WO2026037344A2 - Molécule d'acide nucléique pour inhiber ou réduire l'expression du gène cible angptl3 et utilisation associée - Google Patents

Molécule d'acide nucléique pour inhiber ou réduire l'expression du gène cible angptl3 et utilisation associée

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Publication number
WO2026037344A2
WO2026037344A2 PCT/CN2025/114467 CN2025114467W WO2026037344A2 WO 2026037344 A2 WO2026037344 A2 WO 2026037344A2 CN 2025114467 W CN2025114467 W CN 2025114467W WO 2026037344 A2 WO2026037344 A2 WO 2026037344A2
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Prior art keywords
seq
angptl3
diseases
nucleic acid
expression
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WO2026037344A3 (fr
Inventor
Bill Biliang Zhang
Man Li
Hong Zhang
Nan Cheng
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Argorna Pharmaceuticals Co Ltd
Guangzhou RiboBio Co Ltd
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Argorna Pharmaceuticals Co Ltd
Guangzhou RiboBio Co Ltd
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Publication of WO2026037344A2 publication Critical patent/WO2026037344A2/fr
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate

Definitions

  • the present application relates to the field of siRNAs, and in particular, to a nucleic acid molecule for inhibiting or reducing the expression of target gene ANGPTL3 and use thereof.
  • Angiopoietin-like 3 is a key regulator of serum lipid and lipoprotein metabolism, is mainly expressed in the liver, and plays a role in regulating lipid metabolism by inhibiting lipoprotein lipase (LPL) and endothelial lipase (EL) .
  • LPL and EL play a key role in the catabolism of triglyceride (TG) , high-density lipoprotein cholesterol (HDL-C) and very low-density lipoprotein cholesterol (VLDL-C) .
  • Inactivating mutations of the ANGPTL3 gene may lead to a decrease in TG, HDL-C, and LDL-C, thereby reducing the risk of developing atherosclerotic cardiovascular disease (ASCVD) .
  • ASCVD atherosclerotic cardiovascular disease
  • inhibiting the activity or expression of ANGPTL3 can be used as a lipid-lowering strategy.
  • the object of the present application is to provide a nucleic acid molecule and a use thereof.
  • the nucleic acid molecule has biological activity of effectively inhibiting or reducing the expression of target gene ANGPTL3.
  • nucleic acid molecule for inhibiting or reducing the expression of a target gene ANGPTL3, comprising a sense strand and an antisense strand, wherein the siRNA is at least one pair selected from the following sequences or modified sequences thereof:
  • A3-038 the sense strand thereof is as shown in SEQ ID NO: 75, and the antisense strand thereof is as shown in SEQ ID NO: 76;
  • A3-041 the sense strand thereof is as shown in SEQ ID NO: 81, and the antisense strand thereof is as shown in SEQ ID NO: 82; or
  • A3-043 the sense strand thereof is as shown in SEQ ID NO: 118, and the antisense strand thereof is as shown in SEQ ID NO: 119; or
  • A3-044 the sense strand thereof is as shown in SEQ ID NO: 120, and the antisense strand thereof is as shown in SEQ ID NO: 121.
  • SEQ ID NO: 118 UCAAACCAAUGAAAUCAAAGA
  • SEQ ID NO: 119 UCUUUGAUUUCAUUGGUUUGAAG.
  • SEQ ID NO: 120 UCUUUGAUUUCACUGGUUACUCAACUCAAA,
  • SEQ ID NO: 121 UUUGAGUUGAGUUCAAGUUGAAAUCAAAGA.
  • bases of the sense strand are modified, the modifications comprises 2’-O-methyl modification on bases at positions 1 to 6, 8, 12, 14, and 1 to 6 from the 3’ end, 2’ -fluoro modification on bases at positions 7, 9, and 11, and both phosphodiester linkages between positions 1 and 2, between position 2 and 3 from the 3’ end are replaced by phosphorothioate linkages.
  • bases of the antisense strand are modified, the modifications in the antisense strand comprise 2’ -O-methyl modification on bases at positions 1, 3, 5, 7, 11, 13, 15, 1 to 5 from the 3’ end, and 7 from the 3’ end, 2’ -fluoro modification on bases at positions 2, 6, 8, 14, and 8 from the 3’ end, and both phosphodiester linkages between position 1 and 2, between position 2 and 3, and between positions 1 and 2, between position 2 and 3 from the 3’ end are replaced by phosphorothioate linkages.
  • the sense strand is any one selected from:
  • the hydroxyl protecting group is selected from silyl, monomethoxytrityl (MMTr) , 4, 4’ -dimethoxytrityl (DMTr) , and trityl; and optionally, the silyl is selected from t-butyldimethylsilyl ether (TBMDS) , t-butyldiphenylsilyl (TBDPS) , and triisopropylsilyl ether (TIPS) .
  • TBMDS t-butyldimethylsilyl ether
  • TDPS t-butyldiphenylsilyl
  • TIPS triisopropylsilyl ether
  • R 2 is -CH 2 -, C 6-14 aryl, C 2-6 alkenyl, or C 2-6 alkynyl;
  • R 3 is -O-, C 6-14 aryl, or 3 to 10-membered heterocyclyl
  • R 4 is -CH 2 -, C 6-14 aryl, or 3 to 10-membered heterocyclyl
  • R is O or S
  • Rx is an siRNA
  • a, b, c, and d are each independently any integer of 1-10;
  • n is any integer of 1-3.
  • a fourth aspect provided is use of any of the described conjugates in the preparation of a product for preventing or alleviating or treating diseases related to the expression of target gene ANGPTL3.
  • the product is a biological formulation or pharmaceutical formulation.
  • a pharmaceutical composition wherein active ingredients of the pharmaceutical composition comprise any of the described conjugates for inhibiting or reducing the expression of target gene ANGPTL3 and a pharmaceutically acceptable excipient.
  • a method for inhibiting or reducing the expression of the target gene ANGPTL3 comprises administering to a subject a suitable dose of any of the described nucleic acid molecule, any of the described conjugate, and/or any of the described pharmaceutical composition.
  • a seventh aspect of the present application provided is a method for inhibiting or reducing the expression of target gene ANGPTL3 in cells, comprising the steps of:
  • the cells are in a subject, or the cells are cells in vitro of a subject.
  • the subject is a mammal, and preferably, the mammal is human.
  • target gene ANGPTL3 expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100%.
  • the nucleic acid molecule or conjugate is used for preparing a product for preventing or treating diseases mediated by target gene ANGPTL3.
  • the diseases mediated by target gene ANGPTL3s include, but are not limited to, dyslipidemia diseases, cerebrovascular diseases, and cardiometabolic diseases.
  • the nucleic acid molecule or conjugate or the biological formulation or the pharmaceutical formulation comprising these substances are used to reduce the level of LDL-C, VLDL-C, HDL-C, and triglycerides (TG) in vivo of a subject or in cells.
  • the dyslipidemia diseases include hyperlipidemia, hypertriglyceridemia or high low-density lipoprotein (LDL) .
  • cardiometabolic diseases associated with dyslipidemia are prevented or treated or preventively treated, such as hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid, cholesterol metabolism, atherosclerosis, type II diabetes, thrombosis, coronary heart disease, heart stroke, brain stroke, aortic stenosis, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, homozygous or heterozygous familial hypercholesterolemia, statins resistant hypercholesterolemia, or other metabolic related disorders and diseases.
  • hypertriglyceridemia such as hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid, cholesterol metabolism, atherosclerosis, type II diabetes, thrombosis, coronary heart disease, heart stroke, brain stroke, aortic stenosis, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, homozygous or heterozygous familial hypercholesterolemia, statins resistant hyper
  • a plurality of siRNAs capable of inhibiting the expression of target gene ANGPTL3 are screened through extensive research and experiments on nucleic acid molecules (siRNA sequence) , and on this basis, appropriate modifications are made to improve the silencing capability of the target and reduce the activity of non-targets.
  • a self-developed delivery system enables the siRNA to be delivered to the liver for targeted effects.
  • the nucleic acid molecules and conjugates are expected to be used in the clinical prevention and treatment of diseases such as dyslipidemia diseases, cerebrovascular diseases, and cardiometabolic diseases associated with ANGPTL3 target gene.
  • Fig. 1 is a schematic representation of the results of expression inhibition of ANGPTL3 gene by A3-041-2 siRNA.
  • Fig. 2 is a schematic representation of the results of expression inhibition of ANGPTL3 gene by A3-041-4 siRNA.
  • Fig. 3 is a schematic representation of the results of expression inhibition of ANGPTL3 gene by A3-038-6 siRNA.
  • Fig. 4 is a schematic representation of the results of expression inhibition of ANGPTL3 gene by A3-038-6-1 siRNA.
  • Fig. 5 is a schematic representation of the results of expression inhibition of ANGPTL3 gene by A3-038-6-2 siRNA.
  • Fig. 6 shows the serum TG change following administration in Experiment 1 of Example 4.
  • Fig. 7 shows the ANGPTL3 mRNA change in liver following administration in Experiment 1 of Example 4.
  • Fig. 8 shows the serum TG change following administration in Experiment 2 of Example 4.
  • Fig. 9 shows the ANGPTL3 mRNA change in liver following administration in Experiment 2 of Example 4.
  • Fig. 10 shows the serum TG change following administration of Example 5.
  • Fig. 11 shows the ANGPTL3 mRNA change in liver following administration of Example 5.
  • Fig. 12 shows the serum TG change following administration in Experiment 3 of Example 4.
  • Fig. 13 shows the serum TG change following administration in Experiment 3 of Example 4.
  • Fig. 14 shows the ANGPTL3 mRNA change in liver following administration in Experiment 3 of Example 4.
  • Fig. 15 shows the serum ANGPTL3 protein variation 18 days post-administration in Experiment 3 of Example 4.
  • an amount effective to inhibit the activity or expression of ANGPTL3 means that the level of activity or expression of ANGPTL3 in a treated sample will be lower than the level of activity or expression of ANGPTL3 in an untreated sample.
  • the terms “decrease” and “reduce” can be used interchangeably and generally mean any change less than the original. “Decrease” and “reduce” are relative terms that require comparison before and after measurement, “Decrease” and “reduce” include complete exhaustion.
  • the term “reduce” can be detected by standard methods known in the prior art, such as those described herein, and the expression level/amount of a gene or a gene product, for example, a protein or a biomarker, in a first sample is reduced by about 5%to 95%or total of 100%as compared to the expression level/amount of the corresponding gene or gene product, for example, protein or biomarker, in a second sample.
  • the term “reduce” refers to a decrease in the expression level/amount of a gene or biomarker in a test sample, wherein the decrease refers to at least about 0.9 -to 0.01-fold the expression level/amount of the corresponding gene or biomarker.
  • expression generally means the process by which a gene eventually produces a protein.
  • Expression includes, but is not limited to, transcription, post-transcriptional modification (for example, 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 an active ingredient. Also included are sterile water, saline, and the like. Such formulations may typically comprises a salt, an excipient, a buffer, a preservative, a compatible carrier, and optionally other therapeutic agent. When used in medicine, the salt should be a pharmaceutically acceptable salt, but pharmaceutically acceptable salts can conveniently be prepared using non-pharmaceutically acceptable salts, which cannot be excluded from the scope of the application.
  • Such pharmacologically and pharmaceutically acceptable salt includes salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, boric acid, formic acid, malonic acid, succinic acid, and the like.
  • the pharmaceutically acceptable salt can also be prepared as an alkali metal or alkaline earth metal salt, such as sodium, potassium or calcium salt.
  • the term “preventing and/or treating” not only includes preventing and/or treating diseases, but also generally includes preventing the onset of diseases, slowing or reversing the progression of the diseases, preventing or slowing the onset of one or more symptoms associated with the diseases, reducing and/or alleviating one or more symptoms associated with the diseases, reducing the severity and/or duration of the diseases and/or any symptoms associated therewith and/or preventing further increases in the severity of the diseases and/or any symptoms associated therewith, preventing, reducing or reversing any physiological injuries caused by the diseases, and generally any pharmacological effects that are beneficial to the patient being treated.
  • RNAi agent or pharmaceutical composition of the present application forms a viable therapeutic agent which does not need to complete cure or eradicate any symptoms or manifestations of the diseases.
  • medicaments used as therapeutic agents can reduce the severity of given disease states, but do not need to eliminate every manifestation of the diseases to be considered useful therapeutic agents.
  • a prophylactically administered treatment constitutes a viable prophylactic agent that does not require complete efficacy to prevent the onset of conditions. Simply reducing the effects of the diseases in the subject (for example, by reducing the number or severity of symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect) , or reducing the likelihood that the disease will occur or worsen, is sufficient
  • the term “administration” generally means introducing a pharmaceutical formulation of the present application into the body of a subject by any route of introduction or delivery. Any method known to a person skilled in the art for contacting a cell, organ or tissue with the medicament may be employed. Such administration may include, without limitation, intravenous, intraarterial, intranasal, intraperitoneal, intramuscular, subcutaneous, or oral administration.
  • the daily dose may be divided into one, two or more suitable forms of dose to administer at one, two or more times during a certain period of time.
  • the term “contacting” generally means that two or more different types of substances are contacted together in any order, in any manner and for any time. Contacting can occur in vivo, ex vivo, or in vitro. In some embodiments, it can mean that the RNAi agent or composition of the present application is in direct contact with a cell or tissue. In other embodiments, the term refers to contacting a cell or tissue indirectly with the RNAi agent or composition of the present application.
  • the method of the present application includes a method wherein a subject contacts the RNAi agent or composition of the present application, and then the RNAi agent or composition contacts a cell or tissue by diffusion or any other active or passive transport process known in the art by which the compound circulates in vivo.
  • the term “effective amount” or “effective dosage” generally refers to an amount sufficient to achieve, or at least partially achieve, the desired effect.
  • a “therapeutically effective amount” or “therapeutically effective dosage” of the medicament or therapeutic agent is generally any amount of medicament that, when used alone or in combination with another therapeutic agent, promotes disease regression as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or by prevention of damage or disability due to illness.
  • a “prophylactically effective amount” or “prophylactically effective dosage” of a medicament generally refers to an amount of the medicament that inhibits the development or recurrence of a disease when administered, alone or in combination with another therapeutic agent, to a subject at risk of developing or recurrence of the disease.
  • an “effective amount” refers to the amount of the RNAi agent that produces intended pharmacologic, curative, or prophylactic results.
  • the term “subject” generally refers to human or non-human animals (including mammals) such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, pongidae, and macaques) , domestic animals (dogs and cats) , farm animals (horses, cattle, goats, sheep and pigs) , and laboratory animals (mice, rats, rabbits, and guinea pigs) in need of diagnosis, prognosis, amelioration, prevention and/or treatment of diseases.
  • Human subjects include fetal, neonatal, infant, adolescent and adult subjects.
  • Subjects include animal disease models.
  • HuH7 cells HuH7 cells (SCSP-526, National Collection of Authenticated Cell Cultures, Chinese Academy of Sciences) , Lipofectamine 3000 Transfection Reagent (Thermo Fisher, L3000015) , Serum-Reduction Medium Optim-MEM (Gibco, 31985-070) , DMEM (Gibco, C11995500BT) , FBS (Gibco, 10270-106) , negative control siRNA NC (Ribobio, siM211215011852) and NC002 (Ribobio, RD20221011171701) , siRNA samples (having sequences as shown in Table 1) , MagZol Reagent (Magen, R4801) , Bugle-Loop miRNA qRT-PCR Starter kit (RiboBio, C10211-2) , 24-well plate (Corning, 3524) , CO2 incubator (Memmert, ICO150) , fluorescence quantitative PCR instrument (BioRad, CF
  • HuH7 cells (SCSP-526, National Collection of Authenticated Cell Cultures, Chinese Academy of Sciences) were cultured, and the cell density before transfection was about 30-50%, which is relatively uniform. All siRNA samples were each formulated into 20 ⁇ M stock solutions and set up as an experimental group. In addition to the experimental group, a normal cell control group (Blank) , a transfection reagent control group (Mock) , a negative control group 1 (NC, Ribobio, siM211215011852) , and a negative control group 2 (NC002, Ribobio, RD20221011171701) were set up. Cells were transfected with reference to Lipofectamine 3000 transfection reagent (Thermo Fisher, L3000015) instructions. There were 3 replicates for both the experimental and control groups.
  • RNAs were extracted following MagZol Reagent (Magen, R4801) instructions.
  • Reverse transcription was performed with reference to Bugle-Loop miRNA qRT-PCR Starter kit (RiboBio, C10211-2) instructions.
  • Human housekeeping gene actin was used as an internal reference gene, the upstream primer sequence of the actin gene was 5’ -TCAAGATCATTGCTCCTCCTGAG-3’ (SEQ ID NO: 109) , and the downstream primer sequence was 5’ -ACATCTGCTGGAAGGTGGACA-3’ (SEQ ID NO: 110) .
  • the upstream primer sequence of the human ANGPTL3 gene was 5’-CTTCCAAGCCAAGAGCACCA-3’ (SEQ ID NO: 111) , and the downstream primer sequence was 5’-CATGAAAAACTTGAGAGTTGCTGG-3’ (SEQ ID NO: 112) .
  • N a general term for unmodified ribonucleotides, including A: adenine ribonucleotide, G: guanine ribonucleotide, C: cytosine ribonucleotide, U: uracil ribonucleotide.
  • dN a general term of unmodified deoxyribonucleotides, including dA: deoxyadenosine nucleotide, dG: deoxyguanosine nucleotide, dC: deoxycytidine nucleotide, and dT: deoxythymine nucleotide.
  • Modified double-stranded siRNAs were transfected into HuH-7 cells for in vitro effectiveness screening, three experiments were repeated, and reference can be made to example 1 for the experiment method.
  • the experimental results were as follows.
  • N a general term for unmodified ribonucleotides, including A: adenine ribonucleotide, G: guanine ribonucleotide, C: cytosine ribonucleotide, U: uracil ribonucleotide.
  • dN a general term of unmodified deoxyribonucleotides, including dA: deoxyadenosine nucleotide, dG: deoxyguanosine nucleotide, dC: deoxycytidine nucleotide, and dT: deoxythymine nucleotide
  • IC50 values of A3-041-2, A3-041-4, A3-038-6, A3-038-6-1, and A3-038-6-2 were determined as follows: HuH-7 cells were digested with 0.25%trypsin and adjusted to an appropriate concentration for seeding into a 24-well cell culture plate. Cell transfection was performed using LipofectamineTM 3000 Transfection Reagent (Thermo Fisher, L3000015) following the procedure instructions provided by the manufacturer. siRNA transfection concentrations were 100, 50, 10, 2, 0.4, 0.08, 0.016, 0.0032, and 0.00064 nM.
  • RNA transfection group In addition to the siRNA transfection group, a normal cell control group (Blank) , a transfection reagent control group (Mock) , and a negative control group (NC, Ribobio, siM211215011852) were set. Three transfection replicate wells were provided for each siRNA and control group. After 48 hours of transfection, the culture medium was removed, and cells were collected for RNA extraction. The RNA extraction was performed using the MagZol Reagent (Magen, R4801) extraction reagent, and the procedure was performed following the instructions of the manufacturer.
  • MagZol Reagent Magen, R4801
  • cDNA was synthesized using the Bugle-Loop miRNA qRT-PCR Starter kit (RiboBio, C10211-2) according to the instructions, and ANGPTL3 gene relative expression assays were performed using the Bugle-Loop miRNA qRT-PCR Starter kit (RiboBio, C10211-2) using a fluorescent quantitative PCR instrument (BioRad, CFX DUET) . Normalization was performed on the negative control group, and the expression inhibition percentage of ANGPTL3 gene was calculated for different concentrations of siRNAs compared with the negative control group. A dosage-effect curve was plotted, and the half inhibition rate, i.e. IC50, was calculated. Please refer to Figs. 1-5 for the results.
  • ligand compounds were selected from GalNac-5, GalNac-13, and L96.
  • ligand compounds were selected from GalNac-5, GalNac-13, and L96.
  • specific preparation steps of GalNac-5 and GalNac-13 reference was made to Chinese patent application 202110073911.7.
  • N a general term for unmodified ribonucleotides, including A: adenine ribonucleotide, G: guanine ribonucleotide, C: cytosine ribonucleotide, U: uracil ribonucleotide.
  • dN a general term of unmodified deoxyribonucleotides, including dA: deoxyadenosine nucleotide, dG: deoxyguanosine nucleotide, dC: deoxycytidine nucleotide, and dT: deoxythymine nucleotide
  • VP VP modified mU.
  • mice Male SD rats aged 7-9 weeks (Guangdong Charles River Experimental Animal Technology Co., Ltd. ) were selected and randomly divided into 5 groups (A1, B1, C1, D1, and E1) according to the body weight of the animals, with 15 rats in each group. After grouping, groups B1 and C1 were subcutaneously injected with A3-041-2a-5 once, and groups D1 and E1 were subcutaneously injected with A3-041-4g-5 once, the dosage of groups B1 and D1 was 10 mg/kg, the dosage of groups C1 and E1 was 20 mg/kg, the administration volume was 5 mL/kg, and group A1 was subcutaneously injected with an equal volume of normal saline.
  • groups B1 and C1 were subcutaneously injected with A3-041-2a-5 once
  • groups D1 and E1 were subcutaneously injected with A3-041-4g-5 once
  • the dosage of groups B1 and D1 was 10 mg/kg
  • the dosage of groups C1 and E1 was 20 mg/kg
  • the administration volume was 5
  • Serum ANGPTL3 protein was detected using an ANGPTL3 ELISA Kit (arigo, ARG81898) according to the kit instructions.
  • the mRNA expression of ANGPTL3 in liver was detected by fluorescence quantitative PCR.
  • the total RNAs were extracted from liver tissue using MagZol Reagent (Magen, R4801) according to the manufacturer's instructions.
  • cDNA and PCR were synthesized using Bugle-Loop miRNA qRT-PCR Starter kit (RiboBio, C10211-2) according to the instructions, and were detected by fluorescence quantitative PCR instrument (BioRad, CFX DUET) .
  • mice Male SD rats aged 7-9 weeks (Guangdong Charles River Experimental Animal Technology Co., Ltd. ) were selected and randomly divided into 8 groups (A2, B2, C2, D2, E2, F2, G2, and H2 groups) according to the body weight of the animals, with 15 rats in each group. After grouping, groups B2, C2, D2, E2, F2, G2, and H2 were subcutaneously injected with A3-041-4a-13, A3-041-4b-13, A3-041-4c-13, A3-041-4d-13, A3-041-4e-13, A3-041-4f-13, and A3-041-4-5 once, the dosage of administration was 10 mg/kg, and the administration volume was 5 mL/kg, and group A2 was subcutaneously injected with an equal volume of normal saline.
  • groups B2, C2, D2, E2, F2, G2, and H2 were subcutaneously injected with A3-041-4a-13, A3-041-4b-13, A3-041-4c-13, A3-041-4d-13, A3-041-4e-13, A3-041-4f
  • RNAs were extracted from liver tissue using MagZol Reagent (Magen, R4801) according to the manufacturer's instructions.
  • Magen Magen, R4801
  • cDNA and PCR were synthesized using Bugle-Loop miRNA qRT-PCR Starter kit (RiboBio, C10211-2) according to the instructions, and were detected by fluorescence quantitative PCR instrument (BioRad, CFX DUET) .
  • mice Male SD rats aged 7-9 weeks (Guangdong Charles River Experimental Animal Technology Co., Ltd. ) were selected and randomly divided into 4 groups (A, B, C and D groups) according to the body weight of the animals, with 9 rats in each group. After grouping, groups B, C and D were subcutaneously injected with A3-038-6a-L96, A3-041-4a-L96 and A3-041-2a-L96 once, the dosage of administration was 9 mg/kg, and the administration volume was 5 mL/kg, and group A was subcutaneously injected with an equal volume of normal saline.
  • Serum TC was detected using a total cholesterol (TCH/T-CHO) assay kit (Nanjing Jiancheng Bioengineering Institute, A111-1-1) according to the kit instructions.
  • Serum ANGPTL3 protein was detected using the Mouse/Rat Angiopoietin-Like Protein 3 ELISA (BioVendor, RAG011 R) according to the kit instructions.
  • the mRNA expression of ANGPTL3 in liver was detected by fluorescence quantitative PCR.
  • the total RNAs were extracted from liver tissue using MagZol Reagent (Magen, R4801) according to the manufacturer's instructions.
  • cDNA and PCR were synthesized using Bugle-Loop miRNA qRT-PCR Starter kit (RiboBio, C10211-2) according to the instructions, and were detected by fluorescence quantitative PCR instrument (BioRad, CFX DUET) .
  • mice Male SD rats aged 7-9 weeks were selected and randomly divided into 4 groups according to the body weight of the animals, with 15 rats in each group. After grouping, groups B, C and D were respectively subcutaneously injected with A3-041-2a-5, A3-041-4g-5 and AD-52720-L96 once (using the sequence in US Patent US11525138B2 as a positive control: GfuCfaCfuUfgAfAfCfuCfaAfcUfcAfaAf (SEQ ID NO: 117) , the ligand being L96) , the dosage of administration was 10 mg/kg, and the administration volume was 5 mL/kg, and group A was subcutaneously injected with an equal volume of normal saline.
  • RNAs were extracted from liver tissue using MagZol Reagent (Magen, R4801) according to the manufacturer's instructions.
  • Magen Magen, R4801
  • cDNA and PCR were synthesized using Bugle-Loop miRNA qRT-PCR Starter kit (RiboBio, C10211-2) according to the instructions, and were detected by fluorescence quantitative PCR instrument (BioRad, CFX DUET) .
  • Both A3-041-2a-5 and A3-041-4g-5 can significantly inhibit the expression of ANGPTL3 after administration, can reduce the content of serum TG, and have a better effect than the positive control AD-52720-L96.

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Abstract

La présente demande concerne une molécule d'acide nucléique pour inhiber ou réduire l'expression du gène cible ANGPTL3, qui est un siRNA comprenant un brin sens et un brin antisens, le siRNA étant une molécule d'acide nucléique présentant un brin sens et un brin antisens comme dans l'une quelconque des paires SEQ ID NO: 75 et 76, SEQ ID NO: 81 et 82, SEQ ID NO: 118 et 119, ou SEQ ID NO: 120 et 121, ou des séquences modifiées associées. La présente demande concerne également un conjugué de la molécule d'acide nucléique décrite et d'un ligand.
PCT/CN2025/114467 2024-08-14 2025-08-13 Molécule d'acide nucléique pour inhiber ou réduire l'expression du gène cible angptl3 et utilisation associée Pending WO2026037344A2 (fr)

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US11525138B2 (en) 2011-06-21 2022-12-13 Alnylam Pharmaceuticals, Inc. Angiopoietin-like 3 (ANGPTL3) iRNA compositions and methods of use thereof

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WO2022079222A1 (fr) * 2020-10-16 2022-04-21 Sanofi Nouvelles compositions d'arn et méthodes d'inhibition d'angptl3
KR20230133836A (ko) * 2021-01-20 2023-09-19 아르고나 파마슈티컬스 컴퍼니 리미티드 리간드 화합물, 접합체 및 이의 응용
JP2024508896A (ja) * 2021-03-04 2024-02-28 アルナイラム ファーマシューティカルズ， インコーポレイテッド アンジオポエチン様３（ＡＮＧＰＴＬ３）ｉＲＮＡ組成物およびその使用方法
TW202521697A (zh) * 2021-09-23 2025-06-01 大陸商上海舶望製藥有限公司 用於抑制血管生成素樣3（angptl3）蛋白表達的組合物和方法
WO2023092102A1 (fr) * 2021-11-19 2023-05-25 Sanegene Bio Usa Inc. Arn double brin ciblant la protéine de type angiopoïétine 3 (angptl-3) et ses procédés d'utilisation
WO2023134705A1 (fr) * 2022-01-11 2023-07-20 上海金中锘美生物医药科技有限公司 Agent d'interférence d'arn pour inhiber l'expression d'angptl3, et son utilisation
CN116987696A (zh) * 2022-08-04 2023-11-03 北京福元医药股份有限公司 用于抑制angptl3基因表达的双链核糖核酸及其修饰物、缀合物和用途
CN118185932A (zh) * 2022-12-12 2024-06-14 深圳信立泰药业股份有限公司 用于抑制血管素样蛋白3表达的双链siRNA类似物及其制备方法和用途

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11525138B2 (en) 2011-06-21 2022-12-13 Alnylam Pharmaceuticals, Inc. Angiopoietin-like 3 (ANGPTL3) iRNA compositions and methods of use thereof

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