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WO2024222686A1 - Agent arni pour inhiber l'expression de lpa et utilisation d'agent arni - Google Patents

Agent arni pour inhiber l'expression de lpa et utilisation d'agent arni Download PDF

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Publication number
WO2024222686A1
WO2024222686A1 PCT/CN2024/089357 CN2024089357W WO2024222686A1 WO 2024222686 A1 WO2024222686 A1 WO 2024222686A1 CN 2024089357 W CN2024089357 W CN 2024089357W WO 2024222686 A1 WO2024222686 A1 WO 2024222686A1
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seq
sequence shown
strand comprises
sense strand
antisense strand
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Chinese (zh)
Inventor
倪帅健
宋颖
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SHANGHAI JINGXIN BIOMEDICAL CO Ltd
Zhejiang Jingxin Pharmaceutical Co Ltd
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SHANGHAI JINGXIN BIOMEDICAL CO Ltd
Zhejiang Jingxin Pharmaceutical Co Ltd
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Priority to CN202480025966.2A priority Critical patent/CN121127587A/zh
Publication of WO2024222686A1 publication Critical patent/WO2024222686A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present invention relates to an RNAi agent for inhibiting the expression of apolipoprotein (a) gene LPA in cells and a pharmaceutical composition thereof.
  • the present invention also relates to the therapeutic use of the RNAi agent.
  • Lipoprotein (a) [lipoprotein (a), Lp (a)] is a special lipoprotein present in the circulatory system of primates.
  • Lp (a) is a high molecular weight complex in the circulatory system with a diameter of about 25nm and a density of 1.05-1.12g/mL. It is composed of low-density lipoprotein (LDL)-like particles and apolipoprotein (a) [apolipoprotein (a), apo (a)].
  • LDL low-density lipoprotein
  • apolipoprotein (a) [apolipoprotein (a), apo (a)].
  • Apo(a) is a hydrophilic glycoprotein secreted by hepatocytes. It is highly homologous to plasminogen and is composed of an inactive serine protease and a highly glycosylated tricyclic kringle domain.
  • the amino acid sequence homology between the serine protease of apo(a) and the serine protease in plasminogen is as high as 94%, but since the serine in the active site of apo(a) protease is replaced by arginine, it cannot be activated by plasminogen activators.
  • a plasminogen molecule contains five kringle domains, named KI to KV (one of each), while apo(a) only includes multiple repetitive KIV and one KV domain.
  • KIV-1 to KIV-10 There are 10 subtypes of the KIV structure of apo(a) (KIV-1 to KIV-10). Except for KIV-2, which is multi-copy, the rest are single copies. The number of copies of KIV-2 is determined by the encoding LPA of apo(a), ranging from 2 to more than 40 copies, resulting in a high degree of heterogeneity in the length of the apo(a) polypeptide chain, with a relative molecular mass of 400 000 to 800 000, which is also the main reason for the differences in Lp(a) levels among races, regions and individuals.
  • Lp(a) has been considered as one of the high-risk factors for cardiovascular and cerebrovascular diseases.
  • Lp(a) can enter and deposit on the blood vessel wall, which promotes atherosclerosis.
  • Lp(a) is homologous to plasminogen (PLG) in structure and can compete with plasminogen for binding to fibrin sites, thereby inhibiting fibrinogen hydrolysis and promoting thrombosis. Therefore, Lp(a) is closely related to atherosclerosis and thrombosis. Studies have shown that the level of Lp(a) in the blood is an independent risk factor for cardiovascular and cerebrovascular diseases, stroke and atherosclerotic stenosis.
  • the new lipid-lowering drug Mipomersen is an inhibitor of apo B synthesis. It indirectly reduces the synthesis of Lp(a) by reducing the synthesis of apo B, and can significantly reduce the LDL, apo B and Lp(a) levels in patients with hypercholesterolemia and coronary heart disease.
  • PCSK9 inhibitors can reduce Lp(a) levels by increasing the number of LDLR.
  • these two drugs are only effective for patients with significantly increased Lp(a) levels.
  • Lp(a) or Lp(a) slightly above the critical value their reduction effect is not obvious, and they can only be used for some people.
  • antisense oligonucleotides have been designed for the mRNA transcribed from the LPA gene of hepatocytes. They can significantly reduce Lp(a) levels by directly inhibiting the synthesis of apo(a). This is very effective, but the patient's tolerance is still unknown.
  • RNAi agent for inhibiting the expression of apolipoprotein (a) gene (LPA) in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand is no longer than 23 nucleotides and comprises any one nucleotide sequence selected from SEQ ID NO: 265 to 528.
  • LPA apolipoprotein
  • the length of the double-stranded region of the RNAi agent of the present invention is 17 to 23 base pairs, preferably 18 to 21 base pairs, and more preferably 19 base pairs.
  • the sense strand and the antisense strand of the RNAi agent of the present invention are each 17 to 23 nucleotides in length, preferably 19 to 21 nucleotides in length.
  • the RNAi agent of the present invention includes one or two blunt ends, preferably one blunt end. In some embodiments, the RNAi agent of the present invention includes one or two overhangs, preferably one overhang. In a preferred embodiment, each overhang has 1 to 4 unpaired nucleotides, preferably 2 unpaired nucleotides.
  • the overhang is located at the 3' end of the sense strand, the 3' end of the antisense strand, or at both the 3' end of the sense strand and the 3' end of the antisense strand; preferably, the overhang is located at the 3' end of the antisense strand, and further preferably, the RNAi agent has a blunt end.
  • the modified nucleotides of the sense strand are selected from 3'-terminal deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy-modified nucleotides, non-locked nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-methoxyethyl modified nucleotides, morpholino nucleotides, phosphoramidates, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing thiophosphate groups,
  • the sense strand comprises two consecutive phosphorothioate internucleotide linkages starting from the terminal nucleotide at the 5' end.
  • the structure of the sense strand is mN*mN*mNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmN, wherein mN represents a 2'-O-methyl modified nucleotide, fN represents a 2'-fluorine modified nucleotide, and * represents a phosphorothioate internucleotide linkage.
  • the sense strand is no longer than 21 nucleotides and comprises any nucleotide sequence selected from SEQ ID NO: 1 to 264. More preferably, the sense strand comprises any nucleotide sequence selected from SEQ ID NO: 19, 25, 31, 46, 81, 101, 113, 120, 155, 168, 175, 177, 179, 188, 189, 190, 191, 193, 194, 197, 198, 200, 202, 203, 204, 210, 212, 213, 228, 230 and 231.
  • the antisense strand preferably comprises any nucleotide sequence selected from: SEQ ID NO: 283, 289, 295, 310, 345, 365, 377, 384, 419, 432, 439, 441, 443, 452, 453, 454, 455, 457, 458, 461, 462, 464, 466, 467, 468, 474, 476, 477, 492, 494 and 495.
  • RNAi agent provided by the present invention, wherein:
  • the sense strand comprises the sequence shown in SEQ ID NO:19, and the antisense strand comprises the sequence shown in SEQ ID NO:283;
  • the sense strand comprises the sequence shown in SEQ ID NO:25, and the antisense strand comprises the sequence shown in SEQ ID NO:289;
  • the sense strand comprises the sequence shown in SEQ ID NO:31, and the antisense strand comprises the sequence shown in SEQ ID NO:295;
  • the sense strand comprises the sequence shown in SEQ ID NO:46, and the antisense strand comprises the sequence shown in SEQ ID NO:310;
  • the sense strand comprises the sequence shown in SEQ ID NO:81, and the antisense strand comprises the sequence shown in SEQ ID NO:345;
  • the sense strand comprises the sequence shown in SEQ ID NO:101, and the antisense strand comprises the sequence shown in SEQ ID NO:365;
  • the sense strand comprises the sequence shown in SEQ ID NO:113, and the antisense strand comprises the sequence shown in SEQ ID NO:377;
  • the sense strand comprises the sequence shown in SEQ ID NO:120, and the antisense strand comprises the sequence shown in SEQ ID NO:384;
  • the sense strand comprises the sequence shown in SEQ ID NO:155, and the antisense strand comprises the sequence shown in SEQ ID NO:419;
  • the sense strand comprises the sequence shown in SEQ ID NO:168, and the antisense strand comprises the sequence shown in SEQ ID NO:432;
  • the sense strand comprises the sequence shown in SEQ ID NO:175, and the antisense strand comprises the sequence shown in SEQ ID NO:439;
  • the sense strand comprises the sequence shown in SEQ ID NO:177, and the antisense strand comprises the sequence shown in SEQ ID NO:441;
  • the sense strand comprises the sequence shown in SEQ ID NO:179, and the antisense strand comprises the sequence shown in SEQ ID NO:443;
  • the sense strand comprises the sequence shown in SEQ ID NO:188, and the antisense strand comprises the sequence shown in SEQ ID NO:452;
  • the sense strand comprises the sequence shown in SEQ ID NO:189, and the antisense strand comprises the sequence shown in SEQ ID NO:453;
  • the sense strand comprises the sequence shown in SEQ ID NO:190, and the antisense strand comprises the sequence shown in SEQ ID NO:454;
  • the sense strand comprises the sequence shown in SEQ ID NO:191, and the antisense strand comprises the sequence shown in SEQ ID NO:455;
  • the sense strand comprises the sequence shown in SEQ ID NO:193, and the antisense strand comprises the sequence shown in SEQ ID NO:457;
  • the sense strand comprises the sequence shown in SEQ ID NO:194, and the antisense strand comprises the sequence shown in SEQ ID NO:458;
  • the sense strand comprises the sequence shown in SEQ ID NO:197, and the antisense strand comprises the sequence shown in SEQ ID NO:461;
  • the sense strand comprises the sequence shown in SEQ ID NO:198, and the antisense strand comprises the sequence shown in SEQ ID NO:462;
  • the sense strand comprises the sequence shown in SEQ ID NO:200, and the antisense strand comprises the sequence shown in SEQ ID NO:464;
  • the sense strand comprises the sequence shown in SEQ ID NO:202, and the antisense strand comprises the sequence shown in SEQ ID NO:466;
  • the sense strand comprises the sequence shown in SEQ ID NO:203, and the antisense strand comprises the sequence shown in SEQ ID NO:467;
  • the sense strand comprises the sequence shown in SEQ ID NO:204, and the antisense strand comprises the sequence shown in SEQ ID NO:468;
  • the sense strand comprises the sequence shown in SEQ ID NO:210, and the antisense strand comprises the sequence shown in SEQ ID NO:474. sequence;
  • the sense strand comprises the sequence shown in SEQ ID NO:212, and the antisense strand comprises the sequence shown in SEQ ID NO:476;
  • the sense strand comprises the sequence shown in SEQ ID NO:213, and the antisense strand comprises the sequence shown in SEQ ID NO:477;
  • the sense strand comprises the sequence shown in SEQ ID NO:228, and the antisense strand comprises the sequence shown in SEQ ID NO:492;
  • the sense strand comprises the sequence shown in SEQ ID NO:230, and the antisense strand comprises the sequence shown in SEQ ID NO:494; or
  • the sense strand comprises the sequence shown in SEQ ID NO:231, and the antisense strand comprises the sequence shown in SEQ ID NO:495.
  • RNAi agent provided by the present invention, wherein:
  • the sense strand comprises the sequence shown in SEQ ID NO:81, and the antisense strand comprises the sequence shown in SEQ ID NO:345;
  • the sense strand comprises the sequence shown in SEQ ID NO:120, and the antisense strand comprises the sequence shown in SEQ ID NO:384;
  • the sense strand comprises the sequence shown in SEQ ID NO:203, and the antisense strand comprises the sequence shown in SEQ ID NO:467;
  • the sense strand comprises the sequence shown in SEQ ID NO:204, and the antisense strand comprises the sequence shown in SEQ ID NO:468;
  • the sense strand comprises the sequence shown in SEQ ID NO:212, and the antisense strand comprises the sequence shown in SEQ ID NO:476;
  • the sense strand comprises the sequence shown in SEQ ID NO:228, and the antisense strand comprises the sequence shown in SEQ ID NO:492; or
  • the sense strand comprises the sequence shown in SEQ ID NO:230, and the antisense strand comprises the sequence shown in SEQ ID NO:494.
  • the RNAi agent of the present invention further comprises a ligand targeting hepatocytes, preferably, the ligand comprises a galactose moiety, a galactosamine moiety or an N-acetylgalactosamine moiety, and further preferably, the ligand is a trivalent or tetravalent N-acetylgalactosamine moiety.
  • Another aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising any RNAi agent described herein and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition can be formulated as an intravenous or subcutaneous injection.
  • RNAi agent described herein or a pharmaceutical composition comprising any RNAi agent of the present invention in the preparation of the following drugs: (i) a drug for reducing the expression level of LPA in cells; (ii) a drug for treating a disease caused by increased expression level of LPA; or (iii) a drug for treating cardiovascular and cerebrovascular diseases, such as myocardial infarction, heart failure, stroke (ischemic and hemorrhagic), atherosclerosis, coronary artery disease, peripheral vascular disease (such as peripheral arterial disease); cerebrovascular disease, vulnerable plaque and aortic valve stenosis; familial hypercholesterolemia; venous thrombosis; hypercholesterolemia; hyperlipidemia; and dyslipidemia.
  • cardiovascular and cerebrovascular diseases such as myocardial infarction, heart failure, stroke (ischemic and hemorrhagic), atherosclerosis, coronary artery disease, peripheral vascular disease (such as peripheral arterial disease); cerebrovascular disease, vulnerable plaque and aortic valve
  • Another aspect of the present invention provides a method for preventing or treating the following diseases, the method comprising administering to a subject in need thereof a therapeutically effective amount of any RNAi agent of the present invention or a pharmaceutical composition comprising any RNAi agent of the present invention: (i) a disease caused by increased LPA expression level; (ii) a disease selected from cardiovascular and cerebrovascular diseases, such as myocardial infarction, heart failure
  • a disease selected from cardiovascular and cerebrovascular diseases, such as myocardial infarction, heart failure
  • the diseases include heart failure, stroke (ischemic and hemorrhagic), atherosclerosis, coronary artery disease, peripheral vascular disease (e.g., peripheral arterial disease); cerebrovascular disease, vulnerable plaque and aortic valve stenosis; familial hypercholesterolemia; venous thrombosis; hypercholesterolemia; hyperlipidemia; and dyslipidemia.
  • RNAi agent described herein for reducing the expression level of LPA in cells; (ii) for treating diseases caused by increased expression levels of LPA; (iii) for treating cardiovascular and cerebrovascular diseases, such as myocardial infarction, heart failure, stroke (ischemic and hemorrhagic), atherosclerosis, coronary artery disease, peripheral vascular disease (such as peripheral arterial disease); cerebrovascular disease, vulnerable plaque and aortic valve stenosis; familial hypercholesterolemia; venous thrombosis; hypercholesterolemia; hyperlipidemia; and dyslipidemia.
  • diseases caused by increased expression levels of LPA for treating cardiovascular and cerebrovascular diseases, such as myocardial infarction, heart failure, stroke (ischemic and hemorrhagic), atherosclerosis, coronary artery disease, peripheral vascular disease (such as peripheral arterial disease); cerebrovascular disease, vulnerable plaque and aortic valve stenosis; familial hypercholesterolemia; venous thrombosis; hypercholesterol
  • LPA refers to the gene encoding apolipoprotein(a) (apo(a)) or the LPA messenger RNA (LPA mRNA) transcribed from the gene encoding it.
  • the LPA gene encodes the apo(a) protein, which is the major component of low-density lipoprotein particles known as lipoprotein(a) or LP(a).
  • the LPA gene is located on chromosome 6 at locus 6q25.3-q26.
  • the LPA gene is highly polymorphic, and alleles of the gene differ in the number of copies of the Kringle IV type 2 (KIV-2) domain, which may range from 2 to more than 40 copies between individuals.
  • LPA mRNA refers to any LPA messenger RNA encoding the apo(a) protein, including allelic variants and splice variants.
  • the NCBI reference sequence number for human LPA mRNA is NM_005577.4.
  • RNAi agent refers to an agent comprising an RNA molecule that can downregulate the expression of a target gene (LPA gene in this article) by an RNA interference mechanism when introduced into a cell.
  • RNA interference refers to a process in which a nucleic acid molecule induces the cutting and degradation of a target RNA molecule (such as an mRNA molecule) in a sequence-specific manner, such as by an RNA-induced silencing complex (RISC) pathway.
  • RISC RNA-induced silencing complex
  • RNAi agents include siRNA, shRNA, and DNA/RNA hybrid molecules herein, which are sometimes collectively referred to as double-stranded RNA (dsRNA) herein, and include two antiparallel continuous nucleotide chains that are fully complementary to each other to hybridize to form a double-stranded region.
  • Hybridization refers to the pairing of complementary polynucleotides, typically by hydrogen bonds (such as Watson-Crick hydrogen bonds, Hoogsteen hydrogen bonds, or reverse Hoogsteen hydrogen bonds) between complementary bases in two polynucleotides.
  • Double-stranded region refers to a region in two complementary or substantially complementary polynucleotides that form base pairs by hybridization, thereby forming a double strand between two polynucleotide chains.
  • antisense strand refers to a strand in a dsRNA that includes a region that is substantially complementary to a target sequence.
  • sense strand refers to a strand in a dsRNA that includes a region that is substantially complementary to an antisense strand region as defined herein.
  • substantially complementary region refers to a region that is fully complementary or incompletely complementary. When the complementary region is not fully complementary to the target sequence, mismatches may be located in the interior or terminal regions of the molecule. Typically, the most tolerable mismatches are located in the terminal regions, such as 5, 4, 3, or 2 at the 5'- and/or 3' ends of the dsRNA.
  • siRNA refers to a nucleic acid that forms double-stranded RNA that has the ability to reduce or inhibit the expression of a target gene when the siRNA and the target gene are present in the same cell.
  • the siRNA is typically about 15 to about 30 base pairs in length, most typically about 19 to 25 base pairs in length, such as 19, 20, 21, 22, 23, 24 or 25 nucleotide pairs in length.
  • shRNA refers to short hairpin RNA, which includes two short inverted repeat sequences and an intermediate stem-loop structure connecting the two.
  • the stem-loop may contain at least one unpaired nucleotide, for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides.
  • the stem-loop may be 10 or less nucleotides.
  • the stem-loop may be 8 or less unpaired nucleotides.
  • the stem-loop may be 4 to 10 unpaired nucleotides.
  • the stem-loop may be 4 to 8 nucleotides.
  • the two substantially complementary strands of a dsRNA need not be but may be covalently linked.
  • the maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs present in the duplex.
  • a dsRNA may also contain one or more nucleotide overhangs. Overhanging nucleotides refer to one or more unpaired nucleotides extending beyond the double-stranded region at the end of a strand.
  • a nucleotide overhang is typically generated.
  • at least one strand comprises a 3' overhang of at least 1 nucleotide, for example, 1 to 4 nucleotides.
  • at least one strand comprises a 5' overhang of at least 1 nucleotide, for example, 1 to 4 nucleotides.
  • both the 3' end and the 5' end of one strand of the dsRNA comprise an overhang of at least 1 nucleotide.
  • the term "blunt end” or “blunt end” with respect to dsRNA refers to the absence of unpaired nucleotides or nucleotide analogs at a given end of the dsRNA, i.e., no nucleotide overhangs.
  • One or both ends of the dsRNA may be flat. If both ends of the dsRNA are flat ends, the dsRNA is said to be flat-ended.
  • a "blunt-ended" dsRNA is a dsRNA with both ends being flat, i.e., there is no nucleotide overhang at either end of the molecule. In most cases, such molecules are double-stranded over their entire length.
  • nucleotide overhang refers to at least one unpaired nucleotide protruding from the duplex structure of the dsRNA. For example, when the 3' end of one strand of the dsRNA extends beyond the 5' end of the other strand, or vice versa, there is a nucleotide overhang.
  • the nucleotide overhang may comprise or consist of nucleotide/nucleoside analogs, including deoxynucleotides/nucleosides.
  • the overhang may be on the sense strand, the antisense strand, or any combination thereof.
  • the overhanging nucleotides may be present at the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of the dsRNA.
  • the dsRNA molecule may include chemical modifications to ribonucleotides, including modifications to the ribose sugar, bases or backbone components of ribonucleic acid, as described herein or modifications known in the art. Any such modification, such as used in double-stranded ribonucleic acid molecules (such as siRNA, shRNA, etc.), is encompassed by the term “dsRNA” for the purposes of this disclosure.
  • Modified nucleotides refer to nucleotides independently having modified sugar moieties, modified internucleotide linkages and/or modified nucleobases.
  • modified nucleotides includes substitutions, additions or removals of, for example, functional groups or atoms of internucleoside linkages, sugar moieties or nucleobases. Modifications suitable for use in the present invention include all types of modifications disclosed herein or known in the art.
  • the modified nucleotides are selected from 3'-terminal deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy-modified nucleotides, non-locked nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-methoxyethyl modified nucleotides, morpholino nucleotides, phosphoramidates, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing
  • ligand refers to a cell or tissue targeting agent that binds to a specified cell type (such as a hepatocyte), such as a lectin, glycoprotein, lipid or protein (such as an antibody).
  • exemplary targeting agents include thyrotropin, melanocyte stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrates, multivalent lactose, multivalent galactose, N-acetylgalactosamine (GalNAc), multivalent (such as divalent or trivalent) GalNAc, N-acetylglucosamine, multivalent mannose, multivalent trehalose, glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonates, polyglutamate, polyaspartate, cholesterol, steroids, bile acid, folate, vitamin B12, biotin, RGD peptide and RGD peptide mimetic.
  • the ligand is a carbohydrate, such as a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, a polysaccharide.
  • the ligand can be a derivative comprising GalNAc.
  • the ligand is a ligand comprising one or more N-acetylgalactosamine derivatives attached via a divalent or trivalent branched linker, such as L96.
  • RNAi agent of the invention refers to an amount of a RNAi agent of the invention or composition thereof effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms which are within the scope of sound medical judgment and suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a pharmaceutically acceptable material, composition or vehicle that participates in carrying or delivering the RNAi agent from one organ or part of the body to another organ or part of the body, such as a liquid or solid filler, diluent, excipient, manufacturing aid or solvent encapsulating material.
  • a pharmaceutically acceptable material, composition or vehicle that participates in carrying or delivering the RNAi agent from one organ or part of the body to another organ or part of the body, such as a liquid or solid filler, diluent, excipient, manufacturing aid or solvent encapsulating material.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the composition and not injurious to the patient.
  • treatment covers prevention, therapy and cure.
  • Patients receiving this treatment are usually any animal in need. animals, including primates (especially humans) and other mammals such as horses, cattle, pigs, sheep, poultry and pets.
  • RNAi agent for inhibiting the expression of the LPA gene which comprises a sense chain and an antisense chain that form complementary double-stranded regions, wherein the length of the antisense chain does not exceed 23 nucleotides and comprises any one nucleotide sequence selected from SEQ ID NO: 265 to 528.
  • the double-stranded region of RNAi agent should have enough length to allow RNAi agent to enter RNA interference pathway, for example, by engaging Dicer enzyme and/or RISC complex.
  • the length of double-stranded region is about 17 to about 23 base pairs.
  • the double-stranded region of suitable length is about 17 to about 22 base pairs, about 17 to about 21 base pairs, about 17 to about 20 base pairs, about 17 to about 19 base pairs, about 17 to about 18 base pairs, about 18 to about 23 base pairs, about 18 to about 22 base pairs, about 18 to about 21 base pairs, about 18 to about 20 base pairs, about 19 to 23 base pairs, about 19 to 22 base pairs, about 19 to 21 base pairs, about 20 to 23 base pairs, about 20 to 22 base pairs or about 21 to 23 base pairs.
  • the length of double-stranded region is about 18 to about 21 base pairs. In other embodiments, the double-stranded region is about 19 base pairs in length.
  • RNAi agent for inhibiting LPA gene expression which comprises a sense strand and an antisense strand that form a complementary double-stranded region, the length of the double-stranded region is about 17 to about 23 base pairs, and the length of the antisense strand does not exceed 23 nucleotides and comprises any one nucleotide sequence selected from SEQ ID NO: 265 to 528.
  • an RNAi agent for inhibiting LPA gene expression which comprises a sense strand and an antisense strand that form a complementary double-stranded region, the length of the double-stranded region is about 18 to about 21 base pairs, and the length of the antisense strand does not exceed 23 nucleotides and comprises any one nucleotide sequence selected from SEQ ID NO: 265 to 528.
  • an RNAi agent for inhibiting LPA gene expression which comprises a sense strand and an antisense strand that form a complementary double-stranded region, the length of the double-stranded region is about 19 base pairs, and the length of the antisense strand does not exceed 23 nucleotides and comprises any one nucleotide sequence selected from SEQ ID NO: 265 to 528.
  • the length of the sense strand and the antisense strand in the RNAi agent of the invention is each independently about 17 to about 23 nucleotides, such as about 18 to about 23 nucleotides, about 19 to about 23 nucleotides, about 20 to about 23 nucleotides, about 21 to about 23 nucleotides, about 17 to about 22 nucleotides, about 17 to about 21 nucleotides, about 17 to about 20 nucleotides, about 17 to about 19 nucleotides, about 18 to about 22 nucleotides, about 18 to about 21 nucleotides, about 18 to about 20 nucleotides, about 19 to about 22 nucleotides, about 19 to about 21 nucleotides, or about 20 to about 22 nucleotides.
  • the length of the sense strand and the antisense strand is each independently about 17, about 18, about 19, about 20, about 21, about 22, about 23 nucleotides.
  • the sense strand and the antisense strand have the same length, but form a double-stranded region that is shorter than the strand, so that the RNAi agent has two nucleotide overhangs.
  • the RNAi agent includes (i) a sense strand and an antisense strand that are 21 nucleotides in length, (ii) a double-stranded region that is 19 base pairs in length, and (iii) a nucleotide overhang of 1 unpaired nucleotide at the 3' end of the sense strand and the 3' end of the antisense strand.
  • the RNAi agent includes (i) a sense strand and an antisense strand that are 23 nucleotides in length, (ii) a double-stranded region that is 21 base pairs in length, and (iii) a nucleotide overhang of 1 unpaired nucleotide at the 3' end of the sense strand and the 3' end of the antisense strand.
  • the sense strand and the antisense strand have the same length and form a double-stranded region over their entire length so that no nucleotides protrude from either end of the double-stranded molecule.
  • the RNAi agent is flat-ended and includes (i) a sense strand and an antisense strand each having a length of 21 nucleotides, and (ii) a double-stranded region having a length of 21 base pairs.
  • the RNAi agent is flat-ended and includes (i) a sense strand and an antisense strand each having a length of 23 nucleotides, and (ii) a double-stranded region having a length of 23 base pairs.
  • the RNAi agent is flat-ended and includes (i) a sense strand and an antisense strand each having a length of 19 nucleotides, and (ii) a double-stranded region having a length of 19 base pairs.
  • the sense strand or the antisense strand is longer than the other strand, and the two strands form a double-stranded region having a length equal to the length of the shorter strand, such that the RNAi agent includes at least one nucleotide overhang.
  • the antisense strand is 1 to 4 nucleotides longer than the antisense strand, and the double-stranded region formed by the two strands is equal to the length of the antisense strand, so that the sense strand forms an overhang with 1 to 4 unpaired nucleotides.
  • the antisense strand is 1 to 4 nucleotides longer than the sense strand, and the double-stranded region formed by the two strands is equal to the length of the sense strand, so that the antisense strand forms an overhang with 1 to 4 unpaired nucleotides.
  • the length of the nucleotide overhang is 1, 2, 3 or 4 nucleotides.
  • the overhang includes 2 nucleotides.
  • the overhang includes a single nucleotide.
  • the protruding nucleotides can be ribonucleotides or modified nucleotides as described herein.
  • the protruding nucleotides are 2'-modified nucleotides (e.g., 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides) or combinations thereof.
  • the protruding nucleotides are deoxyribonucleotides, such as deoxythymidine.
  • the protruding nucleotides are 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-methoxyethyl modified nucleotides or combinations thereof.
  • the protruding comprises 5'-uridine-uridine-3' (5'-UU-3') dinucleotides.
  • the UU dinucleotides can include ribonucleotides or modified nucleotides, such as 2'-modified nucleotides.
  • the protruding comprises 5'-deoxythymidine-deoxythymidine-3' (5'-dTdT-3') dinucleotides.
  • the nucleotides in the overhang may be complementary to the target gene sequence, form a mismatch with the target gene sequence, or contain some other sequence (e.g., polypeptide pyrimidine or polypeptide sequence, such as UU, TT, AA, GG, etc.).
  • the nucleotide overhang may be at the 5' end or the 3' end of one or both strands.
  • the RNAi agent comprises nucleotide overhangs at the 5' end and the 3' end of the antisense strand.
  • the RNAi agent comprises nucleotide overhangs at the 5' end and the 3' end of the sense strand.
  • the RNAi agent includes nucleotide overhangs at the 5' end of the sense strand and the 5' end of the antisense strand.
  • the RNAi agent comprises nucleotide overhangs at the 3' end of the sense strand and the 3' end of the antisense strand.
  • the RNAi agent includes only nucleotide overhangs at the 5' end of the sense strand. In some embodiments, the RNAi agent includes only nucleotide overhangs at the 3' end of the sense strand. In some embodiments, the RNAi agent includes only nucleotide overhangs at the 3' end of the antisense strand. In some embodiments, the RNAi agent includes only nucleotide overhangs at the 5' end of the antisense strand. In some embodiments, the RNAi agent includes only nucleotide overhangs at the 5' end of the sense strand.
  • the RNAi agent can include a nucleotide overhang at one end of the double-stranded RNA molecule and a flat end at the other end.
  • "Blunt end” means that the sense strand and the antisense strand are completely base paired at the ends of the molecule, and no unpaired nucleotides extend beyond the double-stranded region.
  • the RNAi agent includes a nucleotide overhang at the 3' end of the sense strand and a flat end at the 5' end of the sense strand and the 3' end of the antisense strand.
  • the RNAi agent includes a nucleotide overhang at the 3' end of the antisense strand and a flat end at the 5' end of the antisense strand and the 3' end of the sense strand.
  • the RNAi agent includes (i) a sense strand of 19 nucleotides in length, (ii) an antisense strand of 21 nucleotides in length, and the two strands form a double-stranded region whose length is equal to the strand length of the sense strand.
  • the RNAi agent comprises (i) a sense strand of 21 nucleotides in length, (ii) an antisense strand of 23 nucleotides in length, and the two strands form a double-stranded region whose length is equal to the strand length of the sense strand.
  • an RNAi agent for inhibiting the expression of apolipoprotein (a) gene (LPA) in a cell comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand being no longer than 23 nucleotides and comprising any nucleotide sequence selected from SEQ ID NO: 265 to 528 (preferably comprising a nucleotide sequence selected from SEQ ID NO: 283, 289, 295, 310, 345, 365, 377, 384, 419, 432, 439, 441, 443, 452, 453, 454, 455, 457, 458, 461, 462, 464, 466, 467, 468, 474, 476, 477, 492, 494 and 495), and the RNAi agent includes an overhang and a blunt end, the overhang preferably has 2 unpaired nucleotides, wherein the overhang is formed at the 3' end of the antisense strand, and the blunt
  • an RNAi agent for inhibiting the expression of apolipoprotein (a) gene (LPA) in a cell comprising a sense strand and an antisense strand forming a double-stranded region, wherein the length of the double-stranded region is 19 base pairs, and the length of the antisense strand is
  • the RNAi agent comprises an overhang and a blunt end, the overhang preferably having 2 unpaired nucleotides, wherein the overhang is formed at the 3' end of the antisense strand, and the blunt end is formed at the 3' end of the sense strand and the 5' end of the antisense strand.
  • an RNAi agent for inhibiting the expression of apolipoprotein (a) gene (LPA) in a cell comprising a sense strand and an antisense strand forming a double-stranded region, wherein the length of the double-stranded region is 19 base pairs, the length of the antisense strand does not exceed 23 nucleotides and comprises any nucleotide sequence selected from SEQ ID NO: 265 to 528 (preferably comprising a nucleotide sequence selected from SEQ ID NO: 283, 289, 295, 310, 345, 365, 377, 384, 419, 432, 439, 441, 452, 461, 473, 483, 491, 500, 512, 524, 530, 531, 532, 533, 534, 536, 537, 538, 540, 541, 542, 543, 544, 545, 546, 547, 550, 551, 552, 553, 554, 555 43, 452, 453,
  • an RNAi agent for inhibiting the expression of apolipoprotein (a) gene (LPA) in a cell comprising a sense strand and an antisense strand forming a double-stranded region, wherein the double-stranded region is 19 base pairs in length, the antisense strand is 21 nucleotides in length and is any nucleotide sequence selected from SEQ ID NO: 265 to 528 (preferably comprising a nucleotide sequence selected from SEQ ID NO: 283, 289, 295, 310, 345, 365, 377, 384, 419, 432, 439, 441, 452, 461, 470, 483, 491, 508, 510, 521, 530, 531, 532, 533, 534, 536, 537, 538, 540, 541, 542, 543, 544, 545, 546, 547, 548, 550, 551, 552, 553, 554, 555 43, 452, 453, 45
  • the sense strand has a length of 17 to 23 nucleotides, preferably 19 to 21 nucleotides, and the sense strand comprises two consecutive phosphorothioate internucleotide linkages starting from the terminal nucleotide at the 5' end.
  • the structure of the sense strand is mN*mN*mNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmN, wherein mN represents a 2'-O-methyl modified nucleotide, fN represents a 2'-fluoro modified nucleotide, * represents a phosphorothioate internucleotide linkage, and each N can independently be U, C, A or G.
  • the sense strand is no longer than 21 nucleotides and comprises any nucleotide sequence selected from SEQ ID NO: 1 to 264, preferably any nucleotide sequence selected from SEQ ID NO: 19, 25, 31, 46, 81, 101, 113, 120, 155, 168, 175, 177, 179, 188, 189, 190, 191, 193, 194, 197, 198, 200, 202, 203, 204, 210, 212, 213, 228, 230 and 231.
  • the sense strand is 20 nucleotides in length and comprises any nucleotide sequence selected from SEQ ID NO: 1 to 264, preferably any nucleotide sequence selected from SEQ ID NO: 19, 25, 31, 46, 81, 101, 113, 120, 155, 168, 175, 177, 179, 188, 189, 190, 191, 193, 194, 197, 198, 200, 202, 203, 204, 210, 212, 213, 228, 230 and 231.
  • the sense strand is any nucleotide sequence selected from SEQ ID NO: 1 to 264, preferably any nucleotide sequence selected from SEQ ID NO: 19, 25, 31, 46, 81, 101, 113, 120, 155, 168, 175, 177, 179, 188, 189, 190, 191, 193, 194, 197, 198, 200, 202, 203, 204, 210, 212, 213, 228, 230 and 231.
  • a RNAi agent for inhibiting LPA gene expression which comprises a sense chain and an antisense chain that form complementary double-stranded regions, the antisense chain is no longer than 23 nucleotides and comprises any nucleotide sequence selected from SEQ ID NO: 265 to 528, and the sense chain is no longer than 21 nucleotides and comprises any nucleotide sequence selected from SEQ ID NO: 1 to 264.
  • a RNAi agent for inhibiting LPA gene expression which comprises a sense strand and an antisense strand that form complementary double-stranded regions, the antisense strand is any one nucleotide sequence selected from SEQ ID NOs: 265 to 528, and the sense strand is any one nucleotide sequence selected from SEQ ID NOs: 1 to 264.
  • an RNAi agent for inhibiting LPA gene expression which comprises a sense chain and an antisense chain that form complementary double-stranded regions, the antisense chain is a nucleotide sequence selected from SEQ ID NO: 265 to 528, and the sense chain is a nucleotide sequence selected from SEQ ID NO: 1 to 264, and they are paired in the manner shown in Table 1 to form a double-stranded body of D1 to D264.
  • RNAi agent for inhibiting LPA gene expression which comprises a sense strand and an antisense strand forming complementary double-stranded regions, the length of each of the sense strand and the antisense strand does not exceed 23 nucleotides, and wherein:
  • the sense strand comprises the sequence shown in SEQ ID NO:19, and the antisense strand comprises the sequence shown in SEQ ID NO:283;
  • the sense strand comprises the sequence shown in SEQ ID NO:25, and the antisense strand comprises the sequence shown in SEQ ID NO:289;
  • the sense strand comprises the sequence shown in SEQ ID NO:31, and the antisense strand comprises the sequence shown in SEQ ID NO:295;
  • the sense strand comprises the sequence shown in SEQ ID NO:46, and the antisense strand comprises the sequence shown in SEQ ID NO:310;
  • the sense strand comprises the sequence shown in SEQ ID NO:81, and the antisense strand comprises the sequence shown in SEQ ID NO:345;
  • the sense strand comprises the sequence shown in SEQ ID NO:101, and the antisense strand comprises the sequence shown in SEQ ID NO:365;
  • the sense strand comprises the sequence shown in SEQ ID NO:113, and the antisense strand comprises the sequence shown in SEQ ID NO:377;
  • the sense strand comprises the sequence shown in SEQ ID NO:120, and the antisense strand comprises the sequence shown in SEQ ID NO:384;
  • the sense strand comprises the sequence shown in SEQ ID NO:155, and the antisense strand comprises the sequence shown in SEQ ID NO:419;
  • the sense strand comprises the sequence shown in SEQ ID NO:168, and the antisense strand comprises the sequence shown in SEQ ID NO:432;
  • the sense strand comprises the sequence shown in SEQ ID NO:175, and the antisense strand comprises the sequence shown in SEQ ID NO:439;
  • the sense strand comprises the sequence shown in SEQ ID NO:177, and the antisense strand comprises the sequence shown in SEQ ID NO:441;
  • the sense strand comprises the sequence shown in SEQ ID NO:179, and the antisense strand comprises the sequence shown in SEQ ID NO:443;
  • the sense strand comprises the sequence shown in SEQ ID NO:188, and the antisense strand comprises the sequence shown in SEQ ID NO:452;
  • the sense strand comprises the sequence shown in SEQ ID NO:189, and the antisense strand comprises the sequence shown in SEQ ID NO:453;
  • the sense strand comprises the sequence shown in SEQ ID NO:190, and the antisense strand comprises the sequence shown in SEQ ID NO:454;
  • the sense strand comprises the sequence shown in SEQ ID NO: 191, and the antisense strand comprises the sequence shown in SEQ ID NO: 455;
  • the sense strand comprises the sequence shown in SEQ ID NO:193, and the antisense strand comprises the sequence shown in SEQ ID NO:457;
  • the sense strand comprises the sequence shown in SEQ ID NO:194, and the antisense strand comprises the sequence shown in SEQ ID NO:458;
  • the sense strand comprises the sequence shown in SEQ ID NO:197, and the antisense strand comprises the sequence shown in SEQ ID NO:461;
  • the sense strand comprises the sequence shown in SEQ ID NO:198, and the antisense strand comprises the sequence shown in SEQ ID NO:462;
  • the sense strand comprises the sequence shown in SEQ ID NO:200, and the antisense strand comprises the sequence shown in SEQ ID NO:464;
  • the sense strand comprises the sequence shown in SEQ ID NO:202, and the antisense strand comprises the sequence shown in SEQ ID NO:466;
  • the sense strand comprises the sequence shown in SEQ ID NO:203, and the antisense strand comprises the sequence shown in SEQ ID NO:467;
  • the sense strand comprises the sequence shown in SEQ ID NO:204, and the antisense strand comprises the sequence shown in SEQ ID NO:468;
  • the sense strand comprises the sequence shown in SEQ ID NO:210, and the antisense strand comprises the sequence shown in SEQ ID NO:474;
  • the sense strand comprises the sequence shown in SEQ ID NO:212, and the antisense strand comprises the sequence shown in SEQ ID NO:476;
  • the sense strand comprises the sequence shown in SEQ ID NO:213, and the antisense strand comprises the sequence shown in SEQ ID NO:477;
  • the sense strand comprises the sequence shown in SEQ ID NO:228, and the antisense strand comprises the sequence shown in SEQ ID NO:492;
  • the sense strand comprises the sequence shown in SEQ ID NO:230, and the antisense strand comprises the sequence shown in SEQ ID NO:494; or
  • the sense strand comprises the sequence shown in SEQ ID NO:231, and the antisense strand comprises the sequence shown in SEQ ID NO:495.
  • RNAi agent for inhibiting LPA gene expression which comprises a sense strand and an antisense strand forming complementary double-stranded regions, the length of each of the sense strand and the antisense strand does not exceed 23 nucleotides, and wherein:
  • the sense strand comprises the sequence shown in SEQ ID NO:81, and the antisense strand comprises the sequence shown in SEQ ID NO:345;
  • the sense strand comprises the sequence shown in SEQ ID NO:120, and the antisense strand comprises the sequence shown in SEQ ID NO:384;
  • the sense strand comprises the sequence shown in SEQ ID NO:203, and the antisense strand comprises the sequence shown in SEQ ID NO:467;
  • the sense strand comprises the sequence shown in SEQ ID NO:204, and the antisense strand comprises the sequence shown in SEQ ID NO:468;
  • the sense strand comprises the sequence shown in SEQ ID NO:212, and the antisense strand comprises the sequence shown in SEQ ID NO:476;
  • the sense strand comprises the sequence shown in SEQ ID NO:228, and the antisense strand comprises the sequence shown in SEQ ID NO:492; or
  • the sense strand comprises the sequence shown in SEQ ID NO:230, and the antisense strand comprises the sequence shown in SEQ ID NO:494.
  • RNAi agent for inhibiting LPA gene expression is provided, which is a sense strand and an antisense strand forming complementary double-stranded regions, wherein:
  • the sense strand is the sequence shown in SEQ ID NO: 19, and the antisense strand is the sequence shown in SEQ ID NO: 283;
  • the sense strand is the sequence shown in SEQ ID NO:25, and the antisense strand is the sequence shown in SEQ ID NO:289;
  • the sense strand is the sequence shown in SEQ ID NO:31, and the antisense strand is the sequence shown in SEQ ID NO:295;
  • the sense strand is the sequence shown in SEQ ID NO:46, and the antisense strand is the sequence shown in SEQ ID NO:310;
  • the sense strand is the sequence shown in SEQ ID NO:81, and the antisense strand is the sequence shown in SEQ ID NO:345;
  • the sense strand is the sequence shown in SEQ ID NO: 101, and the antisense strand is the sequence shown in SEQ ID NO: 365;
  • the sense strand is the sequence shown in SEQ ID NO: 113, and the antisense strand is the sequence shown in SEQ ID NO: 377;
  • the sense strand is the sequence shown in SEQ ID NO: 120, and the antisense strand is the sequence shown in SEQ ID NO: 384;
  • the sense strand is the sequence shown in SEQ ID NO: 155, and the antisense strand is the sequence shown in SEQ ID NO: 419;
  • the sense strand is the sequence shown in SEQ ID NO: 168, and the antisense strand is the sequence shown in SEQ ID NO: 432;
  • the sense strand is the sequence shown in SEQ ID NO: 175, and the antisense strand is the sequence shown in SEQ ID NO: 439;
  • the sense strand is the sequence shown in SEQ ID NO: 177, and the antisense strand is the sequence shown in SEQ ID NO: 441;
  • the sense strand is the sequence shown in SEQ ID NO: 179, and the antisense strand is the sequence shown in SEQ ID NO: 443;
  • the sense strand is the sequence shown in SEQ ID NO: 188, and the antisense strand is the sequence shown in SEQ ID NO: 452;
  • the sense strand is the sequence shown in SEQ ID NO: 189, and the antisense strand is the sequence shown in SEQ ID NO: 453;
  • the sense strand is the sequence shown in SEQ ID NO: 190, and the antisense strand is the sequence shown in SEQ ID NO: 454;
  • the sense strand is the sequence shown in SEQ ID NO: 191, and the antisense strand is the sequence shown in SEQ ID NO: 455;
  • the sense strand is the sequence shown in SEQ ID NO: 193, and the antisense strand is the sequence shown in SEQ ID NO: 457;
  • the sense strand is the sequence shown in SEQ ID NO: 194, and the antisense strand is the sequence shown in SEQ ID NO: 458;
  • the sense strand is the sequence shown in SEQ ID NO: 197, and the antisense strand is the sequence shown in SEQ ID NO: 461;
  • the sense strand is the sequence shown in SEQ ID NO: 198, and the antisense strand is the sequence shown in SEQ ID NO: 462;
  • the sense strand is the sequence shown in SEQ ID NO:200, and the antisense strand is the sequence shown in SEQ ID NO:464;
  • the sense strand is the sequence shown in SEQ ID NO:202, and the antisense strand is the sequence shown in SEQ ID NO:466;
  • the sense strand is the sequence shown in SEQ ID NO:203, and the antisense strand is the sequence shown in SEQ ID NO:467;
  • the sense strand is the sequence shown in SEQ ID NO:204, and the antisense strand is the sequence shown in SEQ ID NO:468;
  • the sense strand is the sequence shown in SEQ ID NO:210, and the antisense strand is the sequence shown in SEQ ID NO:474;
  • the sense strand is the sequence shown in SEQ ID NO:212, and the antisense strand is the sequence shown in SEQ ID NO:476;
  • the sense strand is the sequence shown in SEQ ID NO:213, and the antisense strand is the sequence shown in SEQ ID NO:477;
  • the sense strand is the sequence shown in SEQ ID NO:228, and the antisense strand is the sequence shown in SEQ ID NO:492;
  • the sense strand is the sequence shown in SEQ ID NO:230, and the antisense strand is the sequence shown in SEQ ID NO:494; or
  • the sense strand is the sequence shown in SEQ ID NO:231, and the antisense strand is the sequence shown in SEQ ID NO:495.
  • RNAi agent for inhibiting LPA gene expression which comprises a sense strand and an antisense strand forming complementary double-stranded regions, wherein:
  • the sense strand is the sequence shown in SEQ ID NO:81, and the antisense strand is the sequence shown in SEQ ID NO:345;
  • the sense strand is the sequence shown in SEQ ID NO: 120, and the antisense strand is the sequence shown in SEQ ID NO: 384;
  • the sense strand is the sequence shown in SEQ ID NO:203, and the antisense strand is the sequence shown in SEQ ID NO:467;
  • the sense strand is the sequence shown in SEQ ID NO:204, and the antisense strand is the sequence shown in SEQ ID NO:468;
  • the sense strand is the sequence shown in SEQ ID NO:212, and the antisense strand is the sequence shown in SEQ ID NO:476;
  • the sense strand is the sequence shown in SEQ ID NO:228, and the antisense strand is the sequence shown in SEQ ID NO:492; or
  • the sense strand is the sequence shown in SEQ ID NO:230, and the antisense strand is the sequence shown in SEQ ID NO:494.
  • the RNAi agent of the present invention may include a ligand.
  • a "ligand” refers to any compound or molecule that can interact directly or indirectly with another compound or molecule.
  • the interaction of a ligand with another compound or molecule may trigger a biological response (e.g., initiate a signal transduction cascade, induce receptor-mediated endocytosis), or may be just a physical connection.
  • the ligand may change one or more properties of the attached double-stranded RNA molecule, such as the pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance of the RNA molecule.
  • the LPA gene is primarily expressed in the liver. Therefore, in certain embodiments, it is desirable to specifically deliver the RNAi agent of the present invention to hepatocytes. Therefore, in certain embodiments, the ligands are targeted to specifically deliver RNAi agents to hepatocytes using various methods described in more detail below. In certain embodiments, the RNAi agent is targeted to hepatocytes with a ligand that binds to a surface-expressed asialoglycoprotein receptor (ASGR) or a component thereof (e.g., ASGR1, ASGR2).
  • ASGR asialoglycoprotein receptor
  • RNAi agents can be specifically targeted to the liver by using ligands that bind to or interact with proteins expressed on the surface of hepatocytes.
  • the ligand may include an antigen binding protein (e.g., an antibody or a binding fragment thereof (e.g., Fab, scFv)) that specifically binds to receptors expressed on hepatocytes, such as asialoglycoprotein receptors and LDL receptors.
  • the ligand includes an antibody or a binding fragment thereof that specifically binds to ASGR1 and/or ASGR2.
  • the ligand includes a Fab fragment of an antibody that specifically binds to ASGR1 and/or ASGR2.
  • the ligand includes a single-chain variable antibody fragment (scFv fragment) of an antibody that specifically binds to ASGR1 and/or ASGR2.
  • scFv fragment single-chain variable antibody fragment
  • Exemplary antibodies and binding fragments thereof that can be used as ligands for targeting the RNAi agents of the present invention to the liver are described in WO 2017/058944, which is incorporated herein by reference in its entirety.
  • Other antibodies or binding fragments thereof that specifically bind to ASGR1, LDL receptors, or other liver surface expressed proteins suitable for use as ligands in the RNAi agents of the present invention are purchased from commercial sources.
  • the ligand comprises a carbohydrate.
  • Carbohydrate refers to a compound composed of one or more monosaccharide units having at least 6 carbon atoms (which may be linear, branched, or cyclic), each of which has an oxygen, nitrogen, or sulfur atom attached to it.
  • Carbohydrates include, but are not limited to, sugars (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides (e.g., starch, glycogen, cellulose, and polysaccharide gums).
  • the carbohydrate incorporated into the ligand is selected from pentose, hexose or heptose and disaccharides and trisaccharides comprising such monosaccharide units.
  • the carbohydrate incorporated into the ligand is an amino sugar, such as galactosamine, glucosamine, N-acetylgalactosamine and N-acetylglucosamine.
  • the ligand comprises a hexose or a hexosamine.
  • the hexose may be selected from glucose, galactose, mannose, fucose or fructose.
  • the hexosamine may be selected from fructosamine, galactosamine, glucosamine or mannosamine.
  • the ligand comprises glucose, galactose, galactosamine or glucosamine.
  • the ligand comprises glucose, glucosamine or N-acetylglucosamine.
  • the ligand comprises galactose, galactosamine or N-acetylgalactosamine.
  • ligands comprising glucose, galactose and N-acetylgalactosamine (GalNAc) are particularly effective in targeting RNA to hepatocytes because these ligands bind to ASGR expressed on the surface of hepatocytes.
  • GalNAc- or galactose-containing ligands that can be incorporated into the RNAi agents of the present invention are described in USP 7,491,805, 8,106,022 and 8,877,917; U.S. Patent Publication No. US20030130186; and WIPO Publication No. WO 2013/166155, all of which are incorporated herein by reference in their entirety.
  • the ligand includes a multivalent carbohydrate moiety.
  • a “multivalent carbohydrate moiety” refers to a moiety comprising two or more carbohydrate units that can independently bind or interact with other molecules.
  • a multivalent carbohydrate moiety includes two or more binding domains consisting of carbohydrates, which can bind to two or more different molecules or two or more different sites on the same molecule.
  • the "valence" of a carbohydrate moiety indicates the number of individual binding domains within the carbohydrate moiety.
  • the terms “monovalent,” “divalent,” “trivalent,” and “tetravalent” refer to carbohydrate moieties having one, two, three, and four binding domains, respectively, relative to a carbohydrate moiety.
  • a multivalent carbohydrate moiety can include a multivalent lactose moiety, a multivalent galactose moiety, a multivalent glucose moiety, a multivalent N-acetylgalactosamine moiety, a multivalent N-acetylglucosamine moiety, a multivalent mannose moiety, or a multivalent fucose moiety.
  • the ligand includes a multivalent galactose moiety.
  • the ligand includes a multivalent N-acetylgalactosamine moiety.
  • the multivalent carbohydrate moiety can be divalent, trivalent, or tetravalent. In such embodiments, the multivalent carbohydrate moiety can be bi-branched or tri-branched.
  • the multivalent N-acetylgalactosamine moiety is trivalent or tetravalent. In another specific embodiment, the multivalent galactose moiety is trivalent or tetravalent. Exemplary trivalent and tetravalent GalNAc-containing ligands for incorporation into the RNAi agents of the present invention are described in detail below.
  • the ligand can be directly or indirectly connected or conjugated to the RNA molecule of the RNAi agent.
  • the ligand is directly covalently connected to the sense strand or antisense strand of the RNAi agent.
  • the ligand is covalently connected to the sense strand or antisense strand of the RNAi agent through a joint.
  • the ligand can be connected to the core base, sugar moiety or internucleotide connection of the sense strand or antisense strand of the RNAi agent of the present invention.
  • the ligand can be attached to the 3' or 5' end of the sense strand or antisense strand. In certain embodiments, the ligand is covalently attached to the 5' end of the sense strand. In such embodiments, the ligand is attached to the 5'-terminal nucleotide of the sense strand. In these and other embodiments, the ligand is attached at the 5'-position of the 5'-terminal nucleotide of the sense strand. In other embodiments, the ligand is covalently attached to the 3' end of the sense strand. For example, in some embodiments, the ligand is attached to the 3'-terminal nucleotide of the sense strand.
  • the ligand is attached to the 3'-position of the 3'-terminal nucleotide of the sense strand. In alternative embodiments, the ligand is attached near the 3' end of the sense strand, but before one or more terminal nucleotides (i.e., before 1, 2, 3, or 4 terminal nucleotides). In some embodiments, the ligand is attached to the 2'-position of the sugar of the 3'-terminal nucleotide of the sense strand. In other embodiments, the ligand is attached to the 2'-position of the sugar of the 5'-terminal nucleotide of the sense strand.
  • the ligand is connected to the sense strand or antisense strand through a joint.
  • "Joint" refers to an atom or a group of atoms that covalently connects the ligand to the polynucleotide component of the RNAi agent.
  • the length of the joint can be about 1 to about 30 atoms, about 2 to about 28 atoms, about 3 to about 26 atoms, about 4 to about 24 atoms, about 6 to about 20 atoms, about 7 to about 20 atoms, about 8 to about 20 atoms, about 8 to about 18 atoms, and about 12 to about 18 atoms.
  • the joint may include a bifunctional linking portion, which generally includes an alkyl portion with two functional groups.
  • the joint includes an oligomer of a chain structure or a repeating unit, such as ethylene glycol or an amino acid unit.
  • functional groups commonly used in bifunctional linking portions include, but are not limited to, electrophilic reagents for reacting with nucleophilic groups and electrophilic reagents for reacting with electrophilic groups.
  • the bifunctional linking moiety includes an amino group, a hydroxyl group, a carboxylic acid, a thiol, an unsaturated bond (eg, a double bond or a triple bond), and the like.
  • Linkers that can be used to link the ligand to the sense or antisense strand of the RNAi agent of the invention include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxooctanoic acid, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid, 6-aminohexanoic acid, substituted C 1 -C 10 alkyl, substituted or unsubstituted C 2 -C 10 alkenyl, or substituted or unsubstituted C 2 -C 10 alkynyl.
  • Preferred substituents for such linkers include, but are not limited to, hydroxyl, amino, alkoxy, carboxyl, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.
  • the joint is cleavable.
  • a cleavable joint is one that is sufficiently stable outside the cell but is cleaved to release two parts that are combined together by a joint when entering the target cell.
  • a cleavable joint is at least 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least 100 times faster than cracking in the blood of the experimenter in the target cell or under the first reference condition (it can, for example, be selected as a simulation or represent intracellular conditions).
  • Cleavable joints are susceptible to the effects of cleavage agents, such as pH, redox potential, or the presence of degradation molecules.
  • cleavage agents are more prevalent in cells than in serum or blood, or are found at higher levels or activities in cells.
  • cleavage agents include: redox agents selected for specific substrates or without substrate specificity, including, for example, oxidases or reductases present in cells; esterases; endosomes or reagents that can produce an acidic environment, such as those that result in a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable joints as general acids, peptidases (which can be substrate-specific), and phosphatases.
  • Cleaving joints may include pH-sensitive parts.
  • the pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3.
  • Endosomes have a more acidic pH
  • lysosomes have a more acidic pH (about 5.0) in the range of 5.5-6.0.
  • Some joints will have a cleavable group that is cleaved at a preferred pH, thereby releasing the RNA molecule from the ligand in the cell, or releasing it into the desired organelle of the cell.
  • the joint may include a cleavable group that can be cleaved by a specific enzyme. The type of cleavable group incorporated into the joint may depend on the cell to be targeted.
  • a liver targeting ligand may be connected to an RNA molecule by a joint including an ester group.
  • Hepatocytes are rich in esterases, so joints are more effectively cleaved in hepatocytes than in cell types that are not rich in esterases.
  • Other types of cells rich in esterases include cells of the lung, renal cortex, and testis.
  • a joint containing a peptide bond may be used.
  • linkers suitable for attaching ligands to the sense or antisense strands in the RNAi agents of the invention are known in the art, such as those described in U.S. Patents 7,723,509, 8,017,762, 8,828,956, 8,877,917, and 9,181,551, all of which are incorporated herein by reference in their entirety.
  • the ligand covalently attached to the sense strand or antisense strand of the RNAi agent of the invention includes a GalNAc moiety, such as a multivalent GalNAc.
  • the multivalent GalNAc moiety is a trivalent GalNAc and is attached to the 3' end of the sense strand.
  • the multivalent GalNAc moiety is a trivalent GalNAc and is attached to the 5' end of the sense strand.
  • the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 3' end of the sense strand.
  • the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 5' end of the sense strand.
  • the ligand of the RNAi agent of the invention comprises the structure of Formula I (the wavy line indicates the position of attachment to the sense strand or the antisense strand):
  • the ligand having this structure is covalently linked to the 5' end of the sense strand via a linker.
  • the linker is an aminohexyl linker.
  • GalNAc moieties and linkers that can be attached to double-stranded RNA molecules in the RNAi agents of the invention are provided below in Structural Formulas II-X.
  • "Ac” in the formulas listed herein represents an acetyl group.
  • the RNAi agent comprises a ligand and a linker having the structure of Formula II below, wherein each n is independently 1-3, k is 1-3, m is 1 or 2, j is 1 or 2, and the ligand is linked to the 3' end of the sense strand of the double-stranded RNA molecule (represented by a solid wavy line):
  • the RNAi agent comprises a ligand and a linker having the following structure of Formula III, wherein each n is independently 1-3, k is 1-3, m is 1 or 2, j is 1 or 2, and the ligand is attached to the 3' end of the sense strand of the double-stranded RNA molecule (represented by a solid wavy line):
  • the RNAi agent comprises a ligand having the structure of Formula IV below and a linker, wherein the ligand is attached to the 3' end of the sense strand of the double-stranded RNA molecule (represented by the solid wavy line):
  • the RNAi agent comprises a ligand and a linker having the structure of the following Formula V, wherein the ligand is attached to the 3' end of the sense strand of the double-stranded RNA molecule (represented by the solid wavy line):
  • the RNAi agent comprises a ligand and a linker having the structure of Formula VI below, wherein each n is independently 1-3, k is 1-3, and the ligand is attached to the 5' end of the sense strand of the double-stranded RNA molecule (represented by a solid wavy line):
  • the RNAi agent comprises a ligand and a linker having the structure of Formula VII below, wherein each n is independently 1-3, k is 1-3, and the ligand is attached to the 5' end of the sense strand of the double-stranded RNA molecule (represented by a solid wavy line):
  • the RNAi agent comprises a ligand and a linker having the following structure of Formula IX, wherein each n is independently 1-3, and the ligand is attached to the 5' end of the sense strand of the double-stranded RNA molecule (represented by a solid wavy line):
  • the RNAi agent comprises a ligand and a linker having the structure of the following Formula X, wherein the ligand is attached to the 5' end of the sense strand of the double-stranded RNA molecule (represented by the solid wavy line):
  • Phosphorothioate bonds can be substituted for the phosphodiesterase bonds shown in any of Formulas I-X to covalently attach the ligand and linker to the nucleic acid strand.
  • the present invention also includes pharmaceutical compositions and preparations comprising the RNAi agents described herein and pharmaceutically acceptable carriers, excipients or diluents.
  • Such compositions and preparations can be used to reduce the expression of LPA genes in patients in need.
  • pharmaceutical compositions and preparations will be prepared in a form suitable for the intended application. Typically, this will require the preparation of a composition that is substantially free of pyrogens and other impurities that may be harmful to humans or animals.
  • the composition and method for preparing the pharmaceutical composition depend on many standards, including but not limited to route of administration, the type and degree of the disease to be treated or the condition or the dosage to be administered.
  • the pharmaceutical composition is prepared based on the expected delivery route.
  • the pharmaceutical composition is formulated for parenteral delivery.
  • Parenteral administration forms include intravenous, intraarterial, subcutaneous, intrathecal, intraperitoneal or intramuscular injection or infusion.
  • the pharmaceutical composition is formulated for intravenous delivery.
  • the pharmaceutical composition may include a lipid-based delivery vehicle.
  • the pharmaceutical composition is formulated for subcutaneous delivery.
  • the pharmaceutical composition may include a targeting ligand (such as a ligand containing GalNAc or an antibody as described herein).
  • the pharmaceutical composition comprises an effective amount of the RNAi agent described herein.
  • An "effective amount” refers to an amount sufficient to produce a beneficial or desired clinical outcome.
  • an effective amount is an amount sufficient to reduce the expression of the LPA gene in a particular tissue or cell type (e.g., liver or hepatocyte) of the patient.
  • the effective amount of the RNAi agent of the present invention can be from about 0.01 mg/kg body weight to about 100 mg/kg body weight, and can be administered daily, weekly, monthly, or at longer intervals.
  • Accurately determining a specific effective dosage and dosing frequency may be based on several factors, including the patient's size, age, and general condition, the type of disease to be treated (e.g., myocardial infarction, coronary artery disease, peripheral artery disease, stroke), the specific RNAi agent used, and the route of administration.
  • Administration of the pharmaceutical composition of the present invention can be carried out by any common route, as long as the target tissue is accessible by the route.
  • routes include, but are not limited to, parenteral (e.g., subcutaneous, intramuscular, intraperitoneal or intravenous), oral, nasal
  • parenteral e.g., subcutaneous, intramuscular, intraperitoneal or intravenous
  • oral nasal
  • nasal e.g., subcutaneous, intramuscular, intraperitoneal or intravenous
  • the pharmaceutical composition is administered parenterally.
  • the pharmaceutical composition is administered intravenously.
  • the pharmaceutical composition is administered subcutaneously.
  • Colloidal dispersion systems can be used as delivery vehicles for the RNAi agents of the present invention, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • Commercially available fat emulsions suitable for delivering nucleic acids of the present invention include (Baxter International Inc.), (Abbott Pharmaceuticals), (Hospira), (Hospire), Nutrilipid (B.Braun Medical Inc.) and other similar fat emulsions.
  • the preferred colloidal system used as a delivery vehicle in vivo is a liposome (i.e., an artificial membrane vesicle).
  • RNAi agent of the present invention can be encapsulated in a liposome, or can form a complex therewith, particularly with a cationic liposome.
  • the RNAi agent of the present invention can be complexed with lipids, particularly with cationic lipids.
  • Suitable cationic lipids are, for example, diol tetramethylaminopropyl (DOTAP) and diol phosphatidylethanolamine (DOTMA).
  • compositions suitable for injection include, for example, sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injection solutions or dispersions.
  • these preparations are sterile and, to a certain extent, fluid and easy to inject.
  • the preparation should remain stable under production and storage conditions and should be preserved to prevent the contamination of microorganisms such as bacteria and fungi.
  • Suitable solvents or dispersion media may include, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.), their suitable mixtures and vegetable oils.
  • appropriate fluidity can be maintained by using a coating such as lecithin, by maintaining the desired particle size in the case of dispersion, and by using a surfactant.
  • a coating such as lecithin
  • surfactant for example, a surfactant for maintaining the desired particle size in the case of dispersion
  • the effects of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.
  • isotonic agents are preferably included, such as sugar or sodium chloride.
  • the extended absorption of injectable compositions can be achieved by using agents that delay absorption in the composition, such as aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by adding an appropriate amount of the active compound to a solvent together with any other ingredients (e.g., those listed above) and then filtering and sterilizing.
  • dispersions are prepared by adding various sterilized active ingredients to a dispersion medium containing an alkaline dispersion medium and other desired ingredients, e.g., as described above.
  • preferred preparation methods include vacuum drying and freeze drying techniques, which produce a powder of the active ingredient and any additional desired ingredients from a previously sterile filtered solution thereof.
  • compositions of the present invention can generally be formulated in neutral form or salt form.
  • Pharmaceutically acceptable salts include, for example, acid addition salts (formed by free amino groups) derived from inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium or iron oxide) or organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.).
  • the RNAi agent of the present invention is formulated as a sodium salt.
  • an aqueous solution for parenteral administration in the form of an aqueous solution, the solution is usually appropriately buffered, and the liquid diluent is first made isotonic with, for example, enough saline or glucose.
  • an aqueous solution can be used for, for example, intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • a sterile aqueous medium is used.
  • a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion liquid, or injected at the infusion site of the suggestion.
  • the preparation should meet the sterility, pyrogenicity, general safety and purity standards required by the local Food and Drug Administration standards.
  • the pharmaceutical composition of the present invention comprises sterile saline solution and RNAi agent as described herein or consists of the two.
  • the pharmaceutical composition of the present invention comprises RNAi agent as described herein and sterile water (e.g., water for injection, WFI) or consists of the two.
  • the pharmaceutical composition of the present invention comprises RNAi agent as described herein and phosphate buffered saline (PBS) or consists of it.
  • PBS phosphate buffered saline
  • the pharmaceutical composition of the present invention is packaged with or stored in a drug delivery device.
  • Devices for injectable formulations include, but are not limited to, injection ports, prefilled syringes, automatic injectors, injection pumps, in vivo injectors, and injection pens.
  • Equipment for atomized or powdered formulations includes, but is not limited to, inhalers, insufflators, aspirators, and the like. Therefore, the present invention includes a drug delivery device containing a pharmaceutical composition of the present invention for treating or preventing one or more diseases or conditions described herein.
  • the present invention provides a method for reducing or inhibiting the expression of a gene by contacting a cell with any of the RNAi agents described herein.
  • a method for reducing or inhibiting the production of apo(a) protein by expressing the LPA gene in a cell e.g., a hepatocyte.
  • the cell may be in vitro or in vivo.
  • LPA gene expression can be assessed by measuring the amount or level of LPA mRNA, apo(a) protein, or another biomarker associated with LPA expression (e.g., serum Lp(a) level).
  • the reduction in LPA expression in cells or animals treated with the RNAi agent of the present invention can be determined relative to LPA expression in cells and animals not treated with the RNAi agent or treated with a control RNAi agent.
  • the reduction in LPA expression is assessed by (a) measuring the amount or level of LPA mRNA in hepatocytes treated with the RNAi agent of the present invention, (b) measuring the amount or level of LPA mRNA in hepatocytes treated with a control RNAi agent (e.g., an RNAi agent directed to an RNA molecule not expressed in hepatocytes or an RNAi agent with a nonsense or scrambled sequence) or no RNAi agent, and (c) comparing the LPA mRNA level measured in the treated cells in (a) with the LPA mRNA level in the control cells in (b).
  • a control RNAi agent e.g., an RNAi agent directed to an RNA molecule not expressed in hepatocytes or an RNAi agent
  • LPA mRNA levels in treated and control cells can be normalized to the RNA level of a control gene (e.g., 18S ribosomal RNA or a housekeeping gene).
  • LPA mRNA levels can be measured by a variety of methods, including Northern blot analysis, nuclease protection assays, fluorescence in situ hybridization (FISH), reverse transcriptase (RT)-PCR, real-time RT-PCR, quantitative PCR, droplet digital PCR, etc.
  • reduction in LPA expression is assessed by (a) measuring the amount or level of apo(a) protein in hepatocytes treated with an RNAi agent of the invention, (b) measuring the amount or level of apo(a) protein in hepatocytes treated with a control RNAi agent (e.g., an RNAi agent directed to an RNA molecule not expressed in hepatocytes or an RNAi construct having a nonsense or scrambled sequence) or no RNAi agent, and (c) comparing the measured apo(a) protein levels from treated cells in (a) with the measured apo(a) protein levels from control cells in (b).
  • a control RNAi agent e.g., an RNAi agent directed to an RNA molecule not expressed in hepatocytes or an RNAi construct having a nonsense or scrambled sequence
  • RNAi agents for measuring apo(a) protein levels are known in the art to those skilled in the art and include western blots, immunoassays (e.g., ELISAs), and flow cytometry. Any method capable of measuring LPA mRNA or apo(a) protein can be used to assess the efficacy of the RNAi agents of the invention.
  • the method for assessing the expression level of LPA is performed in vitro in cells (e.g., hepatocytes) that naturally express the LPA gene or in cells that have been engineered to express LPA.
  • the method is performed in hepatocytes in vitro.
  • Suitable hepatocytes include, but are not limited to, primary hepatocytes (e.g., human or non-human primate hepatocytes), HepAD38 cells, HuH-6 cells, HuH-7 cells, HuH-5-2 cells, BNLCL2 cells, Hep3B cells, or HepG2 cells.
  • the hepatocyte is a HuH-7 cell.
  • the hepatocyte is a human primary hepatocyte.
  • the method of assessing LPA expression levels is performed in vivo.
  • the RNAi agent and any control RNAi agent can be administered to an animal (e.g., a transgenic animal or non-human primate expressing an LPA gene), and LPA mRNA or apo(a) protein levels can be assessed in liver tissue harvested from the animal after treatment.
  • an animal e.g., a transgenic animal or non-human primate expressing an LPA gene
  • LPA mRNA or apo(a) protein levels can be assessed in liver tissue harvested from the animal after treatment.
  • biomarkers or functional phenotypes associated with LPA expression can be assessed in treated animals.
  • apo(a) protein is the major component of Lp(a) in serum or plasma. Therefore, serum or plasma levels of Lp(a) can be measured in animals treated with the RNAi agents of the present invention to assess the functional efficacy of reducing LPA expression.
  • the expression of LPA in hepatocytes is reduced by at least 40%, at least 45%, or at least 50% by the RNAi agents of the invention. In some embodiments, the expression of LPA in hepatocytes is reduced by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% by the RNAi agents of the invention. In other embodiments, the expression of LPA in hepatocytes is reduced by about 90% or more, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more by the RNAi agents of the invention. The percentage reduction in LPA expression can be measured by any of the methods described herein and other methods known in the art.
  • the present invention provides methods for reducing or inhibiting the expression of the LPA gene in patients in need, thereby reducing or inhibiting the production of apo(a) protein, and methods for treating or preventing diseases or conditions associated with LPA expression or apo(a) activity.
  • Diseases or conditions associated with LPA expression refer to diseases or conditions in which the level of LPA expression is altered, or conditions or conditions in which increased levels of LPA expression are associated with an increased risk of developing the disease and condition.
  • Diseases or conditions associated with LPA expression may also include diseases or conditions caused by abnormal changes in lipoprotein metabolism, such as changes that result in abnormal or increased levels of Lp(a), cholesterol, lipids, triglycerides, etc., or impaired clearance of these molecules.
  • RNAi agents of the present invention are particularly suitable for treating or preventing cardiovascular and cerebrovascular diseases (e.g., coronary artery disease and myocardial infarction) and reducing circulating levels of Lp(a).
  • cardiovascular and cerebrovascular diseases e.g., coronary artery disease and myocardial infarction
  • Diseases and conditions associated with LPA expression that can be treated or prevented according to the methods of the present invention include, but are not limited to, cardiovascular and cerebrovascular diseases, such as myocardial infarction, heart failure, stroke (ischemic and hemorrhagic), atherosclerosis, coronary artery disease, peripheral vascular disease (e.g., peripheral arterial disease); cerebrovascular disease, vulnerable plaques and aortic valve stenosis; familial hypercholesterolemia; venous thrombosis; hypercholesterolemia; hyperlipidemia; and dyslipidemia.
  • cardiovascular and cerebrovascular diseases such as myocardial infarction, heart failure, stroke (ischemic and hemorrhagic), atherosclerosis, coronary artery disease, peripheral vascular disease (e.g., peripheral arterial disease); cerebrovascular disease, vulnerable plaques and aortic valve stenosis; familial hypercholesterolemia; venous thrombosis; hypercholesterolemia; hyperlipidemia; and dyslipidemia.
  • the present invention provides a method for reducing LPA expression in a patient in need of reducing LPA expression, comprising administering any RNAi agent described herein to the patient.
  • the expression level of LPA in the patient's hepatocytes is reduced compared to the expression level of LPA in a patient who has not received the RNAi agent, or compared to the expression level of LPA in a patient before administering the RNAi agent.
  • the expression of LPA in the patient is reduced by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90%, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
  • the percentage reduction in LPA expression can be measured by any method described herein and other methods known in the art. In certain embodiments, the percentage reduction in LPA expression is determined by assessing the level of Lp(a) in the patient's serum or plasma according to the methods described herein.
  • patients who need to reduce LPA expression are patients at risk of myocardial infarction.
  • Patients at risk of myocardial infarction may be patients with a history of myocardial infarction (e.g., having had a myocardial infarction before).
  • Patients at risk of myocardial infarction may also be patients with a family history of myocardial infarction or with one or more risk factors for myocardial infarction.
  • risk factors include, but are not limited to, hypertension, elevated levels of non-high-density lipoprotein cholesterol, elevated levels of triglycerides, diabetes, obesity, or a history of autoimmune diseases (such as rheumatoid arthritis, lupus).
  • patients at risk of myocardial infarction are patients suffering from or diagnosed with coronary artery disease.
  • the risk of myocardial infarction in these patients and other patients can be reduced by administering any RNAi agent described herein to the patient.
  • the present invention provides a method for reducing the risk of myocardial infarction in patients in need, comprising administering an RNAi agent described herein to the patient.
  • the present invention includes the use of any RNAi agent described herein in the preparation of a medicament for reducing the risk of myocardial infarction in patients in need.
  • the present invention provides an LPA-targeted RNAi agent for a method of reducing the risk of myocardial infarction in patients in need.
  • the present invention includes a method for treating or preventing cardiovascular and cerebrovascular diseases in patients in need by administering any RNAi agent of the present invention.
  • the present invention includes the use of any RNAi agent described herein in the preparation of a medicament for treating or preventing cardiovascular and cerebrovascular diseases in patients in need.
  • the present invention provides LPA-targeted RNAi agents in methods for treating or preventing cardiovascular and cerebrovascular diseases in patients in need.
  • Cardiovascular and cerebrovascular diseases include, but are not limited to, myocardial infarction, heart failure, stroke (ischemic and hemorrhagic), atherosclerosis, coronary artery disease, peripheral vascular disease (such as peripheral artery disease), cerebrovascular disease, vulnerable plaques, and aortic stenosis.
  • the cardiovascular and cerebrovascular disease to be treated or prevented according to the methods of the present invention is coronary artery disease.
  • the cardiovascular and cerebrovascular disease to be treated or prevented according to the methods of the present invention is myocardial infarction.
  • the cardiovascular and cerebrovascular disease to be treated or prevented according to the methods of the present invention is stroke.
  • the cardiovascular and cerebrovascular disease to be treated or prevented according to the methods of the present invention is peripheral artery disease.
  • administration of the RNAi agents described herein reduces the risk of non-fatal myocardial infarction, fatal and non-fatal stroke, certain types of cardiac surgery (e.g., angioplasty, bypass surgery), hospitalization for heart failure, chest pain in patients with heart disease, and/or cardiovascular and cerebrovascular events in patients with established heart disease (e.g., previous myocardial infarction, previous cardiac surgery, and/or chest pain with evidence of arterial blockage).
  • administration of the RNAi agents described herein according to the methods of the invention can be used to reduce the risk of recurrent cardiovascular and cerebrovascular events.
  • the patient in need of reducing LPA expression is a patient with elevated circulating Lp(a) levels. Therefore, in some embodiments, the present invention provides a method of reducing serum or plasma levels of Lp(a) in a patient in need thereof by administering any RNAi agent described herein to the patient. In some embodiments, the present invention includes the use of any RNAi agent described herein in the preparation of a medicament for reducing serum or plasma levels of Lp(a) in a patient in need thereof. In other embodiments, the present invention provides an LPA-targeted RNAi agent for a method of reducing serum or plasma levels of Lp(a) in a patient in need thereof. As described above, elevated circulating Lp(a) levels are associated with an increased risk of cardiovascular and cerebrovascular disease.
  • the Lp (a) level in the serum or plasma of the patient after the RNAi agent is applied is reduced.
  • the Lp (a) level in the patient's serum or plasma is reduced to about 150nmol/L or lower, about 125nmol/L or lower, about 100nmol/L or lower, about 75nmol/L or lower, about 70nmol/L or lower, about 65nmol/L or lower, about 60nmol/L or lower, about 55nmol/L or lower, about 50nmol/L or lower, about 45nmol/L or lower, about 40nmol/L or lower, about 35nmol/L or lower, or about 30nmol/L or lower.
  • Lp (a) levels can be measured in units of mass concentration (e.g., mg/dL).
  • the RNAi agents of the invention can reduce the Lp(a) level in the patient's serum or plasma to about 100 mg/dL or less, about 90 mg/dL or less, about 80 mg/dL or less, about 70 mg/dL or less, about 60 mg/dL or less, about 50 mg/dL or less, about 45 mg/dL or less, about 40 mg/dL or less, about 35 mg/dL or less, about 30 mg/dL or less, about 25 mg/dL or less, about 20 mg/dL or less, or about 15 mg/dL or less.
  • the Lp(a) level in plasma or serum samples can be measured using a commercial kit, such as Mercodia AB (Uppsala, Sweden).
  • the patient to be treated according to the method of the present invention is a patient with elevated circulating levels of Lp(a) (e.g., elevated serum or plasma levels of Lp(a).
  • the circulating Lp(a) level of the patient to be treated according to the method of the present invention may be about 50nmol/L or higher, about 55nmol/L or higher, about 60nmol/L or higher, about 65nmol/L or higher, about 70nmol/L or higher, about 75nmol/L or higher, about 100nmol/L or higher, about 125nmol/L or higher, about 150nmol/L or higher, about 175nmol/L or higher, or about 200nmol/L or higher.
  • the patient's serum or plasma Lp(a) level is about 100nmol/L or higher, the patient is given the RNAi agent of the present invention. In one embodiment, if the patient's serum or plasma Lp(a) level is about 125nmol/L or higher, the patient is given the RNAi agent of the present invention. In another embodiment, if the patient's serum or plasma Lp (a) level is about 150nmol / L or higher, the RNAi agent of the present invention is administered to the patient.
  • the circulating Lp (a) level of the patient to be treated according to the method of the present invention may be about 30mg / dL or greater, about 35mg / dL or greater, about 40mg / dL or greater, about 45mg / dL or greater, about 50mg / dL or greater, about 55mg / dL or greater, about 60mg / d or greater, about 65mg / dL or greater, about 70mg / dL or greater, about 75mg / dL or greater, or about 100mg / dL or greater.
  • the RNAi agent of the present invention is administered to the patient. In another embodiment, if the patient's serum or plasma Lp (a) level is about 70mg / dL or higher, the RNAi agent of the present invention is administered to the patient.
  • the patient to be treated according to the methods of the present invention is a patient with vulnerable plaques (also referred to as unstable plaques).
  • vulnerable plaques also referred to as unstable plaques.
  • Vulnerable plaques are accumulations of macrophages and lipids containing primarily cholesterol located below the endothelial layer of the arterial wall. These vulnerable plaques may rupture, leading to the formation of thrombi, which may impede the flow of blood through the artery and lead to myocardial infarction or stroke.
  • Vulnerable plaques can be identified by methods known in the art, including but not limited to intravascular ultrasound and computed tomography.
  • RNAi agent of the present invention has a better effect of inhibiting LPA expression and has lower toxicity.
  • siRNA was synthesized according to the phosphoramidite technique on a solid phase for oligonucleotide synthesis. Depending on the scale, ABI394 synthesis was used. The synthesis was performed on a solid support made of controlled pore glass (CPG, obtained from LGC Biosearch Technologies).
  • CPG controlled pore glass
  • RNAs All 2′-modified RNAs were purchased from Shanghai Zhaowei Technology Development Co., Ltd.
  • 2'-O-methyl phosphoramidite N-benzoyl-5'-O-(4,4-dimethoxytrityl)-2'-O-methyladenosine-3'-(2-cyanoethyl-N,N-diisopropyl) phosphoramidite; 5'-O-(4,4-dimethoxytrityl)-2'-O-methyl-N-isobutyrylguanosine-3'-(2-cyanoethyl-N,N-diisopropyl) phosphoramidite; N-acetyl-5'-O-(4,4-dimethoxytrityl)-2'-O-methylcytidine-3'-(2-cyanoethyl-N,N-diisopropyl) phosphoramidite; N3-benzoyl-2'-methoxyuridine phosphoramidite.
  • 2'-Fluorophosphoramidite N-benzoyl-5'-O-[bis(4-methoxyphenyl)phenylmethyl]-2'-deoxy-2'-fluoroadenosine 3'- [2-Cyanoethyl N,N-diisopropylamidite; 2'-Fluoro-N2-dimethylformamidine-5'-O-DMT-2'-deoxyguanosine-3'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite;N4-benzoyl-5'-O-DMT-2'-fluoro-deoxycytidine-3'-cyanoethoxyphosphoramidite;2'-Fluoro-deoxyuridine phosphoramidite.
  • the complementary strands were mixed by combining equimolar solutions (sense and antisense) in 0.1 ⁇ PBS (phosphate-buffered saline, adamas life) to form siRNA.
  • the solution was placed in a 70°C thermomixer, heated to 90°C, kept at 90°C for 5 minutes, and slowly cooled to room temperature.
  • the siRNA was lyophilized and stored at -15 to -25°C.
  • the duplex concentration was determined by measuring the absorbance of the solution on a UV-Vis spectrometer in 0.1 ⁇ PBS. The absorbance of the solution at 260 nm was then multiplied by the conversion factor and the dilution factor to determine the duplex concentration. All conversion factors are 0.04 mg/(mL ⁇ cm) unless otherwise stated.
  • the duplex was tested for single concentration activity (1 nM) in the HEK293-LPA stable cell line.
  • the duplex was dissolved in DEPC water, the concentration was measured by Nanodrop 2000, and it was diluted to 266 ng/ ⁇ L, recorded as 20 ⁇ M, as the stock solution.
  • RNAiMAX LipofectamineTM RNAiMAX Transfection Reagent, Invitrogen
  • RNA concentration was adjusted, sample concentration was detected using a nanophotometer, and water was added to adjust all samples to the same concentration; all samples were reverse transcribed into cDNA using a SuperScript TM IV VILO TM premix (containing ezDNase TM enzyme) reverse transcription kit (Thermo Fisher); real-time fluorescence quantitative qPCR was performed on a LightCycler 480II (Roche), and LPA and GAPDH mRNA in cell samples were relatively quantified using a TaqMan TM Fast advanced premix qPCR kit (Thermo Fisher), and each sample was quantitatively analyzed three times. The results are shown in Table 2.
  • HEK293-LPA stable cell line was used, and the transfection conditions were the same as in Example 2, where the duplex concentration range was 0.005-10 nM, and the IC50 curve was drawn using GraphPad Prism software. The results are shown in Table 3.
  • the duplex was tested for single concentration activity (1 nM) in the RT4 cell line.
  • the duplex was dissolved in DEPC water, and the concentration was measured by Nanodrop 2000.
  • the duplex was diluted to 266 ng/ ⁇ L, recorded as 20 ⁇ M, as the stock solution.
  • RNAiMAX LipofectamineTM RNAiMAX Transfection Reagent, Invitrogen
  • RNA concentration was adjusted, sample concentration was detected using a nanophotometer, and water was added to adjust all samples to the same concentration; all samples were reverse transcribed into cDNA using a SuperScript TM IV VILO TM premix (containing ezDNase TM enzyme) reverse transcription kit (Thermo Fisher); real-time fluorescence quantitative qPCR was performed on a LightCycler 480II (Roche), and LPA and GAPDH mRNA in cell samples were relatively quantified using a TaqMan TM Fast advanced premix qPCR kit (Thermo Fisher), and each sample was quantitatively analyzed three times. The results are shown in Table 4.
  • RT4 cells and the same transfection conditions were used, with duplex concentrations ranging from 0.005-10 nM, 8 concentration points.
  • IC50 was determined using GraphPad Prism software. The results are shown in Table 5.
  • test sample was dissolved in DEPC water at a ratio of 50 ⁇ L DEPC H 2 O per OD, and the concentration was determined using Nanodrop 2000. Based on the measurement results, the compound was diluted to 266 ng/ ⁇ L, recorded as 20 ⁇ M, as the stock solution and stored at -20°C for dilution.
  • Transfection Disperse 5 ⁇ L of diluted test sample in 20 ⁇ L Opti-MEM and 0.3 ⁇ L RNAiMAX in 25 ⁇ L Opti-MEM. Incubate for 5 minutes, mix with the dispersion of the test sample and incubate for 10 minutes.
  • RNAiMAX transfection reagent was diluted in Opti-MEM;
  • Cell seeding 150 ⁇ L cell suspension/well, inoculated in a 96-well plate, cell number: 8*10 3 cells/well.
  • 3/7 3D reagent Prepare according to the instructions 3/7 3D reagent, balance the reagent to room temperature, mix well. Leave 100 ⁇ l of culture medium in the cell culture plate, and then add 100 ⁇ l to each well. 3/7 3D reagent. Use a microplate shaker to mix the contents of the wells at 500 rpm for 30 seconds, incubate at room temperature for 90 minutes, and then transfer to a white ELISA plate. Measure the luminescent signal of each sample in the well plate in an ELISA reader.
  • the cytotoxicity was determined based on the ratio of the Caspase 3/7 expression level of the test sample to that of the control group (NC group, 0.9% saline), namely the relative expression level of Caspase 3/7.

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Abstract

L'invention concerne un agent ARNi pour inhiber l'expression du gène d'apolipoprotéine (a) (LPA) dans une cellule. L'agent ARNi comprend un brin sens et un brin antisens qui forment une région double brin. Le brin antisens a une longueur ne dépassant pas 23 nucléotides et comprend n'importe quelle séquence nucléotidique choisie parmi SEQ ID NO : 265 à 528. L'invention concerne également une utilisation de l'agent ARNi dans le traitement de maladies cardiovasculaires et cérébrovasculaires, etc.
PCT/CN2024/089357 2023-04-24 2024-04-23 Agent arni pour inhiber l'expression de lpa et utilisation d'agent arni Pending WO2024222686A1 (fr)

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Publication number Priority date Publication date Assignee Title
CN108368506A (zh) * 2015-10-01 2018-08-03 箭头药业股份有限公司 用于抑制lpa的基因表达的组合物和方法
WO2022121959A1 (fr) * 2020-12-09 2022-06-16 纳肽得有限公司 Molécule d'arnsi et son application dans le traitement de maladies des artères coronaires
US20230078200A1 (en) * 2019-12-09 2023-03-16 Amgen Inc. RNAi CONSTRUCTS AND METHODS FOR INHIBITING LPA EXPRESSION
WO2023041079A1 (fr) * 2021-09-18 2023-03-23 上海金中锘美生物医药科技有限公司 Inhibiteur de lpa et son utilisation
WO2024035898A2 (fr) * 2022-08-11 2024-02-15 Sirius Therapeutics, Inc. Molécules d'acide polynucléique pour inhiber l'expression de lp(a), compositions pharmaceutiques et leurs utilisations

Patent Citations (5)

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Publication number Priority date Publication date Assignee Title
CN108368506A (zh) * 2015-10-01 2018-08-03 箭头药业股份有限公司 用于抑制lpa的基因表达的组合物和方法
US20230078200A1 (en) * 2019-12-09 2023-03-16 Amgen Inc. RNAi CONSTRUCTS AND METHODS FOR INHIBITING LPA EXPRESSION
WO2022121959A1 (fr) * 2020-12-09 2022-06-16 纳肽得有限公司 Molécule d'arnsi et son application dans le traitement de maladies des artères coronaires
WO2023041079A1 (fr) * 2021-09-18 2023-03-23 上海金中锘美生物医药科技有限公司 Inhibiteur de lpa et son utilisation
WO2024035898A2 (fr) * 2022-08-11 2024-02-15 Sirius Therapeutics, Inc. Molécules d'acide polynucléique pour inhiber l'expression de lp(a), compositions pharmaceutiques et leurs utilisations

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O'DONOGHUE, M.L. ET AL.: "Small Interfering RNA to Reduce Lipoprotein(a) in Cardiovascular Disease", THE NEW ENGLAND JOURNAL OF MEDICINE, vol. 387, no. 20, 6 November 2022 (2022-11-06), XP093171350, DOI: 10.1056/NEJMoa2211023 *

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