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WO2025077916A1 - Oligonucléotide ciblant la transthyrétine et son utilisation - Google Patents

Oligonucléotide ciblant la transthyrétine et son utilisation Download PDF

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Publication number
WO2025077916A1
WO2025077916A1 PCT/CN2024/124662 CN2024124662W WO2025077916A1 WO 2025077916 A1 WO2025077916 A1 WO 2025077916A1 CN 2024124662 W CN2024124662 W CN 2024124662W WO 2025077916 A1 WO2025077916 A1 WO 2025077916A1
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seq
strand represented
represented
antisense strand
sense strand
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Chinese (zh)
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高杰
郑文芝
王浩军
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Anlong Biopharmaceutical Co Ltd
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Anlong Biopharmaceutical Co Ltd
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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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

Definitions

  • TTR After TTR is decomposed into monomers, it can cause amyloidosis.
  • TTR protein is related to infection, inflammation, malnutrition, amyloidosis and tumors.
  • TTR-specific drugs For the treatment of TTR amyloidosis, a variety of TTR-specific drugs have been developed and approved for marketing. At the same time, more and more TTR-targeted drugs are gradually moving towards clinical use for neurological diseases, endocrine and metabolic diseases, etc.
  • Amyloidosis is a disease caused by the accumulation of abnormally folded proteins in tissues. The symptoms caused depend on the site of amyloid accumulation, and the lesions mainly occur in organs such as the kidneys, heart, and nervous system. Amyloid deposition of TTR mutants can cause TTR amyloidosis. Specifically, TTR is a tetrameric structural protein with high stability, but TTR tetramers can decompose and degrade into monomers under pathological conditions or abnormal physiological conditions (such as stress and inflammatory response). TTR monomers can generate complex and diverse types of amyloid fibers, leading to abnormal physiological aggregation of amyloid fibers in cells. Abnormal amyloid deposition in cells can cause abnormal metabolism of the cells themselves and even changes and disorders in the function of the entire tissue, thereby causing related diseases, such as the hereditary transthyretin amyloidosis, which is reported more frequently.
  • TTR As a routine biochemical test indicator, TTR has long been regarded as a sensitive clinical indicator for evaluating the nutritional status of the body. Many studies have shown that the level of TTR in diabetic patients is closely related to their complications and prognosis. In addition, high plasma TTR levels are significantly positively correlated with an increased risk of type 2 diabetes and impaired glucose regulation. TTR can bind to retinol transporter to form macromolecular compounds to reduce the renal activity of retinol binding protein. The results showed that TTR can regulate the filtration rate of the liver and maintain the plasma retinol binding protein concentration. As a clear adipokine, retinol binding protein is closely related to the occurrence and development of diabetes. These studies provide a theoretical basis for TTR as a potential biomarker for metabolic disease risk.
  • the purpose of the present disclosure is to provide an inhibitor targeting TTR with good efficacy, high safety and long-lasting efficacy.
  • Figure 5 shows the efficacy of hTTR siRNA in V30 hTTR transgenic mice.
  • nucleotides refers to a structural relationship between nucleotides (e.g., on two nucleotides on a relative nucleic acid or on a relative region of a single nucleic acid chain) that allows nucleotides to form base pairs with each other.
  • a purine nucleotide complementary to a pyrimidine nucleotide of a nucleic acid relative to a nucleic acid can be base-paired together by forming hydrogen bonds with each other.
  • complementary nucleotides can be base-paired in a Watson-Crick manner or in any other manner that allows the formation of a stable duplex.
  • two nucleic acids can have a nucleotide sequence that is complementary to each other so as to form a complementary region, as described herein.
  • deoxyribonucleotide refers to a nucleotide having a hydrogen at the 2' position of its pentose compared to a ribonucleotide.
  • a modified deoxyribonucleotide is a deoxyribonucleotide having a modification or substitution of one or more atoms other than at the 2' position (including a modification or substitution in or of a sugar, a phosphate group, or a base).
  • oligonucleotide refers to a short nucleic acid, such as a short nucleic acid less than 100 nucleotides in length.
  • the oligonucleotide may comprise ribonucleotides, deoxyribonucleotides and/or modified nucleotides, including, for example, modified ribonucleotides.
  • the oligonucleotide may be single-stranded or double-stranded.
  • the oligonucleotide may or may not have a duplex region.
  • the oligonucleotide may be, but is not limited to, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), Dicer substrate interfering RNA (dsiRNA), antisense oligonucleotide, short siRNA or single-stranded siRNA.
  • the double-stranded oligonucleotide is an RNAi oligonucleotide.
  • RNAi agent As used herein, the terms “iRNA”, “RNAi agent”, “iRNA agent”, “RNA interfering agent” are used interchangeably herein and refer to an agent that comprises RNA as such terms are defined herein and mediates targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway.
  • RISC RNA-induced silencing complex
  • RNA interference is a process that directs sequence-specific degradation of mRNA. RNAi modulates, for example, inhibits expression of TTR in a cell, for example, a cell within an individual, for example, a mammalian individual.
  • conjugation refers to the covalent connection between two or more chemical moieties each having a specific function; accordingly, “conjugate” refers to a compound formed by covalent connection between the chemical moieties.
  • siRNA conjugate means a compound formed by covalently connecting one or more chemical moieties having a specific function to siRNA.
  • the siRNA conjugate of the present invention is sometimes referred to as a "conjugate”.
  • the siRNA conjugate should be understood as a general term for siRNA conjugates, the first siRNA conjugate or the second siRNA conjugate, or the siRNA sense chain conjugate or the siRNA antisense chain conjugate, depending on the context.
  • modified nucleotide refers to a nucleotide independently having a modified sugar moiety, a modified internucleotide bond, or a modified nucleobase, or any combination thereof.
  • modified nucleotide encompasses substitutions, additions, or removals of internucleoside bonds, sugar moieties, or nucleobases, such as functional groups or atoms. Modifications suitable for use with the disclosed agents include all types of modifications disclosed herein or known in the art.
  • nucleotide overhang refers to at least one unpaired nucleotide protruding from the duplex structure of a double-stranded iRNA. For example, when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa, there is a nucleotide overhang.
  • the dsRNA may include an overhang of at least one nucleotide; alternatively, the overhang may include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more.
  • naked sequence refers to an unmodified nucleotide sequence.
  • inhibitor is used interchangeably with “knockdown,” “reduction,” “silencing,” “downregulate,” “suppression,” and other similar terms, and includes any degree of inhibition.
  • TTR inhibiting the expression of any TTR gene (such as, for example, a mouse TTR gene, a rat TTR gene, a monkey TTR gene, or a human TTR gene) as well as variants or mutants of a TTR gene.
  • TTR gene can be a wild-type TTR gene, a mutant TTR gene, or a transgenic TTR gene.
  • salts derived from inorganic bases include but are not limited to alkali metal salts (such as sodium salts, potassium salts and lithium salts), ammonium salts, alkaline earth metal salts (such as calcium salts and magnesium salts).
  • Salts derived from organic bases include, but are not limited to, salts formed with the following organic bases (e.g., organic amines): primary amines, secondary amines, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins.
  • the oligonucleotides of the present invention may also exist in the form of zwitterions.
  • Particularly preferred pharmaceutical salts of the present invention are sodium salts, lithium salts, potassium salts, and trialkylammonium salts.
  • each strand is independently 19 to 25 nucleotides in length.
  • the sense strand and the antisense strand are respectively in a 19/21 paired, 21/21 paired, 21/23 paired, or 23/23 paired duplex structure.
  • the oligonucleotide comprises a 3'-overhang sequence that is two nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand and the sense strand, and wherein the sense strand is 23 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense strand and the antisense strand form a duplex that is 21 nucleotides in length.
  • Salts derived from organic bases include, but are not limited to, salts formed with the following organic bases: primary amines, secondary amines and tertiary amines, substituted amines include naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins.
  • organic bases such as organic amines
  • substituted amines include naturally occurring substituted amines
  • cyclic amines and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins.
  • the aforementioned oligonucleotides comprise at least one modified nucleotide.
  • the aforementioned 2'-modified nucleotides are selected from one or more of 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, 2'-fluoro-modified nucleotides, and 2'-deoxynucleotides.
  • the aforementioned 2'-modification is a modification selected from the group consisting of 2'-methoxy, 2'-acetylamino, 2'-aminoethyl, 2'-fluoro, 2'-O-methoxyethyl.
  • the aforementioned '-phosphate analog modified nucleotide has a vinyl phosphate modified nucleotide shown in formula (II), wherein R is selected from H, OH, fluorine, 2'-methoxy, 2'-acetylamino, 2'-aminoethyl and 2'-O-methoxyethyl.
  • At least one modified internucleotide bond is a phosphorothioate bond.
  • the phosphorothioate internucleotide bond modification can occur on any nucleotide of the sense strand, antisense strand, or two strands in any position of the strand.
  • the internucleotide bond modification can occur on each nucleotide on the sense strand or antisense strand; each internucleotide bond modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand can contain two internucleotide bond modifications in an alternating pattern.
  • the antisense strand includes two phosphorothioate internucleotide bonds at the 5' end, and includes two phosphorothioate internucleotide bonds at the 3' end, and the sense strand includes at least two phosphorothioate internucleotide bonds at the 5' end or the 3' end.
  • the aforementioned oligonucleotide is selected from any one of the following sense strand and antisense strand combinations:
  • the aforementioned siRNA conjugate contains the aforementioned siRNA and a conjugated group conjugated to the siRNA.
  • oligonucleotide salt refers to an oligonucleotide compound in the form of a salt. Oligonucleotide salts include salts of oligonucleotide conjugated compounds and salts of unconjugated oligonucleotide compounds. Oligonucleotide salts are advantageously present in the form of solid powders.
  • ASGPR ligands facilitates cell-specific targeting to hepatocytes and endocytosis of molecules into hepatocytes.
  • ASGPR ligands can be monomeric (eg, with a single galactose derivative) or polymeric (eg, with multiple galactose derivatives).
  • Galactose derivatives or clusters of galactose derivatives can be linked to the 3' or 5' end of the siRNA using methods known in the art.
  • the aforementioned targeting ligand is L96 represented by formula (IV):
  • the composition is in the form of an oral agent, an intravenous injection, a subcutaneous injection or an intramuscular injection, preferably a subcutaneous injection.
  • the aforementioned amyloidosis is Alzheimer's disease.
  • inhibition of in vivo expression is determined by knocking down a human gene in a rodent expressing a human gene, e.g., an AAV-infected mouse expressing a human target gene (i.e., TTR), e.g., when administered as a single dose, e.g., at a nadir of TTR expression following subcutaneous injection at 3 mg/kg to confirm the inhibitory effect on the human gene.
  • a human target gene i.e., TTR
  • TTR human target gene
  • ⁇ CT ⁇ CT treated cells- ⁇ CT control cells
  • mRNA inhibition percentage (protein expression level treated cells - protein expression level control cells ) / protein expression level control cells * 100%
  • the present disclosure also provides methods of using the iRNA of the present disclosure or compositions comprising the iRNA of the present disclosure to inhibit TTR expression, thereby preventing or treating
  • the invention relates to the treatment of the following diseases, disorders and/or conditions, including but not limited to: amyloidosis (e.g., familial amyloid polyneuropathy (FAP), Alzheimer's disease), metabolic diseases (e.g., diabetes, impaired glucose regulation) and tumors (e.g., lung cancer, pancreatic ductal carcinoma), as well as complications of each of the above conditions.
  • amyloidosis e.g., familial amyloid polyneuropathy (FAP), Alzheimer's disease
  • metabolic diseases e.g., diabetes, impaired glucose regulation
  • tumors e.g., lung cancer, pancreatic ductal carcinoma
  • the formulation comprises lipid nanoparticles.
  • the excipient comprises a liposome, lipid, lipid complex, microsphere, microparticle, nanosphere or nanoparticle, or may be otherwise formulated for administration to a cell, tissue, organ or body of a subject in need thereof.
  • the composition may contain at least about 0.1% or more of a therapeutic agent (e.g., an oligonucleotide for reducing TTR expression, a salt thereof, or a conjugate thereof, or a salt of a conjugate thereof), although the percentage of one or more active ingredients may be between about 1% and about 80% or more by weight or volume of the total composition.
  • a therapeutic agent e.g., an oligonucleotide for reducing TTR expression, a salt thereof, or a conjugate thereof, or a salt of a conjugate thereof
  • the percentage of one or more active ingredients may be between about 1% and about 80% or more by weight or volume of the total composition.
  • Treatment can be regularly repeated administration.
  • treatment can be carried out at a lower frequency.
  • Repeated dosing regimens may include regular administration of the iRNA of the therapeutic amount, such as once a month to once a year.
  • the iRNA is administered about once a month to about once every three months, or about once every three months to about once every six months, or even once a year.
  • a subject is administered a fixed dose of about 150 mg approximately once every six months. In some embodiments, a subject is administered a fixed dose of about 200 mg approximately once every six months. In some embodiments, a subject is administered a fixed dose of about 300 mg approximately once every six months. In some embodiments, a subject is administered a fixed dose of about 600 mg approximately once every six months. In some embodiments, a subject is administered a fixed dose of about 800 mg approximately once every six months. In some embodiments, a subject is administered a fixed dose of about 800 mg approximately once every six months.
  • Intermediate E1 was treated with succinic anhydride in the presence of DMAP and Et3N to obtain the hemisuccinate intermediate E1-1 in quantitative yield.
  • the hemisuccinate intermediate E1-1 was coupled with the amino group of a 60% divinylbenzene (DVB) cross-linked (aminomethyl) polystyrene resin having an amine content of 250 mol/g to obtain a solid support A1 having a loading of 100 mol/g and having a protective group.
  • DVD 60% divinylbenzene
  • the phosphoramidite monomers, reagents, and purification consumables used are all commercial circulating reagents and consumables, such as various phosphoramidite monomers (such as 5'-O-(4,4'-Dimethoxytrityl)-2'-O-methyl-Uridine-3'-CE-Phosphoramidite) purchased from Shanghai Zhaowei Technology Development Co., Ltd., and reaction reagents (such as 40wt% methylamine aqueous solution, 28wt% ammonium hydroxide aqueous solution, etc.) purchased from Sigma-Aldrich LLC.
  • various phosphoramidite monomers such as 5'-O-(4,4'-Dimethoxytrityl)-2'-O-methyl-Uridine-3'-CE-Phosphoramidite
  • reaction reagents such as 40wt% methylamine aqueous solution, 28wt% ammonium hydroxide aqueous solution, etc.
  • the solid phase phosphoramidite method is a mature oligonucleotide synthesis method.
  • the reaction is carried out in a stainless steel synthesis column using a computer-controlled synthesizer.
  • a solid phase support loaded with a targeting ligand such as L96 and A1 is used as the starting point, or the solid phase support is used directly as the starting point.
  • Different raw materials, reagents and solvents are injected into different pipelines in the order of sequence 3' to 5' through the solid phase synthesizer to connect the phosphoramidite nucleoside monomers one by one.
  • the reaction process includes four cycles of DMT protection group removal reaction, condensation reaction, oxidation or thiolation reaction, and end-capping reaction.
  • Each cycle connects a nucleotide unit to obtain an oligonucleotide sequence of 19 or 21 nucleotide units.
  • the protecting group (2-cyanoethyl) is removed on the solid phase synthesis column, and the synthesized sequence is cut from the solid phase support by aminolysis reaction, filtered, the filter cake is washed with ethanol, the filtrate and washing liquid are collected, and concentrated to obtain the crude positive chain.
  • the crude product is purified by chromatography (SOURCE 15Q) and freeze-dried to obtain the target product positive chain.
  • the siRNA positive chain conjugate is synthesized starting from the solid support loaded with the targeting ligand (such as L96); and the siRNA is synthesized directly starting from the solid support.
  • the synthesis of the antisense strand is similar to that of the sense strand.
  • Different raw materials, reagents and solvents are injected into different pipelines in the order of 3' to 5' of the sequence through a solid phase synthesizer to connect phosphoramidite nucleoside monomers one by one.
  • the reaction process includes four cycles of DMT protection group removal reaction, condensation reaction, oxidation or thiolation reaction, and end-capping reaction. Each cycle connects a nucleotide unit to obtain an oligonucleotide sequence of 21 or 23 nucleotide units.
  • the protecting group (2-cyanoethyl) is removed on the solid phase synthesis column, and then the synthesized sequence is cut from the solid phase support by aminolysis reaction, filtered, and the filter cake is washed with ethanol. The filtrate and washing liquid are collected and concentrated to obtain the crude antisense strand.
  • the crude product is purified by chromatography (SOURCE 15Q), ultrafiltered, and freeze-dried to obtain the target product antisense strand siRNA.
  • a computer-based algorithm was used to generate candidate oligonucleotide sequences complementary to human TTR mRNA (NM_000371.4, Table 1), some of which were also complementary to cynomolgus monkey TTR mRNA (XM_045377903.1, Table 1) or had no more than 2 mismatches.
  • Some of them were designed as double-stranded siRNAs with 19/21 pairings of the sense and antisense strands, respectively, and the antisense strand had two overhangs complementary to the mRNA sequence, and in some cases the overhangs of the antisense strand were non-complementary UU; some of them were designed as double-stranded siRNAs with 21/23 pairings of the sense and antisense strands, respectively, and the antisense strand had two overhangs complementary to the mRNA sequence; some of them were designed as double-stranded siRNAs with 21/21 and 23/23 pairings. In some of the complementary paired sequences, the first base at the 5' end of the antisense strand (the last base at the 3' end of the sense strand) was replaced with a base that did not match the TTR mRNA.
  • m represents 2'-methoxy
  • f represents 2'-deoxy-2'-fluoro
  • s represents thiophosphate
  • APU is uridine acid (2'-acetylamino-5'-vinylphosphonate-uridine acid) modified by a 5'-phosphate analogue as shown in formula (II-1)
  • A1 is a GalNAc targeting ligand as shown in formula (III)
  • L96 is N-[tri(GalNAc-alkyl)amidodecanoyl]-4-hydroxyprolinol (Hyp-(GalNAc-alkyl)3) as shown in formula (IV).
  • RNA-Quick Purification Kit (RN001, Yishan Biotechnology):
  • RNA column Take out the centrifuge tube and add 500 ⁇ l of wash buffer to the column, centrifuge at 12000 ⁇ g for 1min, take out the column after the centrifugation, pour out the waste liquid, put the RNA column back into the collection tube, centrifuge the empty tube once to remove the residual wash buffer. Place the column in a clean 1.5ml centrifuge tube without RNase, open the lid and dry for 2min. Add 30 ⁇ l of elution buffer to the center of the membrane of the RNA column, let it stand at room temperature for 2min, centrifuge at 2000 ⁇ g for 1min, and the RNA is eluted. After eluting, place on ice. Measure the concentration of eluted RNA for subsequent experiments. The extracted RNA can be used immediately for subsequent experiments or stored at -80°C for later use.
  • a mixed solution of 10 ⁇ l 2 ⁇ ChamQ SYBR qPCR Master Mix, 0.5 ⁇ l Forword primer (Ruiboxin), 0.5 ⁇ l Reverse primer (Ruiboxin), 1 ⁇ l Template cDNA, and 8 ⁇ l ddH 2 O was prepared to a 20 ⁇ l system. Each sample was replicated three times.
  • the 96-well plate was placed in a qPCR instrument (ROCGENE, Archimed) and the following program was executed: pre-denaturation, 95°C, 30 sec; amplification, 95°C, 10 sec, 60°C, 30 sec, 40 cycles; melting curve, 95°C, 15 sec, 60°C, 60 sec, 95°C, 15 sec.
  • System 1 was diluted with 50 ⁇ l Opti-MEM (thermofisher, 1105821) to a concentration of 1 ⁇ M, 0.2 ⁇ M or 20 nM siRNA (Suzhou Ouli Biopharmaceutical Technology Co., Ltd.), and system 2 was diluted with 50 ⁇ l Opti-MEM to 3 ⁇ l Lipo3000. After standing for 5 min, system 1 and 2 were mixed. The final concentration of siRNA was 0.5 ⁇ M or 0.1 ⁇ M. After standing for another 15 min, the cells were added dropwise to a 12-well plate. The final concentration of siRNA was 50 nM, 10 nM or 1 nM. DMEM/F12 complete medium was replaced 4 h after transfection, and the 12-well plate was placed in an incubator and incubated for 24 h.
  • the mRNA sequence of the human TTR gene was obtained from the NCBI database (GENBANK NO.NM_000371.4).
  • the TTR gene 27-470 base sequence (SEQ ID NO.258) was selected to obtain a recombinant plasmid by conventional molecular biology techniques such as restriction digestion and ligation.
  • the sequence containing the CAG promoter (SEQ ID NO.259) was inserted between EcoRI and XhoI of the pFB vector (purchased from Agilent, catalog number 013001) to obtain pFB-AAV-CAG, and then the target gene (27-470 of TTR mRNA) was inserted between EcoRI and BamHI by restriction digestion and ligation to obtain pFB-AAV-CAG-TTR-MM recombinant plasmid ( Figure 2).
  • the plasmid was injected into the tail vein by hydrodynamics for transient transfection in vivo, which was administered at least 29 days before the administration of the TTR RNAi reagent or control.
  • mice were subcutaneously administered a single dose of the corresponding TTR RNAi agent and vehicle control. Blood was collected from the mice on the 8th and 15th days after administration, and the serum was separated to detect the TTR concentration. The inhibitory effect of siRNA on exogenous gene mRNA was evaluated. The results are shown in Figure 3. As shown in Figure 3, although the concentration of TTR decreased over time, the knockdown effect of TTR in the AL0057002 and AL0057003 groups was better than that in the N group on the 15th day after administration.
  • the hTTR expression level was measured, and the level of hTTR knockdown was detected (the log value of TTR content in serum) with the control before administration. During the experiment, no animals showed death or dying symptoms. No obvious abnormalities were observed in all animals during clinical observation. The level of TTR changes is shown in Figures 4 and 5.

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Abstract

L'invention concerne un oligonucléotide ciblant la transthyrétine et son utilisation. L'oligonucléotide de ciblage peut inhiber de manière significative le niveau d'expression génique de la transthyrétine, et présente un effet médicamenteux durable.
PCT/CN2024/124662 2023-10-13 2024-10-14 Oligonucléotide ciblant la transthyrétine et son utilisation Pending WO2025077916A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011056883A1 (fr) * 2009-11-03 2011-05-12 Alnylam Pharmaceuticals, Inc. Compositions de lipides formulés et procédés pour l'inhibition de l'expression de transthyrétine (ttr)
US20140315835A1 (en) * 2011-11-18 2014-10-23 Alnylam Pharmaceuticals, Inc. Rnai agents, compositions and methods of use thereof for treating transthyretin (ttr) associated diseases
US20160304870A1 (en) * 2013-12-06 2016-10-20 Dicerna Pharmaceuticals, Inc. Methods and compositions for the specific inhibition of transthyretin (ttr) by double-stranded rna
US20170029817A1 (en) * 2015-07-31 2017-02-02 Alnylam Pharmaceuticals, Inc. TRANSTHYRETIN (TTR) iRNA COMPOSITIONS AND METHODS OF USE THEREOF FOR TREATING OR PREVENTING TTR-ASSOCIATED DISEASES
CN115461460A (zh) * 2020-03-06 2022-12-09 阿尔尼拉姆医药品有限公司 用于抑制转甲状腺素蛋白(ttr)的表达的组合物和方法
CN116003487A (zh) * 2021-10-22 2023-04-25 苏州时安生物技术有限公司 寡核苷酸的缀合物及其制备方法和应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011056883A1 (fr) * 2009-11-03 2011-05-12 Alnylam Pharmaceuticals, Inc. Compositions de lipides formulés et procédés pour l'inhibition de l'expression de transthyrétine (ttr)
US20140315835A1 (en) * 2011-11-18 2014-10-23 Alnylam Pharmaceuticals, Inc. Rnai agents, compositions and methods of use thereof for treating transthyretin (ttr) associated diseases
US20160304870A1 (en) * 2013-12-06 2016-10-20 Dicerna Pharmaceuticals, Inc. Methods and compositions for the specific inhibition of transthyretin (ttr) by double-stranded rna
US20170029817A1 (en) * 2015-07-31 2017-02-02 Alnylam Pharmaceuticals, Inc. TRANSTHYRETIN (TTR) iRNA COMPOSITIONS AND METHODS OF USE THEREOF FOR TREATING OR PREVENTING TTR-ASSOCIATED DISEASES
CN115461460A (zh) * 2020-03-06 2022-12-09 阿尔尼拉姆医药品有限公司 用于抑制转甲状腺素蛋白(ttr)的表达的组合物和方法
CN116003487A (zh) * 2021-10-22 2023-04-25 苏州时安生物技术有限公司 寡核苷酸的缀合物及其制备方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NIEMIETZ CHRISTOPH, CHANDHOK GURSIMRAN, SCHMIDT HARTMUT: "Therapeutic Oligonucleotides Targeting Liver Disease: TTR Amyloidosis", MOLECULES, MDPI AG, CH, vol. 20, no. 10, CH , pages 17944 - 17975, XP093304283, ISSN: 1420-3049, DOI: 10.3390/molecules201017944 *

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