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WO2024245410A1 - 一种靶向LPA的dsRNA及其应用 - Google Patents

一种靶向LPA的dsRNA及其应用 Download PDF

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Publication number
WO2024245410A1
WO2024245410A1 PCT/CN2024/096731 CN2024096731W WO2024245410A1 WO 2024245410 A1 WO2024245410 A1 WO 2024245410A1 CN 2024096731 W CN2024096731 W CN 2024096731W WO 2024245410 A1 WO2024245410 A1 WO 2024245410A1
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Prior art keywords
nucleotide
dsrna
seq
antisense strand
nucleotide sequence
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French (fr)
Inventor
李云飞
张瑱
林晓燕
王艳辉
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Tuojie Biotech Shanghai Co Ltd
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Tuojie Biotech Shanghai Co Ltd
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Priority to CN202480028437.8A priority Critical patent/CN121079106A/zh
Priority to AU2024280186A priority patent/AU2024280186A1/en
Publication of WO2024245410A1 publication Critical patent/WO2024245410A1/zh
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
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • 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 belongs to the field of biomedicine, and specifically relates to a dsRNA targeting LPA, and its application and preparation method.
  • Lp(a) Lipoprotein (a) [Lp(a)], first discovered by Norwegian geneticist Berg in 1963, was identified as a unique lipoprotein (Berg K. A new serum type system in man-the Lp system. Acta Pathol Microbiol Scand 1963; 59: 369–82.).
  • Lp(a) consists of two parts: lipid and protein.
  • the lipid part is mainly LDL-like particles located in the core; the protein part is located in the periphery and is composed of apolipoprotein (a) [apo(a)] and apoB100 connected by disulfide bonds.
  • Apo(a) is mainly expressed in the liver, and its expression is limited to humans and non-human primates.
  • Kringle three internal disulfide bond-stabilized three-ring structure domains
  • KIV-2 CNV leads to the size polymorphism of the encoded apo(a), whose expression is inversely proportional to the number of KIV-2 domains present.
  • the Lp(a) content in plasma will increase significantly.
  • Lp(a) may cause adverse atherosclerotic cardiovascular disease (ASCVD) through the following two mechanisms: on the one hand, since apo(a) has been shown to inhibit fibrinolysis in vitro, it may promote thrombosis at plaque rupture or turbulence at vascular stenosis, leading to vascular obstruction or thrombosis; on the other hand, LDL-like particles can promote intimal cholesterol deposition, inflammation or oxidized phospholipids, leading to atherosclerotic stenosis or aortic valve stenosis (Albert Youngwoo Jang, Seung Hwan Han, Il Suk Sohn, et al. Lipoprotein(a) and Cardiovascular Diseases[J]. Circulation Journal, 2020, 84:867–874). However, even at very high levels of Lp(a), the cholesterol content is lower than the traditional LDL threshold, so the pathogenicity of this part of LDL-like particles may be relatively low.
  • ASCVD adverse atherosclerotic cardiovascular disease
  • Lp(a) abnormality As >30mg/dl. Based on this standard, about 30% of patients with previous cardiovascular events in China have abnormal Lp(a). In 2019, the National Lipid Association of the United States recommended that Lp(a) ⁇ 50mg/dl be considered elevated. According to this standard, 20% of the global population has elevated Lp(a) levels. Although elevated Lp(a) levels are common, there is a lack of targeted therapeutic drugs. So far, no drugs that target the reduction of Lp(a) have been approved for clinical use. The Lp(a) protein has a similar structure to many lipoproteins and is difficult to be a direct target for small and large molecule drugs.
  • siRNA targeting the apo(a) gene is designed to weaken its expression, thereby reducing the level of Lp(a) in serum and reducing adverse cardiovascular events.
  • the present disclosure provides a double-stranded ribonucleic acid, which comprises a sense strand and an antisense strand; the sense strand and the antisense strand form a double-stranded region (preferably the sense strand and the antisense strand are reverse complementary); the double-stranded ribonucleic acid targets the LPA gene or its expression product.
  • the dsRNA comprises at least one modified nucleotide in the sense strand and/or the antisense strand.
  • dsRNA double-stranded ribonucleic acid
  • the naked nucleotide sequence of the positive strand comprises at least 17 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides;
  • the naked nucleotide sequence of the antisense strand comprises at least 19 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2 by no more than 3 nucleotides; wherein, from the 5' end to the 3' end,
  • nucleotides at positions 7, 8 and 9 of the sense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are 2'-methoxy-modified nucleotides;
  • nucleotides at positions 2 and 14 of the antisense strand are 2'-fluoro-modified nucleotides
  • nucleotides at positions 4, 6, 10, 12, 16 and 18 are independently 2'-methoxy or 2'-fluoro-modified nucleotides
  • nucleotides at the remaining positions are 2'-methoxy-modified nucleotides
  • the number of 2'-fluoro-modified nucleotides in the antisense strand is 2-7 (eg, 2, 3, 4, 5, 6, 7).
  • the naked nucleotide sequence of the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO: 1
  • the naked nucleotide sequence of the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO: 2; wherein, in the direction from the 5' end to the 3' end,
  • nucleotides at positions 7, 8 and 9 of the sense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are 2'-methoxy-modified nucleotides;
  • the nucleotides at positions 2 and 14 of the antisense strand are 2'-fluoro-modified nucleotides, the nucleotides at positions 4, 6, 10, 12, 16 and 18 are independently 2'-methoxy or 2'-fluoro-modified nucleotides, and the nucleosides at the remaining positions are
  • the acid is a 2'-methoxy modified nucleotide
  • the number of 2'-fluoro-modified nucleotides in the antisense strand is 2-7 (eg, 2, 3, 4, 5, 6, 7).
  • the sense strand and antisense strand are identical to each other.
  • the positive strand contains 5'-GCUCCUUAUUGUUAUACGA-3',
  • the antisense strand contains 5’-UCGUAUAACAAUAAGGAGCUG-3’.
  • the sense strand and antisense strand are identical to each other.
  • the naked nucleotide sequence of the positive strand is 5’-GCUCCUUAUUGUUAUACGA-3’,
  • the naked nucleotide sequence of the antisense strand is 5′-UCGUAUAACAAUAAGGAGCUG-3′;
  • Naked sequence refers to a nucleotide sequence that has not been modified.
  • the nucleotide at position 7 at the 5' end of the antisense strand of the dsRNA is a modified nucleotide, wherein the modified nucleotide is a 2'-methoxy-modified nucleotide.
  • the nucleotide at position 7 at the 5' end of the antisense strand of the dsRNA is a modified nucleotide, wherein the modified nucleotide comprises a chemical modification represented by formula (I), (I-1), (I-2) or a pharmaceutically acceptable salt thereof:
  • B is the same as the base of the antisense strand when the 7th nucleotide at its 5' end is not modified; in some specific embodiments, B is adenine.
  • the chemical modification represented by formula (I'), (I'-1), (I'-2) is selected from:
  • M is O or S;
  • B is the same as the base of the antisense strand when the 7th nucleotide at its 5' end is not modified; in some specific embodiments, B is adenine.
  • M is S. In some specific embodiments, M is O.
  • the first nucleotide at the 5' end of the antisense strand of the dsRNA is a modified nucleotide, wherein the modified nucleotide is a 2'-methoxy-modified nucleotide.
  • the first nucleotide at the 5' end of the antisense strand of the dsRNA is a modified nucleotide, wherein the modified nucleotide is a chemically modified nucleotide represented by formula (IV):
  • R A1 and R A2 are each independently selected from hydrogen or deuterium
  • M 1 and M 2 are each independently selected from -SH or -OH;
  • B is selected from base, hydrogen, deuterium;
  • RA3 is selected from hydrogen, deuterium, hydroxyl, halogen, alkyl (e.g., C1, C2, C3, C4, C5, C6 alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (e.g., C1 alkoxy, C2 alkoxy, C3 alkoxy, C4 alkoxy, C5 alkoxy, C6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), and the hydroxyl, alkyl, alkoxy are each optionally substituted with one or more deuterium;
  • RA4 is selected from hydrogen, deuterium, alkyl (e.g., C1, C2, C3, C4, C5, C6 alkyl, including but not limited to methyl, ethyl, isopropyl), and the alkyl are each optionally substituted with one or more deuterium; provided that formula
  • RA3 is selected from hydrogen, deuterium.
  • RA3 is selected from halogen (eg, fluorine, chlorine, bromine).
  • RA3 is selected from alkyl (e.g., C1, C2, C3, C4, C5, C6 alkyl, including but not limited to methyl, ethyl, isopropyl), each of which is optionally substituted with one or more deuterium.
  • alkyl e.g., C1, C2, C3, C4, C5, C6 alkyl, including but not limited to methyl, ethyl, isopropyl
  • RA3 is selected from alkoxy (e.g., C1 alkoxy, C2 alkoxy, C3 alkoxy, C4 alkoxy, C5 alkoxy, C6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), each of which is optionally substituted with one or more deuterium.
  • alkoxy e.g., C1 alkoxy, C2 alkoxy, C3 alkoxy, C4 alkoxy, C5 alkoxy, C6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy
  • the 5' terminal chemical modification of formula (IV) is:
  • RA5 , RA6 , and RA7 are each independently selected from hydrogen or deuterium;
  • RA1 , RA2 , M1 , M2 , and B are as defined in formula (IV); provided that formula (IV-1) contains at least one deuterium.
  • RA5 , RA6 , and RA7 are all deuterated.
  • RA5 is deuterium and RA6 and RA7 are hydrogen.
  • RA5 , RA6 are deuterium and RA7 is hydrogen.
  • RA1 is hydrogen and RA2 is deuterium.
  • RA1 and RA2 are deuterium.
  • R A1 and R A2 are hydrogen.
  • the 5' terminal chemical modification represented by formula (IV) is selected from: B is selected from a base or hydrogen.
  • B is selected from a base; in some specific embodiments, B is selected from adenine, guanine, cytosine, uracil or thymine.
  • B is the base at the corresponding position of the modified nucleotide of the antisense strand.
  • B is selected from uracil.
  • the 5' terminal chemical modification represented by formula (IV) is selected from: and structures in which uracil is replaced by adenine, guanine, cytosine, or thymine.
  • the first nucleotide at the 5' end of the antisense strand of the dsRNA is the 5' terminal chemically modified nucleotide shown in formula (IV).
  • the 5' terminal chemically modified nucleotide shown in formula (IV) is The B is the base corresponding to the first nucleotide at the 5' end of the antisense strand.
  • the first nucleotide at the 5' end of the antisense strand of the dsRNA is a modified nucleotide, wherein the modified nucleotide is a chemically modified nucleotide represented by formula (II):
  • B represents the base at the position corresponding to the first nucleotide at the 5' end of the antisense strand.
  • B represents uracil.
  • the antisense strand is at least partially reverse complementary to the target sequence to mediate RNA interference. In some embodiments, there are no more than 5, no more than 4, no more than 3, no more than 2, no more than 1 mismatch between the antisense strand and the target sequence; in some embodiments, the antisense strand is completely reverse complementary to the target sequence.
  • the sense strand and the antisense strand are at least partially reverse complementary to form a double-stranded region; in some embodiments, there are no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 mismatch between the sense strand and the antisense strand; in some embodiments, the sense strand and the antisense strand are completely reverse complementary.
  • the sense strand and antisense strand each independently have 16 to 35, 16 to 34, 17 to 34, 17 to 33, 18 to 33, 18 to 32, 18 to 31, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, or 19 to 23 nucleotides.
  • the sense strand and the antisense strand are each independently 19, 20, 21, 22, or 23 nucleotides in length.
  • the sense strand and antisense strand are the same or different in length, the sense strand is 19-23 nucleotides long, and the antisense strand is 19-26 nucleotides long.
  • the length ratio of the sense strand to the antisense strand can be 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/20, 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21/24, 21/25, 21/26, 22/20, 22/21, 22/22, 22/23, 22/24, 22/25, 22/26, 23/20, 23/21, 23/22, 23/23, 23/24, 23/25 or 23/26.
  • the length ratio of the sense strand to the antisense strand is 19/21, 21/23, or 23/25. In some embodiments, the length ratio of the sense strand
  • the dsRNA comprises one or two blunt ends.
  • the dsRNA comprises an overhang of 1 to 4 unpaired nucleotides, eg, 1, 2, 3, 4.
  • the dsRNA comprises an overhang at the 3' end of the antisense strand.
  • the sense strand of the dsRNA contains or is a nucleotide sequence shown in the following formula: 5'-N a N a N a N a N a N b N b N a N a N a N a N a N a N a N a N a -3'; wherein, Na is a 2'-methoxy-modified nucleotide, and Nb is a 2'-fluoro-modified nucleotide.
  • the antisense strand of the dsRNA comprises or is a nucleotide sequence as shown in the following formula:
  • each X' is independently Na ' or Nb '; Na ' is a 2'-methoxy-modified nucleotide, and Nb ' is a 2'-fluoro-modified nucleotide.
  • the antisense strand of the dsRNA comprises or is a nucleotide sequence as shown in the following formula:
  • Na ' is a 2'-methoxy-modified nucleotide
  • Nb ' is a 2'-fluoro-modified nucleotide
  • the antisense strand of the dsRNA comprises or is a nucleotide sequence as shown in the following formula: 5'-N a 'N b 'N a 'X'N a 'X'W'N a 'N a 'X'N a 'X'N a 'N b 'N a 'X'N a 'X'N a 'N a 'N a '-3', wherein each X' is independently Na 'or N b '; Na 'is a 2'-methoxy-modified nucleotide, N b 'is a 2'-fluoro-modified nucleotide, and W'represents a chemically modified nucleotide as shown in formula (I), (I-1), or (I-2), or a pharmaceutically acceptable salt thereof. Nucleotide.
  • the antisense strand of the dsRNA comprises or is a nucleotide sequence as shown in the following formula:
  • Na ' is a 2'-methoxy modified nucleotide
  • Nb ' is a 2'-fluoro modified nucleotide
  • W' represents a nucleotide comprising the chemical modification shown in formula (I), (I-1), (I-2) or a pharmaceutically acceptable salt thereof.
  • the antisense strand of the dsRNA comprises or is a nucleotide sequence shown in the following formula: 5'-V'N b'N a'X'N a'X'N a'N a'N a'X'N a'X'N a'N b'N a'X'N a'X'N a'N a'N a' - 3 ', wherein each X' is independently Na ' or N b ' ; Na ' is a 2'-methoxy-modified nucleotide, and N b ' is a 2'-fluoro-modified nucleotide; V ' represents a chemically modified nucleotide shown in formula (II).
  • the antisense strand of the dsRNA comprises or is a nucleotide sequence as shown in the following formula:
  • Na ' is a 2'-methoxy-modified nucleotide
  • Nb ' is a 2'-fluoro-modified nucleotide
  • V' represents a chemically modified nucleotide represented by formula (II).
  • the chemical modification represented by formula (I), (I-1), (I-2) or a pharmaceutically acceptable salt thereof is selected from:
  • B represents the 7th base at the 5' end of the antisense strand, and in some specific embodiments, B is adenine.
  • the chemical modification represented by formula (I'), (I'-1), (I'-2) or a pharmaceutically acceptable salt thereof is selected from:
  • M is O or S;
  • B represents the 7th base at the 5' end of the antisense strand, and in some specific embodiments, B is adenine.
  • M is S. In some specific embodiments, M is O.
  • the chemically modified nucleotide represented by formula (II) is selected from:
  • B represents the base at the position corresponding to the first nucleotide at the 5' end of the antisense strand.
  • B represents uracil.
  • the chemically modified nucleotide represented by formula (II) is selected from:
  • B represents the base at the position corresponding to the first nucleotide at the 5' end of the antisense strand.
  • B represents uracil.
  • At least one phosphodiester group in the sense strand and/or antisense strand of the dsRNA is a phosphodiester group having a modified group.
  • At least one phosphodiester group in the sense strand and/or the antisense strand of the dsRNA is a phosphorothioate diester group.
  • the phosphorothioate diester group is present in at least one of the following positions:
  • the sense strand and/or antisense strand comprises a plurality of phosphorothioate diester groups, wherein the phosphorothioate diester groups are present in:
  • the sense strand and/or antisense strand comprises a plurality of phosphorothioate diester groups, wherein the phosphorothioate diester groups are present in:
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:6, and the antisense strand comprises or is selected from the nucleotide sequence shown in any one of SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:22 to SEQ ID NO:33, and SEQ ID NO:35.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:6, and the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:16.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:6, and the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:20.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:6, and the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:22.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:6, and the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:23.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:6, and the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:24.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:6, and the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:25.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:6, and the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:26.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:6, and the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:27.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:6, and the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:28.
  • the sense strand of the dsRNA comprises or is selected from SEQ ID NO: 6.
  • the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO: 29.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:6, and the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:30.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:6, and the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:31.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:6, and the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:32.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:6, and the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:33.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:6, and the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:35.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:7
  • the antisense strand comprises or is selected from the nucleotide sequence shown in any one of SEQ ID NO:9 to SEQ ID NO:20, and SEQ ID NO:33 to SEQ ID NO:35.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:7
  • the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:9.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:7
  • the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:10.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:7
  • the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:11.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:7
  • the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:12.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:7
  • the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:13.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:7
  • the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:14.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:7
  • the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:15.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:7
  • the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:16.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:7
  • the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:17.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:7
  • the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:18.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:7
  • the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:19.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:7
  • the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:20.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:7
  • the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:33.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:7
  • the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:34.
  • the sense strand of the dsRNA comprises or is selected from the nucleotide sequence shown in SEQ ID NO:7
  • the antisense strand comprises or is selected from the nucleotide sequence shown in SEQ ID NO:35.
  • the dsRNA is:
  • the nucleotide sequence of the positive chain contains SEQ ID NO:6, and the nucleotide sequence of the antisense chain contains SEQ ID NO:16.
  • the dsRNA is: the nucleotide sequence of the sense strand is shown in SEQ ID NO: 6, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO: 16.
  • the present disclosure also provides a dsRNA conjugate, which comprises any one of the above dsRNAs and a targeting ligand linked to the dsRNA.
  • the dsRNA and the targeting ligand are covalently or non-covalently linked.
  • the targeting ligand targets the liver. In some embodiments, the targeting ligand binds to the asialoglycoprotein receptor (ASGPR). In some embodiments, the targeting ligand comprises a galactose cluster or a galactose derivative cluster, the galactose derivative being selected from N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine or N-isobutyrylgalactosamine.
  • ASGPR asialoglycoprotein receptor
  • the targeting ligand is linked to the 3' end of the sense strand of the dsRNA.
  • the targeting ligand is linked to the dsRNA terminus via a phosphodiester group, a thiophosphodiester group, or a phosphonic acid group. In some embodiments, the targeting ligand is linked to the dsRNA terminus via a phosphodiester group.
  • the targeting ligand is indirectly linked to the dsRNA terminus via a phosphodiester group, a thiophosphodiester group, or a phosphonic acid group. In some embodiments, the targeting ligand is indirectly linked to the dsRNA terminus via a phosphodiester group.
  • the targeting ligand is directly linked to the dsRNA end via a phosphodiester group, a thiophosphodiester group, or a phosphonic acid group. In some embodiments, the targeting ligand is directly linked to the dsRNA end via a phosphodiester group.
  • the targeting ligand is directly linked to the end of the dsRNA sense strand via a phosphodiester group or a thiophosphodiester group. In some embodiments, the targeting ligand is linked to the end of the dsRNA sense strand via a phosphodiester group.
  • the targeting ligand is directly linked to the 3' end of the dsRNA sense strand via a phosphodiester group or a thiophosphodiester group. In some embodiments, the targeting ligand is directly linked to the 3' end of the dsRNA sense strand via a phosphodiester group.
  • a lipophilic group such as cholesterol can be introduced at the end of the dsRNA sense strand, and the lipophilic group can be covalently bonded to the small interfering nucleic acid, such as Cholesterol, lipoprotein, vitamin E, etc. are introduced at the end to facilitate interaction with mRNA in the cell through the cell membrane composed of lipid bilayer.
  • dsRNA can also be modified by non-covalent bonds, such as binding phospholipid molecules, polypeptides, cationic polymers, etc. through hydrophobic bonds or ionic bonds to increase stability and biological activity.
  • the targeting portion of the targeting ligand is composed of one or more targeting groups or targeting moieties, and the targeting ligand assists in guiding the delivery of the therapeutic agent connected thereto to the desired target location.
  • the targeting moiety can bind to cells or cell receptors and initiate endocytosis to promote the entry of the therapeutic agent into the cell.
  • the targeting moiety can include compounds with affinity for cell receptors or cell surface molecules or antibodies.
  • Various targeting ligands containing targeting moieties can be connected to therapeutic agents and other compounds to target the agent to cells and specific cell receptors.
  • the types of targeting moieties include carbohydrates, cholesterol and cholesterol groups or steroids.
  • Targeting moieties that can bind to cell receptors include sugars such as galactose, galactose derivatives (such as N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine), mannose and mannose derivatives).
  • targeting moieties that bind to the asialoglycoprotein receptor can be particularly useful for directing delivery of oligomeric compounds to the liver.
  • the asialoglycoprotein receptor is expressed in large quantities on liver cells (hepatocytes).
  • Cell receptor targeting moieties that target the ASCPR include galactose and galactose derivatives. Specifically, clusters of galactose derivatives, including clusters consisting of 2, 3, 4 or more than 4 N-acetyl-galactosamines (GalNAc or NAG) can promote the uptake of certain compounds in hepatocytes.
  • the GalNAc clusters coupled to the oligomeric compound are used to direct the composition to the liver, where the N-acetyl-galactosamine sugar can bind to the asialoglycoprotein receptor on the surface of the liver cells.
  • the binding of the asialoglycoprotein receptor is believed to initiate receptor-mediated endocytosis, thereby promoting the entry of the compound into the cell interior.
  • a targeting ligand can include 2, 3, 4, or more than 4 targeting moieties.
  • each targeting moiety independently comprises a galactosamine derivative, which is N-acetyl-galactosamine.
  • Other sugars that can be used as targeting moieties and have affinity for asialoglycoprotein receptors can be selected from galactose, galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, N-n-butyryl-galactosamine and N-isobutyryl-galactosamine, etc.
  • the targeting ligands of the present disclosure include N-acetylgalactosamine as a targeting moiety.
  • the targeting ligand comprises three terminal galactosamines or galactosamine derivatives (such as N-acetyl-galactosamine), each of which has affinity for sialoglycoprotein receptors.
  • the targeting ligand comprises three terminal N-acetyl-galactosamines (GalNAc or NAG) as targeting moieties.
  • the targeting ligand comprises four terminal galactosamines or galactosamine derivatives (such as In some embodiments, the targeting ligand comprises four terminal N-acetyl-galactosamine (GalNAc or NAG) as the targeting moiety.
  • the targeting ligand provided by the present disclosure is a compound as shown in formula (III-1) or a pharmaceutically acceptable salt thereof,
  • the targeting ligand provided by the present disclosure is a compound as shown in formula (III-2) or a pharmaceutically acceptable salt thereof,
  • the N-acetyl-galactosamine moiety in the above targeting ligands can be replaced with N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, or N-isobutyrylgalactosamine.
  • the nucleotide sequence of the sense chain of the dsRNA conjugate comprises or is selected from SEQ ID NO:3
  • the nucleotide sequence of the antisense chain comprises or is selected from any one of SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:22 to SEQ ID NO:33, and SEQ ID NO:35.
  • the nucleotide sequence of the sense chain of the dsRNA conjugate comprises or is selected from SEQ ID NO:4, and the nucleotide sequence of the antisense chain comprises or is selected from any one of SEQ ID NO:9 to SEQ ID NO:20, and SEQ ID NO:33 to SEQ ID NO:35.
  • the nucleotide sequence of the sense chain of the dsRNA conjugate comprises or is selected from SEQ ID NO:5
  • the nucleotide sequence of the antisense chain comprises or is selected from any one of SEQ ID NO:16, SEQ ID NO:20, and SEQ ID NO:33.
  • the dsRNA conjugate is:
  • the nucleotide sequence of the sense strand comprises SEQ ID NO: 3
  • the nucleotide sequence of the antisense strand comprises SEQ ID NO:16.
  • the dsRNA conjugate is:
  • the nucleotide sequence of the positive chain is shown in SEQ ID NO:3, and the nucleotide sequence of the antisense chain is shown in SEQ ID NO:16.
  • the dsRNA conjugate is the following structure or a pharmaceutically acceptable salt thereof:
  • Af adenine 2'-F ribonucleoside
  • Cf cytosine 2'-F ribonucleoside
  • Uf uracil 2'-F ribonucleoside
  • Gf guanine 2'-F ribonucleoside
  • Am adenine 2'-OMe ribonucleoside
  • Cm cytosine 2'-OMe ribonucleoside
  • Gm guanine 2'-OMe ribonucleoside
  • Um uracil 2'-OMe ribonucleoside.
  • the dsRNA conjugate is the following structure or a pharmaceutically acceptable salt thereof:
  • Af adenine 2'-F ribonucleoside
  • Cf cytosine 2'-F ribonucleoside
  • Uf uracil 2'-F ribonucleoside
  • Gf guanine 2'-F ribonucleoside
  • Am adenine 2'-OMe ribonucleoside
  • Cm cytosine 2'-OMe ribonucleoside
  • Gm guanine 2'-OMe ribonucleoside
  • Um uracil 2'-OMe ribonucleoside.
  • the pharmaceutically acceptable salt may be a conventional salt in the art, including but not limited to sodium salt, potassium salt, ammonium salt, amine salt, and the like.
  • the dsRNA conjugate is selected from TJR100422, TJR100423, TJR100424, TJR100425, TJR100426, TJR100427, TJR100428, TJR100429, TJR100430, TJR100431, TJR100432, TJR100800, TJR100801, TJR100802, TJR100803, TJR100804, TJR100805, TJR100806, TJR100807, TJR100808 05.
  • the dsRNA conjugate is TJR101079, which has the structure:
  • Af adenine 2'-F ribonucleoside
  • Cf cytosine 2'-F ribonucleoside
  • Uf uracil 2'-F ribonucleoside
  • Gf guanine 2'-F ribonucleoside
  • Am adenine 2'-OMe ribonucleoside
  • Cm cytosine 2'-OMe ribonucleoside
  • Gm guanine 2'-OMe ribonucleoside
  • Um uracil 2'-OMe ribonucleoside.
  • compositions comprising the dsRNA and/or dsRNA conjugate as claimed in claim 1, and one or more pharmaceutically acceptable excipients, such as carriers, vehicles, diluents, and/or delivery polymers.
  • Conjugates for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, constructing the nucleic acid as part of a retroviral or other vector.
  • Another aspect of the present disclosure provides a use of the above-mentioned dsRNA and/or dsRNA conjugate or a composition thereof in the preparation of a medicament for treating a disease in a subject, which in some embodiments is selected from a liver-related disease.
  • Another aspect of the present disclosure provides a method for treating a disease in a subject, comprising administering the above-mentioned dsRNA and/or dsRNA conjugate, or composition to the subject.
  • Another aspect of the present disclosure provides a method for inhibiting mRNA expression of LPA in a subject, the method comprising administering the above-mentioned dsRNA and/or dsRNA conjugate, or composition to the subject.
  • Another aspect of the present disclosure provides a method for delivering an expression inhibitory oligomeric compound to the liver in vivo, by administering the above-mentioned conjugate and/or composition to a subject.
  • the dsRNA, dsRNA conjugates, compositions and methods disclosed herein can reduce the level of a target mRNA in a cell, a cell population, a tissue or a subject, comprising: administering to a subject a therapeutically effective amount of a dsRNA described herein, wherein the dsRNA is linked to a targeting ligand, thereby inhibiting the expression of the target mRNA in the subject.
  • the subject has been previously identified as having aberrant expression of the target gene in the targeted cells or tissues.
  • the subject described in the present disclosure refers to a subject suffering from a disease or disorder that would benefit from reduction or inhibition of target mRNA expression.
  • Delivery can be by local administration (e.g., direct injection, implantation, or topical administration), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration.
  • local administration e.g., direct injection, implantation, or topical administration
  • systemic administration e.g., systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration.
  • intracranial e.g., intraventricular, intraparenchymal and intrathecal
  • intramuscular e.g., intramus
  • compositions provided by the present disclosure can be administered by injection, for example, intravenous, intramuscular, intradermal, subcutaneous, intraduodenal or intraperitoneal injection.
  • the conjugate can be packaged in a kit.
  • the present disclosure provides a pharmaceutical composition comprising the dsRNA and/or dsRNA conjugate described in the present disclosure.
  • the pharmaceutical composition may further include a pharmaceutically acceptable excipient and/or adjuvant, which may be one or more of various preparations or compounds conventionally used in the art.
  • a pharmaceutically acceptable excipient may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.
  • the above-mentioned dsRNA, dsRNA conjugate or pharmaceutical composition thereof described in the present disclosure when contacted with cells expressing the target gene, the above-mentioned dsRNA, dsRNA conjugate or pharmaceutical composition inhibits the expression of the target gene by at least 5%, at least 10%, at least 15%, at least 20%, or more, as determined by, for example, psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR or branched DNA (bDNA) based methods, or protein-based methods such as immunofluorescence analysis, such as Western Blot or flow cytometry.
  • psiCHECK activity screening and luciferase reporter gene assay other methods such as PCR or branched DNA (bDNA) based methods, or protein-based methods such as immunofluorescence analysis, such as Western Blot or flow cytometry.
  • the residual expression percentage of the target gene mRNA caused by the above-mentioned dsRNA, dsRNA conjugate or pharmaceutical composition is not higher than 99%, not higher than 95%, not higher than 90%, not higher than 85%, not higher than 80%, not higher than 75%, not higher than 70%, not higher than 65%, not higher than 60%, not higher than 55%, not higher than 50%, not higher than 45%, not higher than 40%, not higher than 35%, not higher than 30%, not higher than 25%, not higher than 20%, not higher than 15%, or not higher than 10%, as determined by, for example, psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, such as Western Blot or flow cytometry.
  • bDNA branched DNA
  • the dsRNA, dsRNA conjugate or pharmaceutical composition thereof described in the present disclosure when the dsRNA, dsRNA conjugate or pharmaceutical composition thereof described in the present disclosure is contacted with cells expressing the target gene, the dsRNA, dsRNA conjugate or pharmaceutical composition thereof reduces the off-target activity by at least 20%, at least 25%, 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% or at least 75% while maintaining the target activity, as determined by, for example, psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, such as Western Blot, or flow cytometry.
  • bDNA branched DNA
  • immunofluorescence analysis such as Western Blot, or flow cytometry.
  • the dsRNA, dsRNA conjugate or pharmaceutical composition thereof described in the present disclosure when the dsRNA, dsRNA conjugate or pharmaceutical composition thereof described in the present disclosure is contacted with cells expressing the target gene, the dsRNA, dsRNA conjugate or pharmaceutical composition thereof reduces the target activity by at least 20%, at least 25%, 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% or at least 75% while reducing the off-target activity by at least 20%, at most 19%, at most 15%, at most 10%, at most 5% or more than 1%, as determined by, for example, psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, such as Western Blot, or flow cytometry.
  • bDNA branched DNA
  • immunofluorescence analysis such as Western
  • the target gene when the dsRNA and/or dsRNA conjugates or pharmaceutical compositions thereof described in the present disclosure are contacted with cells expressing the target gene, the target gene can be detected by, for example, psiCHECK activity screening and luciferase reporter gene detection, other methods such as PCR or branched DNA (bDNA) based methods, or protein-based methods such as immunofluorescence analysis, for example Western Blot.
  • psiCHECK activity screening and luciferase reporter gene detection other methods such as PCR or branched DNA (bDNA) based methods, or protein-based methods such as immunofluorescence analysis, for example Western Blot.
  • the dsRNA, dsRNA conjugate, or pharmaceutical composition thereof increases the target activity by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, 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%, or at least 80%, while reducing the off-target activity by at least 20%, at least 25%, 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%, or at least 75%, as determined by blot, or flow cytometry.
  • the IC 50 is no higher than 1 nM, no higher than 0.9 nM, no higher than 0.8 nM, no higher than 0.7 nM, no higher than 0.6 nM, no higher than 0.5 nM, no higher than 0.4 nM, no higher than 0.3 nM, no higher than 0.2 nM, no higher than 0.19 nM, no higher than 0.18 nM, no higher than 0.17 nM, no higher than 0.16 nM, no higher than 0.15 nM, no higher than 0.14 nM, no higher than 0.16 nM, no higher than 0.17 nM, no higher than 0.18 nM, no higher than 0.19 nM, no higher than 0.20 nM, no higher than 0.21 nM, no higher than 0.22 nM, no higher than 0.23 nM, no higher than 0.24
  • the present disclosure also provides a cell comprising the dsRNA and/or dsRNA conjugate of the present disclosure.
  • the present disclosure also provides a pharmaceutical kit or a test kit, which comprises one or more containers, wherein the containers independently contain the dsRNA and/or dsRNA conjugate of the present disclosure, or a pharmaceutical composition thereof.
  • the present disclosure also provides a method for silencing a target gene or mRNA of a target gene in a cell, the method comprising the step of introducing a dsRNA and/or a dsRNA conjugate and/or a pharmaceutical composition according to the present disclosure into the cell.
  • the present disclosure also provides a method for silencing a target gene or mRNA of a target gene in a cell in vivo or in vitro, the method comprising the step of introducing a dsRNA and/or a dsRNA conjugate and/or a pharmaceutical composition according to the present disclosure into the cell.
  • the present disclosure also provides a method for inhibiting expression of a target gene or mRNA of a target gene, the method comprising administering an effective amount or effective dose of the dsRNA and/or dsRNA conjugate and/or pharmaceutical composition according to the present disclosure to a subject in need thereof.
  • administration is by a route of administration that includes intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, intralymphatic, intravenous, subcutaneous, cerebrospinal, or a combination thereof.
  • the effective amount or effective dose of the dsRNA and/or dsRNA conjugate and/or pharmaceutical composition is about 0.001 mg/kg body weight to about 200 mg/kg body weight, about 0.01 mg/kg body weight to about 100 mg/kg body weight, or about 0.5 mg/kg body weight to about 50 mg/kg body weight.
  • the target gene is LPA.
  • the present disclosure provides a use of the dsRNA and/or dsRNA conjugate or a pharmaceutical composition containing the dsRNA and/or dsRNA conjugate in preparing a drug.
  • the present disclosure provides the aforementioned dsRNA and/or pharmaceutical composition and/or dsRNA conjugate for treating and/or preventing a subject from a disease associated with elevated levels of lipoprotein (a) and/or apolipoprotein (a).
  • the disease associated with elevated levels of lipoprotein (a) and/or apolipoprotein (a) is selected from cardiovascular disease.
  • the cardiovascular disease is selected from ischemic stroke, atherosclerosis, thrombosis, coronary heart disease, lower extremity arterial disease or aortic valve stenosis, myocardial infarction, coronary artery stenosis, carotid artery stenosis, femoral artery stenosis, heart failure.
  • the present disclosure provides the aforementioned dsRNA and/or pharmaceutical composition and/or dsRNA conjugate for use in treating and/or preventing a disease, wherein the disease is selected from cardiovascular disease.
  • the vascular disease is selected from ischemic stroke, atherosclerosis, thrombosis, coronary heart disease, lower extremity arterial disease or aortic valve stenosis, myocardial infarction, coronary artery stenosis, carotid artery stenosis, femoral artery stenosis, heart failure.
  • the present disclosure provides the aforementioned dsRNA and/or pharmaceutical composition and/or dsRNA conjugate for use in reducing lipoprotein(a) and/or apolipoprotein(a) levels.
  • the present disclosure provides use of the aforementioned dsRNA and/or pharmaceutical composition and/or dsRNA conjugate in the preparation of a medicament for inhibiting the expression of LPA.
  • the present disclosure provides the use of the aforementioned dsRNA and/or pharmaceutical composition and/or dsRNA conjugate in the preparation of a medicament for treating and/or preventing a subject from a disease associated with elevated levels of lipoprotein (a) and/or apolipoprotein (a).
  • the disease associated with elevated levels of lipoprotein (a) and/or apolipoprotein (a) is selected from cardiovascular disease.
  • the cardiovascular disease is selected from ischemic stroke, atherosclerosis, thrombosis, coronary heart disease, lower extremity artery disease or aortic valve stenosis, myocardial infarction, coronary artery stenosis, carotid artery stenosis, femoral artery stenosis, heart failure.
  • the present disclosure provides the use of the aforementioned dsRNA and/or pharmaceutical composition and/or dsRNA conjugate in the preparation of a medicament for treating and/or preventing a disease, wherein the disease is selected from cardiovascular disease.
  • the cardiovascular disease is selected from ischemic stroke, atherosclerosis, thrombosis, coronary heart disease, lower extremity arterial disease or aortic valve stenosis, myocardial infarction, coronary artery stenosis, carotid artery stenosis, femoral artery stenosis, heart failure.
  • the present disclosure provides use of the aforementioned dsRNA and/or pharmaceutical composition and/or dsRNA conjugate in the preparation of a medicament for reducing lipoprotein(a) and/or apolipoprotein(a) levels.
  • the present disclosure provides a method for inhibiting LPA expression, comprising administering an effective amount or an effective dose of the aforementioned dsRNA and/or pharmaceutical composition and/or dsRNA conjugate to a subject.
  • the present disclosure provides a method for treating and/or preventing a subject from a disease associated with elevated levels of lipoprotein (a) and/or apolipoprotein (a), comprising administering to the subject an effective amount or effective dose of the aforementioned dsRNA and/or pharmaceutical composition and/or dsRNA conjugate.
  • the disease associated with elevated levels of lipoprotein (a) and/or apolipoprotein (a) is selected from cardiovascular disease; in some embodiments, the cardiovascular disease is selected from ischemic stroke, atherosclerosis, thrombosis, coronary heart disease, lower extremity arterial disease or aortic valve stenosis, myocardial infarction, coronary artery stenosis, carotid artery stenosis, femoral artery stenosis, heart failure.
  • the present disclosure provides a method for treating and/or preventing a disease, comprising administering to a subject an effective amount or effective dose of the aforementioned dsRNA and/or pharmaceutical composition and/or dsRNA conjugate, wherein the disease is selected from cardiovascular disease.
  • the cardiovascular disease is selected from ischemic stroke, atherosclerosis, thrombosis, coronary heart disease, lower extremity arterial disease or aortic valve stenosis, myocardial infarction, coronary artery stenosis, carotid artery stenosis, femoral artery stenosis, heart failure.
  • the present disclosure provides a method for reducing lipoprotein (a) and/or apolipoprotein (a) levels, comprising administering an effective amount or an effective dose of the aforementioned dsRNA and/or pharmaceutical composition and/or dsRNA conjugate to a subject.
  • the present disclosure provides a method for delivering dsRNA that inhibits LPA expression and/or replication to the liver in vivo, wherein the dsRNA and/or pharmaceutical composition and/or dsRNA conjugate is administered to a subject.
  • the present disclosure also provides a method for preparing the dsRNA and/or dsRNA conjugate or pharmaceutical composition of the present disclosure, which comprises: synthesizing the dsRNA and/or dsRNA conjugate or pharmaceutical composition of the present disclosure.
  • the present disclosure also provides a dsRNA or dsRNA conjugate, characterized in that one or more bases U of any dsRNA or dsRNA conjugate of the present disclosure are replaced with base T, for example, 1, 2, 3, 3, 5, 6, 7, 8, 9, 10. In some embodiments, all bases U in the present disclosure can be replaced by base T.
  • the pharmaceutically acceptable salts of the compounds described in the present disclosure are selected from inorganic salts or organic salts.
  • the compounds described in the present disclosure can react with acidic or basic substances to form corresponding salts.
  • the compounds of the present disclosure may exist in specific geometric or stereoisomeric forms.
  • the present disclosure contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as mixtures enriched in enantiomers or diastereomers, all of which are within the scope of the present disclosure.
  • Additional asymmetric carbon atoms may be present in substituents such as alkyl. All of these isomers and their mixtures are included within the scope of the present disclosure.
  • the compounds of the present disclosure containing asymmetric carbon atoms may be isolated in optically pure form or in racemic form. Optically pure forms may be resolved from racemic mixtures or synthesized by using chiral raw materials or chiral reagents.
  • Optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer.
  • a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are separated by conventional methods known in the art, and then the pure enantiomer is recovered.
  • the separation of enantiomers and diastereomers is usually accomplished by using chromatography, which uses a chiral stationary phase and is optionally combined with a chemical derivatization method (e.g., carbamates are generated from amines).
  • the bond Indicates that the configuration is not specified, that is, if there are chiral isomers in the chemical structure, the bond Can be or or include both and
  • the bond No configuration is specified, i.e., the bond The configuration can be E-type or Z-type, or include both E and Z configurations.
  • the present disclosure also includes those which are the same as those described herein, but one or more atoms are replaced by the atomic mass
  • the isotopically labeled compounds of the present invention are those in which the atoms are replaced with atoms having an atomic weight or mass number different from the atomic weight or mass number usually found in nature.
  • isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I, and 36 Cl, etc., respectively.
  • deuterated drugs Compared with non-deuterated drugs, deuterated drugs have the advantages of reducing toxic side effects, increasing drug stability, enhancing therapeutic effects, and extending drug biological half-life. All isotopic composition changes of the compounds disclosed herein, whether radioactive or not, are included in the scope of the present disclosure.
  • Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom, wherein the replacement of deuterium can be partial or complete, and partial deuterium replacement means that at least one hydrogen is replaced by at least one deuterium.
  • compounds of the present disclosure when a position is specifically designated as “deuterium” or “D", the position is understood to have an abundance of deuterium that is at least 1000 times greater than the natural abundance of deuterium (which is 0.015%) (i.e., at least 15% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 1000 times greater than the natural abundance of deuterium (i.e., at least 15% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 2000 times greater than the natural abundance of deuterium (i.e., at least 30% deuterium incorporation).
  • the abundance of deuterium for each designated deuterium atom is at least 3000 times greater than the natural abundance of deuterium (i.e., at least 45% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 3340 times greater than the natural abundance of deuterium (i.e., at least 50.1% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 3500 times greater than the natural abundance of deuterium (i.e., at least 52.5% deuterium incorporation).
  • the abundance of deuterium for each designated deuterium atom is at least 4000 times greater than the natural abundance of deuterium (i.e., at least 60% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 4500 times greater than the natural abundance of deuterium (i.e., at least 67.5% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 5000 times greater than the natural abundance of deuterium (i.e., at least 75% deuterium incorporation).
  • the abundance of deuterium for each designated deuterium atom is at least 5500 times greater than the natural abundance of deuterium (i.e., at least 82.5% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 6000 times greater than the natural abundance of deuterium (i.e., at least 90% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 6333.3 times greater than the natural abundance of deuterium (i.e., at least 95% deuterium incorporation).
  • the abundance of deuterium for each designated deuterium atom is at least 6466.7 times greater than the natural abundance of deuterium (i.e., at least 97% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 6600 times greater than the natural abundance of deuterium (i.e., at least 99% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 6633.3 times greater than the natural abundance of deuterium (i.e., at least 99.5% deuterium incorporation). Those skilled in the art can synthesize deuterated compounds with reference to the relevant literature.
  • deuterated starting materials can be used when preparing deuterated compounds, or they can be synthesized using conventional techniques using deuterated reagents, including but not limited to deuterated borane, trideuterated borane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated iodoethane and deuterated iodomethane, etc.
  • the present disclosure introduces the full text of WO2022028462A, WO2023274395A, WO2023208023A and WO2023109940A.
  • apolipoprotein (a) gene includes but is not limited to human LPA, cynomolgus monkey LPA, mouse LPA, rat LPA, and its amino acid and complete coding sequence, mRNA sequence can be easily obtained using the public databases, for example, GenBank, UniProt, OMIM and the Macaca genome project website.
  • target sequence refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of LPA, including mRNA that is an RNA processing product of the primary transcription product.
  • the targeted portion of the target sequence should be long enough to serve as a substrate for iRNA-directed cleavage.
  • the target sequence is within the protein coding region of LPA.
  • the sense strand of a dsRNA refers to a strand comprising a sequence that is identical or substantially identical to a target mRNA sequence
  • the antisense strand of a dsRNA also referred to as AS or AS strand
  • the sense strand of a dsRNA refers to a strand having a sequence that is at least partially complementary to a target mRNA sequence.
  • the term "at least 17 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 1 that differs by no more than 3 nucleotides” is intended to mean that the sense strand of the dsRNA described herein comprises at least 17 consecutive nucleotides as shown in SEQ ID NO: 1, or a sequence that differs by no more than 3 nucleotides from at least 17 consecutive nucleotides of the sense strand shown in SEQ ID NO: 1, optionally, differs by no more than 2 nucleotides, optionally, differs by 1 nucleotide.
  • the sense strand of the dsRNA described herein comprises at least 18 consecutive nucleotides of the sense strand shown in SEQ ID NO: 1, or a sequence that differs by no more than 3 nucleotides from at least 18 consecutive nucleotides of the sense strand shown in SEQ ID NO: 1, optionally, differs by no more than 2 nucleotides, optionally, differs by 1 nucleotide.
  • the term "at least 19 consecutive nucleotides that differ by no more than 3 nucleotides from the antisense strand set forth in SEQ ID NO: 2" is intended to mean at least 19 consecutive nucleotides of the antisense strand set forth in SEQ ID NO: 2 described herein, or a sequence that differs by no more than 3 nucleotides from at least 19 consecutive nucleotides of the antisense strand set forth in SEQ ID NO: 2, optionally differs by no more than 2 nucleotides, optionally differs by 1 nucleotide sequence.
  • the "5' region" of the sense strand or antisense strand i.e., the "5' end” or “5' terminal end”
  • the nucleotides from positions 2 to 8 in the 5' region of the antisense strand can also be replaced by the nucleotides from positions 2 to 8 in the 5' terminal end of the antisense strand.
  • the "3' region", "3' terminal end” and “3' terminal” of the sense strand or antisense strand can also be used interchangeably.
  • G represents nucleotides, including guanine, cytosine, adenine, thymidine and uracil bases, respectively.
  • the lowercase letter m indicates that the nucleotide adjacent to the upstream of the letter m is a methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the upstream of the letter f is a fluorine-modified nucleotide; the lowercase letter s indicates that the two nucleotides adjacent to the letter s on both sides are connected by phosphorothioate diester groups.
  • the term "2'-fluoro (2'-F) modified nucleotide” refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine
  • non-fluorinated modified nucleotide refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorine group.
  • 2'-methoxy (2'-OMe) modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.
  • nucleotide difference between a nucleotide sequence and another nucleotide sequence means that the base type of the nucleotide at the same position in the former is changed compared with the latter. For example, when a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G or T, it is considered that there is a nucleotide difference at that position between the two nucleotide sequences. In some embodiments, when a nucleotide at the original position is replaced by an abasic nucleotide or its equivalent, it can also be considered that a nucleotide difference occurs at that position.
  • the terms "complementary" or “reverse complement” are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one chain are paired with the bases on the other chain in a complementary manner.
  • the purine base adenine is always paired with the pyrimidine base thymine (or uracil in RNA); the purine base guanine is always paired with the pyrimidine base cytosine.
  • Each base pair includes a purine and a pyrimidine.
  • mismatch means in the art that the bases at corresponding positions in a double-stranded nucleic acid are not paired in a complementary form.
  • dsRNA refers to a double-stranded RNA molecule capable of RNA interference, comprising a sense strand and an antisense strand.
  • chemical modification or “modification” includes all changes in the nucleotide by chemical means, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety for another.
  • base encompasses any known DNA and RNA base, base analogs such as purine or pyrimidine, and also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs.
  • C 1-6 alkyl optionally substituted by halogen or cyano means that halogen or cyano may but need not be present, and the description includes situations where the alkyl is substituted by halogen or cyano and situations where the alkyl is not substituted by halogen and cyano.
  • alkyl refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, and in some embodiments is selected from alkyl groups containing 1 to 12 carbon atoms.
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl,
  • alkyl groups containing 1 to 6 carbon atoms non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like.
  • the alkyl group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment, the substituent being selected in some embodiments from one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylate.
  • alkoxy refers to -O-(alkyl), wherein alkyl is as defined above.
  • alkoxy include: methoxy, ethoxy, propoxy, butoxy.
  • Alkoxy may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from halogen, hydroxy, oxo, cyano, amino, C 1-6 alkyl, C 1-6 alkoxy, 3 to 7-membered cycloalkyl or 3 to 7-membered heterocycloalkyl, wherein the alkyl, alkoxy, cycloalkyl or heterocycloalkyl is optionally substituted with halogen, hydroxy, nitro, cyano or amino.
  • alkylthio refers to -S-(alkyl), wherein alkyl is as defined above.
  • alkylthio include: methylthio, ethylthio, propylthio, butylthio.
  • Alkylthio may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from C 1-6 alkoxy, 3 to 6-membered cycloalkyl, 3 to 6-membered heterocycloalkyl, 3 to 6-membered cycloalkyloxy, 3 to 6-membered heterocycloalkyloxy, C 1-6 alkylthio, 3 to 6-membered cycloalkylthio, 3 to 6-membered heterocycloalkylthio, wherein the alkoxy, cycloalkyl, heterocycloalkyl, The cycloalkyloxy group, heterocyclooxy group, alkylthio group, cycloalkylthio group and heterocycloalkylthio group are optionally substituted by halogen, hydroxyl group, cyano group or amino group.
  • alkenyl refers to a straight or branched non-aromatic hydrocarbon group containing at least one carbon-carbon double bond and having 2-10 carbon atoms. Up to 5 carbon-carbon double bonds may be present in such a group.
  • a "C2 - C6 " alkenyl is defined as an alkenyl group having 2-6 carbon atoms.
  • alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, and cyclohexenyl.
  • the straight, branched, or cyclic portion of the alkenyl group may contain double bonds and may be optionally substituted by mono-, di-, tri-, tetra-, or penta- in any position permitted by normal valence.
  • alkynyl refers to a straight or branched hydrocarbon group containing 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond. Up to 5 carbon-carbon triple bonds may be present.
  • C2 - C6 alkynyl means an alkynyl group having 2 to 6 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 2-propynyl, and 2-butynyl.
  • the straight and branched portions of the alkynyl group may contain triple bonds as allowed by normal valence, and may be optionally mono-, di-, tri-, tetra- or penta-substituted at any position allowed by normal valence.
  • cycloalkyl or “carbocycle” refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, the cycloalkyl ring containing 3 to 20 carbon atoms, in some embodiments selected from containing 3 to 7 carbon atoms.
  • monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, etc.
  • polycyclic cycloalkyls include spirocyclic, fused and bridged cycloalkyls.
  • the cycloalkyl group may be substituted or unsubstituted.
  • the substituent may be substituted at any available point of attachment and in some embodiments is selected from one or more of the following groups, independently selected from halogen, deuterium, hydroxy, oxo, nitro, cyano, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyloxy, C 2-6 alkynyloxy, C 3-6 cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C 3-8 cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl, wherein the C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyloxy, C 2-6 alkynyloxy, C 3-6 cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C 3-8 cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl are optionally substituted with one or more selected
  • the cycloalkyl ring may be fused to an aryl or heteroaryl ring, wherein the ring attached to the parent structure is a cycloalkyl, non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptanyl, and the like.
  • the cycloalkyl group may be optionally substituted or unsubstituted, and when substituted, the substituents are in some embodiments selected from one or more of the following groups, which are independently selected from halogen, deuterium, hydroxyl, oxo, nitro, cyano, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyloxy, C 2-6 alkynyloxy, C 3-6 cycloalkyloxy, 3 to 6 membered heterocycloalkyloxy, C 3-8 cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl, wherein the C 1-6 alkyl , C 1-6 alkoxy, C 2-6 alkenyloxy, C 2-6 alkynyloxy, C 3-6 cycloalkyloxy, 3 to 6 membered heterocycloalkyloxy, C 3-8 cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl are optionally substituted with one or
  • heterocycloalkyl or “heterocycle” or “heterocyclyl” refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is a heteroatom selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2), but excluding the ring portion of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon. In some embodiments, it is selected from 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; in some embodiments, it is selected from 3 to 7 ring atoms.
  • Non-limiting examples of monocyclic heterocycloalkyls include Pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc.
  • Polycyclic heterocycloalkyl includes spirocyclic, fused and bridged heterocycloalkyl.
  • Non-limiting examples of "heterocycloalkyl" include: wait.
  • heterocycloalkyl ring may be fused to an aryl or heteroaryl ring, wherein the ring attached to the parent structure is a heterocycloalkyl, non-limiting examples of which include:
  • the heterocycloalkyl group may be optionally substituted or unsubstituted, and when substituted, the substituents are in some embodiments selected from one or more of the following groups, which are independently selected from halogen, deuterium, hydroxyl, oxo, nitro, cyano, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyloxy, C 2-6 alkynyloxy, C 3-6 cycloalkyloxy, 3 to 6 membered heterocycloalkyloxy, C 3-8 cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl, wherein the C 1-6 alkyl , C 1-6 alkoxy, C 2-6 alkenyloxy, C 2-6 alkynyloxy, C 3-6 cycloalkyloxy, 3 to 6 membered heterocycloalkyloxy, C 3-8 cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl are optionally substituted with one or
  • aryl refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group having a conjugated ⁇ electron system, selected from 6- to 12-membered in some embodiments, such as phenyl and naphthyl.
  • the aryl ring may be fused to a heteroaryl, heterocycloalkyl or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring, non-limiting examples of which include:
  • the aryl group may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate, preferably phenyl.
  • condensed ring aryl can be an unsaturated aromatic condensed ring structure containing 8-14 ring atoms formed by two or more ring structures sharing two adjacent atoms, preferably 8-12 ring atoms.
  • it includes all unsaturated condensed ring aryl groups, such as naphthalene, phenanthrene, etc., and also includes partially saturated condensed ring aryl groups, such as benzo 3-8 membered saturated monocyclic cycloalkyl, benzo 3-8 membered partially saturated monocyclic cycloalkyl.
  • Condensed aromatic ring refers to the ring system in the condensed ring aryl group.
  • condensed ring aryl groups include 2,3-dihydro-1H-indenyl, 1H-indenyl, 1,2,3,4-tetrahydronaphthyl, 1,4-dihydronaphthyl, etc.
  • heteroaryl refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen.
  • Heteroaryl is preferably 5 to 12 members, such as imidazolyl, furanyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidyl, thiadiazole, pyrazinyl, etc., preferably imidazolyl, pyrazolyl, pyrimidyl or thiazolyl; more preferably pyrazolyl or thiazolyl.
  • heteroaryl ring can be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring.
  • Heteroaromatic ring refers to the ring system in a heteroaryl group.
  • Non-limiting examples of heteroaryl include:
  • the heteroaryl group may be optionally substituted or unsubstituted, and when substituted, the substituents are in some embodiments selected from one or more of the following groups, which are independently selected from halogen, deuterium, hydroxyl, oxo, nitro, cyano, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyloxy, C 2-6 alkynyloxy, C 3-6 cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C 3-8 cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl, wherein the C 1-6 alkyl , C 1-6 alkoxy, C 2-6 alkenyloxy, C 2-6 alkynyloxy, C 3-6 cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C 3-8 cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl are optionally substituted with one or more groups selected from halogen
  • alkylamino refers to a group having the structure -NH(C1-C12 alkyl).
  • hydroxyalkyl refers to an alkyl group substituted with one or more hydroxy groups, wherein alkyl is as defined above.
  • hydroxy refers to an -OH group.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • haloalkyl refers to an alkyl group substituted with a halogen, wherein alkyl is as defined above.
  • haloalkoxy refers to an alkoxy group substituted with a halogen, wherein alkoxy is as defined above.
  • cyano refers to -CN.
  • nitro refers to -NO2 .
  • amino refers to -NH2 .
  • blunt end or “blunt terminus” are used interchangeably and refer to the absence of unpaired nucleotides or nucleotide analogs at a given end of a dsRNA, i.e., no nucleotide overhangs. In most cases, a dsRNA with both ends blunt-ended will be double-stranded throughout its entire length.
  • the phosphodiester group or the modified phosphodiester group in each modification group can be replaced by any group that can achieve the connection with the adjacent nucleotide, such as chemical modification In
  • the moiety can be replaced with any group that can achieve linkage with adjacent nucleotides.
  • linked when referring to the connection between two molecules, means that the two molecules are connected by a covalent bond or the two molecules are associated via a non-covalent bond (eg, a hydrogen bond or an ionic bond), including direct connection and indirect connection.
  • a non-covalent bond eg, a hydrogen bond or an ionic bond
  • directly linked refers to a first compound or group being linked to a second compound or group without any intervening atoms or groups of atoms.
  • directly linked means that a first compound or group is linked to a second compound or group through an intermediate group, compound or molecule (eg, a linking group).
  • substituted means that any one or more hydrogen atoms on a designated atom (usually a carbon, oxygen, and nitrogen atom) are replaced by any group as defined herein, provided that the normal valence of the designated atom is not exceeded and the substitution generates a stable compound.
  • Non-limiting examples of substituents include C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cyano, hydroxyl, oxo, carboxyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, aryl, ketone, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, or halogen (e.g., F, Cl, Br, I).
  • two (2) hydrogens on the atom are replaced.
  • Substituted by one or more means that the compound may be substituted by a single or multiple substituents. When substituted by multiple substituents, the substituents may be the same or a combination of one or more different substituents.
  • “Pharmaceutical composition” means a mixture containing one or more compounds described herein or their physiologically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically acceptable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert its effect. Biological activity.
  • “Pharmaceutically acceptable excipients” include, but are not limited to, any adjuvant, carrier, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier that has been approved for use by humans or livestock animals.
  • the term “inhibition” can be used interchangeably with “reduction”, “silence”, “downregulation”, “suppression” and other similar terms, and includes inhibition at any level. Inhibition can be assessed by the reduction of the absolute or relative level of one or more of these variables compared to the control level.
  • the control level can be any type of control level used in the art, such as baseline level before administration or the level determined from a similar untreated or controlled subject, cell, or sample (such as only buffer control or inert agent control).
  • the remaining expression of mRNA can be used to characterize the degree of inhibition of dsRNA on target gene expression, such as the remaining expression of mRNA is not higher than 99%, not higher than 95%, not higher than 90%, not higher than 85%, not higher than 80%, not higher than 75%, not higher than 70%, not higher than 65%, not higher than 60%, not higher than 55%, not higher than 50%, not higher than 45%, not higher than 40%, not higher than 35%, not higher than 30%, not higher than 25%, not higher than 20%, not higher than 15%, or not higher than 10%.
  • the "compound”, “ligand”, “nucleic acid ligand conjugate”, “dsRNA conjugate”, “nucleic acid”, “conjugate”, “chemical modification”, “targeting ligand”, “dsRNA”, and “RNAi” of the present disclosure may independently exist in the form of salt, mixed salt, or non-salt (e.g., free acid or free base). When it exists in the form of a salt or a mixed salt, it may be a pharmaceutically acceptable salt.
  • pharmaceutically acceptable salt includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.
  • “Pharmaceutically acceptable acid addition salts” refer to salts formed with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects.
  • Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, and the like; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetates, trifluoroacetates, propionates, caproates, caprylates, decanoates, undecylenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartates, benzoates, me
  • “Pharmaceutically acceptable base addition salt” refers to a salt formed with an inorganic base or an organic base that can maintain the biological effectiveness of the free acid without other side effects.
  • Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like.
  • Preferred inorganic salts are ammonium Salt, sodium salt, potassium salt, calcium salt and magnesium salt, preferably sodium salt.
  • Salts derived from organic bases include but are not limited to the following salts: primary amines, secondary amines and tertiary amines, substituted amines, including natural substituted amines, cyclic amines and alkaline ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc.
  • Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine
  • an “effective amount” or “effective dose” encompasses an amount sufficient to ameliorate or prevent the symptoms or symptoms of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's general health, the method, route and dosage of administration, and the severity of side effects.
  • An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • subject As used herein, “subject,” “patient,” “subject,” or “individual” are used interchangeably and include humans or non-human animals, such as mammals, such as humans or monkeys.
  • the dsRNA provided by the present disclosure can be obtained by conventional preparation methods in the art (e.g., solid phase synthesis and liquid phase synthesis methods). Among them, solid phase synthesis has commercial customization services.
  • the modified nucleotide groups can be introduced into the dsRNA described in the present disclosure by using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into dsRNA are also well known to those skilled in the art.
  • DIPEA N-ethyldiisopropylamine
  • HBTU benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate
  • NMI N-methylimidazole
  • Figure 1A shows the ratio of serum Lp(a) concentrations of TJR101079 and TRD007790 on day 28 after administration relative to that before administration.
  • Figure 1B shows the ratio of serum Lp(a) concentrations of TJR101079 and TJR102134 on day 28 after administration relative to that before administration.
  • Figure 1C shows the ratio of serum Lp(a) concentrations of TJR101079 and TJR102136 on day 28 after administration relative to that before administration.
  • NAG0052 as shown in the above structural formula was prepared according to the method described in patent application WO2023274395A;
  • NAG25 shown in the above structural formula was prepared according to the method of patent application WO2017156012A.
  • tert-butyldimethylsilyl chloride (TBSCl) (93.2 g, 618 mmol) was added, and the reaction was allowed to react at 25°C for 18 hours.
  • Benzoyl chloride (32.62 g, 232.0 mmol) was then added, and the reaction was allowed to react at 25°C for 3 hours.
  • compound A1-6 (60.0 g, 100.3 mmol) was dissolved in methanol (600 mL), and acetyl chloride (10.21 g, 130.1 mmol) was added, and the mixture was reacted at 20°C for 2 hours. Then silver carbonate was added, and the mixture was stirred at 20°C for 1 hour. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain compound A1-7 (46.0 g, 94.7 mmol, yield 94.7%).
  • dsRNA dsRNA
  • the phosphoramidite monomer A1 synthesized in Example 3 is used at the corresponding position.
  • nucleoside phosphoramidite monomers are connected one by one according to the synthesis procedure. Except for the nucleoside phosphoramidite monomer A1 described in Example 3, the remaining nucleoside monomer raw materials 2'-F RNA, 2'-O-methyl RNA and other nucleoside phosphoramidite monomers were purchased from Shanghai Zhaowei or Suzhou Jima.
  • ETT 5-Ethylthio-1H-tetrazole
  • 0.22M PADS dissolved in a 1:1 volume ratio of acetonitrile and trimethylpyridine (Suzhou Kelema) solution was used as a sulfurization reagent to introduce thiophosphates at designated positions
  • iodine pyridine/water solution Kerma was used as an oxidant to introduce oxophosphates at designated positions.
  • some embodiments require the use of TMSI to remove the 5'-terminal protecting group.
  • TMSI ethyl group in the oligoribonucleotide is removed after sufficient stirring to obtain an oligoribonucleotide with NA0127.
  • the oligoribonucleotide is then further cleaved from the solid support under the following cleavage conditions: soaking in a 3:1 28% ammonia and ethanol solution at 50°C for 16 hours.
  • the obtained single-stranded oligonucleotides were annealed according to complementary pairing in an equal molar ratio, and the dsRNA obtained was finally dissolved in 1 ⁇ PBS and adjusted to the required concentration for use in the experiment.
  • the compound NAG0052 (157 mg, 0.062 mmol) containing a carboxylic acid group was dissolved in anhydrous DMF (3 mL). After the substrate was completely dissolved, anhydrous acetonitrile (4 mL), DIEA (0.03 mL, 0.154 mmol, 2.5 eq) and HBTU (35 mg, 0.093 mmol, 1.5 eq) were added in sequence. After the reaction solution was mixed evenly, macroporous aminomethyl resin (476 mg, blank loading was 0.41 mmol/g, target loading was 0.1 mmol/g) was added. The reaction solution was placed on a shaker (temperature: 25 ° C, speed: 200 rpm) and shaken overnight. The reaction solution was filtered, and the filter cake was washed with DCM and anhydrous acetonitrile in sequence. The solid was collected and dried in vacuum overnight.
  • NAG0052 already connected to the resin use the resin as a starting point to connect nucleoside monomers one by one from the 3'-5' direction according to the nucleotide arrangement order.
  • Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. The operation is conventional in the art.
  • NAG0052 was connected to the sequence by solid phase synthesis, and then after aminolysis, some functional groups of the NAG0052 structure were removed to become NAG0052'. 3.
  • L96 was used to replace NAG0052 and synthesize the related conjugate according to the above steps. Among them, L96 was prepared according to the method described in patent application WO2014025805A.
  • the prepared dsRNA conjugates had the sense strand and antisense strand shown in Tables 1 and 2.
  • the positive control dsRNA conjugate TRD007790 was prepared according to the method described in patent application WO2017059223A.
  • capital letters G, A, C, and U represent nucleotides containing guanine, adenine, cytosine, and uracil, respectively; lowercase letter m represents 2'-methoxy modification; lowercase letter f represents 2'-fluoro modification; and lowercase letter s represents that the two nucleotides adjacent to the letter s are connected by a phosphorothioate diester group;
  • NAG0052' The structure of NAG0052' is:
  • L96' The structure of L96' is:
  • NAG25s' The structure of NAG25s' is:
  • NA0127, NA0149 and InvdA respectively represent:
  • Example 4 the method for introducing NA0127 into dsRNA is described in Example 4, the synthetic method for introducing NA0149 into dsRNA is described in US20190177729A; the synthetic method for introducing InvdA into dsRNA is described in WO2017059223A.
  • Human LPA (NM_005577.3) was used as the target gene to design 19/21 nt dsRNA to meet the general rules of active dsRNA.
  • the unmodified sense and antisense strand sequences are shown in Tables 4 and 5.
  • Example 7 dsRNA conjugate psiCHECK 9 concentration point on-target activity
  • dsRNA/dsRNA conjugates were screened for on-target activity at the in vitro molecular level using 9 concentration gradients.
  • HEK293A cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO 2. 24 h before transfection, HEK293A cells were seeded in 96-well plates at a seeding density of 8 ⁇ 10 3 cells per well with 100 ⁇ L of culture medium per well.
  • cells were co-transfected with dsRNA conjugates and corresponding plasmids using Lipofectamine 2000 (ThermoFisher, 11668019), with 0.3 ⁇ L Lipofectamine 2000 used per well.
  • the amount of plasmid transfection was 40ng per well.
  • dsRNA/dsRNA conjugates 9 concentration points were set for dsRNA/dsRNA conjugates, with the highest concentration point having a final concentration of 20nM, 3-fold gradient dilution, 20nM, 6.666666667nM, 2.222222222nM, 0.740740741nM, 0.24691358nM, 0.082304527nM, 0.027434842nM, 0.009144947nM, 0.003048316nM. 24h after transfection, the target level was detected using Dual-Luciferase Reporter Assay System (Promega, E2940).
  • Example 8 dsRNA inhibition of human LPA in primary human hepatocytes (PHH) - inhibitory activity at 7 concentration points
  • the dsRNA sequences were screened for PHH activity in primary human hepatocytes (PHH) using 7 concentration gradients.
  • the concentration of each dsRNA sample was 20 nM from the beginning of transfection, with 5-fold gradient dilution and 7 concentration points.
  • PHH were frozen in liquid nitrogen. 24 h before transfection, primary human hepatocytes (PHH) were revived and inoculated in 96-well plates at a density of 3 ⁇ 104 cells per well and 80 ⁇ L of culture medium per well.
  • PHH primary human hepatocytes
  • RNAi MAX Lipofectamine RNAi MAX (ThermoFisher, 13778150) Transfected with dsRNA, the final concentration of dsRNA transfection gradient was 20nM, 4nM, 0.8nM, 0.16nM, 0.032nM, 0.0064nM and 0.00128nM. After 24 hours of treatment, total RNA extraction, RNA reverse transcription experiment and quantitative real-time PCR were performed using a high-throughput cell RNA extraction kit to determine the mRNA level of human LPA, and the mRNA level of human LPA was corrected according to the level of the GAPDH internal reference gene.
  • Inhibition rate (%) (1-remaining expression amount of target gene)*100%.
  • results are expressed as the remaining percentage of human LPA mRNA expression relative to cells treated with control dsRNA.
  • the IC50 results of inhibition are shown in Table 9.
  • TJR100373 had significantly superior activity compared with TJR100374 to TJR100380, indicating that the target gene fragment targeted by TJR100373 was more suitable as a target.
  • Example 9 Determination of in vivo activity of dsRNA conjugates in humanized mice (hu-Lp(a))
  • the humanized mouse (hu-Lp(a)) used in this example was constructed by Shanghai Tuojie Biotechnology Co., Ltd. on behalf of Saiye (Suzhou) Biotechnology Co., Ltd.
  • mice were evenly divided into groups, with 6 mice in each group (2 males and 4 females), for a total of 4 groups. They were given physiological saline, positive control TRD007790, and the dsRNA conjugate TJR101079 disclosed in the present invention by subcutaneous injection.
  • the dosage was 3 mg/kg, and the administration volume was 10 ⁇ L/g. 40 ⁇ L of serum was collected before administration on the day of administration, and the measured serum Lp(a) concentration was used as the basic data.
  • the administration diary was day 1 (D1), day 8 (D8), day 29 (D29), day 57 (D57), day 85 (D85), and day 99 (D99) after administration.
  • serum Lp(a) protein content was determined using Abbott Ci4100 fully automatic biochemical immunoassay, and the inhibition of dsRNA conjugate on serum Lp(a) protein expression in humanized mice (hu-Lp(a)) was calculated.
  • the ratio of serum Lp(a) protein to blank after normalization of each drug-treated group to the blank control group was calculated, and the results are recorded in Table 10.
  • One-way ANOVA was used to analyze the statistical data of each group relative to the blank drug-treated group.
  • Example 10 Determination of in vivo activity of dsRNA conjugates in humanized mice (hu-Lp(a))
  • the humanized mouse (hu-Lp(a)) used in this example was constructed by Shanghai Tuojie Biotechnology Co., Ltd. on behalf of Saiye (Suzhou) Biotechnology Co., Ltd.
  • mice were evenly divided into groups, with 6 mice in each group, and a total of 4 groups were given by subcutaneous injection.
  • the dosage of dsRNA conjugates TRD007790, TJR101079, TJR102134, and TJR102136 was 1 mg/kg, and the administration volume was 10 ⁇ L/g.
  • 40 ⁇ L of serum was collected before administration, and the measured serum Lp(a) concentration was used as the basic data.
  • the administration diary was the first day (D1) and the 28th day (D28) after administration.
  • the serum Lp(a) concentration at D28 of each group was compared with the serum Lp(a) before drug administration to obtain the residual serum Lp(a) ratio, and the paired t test was used to perform statistical comparisons between TJR101079 and TRD007790, TJR102134, and TJR102136.

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Abstract

一种靶向LPA的dsRNA及其应用。一种靶向LPA的双链核糖核酸(dsRNA)及制备方法。包含该dsRNA的药物组合物、细胞或试剂盒。dsRNA可以干扰LPA的表达，预防和/或治疗相关疾病。

Description

一种靶向LPA的dsRNA及其应用
本公开要求申请日为2023年05月31日的中国专利申请202310628559.8的优先权和申请日为2023年10月25日的中国专利申请202311386740.9的优先权，本公开引用上述中国专利申请的全文。
技术领域
本公开属于生物医药领域，具体涉及一种靶向LPA的dsRNA、其应用及制备方法。
背景技术
1963年挪威遗传学家Berg首先发现的脂蛋白(a)[Lp(a)]被鉴定为独特的脂蛋白(Berg K.Anew serum type systemin man-the Lp system.Acta Pathol Microbiol Scand 1963；59:369–82.)。Lp(a)由脂质和蛋白质两部分组成：脂质部分主要是位于核心部位的LDL样微粒；蛋白质部分位于外周，由载脂蛋白(a)[apo(a)]和apoB100通过二硫键连接而成。apo(a)主要在肝脏中表达，其表达仅限于人类和非人类的灵长类动物，它的特征是存在三个内部二硫键稳定的三环结构的结构域(Kringle)。在人类谱系中，apo(a)中Kringle IV结构域的扩增和分化导致十种不同类型的KIV结构域，其中Kringle IV 2型(KIV-2)的进一步扩增产生了多等位基因(1至40拷贝)内拷贝数变异(CNV)，其他Kringle IV编码域(KIV-1和KIV-3至KIV-10)仅作为单拷贝存在(Schmidt K,Noureen A,Kronenberg F,et al.Structure,Function,and Genetics of Lipoprotein(a)[J].Journal of Lipid Research,2016,57(8):1339.)。所有Kringle均被转录和翻译，因此KIV-2CNV导致了编码的apo(a)的大小多态性，其表达与存在KIV-2结构域的数量成反比，当KIV-2拷贝数较少时，血浆中Lp(a)含量会显著增加。
Lp(a)升高的患者其心血管事件的风险是正常人群的2-3倍，引起包括动脉粥样硬化心血管疾病、下肢动脉病变、主动脉瓣膜狭窄等(EnAs EA,Varkey B,Dharmarajan T S,et al.Lipoprotein(a):An independent,genetic,and causal factor for cardiovascular disease and acute myocardial infarction[J].Indian Heart Journal,2019,71(2).)。Lp(a)可能通过以下两种机制导致不良的动脉粥样硬化性心血管疾病(ASCVD)：一方面由于apo(a)在体外被证实可抑制纤溶，因此可能促进斑块破裂处的血栓形成或血管狭窄处的湍流，从而导致血管阻塞或促进血栓形成；另一方面LDL样颗粒可促进内膜胆固醇沉积、炎症或氧化磷脂，从而导致动脉粥样性狭窄或主动脉瓣狭窄(Albert Youngwoo Jang,Seung Hwan Han，Il Suk Sohn，et al.Lipoprotein(a)and Cardiovascular Diseases[J].Circulation Journal，2020，84:867–874)。但即使非常高水平的Lp(a)，其胆固醇含量也低于传统LDL临界值，因而，LDL样颗粒这部分的致病性可能比较少。
2016年中国成人血脂异常防治指南将>30mg/dl定义为Lp(a)异常，以此为标准，在中国约30％的既往有心血管事件的患者存在Lp(a)异常。2019年美国国家脂质协会推荐Lp(a)≥50mg/dl为水平升高，按照这个标准全球人口的20％均存在Lp(a)水平升高。尽管Lp(a)水平升高常见，但缺乏针对性治疗药物，迄今尚无靶向降低Lp(a)的药物获批用于临床。Lp(a)蛋白与多种脂蛋白结构相似，难以成为小分子和大分子药物的直接靶点，而Lp(a)基因转录的mRNA具有高专属性，可以通过siRNA转录后调控机制特异性降解其mRNA，进而抑制Lp(a)表达。因此设计靶向apo(a)基因(LPA)的siRNA使其表达减弱，从而降低血清中Lp(a)水平，进而降低心血管不良事件。
发明内容
本公开提供了一种双链核糖核酸，其包含正义链与反义链；所述正义链和所述反义链形成双链区(优选所述正义链和所述反义链反向互补)；所述双链核糖核酸靶向LPA基因或其表达产物。
在一些实施方案中，所述dsRNA的正义链和/或反义链中包含至少一个修饰的核苷酸。
本公开提供了一种靶向LPA的双链核糖核酸(dsRNA)，其包含形成双链区的正义链和反义链，其中，
所述正义链的裸核苷酸序列包含与SEQ ID NO:1的核苷酸序列相差不超过3个核苷酸的至少17个连续核苷酸；并且，
所述反义链的裸核苷酸序列包含与SEQ ID NO:2的核苷酸序列相差不超过3个核苷酸的至少19个连续核苷酸；其中，按照5'端到3'端的方向，
所述正义链第7、8和9位的核苷酸为2'-氟代修饰的核苷酸，其余位置的核苷酸为2'-甲氧基修饰的核苷酸；
所述反义链第2和14位的核苷酸为2'-氟代修饰的核苷酸，第4、6、10、12、16和18位的核苷酸独立地为2'-甲氧基或2'-氟代修饰的核苷酸，其余位置的核苷酸为2'-甲氧基修饰的核苷酸；
所述反义链中2'-氟代修饰的核苷酸的个数为2-7个(如2个、3个、4个、5个、6个、7个)。
在一些实施方案中，所述的dsRNA的正义链的裸核苷酸序列包含或选自SEQ ID NO:1所示的核苷酸序列，并且，反义链的裸核苷酸序列包含或选自SEQ ID NO:2所示的核苷酸序列；其中，按照5'端到3'端的方向，
所述正义链第7、8和9位的核苷酸为2'-氟代修饰的核苷酸，其余位置的核苷酸为2'-甲氧基修饰的核苷酸；
所述反义链第2和14位的核苷酸为2'-氟代修饰的核苷酸，第4、6、10、12、16和18位的核苷酸独立地为2'-甲氧基或2'-氟代修饰的核苷酸，其余位置的核苷 酸为2'-甲氧基修饰的核苷酸；
所述反义链中2'-氟代修饰的核苷酸的个数为2-7个(如2个、3个、4个、5个、6个、7个)。
在一些实施方案中，所述正义链和反义链：
正义链包含5’-GCUCCUUAUUGUUAUACGA-3’，
反义链包含5’-UCGUAUAACAAUAAGGAGCUG-3’。
在另一些实施方案中，所述正义链和反义链：
正义链的裸核苷酸序列是5’-GCUCCUUAUUGUUAUACGA-3’，
反义链的裸核苷酸序列是5’-UCGUAUAACAAUAAGGAGCUG-3’；
裸序列是指未经修饰时的核苷酸序列。
在一些实施方案中，所述的dsRNA的反义链5’端第7位的核苷酸是修饰的核苷酸，其中，所述修饰的核苷酸是2'-甲氧基修饰的核苷酸。
在一些实施方案中，所述的dsRNA的反义链5’端第7位的核苷酸是修饰的核苷酸，其中，所述修饰的核苷酸包含式(I)、(Ⅰ-1)、(Ⅰ-2)所示的化学修饰或其药学上可接受的盐：
其中，B与所述反义链在其5’端第7位核苷酸未被修饰时的碱基相同；在一些具体的实施方案中，B为腺嘌呤。
在一些实施方案中，式(I‘)、(Ⅰ’-1)、(Ⅰ‘-2)所示的化学修饰选自：
其中：M为O或S；其中：B与所述反义链在其5’端第7位核苷酸未被修饰时的碱基相同；在一些具体的实施方案中，B为腺嘌呤。
一些具体的实施方案中，M为S。一些具体的实施方案中，M为O。
在一些实施方案中，所述的dsRNA的反义链5’端第1位的核苷酸是修饰的核苷酸，其中，所述修饰的核苷酸是2’-甲氧基修饰的核苷酸。
在一些实施方案中，所述的dsRNA的反义链5’端第1位的核苷酸是修饰的核苷酸，其中，所述修饰的核苷酸为包含式(Ⅳ)所示的化学修饰的核苷酸：
其中，
RA1、RA2各自独立地选自氢或氘；
M1、M2各自独立地选自-SH或-OH；
B选自碱基、氢、氘；RA3选自氢、氘、羟基、卤素、烷基(例如C1、C2、C3、C4、C5、C6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(例如C1烷氧基、C2烷氧基、C3烷氧基、C4烷氧基、C5烷氧基、C6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)，所述羟基、烷基、烷氧基各自任选被一个或多个氘所取代；RA4选自氢、氘、烷基(例如C1、C2、C3、C4、C5、C6烷基，包括但不限于甲基、乙基、异丙基)，所述烷基各自任选被一个或多个氘所取代；条件是，式(Ⅳ)中包含至少一个氘。
在一些实施方案中，RA3选自氢、氘。
在另一些实施方案中，RA3选自卤素(如氟、氯、溴)。
在另一些实施方案中，RA3选自烷基(例如C1、C2、C3、C4、C5、C6烷基，包括但不限于甲基、乙基、异丙基)，所述烷基各自任选被一个或多个氘所取代。
在另一些实施方案中，RA3选自烷氧基(例如C1烷氧基、C2烷氧基、C3烷氧基、C4烷氧基、C5烷氧基、C6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)，所述烷氧基各自任选被一个或多个氘所取代。
在一些实施方案中，式(Ⅳ)所示的5’末端化学修饰是：
其中，RA5、RA6、RA7各自独立地选自氢或氘；
RA1、RA2、M1、M2、B如式(Ⅳ)所定义；条件是，式(Ⅳ-1)中包含至少一个氘。
在一些实施方案中，RA5、RA6、RA7全部为氘。
在一些实施方案中，RA5是氘，RA6、RA7是氢。
在一些实施方案中，RA5、RA6是氘，RA7是氢。
在一些实施方案中，RA1是氢，RA2是氘。
在一些实施方案中，RA1和RA2是氘。
在一些实施方案中，RA1和RA2是氢。
在一些实施方案中，式(Ⅳ)所示的5’末端化学修饰选自：
B选自碱基或氢。
在一些实施方案中，B选自碱基；在一些具体的实施方案中，B选自腺嘌呤、鸟嘌呤、胞嘧啶、尿嘧啶或胸腺嘧啶。
在一些具体的实施方案中，B是反义链的修饰的核苷酸对应位置的碱基。
在一些具体的实施方案中，B选自尿嘧啶。
在一些实施方案中，式(Ⅳ)所示的5’末端化学修饰选自：
以及以腺嘌呤、鸟嘌呤、胞嘧啶、或胸腺嘧啶替换尿嘧啶的结构。
在一些实施方案中，按照5'末端到3'末端的方向，所述dsRNA的反义链5’端第1位的核苷酸为所述的式(Ⅳ)所示的5’末端化学修饰的核苷酸。在一些具体的实施方案中，式(Ⅳ)所示的5’末端化学修饰的核苷酸为 所述B为反义链5'末端第1位核苷酸对应位置的碱基。
在一些实施方案中，所述的dsRNA的反义链5’端第1位的核苷酸是修饰的核苷酸，其中，所述修饰的核苷酸为式(Ⅱ)所示的化学修饰的核苷酸：
其中，B表示反义链5’端第1位核苷酸对应位置的碱基。在一些具体的实施方案中，B表示尿嘧啶。
在一些实施方案中，所述反义链与靶序列至少部分地反向互补以介导RNA干扰。在一些实施方案中，所述反义链与靶序列之间存在不多于5个、不多于4个、不多于3个、不多于2个、不多于1个错配；在一些实施方案中，所述反义链与靶序列完全反向互补。
在一些实施方案中，所述正义链与反义链至少部分地反向互补以形成双链区；在一些实施方案中，所述正义链与反义链之间存在不多于5个、不多于4个、不多于3个、不多于2个、不多于1个错配；在一些实施方案中，所述正义链与反义链完全反向互补。
在一些实施方案中，所述正义链和反义链各自独立地具有16至35个、16至34个、17至34个、17至33个、18至33个、18至32个、18至31个、18至30个、18至29个、18至28个、18至27个、18至26个、18至25个、18至24个、18至23个、19至25个、19至24个、或19至23个核苷酸。
在一些具体的实施方案中，所述正义链和反义链的长度各自独立地具有19、20、21、22、或23个核苷酸。
在一些实施方案中，所述正义链和反义链长度相同或不同，所述正义链的长度为19-23个核苷酸，所述反义链的长度为19-26个核苷酸。在一些实施方案中，所 述正义链和反义链的长度比可以是19/20、19/21、19/22、19/23、19/24、19/25、19/26、20/20、20/21、20/22、20/23、20/24、20/25、20/26、21/20、21/21、21/22、21/23、21/24、21/25、21/26、22/20、22/21、22/22、22/23、22/24、22/25、22/26、23/20、23/21、23/22、23/23、23/24、23/25或23/26。在一些实施方案中，所述正义链和反义链的长度比为19/21、21/23或23/25。在一些实施方案中，所述正义链和反义链的长度比为19/21。
在一些实施方案中，所述dsRNA包含一个或两个平端。
在一些实施方案中，所述dsRNA包含具有1至4个未配对核苷酸的突出端，例如1个、2个、3个、4个。
在一些实施方案中，所述dsRNA包含位于所述反义链3’端的突出端。
在一些实施方案中，所述的dsRNA的正义链含有或为如下式所示的核苷酸序列：5’-NaNaNaNaNaNaNbNbNbNaNaNaNaNaNaNaNaNaNa-3’；其中，Na为2'-甲氧基修饰的核苷酸，Nb为2'-氟代修饰的核苷酸。
在一些实施方案中，所述的dsRNA的反义链包含或为如下式所示的核苷酸序列：
5’-Na’Nb’Na’X’Na’X’Na’Na’Na’X’Na’X’Na’Nb’Na’X’Na’X’Na’Na’Na’-3’，
其中，每个X’独立地为Na’或Nb’；Na’为2'-甲氧基修饰的核苷酸，Nb’为2'-氟代修饰的核苷酸。
在一些实施方案中，所述的dsRNA的反义链包含或为如下式所示的核苷酸序列：
5’-Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
5’-Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’Nb’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’-3’，
5’-Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’-3’，
5’-Na’Nb’Na’Na’Na’Nb’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’-3’，
5’-Na’Nb’Na’Na’Na’Nb’Na’Na’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
5’-Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
5’-Na’Nb’Na’Na’Na’Nb’Na’Na’Na’Nb’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
5’-Na’Nb’Na’Na’Na’Nb’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
5’-Na’Nb’Na’Na’Na’Na’Na’Na’Na’Na’Na’Na’Na’Nb’Na’Na’Na’Na’Na’Na’Na’-3’，
5’-Na’Nb’Na’Na’Na’Nb’Na’Na’Na’Na’Na’Na’Na’Nb’Na’Na’Na’Na’Na’Na’Na’-3’，
5’-Na’Nb’Na’Na’Na’Na’Na’Na’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
5’-Na’Nb’Na’Na’Na’Na’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’Na’Na’-3’，
其中，Na’为2'-甲氧基修饰的核苷酸，Nb’为2'-氟代修饰的核苷酸。
在一些实施方案中，所述的dsRNA的反义链包含或为如下式所示的核苷酸序列：5’-Na’Nb’Na’X’Na’X’W’Na’Na’X’Na’X’Na’Nb’Na’X’Na’X’Na’Na’Na’-3’，其中，每个X’独立地为Na’或Nb’；Na’为2'-甲氧基修饰的核苷酸，Nb’为2'-氟代修饰的核苷酸，W’表示包含式(I)、(Ⅰ-1)、(Ⅰ-2)所示的化学修饰或其药学上可接受的盐的 核苷酸。
在一些实施方案中，所述的dsRNA的反义链包含或为如下式所示的核苷酸序列：
5’-Na’Nb’Na’Nb’Na’Nb’W’Na’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
5’-Na’Nb’Na’Nb’Na’Nb’W’Na’Na’Nb’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’-3’，
5’-Na’Nb’Na’Nb’Na’Nb’W’Na’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’-3’，
5’-Na’Nb’Na’Na’Na’Nb’W’Na’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’-3’，
5’-Na’Nb’Na’Na’Na’Nb’W’Na’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
5’-Na’Nb’Na’Nb’Na’Nb’W’Na’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
5’-Na’Nb’Na’Na’Na’Nb’W’Na’Na’Nb’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
5’-Na’Nb’Na’Na’Na’Nb’W’Na’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
5’-Na’Nb’Na’Na’Na’Na’W’Na’Na’Na’Na’Na’Na’Nb’Na’Na’Na’Na’Na’Na’Na’-3’，
5’-Na’Nb’Na’Na’Na’Nb’W’Na’Na’Na’Na’Na’Na’Nb’Na’Na’Na’Na’Na’Na’Na’-3’，
5’-Na’Nb’Na’Na’Na’Na’W’Na’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
其中，Na’为2'-甲氧基修饰的核苷酸，Nb’为2'-氟代修饰的核苷酸；W’表示包含式(I)、(Ⅰ-1)、(Ⅰ-2)所示的化学修饰或其药学上可接受的盐的核苷酸。
在一些实施方案中，所述的dsRNA的反义链包含或为如下式所示的核苷酸序列：5’-V’Nb’Na’X’Na’X’Na’Na’Na’X’Na’X’Na’Nb’Na’X’Na’X’Na’Na’Na’-3’，其中，每个X’独立地为Na’或Nb’；Na’为2'-甲氧基修饰的核苷酸，Nb’为2'-氟代修饰的核苷酸；V’表示式(Ⅱ)所示的化学修饰的核苷酸。
在一些实施方案中，所述的dsRNA的反义链包含或为如下式所示的核苷酸序列：
5’-V’Nb’Na’Na’Na’Nb’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
5’-V’Nb’Na’Na’Na’Na’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’Na’Na’-3’；
其中，Na’为2'-甲氧基修饰的核苷酸，Nb’为2'-氟代修饰的核苷酸；V’表示式(Ⅱ)所示的化学修饰的核苷酸。
在一些实施方案中，式(I)、(Ⅰ-1)、(Ⅰ-2)所示的化学修饰或其药学上可接受的盐选自：
其中：B表示反义链5’端第7位的碱基，在一些具体的实施方案中，B为腺嘌呤。
在一些实施方案中，式(I‘)、(Ⅰ’-1)、(Ⅰ‘-2)所示的化学修饰或其药学上可接受的盐选自：
其中：M为O或S；其中：B表示反义链5’端第7位的碱基，在一些具体的实施方案中，B为腺嘌呤。
在一些实施方案中，M为S。在一些具体的实施方案中，M为O。
在一些实施方案中，式(Ⅱ)所示的化学修饰的核苷酸选自：
其中，B表示反义链5’端第1位核苷酸对应位置的碱基。在一些具体的实施方案中，B表示尿嘧啶。
在一些实施方案中，式(Ⅱ)所示的化学修饰的核苷酸选自：
其中，B表示反义链5’端第1位核苷酸对应位置的碱基。在一些具体的实施方案中，B表示尿嘧啶。
在一些实施方案中，所述的dsRNA的正义链和/或反义链中至少一个磷酸二酯基为具有修饰基团的磷酸二酯基。
在一些实施方案中，所述的dsRNA的正义链和/或反义链中至少一个磷酸二酯基为硫代磷酸二酯基。
在一些实施方案中，所述硫代磷酸二酯基存在于以下位置中的至少一处：
所述正义链的5'端第1个核苷酸和第2个核苷酸之间；
所述正义链的5'端第2个核苷酸和第3个核苷酸之间；
所述正义链的3’端第1个核苷酸和第2个核苷酸之间；
所述反义链的5'端第1个核苷酸和第2个核苷酸之间；
所述反义链的5'端第2个核苷酸和第3个核苷酸之间；
所述反义链的3'端第1个核苷酸和第2个核苷酸之间；以及
所述反义链的3'端第2个核苷酸和第3个核苷酸之间。
在一些实施方案中，所述正义链和/或反义链中包括多个硫代磷酸二酯基，所述硫代磷酸二酯基存在于：
所述正义链的5'端第1个核苷酸和第2个核苷酸之间；和，
所述正义链的5'端第2个核苷酸和第3个核苷酸之间；和，
所述正义链的3’端第1个核苷酸和第2个核苷酸之间；和
所述反义链的5'端第1个核苷酸和第2个核苷酸之间；和，
所述反义链的5'端第2个核苷酸和第3个核苷酸之间；和，
所述反义链的3'端第1个核苷酸和第2个核苷酸之间；和，
所述反义链的3'端第2个核苷酸和第3个核苷酸之间。
在一些实施方案中，所述正义链和/或反义链中包括多个硫代磷酸二酯基，所述硫代磷酸二酯基存在于：
所述正义链的5'端第1个核苷酸和第2个核苷酸之间；和，
所述正义链的5'端第2个核苷酸和第3个核苷酸之间；和，
所述反义链的5'端第1个核苷酸和第2个核苷酸之间；和，
所述反义链的5'端第2个核苷酸和第3个核苷酸之间；和，
所述反义链的3'端第1个核苷酸和第2个核苷酸之间；和，
所述反义链的3'端第2个核苷酸和第3个核苷酸之间。
在一些实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:6所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:16、SEQ ID NO:20、SEQ ID NO:22至SEQ ID NO:33、SEQ ID NO:35任一项所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:6所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:16所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:6所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:20所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:6所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:22所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:6所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:23所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:6所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:24所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:6所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:25所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:6所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:26所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:6所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:27所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:6所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:28所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:6所 示的核苷酸序列，并且反义链包含或选自SEQ ID NO:29所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:6所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:30所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:6所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:31所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:6所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:32所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:6所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:33所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:6所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:35所示的核苷酸序列。
在一些实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:7所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:9至SEQ ID NO:20、SEQ ID NO:33至SEQ ID NO:35任一项所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:7所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:9所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:7所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:10所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:7所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:11所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:7所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:12所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:7所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:13所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:7所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:14所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:7所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:15所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:7所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:16所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:7所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:17所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:7所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:18所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:7所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:19所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:7所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:20所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:7所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:33所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:7所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:34所示的核苷酸序列。
在一些具体的实施方案中，所述dsRNA的正义链包含或选自SEQ ID NO:7所示的核苷酸序列，并且反义链包含或选自SEQ ID NO:35所示的核苷酸序列。
在一些实施方案中，所述dsRNA为：
正义链的核苷酸序列包含SEQ ID NO:6，反义链的核苷酸序列包含SEQ ID NO:16。
在一些实施方案中，所述dsRNA为：正义链的核苷酸序列是SEQ ID NO:6所示，反义链的核苷酸序列是SEQ ID NO:16所示。本公开还提供了一种dsRNA缀合物，其包含上述dsRNA中的任意一种和连接至所述dsRNA的靶向配体。
在一些实施方案中，dsRNA和所述靶向配体共价或非共价连接。
一些实施方案中，所述靶向配体靶向肝脏。一些实施方案中，所述靶向配体结合脱唾液酸糖蛋白受体(ASGPR)。一些实施方案中，所述靶向配体包括半乳糖簇或半乳糖衍生物簇，所述半乳糖衍生物选自N-乙酰基-半乳糖胺、N-三氟乙酰基半乳糖胺、N-丙酰基半乳糖胺、N-正丁酰基半乳糖胺或N-异丁酰基半乳糖胺。
在一些实施方案中，所述靶向配体连接至所述dsRNA的正义链3’末端。
在一些实施方案中，靶向配体通过磷酸二酯基团、硫代磷酸二酯基团或膦酸基团与dsRNA末端连接。一些实施方案中，通过磷酸二酯基团与dsRNA末端连接。
在一些实施方案中，靶向配体通过磷酸二酯基团、硫代磷酸二酯基团或膦酸基团与dsRNA末端间接连接。一些实施方案中，通过磷酸二酯基团与dsRNA末端间接连接。
在一些实施方案中，靶向配体通过磷酸二酯基团、硫代磷酸二酯基团或膦酸基团与dsRNA末端直接连接。一些实施方案中，通过磷酸二酯基团与dsRNA末端直接连接。
在一些实施方案中，靶向配体通过磷酸二酯基团或硫代磷酸二酯基团与dsRNA正义链末端直接连接。一些实施方案中，通过磷酸二酯基团与dsRNA正义链末端连接。
在一些实施方案中，靶向配体通过磷酸二酯基团或硫代磷酸二酯基团与dsRNA正义链3’末端直接连接。一些实施方案中，通过磷酸二酯基团与dsRNA正义链3’末端直接连接。
在一些实施方案中，为了促进dsRNA进入细胞，可以在dsRNA正义链的末端引入胆固醇等亲脂性的基团，亲脂性的基团可以以共价键与小干扰核酸结合，如 末端引入胆固醇、脂蛋白、维生素E等，以利于通过由脂质双分子层构成的细胞膜与细胞内的mRNA发生作用。同时，dsRNA也可以进行非共价键修饰，如通过疏水键或离子键结合磷脂分子、多肽、阳离子聚合物等增加稳定性和生物学活性。
一些实施方案中，靶向配体的靶向部分是由一个或多个靶向基团或靶向部分组成，靶向配体协助引导将与其连接的治疗性试剂递送至所需靶位置。在一些情况中，靶向部分可以结合细胞或细胞受体，并且启动内吞作用以促进治疗性试剂进入细胞。靶向部分可以包括对细胞受体或细胞表面分子或抗体具有亲和性的化合物。含有靶向部分的各种靶向配体可以与治疗性试剂和其它化合物连接以将试剂靶向细胞和特定细胞受体。
一些实施方案中，靶向部分的类型包括碳水化合物、胆固醇和胆甾醇基团或类固醇。可以结合细胞受体的靶向部分包括糖类，诸如半乳糖、半乳糖衍生物(如N-乙酰基-半乳糖胺，N-三氟乙酰基半乳糖胺、N-丙酰基半乳糖胺、N-正丁酰基半乳糖胺、N-异丁酰基半乳糖胺)、甘露糖和甘露糖衍生物)。
已知结合脱唾液酸糖蛋白受体(ASGPR)的靶向部分可特别用于引导递送寡聚化合物至肝脏。脱唾液酸糖蛋白受体在肝脏细胞(肝细胞)上大量表达。靶向ASCPR的细胞受体靶向部分包括半乳糖和半乳糖衍生物。具体而言，半乳糖衍生物的簇，包括由2、3、4或超过4个N-乙酰基-半乳糖胺(GalNAc或NAG)组成的簇可以促进肝细胞中某些化合物的摄取。偶联寡聚化合物的GalNAc簇用于引导组合物至肝脏，在这里N-乙酰基-半乳糖胺糖能够结合肝脏细胞表面的脱唾液酸糖蛋白受体。脱唾液酸糖蛋白受体的结合被认为将启动受体介导的内吞作用，从而促进化合物进入细胞内部。
一些实施方案中，靶向配体可以包括2、3、4或超过4个靶向部分。
在一些实施方案中，各靶向部分各自独立地包括半乳糖胺衍生物，其为N-乙酰基-半乳糖胺。可用作靶向部分且对脱唾液酸糖蛋白受体具有亲和性的其他糖可选自半乳糖、半乳糖胺、N-甲酰基-半乳糖胺、N-乙酰基-半乳糖胺、N-丙酰基-半乳糖胺、N-正丁酰基-半乳糖胺和N-异丁酰基-半乳糖胺等。
在一些实施方案中，本公开中的靶向配体包括N-乙酰半乳糖胺做为靶向部分，
在一些实施方案中，靶向配体包括三个末端半乳糖胺或半乳糖胺衍生物(诸如N-乙酰基-半乳糖胺)，其各自对唾液酸糖蛋白受体均具有亲和性。在一些实施方案中，靶向配体包括三个末端N-乙酰基-半乳糖胺(GalNAc或NAG)作为靶向部分。
在一些实施方案中，靶向配体包括四个末端半乳糖胺或半乳糖胺衍生物(诸如 N-乙酰基-半乳糖胺)，其各自对脱唾液酸糖蛋白受体均具有亲和性。在一些实施方案中，靶向配体包括四个末端N-乙酰基-半乳糖胺(GalNAc或NAG)作为靶向部分。
当述及三个末端N-乙酰基-半乳糖胺时本领域常用的术语包括三触角(tri-antennary)、三价物(tri-valent)和三聚体。
当述及四个末端N-乙酰基-半乳糖胺时本领域常用的术语包括四触角(tetra-antennary)、四价物(tetra-valent)和四聚体。
在一些实施方案中，本公开提供的靶向配体为如式(Ⅲ-1)所示化合物或其药学上可接受的盐，
在一些实施方案中，本公开提供的靶向配体为如式(Ⅲ-2)所示化合物或其药学上可接受的盐，
在一些实施方案中，可以用N-三氟乙酰基半乳糖胺、N-丙酰基半乳糖胺、N-正丁酰基半乳糖胺或N-异丁酰基半乳糖胺替换以上靶向配体中的N-乙酰基-半乳糖胺部分。
在一些实施方案中，所述dsRNA缀合物的正义链的核苷酸序列包含或选自SEQ ID NO:3，反义链的核苷酸序列包含或选自SEQ ID NO:16、SEQ ID NO:20、SEQ ID NO:22至SEQ ID NO:33、SEQ ID NO:35中任一的核苷酸序列。
在一些实施方案中，所述dsRNA缀合物的正义链的核苷酸序列包含或选自SEQ ID NO:4，反义链的核苷酸序列包含或选自SEQ ID NO:9至SEQ ID NO:20、SEQ ID NO:33至SEQ ID NO:35中任一的核苷酸序列。
在一些实施方案中，所述dsRNA缀合物的正义链的核苷酸序列包含或选自SEQ ID NO:5，反义链的核苷酸序列包含或选自SEQ ID NO:16、SEQ ID NO:20、SEQ ID NO:33中任一的核苷酸序列。
在一些实施方案中，所述dsRNA缀合物为：
正义链的核苷酸序列包含SEQ ID NO:3，反义链的核苷酸序列包含SEQ ID  NO:16。
在一些实施方案中，所述dsRNA缀合物为：
正义链的核苷酸序列是SEQ ID NO:3所示，反义链的核苷酸序列是SEQ ID NO:16所示。
在一些实施方案中，所述dsRNA缀合物是如下结构或其药学上可接受的盐：
其中，Af＝腺嘌呤2'-F核糖核苷；Cf＝胞嘧啶2'-F核糖核苷；Uf＝尿嘧啶2'-F核糖核苷；Gf＝鸟嘌呤2'-F核糖核苷；Am＝腺嘌呤2'-OMe核糖核苷；Cm＝胞嘧啶2'-OMe核糖核苷；Gm＝鸟嘌呤2'-OMe核糖核苷；Um＝尿嘧啶2'-OMe核糖核苷。
表示硫代磷酸二酯基；表示磷酸二酯基；
NAG0052’表示
在一些实施方案中，所述dsRNA缀合物是如下结构或其药学上可接受的盐：
其中，Af＝腺嘌呤2'-F核糖核苷；Cf＝胞嘧啶2'-F核糖核苷；Uf＝尿嘧啶2'-F核糖核苷；Gf＝鸟嘌呤2'-F核糖核苷；Am＝腺嘌呤2'-OMe核糖核苷；Cm＝胞嘧啶2'-OMe核糖核苷；Gm＝鸟嘌呤2'-OMe核糖核苷；Um＝尿嘧啶2'-OMe核糖核苷。
表示硫代磷酸二酯基阴离子形式；表示磷酸二酯基阴离子形式；
NAG0052’表示
在一些实施方案中，所述药学上可接受的盐可为本领域常规的盐，包括但不限于：钠盐、钾盐、铵盐、胺盐等。
在一些实施方案中，所述dsRNA缀合物选自TJR100422、TJR100423、TJR100424、TJR100425、TJR100426、TJR100427、TJR100428、TJR100429、TJR100430、TJR100431、TJR100432、TJR100800、TJR100801、TJR100802、TJR100803、TJR100804、TJR100805、TJR100806、TJR100807、TJR100808、TJR100809、TJR100810、TJR100811、TJR101079、TJR101080、TJR101081、TJR101082、TJR101083、TJR101085、TJR101086、TJR101087、TJR101088、TJR101084、TJR102134、TJR102136中的任一项。
在一些实施方案中，所述dsRNA缀合物为TJR101079，其结构为：
其中，Af＝腺嘌呤2'-F核糖核苷；Cf＝胞嘧啶2'-F核糖核苷；Uf＝尿嘧啶2'-F核糖核苷；Gf＝鸟嘌呤2'-F核糖核苷；Am＝腺嘌呤2'-OMe核糖核苷；Cm＝胞嘧啶2'-OMe核糖核苷；Gm＝鸟嘌呤2'-OMe核糖核苷；Um＝尿嘧啶2'-OMe核糖核苷。
表示硫代磷酸二酯基阴离子形式；表示磷酸二酯基阴离子形式；
NAG0052’表示
本公开另一方面提供一种组合物，其包含权利要求上述dsRNA和/或dsRNA缀合物，和一种或多种药学上可接受的赋形剂，例如载剂、运载体、稀释剂、和/或递送聚合物。
本公开中，各种递药系统是已知的并且可以用于本公开dsRNA和/或dsRNA 缀合物，例如封装在脂质体中、微粒、微囊、能够表达该化合物的重组细胞、受体介导的细胞内吞作用、构建核酸作为逆转录病毒或其他载体的一部分。
本公开另一方面提供一种上述dsRNA和/或dsRNA缀合物或其组合物在制备治疗受试者疾病的药物中的用途，在一些实施方案中选自肝源性疾病。
本公开另一方面提供一种治疗受试者疾病的法，包括向受试者给予上述dsRNA和/或dsRNA缀合物，或组合物。
本公开另一方面提供一种抑制受试者体内LPA的mRNA表达的方法，该方法包括给予受试者上述dsRNA和/或dsRNA缀合物，或组合物。
本公开另一方面提供一种体内递送表达抑制性寡聚化合物至肝脏的方法，给与受试者上述缀合物和/或组合物。
本文所公开的dsRNA、dsRNA缀合物、组合物和方法可以在细胞、细胞群、组织或受试者中降低靶mRNA的水平，包括：向受试者给予治疗有效量的本文所述的dsRNA，所述dsRNA与靶向配体连接，从而抑制靶mRNA在受试者中的表达。
在一些实施方案中，所述受试者已在先前被鉴定为在靶向的细胞或组织中具有靶基因的异常表达。
本公开中所述的受试者是指患有将会受益于靶mRNA表达之减少或抑制的疾病或病症的受试者。
递送可以是通过局部给药(例如，直接注射、植入、或局部给予)，全身给药，或皮下，静脉内，腹膜内，或胃肠外途径，包括颅内(例如，心室内，实质内和鞘内)，肌肉内，透皮，气道(气溶胶)，鼻，口服，直肠，或局部(包括口颊和舌下)给药。
可选的实施方案中，本公开提供的药物组合物可以通过注射给予，例如，静脉内、肌内、皮内、皮下、十二指肠内或腹膜内注射。
可选的实施方案中，当靶向配体与dsRNA连接成缀合物后，所述缀合物可被包装在试剂盒中。
另一方面，本公开提供一种药物组合物，包含本公开所述的dsRNA和/或dsRNA缀合物。
在一些实施方案中，所述药物组合物中还可以包含药学上可接受的辅料和/或佐剂，该辅料可以为一种或多种本领域常规采用的各种制剂或化合物。例如，所述药学上可接受的辅料可以包括pH缓冲剂、保护剂和渗透压调节剂中的至少一种。
在一些实施方案中，本公开所述dsRNA、dsRNA缀合物或其药物组合物当接触到表达靶基因的细胞时，由例如：psiCHECK活性筛选和荧光素酶报告基因检测法，其他如PCR或基于分支DNA(bDNA)的方法、或基于蛋白质的方法，如免疫荧光分析法，例如Western Blot或流式细胞术测定的，上述dsRNA、dsRNA缀合物或药物组合物会抑制靶基因的表达至少5％、至少10％、至少15％、至少20％、 至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％。
在一些实施方案中，本公开所述dsRNA、dsRNA缀合物或其药物组合物当接触到表达靶基因的细胞时，由例如：psiCHECK活性筛选和荧光素酶报告基因检测法，其他如PCR或基于分支DNA(bDNA)的方法、或基于蛋白质的方法，如免疫荧光分析法，例如Western Blot或流式细胞术测定的，上述dsRNA、dsRNA缀合物或药物组合物引起的靶基因mRNA剩余表达百分比为不高于99％、不高于95％、不高于90％、不高于85％、不高于80％、不高于75％、不高于70％、不高于65％、不高于60％、不高于55％、不高于50％、不高于45％、不高于40％、不高于35％、不高于30％、不高于25％、不高于20％、不高于15％、或不高于10％。
在一些实施方案中，本公开所述dsRNA、dsRNA缀合物或其药物组合物当接触到表达靶基因的细胞时，由例如：psiCHECK活性筛选和荧光素酶报告基因检测法，其他如PCR或基于分支DNA(bDNA)的方法、或基于蛋白质的方法，如免疫荧光分析法，例如Western Blot、或流式细胞术测定的，dsRNA、dsRNA缀合物或其药物组合物在保持在靶活性的同时，将脱靶活性减少了至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％或至少75％。
在一些实施方案中，本公开所述dsRNA、dsRNA缀合物或其药物组合物当接触到表达靶基因的细胞时，由例如：psiCHECK活性筛选和荧光素酶报告基因检测法，其他如PCR或基于分支DNA(bDNA)的方法、或基于蛋白质的方法，如免疫荧光分析法，例如Western Blot、或流式细胞术测定的，dsRNA、dsRNA缀合物或其药物组合物使在靶活性降低至多20％、至多19％、至多15％、至多10％、至多5％或超过1％的同时，将脱靶活性减少了至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％或至少75％。
在一些实施方案中，本公开所述dsRNA和/或dsRNA缀合物或其药物组合物当接触到表达靶基因的细胞时，由例如：psiCHECK活性筛选和荧光素酶报告基因检测法，其他如PCR或基于分支DNA(bDNA)的方法、或基于蛋白质的方法，如免疫荧光分析法，例如Western Blot、或流式细胞术测定的，dsRNA、dsRNA缀合物或其药物组合物使在靶活性提高至少1％、至少5％、至少10％、至少15％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％或至少80％的同时，将脱靶活性减少了至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％或至少75％。
在又一些具体实施方案中，本公开所述dsRNA和/或dsRNA缀合物或其药物组合物当接触到表达靶基因的细胞时，IC50不高于1nM、不高于0.9nM、不高于0.8nM、不高于0.7nM、不高于0.6nM、不高于0.5nM、不高于0.4nM、不高于0.3nM、不高于0.2nM、不高于0.19nM、不高于0.18nM、不高于0.17nM、不高于0.16nM、不高于0.15nM、不高于0.14nM、不高于0.13nM、不高于0.12nM、不高于0.11nM、不高于0.10nM、不高于0.09nM、不高于0.08nM、不高于0.07nM、不高于0.06nM、不高于0.05nM、不高于0.04nM、不高于0.03nM、不高于0.02nM、不高于0.01nM、或任意两个数值间的范围。
本公开还提供了一种细胞，其包含本公开的dsRNA和/或dsRNA缀合物。
本公开还提供了一种药盒或试剂盒，其包含一个或多个容器，所述容器独立地包含本公开的dsRNA和/或dsRNA缀合物、或其药物组合物。
本公开还提供了一种用于沉默细胞中靶基因或靶基因的mRNA的方法，该方法包括将根据本公开的dsRNA和/或dsRNA缀合物和/或药物组合物引入该细胞中的步骤。
本公开还提供了一种用于在体内或在体外沉默细胞中靶基因或靶基因的mRNA的方法，该方法包括将根据本公开的dsRNA和/或dsRNA缀合物和/或药物组合物引入该细胞中的步骤。
本公开还提供了一种用于抑制靶基因或靶基因的mRNA表达的方法，该方法包括向有其需要的受试者施用有效量或有效剂量的根据本公开的dsRNA和/或dsRNA缀合物和/或药物组合物。
在一些实施方案中，施用通过包括肌肉内、支气管内、胸膜内、腹膜内、动脉内、淋巴、静脉内、皮下、脑脊髓、或其组合的给药方式进行。
在一些实施方案中，dsRNA和/或dsRNA缀合物和/或药物组合物的有效量或有效剂量为约0.001mg/kg体重至约200mg/kg体重、约0.01mg/kg体重至约100mg/kg体重或约0.5mg/kg体重至约50mg/kg体重。
在一些实施方案中，靶基因是LPA。
另一方面，本公开提供一种所述的dsRNA和/或dsRNA缀合物或含有所述dsRNA和/或dsRNA缀合物的药物组合物在制备药物中的应用。
另一方面，本公开提供了前述dsRNA和/或药物组合物和/或dsRNA缀合物，用于治疗和/或预防受试者与脂蛋白(a)和/或载脂蛋白(a)水平升高相关的疾病。在一些实施方案中，所述的与脂蛋白(a)和/或载脂蛋白(a)水平升高相关的疾病选自心血管疾病。在一些实施方案中，所述心血管疾病选自缺血性中风、动脉粥样硬化、血栓形成、冠心病、下肢动脉病变或主动脉瓣狭窄、心肌梗塞、冠状动脉狭窄、颈动脉狭窄、股动脉狭窄、心脏衰竭。
另一方面，本公开提供了前述dsRNA和/或药物组合物和/或dsRNA缀合物，用于治疗和/或预防疾病，所述疾病选自心血管疾病。在一些实施方案中，所述心 血管疾病选自缺血性中风、动脉粥样硬化、血栓形成、冠心病、下肢动脉病变或主动脉瓣狭窄、心肌梗塞、冠状动脉狭窄、颈动脉狭窄、股动脉狭窄、心脏衰竭。
本公开提供了前述dsRNA和/或药物组合物和/或dsRNA缀合物，用于降低脂蛋白(a)和/或载脂蛋白(a)水平。
本公开提供了前述dsRNA和/或药物组合物和/或dsRNA缀合物在制备用于抑制LPA的表达的药物中的用途。
本公开提供了前述dsRNA和/或药物组合物和/或dsRNA缀合物在制备用于治疗和/或预防受试者与脂蛋白(a)和/或载脂蛋白(a)水平升高相关的疾病的药物中的用途。在一些实施方案中，所述与脂蛋白(a)和/或载脂蛋白(a)水平升高相关的疾病选自心血管疾病。在一些实施方案中，所述心血管疾病选自缺血性中风、动脉粥样硬化、血栓形成、冠心病、下肢动脉病变或主动脉瓣狭窄、心肌梗塞、冠状动脉狭窄、颈动脉狭窄、股动脉狭窄、心脏衰竭。
本公开提供了前述dsRNA和/或药物组合物和/或dsRNA缀合物在制备用于治疗和/或预防疾病的药物中的用途，所述疾病选自心血管疾病。在一些实施方案中，述心血管疾病选自缺血性中风、动脉粥样硬化、血栓形成、冠心病、下肢动脉病变或主动脉瓣狭窄、心肌梗塞、冠状动脉狭窄、颈动脉狭窄、股动脉狭窄、心脏衰竭。
本公开提供了前述dsRNA和/或药物组合物和/或dsRNA缀合物在制备用于降低脂蛋白(a)和/或载脂蛋白(a)水平的药物中的用途。
本公开提供了一种抑制LPA表达的方法，包括给予受试者有效量或有效剂量的前述dsRNA和/或药物组合物和/或dsRNA缀合物。
本公开提供了一种治疗和/或预防受试者与脂蛋白(a)和/或载脂蛋白(a)水平升高相关的疾病的方法，包括给予受试者有效量或有效剂量的前述dsRNA和/或药物组合物和/或dsRNA缀合物。在一些实施方案中，所述与脂蛋白(a)和/或载脂蛋白(a)水平升高相关的疾病选自心血管疾病；在一些实施方案中，所述心血管疾病选自缺血性中风、动脉粥样硬化、血栓形成、冠心病、下肢动脉病变或主动脉瓣狭窄、心肌梗塞、冠状动脉狭窄、颈动脉狭窄、股动脉狭窄、心脏衰竭。
本公开提供了一种治疗和/或预防疾病的方法，包括给予受试者有效量或有效剂量的前述dsRNA和/或药物组合物和/或dsRNA缀合物，所述疾病选自心血管疾病。在一些实施方案中，所述心血管疾病选自缺血性中风、动脉粥样硬化、血栓形成、冠心病、下肢动脉病变或主动脉瓣狭窄、心肌梗塞、冠状动脉狭窄、颈动脉狭窄、股动脉狭窄、心脏衰竭。
本公开提供了一种用于降低脂蛋白(a)和/或载脂蛋白(a)水平的方法，包括给予受试者有效量或有效剂量前述dsRNA和/或药物组合物和/或dsRNA缀合物。
本公开提供了一种体内递送抑制LPA表达和/或复制的dsRNA至肝脏的方法，给与受试者前述dsRNA和/或药物组合物和/或dsRNA缀合物。
另一方面，本公开还提供了一种制备本公开所述dsRNA和/或dsRNA缀合物或药物组合物的方法，其包括：合成本公开所述的dsRNA和/或dsRNA缀合物或药物组合物。
本公开还提供了一种dsRNA或dsRNA缀合物，其特征在于以碱基T，替换本公开任一dsRNA或dsRNA缀合物的一个或多个碱基U，例如1个、2个、3个、3个、5个、6个、7个、8个、9个、10个。在一些实施方案中，本公开中的全部碱基U均可被碱基T替换。
本公开中所述化合物可药用盐选自无机盐或有机盐，本公开所述化合物可与酸性或碱性物质反应成相应盐。
本公开化合物可以存在特定的几何或立体异构体形式。本公开设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本公开的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本公开的范围之内。本公开的含有不对称碳原子的化合物可以以光学活性纯的形式或外消旋形式被分离出来。光学活性纯的形式可以从外消旋混合物拆分，或通过使用手性原料或手性试剂合成。
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本公开某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。
本公开所述化合物的化学结构中，键表示未指定构型，即如果化学结构中存在手性异构体，键可以为或或者同时包含和两种构型。本公开所述化合物的化学结构中，键并未指定构型，即键的构型可以为E型或Z型，或者同时包含E和Z两种构型。
在本公开的化学结构式中，可以根据本文所述发明范围连接一个或多个任何基团。
在不指明构型的情况下，本公开的化合物和中间体还可以以不同的互变异构体形式存在，并且所有这样的形式包含于本公开的范围内。术语“互变异构体”或“互变异构体形式”是指可经由低能垒互变的不同能量的结构异构体。
本公开还包括一些与本文中记载的那些相同的，但一个或多个原子被原子量 或质量数不同于自然中通常发现的原子量或质量数的原子置换的同位素标记的本公开化合物。可结合到本公开化合物的同位素的实例包括氢、碳、氮、氧、磷、硫、氟、碘和氯的同位素，诸如分别为2H、3H、11C、13C、14C、13N、15N、15O、17O、18O、31P、32P、35S、18F、123I、125I和36Cl等。
相比于未氘代药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本公开的化合物的所有同位素组成的变换，无论放射性与否，都包括在本公开的范围之内。与碳原子连接的各个可用的氢原子可独立地被氘原子替换，其中氘的替换可以是部分或完全的，部分氘的替换是指至少一个氢被至少一个氘替换。
除另有说明，本公开的化合物，当其一个位置被特别地指定为“氘”或“D”时，该位置应理解为氘的丰度比氘的天然丰度(其为0.015％)大至少1000倍(即至少15％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少1000倍(即至少15％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少2000倍(即至少30％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少3000倍(即至少45％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少3340倍(即至少50.1％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少3500倍(即至少52.5％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少4000倍(即至少60％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少4500倍(即至少67.5％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少5000倍(即至少75％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少5500倍(即至少82.5％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少6000倍(即至少90％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少6333.3倍(即至少95％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少6466.7倍(即至少97％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少6600倍(即至少99％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少6633.3倍(即至少99.5％的氘掺入)。本领域技术人员能够参考相关文献合成氘化形式的化合物。在制备氘代形式的化合物时可使用市售的氘代起始物质，或它们可使用常规技术采用氘代试剂合成，氘代试剂包括但不限于氘代硼烷、三氘代硼烷四氢呋喃溶液、氘代氢化锂铝、氘代碘乙烷和氘代碘甲烷等。
术语“约”、“大约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，“约” 可意味着在1内或超过1的标准差。或者，“约”或“基本上包含”可意味着至多20％的范围，例如1％至15％之间、在1％至10％之间、在1％至5％之间、在0.5％至5％之间、在0.5％至1％之间变化，本公开中，数字或数值范围之前有术语“约”的每种情况也包括给定数的实施方案。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“基本上包含”的含义应该假定为在该具体值的可接受误差范围内。
本公开引入WO2022028462A、WO2023274395A、WO2023208023A、WO2023109940A全文。
术语解释
为了更容易理解本公开，以下具体定义了一些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
如无特别说明，在本公开上下文中，术语“载脂蛋白(a)基因”、Apo(a)基因、LPA、Lp(a)在本公开中可互换使用。LPA包括但不限于人类LPA、食蟹猴LPA、小鼠LPA、大鼠LPA，其氨基酸和完整编码序列、mRNA序列容易利用已公开的数据库取得，例如，GenBank、UniProt、OMIM和猕猴(Macaca)基因组计划网站。
术语“靶序列”指在LPA转录期间所形成的mRNA分子的核苷酸序列的连续部分，包括作为主要转录产物的RNA加工产物的mRNA。所述靶序列中被靶向的部分应当足够长，才能作为iRNA指导的切割(iRNA-directed cleavage)的底物。在一个实施方案中，该靶序列在LPA的蛋白质编码区内。
如本文所使用的，dsRNA的有义链(又称SS、SS链或正义链)是指包含与靶mRNA序列相同或基本上相同的序列的链；dsRNA的反义链(又称AS或AS链)是指具有与靶mRNA序列至少部分互补的序列的链。
在描述本文所述的dsRNA正义链的上下文中，术语“SEQ ID NO:1的核苷酸序列相差不超过3个核苷酸序列的至少17个连续核苷酸”旨在表示本文所述的dsRNA正义链包含如与SEQ ID NO:1所示的正义链的至少17个连续核苷酸，或与SEQ ID NO:1所示的正义链的至少17个连续核苷酸相差不超过3个核苷酸的序列，任选地，相差不超过2个核苷酸序列，任选地，相差1个核苷酸序列。任选地，本文所述的dsRNA正义链包含SEQ ID NO:1所示正义链的至少18个连续核苷酸，或与SEQ ID NO:1所示的正义链的至少18个连续核苷酸相差不超过3个核苷酸的序列，任选地，相差不超过2个核苷酸序列，任选地，相差1个核苷酸的序列。
在描述本文所述的dsRNA反义链的上下文中，术语“与SEQ ID NO:2所示的反义链相差不超过3个核苷酸序列的至少19个连续核苷酸”旨在表示本文所述的SEQ ID NO:2所示的反义链的至少19个连续核苷酸，或与SEQ ID NO:2所示的反义链的至少19个连续核苷酸相差不超过3个核苷酸的序列，任选地，相差不超过2个核苷酸序列，任选地，相差1个核苷酸序列。
本公开中，正义链或反义链的“5’区域”也即“5’端”、“5’末端”，可替换使用。例如反义链5’区域的第2位至第8位的核苷酸，也可替换为反义链5’端的第2位至第8位的核苷酸。同理，正义链或反义链的“3’区域”、“3’末端”和“3’端”也可替换使用。
如无特别说明，在本公开上下文中，“G”、“C”、“A”、“T”与“U”分别代表核苷酸，其分别包含鸟嘌呤、胞嘧啶、腺嘌呤、胸苷与尿嘧啶的碱基。小写字母m表示该字母m上游相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f上游相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示与该字母s两侧相邻的两个核苷酸之间为硫代磷酸二酯基连接。
如本公开所使用的，术语“2'-氟代(2’-F)修饰的核苷酸”指核苷酸的核糖基2'位的羟基被氟取代形成的核苷酸，“非氟代修饰的核苷酸”指核苷酸的核糖基2'位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物。
如本公开所使用的，术语“2'-甲氧基(2’-OMe)修饰的核苷酸”指核糖基的2'-羟基被甲氧基取代而形成的核苷酸。
在本公开的上下文中，一个核苷酸序列与另外一个核苷酸序列存在“核苷酸差异”，是指前者与后者相比，相同位置的核苷酸的碱基种类发生了改变，例如，在后者中一个核苷酸碱基为A时，在前者的相同位置处的对应核苷酸碱基为U、C、G或者T的情况下，认定为两个核苷酸序列之间在该位置处存在核苷酸差异。在一些实施方案中，以无碱基核苷酸或其等同物代替原位置的核苷酸时，也可认为在该位置处产生了核苷酸差异。
如本文所使用的，术语“互补”或“反向互补”一词可互相替代使用，并具有本领域技术人员周知的含义，即，在双链核酸分子中，一条链的碱基与另一条链上的碱基以互补的方式相配对。在DNA中，嘌呤碱基腺嘌呤始终与嘧啶碱基胸腺嘧啶(或者在RNA中为尿嘧啶)相配对；嘌呤碱基鸟嘌呤始终与嘧啶碱基胞嘧啶相配对。每个碱基对都包括一个嘌呤和一个嘧啶。当一条链上的腺嘌呤始终与另一条链上的胸腺嘧啶(或尿嘧啶)配对，以及鸟嘌呤始终与胞嘧啶配对时，两条链被认为是彼此相互补的，以及从其互补链的序列中可以推断出该链的序列。与此相应地，“错配”在本领域中意指在双链核酸中，对应位置上的碱基并未以互补的形式配对存在。
术语“dsRNA”是指能够进行RNA干扰的双链RNA分子，包含正义链和反义链。
术语“化学修饰”或“修饰”包括核苷酸经化学手段的所有改变，例如化学部分的添加或去除、或以一个化学部分取代另一个化学部分。
术语“碱基”包含任何已知的DNA和RNA碱基、碱基类似物，例如嘌呤或嘧啶，其还包括天然化合物腺嘌呤、胸腺嘧啶、鸟嘌呤、胞嘧啶、尿嘧啶、次黄苷和天然类似物。
术语“任选地”或“任选”是指意味着随后所描述的事件或环境可以但不必发生， 该说明包括该事件或环境发生或不发生的场合。例如“任选的被卤素或者氰基取代的C1-6烷基”是指卤素或者氰基可以但不必须存在，该说明包括烷基被卤素或者氰基取代的情形和烷基不被卤素和氰基取代的情形。
术语“烷基”指饱和脂肪族烃基团，其为包含1至20个碳原子的直链或支链基团，在一些实施方案中选自含有1至12个碳原子的烷基。非限制性实例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基、正壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2,2-二乙基戊基、正癸基、3,3-二乙基己基、2,2-二乙基己基，及其各种支链异构体等。在一些实施方案中选自的是含有1至6个碳原子的烷基，非限制性实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基等。烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基在一些实施方案中选自一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基、羧基或羧酸酯基。
术语“烷氧基”指-O-(烷基)，其中烷基的定义如上所述。烷氧基的非限制性实例包括：甲氧基、乙氧基、丙氧基、丁氧基。烷氧基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自卤素、羟基、氧代、氰基、氨基、C1-6烷基、C1-6烷氧基、3至7元环烷基或3至7元杂环烷基，所述烷基、烷氧基、环烷基或杂环烷基任选被卤素、羟基、硝基、氰基或氨基所取代。
术语“烷硫基”指-S-(烷基)，其中烷基的定义如上所述。烷硫基的非限制性实例包括：甲硫基、乙硫基、丙硫基、丁硫基。烷硫基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自C1-6烷氧基、3至6元环烷基、3至6元杂环烷基、3至6元环烷氧基、3至6元杂环烷氧基、C1-6烷硫基、3至6元环烷硫基、3至6元杂环烷硫基，所述烷氧基、环烷基、杂环烷基、 环烷氧基、杂环氧基、烷硫基、环烷硫基、杂环烷硫基任选被卤素、羟基、氰基或氨基所取代。
术语“烯基”指直链或支链的非芳香族烃基，其含有至少一个碳-碳双键，并且具有2-10个碳原子。在这样的基团中可以存在多达5个碳-碳双键。例如，“C2-C6”烯基被定义为具有2-6个碳原子的烯基。烯基的示例包括但不限于：乙烯基、丙烯基、丁烯基和环己烯基。烯基的直链、支链或环状部分可以含有双键，并且在正常化合价所允许的任何位置任选地被单-、二-、三-、四-或五-取代。
术语“炔基”指直链或支链的烃基，其含有2-10个碳原子并且含有至少一个碳-碳三键。可以存在多达5个碳-碳三键。因此，“C2-C6炔基”表示具有2-6个碳原子的炔基。炔基的示例包括但不限于：乙炔基、2-丙炔基和2-丁炔基。炔基的直链、支链部分可以含有正常化合价所允许的三键，并且在正常化合价所允许的任何位置任选地被单-、二-、三-、四-或五-取代。
术语“环烷基”或“碳环”指饱和或部分不饱和单环或多环环状烃取代基，环烷基环包含3至20个碳原子，在一些实施方案中选自包含3至7个碳原子。单环环烷基的非限制性实例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基等；多环环烷基包括螺环、稠环和桥环的环烷基。环烷基可以是取代的或未取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，在一些实施方案中选自一个或多个以下基团，独立地选自卤素、氘、羟基、氧代、硝基、氰基、C1-6烷基、C1-6烷氧基、C2-6烯氧基、C2-6炔氧基、C3-6环烷氧基、3至6元杂环烷氧基、C3-8环烯氧基、5至6元芳基或杂芳基，所述C1-6烷基、C1-6烷氧基、C2-6烯氧基、C2-6炔氧基、C3-6环烷氧基、3至6元杂环烷氧基、C3-8环烯氧基、5至6元芳基或杂芳基任选被一个或多个选自卤素、氘、羟基、氧代、硝基、氰基所取代。
所述环烷基环可以稠合于芳基或杂芳基环上，其中与母体结构连接在一起的环为环烷基，非限制性实例包括茚满基、四氢萘基、苯并环庚烷基等。环烷基可以是任选取代的或非取代的，当被取代时，取代基在一些实施方案中选自一个或多个以下基团，其独立地选自卤素、氘、羟基、氧代、硝基、氰基、C1-6烷基、C1-6烷氧基、C2-6烯氧基、C2-6炔氧基、C3-6环烷氧基、3至6元杂环烷氧基、C3-8环烯氧基、5至6元芳基或杂芳基，所述C1-6烷基、C1-6烷氧基、C2-6烯氧基、C2-6炔氧基、C3-6环烷氧基、3至6元杂环烷氧基、C3-8环烯氧基、5至6元芳基或杂芳基任选被一个或多个选自卤素、氘、羟基、氧代、硝基、氰基所取代。
术语“杂环烷基”或“杂环”或“杂环基”指饱和或部分不饱和单环或多环环状烃取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧或S(O)m(其中m是整数0至2)的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。在一些实施方案中选自包含3至12个环原子，其中1～4个是杂原子；在一些实施方案中选自包含3至7个环原子。单环杂环烷基的非限制性实例包括 吡咯烷基、咪唑烷基、四氢呋喃基、四氢噻吩基、二氢咪唑基、二氢呋喃基、二氢吡唑基、二氢吡咯基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基等。多环杂环烷基包括螺环、稠环和桥环的杂环烷基。“杂环烷基”非限制性实例包括：
等。
所述杂环烷基环可以稠合于芳基或杂芳基环上，其中与母体结构连接在一起的环为杂环烷基，其非限制性实例包括：
等。
杂环烷基可以是任选取代的或非取代的，当被取代时，取代基在一些实施方案中选自一个或多个以下基团，其独立地选自卤素、氘、羟基、氧代、硝基、氰基、C1-6烷基、C1-6烷氧基、C2-6烯氧基、C2-6炔氧基、C3-6环烷氧基、3至6元杂环烷氧基、C3-8环烯氧基、5至6元芳基或杂芳基，所述C1-6烷基、C1-6烷氧基、C2-6烯氧基、C2-6炔氧基、C3-6环烷氧基、3至6元杂环烷氧基、C3-8环烯氧基、5至6元芳基或杂芳基任选被一个或多个选自卤素、氘、羟基、氧代、硝基、氰基所取代。
术语“芳基”指具有共轭的π电子体系的6至14元全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，在一些实施方案中选自6至12元，例如苯基和萘基。所述芳基环可以稠合于杂芳基、杂环烷基或环烷基环上，其中与母体结构连接在一起的环为芳基环，其非限制性实例包括：
芳基可以是取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、羧基或羧酸酯基，优选苯基。
术语“稠环芳基”可以是含有8-14个环原子由两个或两个以上环状结构彼此共用两个相邻的原子连接起来形成的不饱和的具有芳香性的稠环结构，环原子优选8-12个。例如包括全部不饱和稠环芳基，例如萘、菲等，还包括部分饱和稠环芳基，例如苯并3-8元饱和单环环烷基、苯并3-8元部分饱和单环环烷基。“稠芳环”指的是稠环芳基中的环系。稠环芳基具体实例如2,3-二氢-1H-茚基、IH-茚基、1,2,3,4-四氢萘基、1,4-二氢萘基等。
术语“杂芳基”指包含1至4个杂原子、5至14个环原子的杂芳族体系，其中杂原子选自氧、硫和氮。杂芳基优选为5至12元，例如咪唑基、呋喃基、噻吩基、噻唑基、吡唑基、噁唑基、吡咯基、四唑基、吡啶基、嘧啶基、噻二唑、吡嗪基等，优选为咪唑基、吡唑基、嘧啶基或噻唑基；更优选为吡唑基或噻唑基。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环。“杂芳环”指的是杂芳基中的环系。杂芳基非限制性实例包括：
杂芳基可以是任选取代的或非取代的，当被取代时，取代基在一些实施方案中选自一个或多个以下基团，其独立地选自卤素、氘、羟基、氧代、硝基、氰基、C1- 6烷基、C1-6烷氧基、C2-6烯氧基、C2-6炔氧基、C3-6环烷氧基、3至6元杂环烷氧基、C3-8环烯氧基、5至6元芳基或杂芳基，所述C1-6烷基、C1-6烷氧基、C2-6烯氧基、C2-6炔氧基、C3-6环烷氧基、3至6元杂环烷氧基、C3-8环烯氧基、5至6元芳基或杂芳基任选被一个或多个选自卤素、氘、羟基、氧代、硝基、氰基的基团所取代。
术语“烷氨基”指具有结构-NH(C1-C12烷基)的基团。
术语“羟烷基”指烷基被一个或多个羟基取代，其中烷基如上所定义。
术语“羟基”指-OH基团。
术语“卤素”指氟、氯、溴或碘。
术语“卤代烷基”指被卤素取代的烷基，其中烷基如上所定义。
术语“卤代烷氧基”指被卤素取代的烷氧基，其中烷氧基如上所定义。
术语“氰基”指-CN。
术语“硝基”指-NO2。
术语“氧代”指＝O基团，例如，碳原子与氧原子通过双键连接，其中形成酮或醛基。
术语“氨基”指-NH2。
术语“羧基”指-C(O)OH。
术语“平端”或“平末端”可互换使用，是指在dsRNA的给定的末端没有非配对的核苷酸或核苷酸类似物，即，没有核苷酸突出。大多数情况下，两个末端都是平末端的dsRNA将在其整个长度范围内是双链的。
本公开上下文中，各修饰基团中的磷酸二酯基或具有修饰的磷酸二酯基团可以被替换为任何能够实现将其与相邻核苷酸连接的基团，例如化学修饰中的部分可以替换为能够与相邻核苷酸实现连接的任意基团。
术语“连接”，当表示两个分子之间的联系时，指两个分子通过共价键连接或者两个分子经由非共价键(例如，氢键或离子键)关联，包括直接连接、间接连接。
术语“直接连接”指第一化合物或基团与第二化合物或基团在没有任何间插原子或原子基团的情况下连接。
术语“间接连接”指第一化合物或基团与第二化合物或基团通过中间基团、化合物或分子(例如，连接基团)连接。
术语“取代的”表示指定原子(通常是碳、氧和氮原子)上的任何一个或多个氢原子被本文所限定的任何基团所替代，条件是不超过所述指定原子的正常化合价并且取代生成稳定化合物。取代基的非限制性示例包括C1-C6烷基、C2-C6烯基、C2-C6炔基、氰基、羟基、氧代基、羧基、环烷基、环烯基、杂环基、杂芳基、芳基、酮、烷氧基羰基、芳氧基羰基、杂芳氧基羰基或卤素(例如，F、Cl、Br、I)。当取代基是酮或氧代(即，＝O)时，则原子上有两个(2个)氢被替代。
“被一个或多个……取代”是指可以被单个或多个取代基取代。当被多个取代基取代时，可以是复数个相同取代基，也可以是一个或复数个不同取代基的组合。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥 生物活性。
“药学上可接受的赋形剂”包括但不限于任何已经被批准对于人类或家畜动物使用可接受的任何助剂、载体、助流剂、甜味剂、稀释剂、防腐剂、染料/着色剂、增香剂、表面活性剂、润湿剂、分散剂、助悬剂、稳定剂、等渗剂、溶剂或乳化剂。
如本文所使用的，术语“抑制”，可以与“减少”、“沉默”、“下调”、“阻抑”和其他类似术语交替使用，并且包括任何水平的抑制。抑制可通过这些变量中的一个或多个与对照水平相比的绝对或相对水平的减少来评估。该对照水平可以是本领域中使用的任何类型的对照水平，例如给药前基线水平或从类似的未经处理或经对照(例如仅缓冲液对照或惰性剂对照)处理的受试者、细胞、或样品确定的水平。例如，可以采用mRNA剩余表达量来表征dsRNA对靶基因表达的抑制程度，如mRNA剩余表达量为不高于99％、不高于95％、不高于90％、不高于85％、不高于80％、不高于75％、不高于70％、不高于65％、不高于60％、不高于55％、不高于50％、不高于45％、不高于40％、不高于35％、不高于30％、不高于25％、不高于20％、不高于15％、或不高于10％。靶基因表达的抑制率可以采用Luciferase Assay System检测，分别读取萤火虫(Firefly)化学发光值和海肾(Renilla)化学发光值，计算相对值Ratio＝Ren/Fir，抑制率(％)＝1-(Ratio+siRNA/Ratioreporter only)*100％。本公开中，剩余mRNA表达量比例(或剩余活性％)＝100％-抑制率(％)。
如无特殊说明，本公开的“化合物”、“配体”、“核酸配体缀合物”、“dsRNA缀合物”、“核酸”、“缀合物”、化学修饰”、“靶向配体”、“dsRNA”、“RNAi”均可独立地以盐、混合盐或非盐(例如游离酸或游离碱)的形式存在。当以盐或混合盐的形式存在时，其可为药学上可接受的盐。
术语“药学上可接受的盐”包括药学上可接受的酸加成盐和药学上可接受的碱加成盐。
“药学上可接受的酸加成盐”是指能够保留游离碱的生物有效性而无其它副作用的，与无机酸或有机酸所形成的盐。无机酸盐包括但不限于盐酸盐、氢溴酸盐、硫酸盐、硝酸盐、磷酸盐等；有机酸盐包括但不限于甲酸盐、乙酸盐、2,2-二氯乙酸盐、三氟乙酸盐、丙酸盐、己酸盐、辛酸盐、癸酸盐、十一碳烯酸盐、乙醇酸盐、葡糖酸盐、乳酸盐、癸二酸盐、己二酸盐、戊二酸盐、丙二酸盐、草酸盐、马来酸盐、琥珀酸盐、富马酸盐、酒石酸盐、柠檬酸盐、棕榈酸盐、硬脂酸盐、油酸盐、肉桂酸盐、月桂酸盐、苹果酸盐、谷氨酸盐、焦谷氨酸盐、天冬氨酸盐、苯甲酸盐、甲磺酸盐、苯磺酸盐、对甲苯磺酸盐、海藻酸盐、抗坏血酸盐、水杨酸盐、4-氨基水杨酸盐、萘二磺酸盐等。这些盐可通过本领域已知的方法制备。
“药学上可接受的碱加成盐”是指能够保持游离酸的生物有效性而无其它副作用的、与无机碱或有机碱所形成的盐。衍生自无机碱的盐包括但不限于钠盐、钾盐、锂盐、铵盐、钙盐、镁盐、铁盐、锌盐、铜盐、锰盐、铝盐等。优选的无机盐为铵 盐、钠盐、钾盐、钙盐及镁盐，优选钠盐。衍生自有机碱的盐包括但不限于以下的盐：伯胺类、仲胺类及叔胺类，被取代的胺类，包括天然的被取代胺类、环状胺类及碱性离子交换树脂，例如氨、异丙胺、三甲胺、二乙胺、三乙胺、三丙胺、乙醇胺、二乙醇胺、三乙醇胺、二甲基乙醇胺、2-二甲氨基乙醇、2-二乙氨基乙醇、二环己胺、赖氨酸、精氨酸、组氨酸、咖啡因、普鲁卡因、胆碱、甜菜碱、乙二胺、葡萄糖胺、甲基葡萄糖胺、可可碱、嘌呤、哌嗪、哌啶、N-乙基哌啶、聚胺树脂等。优选的有机碱包括异丙胺、二乙胺、乙醇胺、三甲胺、二环己基胺、胆碱及咖啡因。这些盐可通过本领域已知的方法制备。
“有效量”或“有效剂量”包含足以改善或预防医学病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：如待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
如本文所使用的，“对象”、“患者”、“受试者”或“个体”可互换使用，包括人类或者非人类动物，例如哺乳动物，例如人或猴。
本公开中的序数词“第一”、“第二”等不意图限制顺序或等级，仅用于区别开不同的特征、分子、步骤、组成、要素等。
本公开提供的dsRNA可以通过本领域常规的制备方法(例如固相合成和液相合成的方法)得到。其中，固相合成已经有商业化订制服务。可以通过使用具有相应修饰的核苷单体来将修饰的核苷酸基团引入本公开所述的dsRNA中，制备具有相应修饰的核苷单体的方法及将修饰的核苷酸基团引入dsRNA的方法也是本领域技术人员所熟知的。
本公开中的一些缩略语定义如下：
DMF：二甲基甲酰胺；
DIPEA：N-乙基二异丙基胺；
HBTU：苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐；
DMAP：4-二甲氨基吡啶；
NMI：N-甲基咪唑。
附图说明
图1A为TJR101079、TRD007790在给药后第28天血清Lp(a)浓度相对于给药前的比值。
图1B为TJR101079、TJR102134在给药后第28天血清Lp(a)浓度相对于给药前的比值。
图1C为TJR101079、TJR102136在给药后第28天血清Lp(a)浓度相对于给药前的比值。
具体实施方式
以下结合实施例进一步描述本公开，但这些实施例并非限制本公开的范围。本公开实施例中未注明具体条件的实验方法，通常按照常规条件或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，则该试剂可自任意分子生物学试剂的供应商以用于分子生物学应用的质量/纯度而获得。
实施例1：化学修饰
(-)hmpNA(A)
(-)hmpNA(A)的制备方法参考WO2022028462A进行。(-)hmpNA(A)绝对构型为(R)-hmpNA(A)。
实施例2：制备NAG0052、L96、NAG25
按照专利申请WO2023274395A记载的方法制备获得如上结构式所示的NAG0052；
按照专利申请WO2014025805A记载的方法制备获得如上结构式所示的L96；
按照专利申请WO2017156012A的方法制备获得如上结构式所示的NAG25。
实施例3：制备氘代5’末端化学修饰亚磷酰胺

第一步
1-((6aR,8R,9R,9aS)-9-羟基-2,2,4,4-四异丙基四氢-6H-呋喃并[3,2-f][1,3,5,2,4]三氧杂二硅杂环辛烷-8-基)嘧啶-2,4(1H,3H)-二酮A1-2
在氮气氛下，将商购得到的化合物A1-1(100g,409mmol)溶于吡啶(1000mL)中，在0℃加入1,3-二氯-1,1,3,3-四异丙基二甲硅氧醚(TIPDSiCl2)(135.6g,430.0mmol)，20℃反应18小时。将反应液加入乙酸乙酯(1500mL)和水(1000mL)萃取，有机相用饱和食盐水(1000mL X 3)洗涤三次后用无水硫酸钠干燥，浓缩，用柱层析色谱法纯化(石油醚:乙酸乙酯＝2:1)，得到化合物A1-2(185g，380mmol，产率92.8％)。
第二步
3-((苄氧基)甲基)-1-((6aR,8R,9R,9aS)-9-羟基-2,2,4,4-四异丙基四氢-6H-呋喃并[3,2-][1,3,5,2,4]三氧杂二硅杂环辛烷-8-基)嘧啶-2,4(1H,3H)-二酮A1-3
将化合物A1-2(185g,380.1mmol)溶于N,N-二甲基甲酰胺(1850mL)后，加入苄基氯甲基醚(BOMCl)(89.29g,570.1mmol)和1,8-二氮杂二环十一碳-7-烯(DBU)(115.7g,760.2mmol)，20℃反应3小时。将反应液加入乙酸乙酯(3000mL)和水(3000mL)萃取，有机相用饱和食盐水(2000mL X 3)洗涤三次后用无 水硫酸钠干燥，浓缩，用柱层析色谱法纯化(石油醚:乙酸乙酯＝3:1)，得到化合物A1-3(目标化合物，172g，275.6mmol，产率72.5％)。
第三步
3-((苄氧基)甲基)-1-((6aR,8R,9R,9aR)-2,2,4,4-四异丙基-9-(甲氧基-d3)四氢-6H-呋喃并[3,2-][1,3,5,2,4]三氧杂二硅杂环辛烷-8-基)嘧啶-2,4(1H,3H)-二酮A1-4
将化合物A1-3(150g,247.2mmol)溶于乙腈(300mL)中，加入CD3I(107.5g,741.5mmol)和氧化银(114.5g,494.3mmol)，55℃反应24小时。将反应液过滤，减压浓缩，得到粗品化合物A1-4(目标化合物，125g，200.3mmol，产率81％)。
第四步
3-((苄氧基)甲基)-1-((2R,3R,4R,5R)-4-羟基-5-(羟甲基)-3-(甲氧基-d3)四氢呋喃-2-基)嘧啶-2,4(1H,3H)-二酮A1-5
将化合物A1-4(125g,200mmol)溶于四氢呋喃(1250mL)后，加入吡啶氟化氢(158.8g,1.60mol)，25℃反应18小时。将反应液加入乙酸乙酯(1000mL)和水(1000mL)萃取，有机相用饱和食盐水(1000mL)洗涤一次后用无水硫酸钠干燥，过滤，减压浓缩，用柱层析色谱法纯化(二氯甲烷：甲醇＝20:1)，得到化合物A1-5(目标化合物，59g，154.7mmol，产率77％)。
第五步
(2R,3R,4R,5R)-5-(3-((苄氧基)甲基)-2,4-二氧代-3,4-二氢嘧啶-1(2H)-基)-2-(((叔丁基二甲基硅)氧基)甲基)-4-(甲氧基-d3)四氢呋喃-3-基苯甲酸酯A1-6
将化合物A1-5(59g,154.7mmol)溶于吡啶(590mL)后，加入叔丁基二甲基氯硅烷(TBSCl)(93.2g,618mmol)，该反应在25℃反应18小时。然后加入苯甲酰氯(32.62g,232.0mmol)，在25℃反应3小时。将反应液加入乙酸乙酯(1000mL)和水(1000mL)萃取，有机相用饱和食盐水(1000mL X 2)洗涤两次后用无水硫酸镁干燥，浓缩，用柱层析色谱法纯化(石油醚:乙酸乙酯＝5:1)，得到化合物A1-6(目标化合物，60.0g，100.3mmol，产率64.6％)。
第六步
(2R,3R,4R,5R)-5-(3-((苄氧基)甲基)-2,4-二氧代-3,4-二氢嘧啶-1(2H)-基)-2-羟甲基-4-(甲氧基-d3)四氢呋喃-3-基苯甲酸酯A1-7
在0℃，将化合物A1-6(60.0g,100.3mmol)溶于甲醇(600mL)后，加入乙酰氯(10.21g,130.1mmol)，20℃反应2小时。然后加入碳酸银，20℃搅拌1小时。将反应液过滤，滤液减压浓缩，得到化合物A1-7(46.0g，94.7mmol，产率94.7％)。
第七步
(2S,3S,4R,5R)-3-(苄氧基)-5-(3-((苄氧基)甲基)-2,4-二氧代-3,4-二氢嘧啶-1(2H)-基)-4-(甲氧基-d3)四氢呋喃-2-羧酸A1-8
将化合物A1-7(46.0g，94.75mmol)和4-羰基-四甲基哌啶氧化物TEMPO)(6.95g,44.49mmol)溶于CH3CN(230mL)和H2O(230mL)里，加入5(6)-氨基- 1-(4-氨基苯基)-1,3,3-三甲基茚满(PIDA)(67.14g,208.4mmol)，25℃反应18小时。在反应液中加入乙酸乙酯(500mL)和水(300mL)萃取，有机相用无水硫酸钠干燥后浓缩，用柱层析色谱法纯化(二氯甲烷:甲醇＝10:1)，减压浓缩，得到化合物A1-8(目标化合物，43.0g，86mmol，产率90％)。
第八步
(3S,4R,5R)-2-乙酰氧基-5-(3-((苄氧基)甲基)-2,4-二氧代-3,4-二氢嘧啶-1(2H)-基)-4-(甲氧基-d3)四氢呋喃-3-基苯甲酸酯A1-9
向干燥烧瓶中加入化合物A1-8(5g,10.0mmol)，用氩气吹扫，之后添加N,N-二甲基甲酰胺(50mL)。再依次加入吡啶(8mL,100mmol)和醋酸铅(9.7mL,50mmol)。将反应物避光并且在室温搅拌48小时。反应用水(200mL)淬灭并且用乙酸乙酯(200mL)稀释。将所得的悬浮液经由硅藻土垫过滤。将固体用乙酸乙酯冲洗。将有机层分离并且在真空中浓缩。将粗品在C18反向柱上纯化，得到作为α/β混合物的标题产物A1-9(1.5g，产率：29％)。
MS(ESI)：m/z＝514.2[M+H]+。
第九步
(3S,4R,5R)-5-(3-((苄氧基)甲基)-2,4-二氧代-3,4-二氢嘧啶-1(2H)-基)-2-((二乙氧基磷酰)甲氧基)-4-(甲氧基-d3)四氢呋喃-3基苯甲酸酯A1-10
在氩气氛下，将(羟甲基)膦酸二乙酯(2.5g，156.7mmol)和三氟化硼二乙醚配合物(5.7mL，43.8mmol)添加到化合物A1-9(1.5g，2.9mmol)于无水二氯甲烷(15mL)中的溶液中。将反应物在室温搅拌16小时。将反应物用水淬灭并且用乙酸乙酯萃取。将有机层分离，用盐水洗涤，经过无水硫酸钠干燥并且在真空中浓缩。将粗品在C18反向柱上纯化，得到标题化合物A1-10(1g，收率55％)。
MS(ESI)：m/z＝622.3[M+H]+。
第十步
(3S,4R,5R)-2-((二乙氧基磷酰)甲氧基)-5-(2,4-二氧代-3,4-二氢嘧啶-1(2H)-基)-4-(甲氧基-d3)四氢呋喃-3-基苯甲酸酯A1-11
将化合物A1-10(1g，14.6mmol)于TFA(5mL)中的溶液在80℃搅拌30分钟，然后在真空中浓缩。得到标题化合物A1-11(1g，粗品)，直接用于下一步。
MS(ESI):m/z＝502.3[M+H]+。
第十一步
((((3S,4R,5R)-5-(2,4-二氧代-3,4-二氢嘧啶-1(2H)-基)-3-羟基-4-(甲氧基-d3)四氢呋喃-2-基)氧基)甲基)膦酸二乙酯A1-12
将化合物A1-11(1g，2.0mmol)于氨甲醇溶液(7N，10mL)中的溶液在室温搅拌16小时。将反应混合物在真空中浓缩。将粗品在C18反向柱上纯化，得到标题化合物A1-12(300mg，产率：38％)。
MS(ESI):m/z＝398.2[M+H]+。
第十二步
2-氰乙基((2R,3S,4R,5R)-2-((二乙氧基磷酰)甲氧基)-5-(2,4-二氧代-3,4-二氢嘧啶-1(2H)-基)-4-(甲氧基-d3)四氢呋喃-3-基)二异丙基亚磷酰胺A1

将DIPEA(58mg，0.45mmol)添加到化合物A1-12(100mg,0.252mmol)于无水二氯甲烷(2mL)中的溶液中，继而添加3-[氯-(二异丙基氨基)磷烷基]氧基丙腈(83mg，0.35mmol)。将反应混合物在室温搅拌2小时，然后用MeOH淬灭。将反应混合物用乙酸乙酯稀释，用饱和碳酸氢钠、水和盐水洗涤。将有机层在真空中浓缩。将粗品在C18反向柱上纯化，得到亚磷酰胺单体A1(80mg，产率：53％)。
MS(ESI)：m/z＝596.0[M-H]+。
1H NMR(400MHz,CD3OD)δ7.74-7.70(m,1H),6.33-6.30(m,1H),5.84-5.82(m,1H),5.25-5.18(m,1H),4.51-4.44(m,1H),4.27-3.67(m,11H),2.82-2.79(m,2H),1.41-1.36(m,6H),1.29-1.23(m,12H)。
31P NMR(400MHz,CD3OD)δ151.95,150.57,21.29。
实施例4：5’末端含有氘代化学修饰的dsRNA的合成
dsRNA的合成与通常的亚磷酰胺固相合成法无异，在合成反义链末端修饰的核苷酸时，在对应位置使用实施例3合成的亚磷酰胺单体A1。
合成过程简要描述如下：于Dr.Oligo48合成器(Biolytic)上，以Universal CPG载体为起始，根据合成程序逐个连接核苷亚磷酰胺单体。除实施例3描述的核苷亚磷酰胺单体A1外，其余核苷单体原料2’-F RNA、2’-O-甲基RNA等核苷亚磷酰胺单体购自上海兆维或苏州吉玛。采用5-乙基硫-1H-四唑(ETT)作为活化剂(0.6M乙腈溶液)，使用0.22M的PADS溶于1:1体积比的乙腈和三甲基吡啶(苏州柯乐玛)溶液作为硫化试剂，可以在指定位置引入硫代磷酸酯；使用碘吡啶/水溶液(柯乐玛)作为氧化剂，可以在指定位置引入氧代磷酸酯。
固相合成完成后，某些实施例需用TMSI先去除5’-末端保护基团，如采用亚磷酰胺单体A1进行固相合成后，将寡核糖核苷酸粗品悬浮于无水乙腈中，室温下加入TMSI，充分搅拌后脱掉寡核糖核苷酸中的乙基(Et)，得到带有NA0127的寡核糖核苷酸。然后进一步将寡核糖核苷酸自该固体支撑物裂解，裂解条件：采用3:1的28％氨水和乙醇溶液在50℃浸泡16小时。然后离心，将上清液转移到另一个离心管中，浓缩蒸发干后，使用C18反向色谱纯化，流动相为0.1M TEAA和乙 腈，并使用3％三氟乙酸溶液脱除DMTr。目标寡核苷酸收集后冻干，并经LC-MS鉴定为目标产物，再经过UV(260nm)定量。
所得到的单链寡核苷酸，根据等摩尔比，按照互补配对，退火，最后所得到的dsRNA溶于1×PBS中，并调整至实验所需浓度备用。
经过5’末端含有氘代化学修饰、2’-氟代、2’-甲氧基等修饰的正义链和反义链序列详见表1和表2。
实施例5：dsRNA缀合物的合成
1.自制带有载体的树脂
将含有羧酸基团的化合物NAG0052(157mg,0.062mmol)溶于无水DMF(3mL)，待底物完全溶解后，依次加入无水乙腈(4mL),DIEA(0.03mL,0.154mmol，2.5eq)和HBTU(35mg,0.093mmol，1.5eq)。反应液混合均匀后，再加入大孔胺甲基树脂(476mg,空白载量为0.41mmol/g,目标载量为0.1mmol/g)。将反应液放入摇床上(温度：25℃，转速：200rpm)振摇过夜。反应液过滤，滤饼依次分别用DCM，无水乙腈洗涤，收集固体，真空干燥过夜。
将上步固体分散于无水乙腈(5mL)，依次加入吡啶(0.18mL)，DMAP(3mg)，NMI(0.12mL)和CapB1(2.68mL)。将反应液放入摇床上(温度：25℃，转速：200rpm)振摇2h。反应液过滤，滤饼用无水乙腈洗涤，收集固体，真空干燥过夜，得到带有载体的树脂。载量经过测定为0.1mmol/g。
2.对于已经连接在树脂上的NAG0052，使用该树脂作为起始，按照核苷酸排布顺序自3’-5’方向逐一连接核苷单体。每连接一个核苷单体都包括脱保护、偶联、盖帽、氧化或硫化四步反应。操作为本领域常规。
化合物NAG0052经过固相合成连接到序列上，再经过胺解后，NAG0052结构脱去一部分官能团成为NAG0052’。3.用L96代替NAG0052，按照上述步骤合成相关缀合物。其中，L96按照专利申请WO2014025805A记载的方法制备获得。
制得的dsRNA缀合物具有表1和表2中所示的正义链和反义链。
阳性对照dsRNA缀合物TRD007790按照专利申请WO2017059223A中记载的方法制备得到。
表1.dsRNA列表

表2.dsRNA缀合物的正义链和反义链


其中，大写字母G、A、C、U分别表示包含鸟嘌呤、腺嘌呤、胞嘧啶和尿嘧啶的核苷酸，小写字母m表示2'-甲氧基修饰，小写字母f表示2'-氟代修饰，小写字母s表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸二酯基连接；
(-)hmpNA(A)表示
NAG0052’的结构为：
L96’的结构为：
NAG25s’的结构为：
NA0127、NA0149和InvdA分别表示：
其中，将NA0127引入dsRNA的方法如实施例4中所描述，将NA0149引入dsRNA的合成方法参考文献US20190177729A；将InvdA引入dsRNA的合成方法参考文献WO2017059223A。
表3.dsRNA缀合物的正义链和反义链对应的裸序列

实施例6：人LPA dsRNA的设计
以人LPA(NM_005577.3)作为靶基因，以满足活性dsRNA的一般规则设计19/21nt的dsRNA。未经修饰的正义链及反义链序列详见表4、表5。
表4.人LPA dsRNA的未修饰的正义链、反义链
表5.人LPA dsRNA的未修饰的正义链、反义链(对照组)
实施例7：dsRNA缀合物psiCHECK 9浓度点在靶活性
在HEK293A细胞中采用9个浓度梯度对dsRNA/dsRNA缀合物进行体外分子水平模拟在靶活性筛选。
HEK293A细胞培养于含10％胎牛血清的DMEM高糖培养基中，在37℃，5％CO2条件下培养。转染前24h，将HEK293A细胞接种于96孔板，接种密度为每孔8×103个细胞，每孔100μL培养基。
按照说明书，使用Lipofectamine2000(ThermoFisher，11668019)对细胞共转染dsRNA缀合物及对应质粒，每孔使用0.3μL Lipofectamine2000。质粒转染量为 40ng每孔。对于在靶序列质粒，dsRNA/dsRNA缀合物共设置9个浓度点，最高浓度点终浓度为20nM，3倍梯度稀释，20nM，6.666666667nM，2.222222222nM，0.740740741nM，0.24691358nM，0.082304527nM，0.027434842nM，0.009144947nM，0.003048316nM。转染后24h，采用Dual-Luciferase Reporter Assay System(Promega，E2940)检测在靶水平。
结果如表6所示。
表6.dsRNA/dsRNA缀合物在psi-Check系统9点活性结果


实施例8：dsRNA对人原代肝细胞(PHH)中人LPA的抑制-7个浓度点抑制活性
在人原代肝细胞(PHH)中采用7个浓度梯度对dsRNA序列进行PHH活性筛选。各个dsRNA样品转染起始终浓度为20nM，5倍梯度稀释和7个浓度点。
PHH冻存于液氮中，转染前24h，将人原代肝细胞(PHH)复苏后接种于96孔板，接种密度为每孔3×104个细胞，每孔80μL培养基。
参照产品说明手册，使用Lipofectamine RNAi MAX(ThermoFisher，13778150) 转染dsRNA，dsRNA转染的梯度终浓度为20nM，4nM，0.8nM，0.16nM，0.032nM，0.0064nM和0.00128nM。在处理24小时后，使用高通量细胞RNA提取试剂盒进行细胞总RNA提取、RNA逆转录实验和定量实时PCR检测，测定人LPA的mRNA水平，根据GAPDH内参基因水平对人LPA的mRNA水平进行校正。
其中，在定量实时PCR检测时，采用的是探针Q-PCR检测实验，其引物信息如表7和表8所示。
Taqman探针Q-PCR检测实验完毕后，按照系统自动设定的阈值获取相应的Ct值，可以通过Ct值比较，相对定量某个基因的表达：比较Ct指的是通过与内参基因Ct值之间的差值来计算基因表达差异,也称之是2-△△Ct，△△Ct＝[(Ct实验组目的基因-Ct实验组内参)-(Ct对照组目的基因-Ct对照组内参)]。
抑制率(％)＝(1-目的基因表达剩余量)*100％。
结果以相对于经过对照dsRNA处理的细胞的人LPA mRNA表达剩余百分比来表示。抑制率的IC50结果见表9。
结果表明，和TJR100374至TJR100380相比，TJR100373具有显著优势活性，这指示TJR100373所针对的目的基因片段更适合作为靶点。
表7.Taqman LPA探针引物信息表
表8.Taqman GAPDH探针引物信息表
表9.dsRNA对人原代肝细胞(PHH)中人LPA的多剂量抑制活性

实施例9：dsRNA缀合物在人源化小鼠(hu-Lp(a))体内(in vivo)活性的测定
本实施例中使用的人源化小鼠(hu-Lp(a))是由上海拓界生物医药科技有限公司委托赛业(苏州)生物科技有限公司构建。
根据血清Lp(a)蛋白含量均匀分组，每组6只(2雄，4雌)，共4组，分别通过皮下注射的方式给予生理盐水，阳性对照TRD007790、本公开的dsRNA缀合物TJR101079，给药剂量均是3mg/kg，给药体积均为10μL/g。给药当天给药前采集血清40μL，测定的血清Lp(a)浓度作为基础数据，给药日记为第1天(D1)，给药后第8天(D8)，第29天(D29)，第57天(D57)，第85天(D85)，第99天(D99)，每次采集血清40μL，使用Abbott Ci4100全自动生化免疫仪测定血清Lp(a)蛋白含量，计算dsRNA缀合物对人源化小鼠(hu-Lp(a))血清Lp(a)蛋白表达的抑制。计算得到各个给药组相对于空白对照组均一化后的血清Lp(a)蛋白相对于空白的比值，结果记载于表10中。各组相对于空白给药组采用One-way ANOVA进行统计。
根据表10结果可见，在第99天，对于空白对照组和阳性对照组TRD007790，已经无统计学差异，而TJR101079依旧表现出明显的Lp(a)蛋白表达抑制，具有统计学意义p<0.01，即表明第99天，TRD007790已无明显抑制活性，而TJR101079仍具有显著的抑制活性。
由此说明，本公开的dsRNA缀合物TJR101079具有优异的长效抑制Lp(a)蛋白表达的效果。
表10.人源化小鼠(hu-Lp(a))体内血清中Lp(a)蛋白浓度
表10中，*表示p≤0.05；**表示p<0.01；***表示p<0.001；****表示p<0.0001。
实施例10：dsRNA缀合物在人源化小鼠(hu-Lp(a))体内(in vivo)活性的测定
本实施例中使用的人源化小鼠(hu-Lp(a))是由上海拓界生物医药科技有限公司委托赛业(苏州)生物科技有限公司构建。
根据血清Lp(a)蛋白含量均匀分组，每组6只，共4组，分别通过皮下注射的方式给予，dsRNA缀合物TRD007790、TJR101079、TJR102134、TJR102136给药剂量均是1mg/kg，给药体积均为10μL/g。给药当天给药前采集血清40μL，测定的血清Lp(a)浓度作为基础数据，给药日记为第1天(D1)，给药后第28天(D28)，每次采集血清40μL，使用Abbott Ci4100全自动生化免疫仪测定血清Lp(a)蛋白含量，计算dsRNA缀合物对人源化小鼠(hu-Lp(a))血清Lp(a)蛋白表达的抑制。计算得到各个给药组相对于空白对照组均一化后的血清Lp(a)蛋白相对于空白的比值，结果记载于图1A、图1B、图1C中。各组D28的血清Lp(a)浓度相对于给药前血清Lp(a)比较获得剩余血清Lp(a)比值，采用Paired t test进行TJR101079与TRD007790、TJR102134、TJR102136统计比较。
根据图1A、图1B、图1C结果可见，在第28天，各组相较于给药前血清Lp(a)的比值，TJR101079与TRD007790、TJR102134、TJR102136比较均具有显著性差异。TJR101079与TRD007790“*”p<0.05；TJR101079与TJR102134“*”p<0.05；TJR101079与TJR102136“**”p<0.01。
由此说明，TJR101079对Lp(a)蛋白表达长效抑制的效果优于对照组TRD007790、TJR102134和TJR102136，即，相较于对比缀合物，本公开的dsRNA缀合物具有显著更优异Lp(a)蛋白表达长效抑制的效果。

Claims (22)

  1. 一种靶向LPA的双链核糖核酸(dsRNA)，其包含形成双链区的正义链和反义链，其中，
    所述正义链的裸核苷酸序列包含与SEQ ID NO:1的核苷酸序列相差不超过3个核苷酸的至少17个连续核苷酸；并且，
    所述反义链的裸核苷酸序列包含与SEQ ID NO:2的核苷酸序列相差不超过3个核苷酸的至少19个连续核苷酸；
    其中，按照5'端到3'端的方向，
    所述正义链第7、8和9位的核苷酸为2'-氟代修饰的核苷酸，其余位置的核苷酸为2'-甲氧基修饰的核苷酸；
    所述反义链第2和14位的核苷酸为2'-氟代修饰的核苷酸，第4、6、10、12、16和18位的核苷酸独立地为2'-甲氧基或2'-氟代修饰的核苷酸，其余位置的核苷酸为2'-甲氧基修饰的核苷酸；
    所述反义链中2'-氟代修饰的核苷酸的个数为2-7个。
  2. 如权利要求1所述的dsRNA，其中，
    所述正义链的裸核苷酸序列包含SEQ ID NO:1所示的核苷酸序列，并且反义链的裸核苷酸序列包含SEQ ID NO:2所示的核苷酸序列。
  3. 如权利要求1或2所述的dsRNA，其中，所述反义链5’端第7位的核苷酸是修饰的核苷酸，其中，
    -所述修饰的核苷酸是2’-甲氧基修饰的核苷酸，或
    -所述修饰的核苷酸包含式(I)、(Ⅰ-1)、(Ⅰ-2)所示的化学修饰或其药学上可接受的盐：
    其中，B表示反义链5’端第7位核苷酸对应位置的碱基。
  4. 如权利要求1-3中任一项所述的dsRNA，其中，所述反义链5’端第1位的核苷酸是修饰的核苷酸，其中，
    -所述修饰的核苷酸是2’-甲氧基修饰的核苷酸，或
    -所述修饰的核苷酸为式(Ⅱ)所示的化学修饰的核苷酸：
    其中，B表示反义链5’端第1位核苷酸对应位置的碱基。
  5. 如权利要求1-4中任一项所述的dsRNA，其中，
    所述正义链含有如下式所示的核苷酸序列：
    5’-NaNaNaNaNaNaNbNbNbNaNaNaNaNaNaNaNaNaNa-3’；
    其中，Na为2'-甲氧基修饰的核苷酸，Nb为2'-氟代修饰的核苷酸。
  6. 如权利要求1-5中任一项所述的dsRNA，其中，
    所述反义链包含如下式所示的核苷酸序列：
    5’-Na’Nb’Na’X’Na’X’Na’Na’Na’X’Na’X’Na’Nb’Na’X’Na’X’Na’Na’Na’-3’，或
    5’-Na’Nb’Na’X’Na’X’W’Na’Na’X’Na’X’Na’Nb’Na’X’Na’X’Na’Na’Na’-3’，或
    5’-V’Nb’Na’X’Na’X’Na’Na’Na’X’Na’X’Na’Nb’Na’X’Na’X’Na’Na’Na’-3’；
    优选地，所述反义链包含如下式所示的核苷酸序列：
    5’-Na’Nb’Na’Na’Na’Nb’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
    5’-Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
    5’-Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’Nb’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’-3’，
    5’-Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’-3’，
    5’-Na’Nb’Na’Na’Na’Nb’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’-3’，
    5’-Na’Nb’Na’Na’Na’Nb’Na’Na’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
    5’-Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
    5’-Na’Nb’Na’Na’Na’Nb’Na’Na’Na’Nb’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
    5’-Na’Nb’Na’Na’Na’Na’Na’Na’Na’Na’Na’Na’Na’Nb’Na’Na’Na’Na’Na’Na’Na’-3’，
    5’-Na’Nb’Na’Na’Na’Nb’Na’Na’Na’Na’Na’Na’Na’Nb’Na’Na’Na’Na’Na’Na’Na’-3’，
    5’-Na’Nb’Na’Na’Na’Na’Na’Na’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
    5’-Na’Nb’Na’Na’Na’Na’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’Na’Na’-3’，
    5’-Na’Nb’Na’Nb’Na’Nb’W’Na’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
    5’-Na’Nb’Na’Nb’Na’Nb’W’Na’Na’Nb’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’-3’，
    5’-Na’Nb’Na’Nb’Na’Nb’W’Na’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’-3’，
    5’-Na’Nb’Na’Na’Na’Nb’W’Na’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’-3’，
    5’-Na’Nb’Na’Na’Na’Nb’W’Na’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
    5’-Na’Nb’Na’Nb’Na’Nb’W’Na’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
    5’-Na’Nb’Na’Na’Na’Nb’W’Na’Na’Nb’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
    5’-Na’Nb’Na’Na’Na’Nb’W’Na’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
    5’-Na’Nb’Na’Na’Na’Na’W’Na’Na’Na’Na’Na’Na’Nb’Na’Na’Na’Na’Na’Na’Na’-3’，
    5’-Na’Nb’Na’Na’Na’Nb’W’Na’Na’Na’Na’Na’Na’Nb’Na’Na’Na’Na’Na’Na’Na’-3’，
    5’-Na’Nb’Na’Na’Na’Na’W’Na’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
    5’-V’Nb’Na’Na’Na’Nb’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’，
    5’-V’Nb’Na’Na’Na’Na’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’Na’Na’-3’；
    其中，每个X’独立地为Na’或Nb’；Na’为2'-甲氧基修饰的核苷酸，Nb’为2'-氟代修饰的核苷酸；W’表示包含式(I)、(Ⅰ-1)、(Ⅰ-2)所示的化学修饰或其药学上可接受的盐的核苷酸；V’表示式(Ⅱ)所示的化学修饰的核苷酸。
  7. 如权利要求1-6中任一项所述的dsRNA，其中，所述正义链和/或反义链中至少一个磷酸二酯基为具有修饰基团的磷酸二酯基，优选为硫代磷酸二酯基。
  8. 如权利要求7所述的dsRNA，其中，所述硫代磷酸二酯基存在于以下位置中的至少一处：
    所述正义链的5'端第1个核苷酸和第2个核苷酸之间；
    所述正义链的5'端第2个核苷酸和第3个核苷酸之间；
    所述正义链的3’端第1个核苷酸和第2个核苷酸之间；
    所述反义链的5'端第1个核苷酸和第2个核苷酸之间；
    所述反义链的5'端第2个核苷酸和第3个核苷酸之间；
    所述反义链的3'端第1个核苷酸和第2个核苷酸之间；以及
    所述反义链的3'端第2个核苷酸和第3个核苷酸之间；
    优选地，所述正义链和/或反义链中包括多个硫代磷酸二酯基，所述硫代磷酸二酯基存在于：
    所述正义链的5'端第1个核苷酸和第2个核苷酸之间；和，
    所述正义链的5'端第2个核苷酸和第3个核苷酸之间；和，
    所述正义链的3’端第1个核苷酸和第2个核苷酸之间；和
    所述反义链的5'端第1个核苷酸和第2个核苷酸之间；和，
    所述反义链的5'端第2个核苷酸和第3个核苷酸之间；和，
    所述反义链的3'端第1个核苷酸和第2个核苷酸之间；和，
    所述反义链的3'端第2个核苷酸和第3个核苷酸之间；或，
    所述正义链和/或反义链中包括多个硫代磷酸二酯基，所述硫代磷酸二酯基存在于：
    所述正义链的5'端第1个核苷酸和第2个核苷酸之间；和，
    所述正义链的5'端第2个核苷酸和第3个核苷酸之间；和，
    所述反义链的5'端第1个核苷酸和第2个核苷酸之间；和，
    所述反义链的5'端第2个核苷酸和第3个核苷酸之间；和，
    所述反义链的3'端第1个核苷酸和第2个核苷酸之间；和，
    所述反义链的3'端第2个核苷酸和第3个核苷酸之间。
  9. 如权利要求1-8中任一项所述的dsRNA，其中，
    所述正义链包含SEQ ID NO:6所示的核苷酸序列，并且反义链包含SEQ ID NO:16、SEQ ID NO:20、SEQ ID NO:22至SEQ ID NO:33、SEQ ID NO:35任一项所示的核苷酸序列；或者，
    所述正义链包含SEQ ID NO:7所示的核苷酸序列，并且反义链包含SEQ ID NO:9至SEQ ID NO:20、SEQ ID NO:33至SEQ ID NO:35任一项所示的核苷酸序列。
  10. 一种dsRNA缀合物，其包含：
    权利要求1至9中任一项所述的dsRNA，和
    连接至所述dsRNA末端的靶向配体；
    优选地，所述靶向配体连接至所述dsRNA的正义链3’末端。
  11. 如权利要求10所述的dsRNA缀合物，其中：
    所述靶向配体包含至少一个靶向部分，
    所述靶向部分各自独立地选自：半乳糖、半乳糖胺、N-甲酰基-半乳糖胺、N-乙酰基-半乳糖胺、N-丙酰基-半乳糖胺、N-正丁酰基-半乳糖胺和N-异丁酰基-半乳糖胺；
    优选地，所述靶向部分是N-乙酰基-半乳糖胺；
    更优选地，所述靶向配体包含三个相同或不同的靶向部分。
  12. 如权利要求11所述的dsRNA缀合物，所述靶向配体是如式(Ⅲ-1)或(Ⅲ-2)所示化合物或其药学上可接受的盐，其中：
    所述式(Ⅲ-1)为：
    所述式(Ⅲ-2)为：
  13. 如权利要求10-12中任一项所述的dsRNA缀合物，其中，所述配体通过磷酸二酯基团或硫代磷酸二酯基团与所述dsRNA连接；优选通过磷酸二酯基团连接。
  14. 如权利要求10-13中任一项所述的dsRNA缀合物，其中，
    所述正义链包含SEQ ID NO:3所示的核苷酸序列，并且反义链包含SEQ ID NO:16、SEQ ID NO:20、SEQ ID NO:22至SEQ ID NO:33、SEQ ID NO:35任一项所示的核苷酸序列；或者，
    所述正义链包含SEQ ID NO:4所示的核苷酸序列，并且反义链包含SEQ ID NO:9至SEQ ID NO:20、SEQ ID NO:33至SEQ ID NO:35任一项所示的核苷酸序列；或者，
    所述正义链包含SEQ ID NO:5所示的核苷酸序列，并且反义链包含SEQ ID NO:16、SEQ ID NO:20、SEQ ID NO:33任一项所示的核苷酸序列。
  15. 如权利要求10-14中任一项所述的dsRNA缀合物，其中，所述dsRNA缀合物选自如下结构或其药学上可接受的盐：
    其中，
    Af＝腺嘌呤2'-F核糖核苷；
    Cf＝胞嘧啶2'-F核糖核苷；
    Uf＝尿嘧啶2'-F核糖核苷；
    Gf＝鸟嘌呤2'-F核糖核苷；
    Am＝腺嘌呤2'-OMe核糖核苷；
    Cm＝胞嘧啶2'-OMe核糖核苷；
    Gm＝鸟嘌呤2'-OMe核糖核苷；
    Um＝尿嘧啶2'-OMe核糖核苷；
    表示硫代磷酸二酯基阴离子形式；表示磷酸二酯基阴离子形式；NAG0052’表示
  16. 一种药物组合物，其包含：
    如权利要求1-9中任一项所述的dsRNA和/或权利要求10-15中任一项所述的dsRNA缀合物；
    优选地，所述的药物组合物还包含一种或多种药学上可接受的赋形剂。
  17. 如权利要求1-9中任一项所述的dsRNA和/或权利要求10-15中任一项dsRNA缀合物和/或如权利要求16所述的药物组合物在制备药物中的应用；
    所述药物用于预防和/或治疗心血管疾病，或者，
    所述药物用于预防和/或治疗与脂蛋白(a)和/或载脂蛋白(a)水平升高相关的疾病；
    优选地，所述与脂蛋白(a)和/或载脂蛋白(a)水平升高相关的疾病选自：心血管疾病；
    所述心血管疾病选自：缺血性中风、动脉粥样硬化、血栓形成、冠心病、下肢动脉病变、主动脉瓣狭窄、心肌梗塞、冠状动脉狭窄、颈动脉狭窄、股动脉狭窄、心脏衰竭。
  18. 一种抑制LPA表达的方法，其包括向受试者给予有效量或有效剂量的如权利要求1-9中任一项所述的dsRNA和/或如权利要求10-15中任一项所述的dsRNA缀合物和/或如权利要求16所述的药物组合物。
  19. 一种将dsRNA体内递送至肝脏以抑制LPA表达和/或复制的方法，其包括向受试者给予有效量或有效剂量的如权利要求1-9中任一项所述的dsRNA和/或权利要求10-15中任一项dsRNA缀合物和/或如权利要求16所述的药物组合物。
  20. 一种细胞，其包含如权利要求1-9中任一项所述的dsRNA和/或权利要求10-15中任一项dsRNA缀合物和/或如权利要求16所述的药物组合物。
  21. 一种试剂盒，其包含如权利要求1-9中任一项所述的dsRNA和/或权利要 求10-15中任一项dsRNA缀合物和/或如权利要求16所述的药物组合物。
  22. 一种制备dsRNA、dsRNA缀合物或药物组合物的方法，其包括：
    合成如权利要求1-9中任一项所述的dsRNA和/或权利要求10-15中任一项dsRNA缀合物；和/或
    配制权利要求16所述的药物组合物。
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