Targeted oligonucleotides for treating diseases associated with pcsk9

Abstract

The present disclosure relates to a targeting oligonucleotide for treating a disease related to PCSK9, the targeting oligonucleotide comprising an antisense strand having a sequence as shown in any one of SEQ ID NOs: 53-106 or a fragment thereof, or a modified sequence of the sequence or the fragment thereof. The targeting oligonucleotide significantly inhibits the expression level of the PCSK9 gene in the middle, reduces the low-density lipoprotein cholesterol (LDL-C) level in the blood, and shows a good lipid-lowering effect.

Description

Technical Field

[0001] This disclosure relates to an oligonucleotide, and more particularly to a targeted oligonucleotide for the treatment of PCSK9-related diseases. Background Technology

[0002] Blood lipids refer to the total lipids contained in the blood. The blood lipids most closely related to clinical practice mainly include triglycerides and cholesterol. Dyslipidemia usually refers to elevated levels of cholesterol and / or triglycerides (TG) in the serum, commonly known as hyperlipidemia.

[0003] Hypercholesterolemia refers to elevated levels of total cholesterol (TCHO) and / or low-density lipoprotein cholesterol (LDL-C) or non-high-density lipoprotein cholesterol (non-HDL-C) in the blood. Also known as dyslipidemia, it can be divided into familial and non-familial hypercholesterolemia. One of its most important clinical features is elevated LDL-C levels in the blood. Hypercholesterolemia is a significant factor in the development of cardiovascular disease (Guo, Yanan, et al. Archives of Biochemistry and Biophysics (2020):108717). Some cardiovascular and cerebrovascular diseases, such as cerebrovascular diseases, coronary heart disease, and peripheral artery disease, often do not present with obvious or easily noticeable symptoms until severe atherosclerosis develops. Currently, statins are the first-line drugs for lowering blood lipids in clinical practice. These drugs can reduce the risk of cardiovascular disease by lowering LDL-C. However, many patients are intolerant to statins, or even after taking the maximum dose of statins, they are still unable to lower their LDL-C levels 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.

[0004] The human PCSK9 gene is approximately 22 kb long, contains 12 exons, and encodes a glycoprotein of 692 amino acids (Benjannet, Suzanne, et al. Journal of Biological Chemistry 279.47(2004):48865-48875). The liver is the main site of PCSK9 synthesis and secretion. Normal levels of PCSK9 in human plasma range from 30 ng / mL to 4 μg / mL, playing a crucial role in cholesterol metabolism. It primarily controls plasma LDL levels by influencing the expression of LDL receptors on the surface of hepatocytes. Gain-of-function mutations (reversion mutations) in the PCSK9 gene lead to decreased LDL receptor levels on the surface of hepatocytes and increased LDL-C levels in the blood; conversely, loss-of-function mutations lead to increased LDL receptor levels on the surface of hepatocytes and decreased LDL-C levels in the blood. Studies have shown that when maximum doses of statins fail to effectively control LDL-C levels, reducing PCSK9 levels can effectively lower LDL-C levels.

[0005] Intracellular PCSK9 can bind to and guide newly generated LDL receptors from the trans-Golgi apparatus to lysosomes for degradation. Extracellular PCSK9 can bind to LDL receptors on the surface of liver cells, mediating their entry into hepatocyte lysosomes for degradation. This reduces the number of LDL receptors on the surface of liver cells, leading to a decrease in the liver's ability to bind and clear LDL-C, ultimately resulting in elevated LDL-C levels in the blood. Therefore, reducing PCSK9 expression levels can be used to treat hypercholesterolemia (Norata et al. Annu Rev Pharmacol Toxicol (2014) 54:273-93) and prevent cardiovascular diseases associated with high LDL-C.

[0006] In 1998, two American scientists, Andrew Fire and Craig Mello, discovered a biological mechanism in which small RNA molecules can mediate the degradation of specific mRNAs (Fire, Andrew, et al. Nature 391.6669(1998):806-811). This mechanism is activated when RNA molecules appear in the cell in double-stranded form, resulting in RNA interference. This discovery heralded the beginning of a new field of research, and the two scientists were awarded the 2006 Nobel Prize in Physiology or Medicine for this discovery. When double-stranded RNA binds to the protein complex Dicer, Dicer cleaves the dsRNA into fragments. Then, another protein complex, RISC, binds to these fragments. One strand of the siRNA double strand is removed, but the other strand remains bound to the RISC complex. Guided by single-stranded RNA, RISC recognizes and degrades the mRNA of the target gene, thereby inhibiting the expression of the specific protein and specifically leading to gene silencing.

[0007] RNA interference has opened up a new field for the application of gene technology. Double-stranded RNA molecules have been artificially designed to silence specific genes in humans, animals, or plants. These artificially designed and synthesized double-stranded RNA molecules (siRNA) for gene silencing are introduced into cells and activate RNA interference mechanisms to degrade the corresponding mRNA. Currently, this method is an important research tool in biology and biomedicine. Furthermore, numerous siRNA drugs have been developed to treat viral infections, cardiovascular diseases, cancer, endocrine disorders, and many other diseases. Most siRNA therapies, either in the research and development stage or already approved for marketing, have shown promising therapeutic effects. Since the first siRNA drug was launched in 2018, at least four siRNAs have been approved for marketing in the EU or the US. Therefore, using RNA interference technology to inhibit the expression of specific target genes has become an effective approach to disease treatment.

[0008] Currently, there are reports of inhibitors targeting PCSK9, including marketed antibody and small nucleic acid drugs, which are used or intended for the treatment of hypercholesterolemia and related diseases. However, other inhibitors targeting this target need to be developed in order to achieve better efficacy or better safety. Summary of the Invention

[0009] In order to address the problems existing in the prior art, the purpose of this disclosure is to provide a PCSK9-targeting inhibitor that is effective, safe, and has a long-lasting effect.

[0010] In one aspect, this disclosure provides oligonucleotides or pharmaceutically acceptable salts thereof for reducing PCSK9 expression, wherein the oligonucleotides comprise an antisense strand having a sequence or fragment thereof as shown in any of SEQ ID NO:53-106, or a modified sequence thereof.

[0011] In some embodiments of this disclosure, the oligonucleotide further comprises a sequence or fragment thereof as shown in any of SEQ ID NO:1-52, or a modified sequence thereof.

[0012] In some embodiments of this disclosure, the antisense chain consists of a sequence or fragment thereof as shown in any of SEQ ID NO:53-106, or a modified sequence thereof.

[0013] In some embodiments of this disclosure, the modified sequence of the antisense strand comprises a sequence or fragment thereof having any of the sequences shown in SEQ ID NO:138-183, 185-186.

[0014] In some embodiments of this disclosure, the modified sequence of the justice chain comprises a sequence or fragment thereof having any of the sequences shown in SEQ ID NO:107-137, 135, 184.

[0015] In some embodiments of this disclosure, the justice chain and the antisense chain are respectively a double-chain structure with 19 / 21 pairing, 21 / 21 pairing, 21 / 23 pairing, or 23 / 23 pairing.

[0016] In another aspect, this disclosure provides a composition comprising the oligonucleotide or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

[0017] In another aspect, this disclosure provides the use of the aforementioned oligonucleotides or pharmaceutically acceptable salts or compositions thereof in the preparation of medicaments for treating and / or preventing diseases of lipid dysregulation.

[0018] In some embodiments of this disclosure, the lipid-related disorders are used for the treatment of hyperlipidemia, atherosclerosis, and / or one or more symptoms or complications thereof.

[0019] Experiments have demonstrated that the candidate compounds disclosed herein have significant inhibitory effects on PCSK9 gene expression levels in human liver cancer cell lines Huh7, HepG2, and Hle. Some of these candidate compounds showed inhibition rates exceeding 90% in Huh7 and Hle cell lines, while others inhibited PCSK9 gene expression in cynomolgus monkey hepatocytes by 70%, 80%, and even 90%, exhibiting excellent inhibitory effects. After approximately one month (Day 29) of administration to mice, the reduction in PCSK9 protein levels remained around 60% for some of these compounds; others reduced PCSK9 protein levels by over 85% for four consecutive weeks (Day 30) in mice, demonstrating sustained efficacy. Furthermore, some of these compounds significantly reduced low-density lipoprotein cholesterol (LDL-C) levels in C57 mice. Some compounds maintained an inhibition rate of approximately 40% for LDL-C levels in cynomolgus monkeys for 51 consecutive days, and others maintained an inhibition rate of over 40% even on day 79, demonstrating good lipid-lowering effects. This disclosure provides oligonucleotides targeting PCSK9 mRNA that effectively reduce PCSK9 expression in cells, particularly liver cells (e.g., hepatocytes), for the treatment of hypercholesterolemia, atherosclerosis, and / or one or more of their symptoms or complications. Therefore, in a related aspect, this disclosure provides methods for treating hypercholesterolemia, atherosclerosis, and / or one or more of their symptoms or complications, involving the selective reduction of PCSK9 gene expression in the liver. In some embodiments, the PCSK9-targeting oligonucleotides provided herein are designed for delivery to selected cells (e.g., hepatocytes) of a target tissue to treat hypercholesterolemia, atherosclerosis, and / or one or more of their symptoms or complications in a subject. Attached Figure Description

[0020] Figure 1 The graph shows the efficacy results of the first round of in vitro screening of PCSK9-siRNA in hepatocyte cell lines.

[0021] Figure 2 The graph shows the efficacy results of the second round of in vitro screening of PCSK9-siRNA in hepatocyte cell lines.

[0022] Figure 3 The image shows the efficacy results of PCSK9-siRNA after the first round of in vitro screening of primary monkey hepatocytes.

[0023] Figure 4 The image shows the efficacy results of PCSK9-siRNA after the second round of in vitro screening of primary monkey hepatocytes.

[0024] Figure 5 The changes in LDL-C levels in mice after the first round of PCSK9 RNAi administration are shown.

[0025] Figure 6 The changes in LDL-C levels in mice after a second round of PCSK9 RNAi administration are shown.

[0026] Figure 7 The changes in LDL-C levels in mice after the third round of PCSK9 RNAi administration are shown.

[0027] Figure 8 The changes in LDL-C levels in hPCSK9 mice after administration of PCSK9 RNAi are shown.

[0028] Figure 9 The changes in PCSK9 protein levels in hPCSK9 mice after administration of PCSK9 RNAi agent are shown.

[0029] Figure 10 The changes in LDL-C levels in cynomolgus monkeys after administration of PCSK9 RNAi are shown.

[0030] Figure 11 The changes in TCHO levels in cynomolgus monkeys after administration of PCSK9 RNAi were shown.

[0031] Figure 12 The changes in LDL-C levels in mice after the fourth round of PCSK9 RNAi administration are shown.

[0032] Figure 13 The changes in PCSK9 protein levels in mice after the fourth round of administration of PCSK9 RNAi agent are shown. Detailed Implementation

[0033] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0034] Approximately: As used herein, the term “approximately” or “about” when applied to one or more target values ​​refers to a value similar to the reference value. In some embodiments, unless otherwise stated or otherwise apparent from the context, the term “approximately” or “approximately” refers to a range of values ​​falling within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in any direction (unless such a number would exceed 100% of the possible value).

[0035] Atherosclerosis: As used herein, the term "atherosclerosis" refers to a condition involving narrowing of arteries (e.g., coronary, carotid, peripheral, and / or renal arteries) typically due to the buildup of plaque (composed of fat, cholesterol, calcium, and other substances). In some embodiments, narrowing of the coronary arteries can produce symptoms such as angina, shortness of breath, sweating, nausea, dizziness, irregular heartbeat, and / or palpitations. In some embodiments, narrowing of the carotid arteries can lead to stroke (i.e., brain cell death due to insufficient blood and oxygen flow to the brain) and / or can produce symptoms such as weakness, confusion, difficulty speaking, dizziness, difficulty walking or standing, blurred vision, numbness in the face, arms, and legs, severe headache, and / or loss of consciousness. In some embodiments, narrowing of the peripheral arteries can cause numbness or pain in the arms or legs. In some embodiments, narrowing of the renal arteries (leading to 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 (such as chronic kidney disease).

[0036] Complementarity: As used herein, the term "complementarity" refers to a structural relationship between nucleotides (e.g., two nucleotides on opposing nucleic acids or on opposing regions of a single nucleic acid strand) that allows the nucleotides to form base pairs with each other. For example, a purine nucleotide complementary to a pyrimidine nucleotide of an opposing nucleic acid can be base-paired together by forming hydrogen bonds with each other. In some embodiments, complementary nucleotides may be base-paired in a Watson-Crick manner or in any other manner that allows the formation of a stable duplex. In some embodiments, the two nucleic acids may have nucleotide sequences that are complementary to each other to form complementary regions, as described herein.

[0037] Chain: As used herein, the term "chain" refers to a single, continuous sequence of nucleotides linked together by internucleotide bonds (e.g., phosphodiester bonds, thiophosphate bonds). In some embodiments, the chain has two free ends, such as a 5'-end and a 3'-end.

[0038] Deoxyribonucleotide: As used herein, the term "deoxyribonucleotide" refers to a nucleotide that has a hydrogen atom at the 2' position of its pentose sugar compared to a ribonucleotide. Modified deoxyribonucleotides are deoxyribonucleotides that have modifications or substitutions (including modifications or substitutions in sugars, phosphate groups, or bases) that have one or more atoms other than at the 2' position.

[0039] Double-stranded oligonucleotide: As used herein, the term "double-stranded oligonucleotide" refers to an oligonucleotide that is substantially in a double-stranded form. In some embodiments, complementary base pairings are formed between antiparallel sequences of nucleotides in covalently separated nucleic acid chains of a double-stranded oligonucleotide. In some embodiments, complementary base pairings are formed between antiparallel sequences of nucleotides in covalently linked nucleic acid chains of a double-stranded oligonucleotide. In some embodiments, complementary base pairings are formed from one or more double-stranded regions of a double-stranded oligonucleotide from a single nucleic acid chain that is folded (e.g., via a hairpin) to provide complementary antiparallel sequences of nucleotides that are base-paired together. In some embodiments, the double-stranded oligonucleotide comprises two covalently separated nucleic acid chains that are fully double-stranded with each other. However, in some embodiments, the double-stranded oligonucleotide comprises two covalently separated nucleic acid chains that are partially double-stranded, for example, having overhangs at one or both ends. In some embodiments, the double-stranded oligonucleotide comprises antiparallel sequences of nucleotides that are partially complementary, and therefore may have one or more mismatches, which may include internal mismatches or terminal mismatches.

[0040] Oligonucleotide: As used herein, the term "oligonucleotide" refers to a short nucleic acid, such as a short nucleic acid less than 100 nucleotides in length. Oligonucleotides may comprise ribonucleotides, deoxyribonucleotides, and / or modified nucleotides, including, for example, modified ribonucleotides. Oligonucleotides may be single-stranded or double-stranded. Oligonucleotides may or may not have a double-stranded region. As a set of non-limiting examples, oligonucleotides may be, but are not limited to, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), Dicer substrate interfering RNA (dsiRNA), antisense oligonucleotides, short siRNA, or single-stranded siRNA. In some embodiments, the double-stranded oligonucleotide is an RNAi oligonucleotide.

[0041] PCSK9: As used herein, refers to the gene or protein of the proprotein convertase subtilisin / kexin-9, also known as NARC-1, NARC-FH3, HCHOLA3, or NARCI. The term PCSK9 includes human PCSK9, mouse PCSK9, and rat PCSK9. Examples of PCSK9 mRNA sequences are readily available in GenBank.

[0042] In one aspect, this disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof for reducing PCSK9 expression, the oligonucleotide comprising an antisense strand having a sequence or fragment thereof as shown in any of SEQ ID NO:53-106, or a modified sequence thereof.

[0043] In some embodiments of this disclosure, the oligonucleotide or a pharmaceutically acceptable salt thereof is preferably prepared or synthesized in the form of a carboxylate, sodium salt, triethylamine salt or other pharmaceutically acceptable salt.

[0044] In some embodiments of this disclosure, the oligonucleotide or a pharmaceutically acceptable salt thereof is more preferably its sodium salt or triethylamine salt.

[0045] In some embodiments of this disclosure, the oligonucleotide further comprises a sequence or fragment thereof as shown in any of SEQ ID NO:1-52, or a modified sequence thereof.

[0046] In some embodiments of this disclosure, the antisense chain consists of a sequence or fragment thereof as shown in any of SEQ ID NO:53-106, or a modified sequence thereof.

[0047] In some embodiments of this disclosure, the justice chain consists of a sequence or fragment thereof as shown in any of SEQ ID NO:1-52, or a modified sequence thereof.

[0048] In some preferred embodiments of this disclosure, 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 share 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 share a common motif UUCAAGUUACAAAAGCAAA (SEQ ID NO:187).

[0049] In some embodiments of this disclosure, the modified sequence of the antisense strand comprises a sequence or fragment thereof having any of the sequences shown in SEQ ID NO:138-183, 185-186.

[0050] In some embodiments of this disclosure, the modified sequence of the justice chain comprises a sequence or fragment thereof having any of the sequences shown in SEQ ID NO:107-137, 135, 184.

[0051] In some embodiments of this disclosure, the antisense chain consists of a sequence or fragment thereof as shown in any of SEQ ID NO:138-183, 185-186, or a modified sequence thereof.

[0052] In some embodiments of this disclosure, the justice chain consists of a sequence or fragment thereof as shown in any of SEQ ID NO:107-137, 135, 184, or a modified sequence thereof.

[0053] In some embodiments of this disclosure, the antisense strand is 19 to 23 nucleotides in length.

[0054] In some embodiments of this disclosure, the positive chain is 19 to 23 nucleotides in length.

[0055] In some embodiments of this disclosure, the oligonucleotide comprises a 3'-protruding sequence of one or more nucleotides in length, wherein the 3'-protruding sequence is present on the antisense strand and / or the sense strand.

[0056] In some embodiments of this disclosure, the antisense chain has a protruding end.

[0057] In some embodiments of this disclosure, the justice chain has a protruding end.

[0058] In some embodiments of this disclosure, the oligonucleotide comprises a 3'-overhead sequence of two nucleotides in length.

[0059] In some embodiments of this disclosure, the 3'-protruding end sequence is present on the justice chain; preferably, the protruding end sequence is selected from: GG, GA, GC, UC, UG, UU, UA, CA, CC, CG, CU, AA, AG, AU, AC.

[0060] In some embodiments of this disclosure, the 3'-protrusion sequence is present on the antisense chain; preferably, the protrusion sequence is selected from: UU, UC, UA, UG, GA, GG, GU, GC, TT, AG, AU, AA, AC, CA, CC, U; more preferably, the protrusion sequence is UU.

[0061] In some embodiments of this disclosure, the oligonucleotide comprises an antisense strand and a sense strand, each ranging in length from 19 to 23 nucleotides.

[0062] In some embodiments of this disclosure, the justice chain and the antisense chain form a bichain region.

[0063] In some embodiments of this disclosure, the justice chain and the antisense chain are respectively a double-chain structure with 19 / 21 pairing, 21 / 21 pairing, 21 / 23 pairing, or 23 / 23 pairing.

[0064] In some embodiments of this disclosure, the oligonucleotide includes a 3'-protrusion sequence of two nucleotides in length, wherein the 3'-protrusion sequence is present on the antisense strand, and wherein the sense strand is 19 nucleotides in length and the antisense strand is 21 nucleotides in length, such that the sense strand and the antisense strand form a doublet of 19 nucleotides in length.

[0065] The oligonucleotide comprises a 3'-protrusion sequence of two nucleotides in length, wherein the 3'-protrusion sequence is present on the antisense strand and the sense strand, and wherein the sense strand is 21 nucleotides in length and the antisense strand is 21 nucleotides in length, such that the sense strand and the antisense strand form a double helix of 19 nucleotides in length.

[0066] In some embodiments of this disclosure, the oligonucleotide comprises a 3'-protrusion sequence of two nucleotides in length, wherein the 3'-protrusion sequence is present on the antisense strand, and wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense strand and the antisense strand form a doublet of 21 nucleotides in length.

[0067] In some embodiments of this disclosure, the oligonucleotide is selected from any of the unmodified oligonucleotides described in the antisense strands SEQ ID NO:55-57, 61, 66, 70-71, 80, 82, 84, 93-106, and any of the modified oligonucleotides described in the antisense strands SEQ ID NO:138-183.

[0068] In some embodiments of this disclosure, the oligonucleotide is selected from any of the unmodified oligonucleotides described in antisense strands SEQ ID NO:80, 84, 93-95, 99-106, and any of the modified oligonucleotides described in antisense strands SEQ ID NO:145, 147-150, 154-183, 185-186.

[0069] In some embodiments of this disclosure, the oligonucleotide is selected from any of the unmodified oligonucleotides described in the positive strand SEQ ID NO:3-5, 9, 14, 18, 19, 28, 30, 32, 41-52, and any of the modified oligonucleotides described in the positive strand SEQ ID NO:107-137, 135, 184.

[0070] In some embodiments of this disclosure, the oligonucleotide comprises a 3'-protrusion sequence of two nucleotides in length, wherein the 3'-protrusion sequence is present on the antisense strand and the sense strand, and wherein the sense strand is 23 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense strand and the antisense strand form a doublet of 21 nucleotides in length.

[0071] In some embodiments of this disclosure, the oligonucleotide comprises at least one modified nucleotide.

[0072] In some embodiments of this disclosure, the modification is selected from the following: 2'-methoxy (m), 2'-deoxy-2'-fluorine (f), and thiophosphate (s).

[0073] In some embodiments of this disclosure, the modified nucleotide contains a 2'-modification.

[0074] In some embodiments of this disclosure, the 2'-modification is selected from the following modifications: 2'-aminoethyl, 2'-fluorine, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-β-d-arabinonucleotide.

[0075] In some embodiments of this disclosure, all nucleotides of the oligonucleotide are modified.

[0076] In some embodiments of this disclosure, the oligonucleotide comprises at least one modified internucleotide bond.

[0077] In some embodiments of this disclosure, the at least one modified nucleotide inter-bond is a phosphate thioester bond.

[0078] In some embodiments of this disclosure, the 4'-carbon of the sugar in the 5'-nucleotide of the antisense strand comprises a phosphate ester analog.

[0079] In some embodiments of this disclosure, the phosphate ester analog is an oxymethylphosphonate, a vinylphosphonate, or a malonylphosphonate.

[0080] In some embodiments of this disclosure, at least one nucleotide of the oligonucleotide is conjugated to one or more target ligands.

[0081] In some embodiments of this disclosure, each targeting ligand comprises a carbohydrate, amino sugar, cholesterol, peptide, or lipid.

[0082] In some embodiments of this disclosure, each targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

[0083] In some embodiments of this disclosure, the GalNac portion is a monovalent GalNAc portion, a divalent GalNAc portion, a trivalent GalNAc portion, or a tetravalent GalNAc portion.

[0084] In some embodiments of this disclosure, the oligonucleotide is selected from any one of the oligonucleotides described in AL0061001-AL0061054, AL0065001-AL0065051, and AL0067001-AL0067040.

[0085] In some preferred embodiments of this disclosure, the oligonucleotide is selected from any one of the oligonucleotides described in AL0067010, AL0067016, AL0067028, AL0067029, AL0067030, L0067031, L0067032, L0067033, L0067034, L0067035, AL0067036, AL0067037, AL0067038, AL0067039, and AL0067040.

[0086] In some embodiments of this disclosure, the sense chain comprises the motif sequence shown in SEQ ID NO:51; and the antisense chain comprises the motif sequence shown in SEQ ID NO:187.

[0087] In some preferred embodiments of this disclosure, the oligonucleotide comprises any of the following combinations of sense and antisense strands:

[0088] (1) The positive chain shown in SEQ ID NO:52 and the negative chain shown in SEQ ID NO:105;

[0089] (2) The positive chain shown in SEQ ID NO:135 and the negative chain shown in SEQ ID NO:176;

[0090] (3) The positive chain shown in SEQ ID NO:136 and the negative chain shown in SEQ ID NO:177;

[0091] (4) The positive chain shown in SEQ ID NO:137 and the negative chain shown in SEQ ID NO:178;

[0092] (5) The positive chain shown in SEQ ID NO:135 and the negative chain shown in SEQ ID NO:185;

[0093] (6) The positive chain shown in SEQ ID NO:135 and the negative chain shown in SEQ ID NO:186;

[0094] (7) The positive chain shown in SEQ ID NO:184 and the negative chain shown in SEQ ID NO:185;

[0095] (8) The justice chain shown in SEQ ID NO:184, and the antisense chain shown in SEQ ID NO:186; and

[0096] (9) The justice chain shown in SEQ ID NO:184 and the antisense chain shown in SEQ ID NO:176.

[0097] In some preferred embodiments of this disclosure, the oligonucleotide comprises any of the following combinations of sense and antisense strands:

[0098] (1) The positive chain shown in SEQ ID NO:52 and the negative chain shown in SEQ ID NO:106;

[0099] (2) The positive chain shown in SEQ ID NO:134 and the negative chain shown in SEQ ID NO:179;

[0100] (3) The positive chain shown in SEQ ID NO:135 and the negative chain shown in SEQ ID NO:180;

[0101] (4) The positive chain shown in SEQ ID NO:135 and the negative chain shown in SEQ ID NO:181;

[0102] (5) The positive chain shown in SEQ ID NO:136, and the negative chain shown in SEQ ID NO:182; and

[0103] (6) The justice chain shown in SEQ ID NO:137 and the antisense chain shown in SEQ ID NO:183.

[0104] In some preferred embodiments of this disclosure, the oligonucleotide comprises any of the following combinations of sense and antisense strands:

[0105] (1) The positive chain shown in SEQ ID NO:51 and the negative chain shown in SEQ ID NO:103;

[0106] (2) The positive chain shown in SEQ ID NO:127, and the negative chain shown in SEQ ID NO:158; and

[0107] (3) The positive chain shown in SEQ ID NO:132 and the negative chain shown in SEQ ID NO:164.

[0108] In another aspect, this disclosure provides a composition comprising the oligonucleotide or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

[0109] In some embodiments of this disclosure, the dosage form of the composition is an oral, intravenous, subcutaneous, or intramuscular injection.

[0110] In some preferred embodiments of this disclosure, the dosage form of the composition is a subcutaneous injection.

[0111] In some embodiments of this disclosure, the combination also includes other drugs for treating and / or preventing diseases related to lipid dysregulation.

[0112] In some preferred embodiments of this disclosure, the lipid dysregulation is hyperlipidemia, atherosclerosis, and / or one or more of its symptoms or complications.

[0113] In some preferred embodiments of this disclosure, the hyperlipidemia is hypercholesterolemia.

[0114] In some embodiments of this disclosure, the other drugs for treating hyperlipidemia include, but are not limited to, fibrates, statins, bile acid blockers, and niacin that are already used clinically.

[0115] In another aspect, this disclosure provides the use of the oligonucleotide or a pharmaceutically acceptable salt or composition thereof in the preparation of a medicament for treating and / or preventing diseases of lipid dysregulation.

[0116] In some embodiments of this disclosure, the lipid-related disorders are used for the treatment of hyperlipidemia, atherosclerosis, and / or one or more symptoms or complications thereof.

[0117] In some embodiments of this disclosure, the hyperlipidemia is hypercholesterolemia.

[0118] Various formulations have been developed to facilitate the use of oligonucleotides. For example, oligonucleotides can be delivered to a subject or cellular environment using formulations that minimize degradation, facilitate delivery and / or uptake, or provide another beneficial property to the oligonucleotide in the formulation. In some embodiments, compositions comprising oligonucleotides (e.g., single-stranded or double-stranded oligonucleotides) to reduce PCSK9 expression are provided herein. Such compositions can be suitably formulated such that, when administered to a subject (in the direct environment of target cells or systemically), a sufficient fraction of the oligonucleotide enters the cells to reduce PCSK9 expression. Any of a variety of suitable oligonucleotide formulations can be used to deliver oligonucleotides for reducing PCSK9, as disclosed herein. In some embodiments, the oligonucleotide is formulated in a buffer solution, such as an aqueous phosphate-buffered saline solution, liposomes, micelle structures, and shells. In some embodiments, the naked oligonucleotide or its conjugate is formulated in water or an aqueous solution (e.g., pH-adjusted water). In some embodiments, the naked oligonucleotide or its conjugate is formulated in an alkaline buffer solution (e.g., PBS).

[0119] Formulations of oligonucleotides containing cationic lipids can be used to promote the transfection of oligonucleotides into cells. For example, cationic lipids such as lipofectin, cationic glycerol derivatives, and polycationic molecules (e.g., polylysine) can be used.

[0120] Therefore, in some embodiments, the formulation comprises lipid nanoparticles. In some embodiments, the excipient comprises liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, or nanoparticles, or may be otherwise formulated for administration to the cells, tissues, organs, or body of a subject in need.

[0121] In some embodiments, the formulations disclosed herein include excipients. In some embodiments, the excipients impart to the composition enhanced stability, enhanced absorption, enhanced solubility, and / or therapeutic enhancement of the active ingredient. In some embodiments, the excipients are buffers (e.g., sodium citrate, sodium phosphate, tris base, or sodium hydroxide) or mediators (e.g., buffer solutions, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, the oligonucleotides are lyophilized to extend their shelf life and then formulated into solutions prior to use (e.g., administration to a subject). Therefore, the excipients in compositions comprising any of the oligonucleotides described herein may be lyophilization protectants (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or collapse temperature modifiers (e.g., dextran, ficoll, or gelatin).

[0122] In some embodiments, the pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Typically, the route of administration is intravenous or subcutaneous.

[0123] Pharmaceutical compositions suitable for injectable applications include sterile aqueous solutions (in the case of water solubility) or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. Suitable carriers for intravenous or subcutaneous administration include physiological saline, antibacterial water, Cremophor EL.™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. In many cases, isotonic agents, such as sugars, and polyols such as mannitol, sorbitol, and sodium chloride, are preferably included in the composition. Sterile injectable solutions can be prepared by incorporating the desired amount of oligonucleotides with one or a combination of the desired ingredients listed above into a selected solvent, followed by filtration sterilization.

[0124] In some embodiments, the composition may contain at least about 0.1% or more of a therapeutic agent (e.g., an oligonucleotide for reducing PCSK9 expression), although the percentage of one or more active ingredients may be between about 1% and about 80% or more of the total composition by weight or volume. Those skilled in the art of preparing such pharmaceutical formulations will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and therefore various dosages and treatment regimens may be desirable.

[0125] Even though many implementations involve liver-targeted delivery of any oligonucleotides disclosed herein, targeting other tissues is also considered.

[0126] In some embodiments, the administration of the oligonucleotide as described herein results in a reduction in the level of PCSK9 expression in cells. In some embodiments, the reduction in PCSK9 expression can be a reduction to 1% or less, 5% or less, 10% or less, 15% or less, 20% 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, 70% or less, 80% or less, or 90% or less compared to an appropriate control level of PCSK9. An appropriate control level can be the level of PCSK9 expression in cells or cell populations that have not been contacted with the oligonucleotide as described herein. In some embodiments, the effectiveness of delivering the oligonucleotide to cells according to the methods disclosed herein is evaluated after a limited time period. For example, the level of PCSK9 in cells can be analyzed at least 8 hours, 12 hours, 18 hours, 24 hours; or at least one, two, three, four, five, six, seven, or fourteen days after the introduction of the oligonucleotide into the cells.

[0127] In some embodiments, the oligonucleotide is delivered in the form of a transgene engineered to express the oligonucleotide disclosed herein (e.g., in the form of shRNA) in cells. In some embodiments, the oligonucleotide is delivered using a transgene engineered to express any of the oligonucleotides disclosed herein. The transgene can be delivered using a viral vector (e.g., adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, or herpes simplex virus) or a non-viral vector (e.g., plasmid or synthetic mRNA). In some embodiments, the transgene can be injected directly into a subject.

[0128] This disclosure relates to methods for reducing PCSK9 expression in the treatment of hypercholesterolemia, atherosclerosis, and / or one or more of their symptoms or complications in subjects. In some embodiments, the methods may include administering an effective amount of any of the oligonucleotides disclosed herein to a subject in need. In some embodiments, such treatment may be used, for example, to reduce or prevent hypercholesterolemia (high levels of low-density lipoprotein (LDL) cholesterol), atherosclerosis, coronary artery disease (e.g., coronary artery disease), angina, shortness of breath, sweating, nausea, dizziness, arrhythmia, palpitations, stroke (i.e., brain cell death due to insufficient blood and oxygen flow to the brain), weakness, confusion, difficulty speaking, dizziness, difficulty walking or standing, blurred vision, numbness in the face, arms, and legs, severe headache, loss of consciousness, peripheral artery disease, and / or kidney problems (e.g., chronic kidney disease). In some embodiments, such treatment may be used, for example, to treat or prevent one or more symptoms associated with hypercholesterolemia, atherosclerosis, and / or one or more of their symptoms or complications.

[0129] Therefore, in some embodiments, this disclosure provides a method for treating a subject who is at risk (or susceptible to) hypercholesterolemia, atherosclerosis, and / or one or more of its symptoms or complications, including coronary heart disease (e.g., coronary artery disease), angina, shortness of breath, sweating, nausea, dizziness, shortness of breath, arrhythmia, palpitations, stroke (i.e., brain cell death due to insufficient blood and oxygen flow to the brain), weakness, confusion, difficulty speaking, dizziness, difficulty walking or standing, blurred vision, numbness in the face, arms and legs, severe headache, loss of consciousness, peripheral artery disease, and / or kidney problems (e.g., chronic kidney disease)).

[0130] In some aspects, this disclosure provides methods for preventing diseases, conditions, symptoms, or illnesses as described herein in a subject by administering a therapeutic agent (e.g., an oligonucleotide or a vector or transgene encoding it) to the subject. In some embodiments, the subject to be treated is a subject who would benefit from a reduction in, for example, the amount of PCSK9 protein in the liver.

[0131] The methods described herein generally involve administering an effective amount (i.e., an amount capable of producing the desired therapeutic outcome) of an oligonucleotide to a subject. A therapeutically acceptable amount may be an amount capable of treating a disease or condition. The appropriate dose for any subject will depend on several factors, including the subject's body size, body surface area, age, the specific composition to be administered, one or more active ingredients in the composition, the time and route of administration, overall health, and any other medications administered concurrently.

[0132] In some embodiments, any of the compositions disclosed herein are administered to a subject via the enteral (e.g., orally, via a gastric feeding tube, via a duodenal feeding tube, via a gastrostomy, or via the rectum), parenteral (e.g., subcutaneous injection, intravenous injection or infusion, intra-arterial injection or infusion, intramuscular injection), local (e.g., epidermal, inhalation, via eye drops, or via mucous membranes), or by direct injection into a target organ (e.g., the subject's liver). Typically, the oligonucleotides disclosed herein are administered intravenously or subcutaneously.

[0133] In some embodiments, the oligonucleotide is administered at a dose ranging from 0.1 mg / kg to 25 mg / kg (e.g., 1 mg / kg to 5 mg / kg). In some embodiments, the oligonucleotide is administered at a dose ranging from 0.1 mg / kg to 5 mg / kg or from 0.5 mg / kg to 5 mg / kg.

[0134] As a set of non-limiting examples, the oligonucleotides disclosed herein will be applied typically once a year, twice a year, quarterly (once every three months), every two months (once every two months), monthly, or weekly.

[0135] In some implementations, the subject to be treated is a human (e.g., a human patient) or a non-human primate or other mammal subject. Other exemplary subjects include domestic animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and animals such as mice, rats, guinea pigs, and hamsters.

[0136] For the purpose of clarity and concise description, the features are described herein as part of some identical or separate embodiments; however, it will be understood that the scope of this disclosure may include some embodiments having a combination of all or some of the features described.

[0137] The present disclosure will now be described in more detail with reference to specific embodiments. However, the embodiments are for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0138] Example

[0139] Example 1. Screening of PCSK9-siRNA activity in in vitro liver cell lines

[0140] First, a computer-based algorithm was used to generate candidate oligonucleotide sequences complementary to human PCSK9 mRNA (NM_174936.3, Table 1). Some of these sequences were also complementary to cynomolgus monkey PCSK9 mRNA (XM_005543260.2, Table 1), and some were complementary to mouse PCSK9 mRNA (NM_153565.2, Table 1) or had no more than two mismatches. Some were designed as double-stranded siRNAs with 19 / 21 pairs of sense and antisense strands, with the antisense strand having two drooping ends complementary to the mRNA sequence. In some cases, the drooping ends of the antisense strand were non-complementary UU. Some sequences were designed as double-stranded siRNAs with 21 / 23 pairs of sense and antisense strands, with the antisense strand having two drooping ends complementary to the mRNA sequence. Some sequences were designed as double-stranded siRNAs with 21 / 21 and 23 / 23 pairs. In some complementary pairing sequences, the first base at the 5' end of the antisense strand (the last base at the 3' end of the sense strand) is replaced with a base that does not match PCSK9 mRNA. In some sequences, individual ribonucleic acid molecules are replaced with deoxyribonucleic acid molecules, such as T or dA.

[0141] Table 1: PCSK9 mRNA sequences from humans, cynomolgus monkeys, and mice

[0142] people NM_174936.3 Crab-eating macaques XM_005543260.2 mice NM_153565.2

[0143] In Tables 2, 3 and 4, “G”, “C”, “A”, “U” and “T” usually represent nucleotides with guanine, cytosine, adenine, uracil and thymine as bases, respectively.

[0144] Modifications: d represents DNA; dT represents 2'-deoxythymidine; dA represents 2'-deoxyadenosine; m represents 2'-methoxy; f represents 2'-deoxy-2'-fluorine; s represents thiophosphate; L represents ligand, L96 is N-[tris(GalNAc-alkyl)amidodecyl]-4-hydroxyprolylHyp-(GalNAc-alkyl)3.

[0145] Table 2: Naked Oligonucleotide Sequences

[0146]

[0147]

[0148] Table 3: Modified oligonucleotides

[0149]

[0150]

[0151]

[0152] Table 4: siRNA sequences with vectors

[0153]

[0154]

[0155] (1) Huh7 / HepG2 / HLE cell culture and transfection:

[0156] Human liver cancer cell lines Huh7 (CCTCC, GDC0134), HepG2 (Jiangsu Kaiji Biotechnology Co., Ltd., KG020), and HLE (JCRB Cell Bank, JCRB0404) were used and incubated at 37°C in a 5% CO2 incubator. The cells were cultured in DMEM basal medium (Hyclone, SH30022.01) supplemented with 10% FBS (aqlabteech, AQ-MV-06600) and 1% penicillin-streptomycin (Kaiji Biotechnology, KGY0023). When cell confluence reached 90%, the cells were digested with trypsin (Amresco, 0458-250G), and counted using a cell counter (Nexcelom, cellometer Mini). Huh7 cells were diluted to 2*10^5 / ml, and the cell suspension was added to 12-well plates. Transfection was performed the following day after cell adhesion. Lipofectamine was used. TM Transfection was performed using 3000 Transfection Reagent (thermofisher, L3000150). System ① was diluted with 50 μl Opti-MEM (thermofisher, 1105821) to contain 50 nM siRNA, and system ② was diluted with 50 μl Opti-MEM to contain 3 μl Lip3000. After each system was allowed to stand for 5 min, system ① and system ② were mixed and allowed to stand for another 15 min. The mixture was then added to 12-well plates. After 4 h of transfection, the medium was replaced with DMEM / F12 complete medium, and the 12-well plates were incubated in an incubator for 48 h.

[0157] (2) Total RNA was extracted using the RNA-Quick Purification Kit (RN001, Yishan Biotechnology):

[0158] Remove the 12-well plate from the incubator, aspirate the culture medium, wash once with an appropriate amount of PBS, and add 500 μl of lysis buffer to each well. Transfer the supernatant to a new 1.5 ml centrifuge tube. Add 500 μl of anhydrous ethanol to the lysed cells and mix thoroughly (if precipitation occurs, this is normal; continue the procedure). Invert the centrifuge tube several times, or use a pipette to forcefully aspirate and blow 10 times to disperse any precipitate. Then add the liquid to the centrifuge column, place the centrifuge tube symmetrically in a centrifuge (Eppendorf, 5430), and centrifuge at 4000 × g for 1 min. Remove the centrifuge tube, add 500 μl of wash buffer to the column, and centrifuge at 12000 × g for 1 min. After centrifugation, be careful not to let the waste liquid in the collection tube come into contact with the RNA column to avoid contamination. Discard the waste liquid, put the RNA column back into the collection tube, and centrifuge the empty tube once to completely remove any residual wash buffer. Place the column on a clean, RNase-free 1.5 ml centrifuge tube, open the cap, and let it air dry for 2 minutes. Add 30 μl of elution buffer to the center of the RNA column membrane, incubate at room temperature for 2 minutes, centrifuge at 2000×g for 1 min to elute the RNA, and then place on ice. Measure the concentration of the eluted RNA for subsequent experiments. The extracted RNA can be used immediately for subsequent experiments or stored at -80℃ for later use.

[0159] (3) Use IIQ RT SuperMix for qPCR (+gDNAwiper) Reverse Transcription Kit (Novazia, R223-01) for cDNA synthesis:

[0160] Prepare a mixture in an RNase-free centrifuge tube: 4 μl 4×g DNA wiper Mix, 1 μg template RNA, and RNase-free ddH2O to a final volume of 16 μl to remove genomic DNA. Gently pipette to mix and incubate at 42°C for 2 min. Then, directly add 4 μl 5×HiScript II qRT SuperMix II to the reaction tube and gently pipette to mix. Incubate in a PCR instrument (Applied Biosystems, 9700) at 50°C for 15 min, then at 85°C for 5 sec, and finally at 4°C. The product can be used immediately for qPCR or stored at -20°C and used within six months. For long-term storage, aliquot and store at -80°C. Avoid repeated freeze-thaw cycles for cDNA.

[0161] (4) Quantitative PCR was performed using ChamQ SYBR qPCR Master Mix (Novazia, Q311-02):

[0162] Prepare a 20 μl mixture by adding 10 μl 2×ChamQ SYBR qPCR Master Mix, 0.5 μl Forward primer (Ruibo Kexing), 0.5 μl Reverse primer (Ruibo Kexing), 1 μl Template cDNA, and 8 μl ddH2O. Each sample was tested in triplicate. The 96-well plate was placed in a qPCR instrument (ROCGENE, Archimed). The following program was executed: pre-deformation, 95℃, 30 sec; amplification, 95℃, 10 sec, 60℃, 30 sec, 40 cycles; melting curve, 95℃, 15 sec, 60℃, 60 sec, 95℃, 15 sec.

[0163] (5) Perform statistical analysis using GraphPad Prism 8:

[0164] Export the data to Excel format using CT. PCSK9 -CT GAPDH The control group was normalized. To calculate the fold change in relative silencing efficiency, the data were analyzed using the ΔΔCT method. The three parallel replicate datasets were imported into GraphPad Prism 8, and a column model was applied for histogram analysis. The results are shown in [Figure number missing]. Figure 1 and Figure 2 .

[0165] Depend on Figure 1 It is evident that, at a dosage of 50 nM, AL0061001-AL0061040 significantly inhibited the expression level of the PCSK9 gene in Huh7 cell line; AL0061003, AL0061004, AL0061005, AL0061009, AL0061014, AL0061015, AL0061018-AL0061022, AL0061028, AL0061030, AL0061031, AL0061032, and AL0061034 significantly inhibited the expression level of the PCSK9 gene in HepG2 cell line.

[0166] Depend on Figure 2It is evident that, at a dosage of 50 nM, all candidate compounds significantly inhibited the expression level of the PCSK9 gene in Huh7 and Hle cell lines; in particular, AL0061005, AL0061018, AL0061028, AL0061030, AL0061032, AL0061041, AL0061042, AL0061044, AL0061051, AL0061052, AL0061053, and AL0061054 all achieved an inhibition rate of over 90% on the PCSK9 gene expression level in Huh7 and Hle cell lines.

[0167] Example 2. Screening of the in vitro activity of chemically modified PCSK9-siRNA in primary monkey hepatocytes

[0168] (1) Cynomolgus monkey hepatocyte culture and transfection:

[0169] Using cynomolgus monkey hepatocytes (Beijing Ruide Biotechnology Co., Ltd., cmTCSC), the culture medium was preheated. The thawed culture medium (Beijing Ruide Biotechnology Co., Ltd., HEPO24) was then transferred to a biosafety cabinet. 4 mL of FBS was added to 36 mL of thawed culture medium (TPCS, HEPO24) to prepare a completely thawed medium, which was then heated in a 37°C water bath for 10 minutes. The cells were then treated with coating medium (Beijing Ruide Biotechnology Co., Ltd., HEPO44) in a CO2 incubator at 37°C for 0.5 h. Cells were removed from liquid nitrogen and thawed in a 37°C water bath for approximately 2 minutes. The cell suspension was then transferred to 40 mL of preheated thawed culture medium. The cryovials were washed with 2 mL of completely thawed culture medium. The cell suspension was centrifuged at 180 × g for 1 min, the supernatant was discarded, and 2 mL of preheated CM seeding medium (Beijing Ruide Biotechnology Co., Ltd., CMHEP054) was added. The cell suspension was gently dispersed and mixed. 20 μL of the cell suspension was used for cell counting. Based on the counting results, CM seeding medium was inoculated into 12-well plates at 3*10^5 / well and incubated at 37℃ with 5% CO2. After 4-5 hours of adhesion, the medium was aspirated and replaced with preheated medium (Beijing Ruide Biotechnology Co., Ltd., CMHEP064). Transfection was performed 6 hours after adhesion. Lipofectamine was used. TMTransfection was performed using 3000 TransfectionReagent (thermofisher, L3000150). System ① was diluted with 50 μl Opti-MEM (thermofisher, 1105821) to obtain 50 nM modified siRNA (Suzhou Beixin Biotechnology Co., Ltd.). System ② was diluted with 50 μl Opti-MEM to obtain 3 μl Lip3000. After standing for 5 min, systems ① and ② were mixed and stood for another 15 min. The mixture was then added to 12-well plates. After 4 h of transfection, the medium was replaced with DMEM / F12 complete medium, and the 12-well plates were incubated in an incubator for 48 h.

[0170] (2) Total RNA was extracted using the RNA-Quick Purification Kit (RN001, Yishan Biotechnology):

[0171] Remove the 12-well plate from the incubator, aspirate the culture medium, wash once with an appropriate amount of PBS, and add 500 μl of lysis buffer to each well. Transfer the supernatant to a new 1.5 ml centrifuge tube. Add 500 μl of anhydrous ethanol to the lysed cells and mix thoroughly (if precipitation occurs, this is normal; continue the procedure). Invert the centrifuge tube several times, or use a pipette to forcefully aspirate and blow 10 times to disperse any precipitate. Then add the liquid to the centrifuge column, place the centrifuge tube symmetrically in a centrifuge (Eppendorf, 5430), and centrifuge at 4000 × g for 1 min. Remove the centrifuge tube, add 500 μl of wash buffer to the column, and centrifuge at 12000 × g for 1 min. After centrifugation, be careful not to let the waste liquid in the collection tube come into contact with the RNA column to avoid contamination. Discard the waste liquid, put the RNA column back into the collection tube, and centrifuge the empty tube once to completely remove any residual wash buffer. Place the column on a clean, RNase-free 1.5 ml centrifuge tube, open the cap, and let it air dry for 2 minutes. Add 30 μl of elution buffer to the center of the RNA column membrane, incubate at room temperature for 2 minutes, centrifuge at 2000×g for 1 min to elute the RNA, and then place on ice. Measure the concentration of the eluted RNA for subsequent experiments. The extracted RNA can be used immediately for subsequent experiments or stored at -80℃ for later use.

[0172] (3) Use II. Synthesize cDNA using the QRT SuperMix for qPCR (+gDNAwiper) reverse transcription kit (Novazia, R223-01):

[0173] Prepare a mixture in an RNase-free centrifuge tube: 4 μl 4×g DNA wiper Mix, 1 μg template RNA, and RNase-free ddH2O to a final volume of 16 μl to remove genomic DNA. Gently pipette to mix and incubate at 42°C for 2 min. Then, directly add 4 μl 5×HiScript II qRT SuperMix II to the reaction tube and gently pipette to mix. Incubate in a PCR instrument (Applied Biosystems, 9700) at 50°C for 15 min, then at 85°C for 5 sec, and finally at 4°C. The product can be used immediately for qPCR or stored at -20°C and used within six months. For long-term storage, aliquot and store at -80°C. Avoid repeated freeze-thaw cycles for cDNA.

[0174] (4) Quantitative PCR was performed using ChamQ SYBR qPCR Master Mix (Novazia, Q311-02):

[0175] Prepare a 20 μl mixture by adding 10 μl 2×ChamQ SYBR qPCR Master Mix, 0.5 μl Forward primer (Ruibo Kexing), 0.5 μl Reverse primer (Ruibo Kexing), 1 μl Template cDNA, and 8 μl ddH2O. Each sample was tested in triplicate. The 96-well plate was placed in a qPCR instrument (ROCGENE, Archimed). The following program was executed: pre-deformation, 95℃, 30 sec; amplification, 95℃, 10 sec, 60℃, 30 sec, 40 cycles; melting curve, 95℃, 15 sec, 60℃, 60 sec, 95℃, 15 sec.

[0176] (5) Perform statistical analysis using GraphPad Prism 8:

[0177] Export the data to Excel format using CT. PCSK9 -CT GAPDH The control group was normalized. To calculate the fold change in relative silencing efficiency, the data were analyzed using the ΔΔCT method. The three parallel replicate datasets were imported into GraphPad Prism 8, and a column model was applied for histogram analysis. The results are shown in [Figure number missing]. Figure 3 and Figure 4 .

[0178] Depend on Figure 3It is evident that at a dosage of 50 nM, AL0065004, AL0065005, AL0065006, AL0065008, AL0065009, and AL0065010 significantly inhibited PCSK9 gene expression in cynomolgus monkey hepatocytes, with AL0065006, AL0065008, and AL0065010 achieving an inhibition rate of approximately 80%.

[0179] Depend on Figure 4 At a dosage of 50 nM, AL0065004, AL0065006, AL0065008, AL0065009, and AL0065010-AL0065020 showed significant inhibitory effects on PCSK9 gene expression in cynomolgus monkey hepatocytes, with inhibition rates all exceeding 70%. Among them, AL0065012, AL0065017, AL0065018, AL0065019, and AL0065020 achieved inhibition rates exceeding 90%.

[0180] Example 3. In vivo testing of PCSK9 RNAi agent in mice

[0181] SPF-grade male C57BL / 6 mice, aged 6–8 weeks (Speford (Beijing) Biotechnology Co., Ltd.), were used in the experiment. Serum samples were obtained on day 0 of drug administration before administration, and mice were randomly assigned to groups according to LDL-c levels. C57BL / 6 mice were given a single subcutaneous injection of 9 mg / kg PCSK9 RNAi agent and physiological saline (NC, negative control). Blood samples were collected from mice on days 8, 15, 22, and 29 after drug administration (for the NC / PC / AL007002 / AL007010 group, monitoring continued until day 36 after drug administration) (blood was collected from the eyeball and sent for testing within 1 hour after collection). The levels of TCHO (total cholesterol), TG (triglycerides), HDL-C (high-density lipoprotein cholesterol), and LDL-C (low-density lipoprotein cholesterol) in the blood were examined. During the experiment, no animals showed signs of death or impending death. No obvious abnormalities were observed in any animals during clinical observation.

[0182] Experimental groups and data for each test are shown in Tables 5-12; LDL-C change levels are shown in... Figure 5 As shown.

[0183] Table 5: Dosing Group Table

[0184] 1 NC 9mg / kg Single subcutaneous injection 2 AL0067002 9mg / kg Single subcutaneous injection 3 AL0067003 9mg / kg Single subcutaneous injection 4 AL0067004 9mg / kg Single subcutaneous injection 5 AL0067005 9mg / kg Single subcutaneous injection 6 AL0067006 9mg / kg Single subcutaneous injection 7 AL0067007 9mg / kg Single subcutaneous injection 8 AL0067008 9mg / kg Single subcutaneous injection 9 AL0067009 9mg / kg Single subcutaneous injection 10 AL0067010 9mg / kg Single subcutaneous injection

[0185] Table 6: Blood lipid levels in animals grouped (n=7)

[0186]

[0187]

[0188] Note: Compared with the NC group, *: p<0.05; **: p<0.01; ***: p<0.001

[0189] Table 7: Changes in blood lipids in animals on day 8 after drug administration (n=7)

[0190]

[0191] Note: Compared with the NC group, *: p<0.05; **: p<0.01; ***: p<0.001

[0192] Table 8: Changes in blood lipids in animals on day 15 after drug administration (n=7)

[0193]

[0194] Note: Compared with the NC group, *: p<0.05; **: p<0.01; ***: p<0.001

[0195] Table 9: Changes in blood lipids in animals on day 22 after drug administration (n=7)

[0196]

[0197] Note: Compared with the NC group, *: p<0.05; **: p<0.01; ***: p<0.001

[0198] Table 10: Changes in blood lipids in animals on day 29 after drug administration (n=7)

[0199]

[0200]

[0201] Note: Compared with the NC group, *: p<0.05; **: p<0.01; ***: p<0.001

[0202] Table 11: Changes in blood lipids in animals on day 36 after drug administration (n=7)

[0203]

[0204] Note: Compared with the NC group, *: p<0.05; **: p<0.01; ***: p<0.001

[0205] Table 12: Changes in blood lipids in animals on day 43 after drug administration (n=7)

[0206]

[0207] Note: Compared with the NC group, *: p<0.05; **: p<0.01; ***: p<0.001

[0208] From Table 5-12 and Figure 5 It can be concluded that, compared with the solvent control group, the drug intervention groups AL0067002, AL0067006, AL0067007, and AL0067010 all significantly reduced the LDL-C level in the blood of C57 mice on day 8 after drug intervention, with the most significant and longest sustained reduction observed in the AL0067010 group. It can be seen that the AL0067010 drug intervention group significantly reduced the LDL-C level in the blood of C57 mice, reaching the lowest value on Day 8, and then gradually and slowly rising. At Day 8, Day 15, Day 22, Day 29, Day 36, and Day 43, the blood LDL-C levels in the AL0067010 treatment group were significantly different from those in the solvent control group (Day 8, p<0.001; Day 15, p<0.001; Day 22, p<0.001; Day 29, p<0.001; Day 36, p<0.001; Day 43, p<0.01). After drug intervention, the LDL-C levels in the blood of mice in the AL0067010 treatment group showed a good lipid-lowering effect compared with the solvent control group (NC group) in the first three weeks, decreasing by 51.43%, 38.71%, and 40.63%, respectively.

[0209] Example 4. In vivo testing of PCSK9 RNAi agent in mice

[0210] SPF-grade male C57BL / 6 mice, aged 6–8 weeks (Speford (Beijing) Biotechnology Co., Ltd.), were used in the experiment. Serum samples were obtained on day 0 of drug administration before administration, and mice were randomly assigned to groups according to LDL-c levels. C57BL / 6 mice were given a single subcutaneous injection of 9 mg / kg PCSK9 RNAi agent and physiological saline (NC, negative control). Blood samples were collected from mice on days 8, 15, 22, 29, 36, and 43 after drug administration (blood was collected from the eyeball and sent for testing within 1 hour after collection) to examine the levels of TCHO (total cholesterol), TG (triglycerides), HDL-C (high-density lipoprotein cholesterol), and LDL-C (low-density lipoprotein cholesterol). During the experiment, no obvious abnormalities were observed in any of the animals during clinical observation.

[0211] Experimental groups and data for each test are shown in Tables 13-19; LDL-C change levels are shown in... Figure 6 As shown.

[0212] Table 13 Changes in blood lipids in animals grouped (n=5)

[0213]

[0214] Note: There were no significant differences compared with the NC group.

[0215] Table 14 Changes in blood lipids in animals on day 8 after administration of the test substance (n=5)

[0216]

[0217]

[0218] Note: Compared with N groups, *: p < 0.05; **: p < 0.01; ***: p < 0.001

[0219] Table 15 Changes in blood lipids in animals on day 15 after administration of the test substance (n=5)

[0220]

[0221] Note: Compared with the NC group, *: p<0.05; **: p<0.01; ***: p<0.001

[0222] Table 16 Changes in blood lipids in animals on day 22 after administration of the test substance (n=5)

[0223]

[0224] Note: Compared with the NC group, *: p<0.05; **: p<0.01; ***: p<0.001

[0225] Table 17 Changes in blood lipids in animals on day 29 after administration of the test substance (n=5)

[0226]

[0227]

[0228] Note: Compared with the NC group, *: p<0.05; **: p<0.01; ***: p<0.001

[0229] Table 18 Changes in blood lipids in animals on day 36 after administration of the test substance (n=5)

[0230]

[0231] Note: Compared with the NC group, *: p<0.05; **: p<0.01; ***: p<0.001

[0232] Table 19 Changes in blood lipids in animals on day 43 after administration of the test substance (n=5)

[0233]

[0234] Note: Compared with the NC group, *: p<0.05; **: p<0.01; ***: p<0.001

[0235] From Tables 13-19 and Figure 6It can be concluded that, compared with the solvent control group, on day 8 after administration of the test substance, the serum TCHO, HDL-C, and LDL-C levels in groups AL0067010, AL0067011, AL0067012, AL0067013, AL0067015, AL0067016, AL0067017, and AL0067018 were significantly to extremely significantly decreased (p<0.05, p<0.01, or p<0.001), while the serum TCHO and LDL-C levels in groups AL0067002 and AL0067019 were significantly decreased (p<0.01). On day 15, the serum TCHO and LDL-C levels in groups AL0067002, AL0067010, AL0067011, and AL0067018 were significantly decreased (p<0.01). In groups 67012, AL0067013, AL0067015, AL0067016, AL0067017, AL0067018, and AL00670019, serum TCHO, HDL-C, and LDL-C levels were significantly or extremely significantly decreased (p<0.01 or p<0.001). On day 22, in groups AL0067002, AL0067010, AL0067011, AL0067012, AL0067015, AL0067016, AL0067017, AL0067018, and AL00670019, serum TCHO, HDL-C, and LDL-C levels were significantly to extremely significantly decreased (p<0.001). 5 or p<0.01 or p<0.001), serum TCHO and LDL-C in animals in group AL00670013 were significantly or significantly decreased (p<0.05 or p<0.01); serum LDL-C in animals in group AL0067014 was significantly decreased (p<0.05); on day 29, serum TCHO, HDL-C, and LDL-C in animals in groups AL0067002, AL0067010, AL0067011, AL0067015, AL0067016, AL0067017, and AL0067018 were significantly to extremely significantly decreased (p<0.05 or p<0.01 or p<0.001), and serum TCHO, HDL-C, and LDL-C in animals in groups AL0067012 and AL0067018 were significantly to extremely significantly decreased (p<0.05 or p<0.01 or p<0.001), and serum TCHO, HDL-C, and LDL-C in animals in groups AL00670013 were significantly or extremely significantly decreased (p<0.05 or p<0.01 or p<0.001), and serum TCHO, HDL-C, and LDL-C in animals in groups AL00670 ...3 were significantly or extremely significantly decreased (p<0.0 In groups 0013 and AL00670019, serum LDL-C levels were significantly or substantially decreased (p<0.05 or p<0.01); on day 36, serum TCHO (except in groups AL0067013 and AL00670019), TG (except in group AL0067011), HDL-C, and LDL-C levels were significantly to substantially decreased (p<0.05 or p<0.01 or p<0.01).On day 43, serum TCHO, HDL-C, and LDL-C levels in animals in groups AL0067002 and AL0067011 were significantly to extremely significantly decreased (p<0.05, p<0.01, or p<0.001); serum LDL-C levels in animals in groups AL0067010, AL0067016, and AL0067019 were significantly or substantially decreased (p<0.05 or p<0.01); and serum HDL-C and LDL-C levels in animals in groups AL0067012 and AL0067015 were significantly or substantially decreased (p<0.05 or p<0.01).

[0236] Example 5. In vivo testing of PCSK9 RNAi agent in mice

[0237] SPF-grade male C57BL / 6 mice, aged 6–8 weeks (Speford (Beijing) Biotechnology Co., Ltd.), were used in the experiment. Serum samples were obtained on day 0 of drug administration before administration, and mice were randomly assigned to groups according to LDL-c levels. C57BL / 6 mice were administered a single subcutaneous dose of 9 mg / kg PCSK9 RNAi agent and physiological saline (NC, negative control). Blood samples were collected from mice on days 8, 15, 22, 29, 37, 43, and 50 after drug administration (blood was collected from the eyeball and sent for testing within 1 hour of collection) to examine the levels of TCHO (total cholesterol), TG (triglycerides), HDL-C (high-density lipoprotein cholesterol), and LDL-C (low-density lipoprotein cholesterol). During the experiment, no animals showed signs of death or impending death. No significant abnormalities were observed in any animal during clinical observation. Experimental groupings and data for each test are shown in Tables 20-19; changes in LDL-C levels are shown in... Figure 7 As shown.

[0238] Table 20 Changes in blood lipids when animals were grouped

[0239]

[0240] Note: There were no significant differences compared with the NC group.

[0241] Table 21 Changes in blood lipids in animals on day 8 after administration of the test substance.

[0242]

[0243] Note: Compared with the NC group, *: p<0.05; **: p<0.01; ***: p<0.001

[0244] Table 22 Changes in blood lipids in animals on day 15 after administration of the test substance.

[0245]

[0246] Note: Compared with the NC group, *: p<0.05; **: p<0.01; ***: p<0.001

[0247] Table 23 Changes in blood lipids in animals on day 22 after administration of the test substance.

[0248]

[0249] Note: Compared with the NC group, *: p<0.05; **: p<0.01; ***: p<0.001

[0250] Table 24 Changes in blood lipids in animals on day 29 after administration of the test substance.

[0251]

[0252] Note: Compared with the NC group, *: p<0.05; **: p<0.01; ***: p<0.001

[0253] Table 25 Changes in blood lipids in animals on day 37 after administration of the test substance.

[0254]

[0255]

[0256] Note: Compared with the NC group, *: p<0.05; **: p<0.01; ***: p<0.001

[0257] Table 26 Changes in blood lipids in animals on day 43 after administration of the test substance.

[0258]

[0259] Note: Compared with the NC group, *: p<0.05; **: p<0.01; ***: p<0.001

[0260] Table 27 Changes in blood lipids in animals on day 50 after administration of the test substance.

[0261]

[0262] Note: Compared with the NC group, *: p<0.05; **: p<0.01; ***: p<0.001

[0263] From Tables 20-27 and Figure 7It can be concluded that, compared with the solvent control group, the serum TCHO, HDL-C, and LDL-C levels in the test substance groups (AL0067017, AL0067023, AL0067028, AL0067033, AL0067010) were significantly to extremely significantly reduced (p<0.05, p<0.01, or p<0.001) on days 8, 15, 22, 29, 37, and 43 after administration of the test substance. On day 50, the serum TCHO, HDL-C, and LDL-C levels in the AL0067028 and AL0067033 groups were significantly to extremely significantly reduced (p<0.05, p<0.01, or p<0.001), while there was no significant statistical difference in the AL0067010 group.

[0264] Example 6. In vivo testing of PCSK9 RNAi agent in hPCSK9 mice

[0265] SPF-grade male hPCSK9 mice, aged 5–6 weeks (Shanghai Southern Model Biotechnology Development Co., Ltd.), were used in the experiment. Serum samples were obtained on day 0 of drug administration before administration, and mice were randomly assigned to groups according to body weight. hPCSK9 mice were administered a single subcutaneous dose of 9 mg / kg PCSK9 RNAi agent and physiological saline (NC, negative control). Blood samples were collected from mice on days 8, 15, 22, and 29 post-administration (ocular blood samples were collected and sent for testing within 1 hour of collection) to examine LDL-C (low-density lipoprotein cholesterol) levels in the blood, and PCSK9 protein expression was detected using an ELISA (R&D) kit.

[0266] The data from each test are shown in Tables 28 and 29; changes in LDL-C and PCSK9 protein levels are shown in... Figure 8 , Figure 9 middle.

[0267] Table 28 Changes in human PCSK levels in animal serum (ng / mL)

[0268]

[0269] Note: Compared with the NC group, *: p<0.05; **: p<0.01; ***: p<0.001

[0270] Table 29 Changes in serum LDL-C levels in animals (mmol / L)

[0271]

[0272] Note: Compared with the NC group, *: p<0.05; **: p<0.01; ***: p<0.001

[0273] like Figure 8As shown, for PCSK9 protein levels, all RNAi groups (AL0067001, AL0067010, AL0067016, AL0067028) showed significant reductions in the first two weeks (Day 8 and Day 15) compared to before autologous administration (Day 0). In particular, a single dose in the AL0067016 and AL0067028 groups reduced PCSK9 protein by more than 80% for three consecutive weeks, and the reduction in PCSK9 protein remained at around 60% after about one month of administration (Day 29).

[0274] like Figure 9 As shown, regarding LDL-C expression levels, compared to before autologous administration (Day 0), except for the AL0067001 group which showed virtually no significant change, the other RNAi groups all exhibited highly significant decreases throughout the experimental period. Specifically, AL0067010 decreased to 53.79%, 45.45%, 43.94%, and 46.97% on Day 8, Day 15, Day 22, and Day 29, respectively; AL0067016 decreased to 53.16%, 56.96%, 62.03%, and 51.90%, respectively; and AL0068028 decreased to 57.41%, 48.15%, 51.85%, and 65.74%, respectively.

[0275] Example 7. Study on the testing of PCSK9 RNAi agent in cynomolgus monkeys.

[0276] The experiment used male cynomolgus monkeys aged 12-23 years, with LDL-C levels ranging from 0.82 mmol / L to 2.10 mmol / L, essentially in a state of dyslipidemia. Serum samples were obtained 14 days, 7 days before administration, and on the day of administration (day 1), and the monkeys were randomly assigned to groups according to their LDL-C levels. The monkeys received a single subcutaneous administration of 9 mg / kg of PCSK9 RNAi. Blood samples were collected on days 8, 15, 22, 29, 34, 44, 51, 58 (ends at AL0067001 and AL0067010), 65, 72, and 79 post-administration to examine TCHO (total cholesterol) and LDL-C (low-density lipoprotein cholesterol) levels.

[0277] The changes in LDL-C and TCHO levels are shown in Figure 10 , Figure 11 middle.

[0278] like Figure 10As shown, compared with before administration (day 1), RNAi agents AL0067001, AL0067010, AL0067016, and AL0067028 significantly reduced LDL-C levels in cynomolgus monkeys. Among them, AL0067001 and AL0067010 maintained an inhibition efficiency of about 40% for 51 consecutive days. More significantly, AL0067016 and AL0067028 maintained an inhibition efficiency of over 40% for LDL-C in cynomolgus monkeys throughout the entire experimental period (day 79).

[0279] like Figure 11 As shown, RNAi agents AL0067001, AL0067010, AL0067016, and AL0067028 significantly reduced TCHO levels in cynomolgus monkeys. AL0067001 and AL0067010 maintained an inhibition rate of approximately 20% for LDL-C levels in cynomolgus monkeys for 51 consecutive days. More significantly, AL0067016 and AL0067028 maintained an inhibition rate of over 20% for TCHO in cynomolgus monkeys throughout the entire experimental period (day 79). The AL0067028 group maintained an inhibition rate of approximately 40% for TCHO levels from day 29 to day 51.

[0280] Example 8. In vivo testing of PCSK9 RNAi agent in C57 mice

[0281] Based on the research in Example 7, we explored the optimization of the modification combination based on AL0067028 and evaluated the research effect of the new RNAi agent on C57 mice.

[0282] SPF-grade male C57BL / 6 mice, aged 6–8 weeks (Speford (Beijing) Biotechnology Co., Ltd.), were used in the experiment. Serum samples were obtained on day 0 of drug administration before administration, and mice were randomly assigned to groups according to LDL-c levels. C57BL / 6 mice were administered a single subcutaneous dose of 6 mg / kg PCSK9 RNAi agent and physiological saline (NC, negative control). Blood samples were collected from mice on days 7, 16, 23, 30, 37, 44, and 51 after drug administration (blood was collected from the eye and sent for testing within 1 hour after collection) to examine LDL-C (low-density lipoprotein cholesterol) levels in the blood, and PCSK9 protein expression was detected using an ELISA (R&D) kit. During the experiment, no animals showed signs of death or impending death. No obvious abnormalities were observed in any animals during clinical observation.

[0283] Experimental groups and data for each test are shown in Table 30; changes in LDL-C levels and PCSK9 protein levels are shown in... Figure 12 , Figure 13 middle.

[0284] Table 30 Changes in serum LDL-C levels in animals (mmol / L)

[0285]

[0286] Compared with the NC group, *: p<0.05; **: p<0.01; ***: p<0.001

[0287] like Figure 12 As shown, all RNAi groups (AL0067036, AL0067037, AL0067038, AL0067039, AL0067040, AL0067028) significantly reduced LDL-C levels before Day 37. The significant reduction in LDL-C levels in groups AL0067037 and AL0067040 lasted up to Day 51. Furthermore, the reduction in LDL-C levels in groups AL0067037 and AL0067040 was significantly better than that in group AL0067028 throughout the entire experimental period, and the difference was statistically significant (P<0.05).

[0288] like Figure 13 As shown, for PCSK9 protein levels, all RNAi groups (AL0067036, AL0067037, AL0067038, AL0067039, AL0067040, AL0067028) showed significant reductions throughout the entire experimental period (Day 51) compared to pre-administration levels (Day 0). Particularly significant was the reduction of PCSK9 protein by more than 85% with a single dose in group AL0067037 for four consecutive weeks (Day 30), with a reduction exceeding 90% by day 16 post-administration. Throughout the experimental period, the PCSK9 protein reduction effect and duration of reduction in group AL0067037 were significantly superior to those in group AL0067028.

Claims

1. An oligonucleotide or a pharmaceutically acceptable salt thereof for reducing PCSK9 expression, said oligonucleotide comprising an antisense strand and a sense strand, said antisense strand being a modified sequence as shown in SEQ ID NO: 84; said sense strand being a modified sequence as shown in SEQ ID NO: 32; The antisense strand has a 3'-protrusion of two nucleotides in length; the protrusion sequence is GU; The sense strand is 21 nucleotides long and the antisense strand is 23 nucleotides long, such that the sense strand and the antisense strand form a double strand of 21 nucleotides in length; The combination of the sense and antisense strands of the oligonucleotide is selected from any of the following: (1) The positive chain shown in SEQ ID NO: 116 and the negative chain shown in SEQ ID NO: 147; (2) The positive chain shown in SEQ ID NO: 128 and the negative chain shown in SEQ ID NO: 159; (3) The positive chain shown in SEQ ID NO: 128 and the negative chain shown in SEQ ID NO: 160; or (4) The justice chain shown in SEQ ID NO: 129 and the antisense chain shown in SEQ ID NO:

161.

2. The oligonucleotide of claim 1 or a pharmaceutically acceptable salt thereof, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands.

3. The oligonucleotide of claim 2 or a pharmaceutically acceptable salt thereof, wherein the targeting ligand comprises carbohydrates, amino sugars, cholesterol, peptides, or lipids.

4. The oligonucleotide of claim 2 or a pharmaceutically acceptable salt thereof, wherein the targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

5. The oligonucleotide of claim 4 or a pharmaceutically acceptable salt thereof, wherein the GalNAc moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.

6. A composition comprising the oligonucleotide of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

7. The composition of claim 6, wherein the dosage form is an oral preparation, an intravenous injection, a subcutaneous injection, or an intramuscular injection.

8. Use of the oligonucleotide of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof or the composition of claim 6 or 7 in the preparation of a medicament for treating hyperlipidemia.

9. The use as described in claim 8, wherein, The hyperlipidemia mentioned refers to hypercholesterolemia.

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