WO2023051822A1 - 用于治疗与pcsk9相关疾病的靶向寡核苷酸 - Google Patents

用于治疗与pcsk9相关疾病的靶向寡核苷酸 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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PCT/CN2022/123541
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French (fr)
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田宝磊
高杰
赵君竺
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北京安龙生物医药有限公司
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Priority to CN202280066081.8A priority Critical patent/CN118076736A/zh
Publication of WO2023051822A1 publication Critical patent/WO2023051822A1/zh

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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
    • 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
    • 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
    • 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

  • 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

用于治疗与PCSK9相关疾病的靶向寡核苷酸，所述靶向寡核苷酸包含反义链，所述反义链具有如SEQ ID NO:53-106中任一所示的序列或其片段、或所述序列或其片段的修饰序列。所述靶向寡核苷酸显著抑制中PCSK9基因表达水平，降低血液中低密度脂蛋白胆固醇(LDL-C)水平，表现出良好的降脂效果。

Description

用于治疗与PCSK9相关疾病的靶向寡核苷酸 技术领域
本公开涉及一种寡核苷酸，特别是一种用于治疗与PCSK9相关疾病的靶向寡核苷酸。
背景技术
血脂是指血中所含脂质的总称，与临床密切相关的血脂主要包括甘油三酯和胆固醇。血脂异常通常指血清中胆固醇和(或)甘油三酯(TG)水平升高，俗称高脂血症。
高胆固醇血症是指血液中总胆固醇(TCHO)和/或低密度脂蛋白胆固醇(LDL-C)或非高密度脂蛋白胆固醇(non-HDL-C)升高，也被称为血脂异常，可分为家族性胆固醇血症和非家族性胆固醇血症，其最重要的临床特征之一是患者血液低密度脂蛋白胆固醇(LDL-C)水平增高，高胆固醇血症是心血管疾病发生的一个重要因素(Guo,Yanan,et al.Archives of Biochemistry and Biophysics(2020):108717)。一些心脑血管疾病，例如脑血管疾病、冠心病和外周动脉疾病等，通常没有明显的易察觉症状，直至出现严重的动脉粥样硬化。当前，他汀类药物是临床上降血脂的一线药物，该类药物可以通过降低LDL-C来减少心血管疾病风险，但很多患者对以他汀类药物不耐受，或服用最大剂量他汀类药物后，仍无法使LDL-C水平降低至合适水平(Toutouzas et al.Expert Opin Pharmacother(2010)11:1659-72)，因此，有必要研发新型药物来满足患者的需求，具有重要的临床应用价值。
人PCSK9基因长约22kb，包含12个外显子，可编码长692个氨基酸长度的糖蛋白(Benjannet,Suzanne,et al.Journal of Biological Chemistry 279.47(2004):48865-48875)。肝脏是合成和分泌PCSK9的主要部位，人血浆中PCSK9的正常水平从30ng/mL到4μg/mL不等，在胆固醇代谢中发挥重要作用，其主要通过影响肝细胞表面LDL受体的表达水平来控制血浆LDL水平。PCSK9基因功能获得性突变(回复突变)会导致肝细胞表面LDL受体水平降低，血液中LDL-C水平升高；相反，其基因功能缺失性突变造则导致肝细胞表面LDL受体水平升高，血液中LDL-C水平降低。研究表明，在最大剂量他汀类药物无法有效控制LDL-C水平的情况下，通过降低PCSK9水平，可有效降低LDL-C水平。
细胞内的PCSK9可结合并引导新生成的LDL受体从反式高尔基体到溶酶体内进行降解；细胞外循环的PCSK9可与肝脏细胞表面LDL受体的结合，介导其进入肝细胞溶酶体内降解，从而使肝细胞表面LDL受体减少，导致肝脏结合和清除LDL-C的能力降低，最终使血液中LDL-C水平升高。因此，可通过降低PCSK9表达水平来治疗高胆固醇血症(Norata et al.Annu Rev Pharmacol Toxicol(2014)54:273-93)以及预防与高LDL-C相关的心血管疾病。
1998年，美国两位科学家Andrew Fire和Craig Mello发现的一种生物学机制，小RNA分子可以介导特定的mRNA的降解(Fire,Andrew,et al.Nature 391.6669(1998):806-811)，当RNA分子以双链形式出现在细胞中时，这种机制就会被诱发激活，即出现RNA干扰现象，这一发现预示着一个新研究领域的开始，这两位科学家也因此获得了2006年的诺贝尔生理学和医学奖。当双链RNA与蛋白质复合物Dicer结合后，Dicer将dsRNA切割成片段，而后另一种蛋白质复合物RISC结合这些片段，siRNA双链中的一条链被去除，但另一条链仍与RISC复合物结合，RISC通过单链RNA的引导识别并降解靶基因的mRNA，使特定蛋白的表达受到抑制，进而特异性导致基因沉默。
RNA干扰为基因技术的应用开辟了一个新领域。双链RNA分子已被人工设计用于沉默人类、动物或植物中的特定基因。这种人工设计合成的用于基因沉默的双链RNA分子(siRNA)被导入细胞并激活RNA干扰机制以降解对应的mRNA，目前，这种方法是生物学和生物医学的重要研究工具。此外，目前人们已开发了众多的siRNA药物，用以治疗病毒感染、心血管疾病、癌症、内分泌失调和其他多种疾病，多数处于研发阶段或已获批上市的siRNA疗法，显示出很好的治疗效果。从2018年第一款siRNA药物上市至今，已有至少4款siRNA在欧盟或美国获批上市。因此，利用RNA干扰技术抑制特定靶基因表达已经成为一种有效的疾病治疗途径。
目前已有报道靶向PCSK9的抑制剂，包括已上市的抗体类和小核酸类药物，用于或拟用于治疗高胆固醇血症及相关疾病，但需要开发针对该靶点的其他抑制剂，以期有更好的疗效或更好的安全性。
发明内容
为了解决现有技术中存在的问题，本公开的目的在于提供疗效好、安全性高、药效持久的靶向PCSK9的抑制剂。
在一方面，本公开提供了用于降低PCSK9的表达的寡核苷酸或其药学上可接受的盐，所寡核苷酸包含反义链，所述反义链具有如SEQ ID NO:53-106中任一所示的序列或其片段、或所述序列或其片段的修饰序列。
在本公开的一些实施方案中，所述寡核苷酸进一步包含具有如SEQ ID NO:1-52中任一所示的序列或其片段、或所述序列或其片段的修饰序列。
在本公开的一些实施方案中，所述反义链由如SEQ ID NO:53-106中任一所示的序列或其片段、或所述序列或其片段的修饰序列组成。
在本公开的一些实施方案中，所述反义链的修饰序列包含具有如SEQ ID NO:138-183、185-186中任一所示的序列或其片段。
在本公开的一些实施方案中，所述正义链的修饰序列包含具有如SEQ ID NO:107-137、135、184中任一所示的序列或其片段。
在本公开的一些实施方案中，所述正义链和所述反义链分别为19/21配对、21/21配对、21/23配对或23/23配对的双链体结构。
在另一方面，本公开提供了一种组合物，所述组合物包含所述寡核苷酸或其药学上可接受的盐，以及任选地药学上可接受的载体。
在另一方面，本公开提供了所述的寡核苷酸或其药学上可接受的盐或组合物在制备用于治疗或/和预防脂质失调方面的疾病的药物中的用途。
在本公开的一些实施方案中，所述脂质失调方面的疾病为高脂血症、动脉粥样硬化和/或其一种或多种症状或并发症的一种或多种症状的用途。
实验证明，本公开的候选化合物对人类肝癌细胞系Huh7、HepG2和Hle中PCSK9基因表达水平的抑制有显著效果；其中一些候选化合物对Huh7和Hle细胞株的PCSK9基因表达水平抑制效果均达到了90％以上，一些化合物对食蟹猴肝细胞PCSK9基因表达的抑制达到70％、80％、甚至90％以上，表现出良好的抑制效果。其中一些化合物给药小鼠约一月后(Day29)PCSK9蛋白降低还能维持在60％左右；一些化合物可以在小鼠体内连续四周(Day 30)使PCSK9蛋白降低85％以上，药效持久。此外，其中一些化合物可显著降低C57小鼠血液中低密度脂蛋白胆固醇(LDL-C)水平，一些化合物能够连续51天使食蟹猴LDL-C水平抑制效率维持在40％左右，一些化合物在第79天都能够使食蟹猴LDL-C抑制效率维持在40％以上，表现出良好的降脂效果。本公开提供了靶向PCSK9 mRNA的寡核苷酸，其有效降低细胞、尤其是肝脏细胞(例如，肝细胞)中的PCSK9表达，用于治疗高胆固醇血症、动脉粥样硬化和/或其一种或多种症状或并发症。因此，在相关方面，本公开提供了治疗高胆固醇血症、动脉粥样硬化和/或其一种或多种症状或并发症的方法，其涉及选择性降低肝脏中的PCSK9基因表达。在某些实施方案中，本文提供的PCSK9靶向寡核苷酸被设计用于递送至靶标组织的选择细胞(例如，肝脏肝细胞)以治疗受试者中的高胆固醇血症、动脉粥样硬化和/或其一种或多种症状或并发症。
附图说明
图1为肝细胞系第一轮体外筛选PCSK9-siRNA药效结果图。
图2为肝细胞系第二轮体外筛选PCSK9-siRNA药效结果图。
图3为猴原代肝细胞第一轮体外筛选修饰后PCSK9-siRNA药效结果图。
图4为猴原代肝细胞第二轮体外筛选修饰后PCSK9-siRNA药效结果图。
图5示出了第一轮施用PCSK9 RNAi剂后小鼠LDL-C水平的变化。
图6示出了第二轮施用PCSK9 RNAi剂后小鼠LDL-C水平的变化。
图7示出了第三轮施用PCSK9 RNAi剂后小鼠LDL-C水平的变化。
图8示出了施用PCSK9 RNAi剂后hPCSK9小鼠LDL-C水平的变化。
图9示出了施用PCSK9 RNAi剂后hPCSK9小鼠PCSK9蛋白水平的变化。
图10示出了施用PCSK9 RNAi剂后食蟹猴LDL-C水平的变化。
图11示出了施用PCSK9 RNAi剂后食蟹猴TCHO水平的变化。
图12示出了第四轮施用PCSK9 RNAi剂后小鼠LDL-C水平的变化。
图13示出了第四轮施用PCSK9 RNAi剂后小鼠PCSK9蛋白水平的变化。
具体实施方式
在本公开中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本公开，下面提供相关术语的定义和解释。
约：如本文所用，如应用于一种或多种目标值的术语“约”或“近似”是指与所述参考值类似的值。在某些实施方案中，除非另有说明或另外从上下文显而易见，否则术语“近似”或“约”是指落入所述参考值在任一方向(大于或小于)的20％、19％、18％、17％、16％、15％、14％、13％、12％、11％、10％、9％、8％、7％、6％、5％、4％、3％、2％、1％或更少内的值的范围(除非这种数字将超过可能值的100％)。
动脉粥样硬化：如本文所用，术语“动脉粥样硬化”是指涉及通常由于斑块(由脂肪、胆固醇、钙以及其他物质制成)的堆积所导致的动脉(例如冠状、颈动脉、外周和/或肾动脉)狭窄的疾病。在一些实施方案中，冠状动脉的狭窄可产生症状，例如心绞痛、呼吸短促、出汗、恶心、头晕、呼吸短促、心律不齐和/或心悸。在一些实施方案中，颈动脉的狭窄可导致中风(即，由于血液和氧气流向大脑的不足导致脑细胞死亡)和/或可产生症状，例如虚弱感、困惑、说话困难、头晕、难以走路或站直、视力模糊、脸、手臂和腿麻木、严重的头痛和/或意识丧失。在一些实施方案中，***动脉的狭窄可导致手臂或腿内的麻木或疼痛。在一些实施方案中，肾动脉的狭窄(导致肾血流量减少)可导致慢性肾脏疾病。动脉粥样硬化的并发症可包括冠状动脉疾病、中风、外周动脉疾病和肾脏问题(例如慢性肾脏疾病)。
互补：如本文所用，术语“互补”是指核苷酸(例如，在相对的核酸上或在单一核酸链的相对区域上的两个核苷酸上)之间允许核苷酸彼此形成碱基对的结构关系。例如，一个核酸的与相对核酸的嘧啶核苷酸互补的嘌呤核苷酸可以通过彼此形成氢键而碱基配对在一起。在一些实施方案中，互补的核苷酸可以以沃森-克里克(Watson-Crick)方式或 以允许形成稳定双链体的任何其他方式碱基配对。在一些实施方案中，两个核酸可以具有与彼此互补以便形成互补区域的核苷酸序列，如本文所述。
链：如本文所用，术语“链”是指通过核苷酸间键(例如，磷酸二酯键、硫代磷酸酯键)连接在一起的核苷酸的单一连续序列。在一些实施方案中，链具有两个游离末端，例如5'-末端和3'-末端。
脱氧核糖核苷酸：如本文所用，术语“脱氧核糖核苷酸”是指与核糖核苷酸相比在其戊糖的2'位置处具有氢的核苷酸。修饰的脱氧核糖核苷酸是除了在2'位置以外具有一个或多个原子的修饰或取代(包括糖、磷酸酯基团或碱基中的修饰或取代或糖、磷酸酯基团或碱基的修饰或取代)的脱氧核糖核苷酸。
双链寡核苷酸：如本文所用，术语“双链寡核苷酸”是指基本上呈双链体形式的寡核苷酸。在一些实施方案中，在共价分离的核酸链的核苷酸的反向平行序列之间形成双链寡核苷酸的一个或多个双链体区域的互补碱基配对。在一些实施方案中，在共价连接的核酸链的核苷酸的反向平行序列之间形成双链寡核苷酸的一个或多个双链体区域的互补碱基配对。在一些实施方案中，从单一核酸链形成双链寡核苷酸的一个或多个双链体区域的互补碱基配对，所述单一核酸链被折叠(例如，经由发夹)，以提供在一起碱基配对的核苷酸的互补的反向平行序列。在一些实施方案中，双链寡核苷酸包含彼此完全双链体化的两条共价分离的核酸链。然而，在一些实施方案中，双链寡核苷酸包含部分双链体化、例如在一个或两个末端具有突出端的两条共价分离的核酸链。在一些实施方案中，双链寡核苷酸包含核苷酸的反向平行序列，其部分互补，且因此，可以具有一个或多个错配，所述错配可以包括内部错配或末端错配。
寡核苷酸：如本文所用，术语“寡核苷酸”是指短核酸，例如长度小于100个核苷酸的短核酸。寡核苷酸可以包含核糖核苷酸、脱氧核糖核苷酸和/或修饰的核苷酸，包括例如修饰的核糖核苷酸。寡核苷酸可以是单链或双链的。寡核苷酸可以具有或可以不具有双链体区域。作为一组非限制性实例，寡核苷酸可以是，但不限于，小干扰RNA(siRNA)、微RNA(miRNA)、短发夹RNA(shRNA)、Dicer底物干扰RNA(dsiRNA)、反义寡核苷酸、短siRNA或单链siRNA。在一些实施方案中，双链寡核苷酸是RNAi寡核苷酸。
PCSK9：如本文所用，是指“前蛋白转化酶枯草杆菌蛋白酶/kexin-9基因或蛋白，也称为NARC-1、神FH3、HCHOLA3、NARC-1或NARCI。术语PCSK9包括人PCSK9、小鼠PCSK9、大鼠PCSK9，PCSK9 mRNA序列的实例使用可在GenBank易于获得。5
在一方面，本公开提供了一种用于降低PCSK9的表达的寡核苷酸或其药学上可接受的盐，所述寡核苷酸包含反义链，所述反义链具有如SEQ ID NO:53-106中任一所示的序列或其片段、或所述序列或其片段的修饰序列。
在本公开的一些实施方案中，所述寡核苷酸或其药学上可接受的盐优选以羧酸盐、钠盐、三乙胺盐或其它可药用盐的形式制备或合成。
在本公开的一些实施方案中，所述寡核苷酸或其药学上可接受的盐更优选为其钠盐或三乙胺盐。
在本公开的一些实施方案中，所述寡核苷酸进一步包含具有如SEQ ID NO:1-52中任一所示的序列或其片段、或所述序列或其片段的修饰序列。
在本公开的一些实施方案中，所述反义链由如SEQ ID NO:53-106中任一所示的序列或其片段、或所述序列或其片段的修饰序列组成。
在本公开的一些实施方案中，所述正义链由如SEQ ID NO:1-52中任一所示的序列或其片段、或所述序列或其片段的修饰序列组成。
在本公开的一些优选的实施方案中，所述寡核苷酸具有正义链和反义链，其中，所述寡核苷酸包含：(1)SEQ ID NO:51所示的正义链，和SEQ ID NO:103所示的反义链；或(2)SEQ ID NO:52所示的正义链，和SEQ ID NO:105所示的反义链。SEQ ID NO:51所示的正义链和SEQ ID NO:52所示的正义链具有共同的基序UUUGCUUUUGUAACUUGAA(SEQ ID NO:51)。SEQ ID NO:103所示的反义链和SEQ ID NO:105所示的反义链具有共同的基序UUCAAGUUACAAAAGCAAA(SEQ ID NO:187)。
在本公开的一些实施方案中，所述反义链的修饰序列包含具有如SEQ ID NO:138-183、185-186中任一所示的序列或其片段。
在本公开的一些实施方案中，所述正义链的修饰序列包含具有如SEQ ID NO:107-137、135、184中任一所示的序列或其片段。
在本公开的一些实施方案中，所述反义链由如SEQ ID NO:138-183、185-186中任一所示的序列或其片段、或所述序列或其片段的修饰序列组成。
在本公开的一些实施方案中，所述正义链由如SEQ ID NO:107-137、135、184中任一所示的序列或其片段、或所述序列或其片段的修饰序列组成。
在本公开的一些实施方案中，所述反义链长度为19至23个核苷酸。
在本公开的一些实施方案中，所述正义链长度为19至23个核苷酸。
在本公开的一些实施方案中，所述寡核苷酸包含长度为一个或多个核苷酸的3'-突出端序列，其中所述3'-突出端序列存在于反义链和/或正义链上。
在本公开的一些实施方案中，所述反义链带有一个突出端。
在本公开的一些实施方案中，所述正义链带有一个突出端。
在本公开的一些实施方案中，所述寡核苷酸包含长度为两个核苷酸的3'-突出端序列。
在本公开的一些实施方案中，所述3'-突出端序列存在于所述正义链上；优选地，所述突出端序列选自：GG、 GA、GC、UC、UG、UU、UA、CA、CC、CG、CU、AA、AG、AU、AC。
在本公开的一些实施方案中，所述3'-突出端序列存在于所述反义链上；优选地，所述突出端序列选自：UU、UC、UA、UG、GA、GG、GU、GC、TT、AG、AU、AA、AC、CA、CC、U；更优选地，所述突出端序列为UU。
在本公开的一些实施方案中，所述寡核苷酸包含长度各自在19至23个核苷酸的范围内的反义链和正义链。
在本公开的一些实施方案中，所述正义链与所述反义链形成双链体区域。
在本公开的一些实施方案中，所述正义链和所述反义链分别为19/21配对、21/21配对、21/23配对或23/23配对的双链体结构。
在本公开的一些实施方案中，所述寡核苷酸包含长度为两个核苷酸的3'-突出端序列，其中所述3'-突出端序列存在于所述反义链上，且其中所述正义链长度为19个核苷酸且所述反义链长度为21个核苷酸，使得所述正义链和反义链形成长度为19个核苷酸的双链体。
所述寡核苷酸包含长度为两个核苷酸的3'-突出端序列，其中所述3'-突出端序列存在于所述反义链和正义链上，且其中所述正义链长度为21个核苷酸且所述反义链长度为21个核苷酸，使得所述正义链和反义链形成长度为19个核苷酸的双链体。
在本公开的一些实施方案中，所述寡核苷酸包含长度为两个核苷酸的3'-突出端序列，其中所述3'-突出端序列存在于所述反义链上，且其中所述正义链长度为21个核苷酸且所述反义链长度为23个核苷酸，使得所述正义链和反义链形成长度为21个核苷酸的双链体。
在本公开的一些实施方案中，所述寡核苷酸选自反义链SEQ ID NO:55-57、61、66、70-71、80、82、84、93-106中任一所述的未修饰寡核苷酸，以及反义链SEQ ID NO:138-183中任一所述的修饰寡核苷酸。
在本公开的一些实施方案中，所述寡核苷酸选自反义链SEQ ID NO:80、84、93-95、99-106中任一所述的未修饰寡核苷酸，以及反义链SEQ ID NO:145、147-150、154-183、185-186中任一所述的修饰寡核苷酸。
在本公开的一些实施方案中，所述寡核苷酸选自正义链SEQ ID NO:3-5、9、14、18、19、28、30、32、41-52中任一所述的未修饰寡核苷酸，以及正义链SEQ ID NO:107-137、135、184中任一所述的修饰寡核苷酸。
在本公开的一些实施方案中，所述寡核苷酸包含长度为两个核苷酸的3'-突出端序列，其中所述3'-突出端序列存在于所述反义链和正义链上，且其中所述正义链长度为23个核苷酸且所述反义链长度为23个核苷酸，使得所述正义链和反义链形成长度为21个核苷酸的双链体。
在本公开的一些实施方案中，所述寡核苷酸包含至少一个修饰的核苷酸。
在本公开的一些实施方案中，所述修饰是选自以下的修饰：2'-甲氧基(m)、2'-脱氧-2'-氟(f)和硫代磷酸酯(s)。
在本公开的一些实施方案中，所述修饰的核苷酸包含2'-修饰。
在本公开的一些实施方案中，所述2'-修饰是选自以下的修饰：2'-氨基乙基、2'-氟、2'-O-甲基、2'-O-甲氧基乙基和2'-脱氧-2'-氟-β-d-***核糖核酸。
在本公开的一些实施方案中，所述寡核苷酸的所有核苷酸都是修饰的。
在本公开的一些实施方案中，所述寡核苷酸包含至少一个修饰的核苷酸间键。
在本公开的一些实施方案中，所述至少一个修饰的核苷酸间键是硫代磷酸酯键。
在本公开的一些实施方案中，所述反义链的5'-核苷酸的糖的4'-碳包含磷酸酯类似物。
在本公开的一些实施方案中，所述磷酸酯类似物是氧基甲基膦酸酯、乙烯基膦酸酯或丙二酰基膦酸酯。
在本公开的一些实施方案中，所述寡核苷酸的至少一个核苷酸缀合至一个或多个靶向配体。
在本公开的一些实施方案中，各靶向配体包含碳水化合物、氨基糖、胆固醇、多肽或脂质。
在本公开的一些实施方案中，各靶向配体包含N-乙酰基半乳糖胺(GalNAc)部分。
在本公开的一些实施方案中，所述GalNac部分是单价GalNAc部分、二价GalNAc部分、三价GalNAc部分或四价GalNAc部分。
在本公开的一些实施方案中，所述寡核苷酸选自AL0061001-AL0061054、AL0065001-AL0065051、AL0067001-AL0067040中任一所述的寡核苷酸。
在本公开的一些优选的实施方案中，所述寡核苷酸选自AL0067010、AL0067016、AL0067028、AL0067029、AL0067030、L0067031、L0067032、L0067033、L0067034、L0067035、AL0067036、AL0067037、AL0067038、AL0067039、AL0067040中任一所述的寡核苷酸。
在本公开的一些实施方案中，所述正义链包含SEQ ID NO:51所示的基序序列；所述反义链包含SEQ ID NO:187所示的基序序列。
在本公开的一些优选的实施方案中，所述寡核苷酸包含选自下述正义链和反义链组合中的任一：
(1)SEQ ID NO:52所示的正义链，和SEQ ID NO:105所示的反义链；
(2)SEQ ID NO:135所示的正义链，和SEQ ID NO:176所示的反义链；
(3)SEQ ID NO:136所示的正义链，和SEQ ID NO:177所示的反义链；
(4)SEQ ID NO:137所示的正义链，和SEQ ID NO:178所示的反义链；
(5)SEQ ID NO:135所示的正义链，和SEQ ID NO:185所示的反义链；
(6)SEQ ID NO:135所示的正义链，和SEQ ID NO:186所示的反义链；
(7)SEQ ID NO:184所示的正义链，和SEQ ID NO:185所示的反义链；
(8)SEQ ID NO:184所示的正义链，和SEQ ID NO:186所示的反义链；和
(9)SEQ ID NO:184所示的正义链，和SEQ ID NO:176所示的反义链。
在本公开的一些优选的实施方案中，所述寡核苷酸包含选自下述正义链和反义链组合中的任一：
(1)SEQ ID NO:52所示的正义链，和SEQ ID NO:106所示的反义链；
(2)SEQ ID NO:134所示的正义链，和SEQ ID NO:179所示的反义链；
(3)SEQ ID NO:135所示的正义链，和SEQ ID NO:180所示的反义链；
(4)SEQ ID NO:135所示的正义链，和SEQ ID NO:181所示的反义链；
(5)SEQ ID NO:136所示的正义链，和SEQ ID NO:182所示的反义链；和
(6)SEQ ID NO:137所示的正义链，和SEQ ID NO:183所示的反义链。
在本公开的一些优选的实施方案中，所述寡核苷酸包含选自下述正义链和反义链组合中的任一：
(1)SEQ ID NO:51所示的正义链，和SEQ ID NO:103所示的反义链；
(2)SEQ ID NO:127所示的正义链，和SEQ ID NO:158所示的反义链；和
(3)SEQ ID NO:132所示的正义链，和SEQ ID NO:164所示的反义链.。
在另一方面，本公开提供了一种组合物，其包含所述寡核苷酸或其药学上可接受的盐，以及任选地药学上可接受的载体。
在本公开的一些实施方案中，所述组合物的剂型为口服剂、静脉注射剂、皮下注射剂或肌内注射剂。
在本公开的一些优选的实施方案中，所述组合物的剂型为皮下注射剂。
在本公开的一些实施方案中，所述组合还包含其他治疗或/和预防脂质失调方面的疾病的药物。
在本公开的一些优选的实施方案中，所述脂质失调方面的疾病为高脂血症、动脉粥样硬化和/或其一种或多种症状或并发症的一种或多种症状。
在本公开的一些优选的实施方案中，所述高脂血症是高胆固醇血症。
在本公开的一些实施方案中，所述其他治疗高脂血症的药物包括但不限于已在临床上使用的贝特类、他汀类、胆汁酸隔离剂和烟酸类。
在另一方面，本公开提供了一种所述寡核苷酸或其药学上可接受的盐或组合物在制备用于治疗或/和预防脂质失调方面的疾病的药物中的用途。
在本公开的一些实施方案中，所述脂质失调方面的疾病为高脂血症、动脉粥样硬化和/或其一种或多种症状或并发症的一种或多种症状的用途。
在本公开的一些实施方案中，所述高脂血症是高胆固醇血症。
已经开发了各种制剂以促进寡核苷酸使用。例如，可以使用使降解最小化、促进递送和/或摄取或为制剂中的寡核苷酸提供另一种有益特性的制剂将寡核苷酸递送至受试者或细胞环境。在一些实施方案中，本文提供了包含寡核苷酸(例如，单链或双链寡核苷酸)以降低PCSK9的表达的组合物。可以合适地配制此类组合物，使得当施用于受试者(至靶标细胞的直接环境中或全身性地)时，足够部分的寡核苷酸进入细胞以降低PCSK9表达。各种合适的寡核苷酸制剂中的任一种可用于递送寡核苷酸用于减少PCSK9，如本文所公开。在一些实施方案中，将寡核苷酸配制于缓冲溶液、诸如磷酸盐缓冲盐水溶液、脂质体、胶束结构和壳体中。在一些实施方案中，将裸露的寡核苷酸或其缀合物配制于水中或水溶液(例如，pH调节的水)中。在一些实施方案中，将裸露的寡核苷酸或其缀合物配制于碱性缓冲水溶液(例如，PBS)中。
具有阳离子脂质的寡核苷酸的制剂可用于促进寡核苷酸转染至细胞中。例如，可以使用阳离子脂质，诸如脂转染试剂(lipofectin)，阳离子甘油衍生物和聚阳离子分子(例如，聚赖氨酸)。
因此，在一些实施方案中，制剂包含脂质纳米颗粒。在一些实施方案中，赋形剂包含脂质体、脂质、脂质复合物、微球、微粒、纳米球或纳米颗粒，或者可以以其他方式配制用于向有此需要的受试者的细胞、组织、器官或身体施用。
在一些实施方案中，如本文所公开的制剂包含赋形剂。在一些实施方案中，赋形剂向组合物赋予活性成分的提高的稳定性、提高的吸收、提高的溶解度和/或治疗性增强。在一些实施方案中，赋形剂是缓冲剂(例如，柠檬酸钠、磷酸钠、tris碱或氢氧化钠)或媒介物(例如，缓冲溶液、矿脂、二甲基亚砜或矿物油)。在一些实施方案中，将寡核苷酸冻干用于延长其保质期，且然后在使用(例如，施用于受试者)之前制成溶液。因此，包含本文所述的寡核苷酸中的任一种的组合物中的赋形剂可以是冻干保护剂(例如，甘露醇、乳糖、聚乙二醇或聚乙烯吡咯烷酮)或塌陷温度改变剂(例如，葡聚糖、ficoll或明胶)。
在一些实施方案中，将药物组合物配制为与其意欲的施用途径相容。施用途径的实例包括肠胃外，例如静脉内、皮内、皮下、口服(例如，吸入)、经皮(局部)、经粘膜和直肠施用。通常，施用的途径是静脉内或皮下的。
适用于可注射用途的药物组合物包括无菌水溶液(在水溶性的情况下)或分散体以及用于临时制备无菌可注射溶液或分散体的无菌粉末。对于静脉内或皮下施用，合适的载体包括生理盐水、抑菌水、Cremophor EL.TM.(BASF,Parsippany,N.J.)或磷酸盐缓冲盐水(PBS)。所述载体可以是溶剂或分散介质，其含有例如水、乙醇、多元醇(例如，甘油、丙二醇和液体聚乙二醇等)及其合适的混合物。在许多情况下，优选在组合物中包括等渗剂，例如糖，多元醇诸如甘露醇、山梨醇和氯化钠。无菌可注射溶液可以通过如下来制备：将所需量的寡核苷酸与所需的以上列举的成分中的一种或组合并入选择的溶剂中，随后进行过滤灭菌。
在一些实施方案中，组合物可以含有至少约0.1％或更多的治疗剂(例如，用于降低PCSK9表达的寡核苷酸)， 尽管一种或多种活性成分的百分比可以在总组合物的重量或体积的约1％和约80％或更多之间。制备此类药物制剂的领域中的技术人员将考虑因素诸如溶解度、生物利用度、生物半衰期、施用的途径、产品保质期以及其他药理学考量，并且因此各种剂量和治疗方案可以是期望的。
即使许多实施方案涉及本文公开的任何寡核苷酸的肝脏靶向递送，也考虑靶向其他组织。
在一些实施方案中，如本文所述的寡核苷酸的施用导致细胞中的PCSK9表达的水平的降低。在一些实施方案中，PCSK9表达的水平的降低可以是与PCSK9的适当对照水平相比降低至1％或更低、5％或更低、10％或更低、15％或更低、20％或更低、25％或更低、30％或更低、35％或更低、40％或更低、45％或更低、50％或更低、55％或更低、60％或更低、70％或更低、80％或更低或90％或更低。适当的对照水平可以是未与如本文所述的寡核苷酸接触的细胞或细胞群体中的PCSK9表达的水平。在一些实施方案中，在有限的时间段之后评价根据本文公开的方法将寡核苷酸递送至细胞的效果。例如，可以在将所述寡核苷酸引入细胞后至少8小时、12小时、18小时、24小时；或至少一、二、三、四、五、六、七或十四天分析细胞中的PCSK9的水平。
在一些实施方案中，寡核苷酸以转基因的形式递送，所述转基因被工程改造为在细胞中表达本文公开的寡核苷酸(例如，呈shRNA形式)。在一些实施方案中，使用经工程改造以表达本文公开的任何寡核苷酸的转基因递送寡核苷酸。可以使用病毒载体(例如，腺病毒、逆转录病毒、牛痘病毒、痘病毒、腺相关病毒或单纯疱疹病毒)或非病毒载体(例如，质粒或合成的mRNA)递送转基因。在一些实施方案中，转基因可以直接注射给受试者。
本公开的方面涉及用于降低PCSK9表达用于治疗受试者中的高胆固醇血症、动脉粥样硬化和/或其一种或多种症状或并发症的方法。在一些实施方案中，所述方法可以包括向有此需要的受试者施用有效量的本文公开的寡核苷酸中的任一种。在一些实施方案中，此类治疗可用于例如降低或预防高胆固醇血症(高水平的低密度脂蛋白(LDL)-胆固醇)、动脉粥样硬化、冠心病(例如，冠状动脉疾病)、心绞痛、呼吸短促、出汗、恶心、头晕、呼吸短促、心律不齐、心悸、中风(即由于血液和氧气流向大脑的不足导致脑细胞死亡)、虚弱感、困惑、说话困难、头晕、难以走路或站直、视力模糊、脸、手臂和腿麻木、严重的头痛、意识丧失、外周动脉疾病和/或肾脏问题(例如慢性肾脏疾病)。在一些实施方案中，此类治疗可用于例如治疗或预防与高胆固醇血症、动脉粥样硬化和/或其一种或多种症状或并发症相关的一种或多种症状。
因此，在一些实施方案中，本公开提供了治疗处于高胆固醇血症、动脉粥样硬化和/或其一种或多种症状或并发症(包括冠心病(例如，冠状动脉疾病)、心绞痛、呼吸短促、出汗、恶心、头晕、呼吸短促、心律不齐、心悸、中风(即由于血液和氧气流向大脑的不足导致脑细胞死亡)、虚弱感、困惑、说话困难、头晕、难以走路或站直、视力模糊、脸、手臂和腿麻木、严重的头痛、意识丧失、外周动脉疾病和/或肾脏问题(例如慢性肾脏疾病))的风险中(或易患高胆固醇血症、动脉粥样硬化和/或其一种或多种症状或并发症)的受试者的方法。
在某些方面，本公开提供了用于通过向受试者施用治疗剂(例如，寡核苷酸或编码其的载体或转基因)来预防受试者中的如本文所述的疾病、病症、症状或病况的方法。在一些实施方案中，待治疗的受试者是将从例如肝脏中的PCSK9蛋白的量的减少在治疗上受益的受试者。
本文所述的方法通常涉及向受试者施用有效量(即能够产生期望的治疗结果的量)的寡核苷酸。治疗上可接受的量可以是能够治疗疾病或病症的量。任一受试者的适当剂量将取决于某些因素，包括受试者的体型、体表面积、年龄、待施用的特定组合物、组合物中的一种或多种活性成分、施用的时间和途径、总体健康和同时施用的其他药物。
在一些实施方案中，经肠(例如，经口，通过胃喂食管，通过十二指肠喂食管，经由胃造口术或经直肠)，肠胃外(例如，皮下注射，静脉内注射或输注，动脉内注射或输注，肌内注射)，局部(例如，表皮，吸入，经由滴眼或通过粘膜)，或通过直接注射入靶标器官(例如，受试者的肝脏)，向受试者施用本文公开的组合物中的任一种。通常，本文公开的寡核苷酸静脉内或皮下施用。
在一些实施方案中，寡核苷酸以0.1mg/kg至25mg/kg(例如，1mg/kg至5mg/kg)的范围内的剂量施用。在一些实施方案中，寡核苷酸以0.1mg/kg至5mg/kg的范围内或0.5mg/kg至5mg/kg的范围内剂量施用。
作为一组非限制性实例，将通常每年一次、每年两次、每季度(每三个月一次)、每两个月(每两个月一次)、每个月或每周施用本公开的寡核苷酸。
在一些实施方案中，待治疗的受试者是人(例如人患者)或非人灵长类动物或其他哺乳动物受试者。其他示例性受试者包括家养动物诸如狗和猫；牲畜诸如马、牛、猪、绵羊、山羊和鸡；和动物诸如小鼠、大鼠、豚鼠和仓鼠。
为了达到清楚和简洁描述的目的，本文中作为相同的或分开的一些实施方案的一部分来描述特征，然而，将要理解的是，本公开的范围可包括具有所描述的所有或一些特征的组合的一些实施方案。
下面，参考具体实施例更详细地描述本公开，然而，实施例仅用于说明目的，对于本公开不具有限制作用。
实施例
实施例1.PCSK9-siRNA体外肝脏细胞系活性筛选
首先，使用基于计算机的算法来生成了与人PCSK9 mRNA(NM_174936.3，表1)互补的候选寡核苷酸序列，其中某些序列也与食蟹猴的PCSK9 mRNA(XM_005543260.2，表1)互补，某些序列也与小鼠的PCSK9 mRNA(NM_153565.2，表1)互补或不多于2个错配。其中某些设计为正义链和反义链分别为19/21配对的双链siRNA，反义链带有两个与mRNA序列互补的悬垂末端，在某些情况下反义链的悬垂末端为非互补的UU；其中某些序列设计为正义链和反义链分别为21/23配对的双链siRNA，反义链带有两个与mRNA序列互补的悬垂末端；其中某些序 列设计为21/21、23/23配对的双链siRNA。其中某些互补配对序列反义链的5'端第1位(正义链3'端最后1位)碱基替换为与PCSK9 mRNA不匹配的碱基。某些序列中，个别核糖核酸替换为脱氧核糖核酸，比如T或者dA。
表1：人、食蟹猴、小鼠PCSK9 mRNA序列
物种 GenBank RefSeq#
人 NM_174936.3
食蟹猴 XM_005543260.2
小鼠 NM_153565.2
表2、表3和表4中，“G”、“C”、“A”、“U”和“T”通常分别代表以鸟嘌呤、胞嘧啶、腺嘌呤、尿嘧啶和胸腺嘧啶为碱基的核苷酸；
修饰：d代表DNA；dT代表2'-脱氧胸苷；dA代表2'-脱氧腺苷；m代表2'-甲氧基；f代表2'-脱氧-2'-氟；s代表硫代磷酸酯；L代表配体，L96为N-[tris(GalNAc-烷基)酰胺基癸酰基]-4-羟基脯氨醇Hyp-(GalNAc-烷基)3。
表2：寡核苷酸裸序列
Figure PCTCN2022123541-appb-000001
Figure PCTCN2022123541-appb-000002
表3：修饰寡核苷酸
Figure PCTCN2022123541-appb-000003
Figure PCTCN2022123541-appb-000004
Figure PCTCN2022123541-appb-000005
表4：带载体siRNA序列
Figure PCTCN2022123541-appb-000006
Figure PCTCN2022123541-appb-000007
(1)Huh7/HepG2/HLE细胞培养和转染：
使用人类肝癌细胞系Huh7(CCTCC，GDC0134)、HepG2(江苏凯基生物技术股份有限公司，KG020)和HLE(JCRB Cell Bank，JCRB0404)，置于37℃，5％CO 2的培养箱中，使用DMEM基础培养基(Hyclone，SH30022.01)添加10％FBS(aqlabteech，AQ-MV-06600)和1％青霉素-链霉素(凯基生物，KGY0023)培养，待细胞汇合度达 到90％，通过胰酶(amresco，0458-250G)进行消化，用计数仪(Nexcelom，cellometer Mini)计数，将Huh7细胞稀释成2*10^5/ml，混匀细胞将细胞悬液加到12孔板中，待次日贴壁进行转染。使用Lipofectamine TM 3000 Transfection Reagent(thermofisher，L3000150)进行转染，体系①用50μl Opti-MEM(thermofisher，1105821)稀释50nM siRNA，体系②用50μl Opti-MEM稀释3μl Lip3000，分别静置5min后，将体系①与②混合再静置15min，滴加到12孔板，转染4h后更换DMEM/F12完全培养基，将12孔板置于培养箱中孵育48h。
(2)使用RNA-Quick Purification Kit(RNA快速提取试剂盒，奕杉生物科，RN001)提取总RNA：
从培养箱中取出12孔板，吸干培养基，用适量的PBS洗一次，每孔加入500μl的裂解缓冲液，将上清转移到新的1.5ml离心管中。向裂解的细胞中加入500μl的无水乙醇充分混匀(若出现沉淀，这是正常现象，继续进行操作即可)。将离心管颠倒几次，或用移液器用力吹吸10次使产生的沉淀分散开，然后将液体加入离心柱，将离心管对称放入离心机(eppendorf，5430)，4000×g离心1min。取出离心管向柱中加入500μl的洗涤缓冲液，12000×g离心1min，离心结束后取出柱子时注意不要让收集管内的废液接触到RNA柱，以免污染，倒掉废液，将RNA柱装回收集管，空管离心一次，能完全去除可能残留的洗涤缓冲液。将柱子放到干净的无RNA酶的1.5ml离心管上，开盖晾干2分钟。在RNA柱的膜中心部位加入30μl的洗脱缓冲液，室温静置2分钟，2000×g离心1min，RNA洗脱下来后，置于冰上。测定洗脱的RNA浓度，以便于后续实验使用。提取出来的RNA可立即用于后续实验，也可保存在-80℃备用。
(3)使用
Figure PCTCN2022123541-appb-000008
II Q RT SuperMix for qPCR(+gDNA wiper)反转录试剂盒(诺唯赞，R223-01)合成cDNA：
使用无RNA酶的离心管中配制混合液4μl 4×gDNA wiper Mix、1μg模板RNA、加无RNA酶的ddH 2O至16μl去除基因组DNA，用移液器轻轻吹打混匀，42℃2min。然后反应管中直接加入4μl 5×HiScript II qRT SuperMix II用移液器轻轻吹打混匀，置于PCR仪(Applied Biosystems，9700)50℃，15min；85℃，5sec，4℃保持。产物可立即用于qPCR反应，或在-20℃保存，并在半年内使用，长期存放需分装后在-80℃保存，cDNA应避免反复冻融。
(4)使用ChamQ SYBR qPCR Master Mix(诺唯赞，Q311-02)qPCR定量：
配置混合液10μl 2×ChamQ SYBR qPCR Master Mix、0.5μl Forword primer(睿博科兴)、0.5μl Reverse primer(睿博科兴)、1μl Template cDNA、8μl ddH 2O至20μl体系，每个样品3个重复，将96孔板放到qPCR仪(ROCGENE，Archimed)，执行程序：预变形，95℃，30sec；扩增，95℃，10sec，60℃，30sec，40个循环；溶解曲线，95℃，15sec，60℃，60sec，95℃，15sec。
(5)使用GraphPad Prism 8进行数据统计分析：
将数据导出EXCEL格式，用CT PCSK9-CT GAPDH，将对照组进行归一化处理，为了计算相对沉默效率的倍数变化，数据使用△△CT法进行分析，将得到的三个平行重复数据导入GraphPad Prism 8，应用column模型做柱状分析，结果见图1和图2。
由图1可见，在加药量为50nM时，AL0061001-AL0061040对Huh7细胞株的PCSK9基因表达水平的抑制有显著效果；AL0061003、AL0061004、AL0061005、AL0061009、AL0061014、AL0061015、AL0061018-AL0061022、AL0061028、AL0061030、AL0061031、AL0061032、AL0061034对HepG2细胞株的PCSK9基因表达水平的抑制有显著效果。
由图2可见，在加药量为50nM时，所有候选化合物对Huh7和Hle细胞株中PCSK9基因表达水平的抑制有显著效果；尤其是AL0061005、AL0061018、AL0061028、AL0061030、AL0061032、AL0061041、AL0061042、AL0061044、AL0061051、AL0061052、AL0061053、AL0061054对Huh7和Hle细胞株的PCSK9基因表达水平抑制效果均达到了90％以上。
实施例2.化学修饰的PCSK9-siRNA体外猴原代肝细胞活性筛选
(1)食蟹猴肝细胞细胞培养和转染：
使用食蟹猴肝细胞(北京瑞德百奥生物科技有限公司，cmTCSC)，先预热培养基，取出解冻培养基(北京瑞德百奥生物科技有限公司，HEPO24)到生物安全柜，在36ml解冻培养基(TPCS，HEPO24)中加入4mL的FBS，制成完全解冻培养基，在37℃水浴中加热10分钟。用涂层介质(北京瑞德百奥生物科技有限公司，HEPO44)在CO 2培养箱中的37℃处理0.5h。从液氮中取出细胞，37℃水浴锅溶解细胞，约2min后取出，将细胞悬液转至预热的40ml解冻培养基中，用2ml完全解冻培养基清洗细胞冻存管，将细胞悬液在180×g下离心1min，弃去上清，加入2ml提前预热的CM播种培养基(北京瑞德百奥生物科技有限公司，CMHEP054)，轻缓吹散混匀细胞悬液，取20μl细胞悬液计数。根据计数结果，接种12孔板，3*10^5/孔，置于37℃，5％CO 2的培养箱中培养。贴壁4-5h后，吸出CM播种培养基，更换为预热的培养基(北京瑞德百奥生物科技有限公司，CMHEP064)，贴壁6h后转染。使用Lipofectamine TM 3000 Transfection Reagent(thermofisher，L3000150)进行转染，体系①用50μl Opti-MEM(thermofisher，1105821)稀释50nM修饰后siRNA(苏州贝信生物技术有限公司)，体系②用50μl Opti-MEM稀释3μl Lip3000，分别静置5min后，将体系①与②混合再静置15min，滴加到12孔板，转染4h后更换DMEM/F12完全培养基，将12孔板置于培养箱中孵育48h。
(2)使用RNA-Quick Purification Kit(RNA快速提取试剂盒，奕杉生物科，RN001)提取总RNA：
从培养箱中取出12孔板，吸干培养基，用适量的PBS洗一次，每孔加入500μl的裂解缓冲液，将上清转移到新的1.5ml离心管中。向裂解的细胞中加入500μl的无水乙醇充分混匀(若出现沉淀，这是正常现象，继续进行操作即可)。将离心管颠倒几次，或用移液器用力吹吸10次使产生的沉淀分散开，然后将液体加入离心柱，将离心 管对称放入离心机(eppendorf，5430)，4000×g离心1min。取出离心管向柱中加入500μl的洗涤缓冲液，12000×g离心1min，离心结束后取出柱子时注意不要让收集管内的废液接触到RNA柱，以免污染，倒掉废液，将RNA柱装回收集管，空管离心一次，能完全去除可能残留的洗涤缓冲液。将柱子放到干净的无RNA酶的1.5ml离心管上，开盖晾干2分钟。在RNA柱的膜中心部位加入30μl的洗脱缓冲液，室温静置2分钟，2000×g离心1min，RNA洗脱下来后，置于冰上。测定洗脱的RNA浓度，以便于后续实验使用。提取出来的RNA可立即用于后续实验，也可保存在-80℃备用。
(3)使用
Figure PCTCN2022123541-appb-000009
II Q RT SuperMix for qPCR(+gDNA wiper)反转录试剂盒(诺唯赞，R223-01)合成cDNA：
使用无RNA酶的离心管中配制混合液4μl 4×gDNA wiper Mix、1μg模板RNA、加无RNA酶的ddH 2O至16μl去除基因组DNA，用移液器轻轻吹打混匀，42℃2min。然后反应管中直接加入4μl 5×HiScript II qRT SuperMix II用移液器轻轻吹打混匀，置于PCR仪(Applied Biosystems，9700)50℃，15min；85℃，5sec，4℃保持。产物可立即用于qPCR反应，或在-20℃保存，并在半年内使用，长期存放需分装后在-80℃保存，cDNA应避免反复冻融。
(4)使用ChamQ SYBR qPCR Master Mix(诺唯赞，Q311-02)qPCR定量：
配置混合液10μl 2×ChamQ SYBR qPCR Master Mix、0.5μl Forword primer(睿博科兴)、0.5μl Reverse primer(睿博科兴)、1μl Template cDNA、8μl ddH 2O至20μl体系，每个样品3个重复，将96孔板放到qPCR仪(ROCGENE，Archimed)，执行程序：预变形，95℃，30sec；扩增，95℃，10sec，60℃，30sec，40个循环；溶解曲线，95℃，15sec，60℃，60sec，95℃，15sec。
(5)使用GraphPad Prism 8进行数据统计分析：
将数据导出EXCEL格式，用CT PCSK9-CT GAPDH，将对照组进行归一化处理，为了计算相对沉默效率的倍数变化，数据使用△△CT法进行分析，将得到的三个平行重复数据导入GraphPad Prism 8，应用column模型做柱状分析，，结果见图3和图4。
由图3可见，在加药量为50nM时，AL0065004、AL0065005、AL0065006、AL0065008、AL0065009、AL0065010对食蟹猴肝细胞PCSK9基因表达的抑制有显著效果，且AL0065006、AL0065008、AL0065010抑制效果能达到约80％。
由图4，在加药量为50nM时，AL0065004、AL0065006、AL0065008、AL0065009、AL0065010-AL0065020对食蟹猴肝细胞PCSK9基因表达的抑制有显著效果，且抑制效果均达到70％以上；其中，AL0065012、AL0065017、AL0065018、AL0065019、AL0065020抑制效果达到90％以上。
实施例3.PCSK9 RNAi剂在小鼠中的体内测试
试验采用SPF级雄性、6～8周周龄的C57BL/6小鼠(斯贝福(北京)生物技术有限公司)。在给药第0天获得给药前血清样品，按照LDL-c水平随机分组。C57Bl/6小鼠单次皮下给予9mg/kg的PCSK9 RNAi剂、生理盐水(NC，阴性对照)。分别给药后第8天、15天、22天、29天(NC/PC/AL007002/AL007010组持续检测至给药后36天)取小鼠血液(眼球取血，取血后1h内送检)，检查血液中TCHO(总胆固醇)、TG(甘油三酯)、HDL-C(高密度脂蛋白胆固醇)、LDL-C(低密度脂蛋白胆固醇)水平。实验期间，所有动物未见死亡或濒死症状。临床观察所有动物均未见明显异常。
实验分组及各检测数据显示在表5-表12；LDL-C变化水平显示在图5示出。
表5：给药分组表
序号 组别 给药剂量 给药方式
1 NC 9mg/kg 单次皮下注射
2 AL006002 9mg/kg 单次皮下注射
3 AL006003 9mg/kg 单次皮下注射
4 AL006004 9mg/kg 单次皮下注射
5 AL006005 9mg/kg 单次皮下注射
6 AL006006 9mg/kg 单次皮下注射
7 AL006007 9mg/kg 单次皮下注射
8 AL006008 9mg/kg 单次皮下注射
9 AL006009 9mg/kg 单次皮下注射
10 AL006010 9mg/kg 单次皮下注射
表6：动物分组时血脂水平(n＝7)
Figure PCTCN2022123541-appb-000010
Figure PCTCN2022123541-appb-000011
注：与NC组比较，*：p<0.05；**：p<0.01；***：p<0.001
表7：给药后第8天动物血脂变化(n＝7)
Figure PCTCN2022123541-appb-000012
注：与NC组比较，*：p<0.05；**：p<0.01；***：p<0.001
表8：给药后第15天动物血脂变化(n＝7)
Figure PCTCN2022123541-appb-000013
注：与NC组比较，*：p<0.05；**：p<0.01；***：p<0.001
表9：给药后第22天动物血脂变化(n＝7)
Figure PCTCN2022123541-appb-000014
注：与NC组比较，*：p<0.05；**：p<0.01；***：p<0.001
表10：给药后第29天动物血脂变化(n＝7)
Figure PCTCN2022123541-appb-000015
Figure PCTCN2022123541-appb-000016
注：与NC组比较，*：p<0.05；**：p<0.01；***：p<0.001
表11：给药后第36天动物血脂变化(n＝7)
Figure PCTCN2022123541-appb-000017
注：与NC组比较，*：p<0.05；**：p<0.01；***：p<0.001
表12：给药后第43天动物血脂变化(n＝7)
Figure PCTCN2022123541-appb-000018
注：与NC组比较，*：p<0.05；**：p<0.01；***：p<0.001
由表5-12以及图5可以得出，与溶剂对照组相比，在药物干预后第8天AL0067002、AL0067006、AL0067007、AL0067010药物干预组都可显著降低C57小鼠血液中LDL-C水平，而其中最显著也是持续降低时间最长的为AL0067010组；可以看到AL0067010药物干预组可显著降低C57小鼠血液中LDL-C水平，在Day8时LDL-C水平达最低值，随后逐渐缓慢回升。其中在Day8、Day15、Day22、Day29、Day36以及Day43时，AL0067010处理组小鼠血液LDL-C水平与溶剂对照相比差异具有显著性(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)。在药物干预后，AL0067010处理组小鼠血液中LDL-C水平与溶剂对照组(NC组)相比，在前三周表现出良好的降脂效果，分别降低了51.43％、38.71％和40.63％。
实施例4.PCSK9 RNAi剂在小鼠中的体内测试
试验采用SPF级雄性、6～8周周龄的C57BL/6小鼠(斯贝福(北京)生物技术有限公司)。在给药第0天获得给药前血清样品，按照LDL-c水平随机分组。C57Bl/6小鼠单次皮下给予9mg/kg的PCSK9 RNAi剂、生理盐水(NC，阴性对照)。分别给药后第8天、15天、22天、29天、36天、43天取小鼠血液(眼球取血，取血后1h内送检)，检查血液中TCHO(总胆固醇)、TG(甘油三酯)、HDL-C(高密度脂蛋白胆固醇)、LDL-C(低密度脂蛋白胆固醇)水平。实验期间，临床观察所有动物均未见明显异常。
实验分组及各检测数据显示在表13-表19；LDL-C变化水平显示在图6示出。
表13 动物分组时血脂变化(n＝5)
Figure PCTCN2022123541-appb-000019
注：与NC组比较，均无明显差异
表14 给予受试物后第8天动物血脂变化(n＝5)
Figure PCTCN2022123541-appb-000020
Figure PCTCN2022123541-appb-000021
注：与N组比较，*：p<0.05；**：p<0.01；***：p<0.001
表15 给予受试物后第15天动物血脂变化(n＝5)
Figure PCTCN2022123541-appb-000022
注：与NC组比较，*：p<0.05；**：p<0.01；***：p<0.001
表16 给予受试物后第22天动物血脂变化(n＝5)
Figure PCTCN2022123541-appb-000023
注：与NC组比较，*：p<0.05；**：p<0.01；***：p<0.001
表17 给予受试物后第29天动物血脂变化(n＝5)
Figure PCTCN2022123541-appb-000024
Figure PCTCN2022123541-appb-000025
注：与NC组比较，*：p<0.05；**：p<0.01；***：p<0.001
表18 给予受试物后第36天动物血脂变化(n＝5)
Figure PCTCN2022123541-appb-000026
注：与NC组比较，*：p<0.05；**：p<0.01；***：p<0.001
表19 给予受试物后第43天动物血脂变化(n＝5)
Figure PCTCN2022123541-appb-000027
注：与NC组比较，*：p<0.05；**：p<0.01；***：p<0.001
由表13-表19以及图6可以得出，与溶媒对照组比较，给予受试物后第8天AL0067010、AL0067011、AL0067012、AL0067013、AL0067015、AL0067016、AL0067017、AL0067018组动物血清TCHO、HDL-C、LDL-C明显至极显著降低(p<0.05或p<0.01或p<0.001)，AL0067002、AL0067019组动物血清TCHO、LDL-C显著降低(p<0.01)；第15天AL0067002、AL0067010、AL0067011、AL0067012、AL0067013、AL0067015、AL0067016、AL0067017、AL0067018、AL00670019组动物血清TCHO、HDL-C、LDL-C显著或极显著降低(p<0.01或p<0.001)；第22天AL0067002、AL0067010、AL0067011、AL0067012、AL0067015、AL0067016、AL0067017、AL0067018、AL00670019组动物血清TCHO、HDL-C、LDL-C明显至极显著降低(p<0.05或p<0.01或p<0.001)，AL00670013组动物血清TCHO、LDL-C明显或显著降低(p<0.05或p<0.01)；AL0067014组动物血清LDL-C明显降低(p<0.05)；第29天AL0067002、AL0067010、AL0067011、AL0067015、AL0067016、AL0067017、AL0067018组动物血清TCHO、HDL-C、LDL-C明显至极显著降低(p<0.05或p<0.01或p<0.001)，AL0067012、AL00670013、AL00670019组动物血清LDL-C明显或显著降低(p<0.05或p<0.01)；第36天AL0067002、AL0067010、AL0067011、AL0067012、AL0067013、AL0067015、AL0067016、AL0067017、AL0067018、AL0067019组动物血清TCHO(AL0067013、AL00670019组除外)、TG(AL0067011组除外)、HDL-C、LDL-C明显至极显著降低(p<0.05或p<0.01或p<0.001)；第43天AL0067002、AL0067011组动物血清TCHO、HDL-C、LDL-C明显至极显著降低(p<0.05或p<0.01或p<0.001)，AL0067010、AL0067016、AL0067019组动物血清LDL-C明显或显著降低(p<0.05或p<0.01)，AL0067012、AL0067015组动物血清HDL-C、LDL-C明显或显著降低(p<0.05或p<0.01)。
实施例5.PCSK9 RNAi剂在小鼠中的体内测试
试验采用SPF级雄性、6～8周周龄的C57BL/6小鼠(斯贝福(北京)生物技术有限公司)。在给药第0天获得给药前血清样品，按照LDL-c水平随机分组。C57Bl/6小鼠单次皮下给予9mg/kg的PCSK9 RNAi剂、生理盐水(NC，阴性对照)。分别给药后第8天、15天、22天、29天、37天、43天、50天取小鼠血液(眼球取血，取血后1h内送检)，检查血液中TCHO(总胆固醇)、TG(甘油三酯)、HDL-C(高密度脂蛋白胆固醇)、LDL-C(低密度脂蛋白胆固醇)水平。实验期间，所有动物未见死亡或濒死症状。临床观察所有动物均未见明显异常。 实验分组及各检测数据显示在表20-表19；LDL-C变化水平显示在图7示出。
表20 动物分组时血脂变化
Figure PCTCN2022123541-appb-000028
注：与NC组比较，均无明显差异
表21 给予受试物后第8天动物血脂变化
Figure PCTCN2022123541-appb-000029
注：与NC组比较，*：p<0.05；**：p<0.01；***：p<0.001
表22 给予受试物后第15天动物血脂变化
Figure PCTCN2022123541-appb-000030
注：与NC组比较，*：p<0.05；**：p<0.01；***：p<0.001
表23 给予受试物后第22天动物血脂变化
Figure PCTCN2022123541-appb-000031
注：与NC组比较，*：p<0.05；**：p<0.01；***：p<0.001
表24 给予受试物后第29天动物血脂变化
Figure PCTCN2022123541-appb-000032
注：与NC组比较，*：p<0.05；**：p<0.01；***：p<0.001
表25 给予受试物后第37天动物血脂变化
Figure PCTCN2022123541-appb-000033
Figure PCTCN2022123541-appb-000034
注：与NC组比较，*：p<0.05；**：p<0.01；***：p<0.001
表26 给予受试物后第43天动物血脂变化
Figure PCTCN2022123541-appb-000035
注：与NC组比较，*：p<0.05；**：p<0.01；***：p<0.001
表27 给予受试物后第50天动物血脂变化
Figure PCTCN2022123541-appb-000036
注：与NC组比较，*：p<0.05；**：p<0.01；***：p<0.001
由表20-表27以及图7可以得出，与溶媒对照组比较，给予受试物后第8天、第15天、第22天、第29天、第37天、第43天各受试物组(AL0067017、AL0067023、AL0067028、AL0067033、AL0067010)动物血清TCHO、HDL-C、LDL-C均明显至极显著降低(p<0.05或p<0.01或p<0.001)，第50天AL0067028、AL0067033组动物血清TCHO、HDL-C、LDL-C均明显至极显著降低(p<0.05或p<0.01或p<0.001)，AL0067010组无明显统计学差异。
实施例6.PCSK9 RNAi剂在hPCSK9小鼠中的体内测试
试验采用SPF级雄性、5～6周周龄的hPCSK9小鼠(上海南方模式生物科技发展有限公司)。在给药第0天获得给药前血清样品，按照体重水平随机分组。hPCSK9小鼠单次皮下给予9mg/kg的PCSK9 RNAi剂、生理盐水(NC，阴性对照)。分别给药后第8天、15天、22天、29天取小鼠血液(眼球取血，取血后1h内送检)，检查血液中LDL-C(低密度脂蛋白胆固醇)水平，ELISA(R&D)试剂盒检测PCSK9蛋白表达情况。
各检测数据显示在表28-表29；LDL-C及PCSK9蛋白变化水平显示在图8、图9中。
表28 动物血清人PCSK含量变化(ng/mL)
Figure PCTCN2022123541-appb-000037
注：与NC组比较，*：p<0.05；**：p<0.01；***：p<0.001
表29 动物血清LDL-C含量变化(mmol/L)
Figure PCTCN2022123541-appb-000038
注：与NC组比较，*：p<0.05；**：p<0.01；***：p<0.001
如图8所示，对于PCSK9蛋白水平，所有RNAi剂组(AL0067001、AL0067010、AL0067016、AL0067028)与自身给药前(Day 0)相比在前二周(Day 8、Day15)中都有显著降低；尤其明显的是，AL0067016、AL0067028组单次给药可以连续三周使PCSK9蛋白降低80％以上，给药约一月后(Day29)PCSK9蛋白降低还能维持在60％左右。
如图9所示，对于LDL-C表达水平，与自身给药前(Day 0)相比，除AL0067001组基本无显著变化外，其他RNAi剂组在整个实验周期都有极显著下降，其中AL0067010在Day8、Day15、Day22、Day29分别下降到53.79％、45.45％、43.94％、46.97％，AL0067016分别下降到53.16％、56.96％、62.03％、51.90％，AL0068028分别下降到57.41％、48.15％、51.85％、65.74％。
实施例7.PCSK9 RNAi剂在食蟹猴体内测试研究
试验采用12-23岁中老年雄性食蟹猴，LDL-C范围在0.82mmoL-2.10mmoL，基本处于血脂异常状态。在给药前14天、7天以及给药当天(第1天)获得给药前血清样品，按照LDL-C水平随机分组。食蟹猴单次皮下给予9mg/kg的PCSK9 RNAi剂，分别给药后第8天、15天、22天、29天、34天、44天、51天、58天(AL0067001、AL0067010结束)、65天、72天、79天取血液，检查血液中TCHO(总胆固醇)、LDL-C(低密度脂蛋白胆固醇)水平。
LDL-C、TCHO变化水平显示在图10、图11中。
如图10所示，与给药前(第1天)相比，RNAi剂AL0067001、AL0067010、AL0067016、AL0067028都能够显著降低食蟹猴LDL-C水平，其中AL0067001、AL0067010能够连续51天使食蟹猴LDL-C水平抑制效率维持在40％左右，更为显著的是AL0067016与AL0067028组，在整个实验期间(第79天)都能够使食蟹猴LDL-C抑制效率维持在40％以上。
如图11所示，RNAi剂AL0067001、AL0067010、AL0067016、AL0067028都能够显著降低食蟹猴TCHO水平，其中AL0067001、AL0067010能够连续51天使食蟹猴LDL-C水平抑制效率维持在20％左右，更为显著的是AL0067016与AL0067028组，在整个实验期间(第79天)都能够使食蟹猴TCHO抑制效率维持在20％以上。AL0067028组在第29天至第51天能够将TCHO水平抑制率维持在40％左右。
实施例8.PCSK9 RNAi剂在C57小鼠体内测试研究
基于实施例7研究基础，探索AL0067028的基础上进行修饰组合的优化，评估新的RNAi剂在C57小鼠上的研究效果。
试验采用SPF级雄性、6～8周周龄的C57BL/6小鼠(斯贝福(北京)生物技术有限公司)。在给药第0天获得给药前血清样品，按照LDL-c水平随机分组。C57BL/6小鼠单次皮下给予6mg/kg的PCSK9 RNAi剂、生理盐水(NC，阴性对照)。分别给药后第7天、16天、23天、30天、37天、44天、51天取小鼠血液(眼球取血，取血后1h内送检)，检查血液中LDL-C(低密度脂蛋白胆固醇)水平，ELISA(R&D)试剂盒检测PCSK9蛋白表达情况。实验期间，所有动物未见死亡或濒死症状。临床观察所有动物均未见明显异常。
实验分组及各检测数据显示在表30；LDL-C变化水平、PCSK9蛋白变化水平显示在图12、图13中。
表30 动物血清LDL-C含量变化(mmol/L)
Figure PCTCN2022123541-appb-000039
与NC组比较，*：p<0.05；**：p<0.01；***：p<0.001
如图12所示，对于LDL-C表达水平，所有RNAi剂组(AL0067036、AL0067037、AL0067038、AL0067039、AL0067040、AL0067028)在Day37前都有显著降低LDL-C水平作用，尤其明显是AL0067037组与AL0067040组对于LDL-C水平的显著降低能够持续到51天；另外，AL0067037组、AL0067040组降低LDL-C水平在整个试验周期都明显优于AL0067028组，且达到显著差异(P<0.05)。
如图13所示，对于PCSK9蛋白水平，所有RNAi剂组(AL0067036、AL0067037、AL0067038、AL0067039、AL0067040、AL0067028)与自身给药前(Day 0)相比在整个试验周期(Day 51)中都有显著降低；尤其明显的是，AL0067037组单次给药可以连续四周(Day 30)使PCSK9蛋白降低85％以上，在给药后第16天使PCSK9蛋白降低超过90％；在整个试验周期，AL0067037组降低PCSK9蛋白的效果、降低PCSK9蛋白持续的时间都要显著优于AL0067028组。

Claims (10)

  1. 用于降低PCSK9的表达的寡核苷酸或其药学上可接受的盐，所述寡核苷酸包含反义链，所述反义链具有如SEQ ID NO:53-106中任一所示的序列或其片段、或所述序列或其片段的修饰序列；
    优选地，所述寡核苷酸进一步包含正义链，所述正义链具有如SEQ ID NO:1-52中任一所示的序列或其片段、或所述序列或其片段的修饰序列；
    优选地，所述反义链由如SEQ ID NO:53-106中任一所示的序列或其片段、或所述序列或其片段的修饰序列组成；
    优选地，所述正义链由如SEQ ID NO:1-52中任一所示的序列或其片段、或所述序列或其片段的修饰序列组成；
    优选地，所述反义链的修饰序列包含具有如SEQ ID NO:138-183、185-186中任一所示的序列或其片段；
    优选地，所述正义链的修饰序列包含具有如SEQ ID NO:107-137、135、184中任一所示的序列或其片段；
    优选地，所述反义链由如SEQ ID NO:138-183、185-186中任一所示的序列或其片段、或所述序列或其片段的修饰序列组成；
    优选地，所述正义链由如SEQ ID NO:107-137、135、184中任一所示的序列或其片段、或所述序列或其片段的修饰序列组成。
  2. 权利要求1所述的寡核苷酸或其药学上可接受的盐，其中所述反义链长度为19至23个核苷酸。
  3. 权利要求1或2所述的寡核苷酸或其药学上可接受的盐，其中所述正义链长度为19至23个核苷酸。
  4. 权利要求1至3中任一项所述的寡核苷酸或其药学上可接受的盐，其中所述寡核苷酸包含长度为一个或多个核苷酸的3'-突出端序列，其中所述3'-突出端序列存在于反义链和/或正义链上；
    优选地，所述反义链带有一个突出端；
    优选地，所述正义链带有一个突出端；
    优选地，所述寡核苷酸包含长度为两个核苷酸的3'-突出端序列；
    优选地，所述3'-突出端序列存在于所述正义链上；优选地，所述突出端序列选自：GG、GA、GC、UC、UG、UU、UA、CA、CC、CG、CU、AA、AG、AU、AC；
    优选地，所述3'-突出端序列存在于所述反义链上；优选地，所述突出端序列选自：UU、UC、UA、UG、GA、GG、GU、GC、TT、AG、AU、AA、AC、CA、CC、U；更优选地，所述突出端序列为UU。
  5. 权利要求1至4中任一项所述的寡核苷酸或其药学上可接受的盐，优选地，所述寡核苷酸包含长度各自在19至23个核苷酸的范围内的反义链和正义链；
    优选地，所述正义链与所述反义链形成双链体区域；
    优选地，所述正义链和所述反义链分别为19/21配对、21/21配对、21/23配对或23/23配对的双链体结构；
    优选地，所述寡核苷酸包含长度为两个核苷酸的3'-突出端序列，其中所述3'-突出端序列存在于所述反义链上，且其中所述正义链长度为19个核苷酸且所述反义链长度为21个核苷酸，使得所述正义链和反义链形成长度为19个核苷酸的双链体；
    所述寡核苷酸包含长度为两个核苷酸的3'-突出端序列，其中所述3'-突出端序列存在于所述反义链和正义链上，且其中所述正义链长度为21个核苷酸且所述反义链长度为21个核苷酸，使得所述正义链和反义链形成长度为19个核苷酸的双链体；
    优选地，所述寡核苷酸包含长度为两个核苷酸的3'-突出端序列，其中所述3'-突出端序列存在于所述反义链上，且其中所述正义链长度为21个核苷酸且所述反义链长度为23个核苷酸，使得所述正义链和反义链形成长度为21个核苷酸的双链体；
    优选地，所述寡核苷酸包含长度为两个核苷酸的3'-突出端序列，其中所述3'-突出端序列存在于所述反义链和正义链上，且其中所述正义链长度为23个核苷酸且所述反义链长度为23个核苷酸，使得所述正义链和反义链形成长度为21个核苷酸的双链体；
    优选地，所述寡核苷酸选自反义链SEQ ID NO:55-57、61、66、70-71、80、82、84、93-106中任一所述的未修饰寡核苷酸，以及反义链SEQ ID NO:138-183中任一所述的修饰寡核苷酸；
    优选地，所述寡核苷酸选自反义链SEQ ID NO:80、84、93-95、99-106中任一所述的未修饰寡核苷酸，以及反义链SEQ ID NO:145、147-150、154-183、185-186中任一所述的修饰寡核苷酸；
    优选地，所述寡核苷酸选自正义链SEQ ID NO:3-5、9、14、18、19、28、30、32、41-52中任一所述的未修饰寡核苷酸，以及正义链SEQ ID NO:107-137、135、184中任一所述的修饰寡核苷酸。
    优选地，所述寡核苷酸包含至少一个修饰的核苷酸；
    优选地，所述修饰是选自以下的修饰：2'-甲氧基(m)、2'-脱氧-2'-氟(f)和硫代磷酸酯(s)
    优选地，所述修饰的核苷酸包含2'-修饰；
    优选地，所述2'-修饰是选自以下的修饰：2'-氨基乙基、2'-氟、2'-O-甲基、2'-O-甲氧基乙基和2'-脱氧-2'-氟-β-d-***核糖核酸；
    优选地，所述寡核苷酸的所有核苷酸都是修饰的。
  6. 权利要求1至5中任一项所述的寡核苷酸或其药学上可接受的盐，其中所述正义链包含SEQ ID NO:51所示 的基序序列；所述反义链包含SEQ ID NO:187所示的基序序列；
    优选地，所述寡核苷酸包含选自下述正义链和反义链组合中的任一：
    (1)SEQ ID NO:52所示的正义链，和SEQ ID NO:105所示的反义链；
    (2)SEQ ID NO:135所示的正义链，和SEQ ID NO:176所示的反义链；
    (3)SEQ ID NO:136所示的正义链，和SEQ ID NO:177所示的反义链；
    (4)SEQ ID NO:137所示的正义链，和SEQ ID NO:178所示的反义链；
    (5)SEQ ID NO:135所示的正义链，和SEQ ID NO:185所示的反义链；
    (6)SEQ ID NO:135所示的正义链，和SEQ ID NO:186所示的反义链；
    (7)SEQ ID NO:184所示的正义链，和SEQ ID NO:185所示的反义链；
    (8)SEQ ID NO:184所示的正义链，和SEQ ID NO:186所示的反义链；和
    (9)SEQ ID NO:184所示的正义链，和SEQ ID NO:176所示的反义链；
    优选地，所述寡核苷酸包含选自下述正义链和反义链组合中的任一：
    (1)SEQ ID NO:52所示的正义链，和SEQ ID NO:106所示的反义链；
    (2)SEQ ID NO:134所示的正义链，和SEQ ID NO:179所示的反义链；
    (3)SEQ ID NO:135所示的正义链，和SEQ ID NO:180所示的反义链；
    (4)SEQ ID NO:135所示的正义链，和SEQ ID NO:181所示的反义链；
    (5)SEQ ID NO:136所示的正义链，和SEQ ID NO:182所示的反义链；和
    (6)SEQ ID NO:137所示的正义链，和SEQ ID NO:183所示的反义链；
    优选地，所述寡核苷酸包含选自下述正义链和反义链组合中的任一：
    (1)SEQ ID NO:51所示的正义链，和SEQ ID NO:103所示的反义链；
    (2)SEQ ID NO:127所示的正义链，和SEQ ID NO:158所示的反义链；和
    (3)SEQ ID NO:132所示的正义链，和SEQ ID NO:164所示的反义链.。
  7. 权利要求1至6中任一项所述的寡核苷酸或其药学上可接受的盐，其中所述寡核苷酸包含至少一个修饰的核苷酸间键；
    优选地，所述至少一个修饰的核苷酸间键是硫代磷酸酯键；
    优选地，所述反义链的5'-核苷酸的糖的4'-碳包含磷酸酯类似物；
    优选地，所述磷酸酯类似物是氧基甲基膦酸酯、乙烯基膦酸酯或丙二酰基膦酸酯。
  8. 权利要求1至7中任一项所述的寡核苷酸或其药学上可接受的盐，其中所述寡核苷酸的至少一个核苷酸缀合至一个或多个靶向配体；
    优选地，各靶向配体包含碳水化合物、氨基糖、胆固醇、多肽或脂质；
    优选地，各靶向配体包含N-乙酰基半乳糖胺(GalNAc)部分；
    优选地，其中所述GalNac部分是单价GalNAc部分、二价GalNAc部分、三价GalNAc部分或四价GalNAc部分。
  9. 组合物，其包含所述权利要求1至8中任一项所述的寡核苷酸或其药学上可接受的盐，以及任选地药学上可接受的载体；
    优选地，所述组合物的剂型为口服剂、静脉注射剂、皮下注射剂或肌内注射剂，优选为皮下注射剂；
    优选地，所述组合还包含其他治疗或/和预防脂质失调方面的疾病的药物；优选地，所述脂质失调方面的疾病为高脂血症、动脉粥样硬化和/或其一种或多种症状或并发症的一种或多种症状；更优选地，所述高脂血症是高胆固醇血症；
    所述其他治疗高脂血症的药物选自贝特类、他汀类、胆汁酸隔离剂和烟酸类。
  10. 权利要求1至8中任一项所述的寡核苷酸或其药学上可接受的盐或权利要求9所述的组合物在制备用于治疗或/和预防脂质失调方面的疾病的药物中的用途，
    优选地，所述脂质失调方面的疾病为高脂血症、动脉粥样硬化和/或其一种或多种症状或并发症的一种或多种症状；
    优选地，所述高脂血症是高胆固醇血症。
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Publication number Priority date Publication date Assignee Title
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109957567A (zh) * 2017-12-26 2019-07-02 广州市锐博生物科技有限公司 一种抑制PCSK9基因表达的siRNA分子及其应用
WO2019126990A1 (zh) * 2017-12-26 2019-07-04 广州市锐博生物科技有限公司 一种抑制PCSK9基因表达的siRNA分子及其应用
CN110913889A (zh) * 2017-06-09 2020-03-24 赛诺菲生物技术公司 通过施用pcsk9抑制剂治疗糖尿病患者中的高脂血症的方法
WO2021077856A1 (zh) * 2019-10-25 2021-04-29 广州医科大学 一种能够沉默Pcsk9蛋白的siRNA、其纳米递送***及应用
WO2021177518A1 (ko) * 2020-03-02 2021-09-10 서울대학교병원 혈중 콜레스테롤 저하용, 심혈관 대사질환의 예방 또는 치료용 및 항염용 약학적 조성물

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110913889A (zh) * 2017-06-09 2020-03-24 赛诺菲生物技术公司 通过施用pcsk9抑制剂治疗糖尿病患者中的高脂血症的方法
CN109957567A (zh) * 2017-12-26 2019-07-02 广州市锐博生物科技有限公司 一种抑制PCSK9基因表达的siRNA分子及其应用
WO2019126990A1 (zh) * 2017-12-26 2019-07-04 广州市锐博生物科技有限公司 一种抑制PCSK9基因表达的siRNA分子及其应用
WO2021077856A1 (zh) * 2019-10-25 2021-04-29 广州医科大学 一种能够沉默Pcsk9蛋白的siRNA、其纳米递送***及应用
WO2021177518A1 (ko) * 2020-03-02 2021-09-10 서울대학교병원 혈중 콜레스테롤 저하용, 심혈관 대사질환의 예방 또는 치료용 및 항염용 약학적 조성물

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BARALE CRISTINA, MELCHIONDA ELENA, MOROTTI ALESSANDRO, RUSSO ISABELLA: "PCSK9 Biology and Its Role in Atherothrombosis", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 22, no. 11, pages 5880, XP093053560, DOI: 10.3390/ijms22115880 *
FITZGERALD KEVIN, WHITE SUELLEN, BORODOVSKY ANNA, BETTENCOURT BRIAN R., STRAHS ANDREW, CLAUSEN VALERIE, WIJNGAARD PETER, HORTON JA: "A Highly Durable RNAi Therapeutic Inhibitor of PCSK9", THE NEW ENGLAND JOURNAL OF MEDICINE, vol. 376, no. 1, 5 January 2017 (2017-01-05), US , pages 41 - 51, XP093048372, ISSN: 0028-4793, DOI: 10.1056/NEJMoa1609243 *
TAN, XIAOQING ET AL.: "Effect of PCSK9 siRNA on Proliferation of Vascular Smooth Muscle Cells", CHINESE JOURNAL OF GERIATRIC HEART BRAIN AND VESSEL DISEASES, vol. 18, no. 7, 31 July 2016 (2016-07-31), pages 734 - 738, XP009545087 *

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