Targeted oligonucleotides for treating diseases associated with pcsk9

Abstract

The present disclosure relates to a targeting oligonucleotide for the treatment of PCSK9 related diseases, the targeting oligonucleotide comprising an antisense strand having a sequence as shown in any one of SEQ ID NO: 53-106 or a fragment thereof, or a modified sequence of the sequence or fragment thereof. The targeting oligonucleotide significantly inhibits the expression level of the PCSK9 gene, reduces the level of low-density lipoprotein cholesterol (LDL-C) in blood, and shows a good lipid-lowering effect.

Description

Technical Field

[0001] The present disclosure relates to an oligonucleotide, particularly a targeting oligonucleotide for treating PCSK9-related diseases. Background Art

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

[0003] Hypercholesterolemia refers to an increase in total cholesterol (TCHO) and / or low-density lipoprotein cholesterol (LDL-C) or non-high-density lipoprotein cholesterol (non-HDL-C) in the blood, and is also known as dyslipidemia. It can be divided into familial hypercholesterolemia and non-familial hypercholesterolemia. One of its most important clinical features is the increase in the level of low-density lipoprotein cholesterol (LDL-C) in the patient's blood. Hypercholesterolemia is an important factor in the occurrence of cardiovascular diseases (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 diseases, usually do not have obvious detectable symptoms until severe atherosclerosis occurs. Currently, statins are the first-line drugs for lowering blood lipids in clinical practice. These drugs can reduce the risk of cardiovascular diseases by lowering LDL-C. However, many patients are intolerant to statins, or the LDL-C level still cannot be reduced to an appropriate level after taking the maximum dose of statins (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 in length and contains 12 exons, encoding a glycoprotein that is 692 amino acids in length (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 30 ng / mL to 4 μg / mL, and it plays an important role in cholesterol metabolism. It mainly controls the plasma LDL level by affecting the expression level of LDL receptors on the surface of hepatocytes. Gain-of-function mutations (reverse mutations) in the PCSK9 gene lead to a decrease in the level of LDL receptors on the surface of hepatocytes and an increase in the LDL-C level in the blood; conversely, loss-of-function mutations in the gene result in an increase in the level of LDL receptors on the surface of hepatocytes and a decrease in the LDL-C level in the blood. Studies have shown that when the maximum dose of statins fails to effectively control the LDL-C level, reducing the PCSK9 level can effectively lower the LDL-C level.

[0005] Intracellular PCSK9 can bind to and direct newly synthesized LDL receptors from the trans-Golgi to lysosomes for degradation; extracellular circulating PCSK9 can bind to LDL receptors on the surface of liver cells and mediate their entry into lysosomes in liver cells for degradation, thereby reducing the LDL receptors on the surface of hepatocytes, leading to a decrease in the ability of the liver to bind and clear LDL-C, and ultimately increasing the LDL-C level in the blood. Therefore, hypercholesterolemia (Norata et al. Annu Rev Pharmacol Toxicol (2014) 54:273-93) and the prevention of cardiovascular diseases associated with high LDL-C can be treated by reducing the PCSK9 expression level.

[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). When RNA molecules appear in cells in double - stranded form, this mechanism is induced and activated, that is, the RNA interference phenomenon occurs. This discovery heralded the beginning of a new research field, and these two scientists were awarded the 2006 Nobel Prize in Physiology and Medicine. When double - stranded RNA binds to the protein complex Dicer, Dicer cuts 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 still binds to the RISC complex. RISC recognizes and degrades the mRNA of the target gene through the guidance of single - stranded RNA, inhibiting the expression of specific proteins and thus specifically causing 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 the RNA interference mechanism to degrade the corresponding mRNA. Currently, this method is an important research tool in biology and biomedicine. In addition, numerous siRNA drugs have been developed to treat viral infections, cardiovascular diseases, cancer, endocrine disorders, and many other diseases. Most siRNA therapies in the R & D stage or those that have been approved for marketing show good therapeutic effects. 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, using RNA interference technology to inhibit the expression of specific target genes has become an effective disease treatment approach.

[0008] Currently, inhibitors targeting PCSK9 have been reported, including marketed antibody - based and small nucleic acid - based drugs, which are used or intended to be used to treat 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 solve the problems existing in the prior art, the purpose of the present disclosure is to provide an inhibitor targeting PCSK9 with good efficacy, high safety, and long - lasting drug effect.

[0010] In one aspect, 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 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 its fragment.

[0011] In some embodiments of the present disclosure, the oligonucleotide further comprises a sequence as shown in any one of SEQ ID NOs: 1 - 52 or a fragment thereof, or a modified sequence of the sequence or its fragment.

[0012] In some embodiments of the present disclosure, the antisense strand consists of 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 its fragment.

[0013] In some embodiments of the present disclosure, the modified sequence of the antisense strand comprises a sequence as shown in any one of SEQ ID NOs: 138 - 183, 185 - 186 or a fragment thereof.

[0014] In some embodiments of the present disclosure, the modified sequence of the sense strand comprises a sequence as shown in any one of SEQ ID NOs: 107 - 137, 135, 184 or a fragment thereof.

[0015] In some embodiments of the present disclosure, the sense strand and the antisense strand are respectively a double-stranded structure of 19 / 21 pairing, 21 / 21 pairing, 21 / 23 pairing or 23 / 23 pairing.

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

[0017] In another aspect, the present disclosure provides the use of the oligonucleotide or a pharmaceutically acceptable salt thereof or the composition in the preparation of a drug for treating and / or preventing diseases related to lipid disorders.

[0018] In some embodiments of the present disclosure, the diseases related to lipid disorders are hyperlipidemia, atherosclerosis and / or one or more symptoms or complications thereof.

[0019] Experiments have shown that the candidate compounds of the present disclosure have significant effects on inhibiting the expression levels of the PCSK9 gene in human liver cancer cell lines Huh7, HepG2, and Hle; some of these candidate compounds have inhibitory effects on the expression levels of the PCSK9 gene in both the Huh7 and Hle cell lines reaching over 90%, and some compounds have inhibitory effects on the PCSK9 gene expression in cynomolgus monkey hepatocytes reaching 70%, 80%, or even over 90%, showing good inhibitory effects. After administration to mice for about one month (Day 29), the reduction of PCSK9 protein by some of these compounds can still be maintained at about 60%; some compounds can reduce the PCSK9 protein by over 85% in mice for four consecutive weeks (Day 30), with a long-lasting drug effect. In addition, some of these compounds can significantly reduce the levels of low-density lipoprotein cholesterol (LDL-C) in the blood of C57 mice, some compounds can maintain the inhibitory efficiency of LDL-C levels in cynomolgus monkeys at about 40% for 51 consecutive days, and some compounds can maintain the inhibitory efficiency of LDL-C in cynomolgus monkeys above 40% on the 79th day, showing good lipid-lowering effects. The present disclosure provides oligonucleotides targeting PCSK9 mRNA, which effectively reduce the expression of PCSK9 in cells, especially liver cells (e.g., hepatocytes), for the treatment of hypercholesterolemia, atherosclerosis, and / or one or more symptoms or complications thereof. Thus, in related aspects, the present disclosure provides methods for treating hypercholesterolemia, atherosclerosis, and / or one or more symptoms or complications thereof, which involve selectively reducing the expression of the PCSK9 gene in the liver. In certain embodiments, the PCSK9-targeting oligonucleotides provided herein are designed to be delivered to selected cells of the target tissue (e.g., liver hepatocytes) to treat hypercholesterolemia, atherosclerosis, and / or one or more symptoms or complications thereof in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a graph showing the efficacy results of the first round of in vitro screening of PCSK9-siRNA for hepatocyte cell lines.

[0021] Figure 2 It is a graph showing the efficacy results of the second round of in vitro screening of PCSK9-siRNA for hepatocyte cell lines.

[0022] Figure 3 It is a graph showing the efficacy results of the first round of in vitro screening of modified PCSK9-siRNA for primary monkey hepatocytes.

[0023] Figure 4 It is a graph showing the efficacy results of the second round of in vitro screening of modified PCSK9-siRNA for primary monkey hepatocytes.

[0024] Figure 5 It shows the changes in the LDL-C levels in mice after the first administration of PCSK9 RNAi agents.

[0025] Figure 6 Shows the change in LDL-C level in mice after the second round of administration of the PCSK9 RNAi agent.

[0026] Figure 7 Shows the change in LDL-C level in mice after the third round of administration of the PCSK9 RNAi agent.

[0027] Figure 8 Shows the change in LDL-C level in hPCSK9 mice after administration of the PCSK9 RNAi agent.

[0028] Figure 9 Shows the change in PCSK9 protein level in hPCSK9 mice after administration of the PCSK9 RNAi agent.

[0029] Figure 10 Shows the change in LDL-C level in cynomolgus monkeys after administration of the PCSK9 RNAi agent.

[0030] Figure 11 Shows the change in TCHO level in cynomolgus monkeys after administration of the PCSK9 RNAi agent.

[0031] Figure 12 Shows the change in LDL-C level in mice after the fourth round of administration of the PCSK9 RNAi agent.

[0032] Figure 13 Shows the change in PCSK9 protein level in mice after the fourth round of administration of the PCSK9 RNAi agent. Detailed implementation manners

[0033] In the present disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory operation procedures used herein are all terms and conventional procedures widely used in the corresponding fields. Meanwhile, to better understand the present disclosure, the definitions and explanations of relevant terms are provided below.

[0034] About: As used herein, the term "about" or "approximate" as applied to one or more target values refers to a value similar to the reference value. In certain embodiments, unless otherwise specified or otherwise apparent from the context, the term "approximate" or "about" refers to a range of values that fall 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 either direction (greater than or less than) (unless such a number would exceed 100% of the possible value).

[0035] Atherosclerosis: As used herein, the term "atherosclerosis" refers to a disease involving the narrowing of arteries (e.g., coronary, carotid, peripheral, and / or renal arteries) typically due to the accumulation of plaque (made up of fat, cholesterol, calcium, and other substances). In some embodiments, the narrowing of the coronary arteries can produce symptoms such as angina, shortness of breath, sweating, nausea, dizziness, shortness of breath, arrhythmia, and / or palpitations. In some embodiments, the narrowing of the carotid arteries can lead to stroke (i.e., death of brain cells 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 straight, blurred vision, numbness in the face, arms, and legs, severe headache, and / or loss of consciousness. In some embodiments, the narrowing of the peripheral arteries can lead to numbness or pain in the arms or legs. In some embodiments, the 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 (such as chronic kidney disease).

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

[0037] Strand: As used herein, the term "strand" refers to a single continuous sequence of nucleotides linked together by internucleotide bonds (e.g., phosphodiester bonds, phosphorothioate bonds). In some embodiments, a strand 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 at the 2'-position of its pentose sugar as compared to a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide that has one or more atoms modified or substituted (including modifications or substitutions in the sugar, phosphate group, or base or modifications or substitutions in the sugar, phosphate group, or base) 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 duplex form. In some embodiments, complementary base pairing forms one or more duplex regions of the double-stranded oligonucleotide between the antiparallel sequences of the nucleotides of covalently separated nucleic acid strands. In some embodiments, complementary base pairing forms one or more duplex regions of the double-stranded oligonucleotide between the antiparallel sequences of the nucleotides of covalently linked nucleic acid strands. In some embodiments, complementary base pairing forms one or more duplex regions of the double-stranded oligonucleotide from a single nucleic acid strand that is folded (e.g., via a hairpin) to provide complementary antiparallel sequences of nucleotides that base pair together. In some embodiments, the double-stranded oligonucleotide comprises two covalently separated nucleic acid strands that are fully duplexed with each other. However, in some embodiments, the double-stranded oligonucleotide comprises two covalently separated nucleic acid strands that are partially duplexed, e.g., having overhangs at one or both ends. In some embodiments, the double-stranded oligonucleotide comprises antiparallel sequences of nucleotides that are partially complementary and thus 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, e.g., a short nucleic acid having a length of less than 100 nucleotides. Oligonucleotides can comprise ribonucleotides, deoxyribonucleotides, and / or modified nucleotides, including, for example, modified ribonucleotides. Oligonucleotides can be single-stranded or double-stranded. Oligonucleotides can have or can not have duplex regions. As a non-limiting group of examples, oligonucleotides can 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 "proprotein convertase subtilisin / kexin type 9 gene or protein, also known as NARC-1, FH3, HCHOLA3, NARC-1, or NARCI. The term PCSK9 includes human PCSK9, mouse PCSK9, rat PCSK9, and examples of PCSK9 mRNA sequences are available in GenBank.

[0042] In one aspect, 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 a sequence as shown in any of SEQ ID NOs: 53-106 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.

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

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

[0045] In some embodiments of the present disclosure, the oligonucleotide further comprises a sequence as shown in any one of SEQ ID NOs: 1-52 or a fragment thereof, or a modified sequence of the sequence or its fragment.

[0046] In some embodiments of the present disclosure, the antisense strand consists of 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 its fragment.

[0047] In some embodiments of the present disclosure, the sense strand consists of a sequence as shown in any one of SEQ ID NOs: 1-52 or a fragment thereof, or a modified sequence of the sequence or its fragment.

[0048] In some preferred embodiments of the present disclosure, the oligonucleotide has a sense strand and an antisense strand, wherein the oligonucleotide comprises: (1) a sense strand shown in SEQ ID NO: 51 and an antisense strand shown in SEQ ID NO: 103; or (2) a sense strand shown in SEQ ID NO: 52 and an antisense strand shown in SEQ ID NO: 105. The sense strands shown in SEQ ID NO: 51 and SEQ ID NO: 52 have a common motif UUUGCUUUUGUAACUUGAA (SEQ ID NO: 51). The antisense strands shown in SEQ ID NO: 103 and SEQ ID NO: 105 have a common motif UUCAAGUUACAAAAGCAAA (SEQ ID NO: 187).

[0049] In some embodiments of the present disclosure, the modified sequence of the antisense strand comprises a sequence as shown in any one of SEQ ID NOs: 138-183, 185-186 or a fragment thereof.

[0050] In some embodiments of the present disclosure, the modified sequence of the sense strand comprises a sequence as shown in any one of SEQ ID NOs: 107-137, 135, 184 or a fragment thereof.

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

[0052] In some embodiments of the present disclosure, the sense strand consists of a sequence shown in any of SEQ ID NO: 107-137, 135, 184, or a fragment thereof, or a modified sequence of the sequence or its fragment.

[0053] In some embodiments of the present disclosure, the antisense strand has a length of 19 to 23 nucleotides.

[0054] In some embodiments of the present disclosure, the sense strand has a length of 19 to 23 nucleotides.

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

[0056] In some embodiments of the present disclosure, the antisense strand has an overhang.

[0057] In some embodiments of the present disclosure, the sense strand has an overhang.

[0058] In some embodiments of the present disclosure, the oligonucleotide comprises a 3'-overhang sequence having a length of two nucleotides.

[0059] In some embodiments of the present disclosure, 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.

[0060] In some embodiments of the present disclosure, 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.

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

[0062] In some embodiments of the present disclosure, the sense strand forms a duplex region with the antisense strand.

[0063] In some embodiments of the present disclosure, the sense strand and the antisense strand are respectively double-stranded structures with 19 / 21 pairing, 21 / 21 pairing, 21 / 23 pairing, or 23 / 23 pairing.

[0064] In some embodiments of the present disclosure, the oligonucleotide comprises a 3'-overhang sequence of two nucleotides, wherein the 3'-overhang sequence is present on the antisense strand, and wherein the sense strand has a length of 19 nucleotides and the antisense strand has a length of 21 nucleotides, such that the sense strand and the antisense strand form a double-stranded body with a length of 19 nucleotides.

[0065] The oligonucleotide comprises a 3'-overhang sequence of two nucleotides, wherein the 3'-overhang sequence is present on both the antisense strand and the sense strand, and wherein the sense strand has a length of 21 nucleotides and the antisense strand has a length of 21 nucleotides, such that the sense strand and the antisense strand form a double-stranded body with a length of 19 nucleotides.

[0066] In some embodiments of the present disclosure, the oligonucleotide comprises a 3'-overhang sequence of two nucleotides, wherein the 3'-overhang sequence is present on the antisense strand, and wherein the sense strand has a length of 21 nucleotides and the antisense strand has a length of 23 nucleotides, such that the sense strand and the antisense strand form a double-stranded body with a length of 21 nucleotides.

[0067] In some embodiments of the present disclosure, the oligonucleotide is selected from the unmodified oligonucleotides of any one of antisense strand SEQ ID NOs: 55 - 57, 61, 66, 70 - 71, 80, 82, 84, 93 - 106, and the modified oligonucleotides of any one of antisense strand SEQ ID NOs: 138 - 183.

[0068] In some embodiments of the present disclosure, the oligonucleotide is selected from the unmodified oligonucleotides of any one of antisense strand SEQ ID NOs: 80, 84, 93 - 95, 99 - 106, and the modified oligonucleotides of any one of antisense strand SEQ ID NOs: 145, 147 - 150, 154 - 183, 185 - 186.

[0069] In some embodiments of the present disclosure, the oligonucleotide is selected from the unmodified oligonucleotides of any one of sense strand SEQ ID NOs: 3 - 5, 9, 14, 18, 19, 28, 30, 32, 41 - 52, and the modified oligonucleotides of any one of sense strand SEQ ID NOs: 107 - 137, 135, 184.

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

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

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

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

[0074] In some embodiments of the present disclosure, the 2'-modification is a modification selected from the following: 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-β-D-arabinonucleic acid.

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

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

[0077] In some embodiments of the present disclosure, the at least one modified internucleotide bond is a phosphorothioate bond.

[0078] In some embodiments of the present disclosure, the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand comprises a phosphonate analogue.

[0079] In some embodiments of the present disclosure, the phosphonate analogue is oxy-methylphosphonate, vinylphosphonate, or malonylphosphonate.

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

[0081] In some embodiments of the present disclosure, each targeting ligand comprises a carbohydrate, an amino sugar, cholesterol, a polypeptide, or a lipid.

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

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

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

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

[0086] In some embodiments of the present disclosure, 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.

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

[0088] (1) The sense strand shown in SEQ ID NO:52, and the antisense strand shown in SEQ ID NO:105;

[0089] (2) The sense strand shown in SEQ ID NO:135, and the antisense strand shown in SEQ ID NO:176;

[0090] (3) The sense strand shown in SEQ ID NO:136, and the antisense strand shown in SEQ ID NO:177;

[0091] (4) The sense strand shown in SEQ ID NO:137, and the antisense strand shown in SEQ ID NO:178;

[0092] (5) The sense strand shown in SEQ ID NO:135, and the antisense strand shown in SEQ ID NO:185;

[0093] (6) The sense strand shown in SEQ ID NO:135, and the antisense strand shown in SEQ ID NO:186;

[0094] (7) The sense strand shown in SEQ ID NO: 184, and the antisense strand shown in SEQ ID NO: 185;

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

[0096] (9) The sense strand shown in SEQ ID NO: 184, and the antisense strand shown in SEQ ID NO: 176.

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

[0098] (1) The sense strand shown in SEQ ID NO: 52, and the antisense strand shown in SEQ ID NO: 106;

[0099] (2) The sense strand shown in SEQ ID NO: 134, and the antisense strand shown in SEQ ID NO: 179;

[0100] (3) The sense strand shown in SEQ ID NO: 135, and the antisense strand shown in SEQ ID NO: 180;

[0101] (4) The sense strand shown in SEQ ID NO: 135, and the antisense strand shown in SEQ ID NO: 181;

[0102] (5) The sense strand shown in SEQ ID NO: 136, and the antisense strand shown in SEQ ID NO: 182; and

[0103] (6) The sense strand shown in SEQ ID NO: 137, and the antisense strand shown in SEQ ID NO: 183.

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

[0105] (1) The sense strand shown in SEQ ID NO: 51, and the antisense strand shown in SEQ ID NO: 103;

[0106] (2) The sense strand shown in SEQ ID NO: 127, and the antisense strand shown in SEQ ID NO: 158; and

[0107] (3) The sense strand shown in SEQ ID NO: 132, and the antisense strand shown in SEQ ID NO: 164..

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

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

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

[0111] In some embodiments of the present disclosure, the combination further comprises other drugs for treating and / or preventing diseases related to lipid disorders.

[0112] In some preferred embodiments of the present disclosure, the diseases related to lipid disorders are hyperlipidemia, atherosclerosis and / or one or more symptoms or complications thereof.

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

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

[0115] In another aspect, the present disclosure provides the use of the oligonucleotide or a pharmaceutically acceptable salt or composition thereof in the preparation of a drug for treating and / or preventing diseases related to lipid disorders.

[0116] In some embodiments of the present disclosure, the use for diseases related to lipid disorders are hyperlipidemia, atherosclerosis and / or one or more symptoms or complications thereof.

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

[0118] A variety of formulations have been developed to facilitate the use of oligonucleotides. For example, formulations that minimize degradation, promote delivery and / or uptake, or provide another beneficial property to the oligonucleotide in the formulation can be used to deliver the oligonucleotide to a subject or cellular environment. In some embodiments, the present disclosure provides compositions comprising an oligonucleotide (e.g., single-stranded or double-stranded oligonucleotide) to reduce the expression of PCSK9. Such compositions can be suitably formulated such that when administered to a subject (either directly to the environment of the target cell or systemically), a sufficient portion of the oligonucleotide enters the cell to reduce PCSK9 expression. Any of a variety of suitable oligonucleotide formulations can be used to deliver the oligonucleotide for reducing PCSK9, as disclosed herein. In some embodiments, the oligonucleotide is formulated in a buffer solution, such as phosphate buffered saline solution, liposomes, micellar 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 buffered aqueous solution (e.g., PBS).

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

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

[0121] In some embodiments, the formulations disclosed herein comprise an excipient. In some embodiments, the excipient imparts increased stability, increased absorption, increased solubility, and / or therapeutic enhancement of the active ingredient to the composition. In some embodiments, the excipient is a buffer (e.g., sodium citrate, sodium phosphate, tris base, or sodium hydroxide) or a vehicle (e.g., a buffer solution, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, the oligonucleotide is lyophilized to extend its shelf life and then made into a solution prior to use (e.g., administration to a subject). Thus, the excipient in a composition comprising any of the oligonucleotides described herein can be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or a collapse temperature modifier (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. Generally, the route of administration is intravenous or subcutaneous.

[0123] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble), dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous or subcutaneous administration, suitable carriers include 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, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. In many cases, it is preferred to include an isotonic agent, such as sugar, polyols such as mannitol, sorbitol, and sodium chloride, in the composition. Sterile injectable solutions can be prepared by incorporating the required amount of oligonucleotide with one or a combination of the above-listed ingredients in a selected solvent, followed by filtration sterilization.

[0124] In some embodiments, the composition can 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 can be between about 1% and about 80% or more of the total weight or volume of the composition. 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 thus various dosages and treatment regimens can be desirable.

[0125] Even though many embodiments relate to hepatic targeting delivery of any of the oligonucleotides disclosed herein, targeting other tissues is also contemplated.

[0126] In some embodiments, administration of an oligonucleotide as described herein results in a decrease in the level of PCSK9 expression in a cell. In some embodiments, the decrease in the level of PCSK9 expression can be a decrease 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. The appropriate control level can be the level of PCSK9 expression in a cell or cell population that has not been contacted with an oligonucleotide as described herein. In some embodiments, the effect of delivering an oligonucleotide to a cell according to the methods disclosed herein is evaluated after a limited time period. For example, the level of PCSK9 in the cell 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 introducing the oligonucleotide into the cell.

[0127] In some embodiments, the oligonucleotide is delivered in the form of a transgene that is engineered to express an oligonucleotide as disclosed herein (e.g., in the form of shRNA) in a cell. In some embodiments, an oligonucleotide is delivered using a transgene engineered to express any oligonucleotide as 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 directly injected into a subject.

[0128] Aspects of the present disclosure relate to methods for reducing PCSK9 expression for treating hypercholesterolemia, atherosclerosis, and / or one or more symptoms or complications thereof in a subject. In some embodiments, the method can include administering to a subject in need thereof an effective amount of any of the oligonucleotides disclosed herein. In some embodiments, such treatment can be used, 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, shortness of breath, sweating, nausea, dizziness, shortness of breath, arrhythmia, palpitations, stroke (i.e., death of brain cells due to insufficient blood and oxygen flow to the brain), weakness, confusion, difficulty speaking, dizziness, difficulty walking or standing straight, 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 can be used, for example, to treat or prevent one or more symptoms associated with hypercholesterolemia, atherosclerosis, and / or one or more symptoms or complications thereof.

[0129] Thus, in some embodiments, the present disclosure provides methods of treating a subject at risk of (or predisposed to) hypercholesterolemia, atherosclerosis, and / or one or more symptoms or complications thereof, including coronary heart disease (e.g., coronary artery disease), angina, shortness of breath, sweating, nausea, dizziness, shortness of breath, arrhythmia, palpitations, stroke (i.e., death of brain cells due to insufficient blood and oxygen flow to the brain), feelings of weakness, confusion, difficulty speaking, dizziness, difficulty walking or standing straight, 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 certain aspects, the present disclosure provides methods for preventing a disease, disorder, symptom, or condition as described herein in a subject by administering a therapeutic agent (e.g., an oligonucleotide or a vector or transgene encoding the same) to the subject. In some embodiments, the subject to be treated is a subject who would therapeutically benefit from a reduction in the amount of PCSK9 protein, for example, in the liver.

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

[0132] In some embodiments, any one of the compositions disclosed herein is administered to a subject enterally (e.g., orally, via a gastric feeding tube, via a duodenal feeding tube, via a gastrostomy, or rectally), parenterally (e.g., subcutaneous injection, intravenous injection or infusion, intra-arterial injection or infusion, intramuscular injection), topically (e.g., epidermally, by inhalation, via eye drops, or through a mucosa), or by direct injection into a target organ (e.g., the liver of the subject). Generally, the oligonucleotides disclosed herein are administered intravenously or subcutaneously.

[0133] In some embodiments, the oligonucleotide is administered at a dose in the range of 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 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.

[0134] As a set of non-limiting examples, the oligonucleotides of the present disclosure are administered once a year, twice a year, quarterly (once every three months), bi-monthly (once every two months), monthly or weekly.

[0135] In some embodiments, the subject to be treated is a human (e.g., a human patient) or a non-human primate or other mammalian subject. Other exemplary subjects include domestic 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.

[0136] For purposes of clear and concise description, features are described herein as part of the same or separate embodiments, however, it will be understood that the scope of the present disclosure may include embodiments having combinations of all or some of the described features.

[0137] Hereinafter, the present disclosure will be described in more detail with reference to specific examples. However, the examples are for illustrative purposes only and do not limit the present disclosure.

[0138] Examples

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

[0140] First, computer-based algorithms were used to generate candidate oligonucleotide sequences complementary to human PCSK9 mRNA (NM_174936.3, Table 1), where some sequences were also complementary to cynomolgus monkey PCSK9 mRNA (XM_005543260.2, Table 1), and some sequences were also complementary to mouse PCSK9 mRNA (NM_153565.2, Table 1) or had no more than 2 mismatches. Some of them were designed as double-stranded siRNAs with 19 / 21 pairing for the sense and antisense strands, respectively, and the antisense strand had two overhanging ends complementary to the mRNA sequence. In some cases, the overhanging ends of the antisense strand were non-complementary UU; some sequences were designed as double-stranded siRNAs with 21 / 23 pairing for the sense and antisense strands, respectively, and the antisense strand had two overhanging ends complementary to the mRNA sequence; some sequences were designed as double-stranded siRNAs with 21 / 21 or 23 / 23 pairing. In some of the 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) was replaced with a base that did not match the PCSK9 mRNA. In some sequences, individual ribonucleic acids were replaced with deoxyribonucleic acids, such as T or dA.

[0141] Table 1: Human, cynomolgus monkey, and mouse PCSK9 mRNA sequences

[0142] Species GenBank RefSeq# Human NM_174936.3 Cynomolgus monkey XM_005543260.2 Mouse NM_153565.2

[0143] In Tables 2, 3, and 4, "G", "C", "A", "U", and "T" generally 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'-fluoro; s represents phosphorothioate; L represents a ligand, and L96 is N-[tris(GalNAc-alkyl)amido decanoyl]-4-hydroxyprolinol Hyp-(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 Carriers

[0153]

[0154]

[0155] (1) Huh7 / HepG2 / HLE Cell Culture and Transfection:

[0156] Using human liver cancer cell lines Huh7 (CCTCC, GDC0134), HepG2 (Jiangsu KeyGen Biotech Co., Ltd., KG020), and HLE (JCRB Cell Bank, JCRB0404), they were placed in an incubator at 37°C and 5% CO2, and cultured using DMEM basal medium (Hyclone, SH30022.01) supplemented with 10% FBS (aqlabteech, AQ-MV-06600) and 1% penicillin-streptomycin (KeyGen Biotech, KGY0023). When the cell confluence reached 90%, they were digested with trypsin (amresco, 0458-250G), counted with a cell counter (Nexcelom, cellometer Mini), and the Huh7 cells were diluted to 2*10^5 / ml. The cell suspension was added to a 12-well plate and allowed to adhere overnight before transfection. Lipofectamine TM 3000 Transfection Reagent (Thermo Fisher, L3000150) was used for transfection. In system ①, 50 nM siRNA was diluted with 50 μl of Opti-MEM (Thermo Fisher, 1105821), and in system ②, 3 μl of Lip3000 was diluted with 50 μl of Opti-MEM. After standing for 5 min respectively, system ① and ② were mixed and then allowed to stand for another 15 min, and then added dropwise to the 12-well plate. After 4 h of transfection, the DMEM / F12 complete medium was replaced, and the 12-well plate was placed in the incubator and incubated for 48 h.

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

[0158] Take out the 12-well plate from the incubator, aspirate the culture medium, wash it once with an appropriate amount of PBS, add 500 μl of lysis buffer to each well, and transfer the supernatant to a new 1.5-ml centrifuge tube. Add 500 μl of absolute ethanol to the lysed cells and mix well (if precipitation occurs, this is normal and the operation can continue). Invert the centrifuge tube several times or pipette forcefully 10 times to disperse the generated precipitate, then add the liquid to the centrifugal column. Place the centrifuge tube symmetrically in a centrifuge (eppendorf, 5430) and centrifuge at 4000×g for 1 min. Take out the centrifuge tube, add 500 μl of washing buffer to the column, centrifuge at 12000×g for 1 min. When taking out the column after centrifugation, be careful not to let the waste liquid in the collection tube contact the RNA column to avoid contamination. Pour out the waste liquid, reinstall the RNA column into the collection tube, and centrifuge the empty tube once to completely remove any possible residual washing buffer. Place the column on a clean RNase-free 1.5-ml centrifuge tube, open the lid and air-dry for 2 minutes. Add 30 μl of elution buffer to the center of the membrane of the RNA column, let it stand at room temperature for 2 minutes, centrifuge at 2000×g for 1 min. After the RNA is eluted, place it on ice. Measure the concentration of the eluted RNA for subsequent experimental use. The extracted RNA can be immediately used for subsequent experiments or stored at -80 °C for later use.

[0159] (3) Use Synthesize cDNA using the IIQ RT SuperMix for qPCR(+gDNAwiper) reverse transcription kit (Vazyme, R223-01):

[0160] Prepare a mixture in a RNase-free centrifuge tube: 4 μl of 4×gDNA wiper Mix, 1 μg of template RNA, add RNase-free ddH2O to 16 μl to remove genomic DNA, gently pipette and mix well, incubate at 42 °C for 2 min. Then directly add 4 μl of 5×HiScript II qRT SuperMix II to the reaction tube, gently pipette and mix well, place it in a PCR instrument (Applied Biosystems, 9700) at 50 °C for 15 min; 85 °C for 5 sec, hold at 4 °C. The product can be immediately used for qPCR reaction, or stored at -20 °C and used within half a year. For long-term storage, it needs to be aliquoted and stored at -80 °C. cDNA should be avoided from repeated freezing and thawing.

[0161] (4) Perform qPCR quantification using ChamQ SYBR qPCR Master Mix (Vazyme, Q311-02):

[0162] Prepare a 20 μl reaction system by adding 10 μl of 2×ChamQ SYBR qPCR Master Mix, 0.5 μl of Forword primer (Ruibo Kexing), 0.5 μl of Reverse primer (Ruibo Kexing), 1 μl of Template cDNA, and 8 μl of ddH2O. Each sample has 3 replicates. Place the 96-well plate into a qPCR instrument (ROCGENE, Archimed) and execute the program: pre-denaturation at 95°C for 30 sec; amplification at 95°C for 10 sec, 60°C for 30 sec, for 40 cycles; melting curve at 95°C for 15 sec, 60°C for 60 sec, 95°C for 15 sec.

[0163] (5) Use GraphPad Prism 8 for data statistical analysis:

[0164] Export the data in EXCEL format and use CT PCSK9 -CT GAPDH , normalize the control group. To calculate the fold change of relative silencing efficiency, the data is analyzed using the ΔΔCT method. Import the three parallel replicated data obtained into GraphPad Prism 8 and apply the column model for bar analysis. The results are shown in Figure 1 and Figure 2 .

[0165] As Figure 1 can be seen, when the drug dosage is 50 nM, AL0061001 - AL0061040 has a significant inhibitory effect on the expression level of PCSK9 gene in Huh7 cell line; AL0061003, AL0061004, AL0061005, AL0061009, AL0061014, AL0061015, AL0061018 - AL0061022, AL0061028, AL0061030, AL0061031, AL0061032, AL0061034 have a significant inhibitory effect on the expression level of PCSK9 gene in HepG2 cell line.

[0166] As Figure 2It can be seen that when the dosage was 50nM, all candidate compounds had a significant effect on inhibiting the expression level of PCSK9 gene in Huh7 and Hle cell lines; especially AL0061005, AL0061018, AL0061028, AL0061030, AL0061032, AL0061041, AL0061042, AL0061044, AL0061051, AL0061052, AL0061053, and AL0061054 had an inhibitory effect of more than 90% on the expression level of PCSK9 gene in Huh7 and Hle cell lines.

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

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

[0169] Use cynomolgus monkey hepatocytes (Beijing Red Bio Biotechnology Co., Ltd., cmTCSC), preheat the culture medium first, take out the thawing culture medium (Beijing Red Bio Biotechnology Co., Ltd., HEPO24) to the biosafety cabinet, add 4mL of FBS to 36ml of thawing culture medium (TPCS, HEPO24) to make a complete thawing culture medium, and heat it in a 37℃ water bath for 10 minutes. Treat with coating medium (Beijing Red Bio Biotechnology Co., Ltd., HEPO44) in a CO2 incubator at 37℃ for 0.5h. Take out the cells from liquid nitrogen, dissolve the cells in a 37℃ water bath, take them out after about 2min, transfer the cell suspension to preheated 40ml thawing culture medium, wash the cell cryopreservation tube with 2ml of complete thawing culture medium, centrifuge the cell suspension at 180×g for 1min, discard the supernatant, add 2ml of preheated CM seeding culture medium (Beijing Red Bio Biotechnology Co., Ltd., CMHEP054), gently blow to mix the cell suspension, and take 20μl of cell suspension for counting. According to the counting results, 12-well plates were inoculated with 3*10^5 / well and cultured in an incubator at 37°C and 5% CO2. After 4-5 hours of adhesion, the CM seeding medium was aspirated and replaced with preheated medium (Beijing Red Biotech Co., Ltd., CMHEP064). Transfection was performed 6 hours after adhesion. Lipofectamine TMTransfection was carried out using 3000 Transfection Reagent (Thermo Fisher, L3000150). In system ①, 50 nM modified siRNA (Suzhou Beixin Biotechnology Co., Ltd.) was diluted with 50 μl of Opti-MEM (Thermo Fisher, 1105821). In system ②, 3 μl of Lip3000 was diluted with 50 μl of Opti-MEM. After standing for 5 min respectively, system ① and ② were mixed and then stood for another 15 min, and then dropped into a 12-well plate. After 4 h of transfection, the DMEM / F12 complete medium was replaced, and the 12-well plate was placed in an incubator and incubated for 48 h.

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

[0171] The 12-well plate was taken out of the incubator, the medium was aspirated dry, washed once with an appropriate amount of PBS, and 500 μl of lysis buffer was added to each well. The supernatant was transferred to a new 1.5 ml centrifuge tube. 500 μl of absolute ethanol was added to the lysed cells and mixed well (if precipitation occurs, this is normal and the operation can continue). The centrifuge tube was inverted several times, or pipetted forcefully 10 times to disperse the generated precipitate, and then the liquid was added to the centrifugal column. The centrifuge tube was symmetrically placed in a centrifuge (Eppendorf, 5430) and centrifuged at 4000×g for 1 min. The centrifuge tube was taken out and 500 μl of washing buffer was added to the column, and centrifuged at 12000×g for 1 min. When taking out the column after centrifugation, note not to let the waste liquid in the collection tube touch the RNA column to avoid contamination. Pour out the waste liquid, put the RNA column back into the collection tube, and centrifuge the empty tube once to completely remove the possibly remaining washing buffer. The column was placed on a clean RNase-free 1.5 ml centrifuge tube, the lid was opened and air-dried for 2 minutes. 30 μl of elution buffer was added to the center of the membrane of the RNA column, left at room temperature for 2 minutes, and centrifuged at 2000×g for 1 min. After the RNA was eluted, it was placed on ice. The concentration of the eluted RNA was measured for subsequent experiments. The extracted RNA can be immediately used for subsequent experiments or stored at -80 °C for later use.

[0172] (3) Use II Q RT SuperMix for qPCR (+gDNAwiper) reverse transcription kit (Novoprotein, R223-01) to synthesize cDNA:

[0173] Prepare a 4-μl mixture in a RNase-free centrifuge tube: 4 μl of 4× gDNA wiper Mix, 1 μg of template RNA, and add RNase-free ddH2O to 16 μl to remove genomic DNA. Gently pipette to mix well and incubate at 42 °C for 2 min. Then directly add 4 μl of 5× HiScript II qRT SuperMix II to the reaction tube, gently pipette to mix well, place it in a PCR instrument (Applied Biosystems, 9700) at 50 °C for 15 min; 85 °C for 5 sec, and hold at 4 °C. The product can be immediately used for qPCR reaction, or stored at -20 °C and used within half a year. For long-term storage, it needs to be aliquoted and stored at -80 °C. cDNA should be avoided from repeated freezing and thawing.

[0174] (4) qPCR quantification using ChamQ SYBR qPCR Master Mix (Vazyme, Q311-02):

[0175] Prepare a 20-μl mixture: 10 μl of 2× ChamQ SYBR qPCR Master Mix, 0.5 μl of Forword primer (Ruibo Kexing), 0.5 μl of Reverse primer (Ruibo Kexing), 1 μl of Template cDNA, and 8 μl of ddH2O. Each sample has 3 replicates. Place the 96-well plate into a qPCR instrument (ROCGENE, Archimed) and execute the program: pre-denaturation, 95 °C for 30 sec; amplification, 95 °C for 10 sec, 60 °C for 30 sec, 40 cycles; melting curve, 95 °C for 15 sec, 60 °C for 60 sec, 95 °C for 15 sec.

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

[0177] Export the data in EXCEL format and use CT PCSK9 -CT GAPDH , normalize the control group. To calculate the fold change of relative silencing efficiency, the data is analyzed using the ΔΔCT method. Import the three parallel replicate data obtained into GraphPad Prism 8, and apply the column model for column analysis. The results are shown in Figure 3 and Figure 4 .

[0178] By Figure 3It can be seen that when the dosage of the drug added is 50 nM, AL0065004, AL0065005, AL0065006, AL0065008, AL0065009, and AL0065010 have significant effects on inhibiting the expression of the PCSK9 gene in cynomolgus monkey hepatocytes, and the inhibitory effects of AL0065006, AL0065008, and AL0065010 can reach approximately 80%.

[0179] From Figure 4 , when the dosage of the drug added is 50 nM, AL0065004, AL0065006, AL0065008, AL0065009, AL0065010 - AL0065020 have significant effects on inhibiting the expression of the PCSK9 gene in cynomolgus monkey hepatocytes, and the inhibitory effects all reach more than 70%; among them, the inhibitory effects of AL0065012, AL0065017, AL0065018, AL0065019, and AL0065020 reach more than 90%.

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

[0181] SPF-grade male C57BL / 6 mice aged 6 - 8 weeks (Beijing Speveto Biotechnology Co., Ltd.) were used in the experiment. Serum samples were obtained on the 0th day before administration, and the mice were randomly grouped according to the LDL-c level. The C57Bl / 6 mice were subcutaneously administered with 9 mg / kg of the PCSK9 RNAi agent and physiological saline (NC, negative control) once. On the 8th day, 15th day, 22nd day, and 29th day after administration respectively (the NC / PC / AL007002 / AL007010 groups were continuously detected until 36 days after administration), mouse blood was collected (by eye bleeding and sent for inspection within 1 h after blood collection), and the levels of TCHO (total cholesterol), TG (triglyceride), HDL-C (high-density lipoprotein cholesterol), and LDL-C (low-density lipoprotein cholesterol) in the blood were examined. During the experiment, no deaths or near-death symptoms were observed in all animals. No obvious abnormalities were observed in all animals during clinical observation.

[0182] The experimental grouping and each detection data are shown in Tables 5 - 12; the changing levels of LDL-C are shown in Figure 5 shown.

[0183] Table 5: Administration grouping table

[0184] Serial number Group Dosing dose Dosing method 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 at the time of animal grouping (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 of animals on the 8th day 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 of animals on the 15th day 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 of animals on the 22nd day 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 of animals on the 29th day 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 of animals on the 36th day 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 of animals on the 43rd day 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, after drug intervention on the 8th day, the AL0067002, AL0067006, AL0067007, and AL0067010 drug intervention groups could significantly reduce the LDL-C level in the blood of C57 mice. Among them, the most significant and the longest-lasting reduction was in the AL0067010 group. It can be seen that the AL0067010 drug intervention group could significantly reduce the LDL-C level in the blood of C57 mice, reaching the lowest value on Day 8 and then gradually and slowly rising. Among them, on Day 8, Day 15, Day 22, Day 29, Day 36, and Day 43, the difference in the LDL-C level in the blood of mice in the AL0067010 treatment group was significant compared with the solvent control (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 level in the blood of mice in the AL0067010 treatment group showed good lipid-lowering effects compared with the solvent control group (NC group) in the first three weeks, with reductions of 51.43%, 38.71%, and 40.63% respectively.

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

[0210] SPF-grade male C57BL / 6 mice aged 6-8 weeks (Spebefu (Beijing) Biotechnology Co., Ltd.) were used in the experiment. Serum samples were obtained before dosing on Day 0, and the mice were randomly grouped according to the LDL-c level. The C57Bl / 6 mice were subcutaneously administered a single dose of 9 mg / kg of the PCSK9 RNAi agent or normal saline (NC, negative control). Blood samples were taken from the mice on the 8th, 15th, 22nd, 29th, 36th, and 43rd days after administration (blood was taken from the eyeballs and sent for inspection within 1 hour after blood collection), and the levels of TCHO (total cholesterol), TG (triglyceride), HDL-C (high-density lipoprotein cholesterol), and LDL-C (low-density lipoprotein cholesterol) in the blood were examined. During the experiment, no obvious abnormalities were observed in all animals during clinical observation.

[0211] The experimental grouping and each detection data are shown in Tables 13-19; the change level of LDL-C is shown in Figure 6 shown.

[0212] Table 13 Blood lipid changes at the time of animal grouping (n = 5)

[0213]

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

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

[0216]

[0217]

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

[0219] Table 15 Changes in blood lipid levels of animals on the 15th day 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 lipid levels of animals on the 22nd day 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 lipid levels of animals on the 29th day 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 lipid levels of animals on the 36th day 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 lipid levels of animals on the 43rd day 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 Table 13 - Table 19 and Figure 6It can be concluded that compared with the vehicle control group, on the 8th day after administration of the test article, the serum TCHO, HDL-C, and LDL-C in the animals of groups AL0067010, AL0067011, AL0067012, AL0067013, AL0067015, AL0067016, AL0067017, and AL0067018 decreased significantly to extremely significantly (p < 0.05 or p < 0.01 or p < 0.001). The serum TCHO and LDL-C in the animals of groups AL0067002 and AL0067019 decreased significantly (p < 0.01). On the 15th day, the serum TCHO, HDL-C, and LDL-C in the animals of groups AL0067002, AL0067010, AL0067011, AL0067012, AL0067013, AL0067015, AL0067016, AL0067017, AL0067018, and AL00670019 decreased significantly or extremely significantly (p < 0.01 or p < 0.001). On the 22nd day, the serum TCHO, HDL-C, and LDL-C in the animals of groups AL0067002, AL0067010, AL0067011, AL0067012, AL0067015, AL0067016, AL0067017, AL0067018, and AL00670019 decreased significantly to extremely significantly (p < 0.05 or p < 0.01 or p < 0.001). The serum TCHO and LDL-C in the animals of group AL00670013 decreased significantly or markedly (p < 0.05 or p < 0.01). The serum LDL-C in the animals of group AL0067014 decreased significantly (p < 0.05). On the 29th day, the serum TCHO, HDL-C, and LDL-C in the animals of groups AL0067002, AL0067010, AL0067011, AL0067015, AL0067016, AL0067017, and AL0067018 decreased significantly to extremely significantly (p < 0.05 or p < 0.01 or p < 0.001). The serum LDL-C in the animals of groups AL0067012, AL00670013, and AL00670019 decreased significantly or markedly (p < 0.05 or p < 0.01). On the 36th day, the serum TCHO (except for groups AL0067013 and AL00670019), TG (except for group AL0067011), HDL-C, and LDL-C in the animals of groups AL0067002, AL0067010, AL0067011, AL0067012, AL0067013, AL0067015, AL0067016, AL0067017, AL0067018, and AL0067019 decreased significantly to extremely significantly (p < 0.05 or p < 0.01 or p < 0.001); on the 43rd day, serum TCHO, HDL-C, and LDL-C of animals in groups AL0067002 and AL0067011 were significantly or extremely significantly reduced (p<0.05 or p<0.01 or p<0.001), serum LDL-C of animals in groups AL0067010, AL0067016, and AL0067019 were significantly or significantly reduced (p<0.05 or p<0.01), and serum HDL-C and LDL-C of animals in groups AL0067012 and AL0067015 were significantly or significantly reduced (p<0.05 or p<0.01). .

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

[0237] The experiment used SPF-grade male C57BL / 6 mice aged 6 to 8 weeks (Beijing Biotechnology Co., Ltd.). Pre-dose serum samples were obtained on day 0 of administration, and the mice were randomly divided into groups according to the LDL-c level. C57Bl / 6 mice were subcutaneously administered a single 9 mg / kg PCSK9 RNAi agent and normal saline (NC, negative control). Blood was collected from mice on the 8th, 15th, 22nd, 29th, 37th, 43rd, and 50th days after administration (blood was collected from the eyeball and sent for inspection within 1 hour after blood collection) to check the levels of TCHO (total cholesterol), TG (triglycerides), HDL-C (high-density lipoprotein cholesterol), and LDL-C (low-density lipoprotein cholesterol) in the blood. During the experiment, no animals showed signs of death or dying. Clinical observation showed no obvious abnormalities in all animals. The experimental groups and test data are shown in Tables 20-19; the change level of LDL-C is shown in Figure 7 Shown.

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

[0239]

[0240] Note: There is no significant difference compared with the NC group

[0241] Table 21 Changes in blood lipids of animals on the 8th day 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 of animals on the 15th day 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 of animals on the 22nd day 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 of animals on the 29th day 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 of animals on the 37th day 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 of animals on the 43rd day 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 of animals on the 50th day 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 Table 20 - Table 27 and Figure 7It can be concluded that, compared with the vehicle control group, on the 8th, 15th, 22nd, 29th, 37th, and 43rd days after administration of the test substances, the levels of TCHO, HDL-C, and LDL-C in the sera of animals in each test substance group (AL0067017, AL0067023, AL0067028, AL0067033, AL0067010) were significantly to extremely significantly decreased (p < 0.05 or p < 0.01 or p < 0.001). On the 50th day, the levels of TCHO, HDL-C, and LDL-C in the sera of animals in the AL0067028 and AL0067033 groups were significantly to extremely significantly decreased (p < 0.05 or p < 0.01 or p < 0.001), and 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, 5 - 6 weeks old (Shanghai Model Organisms Center, Inc.), were used in the experiment. Serum samples were obtained on the 0th day before administration, and the mice were randomly grouped according to body weight. The hPCSK9 mice were subcutaneously administered a single dose of 9 mg / kg of the PCSK9 RNAi agent or normal saline (NC, negative control). Blood samples were collected from the mice on the 8th, 15th, 22nd, and 29th days after administration (by orbital bleeding and the samples were sent for testing within 1 h after blood collection), and the levels of LDL-C (low-density lipoprotein cholesterol) in the blood were examined. The expression of PCSK9 protein was detected using an ELISA (R&D) kit.

[0266] All the detected data are shown in Tables 28 - 29; the changing levels of LDL-C and PCSK9 protein are shown in Figure 8 、 Figure 9 .

[0267] Table 28 Changes in the Content of Human PCSK in Animal Sera (ng / mL)

[0268]

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

[0270] Table 29 Changes in the Content of LDL-C in Animal Sera (mmol / L)

[0271]

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

[0273] As Figure 8As shown, for the PCSK9 protein level, in all RNAi agent groups (AL0067001, AL0067010, AL0067016, AL0067028), there was a significant decrease in the first two weeks (Day 8, Day 15) compared with before self - administration (Day 0); in particular, in the AL0067016 and AL0067028 groups, a single administration could continuously reduce the PCSK9 protein by more than 80% for three weeks, and about one month after administration (Day 29), the reduction of the PCSK9 protein could still be maintained at about 60%.

[0274] As Figure 9 shown, for the LDL - C expression level, compared with before self - administration (Day 0), except that there was basically no significant change in the AL0067001 group, other RNAi agent groups had a highly significant decrease throughout the experimental period. Among them, 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; AL0068028 decreased to 57.41%, 48.15%, 51.85%, and 65.74% respectively.

[0275] Example 7. Testing and Research of PCSK9 RNAi Agent in Cynomolgus Monkeys

[0276] The test used male cynomolgus monkeys aged 12 - 23 years old, with the LDL - C range of 0.82 mmol - 2.10 mmol, basically in the state of dyslipidemia. Serum samples before administration were obtained 14 days, 7 days before administration, and on the day of administration (Day 1), and the monkeys were randomly grouped according to the LDL - C level. The cynomolgus monkeys were subcutaneously administered 9 mg / kg of the PCSK9 RNAi agent once. Blood was taken on the 8th, 15th, 22nd, 29th, 34th, 44th, 51st, 58th (AL0067001 and AL0067010 ended), 65th, 72nd, and 79th days after administration to examine the levels of TCHO (total cholesterol) and LDL - C (low - density lipoprotein cholesterol) in the blood.

[0277] The changing levels of LDL - C and TCHO are shown in Figure 10 、 Figure 11 .

[0278] As Figure 10As shown, compared with before dosing (day 1), the RNAi agents AL0067001, AL0067010, AL0067016, and AL0067028 can all significantly reduce the LDL-C level in cynomolgus monkeys. Among them, AL0067001 and AL0067010 can maintain the inhibition efficiency of the LDL-C level in cynomolgus monkeys at about 40% for 51 consecutive days. More significantly, in the AL0067016 and AL0067028 groups, the inhibition efficiency of the LDL-C level in cynomolgus monkeys can be maintained above 40% throughout the entire experiment period (day 79).

[0279] As Figure 11 shown, the RNAi agents AL0067001, AL0067010, AL0067016, and AL0067028 can all significantly reduce the TCHO level in cynomolgus monkeys. Among them, AL0067001 and AL0067010 can maintain the inhibition efficiency of the LDL-C level in cynomolgus monkeys at about 20% for 51 consecutive days. More significantly, in the AL0067016 and AL0067028 groups, the inhibition efficiency of the TCHO level in cynomolgus monkeys can be maintained above 20% throughout the entire experiment period (day 79). In the AL0067028 group, the inhibition rate of the TCHO level can be maintained at about 40% from day 29 to day 51.

[0280] Example 8. Test and research of PCSK9 RNAi agent in C57 mice

[0281] Based on the research in Example 7, on the basis of exploring AL0067028, the modification combination was optimized to evaluate the research effect of the new RNAi agent on C57 mice.

[0282] SPF-grade male C57BL / 6 mice aged 6 - 8 weeks (Beijing Speveto Biotechnology Co., Ltd.) were used in the experiment. Serum samples were obtained before dosing on day 0, and the mice were randomly grouped according to the LDL-c level. The C57BL / 6 mice were subcutaneously administered with 6 mg / kg of the PCSK9 RNAi agent or normal saline (NC, negative control) once. Blood samples were taken from the mice on days 7, 16, 23, 30, 37, 44, and 51 after dosing respectively (blood was taken from the eyeballs and sent for inspection within 1 h after blood collection), and the LDL-C (low-density lipoprotein cholesterol) level in the blood was examined, and the expression of PCSK9 protein was detected using an ELISA (R&D) kit. During the experiment, no deaths or near-death symptoms were observed in all animals. No obvious abnormalities were observed in all animals during clinical observation.

[0283] The experimental grouping and each detection data are shown in Table 30; the change levels of LDL-C and the change levels of PCSK9 protein are shown in Figure 12 、 Figure 13 respectively.

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

[0285]

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

[0287] As Figure 12 shown, for the LDL-C expression level, all RNAi agent groups (AL0067036, AL0067037, AL0067038, AL0067039, AL0067040, AL0067028) had a significant effect on reducing the LDL-C level before Day 37. In particular, the significant reduction in the LDL-C level in the AL0067037 group and the AL0067040 group could last until Day 51. In addition, the reduction in the LDL-C level in the AL0067037 group and the AL0067040 group was significantly better than that in the AL0067028 group throughout the entire test period, and the significant difference was reached (P < 0.05).

[0288] As Figure 13 shown, for the PCSK9 protein level, all RNAi agent groups (AL0067036, AL0067037, AL0067038, AL0067039, AL0067040, AL0067028) had a significant reduction throughout the entire test period (Day 51) compared with before self-administration (Day 0). In particular, a single administration of the AL0067037 group could continuously reduce the PCSK9 protein by more than 85% for four weeks (Day 30), and the PCSK9 protein was reduced by more than 90% on the 16th day after administration. Throughout the entire test period, the effect of reducing the PCSK9 protein and the duration of reducing the PCSK9 protein in the AL0067037 group were significantly better than those in the AL0067028 group.

Claims

1. An oligonucleotide or a pharmaceutically acceptable salt thereof for reducing the expression of PCSK9, the oligonucleotide comprising an antisense strand having a sequence as shown in SEQ ID NO: 84 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.

2. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1, wherein the oligonucleotide further comprises a sense strand having a sequence as shown in SEQ ID NO: 32 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.

3. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1, wherein the antisense strand consists of a sequence as shown in SEQ ID NO: 84 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof; Preferably, the sense strand consists of a sequence as shown in any of SEQ ID NO: 32 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof; Preferably, the modified sequence of the antisense strand comprises a sequence as shown in SEQ ID NO: 147 or a fragment thereof; Preferably, the modified sequence of the sense strand comprises a sequence as shown in SEQ ID NO: 116 or a fragment thereof; Preferably, the antisense strand consists of a sequence as shown in SEQ ID NO: 147 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof; Preferably, the sense strand consists of a sequence as shown in SEQ ID NO: 116 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.

4. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the oligonucleotide comprises a 3'-overhang sequence having a length of one or more nucleotides, and the 3'-overhang sequence is present on the antisense strand and / or the sense strand; Preferably, the antisense strand has one overhang; Preferably, the sense strand has one overhang; Preferably, the oligonucleotide comprises a 3'-overhang sequence having a length of two nucleotides; Preferably, 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; Preferably, 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.

5. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein the sense strand and the antisense strand form a duplex region; Preferably, the oligonucleotide comprises a 3'-overhang sequence having a length of two nucleotides, wherein the 3'-overhang sequence is present on the antisense strand, the sense strand has a length of 21 nucleotides and the antisense strand has a length of 23 nucleotides, such that the sense strand and the antisense strand form a duplex having a length of 21 nucleotides. Preferably, the oligonucleotide comprises at least one modified nucleotide; Preferably, the modification is a modification selected from the following: 2'-methoxy (m), 2'-deoxy-2'-fluoro (f), or phosphorothioate (s); Preferably, the modified nucleotide comprises a 2'-modification; Preferably, the 2'-modification is a modification selected from the following: 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, or 2'-deoxy-2'-fluoro-β-D-arabinonucleic acid; Preferably, all nucleotides of the oligonucleotide are modified.

6. The oligonucleotide according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, wherein the sense strand comprises the motif sequence shown in SEQ ID NO: 32; the antisense strand comprises the motif sequence shown in SEQ ID NO: 84; Preferably, the oligonucleotide comprises any one of the following combinations of sense and antisense strands: (1) the sense strand shown in SEQ ID NO: 32, and the antisense strand shown in SEQ ID NO: 84; (2) the sense strand shown in SEQ ID NO: 116, and the antisense strand shown in SEQ ID NO: 147; (3) the sense strand shown in SEQ ID NO: 128, and the antisense strand shown in SEQ ID NO: 159; (4) the sense strand shown in SEQ ID NO: 128, and the antisense strand shown in SEQ ID NO: 160; (5) the sense strand shown in SEQ ID NO: 129, and the antisense strand shown in SEQ ID NO: 161; or (6) the sense strand shown in SEQ ID NO: 130, and the antisense strand shown in SEQ ID NO: 162; Preferably, the oligonucleotide comprises any one of the following combinations of sense and antisense strands: (1) the sense strand shown in SEQ ID NO: 32, and the antisense strand shown in SEQ ID NO: 84; or (2) the sense strand shown in SEQ ID NO: 116, and the antisense strand shown in SEQ ID NO:

147.

7. The oligonucleotide according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide comprises at least one modified internucleotide bond; Preferably, the at least one modified internucleotide bond is a phosphorothioate bond; Preferably, the sugar of the 5'-nucleotide of the antisense strand has a phosphonate analogue at the 4'-carbon; Preferably, the phosphonate analogue is oxy-methylphosphonate, vinylphosphonate, or malonylphosphonate.

8. The oligonucleotide according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands; Preferably, each targeting ligand comprises a carbohydrate, an amino sugar, cholesterol, a polypeptide, or a lipid; Preferably, each targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety; Preferably, the GalNac moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety or a tetravalent GalNAc moiety.

9. A composition comprising the oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, and optionally a pharmaceutically acceptable carrier; Preferably, the dosage form of the composition is an oral preparation, an intravenous injection, a subcutaneous injection or an intramuscular injection, preferably a subcutaneous injection; Preferably, the combination further comprises other drugs for treating and / or preventing diseases related to lipid disorders; preferably, the diseases related to lipid disorders are hyperlipidemia, atherosclerosis and / or one or more symptoms or complications thereof; more preferably, the hyperlipidemia is hypercholesterolemia; The other drugs for treating hyperlipidemia are selected from fibrates, statins, bile acid sequestrants and niacin.

10. Use of the oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8 or the composition according to claim 9 in the preparation of a drug for treating and / or preventing diseases related to lipid disorders, Preferably, the diseases related to lipid disorders are hyperlipidemia, atherosclerosis and / or one or more symptoms or complications thereof; Preferably, the hyperlipidemia is hypercholesterolemia.

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