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WO2023051822A1 - Oligonucléotide de ciblage pour le traitement de maladies associées à pcsk9 - Google Patents

Oligonucléotide de ciblage pour le traitement de maladies associées à pcsk9 Download PDF

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WO2023051822A1
WO2023051822A1 PCT/CN2022/123541 CN2022123541W WO2023051822A1 WO 2023051822 A1 WO2023051822 A1 WO 2023051822A1 CN 2022123541 W CN2022123541 W CN 2022123541W WO 2023051822 A1 WO2023051822 A1 WO 2023051822A1
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seq
sequence
antisense strand
oligonucleotide
sense strand
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田宝磊
高杰
赵君竺
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Anlong Biopharmaceutical Co Ltd
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Anlong Biopharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N15/1137—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 against enzymes
    • 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
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • 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
    • A61P3/06—Antihyperlipidemics
    • 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
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21061—Kexin (3.4.21.61), i.e. proprotein convertase subtilisin/kexin type 9
    • 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]

Definitions

  • the present disclosure relates to an oligonucleotide, especially a targeting oligonucleotide for treating diseases related to PCSK9.
  • Blood lipids refer to the general term for lipids contained in blood, and blood lipids that are closely related to clinical practice mainly include triglycerides and cholesterol.
  • Dyslipidemia usually refers to elevated serum cholesterol and/or triglyceride (TG) levels, commonly known as hyperlipidemia.
  • TG triglyceride
  • Hypercholesterolemia refers to elevated total cholesterol (TCHO) and/or low-density lipoprotein cholesterol (LDL-C) or non-HDL-C (non-HDL-C) in the blood, also known as dyslipidemia, It can be divided into familial cholesterolemia and non-familial cholesterolemia. One of the most important clinical features is the increase of blood low-density lipoprotein cholesterol (LDL-C) level in patients. Hypercholesterolemia is the result of cardiovascular disease. An important factor (Guo, Yanan, et al. Archives of Biochemistry and Biophysics (2020): 108717). Some cardiovascular and cerebrovascular diseases, such as cerebrovascular disease, coronary heart disease and peripheral arterial disease, usually have no obvious symptoms until severe atherosclerosis occurs.
  • statins are the first-line drugs for lowering blood lipids in clinical practice. These drugs can reduce the risk of cardiovascular diseases by reducing LDL-C, but many patients are intolerant to statins, or after taking the maximum dose of statins , still can not reduce the LDL-C level to an appropriate level (Toutouzas et al. Expert Opin Pharmacother (2010) 11:1659-72), therefore, it is necessary to develop new drugs to meet the needs of patients, which has important clinical application value.
  • the human PCSK9 gene is about 22 kb in length, includes 12 exons, and can encode a glycoprotein with a length of 692 amino acids (Benjannet, Suzanne, et al. Journal of Biological Chemistry 279.47 (2004): 48865-48875).
  • the liver is the main site for the synthesis and secretion of PCSK9.
  • the normal level of PCSK9 in human plasma ranges from 30ng/mL to 4 ⁇ g/mL. It plays an important role in cholesterol metabolism, mainly through affecting the expression level of LDL receptors on the surface of liver cells. Control plasma LDL levels.
  • Gain-of-function mutations (reverse mutations) of the PCSK9 gene can lead to decreased levels of LDL receptors on the surface of liver cells and increased levels of LDL-C in the blood; on the contrary, loss-of-function mutations can lead to increased levels of LDL receptors on the surface of liver cells , the level of LDL-C in the blood is reduced. Studies have shown that when the maximum dose of statins cannot effectively control the level of LDL-C, the level of LDL-C can be effectively reduced by reducing the level of PCSK9.
  • Intracellular PCSK9 can bind and guide newly generated LDL receptors from trans-Golgi apparatus to lysosome for degradation; extracellular circulating PCSK9 can bind to LDL receptors on the surface of liver cells and mediate their entry into liver cell lysosomes Degradation in the body reduces the LDL receptors on the surface of liver cells, leading to a decrease in the ability of the liver to bind and clear LDL-C, and ultimately increases the level of LDL-C in the blood. Therefore, it is possible to treat hypercholesterolemia (Norata et al. Annu Rev Pharmacol Toxicol (2014) 54:273-93) and prevent cardiovascular diseases associated with high LDL-C by reducing the expression level of PCSK9.
  • RNA molecules can mediate the degradation of specific mRNAs (Fire, Andrew, et al. Nature 391.6669 (1998): 806-811), When RNA molecules appear in double-stranded form in cells, this mechanism is induced to activate, that is, the phenomenon of RNA interference. Nobel Prize in Physiology and Medicine.
  • Dicer cuts the dsRNA into fragments, and then another protein complex RISC binds these fragments, one strand of the siRNA double strand is removed, but the other strand remains in complex with RISC Combined, RISC recognizes and degrades the mRNA of the target gene through the guidance of single-stranded RNA, inhibits the expression of specific proteins, and then specifically leads to gene silencing.
  • RNA interference has opened up a new field for the application of gene technology.
  • Double-stranded RNA molecules have been artificially engineered to silence specific genes in humans, animals or plants.
  • This artificially designed and synthesized double-stranded RNA molecule (siRNA) for gene silencing is introduced into cells and activates the RNA interference mechanism to degrade the corresponding mRNA.
  • siRNA double-stranded RNA molecule
  • this method is an important research tool in biology and biomedicine.
  • people have developed a large number of siRNA drugs to treat viral infections, cardiovascular diseases, cancer, endocrine disorders and other diseases. treatment effect. Since the first siRNA drug was launched in 2018, at least 4 siRNAs have been approved for marketing in the EU or the US. Therefore, the use of RNA interference technology to inhibit the expression of specific target genes has become an effective way to treat diseases.
  • inhibitors targeting PCSK9 have been reported, including marketed antibodies and small nucleic acid drugs, which are used or proposed to be used in the treatment of hypercholesterolemia and related diseases, but other inhibitors targeting this target need to be developed. In order to have better curative effect or better security.
  • the purpose of the present disclosure is to provide an inhibitor targeting PCSK9 with good curative effect, high safety and long-lasting drug effect.
  • the present disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof for reducing the expression of PCSK9, the oligonucleotide comprising an antisense strand having an expression as SEQ ID NO:53 - any of the sequences shown in 106 or fragments thereof, or modified sequences of said sequences or fragments thereof.
  • the oligonucleotide further comprises a sequence as shown in any one of SEQ ID NO: 1-52 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.
  • the antisense strand consists of a sequence as shown in any of SEQ ID NO: 53-106 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.
  • the modified sequence of the antisense strand comprises a sequence as shown in any one of SEQ ID NO: 138-183, 185-186 or a fragment thereof.
  • the modified sequence of the sense strand comprises a sequence as shown in any one of SEQ ID NO: 107-137, 135, 184 or a fragment thereof.
  • the sense strand and the antisense strand are respectively a duplex structure of 19/21 pairing, 21/21 pairing, 21/23 pairing or 23/23 pairing.
  • the present disclosure provides a composition comprising said oligonucleotide or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.
  • the present disclosure provides the use of the oligonucleotide or a pharmaceutically acceptable salt or composition thereof in the preparation of a medicament for treating or/and preventing diseases related to lipid disorders.
  • the disease in lipid dysregulation is the use of one or more symptoms of hyperlipidemia, atherosclerosis, and/or one or more symptoms or complications thereof.
  • the candidate compounds of the present disclosure have significant effects on the inhibition of PCSK9 gene expression levels in human liver cancer cell lines Huh7, HepG2 and Hle; some of the candidate compounds have an inhibitory effect of 90% on the PCSK9 gene expression levels of Huh7 and Hle cell lines. % or more, and some compounds can inhibit the expression of PCSK9 gene in cynomolgus monkey liver cells by 70%, 80%, or even 90%, showing a good inhibitory effect. Some of the compounds were administered to mice for about one month (Day 29) and the reduction of PCSK9 protein could still be maintained at about 60%; some compounds could reduce PCSK9 protein by more than 85% in mice for four consecutive weeks (Day 30), and the drug effect was long-lasting.
  • LDL-C low-density lipoprotein cholesterol
  • the present disclosure provides oligonucleotides targeting PCSK9 mRNA, which effectively reduce PCSK9 expression in cells, especially liver cells (e.g., hepatocytes), for the treatment of hypercholesterolemia, atherosclerosis, and/or its One or more symptoms or complications.
  • the present disclosure provides methods of treating hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof involving selectively reducing PCSK9 gene expression in the liver.
  • the PCSK9 targeting oligonucleotides provided herein are designed for delivery to selected cells of a target tissue (e.g., liver hepatocytes) to treat hypercholesterolemia, atherosclerosis, Cirrhosis and/or one or more of its symptoms or complications.
  • Fig. 1 is a graph showing the drug efficacy results of the first round of in vitro screening of PCSK9-siRNA in hepatic cell lines.
  • Fig. 2 is a diagram showing the drug efficacy results of the second round of in vitro screening of PCSK9-siRNA on liver cell lines.
  • Fig. 3 is a graph showing the pharmacodynamic results of PCSK9-siRNA after the first round of in vitro screening and modification of monkey primary hepatocytes.
  • Fig. 4 is a graph showing the pharmacodynamic results of PCSK9-siRNA after the second round of in vitro screening and modification of monkey primary hepatocytes.
  • Figure 5 shows the changes in LDL-C levels in mice after the first round of administration of PCSK9 RNAi agents.
  • Figure 6 shows the changes in mouse LDL-C levels after the second round of administration of PCSK9 RNAi agents.
  • Figure 7 shows the changes in mouse LDL-C levels after the third round of administration of PCSK9 RNAi agents.
  • Figure 8 shows the changes in LDL-C levels in hPCSK9 mice after administration of PCSK9 RNAi agents.
  • Figure 9 shows the change of PCSK9 protein level in hPCSK9 mice after administration of PCSK9 RNAi agent.
  • Figure 10 shows the changes in LDL-C levels in cynomolgus monkeys after administration of PCSK9 RNAi agents.
  • Figure 11 shows the changes in TCHO levels in cynomolgus monkeys after administration of PCSK9 RNAi agents.
  • Figure 12 shows the changes in mouse LDL-C levels after the fourth round of administration of PCSK9 RNAi agents.
  • Figure 13 shows the changes in mouse PCSK9 protein levels after the fourth round of administration of PCSK9 RNAi agents.
  • the term “about” or “approximately” as applied to one or more target values refers to a value that is similar to the stated reference value. In certain embodiments, the term “approximately” or “about” refers to falling within 20%, 19%, 18% in either direction (greater than or less than) of the stated reference value, unless otherwise stated or otherwise apparent from the context. %, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, range of values within 1% or less (unless such figures would exceed 100% of the possible values).
  • Atherosclerosis refers to a disease involving arteries (e.g., coronary, carotid, peripheral and/or renal artery) stenosis.
  • arteries e.g., coronary, carotid, peripheral and/or renal artery
  • narrowing of the coronary arteries can produce symptoms such as angina, shortness of breath, sweating, nausea, dizziness, shortness of breath, irregular heartbeat, and/or palpitations.
  • narrowing of the carotid arteries can lead to stroke (ie, death of brain cells due to insufficient blood and oxygen flow to the brain) and/or can produce symptoms such as weakness, confusion, trouble speaking, dizziness, difficulty walking or Standing up straight, blurred vision, numbness of face, arms and legs, severe headache and/or loss of consciousness.
  • stroke ie, death of brain cells due to insufficient blood and oxygen flow to the brain
  • narrowing of peripheral arteries can cause numbness or pain in the arms or legs.
  • narrowing of the renal arteries (resulting in reduced renal blood flow) can lead to chronic kidney disease.
  • Complications of atherosclerosis can include coronary artery disease, stroke, peripheral artery disease, and kidney problems (eg, chronic kidney disease).
  • nucleotides e.g., two nucleotides on opposing nucleic acids or on opposing regions of a single nucleic acid strand
  • nucleotides e.g., purine nucleotides of one nucleic acid that are complementary to pyrimidine nucleotides of an opposing nucleic acid can base pair together by forming hydrogen bonds with each other.
  • complementary nucleotides can base pair in a Watson-Crick fashion or in any other fashion that allows the formation of stable duplexes.
  • two nucleic acids can have nucleotide sequences that are complementary to each other so as to form a region of complementarity, as described herein.
  • Strand refers to a single contiguous sequence of nucleotides linked together by internucleotide bonds (eg, phosphodiester bonds, phosphorothioate bonds). In some embodiments, a strand has two free ends, eg, a 5'-end and a 3'-end.
  • Deoxyribonucleotide refers to a nucleotide having a hydrogen at the 2' position of its pentose sugar compared to a ribonucleotide.
  • Modified deoxyribonucleotides are modifications or substitutions having one or more atoms other than at the 2' position (including modifications or substitutions in sugars, phosphate groups or bases or sugars, phosphate groups or bases modified or substituted) deoxyribonucleotides.
  • Double-stranded oligonucleotide refers to an oligonucleotide that is substantially in the form of a duplex.
  • complementary base pairing of one or more duplex regions of a double-stranded oligonucleotide is formed between antiparallel sequences of nucleotides of the covalently separated nucleic acid strands.
  • complementary base pairing of one or more duplex regions of a double-stranded oligonucleotide is formed between antiparallel sequences of nucleotides of covalently linked nucleic acid strands.
  • complementary base pairing of one or more duplex regions of a double-stranded oligonucleotide is formed from a single nucleic acid strand that is folded (e.g., via a hairpin) to provide Complementary antiparallel sequence of nucleotides that base pair together.
  • a double-stranded oligonucleotide comprises two covalently separated nucleic acid strands that are fully duplexed with each other.
  • double-stranded oligonucleotides comprise two covalently separated nucleic acid strands that are partially duplexed, eg, having overhangs at one or both ends.
  • a double-stranded oligonucleotide comprises an antiparallel sequence of nucleotides that is partially complementary and, therefore, may have one or more mismatches, which may include internal or terminal mismatches match.
  • Oligonucleotide refers to a short nucleic acid, eg, less than 100 nucleotides in length.
  • An oligonucleotide may comprise ribonucleotides, deoxyribonucleotides, and/or modified nucleotides, including, for example, modified ribonucleotides.
  • Oligonucleotides can be single-stranded or double-stranded.
  • An oligonucleotide may or may not have a duplex region.
  • oligonucleotides can be, but are not limited to, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), Dicer substrate interfering RNA (dsiRNA), anti- sense oligonucleotides, short siRNA, or single-stranded siRNA.
  • the double-stranded oligonucleotide is an RNAi oligonucleotide.
  • PCSK9 refers to the "proprotein convertase subtilisin/kexin-9 gene or protein, also known as NARC-1, ShenFH3, HCHOLA3, NARC-1 or NARCI.
  • the term PCSK9 includes human PCSK9, mouse PCSK9, Rat PCSK9, Example uses of PCSK9 mRNA sequences are readily available at GenBank.5
  • the present disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof for reducing the expression of PCSK9, said oligonucleotide comprising an antisense strand having an expression as SEQ ID A sequence or a fragment thereof shown in any one of NO:53-106, or a modified sequence of said sequence or a fragment thereof.
  • the oligonucleotide or the pharmaceutically acceptable salt thereof is preferably prepared or synthesized in the form of carboxylate, sodium salt, triethylamine salt or other pharmaceutically acceptable salts.
  • the oligonucleotide or its pharmaceutically acceptable salt is more preferably its sodium salt or triethylamine salt.
  • the oligonucleotide further comprises a sequence as shown in any one of SEQ ID NO: 1-52 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.
  • the antisense strand consists of a sequence as shown in any of SEQ ID NO: 53-106 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.
  • the sense strand consists of a sequence as shown in any of SEQ ID NO: 1-52 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.
  • the oligonucleotide has a sense strand and an antisense strand, wherein the oligonucleotide comprises: (1) the sense strand shown in SEQ ID NO:51, and The antisense strand shown in SEQ ID NO:103; or (2) the sense strand shown in SEQ ID NO:52, and the antisense strand shown in SEQ ID NO:105.
  • the sense strand shown in SEQ ID NO:51 and the sense strand shown in SEQ ID NO:52 have a common motif UUUGCUUUUGUAACUUGAA (SEQ ID NO:51).
  • the antisense strand shown in SEQ ID NO: 103 and the antisense strand shown in SEQ ID NO: 105 have a common motif UUCAAGUUACAAAAGCAAA (SEQ ID NO: 187).
  • the modified sequence of the antisense strand comprises a sequence as shown in any one of SEQ ID NO: 138-183, 185-186 or a fragment thereof.
  • the modified sequence of the sense strand comprises a sequence as shown in any one of SEQ ID NO: 107-137, 135, 184 or a fragment thereof.
  • the antisense strand consists of a sequence as shown in any of SEQ ID NO: 138-183, 185-186 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.
  • the sense strand is composed of a sequence or a fragment thereof as shown in any of SEQ ID NO: 107-137, 135, 184, or a modified sequence of the sequence or a fragment thereof.
  • the antisense strand is 19 to 23 nucleotides in length.
  • the sense strand is 19 to 23 nucleotides in length.
  • the oligonucleotide comprises a 3'-overhang sequence of one or more nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand and/or chain of justice.
  • the antisense strand has an overhang.
  • the sense strand has an overhang.
  • the oligonucleotide comprises a 3'-overhang sequence that is two nucleotides in length.
  • the 3'-overhang sequence is present on the sense strand; preferably, the overhang sequence is selected from: GG, GA, GC, UC, UG, UU, UA, CA, CC, CG, CU, AA, AG, AU, AC.
  • the 3'-overhang sequence is present on the antisense strand; preferably, the overhang sequence is selected from: UU, UC, UA, UG, GA, GG, GU , GC, TT, AG, AU, AA, AC, CA, CC, U; more preferably, the overhang sequence is UU.
  • the oligonucleotide comprises an antisense strand and a sense strand each ranging in length from 19 to 23 nucleotides.
  • the sense strand and the antisense strand form a duplex region.
  • the sense strand and the antisense strand are respectively a duplex structure of 19/21 pairing, 21/21 pairing, 21/23 pairing or 23/23 pairing.
  • the oligonucleotide comprises a 3'-overhang sequence of two nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand, and wherein the length of the sense strand is 19 nucleotides and the length of the antisense strand is 21 nucleotides, so that the sense strand and the antisense strand form a duplex with a length of 19 nucleotides.
  • the oligonucleotide comprises a 3'-overhang sequence of two nucleotides in length, wherein the 3'-overhang sequence is present on the antisense and sense strands, and wherein the sense strand is of length is 21 nucleotides and the antisense strand is 21 nucleotides in length, so that the sense and antisense strands form a duplex of 19 nucleotides in length.
  • the oligonucleotide comprises a 3'-overhang sequence of two nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand, and wherein the length of the sense strand is 21 nucleotides and the length of the antisense strand is 23 nucleotides, so that the sense strand and the antisense strand form a duplex with a length of 21 nucleotides.
  • the oligonucleotide is selected from any one of the antisense strand SEQ ID NO: 55-57, 61, 66, 70-71, 80, 82, 84, 93-106
  • the oligonucleotide is selected from the unmodified oligonucleotide described in any one of the antisense strand SEQ ID NO:80,84,93-95,99-106, and the antisense strand The modified oligonucleotide described in any one of sense strand SEQ ID NO:145, 147-150, 154-183, 185-186.
  • the oligonucleotide is selected from any one of the sense strand SEQ ID NO: 3-5, 9, 14, 18, 19, 28, 30, 32, 41-52 Unmodified oligonucleotide, and the modified oligonucleotide described in any one of the sense strand SEQ ID NO:107-137,135,184.
  • the oligonucleotide comprises a 3'-overhang sequence of two nucleotides in length, wherein the 3'-overhang sequence is present on the antisense and sense strands above, and wherein the sense strand is 23 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense and antisense strands form a duplex of 21 nucleotides in length .
  • the oligonucleotide comprises at least one modified nucleotide.
  • the modification is a modification selected from the group consisting of 2'-methoxy (m), 2'-deoxy-2'-fluoro (f), and phosphorothioate (s).
  • the modified nucleotides comprise a 2'-modification.
  • the 2'-modification is a modification selected from the group consisting of 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxy Ethyl and 2'-deoxy-2'-fluoro- ⁇ -d-arabinucleic ribonucleic acid.
  • all nucleotides of the oligonucleotide are modified.
  • the oligonucleotide comprises at least one modified internucleotide linkage.
  • the at least one modified internucleotide linkage is a phosphorothioate linkage.
  • the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand comprises a phosphate analog.
  • the phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.
  • At least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands.
  • each targeting ligand comprises a carbohydrate, amino sugar, cholesterol, polypeptide or lipid.
  • each targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety.
  • the GalNac moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.
  • the oligonucleotides are selected from the oligonucleotides described in any one of AL0061001-AL0061054, AL0065001-AL0065051, AL0067001-AL0067040.
  • the sense strand comprises the motif sequence shown in SEQ ID NO:51; the antisense strand comprises the motif sequence shown in SEQ ID NO:187.
  • the oligonucleotide comprises any one selected from the following combinations of sense strand and antisense strand:
  • the oligonucleotide comprises any one selected from the following combinations of sense strand and antisense strand:
  • the oligonucleotide comprises any one selected from the following combinations of sense strand and antisense strand:
  • the present disclosure provides a composition comprising said oligonucleotide or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.
  • the dosage form of the composition is oral, intravenous, subcutaneous or intramuscular injection.
  • the dosage form of the composition is subcutaneous injection.
  • the combination further comprises other drugs for the treatment and/or prevention of diseases related to lipid imbalance.
  • the disease in terms of lipid imbalance is one or more symptoms of hyperlipidemia, atherosclerosis and/or one or more symptoms or complications thereof.
  • the hyperlipidemia is hypercholesterolemia.
  • the other drugs for treating hyperlipidemia include but are not limited to fibrates, statins, bile acid sequestering agents and niacin that have been used clinically.
  • the present disclosure provides a use of the oligonucleotide or a pharmaceutically acceptable salt or composition thereof in the manufacture of a medicament for treating or/and preventing diseases related to lipid disorders.
  • the disease in lipid dysregulation is the use of one or more symptoms of hyperlipidemia, atherosclerosis, and/or one or more symptoms or complications thereof.
  • the hyperlipidemia is hypercholesterolemia.
  • compositions comprising oligonucleotides (eg, single-stranded or double-stranded oligonucleotides) to reduce expression of PCSK9.
  • oligonucleotides eg, single-stranded or double-stranded oligonucleotides
  • Such compositions may be suitably formulated such that when administered to a subject (either into the immediate environment of the target cell or systemically), a sufficient portion of the oligonucleotide enters the cell to reduce PCSK9 expression.
  • oligonucleotide formulations can be used to deliver oligonucleotides for reducing PCSK9, as disclosed herein.
  • oligonucleotides are formulated in buffered solutions, such as phosphate-buffered saline, liposomes, micellar structures, and capsids.
  • naked oligonucleotides or conjugates thereof are formulated in water or an aqueous solution (eg, pH adjusted water).
  • naked oligonucleotides or conjugates thereof are formulated in aqueous alkaline buffer (eg, PBS).
  • Formulation of oligonucleotides with cationic lipids can be used to facilitate transfection of oligonucleotides into cells.
  • cationic lipids such as lipofectin, cationic glycerol derivatives and polycationic molecules (eg, polylysine) can be used.
  • the formulation comprises lipid nanoparticles.
  • the excipient comprises liposomes, lipids, lipoplexes, microspheres, microparticles, nanospheres, or nanoparticles, or can be otherwise formulated for administration to a subject in need thereof. Cell, tissue, organ or body administration.
  • formulations as disclosed herein comprise excipients.
  • excipients confer increased stability, increased absorption, increased solubility, and/or therapeutic enhancement of the active ingredient to the composition.
  • the excipient is a buffer (e.g., sodium citrate, sodium phosphate, tris base, or sodium hydroxide) or vehicle (e.g., buffer solution, petrolatum, dimethylsulfoxide, or mineral oil) .
  • oligonucleotides are lyophilized to extend their shelf life, and then made into solutions prior to use (eg, administration to a subject).
  • an excipient in a composition comprising any of the oligonucleotides described herein may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or a collapse temperature Altering agents (eg, dextran, ficoll, or gelatin).
  • a lyoprotectant e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone
  • a collapse temperature Altering agents eg, dextran, ficoll, or gelatin.
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (topical), transmucosal, and rectal administration.
  • the route of administration is intravenous or subcutaneous.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol and sodium chloride in the compositions.
  • Sterile injectable solutions can be prepared by incorporating the oligonucleotide in the required amount in the solvent of choice with one or a combination of ingredients enumerated above as required, followed by filtered sterilization.
  • the composition may contain at least about 0.1% or more of a therapeutic agent (e.g., an oligonucleotide for reducing expression of PCSK9), although the percentage of one or more active ingredients may vary in the total composition Between about 1% and about 80% or more by weight or volume.
  • a therapeutic agent e.g., an oligonucleotide for reducing expression of PCSK9
  • the percentage of one or more active ingredients may vary in the total composition
  • a therapeutic agent e.g., an oligonucleotide for reducing expression of PCSK9
  • administration of an oligonucleotide as described herein results in a decrease in the level of PCSK9 expression in the cell.
  • the reduction in the level of PCSK9 expression may be to 1% or less, 5% or less, 10% or less, 15% or less, 20% compared to an appropriate control level of PCSK9 or less, 25% or less, 30% or less, 35% or less, 40% or less, 45% or less, 50% or less, 55% or less, 60% or less Low, 70% or less, 80% or less, or 90% or less.
  • a suitable control level may be the level of PCSK9 expression in a cell or population of cells not contacted with an oligonucleotide as described herein.
  • the effect of delivering oligonucleotides to cells according to the methods disclosed herein is assessed after a limited period of time.
  • the cells can be assayed for oligonucleotides at least 8 hours, 12 hours, 18 hours, 24 hours; or at least one, two, three, four, five, six, seven, or fourteen days after introducing the oligonucleotide into the cells.
  • PCSK9 level is assessed after a limited period of time.
  • the oligonucleotides are delivered in the form of a transgene engineered to express the oligonucleotides disclosed herein (eg, in the form of shRNA) in the cell.
  • the oligonucleotide is delivered using a transgene engineered to express any of the oligonucleotides disclosed herein.
  • Transgenes can be delivered using viral vectors (eg, adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, or herpes simplex virus) or non-viral vectors (eg, plasmids or synthetic mRNA).
  • the transgene can be injected directly into the subject.
  • aspects of the disclosure relate to methods for reducing PCSK9 expression for treating hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof in a subject.
  • the method can comprise administering to a subject in need thereof an effective amount of any of the oligonucleotides disclosed herein.
  • such treatments are useful, for example, to reduce or prevent hypercholesterolemia (high levels of low-density lipoprotein (LDL)-cholesterol), atherosclerosis, coronary heart disease (e.g., coronary artery disease), angina pectoris , shortness of breath, sweating, nausea, dizziness, shortness of breath, irregular heartbeat, palpitations, stroke (death of brain cells due to lack of blood and oxygen flow to the brain), weakness, confusion, trouble speaking, dizziness, difficulty walking, or standing up straight, blurred vision, numbness of the face, arms, and legs, severe headache, loss of consciousness, peripheral artery disease, and/or kidney problems (such as chronic kidney disease).
  • such treatment is useful, for example, to treat or prevent one or more symptoms associated with hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof.
  • the present disclosure provides treatment for patients with hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof (including coronary artery disease (eg, coronary artery disease), angina, Shortness of breath, sweating, nausea, dizziness, shortness of breath, irregular heartbeat, heart palpitations, stroke (death of brain cells due to lack of blood and oxygen flow to the brain), feeling of weakness, confusion, trouble speaking, dizziness, difficulty walking or standing straight, blurred vision, numbness of the face, arms, and legs, severe headache, loss of consciousness, peripheral artery disease, and/or kidney problems (such as chronic kidney disease) (or predisposed to hypercholesterolemia, atherosclerosis and/or one or more symptoms or complications thereof).
  • coronary artery disease eg, coronary artery disease
  • angina Shortness of breath, sweating, nausea, dizziness, shortness of breath, irregular heartbeat, heart palpitations, stroke (death of brain cells due to lack of blood and oxygen flow to the brain), feeling of weakness
  • the present disclosure provides methods for preventing a disease, disorder, disorder, or disease as described herein in a subject by administering to the subject a therapeutic agent (e.g., an oligonucleotide or a vector encoding the same or a transgene). Symptoms or Conditions.
  • a therapeutic agent e.g., an oligonucleotide or a vector encoding the same or a transgene.
  • the subject to be treated is one that would therapeutically benefit from a reduction in the amount of PCSK9 protein, eg, in the liver.
  • the methods described herein generally involve administering to a subject an effective amount (ie, an amount capable of producing a desired therapeutic result) of an oligonucleotide.
  • a therapeutically acceptable amount may be an amount capable of treating a disease or condition.
  • the appropriate dosage for any subject will depend on certain factors, including the subject's size, body surface area, age, the particular composition to be administered, the active ingredient(s) in the composition, the time of administration and route, general health, and other concomitant medications.
  • enteral e.g., orally, via a gastric feeding tube, via a duodenal feeding tube, via gastrostomy, or rectally
  • parenterally e.g., subcutaneous injection, intravenous injection, or infusion
  • injection intraarterial injection or infusion
  • intramuscular injection intramuscular injection
  • topically e.g., epidermis, inhalation, via eye drops, or through the mucosa
  • the oligonucleotides disclosed herein are administered intravenously or subcutaneously.
  • the oligonucleotide is administered at a dose ranging from 0.1 mg/kg to 25 mg/kg (eg, 1 mg/kg to 5 mg/kg). In some embodiments, the oligonucleotide is administered at a dose in the range of 0.1 mg/kg to 5 mg/kg or in the range of 0.5 mg/kg to 5 mg/kg.
  • the oligosaccharides of the present disclosure will typically be administered annually, twice a year, quarterly (every three months), bimonthly (every two months), monthly, or weekly. Nucleotides.
  • the subject to be treated is a human (eg, a human patient) or a non-human primate or other mammalian subject.
  • Other exemplary subjects include domesticated animals such as dogs and cats; livestock such as horses, cows, pigs, sheep, goats, and chickens; and animals such as mice, rats, guinea pigs, and hamsters.
  • a computer-based algorithm was used to generate candidate oligonucleotide sequences complementary to human PCSK9 mRNA (NM_174936.3, Table 1), some of which were also complementary to PCSK9 mRNA from cynomolgus monkeys (XM_005543260.2, Table 1 ), some sequences are also complementary to mouse PCSK9 mRNA (NM_153565.2, Table 1) or have no more than 2 mismatches. Some of them are designed as double-stranded siRNAs with 19/21 pairs of sense and antisense strands, respectively. The antisense strand has two overhanging ends that are complementary to the mRNA sequence.
  • Complementary UU some of the sequences are designed as double-stranded siRNA with sense strand and antisense strand being 21/23 pairs respectively, and the antisense strand has two overhanging ends complementary to the mRNA sequence; some of the sequences are designed as 21/23 21, 23/23 paired double-stranded siRNA. Among them, the first base at the 5' end of the antisense strand (the last base at the 3' end of the sense strand) of some complementary paired sequences was replaced with a base that did not match PCSK9 mRNA. In some sequences, individual ribonucleic acid is replaced by deoxyribonucleic acid, such as T or dA.
  • Table 1 Human, cynomolgus monkey, and mouse PCSK9 mRNA sequences
  • d stands for DNA
  • dT stands for 2'-deoxythymidine
  • dA stands for 2'-deoxyadenosine
  • m stands for 2'-methoxy
  • f stands for 2'-deoxy-2'-fluoro
  • s stands for phosphorothioate Ester
  • L represents ligand
  • L96 is N-[tris(GalNAc-alkyl)amidodecanoyl]-4-hydroxyprolinol Hyp-(GalNAc-alkyl)3.
  • Human liver cancer cell lines Huh7 (CCTCC, GDC0134), HepG2 (Jiangsu KGI Biotechnology Co., Ltd., KG020) and HLE (JCRB Cell Bank, JCRB0404) were used in an incubator at 37°C and 5% CO 2 .
  • DMEM basal medium (Hyclone, SH30022.01) was cultured with 10% FBS (aqlabteech, AQ-MV-06600) and 1% penicillin-streptomycin (KGY0023) until the confluence of the cells reached 90%.
  • RNA-Quick Purification Kit RNA Quick Purification Kit, Yishan Biological Sciences, RN001
  • cynomolgus monkey hepatocytes (Beijing Ruide Biotechnology Co., Ltd., cmTCSC), preheat the medium first, take out the thawed medium (Beijing Ruide Biotechnology Co., Ltd., HEPO24) into a biological safety cabinet, and store in 36ml Add 4 mL of FBS to the thawed medium (TPCS, HEPO24) to make a completely thawed medium, and heat it in a water bath at 37°C for 10 minutes. Treat with coating medium (Beijing Ruide Biotechnology Co., Ltd., HEPO44) at 37°C in a CO 2 incubator for 0.5h.
  • TPCS thawed medium
  • HEPO24 thawed medium
  • Treat with coating medium (Beijing Ruide Biotechnology Co., Ltd., HEPO44) at 37°C in a CO 2 incubator for 0.5h.
  • CM seeding medium was sucked out, replaced with preheated medium (Beijing Red Biotech Co., Ltd., CMHEP064), and transfected after 6 hours of adherence.
  • Use Lipofectamine TM 3000 Transfection Reagent (thermofisher, L3000150) for transfection system 1 was diluted with 50 ⁇ l Opti-MEM (thermofisher, 1105821) to 50 nM modified siRNA (Suzhou Beixin Biotechnology Co., Ltd.), system 2 was diluted with 50 ⁇ l Opti-MEM 3 ⁇ l Lip3000, after standing for 5 minutes, mix the system 1 and 2 and let stand for 15 minutes, add dropwise to the 12-well plate, replace the DMEM/F12 complete medium 4 hours after transfection, place the 12-well plate in the incubator and incubate for 48 hours .
  • RNA-Quick Purification Kit RNA Quick Purification Kit, Yishan Biological Sciences, RN001
  • AL0065004, AL0065006, AL0065008, AL0065009, AL0065010-AL0065020 have a significant effect on the inhibition of PCSK9 gene expression in cynomolgus monkey hepatocytes, and the inhibitory effects all reach more than 70%; among them, AL0065012 , AL0065017, AL0065018, AL0065019, and AL0065020 have an inhibitory effect of more than 90%.
  • C57BL/6 mice SPF grade male C57BL/6 mice aged 6-8 weeks (Speyford (Beijing) Biotechnology Co., Ltd.) were used in the experiment. Serum samples before administration were obtained on the 0th day of administration, and were randomly divided into groups according to LDL-c levels. C57Bl/6 mice were subcutaneously given 9 mg/kg of PCSK9 RNAi agent and normal saline (NC, negative control).
  • the NC/PC/AL007002/AL007010 group continued to detect until 36 days after administration
  • the blood of the mice was taken (blood was taken from the eyeball, and sent for inspection within 1 hour after the blood was taken) ), check blood TCHO (total cholesterol), TG (triglycerides), HDL-C (high-density lipoprotein cholesterol), LDL-C (low-density lipoprotein cholesterol) levels.
  • TCHO total cholesterol
  • TG total cholesterol
  • TG total cholesterol
  • HDL-C high-density lipoprotein cholesterol
  • LDL-C low-density lipoprotein cholesterol
  • the AL0067002, AL0067006, AL0067007, and AL0067010 drug intervention groups can significantly reduce the LDL-C level in the blood of C57 mice on the 8th day after drug intervention.
  • the AL0067010 group was the most significant and lasted for the longest time; it can be seen that the AL0067010 drug intervention group can significantly reduce the LDL-C level in the blood of C57 mice, and the LDL-C level reached the lowest value on Day 8, and then gradually rose slowly .
  • the blood LDL-C level of mice treated with AL0067010 was significantly different from that of the solvent control group (Day8, p ⁇ 0.001; Day15, p ⁇ 0.001; Day22, p ⁇ 0.001; Day29, p ⁇ 0.001; Day 36, p ⁇ 0.001; Day 43, p ⁇ 0.01).
  • the blood LDL-C level of mice treated with AL0067010 showed a good lipid-lowering effect in the first three weeks compared with the vehicle control group (NC group), which decreased by 51.43%, 38.71% and 40.63% respectively .
  • mice SPF grade male C57BL/6 mice aged 6-8 weeks (Speyford (Beijing) Biotechnology Co., Ltd.) were used in the experiment. Serum samples before administration were obtained on the 0th day of administration, and were randomly divided into groups according to LDL-c levels. C57Bl/6 mice were subcutaneously given 9 mg/kg of PCSK9 RNAi agent and normal saline (NC, negative control). On the 8th day, 15th day, 22nd day, 29th day, 36th day, and 43rd day after the administration respectively, the blood of the mice was taken (the blood was taken from the eyeball, and sent for inspection within 1 hour after the blood was taken), and the TCHO (total cholesterol) and TG in the blood were checked. (triglycerides), HDL-C (high-density lipoprotein cholesterol), LDL-C (low-density lipoprotein cholesterol) levels. During the experiment, no obvious abnormalities were found in all animals in clinical observation.
  • TCHO, HDL-C , LDL-C decreased significantly (p ⁇ 0.05 or p ⁇ 0.01 or p ⁇ 0.001)
  • serum TCHO and LDL-C of animals in AL0067002 and AL0067019 groups decreased significantly (p ⁇ 0.01)
  • mice SPF grade male C57BL/6 mice aged 6-8 weeks (Speyford (Beijing) Biotechnology Co., Ltd.) were used in the experiment. Serum samples before administration were obtained on the 0th day of administration, and were randomly divided into groups according to LDL-c levels. C57Bl/6 mice were subcutaneously given 9 mg/kg of PCSK9 RNAi agent and normal saline (NC, negative control). On the 8th day, 15th day, 22nd day, 29th day, 37th day, 43rd day, and 50th day after the administration respectively, the mouse blood was taken (the blood was taken from the eyeball, and sent for inspection within 1 hour after the blood was taken), and the TCHO (total cholesterol) in the blood was checked.
  • PCSK9 RNAi agent normal saline
  • hPCSK9 mice SPF grade male, 5-6 week old hPCSK9 mice (Shanghai Southern Model Biotechnology Development Co., Ltd.) were used in the experiment. Pre-dose serum samples were obtained on day 0 of dosing, and randomized according to body weight level. hPCSK9 mice were subcutaneously administered 9 mg/kg of PCSK9 RNAi agent and normal saline (NC, negative control).
  • the blood of the mice was taken (take the blood from the eyeball, and send it for inspection within 1 hour after taking the blood), and check the level of LDL-C (low-density lipoprotein cholesterol) in the blood, ELISA (R&D) kits were used to detect the expression of PCSK9 protein.
  • LDL-C low-density lipoprotein cholesterol
  • the test uses 12-23-year-old middle-aged and elderly male cynomolgus monkeys, whose LDL-C ranges from 0.82mmoL to 2.10mmoL, and is basically in a state of dyslipidemia. Serum samples before administration were obtained 14 days, 7 days before administration, and on the day of administration (day 1), and were randomly divided into groups according to LDL-C levels.
  • Cynomolgus monkeys were given a single subcutaneous administration of 9 mg/kg of PCSK9 RNAi agent, respectively on the 8th day, 15th day, 22th day, 29th day, 34th day, 44th day, 51st day, 58th day after administration (AL0067001, AL0067010 end), Blood was collected on days 65, 72, and 79 to check the levels of TCHO (total cholesterol) and LDL-C (low-density lipoprotein cholesterol) in the blood.
  • TCHO total cholesterol
  • LDL-C low-density lipoprotein cholesterol
  • RNAi agents AL0067001, AL0067010, AL0067016, and AL0067028 can all significantly reduce LDL-C levels in cynomolgus monkeys, and AL0067001 and AL0067010 can significantly reduce LDL-C levels in cynomolgus monkeys for 51 consecutive days.
  • the inhibition efficiency of LDL-C level was maintained at about 40%. More significantly, the AL0067016 and AL0067028 groups could maintain the inhibition efficiency of LDL-C in cynomolgus monkeys above 40% throughout the experimental period (day 79).
  • RNAi agents AL0067001, AL0067010, AL0067016, and AL0067028 can significantly reduce the level of TCHO in cynomolgus monkeys, among which AL0067001 and AL0067010 can maintain the inhibition efficiency of LDL-C levels in cynomolgus monkeys at about 20% for 51 consecutive days, and even more It is remarkable that the AL0067016 and AL0067028 groups can maintain the TCHO inhibition efficiency of cynomolgus monkeys above 20% throughout the experiment period (day 79). The AL0067028 group was able to maintain the inhibition rate of TCHO level at about 40% from the 29th day to the 51st day.
  • Example 7 Based on the research basis in Example 7, the modification combination was optimized on the basis of exploring AL0067028, and the research effect of the new RNAi agent on C57 mice was evaluated.
  • C57BL/6 mice SPF grade male C57BL/6 mice aged 6-8 weeks (Speyford (Beijing) Biotechnology Co., Ltd.) were used in the experiment. Serum samples before administration were obtained on the 0th day of administration, and were randomly divided into groups according to LDL-c levels. C57BL/6 mice were subcutaneously administered 6 mg/kg of PCSK9 RNAi agent and normal saline (NC, negative control).
  • the blood of the mice was taken (the blood was taken from the eyeball, and sent for inspection within 1 hour after the blood was taken), and the LDL-C in the blood was checked ( Low-density lipoprotein cholesterol) level, and ELISA (R&D) kit to detect the expression of PCSK9 protein.
  • the rats showed signs of death or dying. All animals showed no obvious abnormality in clinical observation.
  • RNAi agent groups (AL0067036, AL0067037, AL0067038, AL0067039, AL0067040, AL0067028) had a significant effect on reducing LDL-C levels before Day37, especially the AL0067037 group and AL0067040 group
  • the significant reduction of LDL-C level in the AL0067037 group and AL0067040 group was significantly better than that in the AL0067028 group throughout the test period, and the difference was significant (P ⁇ 0.05).
  • RNAi agent groups (AL0067036, AL0067037, AL0067038, AL0067039, AL0067040, AL0067028) were significantly higher throughout the test period (Day 51) compared with before self-administration (Day 0). Significantly reduced; especially obvious, a single administration of AL0067037 group can reduce PCSK9 protein by more than 85% for four consecutive weeks (Day 30), and reduce PCSK9 protein by more than 90% on the 16th day after administration; The effect of reducing PCSK9 protein and the duration of reducing PCSK9 protein were significantly better than those of the AL0067028 group.

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Abstract

L'invention concerne un oligonucléotide de ciblage pour le traitement de maladies associées à PCSK9. L'oligonucléotide de ciblage comprend un brin antisens, le brin antisens ayant une séquence telle que représentée dans l'une quelconque des SEQ. ID NO : 53 à 106 ou un fragment de celle-ci, ou une séquence de modification de la séquence ou du fragment de celle-ci. Le présent oligonucléotide de ciblage inhibe de manière significative le niveau d'expression du gène PCSK9, réduit le niveau de cholestérol à lipoprotéines de basse densité (LDL-C) dans le sang, et présente un bon effet de réduction des lipides.
PCT/CN2022/123541 2021-09-30 2022-09-30 Oligonucléotide de ciblage pour le traitement de maladies associées à pcsk9 Ceased WO2023051822A1 (fr)

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