18中任意一个或与其相差不超过3个核苷酸的序列具有基本上同源性。
22.根据实施方式1-21中任一项所述的RNAi剂或其药学上可接受的盐，其中所述反义链包含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和SEQ ID NO:36中任意一个或与其相差不超过3个核苷酸的序列。
23.根据实施方式1-22中任一项所述的RNAi剂或其药学上可接受的盐，其中所述正义链包含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和SEQ ID NO:54中任意一个或与其相差不超过3个核苷酸的序列。
24.根据实施方式1-23中任一项所述的RNAi剂或其药学上可接受的盐，其中所述反义链包含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和SEQ ID NO:72中任意一个或与其相差不超过3个核苷酸的序列。
25.根据实施方式1-24中任一项所述的RNAi剂或其药学上可接受的盐，其中所述正义链和/或反义链上的一个或多个核苷酸被修饰以形成修饰的核苷酸。
26.根据实施方式25所述的RNAi剂或其药学上可接受的盐，其中所述修饰的核苷酸选自：脱氧核糖核苷酸、核苷酸模拟物、脱碱基核苷酸、2’-修饰核苷酸、3’至3’连接(倒置)核苷酸、含非天然碱基的核苷酸、桥接核苷酸、肽核酸(PNA)、解锁的核碱基类似物、锁定核苷酸、3’-O-甲氧基(2’核苷间连接)核苷酸、2’-氟-阿拉伯糖核苷酸、5’-甲基/2’-氟带核苷酸、吗啉代核苷酸、乙烯基膦酸酯脱氧核糖核苷酸、含乙烯基膦酸酯的核苷酸和含环丙基膦酸酯的核苷酸。
27.根据实施方式1-26中任一项所述的RNAi剂或其药学上可接受的盐，其中所述正义链和反义链的部分或全部核苷酸糖基2’位是氟或甲氧基。
28.根据实施方式1-27中任一项所述的RNAi剂或其药学上可接受的盐，其中所述正义链末端和/或反义链末端的3个连续的核苷酸之间存在至少连续两个硫代磷酸酯键。
29.根据实施方式1-28中任一项所述的RNAi剂或其药学上可接受的盐，其中所述反义链5’末端开始的第7、12、14位核苷酸糖基2’位是氟，反义链其余的核苷酸糖基的2’位是甲氧基，且所述反义链末端的3个连续的核苷酸之间存在至少连续两个硫代磷酸酯键；所述正义链5’末端开始的第5、7、8、9位核苷酸糖基2’位是氟，正义链其余的核苷酸糖基的2’位是甲氧基；且所述正义链末端的3个连续的核苷酸之间存在至少连续两个硫代磷酸酯键。
30.根据实施方式1-29中任一项所述的RNAi剂或其药学上可接受的盐，其中所述反义链包含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和SEQ ID NO:126中任意一个或与其相差不超过3个核苷酸的序列。
31.根据实施方式1-30中任一项所述的RNAi剂或其药学上可接受的盐，其中所述正义链包含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和SEQ ID NO:202中任意一个或与其相差不超过3个核苷酸的序列。
32.根据实施方式1-31中任一项所述的RNAi剂或其药学上可接受的盐，其中所述干扰核酸包括Kylo-09-DS01至Kylo-09-DS112中任意一个。
33.一种抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其包含反义链，其中所述反义链包含至少12个与选自以下序列相应位置的核苷酸基本上互补的连续核苷酸或与其相差不超过3个核苷酸的序列：AGT mRNA NM_001382817.3中起始位置为1854-1874、1907-1927、1895-1915、1352-1372、1903-1923、2019-2039、1853-1873和1818-1838的至少连续12个核苷酸或与其相差不超过3个核苷酸的序列或NM_001384479.1中起始位置为 1822-1842、1875-1895、1863-1883、1320-1340、1871-1891、1987-2007、1821-1841和1786-1806的至少连续12个核苷酸或与其相差不超过3个核苷酸的序列。
34.根据实施方式33所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述反义链长度为15-30个核苷酸，优选17-25个核苷酸，更优选19-23个核苷酸。
35.根据实施方式33-34中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述反义链包含以下的核苷酸序列：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和SEQ ID NO:36中任意一个或与其相差不超过3个核苷酸的序列。
36.根据实施方式33-35中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述RNAi剂包括单链或双链核酸分子。
37.根据实施方式33-36中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述RNAi剂包括siRNA或miRNA。
38.根据实施方式33-37中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其还包含正义链，其中所述反义链和正义链形成的互补区，所述互补区包括至少12个连续核苷酸。
39.根据实施方式33-38中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述互补区包含12-25个连续核苷酸碱基对。
40.根据实施方式33-39中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述正义链和反义链之间至少有约80％的碱基互补。
41.根据实施方式33-40中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述正义链包含AGT mRNA NM_001382817.3中起始位置为1854-1874、1907-1927、1895-1915、1352-1372、1903-1923、2019-2039、1853-1873和1818-1838的至少连续12个核苷酸或与其相差不超过3个核苷酸的序列或NM_001384479.1中起始位置为1822-1842、1875-1895、1863-1883、1320-1340、1871-1891、1987-2007、1821-1841和1786-1806的至少连续12个核苷酸或与其相差不超过3个核苷酸的序列。
42.根据实施方式33-41中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述正义链长度为15-30个核苷酸，优选17-25个核苷酸，更优选19-23个核苷酸。
43.根据实施方式33-43中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述正义链与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和SEQ ID NO:18中任意一个或与其相差不超过3个核苷酸的序列具有基本上同源性。
44.根据实施方式33-43中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述正义链与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和SEQ ID NO:18中任意一个或与其相差不超过3个核苷酸的序列具有基本上同源性，且所述反义链包含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和SEQ ID NO:36中任意一个或与其相差不超过3个核苷酸的序列。
45.根据实施方式33-44中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述反义链包含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和SEQ ID NO:72中任意一个或与其相差不超过3个核苷酸的序列。
46.根据实施方式33-45中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述正义链包含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和SEQ ID NO:54中任意一个或与其相差不超过3个核苷酸的序列。
47.根据实施方式33-46中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述反义链包含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和SEQ ID NO:72中任意一个或与其相差不超过3个核苷酸的序列，且所述正义链包含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和SEQ ID NO:54中任意一个或与其相差不超过3个核苷酸的序列。
48.根据实施方式33-47中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述正义链和/或反义链上的一个或多个核苷酸被修饰以形成修饰的核苷酸。
49.根据实施方式48所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述修饰的核苷酸选自：脱氧核糖核苷酸、核苷酸模拟物、脱碱基核苷酸、2’-修饰核苷酸、3’至3’连接(倒置)核苷酸、含非天然碱基的核苷酸、桥接核苷酸、肽核酸(PNA)、解锁的核碱基类似物、锁定核苷酸、3’-O-甲氧基(2’核苷间连接)核苷酸、2’-氟-阿拉伯糖核苷酸、5’-甲基/2’-氟带核苷酸、吗啉代核苷酸、乙烯基膦酸酯脱氧核糖核苷酸、含乙烯基膦酸酯的核苷酸和含环丙基膦酸酯的核苷酸。
50.根据实施方式49所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述2’-修饰核苷酸包括：2’-O-甲基核苷酸、2’-脱氧-2’-氟核苷酸、2’-脱氧核苷酸、2’-甲氧基乙基核苷酸、2’-氨基核苷酸和/或2’-烷基核苷酸。
51.根据实施方式33-50中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述正义链和反义链的部分或全部核苷酸糖基2’位取代基团为氟或甲氧基。
52.根据实施方式33-51中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述正义链末端和/或反义链末端的3个连续的核苷酸之间存在至少连续两个硫代磷酸酯键。
53.根据实施方式33-52中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述反义链5’末端开始的第7、12、14位核苷酸糖基2’位是氟，反义链其余的核苷酸糖基的2’位是甲氧基，且所述反义链末端的3个连续的核苷酸之间存在至少连续两个硫代磷酸酯键；所述正义链5’末端开始的第5、7、8、9位核苷酸糖基2’位是氟，正义链其余的核苷酸糖基的2’位是甲氧基；且所述正义链末端的3个连续的核苷酸之间存在至少连续两个硫代磷酸酯键。
54.根据实施方式33-53中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述反义链包含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和SEQ ID NO:126中任意一个或与其相差不超过3个核苷酸的序列。
55.根据实施方式33-54中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述正义链包含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和SEQ ID NO:202中任意一个或与其相差不超过3个核苷酸的序列。
56.根据实施方式33-55中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述干扰核酸包括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～Kylo-09-DS54中任意任一种或多种。
57.根据实施方式33-56中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，还包含配体，所述配体通过载体结构偶联于正义链和/或反义链。
58.根据实施方式33-57中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述配体包含靶向配体。
59.根据实施方式33-58中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述靶向配体通过载体结构偶联于反义链的5’末端和/或3’末端。
60.根据实施方式33-59中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述载体结构合于正义链的5’末端和/或3’末端。
61.根据实施方式57-60中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述载体结构包括5’MVIP和3’MVIP，其中，所述5’MVIP偶联在所述正义链和/或反义链5’末端，所述3’MVIP偶联在所述反义链和/或正义链3’末端，所述5’MVIP的结构如式I所示，所述3’MVIP结构如式II所示，
(X-L) n-B-D-R 1-，
      I
(X-L) m-B-D-R 2-，
      II
其中，
X为靶向特异性配体；
L为支链；
B为接头；
D为连接链；
R 1和R 2为转接点；
所述5’MVIP通过转接点R 1与正义链5’端或反义链5’端连接，所述3’MVIP通过转接点R 2与正义链3’端或反义链3’端连接，n和m各自独立地为0-4的任意整数。
62.根据实施方式61所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述且n+m＝2-6的整数，优选n+m＝2、3或4，更优选为4。
63.根据实施方式61-62中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，其中所述R 1或R 2与所述正义链或反义链通过磷酸酯或经修饰的磷酸酯，优选地通过磷酸酯或硫代磷酸酯相连接。
64.根据实施方式61-63中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述R 1为含有N、S或O的杂环或碳环结构：
Figure PCTCN2022125877-appb-000098
或者，所述R 1为-NH(CH 2) xCH 2O-，其中x为3-12的任意整数，优选为4-6的任意整数。
65.根据实施方式61-64中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述R 2为含有N、S或O的杂环或碳环结构：
Figure PCTCN2022125877-appb-000099
或者，所述R 2为-NH(CH 2) x1CH(OH)(CH 2) x2CH 2O-，其中x1为1-4的任意整数，x2为0-4的任意整数。
66.根据实施方式61-65中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述X为靶向配体选自用于增强肝细胞对RNAi剂的摄取的结构。
67.根据实施方式61-66中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述X选自单糖及其衍生物。
68.根据实施方式61-67中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述X选自半乳糖、半乳糖胺、N-乙酰半乳糖胺及其衍生物。
69.根据实施方式61-68中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述X为N-乙酰半乳糖胺及其衍生物。
70.根据实施方式61-69中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述X选自以下结构：
Figure PCTCN2022125877-appb-000100
其中，W选自-OH、-NHCOOH或-NHCO(CH 2) qCH 3中的一种或两种，其中q为0-4的整数。
71.根据实施方式61-70中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述L选自如下结构中的一种或多种：
Figure PCTCN2022125877-appb-000101
其中，r1是1-12的任意整数，r2为0-20的任意整数，Z为H、烷基或酰胺基。
72.根据实施方式61-71中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述B选自以下结构：
Figure PCTCN2022125877-appb-000102
其中，A 1和A 2各自独立地是C、O、S、-NH-、羰基、酰胺基、磷酰基或硫代磷酰基，r为0-4的整数。
73.根据实施方式61-72中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述D选自以下结构：
Figure PCTCN2022125877-appb-000103
Figure PCTCN2022125877-appb-000104
其中，每个p各自独立地为1-20的任意整数；s为2-13的整数；Z 1和Z 2为相同或者不同的取代基团。
74.根据实施方式61-73中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述5’MVIP结构中的(X-L) n-B-D-和3’MVIP结构中的(X-L) m-B-D-选自以下结构中的一种或多种：
Figure PCTCN2022125877-appb-000105
Figure PCTCN2022125877-appb-000106
Figure PCTCN2022125877-appb-000107
Figure PCTCN2022125877-appb-000108
Figure PCTCN2022125877-appb-000109
Figure PCTCN2022125877-appb-000110
Figure PCTCN2022125877-appb-000111
Figure PCTCN2022125877-appb-000112
Figure PCTCN2022125877-appb-000113
Figure PCTCN2022125877-appb-000114
Figure PCTCN2022125877-appb-000115
Figure PCTCN2022125877-appb-000116
Figure PCTCN2022125877-appb-000117
75.根据实施方式61-74中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述X、L、D、及B在5’MVIP与3’MVIP各自的内部或5’MVIP与3’MVIP之间相同或不同。
76.根据实施方式61-75中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述5’MVIP选自表10中5’MVIP01至5’MVIP22中的任一个。
77.根据实施方式61-76中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述3’MVIP选自表11中3’MVIP01至3’MVIP27的任一个。
78.根据实施方式61-77中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述正义链5’MVIP和反义链3’MVIP的组合为5’MVIP01/3’MVIP01、5’MVIP01/3’MVIP17或5’MVIP09/3’MVIP09；或者所述正义链5’MVIP和正义链3’MVIP的组合为5’MVIP01/3’MVIP09或5’MVIP09/3’MVIP01。
79.根据实施方式33-78中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述反义链包括AS131、AS137、AS138、AS140、AS141、AS142、AS147和AS148的任一种或多种。
80.根据实施方式33-79中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述正义链包括S131、S137、S138、S140、S141、S142、S147和S148中的任一种或多种。
81.根据实施方式33-80中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述RNAi剂或其药学上可接受的盐为表15中Kylo-09-DS113、Kylo-09-DS119、Kylo-09-DS120、Kylo-09-DS122、Kylo-09-DS123、Kylo-09-DS124、Kylo-09-DS129和Kylo-09-DS130中的任一种或多种。
82.根据实施方式33-81中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述反义链选自AS140、AS207～AS266中的任一种或多种。
83.根据实施方式33-82中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述正义链选自S140、S207～S264中的任一种或多种。
84.根据实施方式33-83中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，所述反义链选自AS140、AS207SEQ ID NO:413～AS266中的任一种或多种，且所述正义链选自S140、S207～S264中的任一种或多种。
85.根据实施方式33-84中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，包括表18中Kylo-09-DS122，Kylo-09-DS131，Kylo-09-DS141，Kylo-09-DS142和Kylo-09-DS147中的任一种或多种。
86.细胞，其包含实施方式1-32中任一项所述的RNAi剂或其药学上可接受的盐，或者实施方式33-85中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐。
87.药物组合物，其包含实施方式1-32中任一项所述的RNAi剂或其药学上可接受的盐，或者实施方式33-85中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，以及任选地药学上可接受的赋形剂、载体和/或稀释剂。
88.根据权要求87所述的药物组合物，其还包含递送媒介物。
89.根据权要求88所述的药物组合物，其中所述递送媒介物包括脂质体。
90.根据权要求89所述的药物组合物，其中所述递送媒介物包括纳米脂质。
91.一种减少细胞或组织中AGT mRNA或蛋白质表达的方法，其包括使细胞或组织与有效量的实施方式1-32中任一项所述的RNAi剂或其药学上可接受的盐、实施方式33-85中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，和/或实施方式87-90中任一项所述的药物组合物接触。
92.根据实施方式91所述的方法，其中所述细胞是肝细胞。
93.根据实施方式91所述的方法，其中所述组织是肝脏组织。
94.根据实施方式91所述的方法，其中所述细胞和组织是离体的。
95.根据实施方式91所述的方法，其中所述细胞和组织在受试者体内。
96.实施方式1-32中任一项所述的RNAi剂或其药学上可接受的盐、实施方式33-85中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，或实施方式87-90中任一项所述的药物组合物在制备药物中的用途，所述药物用于预防和/或治疗疾病或病症或者降低疾病或病症的风险。
97.根据实施方式96所述的用途，其中所述疾病或病症包括与AGT相关的疾病或病症。
98.根据实施方式96所述的用途，其中所述疾病或病症选自：高血压病、高血压、临界性高血压、原发性高血压、继发性高血压、孤立性收缩期或舒张期高血压、妊娠相关高血压、糖尿病性高血压、顽固性高血压、难治性高血压、阵发性高血压、肾血管性高血压、戈德布拉特氏高血压、低血浆肾素活性或血浆肾素浓度相关的高血压、眼高血压、青光眼、肺动脉高血压、门静脉高血压、系统性静脉高血压、收缩期高血压、不稳定性高血压；高血压性心脏病、高血压性肾病、动脉粥样硬化、动脉硬化、血管病变、糖尿病性肾病、糖尿病性视网膜病、慢性心力衰竭、心肌病、糖尿病性心肌病、肾小球硬化症、主动脉缩窄、主动脉瘤、心室纤维化、心力衰竭、心肌梗塞、心绞痛、中风、肾疾病、肾衰竭、系统性硬化症、宫内发育迟缓(IUGR)、胎儿生长受限、肥胖、肝脂肪变性/脂肪肝、非酒精性脂肪性肝炎(NASH)、非酒精性脂肪肝病(NAFLD)；葡萄糖耐受不良、2型糖尿病(非胰岛素依赖型糖尿病)和代谢综合征。
99.一种预防和/或治疗疾病或病症的方法，所述方法包括向有此需要的受试者施用有效量的实施方式1-32中任一项所述的RNAi剂或其药学上可接受的盐、实施方式33-85中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，和/或实施方式87-90中任一项所述的药物组合物。
100.根据实施方式99所述的方法，其中所述施用包括经皮下方式、静脉内方式、口服、经直肠或腹膜内施加途径向受试者施用。
101.根据实施方式99所述的方法，其中所述的RNAi剂、抑制AGT基因表达的RNAi剂或药物组合物以约0.01mg/kg至约50mg/kg的剂量施用于所述受试者。
102.根据实施方式99所述的方法，其进一步包括测定来自所述受试者的样品中的AGT水平。
103.根据实施方式99所述的方法，其中所述受试者样品中的AGT水平为血液样品、血清样品或尿液样品中的AGT蛋白质水平。
104.根据实施方式99所述的方法，其进一步包括对所述受试者施用用于治疗高血压的另外的治疗剂。
105.根据实施方式104所述的方法，其中该另外的治疗剂选自：利尿剂、血管紧张素转化酶(ACE)抑制剂、血管紧张素II受体拮抗剂、β-阻滞剂、血管扩张剂、钙通道阻滞剂、醛固酮拮抗剂、α2-激动剂、肾素抑制剂、α-阻滞剂、外周作用肾上腺素能剂、选择性D1受体部分激动剂、非选择性α-肾上腺素能拮抗剂、合成的甾体抗盐皮质激素剂、血管紧张素受体-脑啡肽酶抑制剂(ARNi)、沙库比曲/缬沙坦；或内皮素受体拮抗剂(ERA)、西他生坦、安贝生坦、阿曲生坦、BQ-123、齐泊腾坦、波生坦、马昔腾坦和替唑生坦；上述任何治疗剂的组合；及配制成药剂组合的高血压治疗剂。
106.根据实施方式104所述的方法，其中该另外的治疗剂包括血管紧张素II受体拮抗剂。
107.根据实施方式106所述的方法，其中该血管紧张素II受体拮抗剂选自：氯沙坦、缬沙坦、奥美沙坦、依普沙坦和阿齐沙坦。
108.药盒，其包含实施方式1-32中任一项所述的RNAi剂或其药学上可接受的盐、实施方式33-85中任一项所述的抑制AGT基因表达的RNAi剂或其药学上可接受的盐，或实施方式87-90中任一项所述的药物组合物。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的RNAi剂、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
说明：
DMSO的中文名称为二甲基亚砜；
DMF的中文名称为N,N-二甲基甲酰胺；
HOBt的中文名称为1-羟基苯并三氮唑；
HBTU的中文名称为O-苯并三氮唑-四甲基脲六氟磷酸酯；
DIPEA(DIEA)的中文名称为N,N-二异丙基乙胺；
DCM的中文名称为二氯甲烷；
DMAP的中文名称为4-二甲氨基吡啶；
DMT-CL的中文名称为4,4'-二甲氧基三苯基氯甲烷；
MEOH的中文名称为甲醇；
TBTU的中文名称为O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸；
Figure PCTCN2022125877-appb-000118
的名称为固相载体，如大孔氨甲基树脂(Resin)。
实施例1 合成siRNA的固相亚磷酰胺法
根据标准的固相亚磷酰胺法合成表1中的正义链和表2中的反义链，正义链与对应的反义链互补退火得到siRNA。
固相亚磷酰胺法基本步骤包括：
1)脱保护：脱掉起始单体Solid Support羟基保护基(DMTr)；
2)偶联：加上第一个亚磷酰胺单体，通过3’至5’方向发生偶联反应；
3)氧化：将所得的核苷亚磷酸酯氧化成更稳定的核苷磷酸酯(即三价磷氧化成五价磷)；
4)封闭：将前步骤失败核苷酸序列5’-OH加冒封死，使其不再进一步参与反应；重复上述步骤，直至最后一个亚磷酰胺单体的接入；然后用甲胺水溶液和氨水裂解Solid Support与起始单体之间酯键，并将所得核苷酸序列上的各个碱基与磷酸上的保护基脱掉；经HPLC分离纯化后，过滤除菌，冻干得到相应的正义链或反义链。
退火工艺描述：
将正义链和反义链冻干粉分别复溶、等摩尔混合，加入注射用水适量，投入适量的TRIS缓冲溶液。轻晃约1～2min，使溶液混合均匀。将水浴锅升温至92℃～95℃。将上述反应液置水浴锅中加热3min～5min,轻晃使溶液受热均匀。自然冷却至室温。得到无色或微黄色透明液体，取样送检，测浓度。本申请所述的RNAi剂是通过固相亚磷酰胺法得到各自的正义链和反义链，正义链与对应的反义链互补退火得到终产品。
实施例2 RNAi剂体外抑制AGT基因表达试验
取按实施例1所述的方法制备得到的RNAi剂(表3中的Kylo-09-DS01～Kylo-09-DS18)。将RNAi剂水溶液和DOTMA的有机溶液混合形成不溶于水的沉淀，沉淀分离干燥后溶于氯仿，进一步和其他脂质氯仿溶液混合，其他脂质包括M10C1和PEG600-胆固醇。混合物真空离心蒸发干燥过夜，得到纳米脂质包裹的RNAi剂，其中DOTMA、M10C1和PEG600-胆固醇与RNAi剂的重量配比为1～1.6，1.5～2.5和2.5～3.5。
含10％胎牛血清的DMEM配制相应浓度的纳米脂质包裹的RNAi剂(表3中RNAi剂：Kylo-09-DS01～Kylo-09-DS18)样品溶液。以10 5细胞密度接种Hep3B细胞，10％胎牛血清DMEM培养基，37℃，5％CO2，培养24h后，加不同浓度(10nM、1nM、0.1nM)的样品干预，孵育72h后，收集细胞样品，往收集完毕的细胞样品中加入1ml Ezol裂解液，漩涡震荡混匀。加入0.2ml三氯甲烷，剧烈摇动10s，室温放置1分钟。4℃，12,000x g离心15min。将上清水相转移至另一新的无RNA酶离心管中，并加入等体积的100％乙醇。吸取全部样品，加入带有2ml收集管的mini-spin离心柱。8,000x g，室温离心15s，弃尽流穿液。将剩余的样品转移至离心柱，重复上一步骤。往离心柱中加入700μl WB，轻盖盖子，8,000×g，室温离心15秒，弃尽流穿液，重复上一步骤，用500μl WB洗涤离心柱两次。通过QRT-PCR测定AGT mRNA水平。与未干预的Hep3B细胞上清对比，标定样品干预组的AGT mRNA相对百分率。所得试验结果见下表19和图1A。
表19 RNAi干预后Hep3B细胞中AGT mRNA的水平
Figure PCTCN2022125877-appb-000119
Figure PCTCN2022125877-appb-000120
所得试验结果显示，由表1正义链和表2反义链退火形成表3中RNAi剂，在不同浓度下对Hep3B细胞中AGTmRNA表达水平都呈现出不同程度的抑制效果，其中Kylo-09-DS01，Kylo-09-DS07，Kylo-09-DS08，Kylo-09-DS10，Kylo-09-DS11，Kylo-09-DS12，Kylo-09-DS17和Kylo-09-DS18对Hep3B细胞中AGTmRNA表达水平的抑制呈现明显的剂量依耐性，其分别对应的正义链序列为SEQ ID NO.37，43，44，46，47，48，53和54及对应的反义链序列SEQ ID NO.55，61，62，64，65，66，71和72。
将筛选出来的Kylo-09-DS01，Kylo-09-DS07，Kylo-09-DS08，Kylo-09-DS10，Kylo-09-DS11，Kylo-09-DS12，Kylo-09-DS17和Kylo-09-DS18参照Hep 3B细胞实验操作，考察RNAi剂不同浓度(10nM、0.1nM)对HepG 2细胞的干预效果，通过QRT-PCR测定AGT mRNA水平。与未干预的HepG 2细胞上清对比，标定样品干预组的AGT mRNA相对百分率。所得试验结果见下表20和图1B。
表20 RNAi干预后HepG 2细胞中AGT mRNA水平
Figure PCTCN2022125877-appb-000121
实施例3 修饰的siRNA在血浆中稳定性研究
本实施例所涉及的RNAi剂选自表7，母链为实施例2优选出来的序列(正义链序列为SEQ ID NO.37，43，44，46，47，48，53和54及对应的反义链序列SEQ ID NO.55，61，62，64，65，66，71和72)。从反义链5’末端开始的第7、12、14位的核苷酸2’位是氟和从其余的核苷酸为2’位是甲氧基，且所述反义链的末端至少有3个相邻核苷酸之间的磷酸酯键可以硫代，从正义链5’末端开始的第5、7、8、9位的核苷酸2’位是氟和从其余的核苷酸为2’位是甲氧基，且所述反义链的末端至少有3个相邻核苷酸之间的磷酸酯键可以硫代。
本实施例目的是验证上述的修饰方式可以增强RNAi剂在人血清中的稳定性，试验结果见表21和图2。
其中，图2显示的是RNAi Kylo-09-DS46与其母链Kylo-09-DS10不同时段的稳定性检测的HPLC图。
表21 RNAi剂在人血清中不同时长，全长双链相对0h的峰面积比％。
Figure PCTCN2022125877-appb-000122
上述试验结果显示，与各自未修饰的母链反义链5’末端开始的第7、12、14位的核苷酸2’位是氟和从其余的核苷酸为2’位是甲氧基，且所述反义链的末端至少有3个相邻核苷酸之间的磷酸酯键可以硫代，从正义链5’末端开始的第5、7、8、9位的核苷酸2’位是氟和从其余的核苷酸为2’位是甲氧基，且所述反义链的末端至少有3个相邻核苷酸之间的磷酸酯键可以硫代的修饰方式可以有效增强RNAi剂在人血清中稳定性，增强的效果存在序列的特异性，不同的序列稳定性的增强效果不同。
实施例4 5’MVIP和3’MVIP化合物的合成
当本申请的RNAi剂的正义链或反义链的3'端偶联有载体结构3'MVIP时，3'MVIP的solid support作为固相合成的起始单体。当本申请的RNAi剂的正义链或反义链的5端偶联有载体结构5'MVIP时，5’MVIP亚磷酰胺单体作为固相合成的最后一个单体。
当本申请的RNAi剂的正义链或反义链的3'端偶联有3'MVIP时，3'MVIP的solid support作为固相合成的起始单体，3’MVIP的solid spport通式如下：
Figure PCTCN2022125877-appb-000123
m为1-4时，通式中接头B部分分别支化1至4次，以获得对应的3’MVIP的Solid Support。
例如，当m为1时，所得的Solid Support作为RNAi剂Kylo-09-DS142(结构式见图9D)的正义链和Kylo-09-DS133反义链固相合成的起始单体；当m为2时，所得的Solid Support作为RNAi剂Kylo-09-DS141(结构式见9C)的正义链和Kylo-09-DS122的反义链的固相合成起始单体；
当m为3时，所得的Solid Support作为RNAi剂Kylo-09-DS147的反义链固相合成起始单体。
当本申请的RNAi剂的正义链或反义链的5'端有5’MVIP时，5’MVIP亚磷酰胺单体是作为正义链或反义链固相合成的最后一个亚磷酰胺单体。5’MVIP亚磷酰胺单体通式如下：
Figure PCTCN2022125877-appb-000124
n为1-4时，通式中接头B部分分别支化1至4次，以获得对应的5’MVIP亚磷酰胺单体。
例如，当n为1时，所得的5’MVIP亚磷酰胺单体作为RNAi剂Kylo-09-DS141、Kylo-09-DS131(结构式加9B)以及Kylo-09-DS147(结构式见9E)的正义链固相合成的最后一个单体；当n等于2时，所得的5’MVIP亚磷酰胺单体作为Kylo-09-DS122(结构式见9A)和Kylo-09-DS142的正义链固相合成的最后一个单体。
当n等于3时，所得的带3个配体X的5’MVIP亚磷酰胺单体可作为正义链或反义链固相合成的最后一个单体。
本申请所述的这些RNAi剂的正义链和反义链在进行亚磷酰胺固相合成之前，需要先化学合成相应的3’MVIP Solid Support和5’MVIP亚磷酰胺单体。
本实施示例性叙述了如下RNAi剂的3’MVIP Solid Support和5’MVIP亚磷酰胺单体化学合成过程，叙述如下：
4.1 3’MVIP的Solid Support的合成
4.1.1 3'MVIP09的Solid Support的合成
Figure PCTCN2022125877-appb-000125
合成过程描述：
4.1.1.1 ERC-01-c1的合成
Figure PCTCN2022125877-appb-000126
称取2-氨基-1,3-丙二醇(5.0g，54.9mmol)加入DMSO 50mL，氢氧化钠溶液(1g/mL)5mL，降温到0℃，滴加丙烯酸叔丁酯(20mL，137.8mol)2小时加完，室温反应48h，加石油醚(100mL)，饱和食盐水洗2次，有机层干燥。过层析柱(洗脱液：乙酸乙酯:石油醚＝25％-75％)，上柱加0.05％的三乙胺，得无色油状物6.2g。
4.1.1.2 ERC-01-c2的合成
Figure PCTCN2022125877-appb-000127
称取ERC-01-c1(6.2g，17.9mmol)，加二氯甲烷50mL，碳酸钠溶液(25％)23mL，室温滴加氯甲酸苄酯(8.2mL，57.4mmol)，2小时滴加完，室温反应过夜，饱和食盐水洗涤3次，无水硫酸钠干燥，蒸干溶剂，过层析柱(乙酸乙酯:石油醚＝5％-30％)得油状物4.0g。
4.1.1.3 ERC-01-c3的合成
Figure PCTCN2022125877-appb-000128
取ERC-01-c2(4.0g，8.3mmol)加甲酸12mL，室温反应过夜，减压蒸干溶剂，得产品2.8g。
4.1.1.4 ERCd-01-c1的合成
Figure PCTCN2022125877-appb-000129
将化合物ERC-01-c3(1.11g，3.0mmol)和dlSANC-c4(3.6g，8.04mmol)加到DMF(60mL)中，然后加入HOBt(2.24g)和HBTU(3.36g)，然后缓慢加入DIEA(4.16mL)。反应液室温下搅拌反应3小时。然后加入水，水层用二氯甲烷萃取(2x10mL)。合并有机层，然后依次用饱和碳酸氢钠(80mL)、水(2x60mL)、饱和食盐水(60mL)洗。用无水硫酸钠干燥，减压蒸干，用硅胶柱层析纯化(洗脱液：3-15％MeOH in DCM)。得淡黄色固3.24g。
4.1.1.5 ERCd-01-c2的合成
Figure PCTCN2022125877-appb-000130
ERCd-01-c1(3.24g，2.6mmol)用甲醇(60mL)溶解，加入10％钯碳(0.3g)，乙酸(2.0mL)。然后常压下加氢，反应过夜。反应液用硅藻土过滤，滤液减压蒸干，得油状物ERCd-01-c2 2.9g，其高分辨质谱图见图3。
4.1.1.6 3’MVIP09-c1的合成
Figure PCTCN2022125877-appb-000131
向反应瓶内依次加入SANCd-01-c0(0.824g，1.5mmol)和ERCd-01-c2(1.09g，1.0mmol)，再加入10mL的DCM，搅拌溶解，再依次加入TBTU(0.963g)和DIPEA(0.517g)，反应过夜，加水，用DCM萃取，有机相再用饱和食盐水洗涤，干燥、过滤、浓缩，最后过硅胶柱进行纯化，得产品1.3g。
4.1.1.7 3’MVIP09-c2的合成
Figure PCTCN2022125877-appb-000132
向反应瓶内依次加入3’MVIP09-c1(1.62g，1μmol)和10mL的DCM，室温搅拌溶解，再依次加入DMAP(0.366g)和丁二酸酐(0.2g，3μmol)，室温搅拌反应，TLC分析，反应合格浓缩掉DCM，加水，用DCM萃取，有机相再用饱和食盐水洗涤，有机相经无水硫酸钠干燥、过滤、浓缩，最后过硅胶柱进行纯化，得到产品为1.55g。
4.1.1.8 3’MVIP09的Solid Support合成
Figure PCTCN2022125877-appb-000133
向反应瓶内依次加入3’MVIP09-c2(0.86g，0.5μmol)和10mL DMF，溶解，再依次加入HBTU(0.19g)、DIPEA(0.194g)和大孔氨甲基树脂(2.0g)，摇床24h，过滤，树脂用10％甲醇/DCM洗涤，再用25％醋酸/吡啶进行封端，取代度150μmol/g。
4.1.2 3'MVIP17的Solid Support的合成
Figure PCTCN2022125877-appb-000134
Figure PCTCN2022125877-appb-000135
4.1.2.1 SANC-01-c1的合成
Figure PCTCN2022125877-appb-000136
合成步骤参照4.1.1.1.ERC-01-c1的合成。
4.1.2.2 SANC-01-c2的合成
Figure PCTCN2022125877-appb-000137
合成步骤参照4.1.1.2.ERC-01-c2的合成。
4.1.2.3 SANC-01-c3的合成
Figure PCTCN2022125877-appb-000138
合成步骤参照4.1.1.3.ERC-01-c3的合成。
4.1.2.4 SANCd-01-c1的合成
Figure PCTCN2022125877-appb-000139
合成步骤参照4.1.1.4.ERCd-01-c1的合成。
4.1.2.5 SANCd-01-c2的合成
Figure PCTCN2022125877-appb-000140
合成步骤参照4.1.1.5.ERCd-01-c2的合成。
4.1.2.6 3’MVIP17-c1的合成
Figure PCTCN2022125877-appb-000141
合成步骤参照4.1.1.6.3’MVIP09-c1的合成，合成所得3’MVIP17-c1的高分辨质谱图见图4。
4.1.2.7 3’MVIP17-c2的合成
Figure PCTCN2022125877-appb-000142
合成步骤参照4.1.1.7.3’MVIP09-c2的合成。
4.1.2.8 3’MVIP17的Solid Support合成
Figure PCTCN2022125877-appb-000143
合成步骤参照4.1.1.8 3’MVIP09的Solid Support合成。
4.1.3 3'MVIP01的Solid Support的合成：
Figure PCTCN2022125877-appb-000144
合成过程描述：
4.1.3.1 3’MVIP01-c1的合成
Figure PCTCN2022125877-appb-000145
合成步骤参照4.1.1.6.3’MVIP09-c1的合成。
4.1.3.2 3’MVIP01-c2的合成
Figure PCTCN2022125877-appb-000146
合成步骤参照5.1.1.7.3’MVIP09-c2的合成。
4.1.3.3 3’MVIP01的Solid Support合成
Figure PCTCN2022125877-appb-000147
合成步骤参照4.1.1.8.3’MVIP09的Solid Support合成。
4.2.5’MVIP亚磷酰胺单体的合成
4.2.1当n为2时，所得的5’MVIP亚磷酰胺单体作为Kylo-09-DS122、Kylo-09-DS142正义链固相合成的最后一个单体5’MVIP09亚磷酰胺单体的合成：
Figure PCTCN2022125877-appb-000148
4.2.1.1 5’MVIP09-ERCd-PFP-c1的合成
Figure PCTCN2022125877-appb-000149
称量ERCd-01-c2(2.18g，2.0mmol)溶于DMF(50mL)，加戊二酸单苄酯(0.53g，2.4mmol)、DIPEA(0.78g)与TBTU(0.84g)，室温搅拌过夜，加水淬灭(50mL)，DCM(30mL*3)萃取，10％柠檬酸(50mL*3)、饱和碳酸氢钠50mL和吡啶100mL洗涤，无水硫酸钠干燥，过滤，旋蒸，过柱纯化得产品5’MVIP09-ERCd-PFP-c1(2.15g)。
4.2.1.2 5’MVIP09-ERCd-PFP-c2的合成
Figure PCTCN2022125877-appb-000150
称量5’MVIP09-ERCd-PFP-c1(2.15g，1.66mmol)和10％钯碳(0.21g)，加甲醇(50mL)，室温搅拌加氢过夜，反应结束后硅藻土过滤钯碳，旋蒸得5’MVIP09-ERCd-PFP-c2粗品(1.9g)，其高分辨率质谱图如图5所示。
4.2.1.3 5’MVIP09-ERCd-PFP的合成
Figure PCTCN2022125877-appb-000151
称量5’MVIP09-ERCd-PFP-c2粗品(1.9g，1.58mmol)溶于DCM(60mL)，加DIPEA(1.33g)，冷却，加三氟乙酸五氟苯酚酯(2.21g，7.9mmol)，室温搅拌反应2h后旋蒸，再溶于DCM(60mL)，饱和碳酸氢钠(30mL*3)、10％柠檬酸(30mL*1)、饱和食盐水(50mL*1)洗涤，无水硫酸钠干燥，过滤，旋蒸得5’MVIP09-ERCd-PFP粗品(2.35g)，抽干后无纯化直接用于下一步反应。
4.2.1.4 5’MVIP09亚磷酰胺单体-c1的合成
Figure PCTCN2022125877-appb-000152
5’MVIP09-ERCd-PFP粗品(2.35g，1.58mmol)溶于DCM(60mL)，加DIPEA(0.82g，6.32mmol)、6-氨基-1-己醇(0.37g，3.16mmol)，室温搅拌反应过夜。加10％柠檬酸(30mL)，DCM(30mL*3)萃取，饱和食盐水(50mL)洗涤，无水硫酸钠干燥，过滤、旋蒸，过柱纯化得产品5’MVIP09单体-c1(1.73g)。
4.2.1.5 5’MVIP09亚磷酰胺单体
Figure PCTCN2022125877-appb-000153
称量5’MVIP09亚磷酰胺单体-c1(1.3g、1.0mmol)溶于乙腈(30mL)，加入二异丙胺三氮唑(0.22g)，冰浴下滴加双-(二异丙基氨基)(2-氰基乙氧基)膦(0.36g，1.2mmol)，室温反应4h，HPLC中控，反应合格后，浓缩过柱纯化得到产品5’MVIP09单体(1.2g)。
当n为1时，所得的5’MVIP亚磷酰胺单体作为Kylo-09-DS141、Kylo-09-DS131、Kylo-09-DS147的正义链固相合成的最后一个单体，代码为5'MVIP01：
Figure PCTCN2022125877-appb-000154
5'MVIP01的亚磷酰胺单体称量YICd-01-c2(1.12g，2.0mmol)，剩余操作参照4.2.1.1.～4.2.1.5。
实施例5 5’MVIP09/3’MVIP 09偶联不同的siRNA的RNAi剂的合成
表13中反义链合成描述：用氩气吹扫试剂瓶至少2min。向试剂瓶中依次加入亚磷酰胺单体和乙腈，拧紧瓶盖后，震荡直至目测固体全溶。然后加入3A分子筛，静置8h以上待用。用氩气吹扫试剂瓶至少2min。向试剂瓶中依次加入氢化黄原素和干燥的吡啶，拧紧瓶盖后，震荡直至目测固体全溶，暂存待用。确认在室温20～30℃环境条件下进行以下操作:称取3’MVIP载体,加入到试剂瓶中，再加入乙腈，震荡混合均匀。将载体转移至合成柱内，并用乙腈将试剂瓶中残余的载体淋洗转移至合成柱内。淋洗完毕后加乙腈充满合成柱，记录使用乙腈的用量。按仪器操作安装固定合成柱。
将上述配制的单体溶液、CAP A、CAP B、氧化剂、硫代试剂、活化剂、脱帽剂以及乙腈，连接至AKTA PILOT100对应的管路，确保管路插入试剂瓶底。
合成方法设置完毕后，仪器各项工作准备就绪，点击运行，开始合成。在线观察记录每个detritylation峰面积。在合成过程中根据脱保护试剂实际使用量，进行补加操作。
合成结束后，氩气吹扫合成柱≥2h，按操作规程卸载合成柱。向合成柱内固相载体转移至反应瓶，加入甲胺水溶液和氨水，将反应瓶放入摇床中，35℃，2-3小时。将溶液过滤至圆底烧瓶中，再使用50％乙醇水溶液洗涤残留固相，再次过滤与之前滤液合并，将圆底烧瓶连接旋转蒸发仪，设置水温50℃蒸至无馏出，向圆底烧瓶内加入乙醇，混匀，再次蒸至无馏出，重复操作至瓶底出现白色粉末。将得到的白色粉末配制成溶液，使用反向层析柱进行纯化，取样检测OD260、纯度。将纯化的反义链溶液分装于西林瓶中冻干备用，并将产品密封储存于-20℃冰箱中。
表14中正义链的合成操作同反义链，其中装柱的载体为Universal载体。所得中间体加DIPEA配制成溶液，加入5’MVIP亚磷酰胺单体，混匀，将反应瓶放入摇床中，35℃，2-3小时。
表15中RNAi剂的合成退火工艺描述：
取表13中反义链，取与表13中反义链碱基配对的表14中反正义链，1:1等摩尔混合在反应瓶中，水浴95℃5分钟后，关闭水浴锅电源，使其自然降温至40℃以下。向双链溶液中加入3M醋酸钠水溶液，混合均匀后，再加入适量体积的无水乙醇，混合均匀，将反应液放入-20℃冰箱内45min。冷冻高速离心机设置4℃预制冷，温度达到后，放入双链溶液，启动离心机。取出离心后的双链溶液，去除上清液，加入超纯水使固体完全溶解，取样检测OD260、纯度。将纯化的双链溶液分装于西林瓶中冻干备用，并将产品密封储存于-20℃冰箱中。
实施例6 5’MVIP09/3’MVIP09偶联不同siRNA的RNAi剂活性研究
取适龄的雌性AGT转基因小鼠用于实验评估。供试品为表15中RNAi剂Kylo-09-DS113～Kylo-09-DS130。在Day0分别通过皮下注射给药3mg/kg；给药体积为100-200μL。在给药后第0，7，14，21，28和第35天取血，分离血清，-80℃储存。用Elisa方法测定血清中hAGT水平，试验结果见表22和图6。
表22转基因小鼠给药后血清中hAGT平均水平
Figure PCTCN2022125877-appb-000155
结果显示5’MVIP09/3’MVIP09偶联的实施例2优选出来的序列(正义链序列为SEQ ID NO.37，43，44，46，47，48，53和54及对应的反义链序列SEQ ID NO.55，61，62，64，65，66，71和72)所得到的RNAi剂Kylo-09-DS113，119，120，122，123，124，129和130在体内活性仍旧是显著且持续性好。本实施例验证了5’MVIP09/3’MVIP09载体结构可以实现siRNA的安全递送，且作用效果显著。
实施例7 不同结构5’MVIP和3’MVIP偶联同一种siRNA对RNAi剂的活性影响研究
按照实施例5所述的方法合成表16中的反义链和表17中的正义链，配对退火合成表18中本实施例供试品RNAi剂Kylo-09-DS122，Kylo-09-DS147～160，Kylo-09-DS161和Kylo-09-171。取适龄的雌性人AGT转基因小鼠用于实验评估。在Day0分别通过皮下注射给药：3mg/kg；给药体积为100-200μL。在给药后14取血，分离血清，用Elisa方法测定血清中hAGT水平，试验结果见表23及图7。
表23转基因小鼠血清中hAGT平均水平
Figure PCTCN2022125877-appb-000156
Figure PCTCN2022125877-appb-000157
上述试验结果显示，载体结构5’MVIP和/或3’MVIP偶联的位置包括反义链的5’末端和/或3’末端、正义链的5’末端和/或3’末端、反义链的5’末端和正义链的3’末端、正义链的5’末端和3’末端，各种载体结构与同一AGT siRNA进行不同位置偶联，所得的RNAi剂对转基因小鼠hAGT水平抑制效果呈现出差异性，其中n和m分别为2的5’MVIP09，20，19和10偶联正义链的同时，3’MVIP09，10，15和12分别对应偶联在反义链上，退火配对所得RNAi剂Kylo-09-DS122，151，152和153对转基因小鼠hAGT抑制效果都优于其它组合；n和m不同，n+m等于4的RNAi剂Kylo-09-DS147，148，149和150的抑制效果其次。反义链5’末端偶联有载体结构所得到的RNAi剂中，5’MVIP21与3’MVIP18组合中得到的Kylo-09-DS160抑制效果也达到67.3％。Kylo-09-DS161剂和Kylo-09-DS171虽然n+m分别为5和6，但在抑制效果上并未显示出支化多的优势，推测与序列的特异性或引入的载体结构的空间位阻有关。试验结果显示，选自表10中的5’MVIP和/或选自表11中3’MVIP组合成载体结构所得的RNAi剂对转基因小鼠体内的hAGT表达水平都有一定的抑制效果。
实施例8 5'MVIP09/3'MVIP09组合形成的RNAi剂的在食蟹猴体内药效探索研究
本实施例的目的是研究不同结构5’MVIP和3’MVIP偶联同一种AGT siRNA所得RNAi在食蟹猴体内抑制活性的影响。按实施例6所述的方法制备相应的RNAi剂Kylo-09-DS131、Kylo-09-DS141、Kylo-09-DS142、Kylo-09-DS147或Kylo-09-DS122，选取18只3～5岁雄性洗脱猴适应性饲养结束后根据体重随机分为对照组(n＝3)以及给药组(n＝3)。给药剂量为3mg/kg，给药体积3ml/kg。分组给药当天定义为Day0，在首次给药后7天、14天、21天、28天、35天、42天和49天采集血液，分离血浆后，用Elisa方法测定血清中AGT水平，试验结果见表24和图8。
表24食蟹猴血清中AGT平均水平
Figure PCTCN2022125877-appb-000158
实验结果显示，正义链5’MVIP和反义链3’MVIP的组合为5’MVIP01/3’MVIP01、5’MVIP01/3’MVIP17或5’MVIP09/3’MVIP09或正义链5’MVIP和正义链3’MVIP的组合5’MVIP01/3’MVIP09或5’MVIP09/3’MVIP01的偶联方式都对食蟹猴体内AGT水平的抑制都呈现了显著的效果且持续性好。

Claims (10)

  1. 一种RNAi剂或其药学上可接受的盐，所述RNAi剂的结构中含有载体结构和干扰核酸，其结构如式IIIa、IIIb或IIIc所示：
    Figure PCTCN2022125877-appb-100001
    其中，
    所述干扰核酸靶向AGT基因，其包括反义链和正义链；
    所述载体结构包括5’MVIP(5’MultiValent Import Platform)和/或3’MVIP(3’MultiValent Import Platform)；
    所述5’MVIP由转接点R 1、连接链D、接头B、支链L和肝靶向特异性配体X组成，所述3’MVIP由转接点R 2、连接链D、接头B、支链L和肝靶向特异性配体X组成，所述5’MVIP 通过转接点R 1与正义链5’端或反义链5’端连接，所述3’MVIP通过转接点R 2与正义链3’端或反义链3’端连接，n和m各自独立地为0-4的任意整数。
  2. 根据权利要求1所述的RNAi剂或其药学上可接受的盐，其中所述n+m＝2-6的整数，优选n+m＝2、3或4，更优选为4。
  3. 根据权利要求1-2中任一项所述的RNAi剂或其药学上可接受的盐，所述5’MVIP选自表10中5’MVIP01至5’MVIP22中的任一个，和/或所述3’MVIP选自表11中3’MVIP01至3’MVIP27的任一个。
  4. 根据权利要求1-3中任一项所述的RNAi剂或其药学上可接受的盐，其中所述正义链与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和SEQ ID NO:18中任意一个或与其相差不超过3个核苷酸的序列具有基本上同源性。
  5. 根据权利要求1-4中任一项所述的RNAi剂或其药学上可接受的盐，所述反义链包含以下的核苷酸序列：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和SEQ ID NO:36中任意一个或与其相差不超过3个核苷酸的序列。
  6. 根据权利要求1-5中任一项所述的RNAi剂或其药学上可接受的盐，其中所述正义链包含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和SEQ ID NO:54中任意一个或与其相差不超过3个核苷酸的序列，且所述反义链包含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和SEQ ID NO:72中任意一个或与其相差不超过3个核苷酸的序列。
  7. 根据权利要求1-6中任一项所述的RNAi剂或其药学上可接受的盐，其中所述干扰核酸包括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～Kylo-09-DS54中任意任一种或多种。
  8. 根据权利要求1-7中任一项所述的RNAi剂或其药学上可接受的盐，其包括表18中Kylo-09-DS122，Kylo-09-DS131至Kylo-09-DS147中的任一种或多种。
  9. 药物组合物，其包含权利要求1-8中任一项所述的RNAi剂或其药学上可接受的盐，以及任选地药学上可接受的赋形剂、载体和/或稀释剂。
  10. 权利要求1-8中任一项所述的RNAi剂或其药学上可接受的盐，或权利要求9所述的药物组合物在制备药物中的用途，所述药物用于预防和/或治疗疾病或病症或者降低疾病或病症的风险。
PCT/CN2022/125877 2021-10-20 2022-10-18 Agt抑制剂及其用途 WO2023066236A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111222428.7A CN113862268A (zh) 2021-10-20 2021-10-20 Agt抑制剂及其用途
CN202111222428.7 2021-10-20

Publications (1)

Publication Number Publication Date
WO2023066236A1 true WO2023066236A1 (zh) 2023-04-27

Family

ID=78996852

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/125877 WO2023066236A1 (zh) 2021-10-20 2022-10-18 Agt抑制剂及其用途

Country Status (3)

Country Link
CN (1) CN113862268A (zh)
TW (1) TW202328450A (zh)
WO (1) WO2023066236A1 (zh)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113862268A (zh) * 2021-10-20 2021-12-31 厦门甘宝利生物医药有限公司 Agt抑制剂及其用途
TW202339773A (zh) * 2022-01-20 2023-10-16 大陸商上海拓界生物醫藥科技有限公司 一種ｄｓＲＮＡ、其應用及製備方法
CN114703184A (zh) * 2022-03-11 2022-07-05 厦门甘宝利生物医药有限公司 Lpa抑制剂及其用途
WO2024032679A1 (zh) * 2022-08-11 2024-02-15 益杰立科(上海)生物科技有限公司 一种表观编辑靶点的方法及用途

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997033623A1 (en) * 1996-03-15 1997-09-18 University Of Florida OLIGONUCLEOTIDES TARGETED TO ANGIOTENSINOGEN mRNA
WO2004091515A2 (en) * 2003-04-09 2004-10-28 Alnylam Pharmaceuticals, Inc. iRNA CONJUGATES
CN106574268A (zh) * 2014-05-22 2017-04-19 阿尔尼拉姆医药品有限公司 血管紧张素原（AGT）iRNA组合物及其使用方法
CN108271351A (zh) * 2015-10-08 2018-07-10 Ionis 制药公司 用于调节血管紧张素原表达的化合物和方法
CN112313335A (zh) * 2018-05-14 2021-02-02 阿尔尼拉姆医药品有限公司 血管紧张素原(AGT)iRNA组合物及其使用方法
CN113171371A (zh) * 2021-04-13 2021-07-27 厦门甘宝利生物医药有限公司 一种抑制乙型肝炎病毒基因表达的rna抑制剂及其应用
CN113234725A (zh) * 2021-05-28 2021-08-10 厦门甘宝利生物医药有限公司 一种抑制pcsk9基因表达的rna抑制剂及其应用
CN113862268A (zh) * 2021-10-20 2021-12-31 厦门甘宝利生物医药有限公司 Agt抑制剂及其用途

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110218728A (zh) * 2019-06-28 2019-09-10 厦门甘宝利生物医药有限公司 一种新化合物及其应用

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997033623A1 (en) * 1996-03-15 1997-09-18 University Of Florida OLIGONUCLEOTIDES TARGETED TO ANGIOTENSINOGEN mRNA
WO2004091515A2 (en) * 2003-04-09 2004-10-28 Alnylam Pharmaceuticals, Inc. iRNA CONJUGATES
CN106574268A (zh) * 2014-05-22 2017-04-19 阿尔尼拉姆医药品有限公司 血管紧张素原（AGT）iRNA组合物及其使用方法
CN108271351A (zh) * 2015-10-08 2018-07-10 Ionis 制药公司 用于调节血管紧张素原表达的化合物和方法
CN112313335A (zh) * 2018-05-14 2021-02-02 阿尔尼拉姆医药品有限公司 血管紧张素原(AGT)iRNA组合物及其使用方法
CN113171371A (zh) * 2021-04-13 2021-07-27 厦门甘宝利生物医药有限公司 一种抑制乙型肝炎病毒基因表达的rna抑制剂及其应用
CN113234725A (zh) * 2021-05-28 2021-08-10 厦门甘宝利生物医药有限公司 一种抑制pcsk9基因表达的rna抑制剂及其应用
CN113862268A (zh) * 2021-10-20 2021-12-31 厦门甘宝利生物医药有限公司 Agt抑制剂及其用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
UIJL ESTRELLITA, MIRABITO COLAFELLA KATRINA M., SUN YUAN, REN LIWEI, VAN VEGHEL RICHARD, GARRELDS INGRID M., DE VRIES RENÉ, POGLIT: "Strong and Sustained Antihypertensive Effect of Small Interfering RNA Targeting Liver Angiotensinogen", HYPERTENSION, vol. 73, no. 6, 1 June 2019 (2019-06-01), US , pages 1249 - 1257, XP093058451, ISSN: 0194-911X, DOI: 10.1161/HYPERTENSIONAHA.119.12703 *

Also Published As

Publication number Publication date
CN113862268A (zh) 2021-12-31
TW202328450A (zh) 2023-07-16

Similar Documents

Publication Publication Date Title
WO2023066236A1 (zh) Agt抑制剂及其用途
TWI823866B (zh) 用於抑制類血管生成素蛋白-3(ANGPTL3)表現的RNAi藥劑及組合物及使用方法
WO2023169548A1 (zh) Lpa抑制剂及其用途
JP7383308B2 (ja) 新規化合物及びその適用
CN111107853A (zh) 用于抑制载脂蛋白C-III (APOC3)的表达的RNAi试剂和组合物
CN113164509A (zh) 用于抑制17β-HSD 13型(HSD17B13)表达的RNAi试剂、其组合物和使用方法
TW201827594A (zh) 用於抑制b型肝炎病毒基因表現之組合物及方法
CA2917299C (en) Improved nanoparticle type oligonucleotide structure having high efficiency and method for preparing same
CN113234725B (zh) 一种抑制pcsk9基因表达的rna抑制剂及其应用
US20220389430A1 (en) Chemical modifications of small interfering rna with minimal fluorine content
WO2022218163A1 (zh) 一种抑制乙型肝炎病毒基因表达的rna抑制剂及其应用
US20220340909A1 (en) Compositions and methods for inhibiting ketohexokinase (khk)
TW202200163A (zh) 用於抑制angptl3表現之組合物及方法
WO2023134705A1 (zh) 抑制angptl3表达的rna干扰剂及其用途
CN115851723B (zh) 一种抑制lpa基因表达的rna抑制剂及其应用
CN111212909A (zh) 用于抑制去唾液酸糖蛋白受体1的表达的RNAi试剂和组合物
WO2023041079A9 (zh) Lpa抑制剂及其用途
WO2011135140A1 (es) Método para la administración de oligonucleótidos
WO2024027809A1 (zh) 一种抑制apoc3基因表达的rna抑制剂及其应用
WO2024088190A1 (zh) 一种抑制lpa基因表达的rna抑制剂及其应用
CN112055597A (zh) 用于治疗胆管缺乏相关病况的方法和组合物
CA3235994A1 (en) Agt inhibitors and use thereof
TWI835116B (zh) 一種抑制b型肝炎病毒基因表達的rna抑制劑及其應用
US20220364098A1 (en) Compositions and methods for modulating pnpla3 expression
CA3218815A1 (en) Rnai agents for inhibiting expression of mucin 5ac (muc5ac), compositions thereof, and methods of use

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22882841

Country of ref document: EP

Kind code of ref document: A1