WO2024179573A1 - 靶向抑制素βE的siRNA、siRNA缀合物及其医药用途 - Google Patents

靶向抑制素βE的siRNA、siRNA缀合物及其医药用途 Download PDF

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WO2024179573A1
WO2024179573A1 PCT/CN2024/079576 CN2024079576W WO2024179573A1 WO 2024179573 A1 WO2024179573 A1 WO 2024179573A1 CN 2024079576 W CN2024079576 W CN 2024079576W WO 2024179573 A1 WO2024179573 A1 WO 2024179573A1
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seq
sirna
antisense strand
sense strand
nucleotide
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English (en)
French (fr)
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严翠翠
温宇豪
林晓燕
李云飞
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上海拓界生物医药科技有限公司
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present disclosure belongs to the field of biomedicine, and specifically relates to siRNA, conjugates, compositions and medical uses thereof targeting inhibin ⁇ E gene (INHBE).
  • the inhibin beta E gene is a member of the TGF- ⁇ (transforming growth factor- ⁇ ) superfamily of proteins that is involved in regulating many cellular processes, including cell proliferation, apoptosis, immune response, and hormone secretion. Inhibin is responsible for inhibiting the secretion of follicle-stimulating hormone from the pituitary gland and can be involved in regulating many different functions, such as hypothalamic and pituitary hormone secretion, gonadal hormone secretion, germ cell development and maturation, erythrocyte differentiation, insulin secretion, neural cell survival, embryonic axial development, or bone growth. In addition, INHBE may be upregulated under endoplasmic reticulum stress conditions, so that the protein may inhibit cell proliferation and growth on the pancreas and liver.
  • TGF- ⁇ transforming growth factor- ⁇
  • INHBE may be upregulated under endoplasmic reticulum stress conditions, so that the protein may inhibit cell proliferation and growth on the pancreas and liver.
  • Body fat distribution is an important basis for judging cardiovascular and metabolic diseases.
  • various body fat distributions there is a special body fat distribution that is prone to cause serious cardiovascular or metabolic diseases. It is characterized by more fat distribution in the waist and/or less fat accumulation in the buttocks, resulting in a larger waist-to-hip ratio (WHR).
  • WHR waist-to-hip ratio
  • Metabolic diseases caused by abnormal body fat distribution include: type 2 diabetes, hyperlipidemia, dyslipidemia (increased or altered circulating levels of low-density lipoprotein cholesterol LDL-C, triglycerides, very low-density lipoprotein cholesterol VLDL-C, apolipoprotein B or other lipid moieties), obesity (especially abdominal obesity), dyslipidemia, insulin resistance, non-alcoholic steatohepatitis, hypertension and/or high blood sugar or glucose or hyperglycemia, metabolic syndrome, coronary artery disease and other atherosclerotic diseases.
  • dyslipidemia increased or altered circulating levels of low-density lipoprotein cholesterol LDL-C, triglycerides, very low-density lipoprotein cholesterol VLDL-C, apolipoprotein B or other lipid moieties
  • obesity especially abdominal obesity
  • dyslipidemia insulin resistance
  • non-alcoholic steatohepatitis hypertension and/or high blood sugar or glucose or hyperglycemia
  • the main methods for treating metabolic diseases include changing lifestyle habits, adjusting diet, exercising, and taking lipid-lowering drugs (such as statins), but these methods are less effective and taking lipid-lowering drugs will cause some side effects.
  • Interfering RNAi is an effective way to silence gene expression. Through post-transcriptional regulatory mechanisms, it can specifically degrade target gene mRNA related to metabolic diseases, such as INHBE, thereby effectively inhibiting the expression of the target gene and achieving the purpose of effectively treating such diseases.
  • WO2022028462A relates to an anti-off-target modified siRNA, and the present disclosure cites the full text of the above patent application.
  • WO2023274395A relates to a nucleic acid ligand and a conjugate thereof, and the present disclosure cites the full text of the above patent application.
  • the present disclosure provides a siRNA targeting INHBE.
  • the present disclosure provides a siRNA comprising a sense strand and an antisense strand forming a double-stranded region
  • the positive strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21) consecutive nucleotides and differs from any nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 9 by no more than 3 (e.g., 0, 1, 2, 3) nucleotides;
  • the antisense strand contains at least 15 (e.g., 16, 17, 18, 19, 20, 21, 22, 23) consecutive nucleotides and differs from the nucleotide sequence of any one of SEQ ID NO: 10 or SEQ ID NO: 18 by no more than 3 (e.g., 0, 1, 2, 3) nucleotides.
  • 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, 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 disclosed siRNAs comprise one or two blunt ends.
  • each strand of the siRNA independently comprises 1 to 2 unpaired nucleotides.
  • the disclosed siRNA comprises an overhang at the 3' end of the antisense strand.
  • the sense strand and the 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 (e.g., 19, 20, 21, 22, 23 nucleotides).
  • the sense strand and the antisense strand are the same or different in length, wherein the sense strand is 19-23 nucleotides in length and the antisense strand is 19-26 nucleotides in length.
  • the length ratio of the sense strand and the antisense strand of the siRNA provided by the present disclosure can be 19/19, 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/19, 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 of the siRNA is 19/21, 21/21, 21/23, or 23/25. In some embodiments, the length ratio of the sense strand to the antisense strand of the siRNA is 19/21, 21/21 or 21/23. In some embodiments, the length ratio of the sense strand to the antisense strand of the siRNA is 19/21.
  • the sense strand comprises at least 15 consecutive nucleotides and differs by no more than 2 nucleotides from the nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 9, SEQ ID NO: 19 to SEQ ID NO: 122.
  • the nucleotide sequence differs by no more than 1 nucleotide; in some embodiments, the difference is 1 nucleotide;
  • the antisense chain comprises at least 15 consecutive nucleotide sequences and differs from any one of SEQ ID NO: 10 to SEQ ID NO: 18, SEQ ID NO: 123 to SEQ ID NO: 226 by no more than 2 nucleotides; in some embodiments, the nucleotide sequence differs by no more than 1 nucleotide; in some embodiments, the difference is 1 nucleotide.
  • the sense strand comprises at least 15 consecutive nucleotides of a nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 9, SEQ ID NO: 19 to SEQ ID NO: 122. In some embodiments, the sense strand comprises at least 16 consecutive nucleotides of a nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 9, SEQ ID NO: 19 to SEQ ID NO: 122. In some embodiments, the sense strand comprises at least 17 consecutive nucleotides of a nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 9, SEQ ID NO: 19 to SEQ ID NO: 122. In some embodiments, the sense strand comprises at least 19 consecutive nucleotides of a nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 9, SEQ ID NO: 19 to SEQ ID NO: 122.
  • the antisense strand comprises at least 15 consecutive nucleotides of any one of the nucleotide sequences of SEQ ID NO:10 to SEQ ID NO:18, SEQ ID NO:123 to SEQ ID NO:226. In some embodiments, the antisense strand comprises at least 17 consecutive nucleotides of any one of the nucleotide sequences of SEQ ID NO:10 to SEQ ID NO:18, SEQ ID NO:123 to SEQ ID NO:226. In some embodiments, the antisense strand comprises at least 19 consecutive nucleotides of any one of the nucleotide sequences of SEQ ID NO:10 to SEQ ID NO:18, SEQ ID NO:123 to SEQ ID NO:226.
  • the antisense strand comprises at least 20 consecutive nucleotides of any one of the nucleotide sequences of SEQ ID NO:10 to SEQ ID NO:18, SEQ ID NO:123 to SEQ ID NO:226. In some embodiments, the antisense strand comprises at least 21 consecutive nucleotides of any one of the nucleotide sequences of SEQ ID NO:10 to SEQ ID NO:18, and SEQ ID NO:123 to SEQ ID NO:226.
  • the positive chain comprises or is selected from any one of the following nucleotide sequences: SEQ ID NO: 1 to SEQ ID NO: 9, SEQ ID NO: 19 to SEQ ID NO: 122.
  • the antisense strand comprises or is selected from any of the following nucleotide sequences: SEQ ID NO: 10 to SEQ ID NO: 18, SEQ ID NO: 123 to SEQ ID NO: 226.
  • the present invention provides a siRNA targeting inhibin ⁇ E, which comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the antisense strand comprise or are selected from the following combinations:
  • At least one nucleotide in the sense strand and/or the antisense strand is a modified nucleotide.
  • all nucleotides are modified nucleotides.
  • the modified nucleotide is a 2'-methoxy modified nucleotide or a 2'-fluoro modified nucleotide.
  • three consecutive nucleotides in the sense strand of the siRNA are 2'-fluoro-modified nucleotides.
  • three consecutive nucleotides at positions 7-9 at the 5' end are 2'-fluoro-modified nucleotides.
  • the three consecutive nucleotides at positions 7-9 of the 5' end of the sense strand of the siRNA are 2'-fluoro-modified nucleotides, and the remaining positions of the sense strand are 2'-methoxy-modified nucleotides.
  • the four nucleotides at positions 5 and 7-9 of the 5' end of the sense strand are each independently 2'-fluoro-modified nucleotides, and the remaining positions of the sense strand are 2'-methoxy-modified nucleotides.
  • the nucleotides at positions 2, 6, 12, 14 and 16 of the antisense strand are each independently a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions are 2'-methoxy-modified nucleotides.
  • the nucleotides at positions 2, 4, 6, 10, 12, 14, 16, and 18 of the antisense strand are each independently a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions are 2'-methoxy-modified nucleotides.
  • the nucleotides at positions 2, 6, 14 and 16 of the antisense strand are each independently a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions are 2'-methoxy-modified nucleotides.
  • At least one phosphodiester group in the sense strand and/or the antisense strand is a phosphodiester group with a modifying group.
  • the modifying group enables the siRNA to have increased stability in a biological sample or environment.
  • the phosphodiester group with a modifying group is a thiophosphate diester group.
  • the phosphorothioate diester group is present in at least one of the following positions:
  • the 3' end of the antisense strand is between the second nucleotide and the third nucleotide.
  • the sense strand and/or the antisense strand includes a plurality of phosphorothioate diester groups, wherein the phosphorothioate Phosphodiester groups are found in:
  • the 3' end of the antisense strand is between the second nucleotide and the third nucleotide.
  • the present disclosure also provides a siRNA conjugate, which comprises any one of the above siRNAs and a targeting ligand linked to a terminal of the siRNA.
  • the siRNA 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, wherein the galactose derivative is selected from N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine or N-isobutyrylgalactosamine.
  • ASGPR asialoglycoprotein receptor
  • the targeting ligand comprises one or more of N-acetyl-galactosamine, N-trifluoroacetyl-galactosamine, N-propionyl-galactosamine, N-n-butyryl-galactosamine or N-isobutyryl-galactosamine, preferably, the targeting ligand comprises N-acetyl-galactosamine. In some embodiments, the targeting ligand is attached to the 3' end of the sense strand of the siRNA.
  • the targeting ligand is linked to the 3' end or 5' end of the sense strand or antisense strand in the siRNA through a phosphodiester group, a phosphorothioate diester group, or a phosphonic acid group.
  • the targeting ligand is linked to the end of the siRNA via a phosphodiester group.
  • the targeting ligand is indirectly linked to the siRNA end via a phosphodiester group, a thiophosphodiester group, or a phosphonic acid group. In some embodiments, the targeting ligand is indirectly linked to the siRNA end via a phosphodiester group.
  • the targeting ligand is directly linked to the end of the siRNA via a phosphodiester group, a phosphorothioate diester group, or a phosphonic acid group; in some embodiments, the targeting ligand is directly linked to the end of the siRNA via a phosphodiester group.
  • the targeting ligand is directly linked to the 3' end of the siRNA 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 siRNA sense strand via a phosphodiester group.
  • the targeting portion of the targeting ligand is composed of one or more targeting groups or targeting moieties, and the targeting ligand assists in directing 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.
  • the targeting ligand may include 2, 3, 4 or more than 4 targeting moieties. In some embodiments, the targeting ligand disclosed herein may include 1, 2, 3, 4 or more than 4 targeting moieties connected to the branching group via L2 .
  • each targeting moiety 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 ligand of the present disclosure includes N-acetylgalactosamine as a targeting moiety.
  • the targeting ligand comprises three terminal galactosamine or galactosamine derivatives (such as N-acetyl-galactosamine), each of which has affinity for sialoglycoprotein receptors.
  • the targeting ligand comprises three terminal N-acetyl-galactosamine (GalNAc or NAG) as targeting moieties.
  • the targeting ligand comprises four terminal galactosamines or galactosamine derivatives (such as N-acetyl-galactosamine), each of which has affinity for asialoglycoprotein receptors.
  • the targeting ligand comprises four terminal N-acetyl-galactosamines (GalNAc or NAG) as targeting moieties.
  • the targeting ligand provided by the present disclosure is a compound represented by formula (II) or formula (III): 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 siRNA and/or siRNA conjugate of the present disclosure has a nucleotide sequence as shown in any pair of sense strands and antisense strands in Table 1a, 1b or Table 2a, 2b.
  • the siRNA and/or siRNA conjugate disclosed herein comprises or is selected from any one of SEQ ID NO: 275 to SEQ ID NO: 383.
  • the sense strand in the siRNA and/or siRNA conjugate disclosed herein comprises or is selected from a nucleotide sequence as shown in any one of SEQ ID NO:227 to SEQ ID NO:242, SEQ ID NO:243 to SEQ ID NO:250, and SEQ ID NO:275 to SEQ ID NO:383; and/or, the antisense strand comprises or is selected from a nucleotide sequence as shown in any one of SEQ ID NO:251 to SEQ ID NO:266, SEQ ID NO:267 to SEQ ID NO:274, and SEQ ID NO:384 to SEQ ID NO:492.
  • the sense strand and the antisense strand comprise or are selected from the following combinations: 1) the sense strand shown in SEQ ID NO: 227 and the antisense strand shown in SEQ ID NO: 251;
  • the present disclosure provides a composition comprising the siRNA, siRNA conjugate described in the present disclosure, and one or more pharmaceutically acceptable excipients, such as a vehicle, a carrier, a diluent, and/or a delivery polymer.
  • a pharmaceutically acceptable excipients such as a vehicle, a carrier, a diluent, and/or a delivery polymer.
  • siRNA or siRNA conjugates such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing siRNA or siRNA conjugates, receptor-mediated endocytosis, constructing the nucleic acid as part of a retroviral or other vector.
  • the present disclosure provides a use of the above-mentioned siRNA, siRNA conjugate or composition in the preparation of a medicament for treating a disease in a subject, which in some embodiments is selected from a liver-related disease.
  • the present disclosure provides a method for treating a disease in a subject, comprising administering the above-mentioned siRNA, siRNA conjugate or composition to the subject.
  • the present disclosure provides a method for inhibiting mRNA expression in a subject, the method comprising administering the above-mentioned siRNA, siRNA conjugate or composition to the subject.
  • the present disclosure provides a method for delivering an expression inhibitory oligomeric compound to the liver in vivo, the method comprising administering the above-mentioned siRNA, siRNA conjugate or composition to a subject.
  • siRNA, siRNA conjugates or compositions and methods disclosed herein can reduce the level of target mRNA in a cell, a cell population, a cell population, a tissue or a subject, comprising: administering to the subject a therapeutically effective amount of the siRNA, siRNA conjugate or composition described in the present disclosure, wherein the siRNA 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 pathological upregulation of the target gene in the targeted cells or tissues.
  • the subject described in the present disclosure refers to a subject diagnosed with (or suspected of having, or susceptible to) 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 also provides a pharmaceutical composition comprising the siRNA or siRNA conjugate of 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 unit dose of the pharmaceutical composition is 0.001 mg-1000 mg.
  • the pharmaceutical composition contains 0.01-99.99% of the aforementioned compound or its pharmaceutically acceptable salt or its isotope substitution, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1-99.9% of the aforementioned compound or its pharmaceutically acceptable salt or its isotope substitution. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned compound or its pharmaceutically acceptable salt or its isotope substitution. In certain embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned compound or its pharmaceutically acceptable salt or its isotope substitution. In certain embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned compound or its pharmaceutically acceptable salt or its isotope substitution.
  • the pharmaceutical composition contains 0.01%-99.99% of a pharmaceutically acceptable excipient based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1%-99.9% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition contains 1%-99% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition contains 2%-98% of a pharmaceutically acceptable excipient.
  • the siRNA, siRNA conjugate or pharmaceutical composition when contacted with a cell expressing the target gene, inhibits the expression of the target gene by 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%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least
  • the residual expression percentage of the target gene mRNA caused by the above-mentioned siRNA 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 siRNA conjugate when the above-mentioned siRNA, siRNA conjugate or pharmaceutical composition is contacted with cells expressing the target gene, the siRNA conjugate 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
  • the siRNA conjugate when the above-mentioned siRNA, siRNA conjugate or pharmaceutical composition is contacted with cells expressing the target gene, the siRNA conjugate 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 least 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 Blot, or flow cytometry.
  • the siRNA conjugate when the above-mentioned siRNA, siRNA conjugate or pharmaceutical composition is contacted with a cell expressing the target gene, the siRNA conjugate increases the on-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, 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
  • the present disclosure also provides a cell, which comprises the siRNA or siRNA conjugate of the present disclosure.
  • the present disclosure also provides a kit comprising the siRNA, siRNA conjugate or pharmaceutical composition of the present disclosure.
  • 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 steps of introducing the siRNA, siRNA conjugate and/or pharmaceutical composition according to the present disclosure into the cell. Steps.
  • 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 the siRNA, siRNA conjugate and/or pharmaceutical composition according to the present disclosure into the cell.
  • the present disclosure also provides a method for inhibiting the expression of a target gene or mRNA of a target gene, the method comprising administering an effective amount or an effective dose of the siRNA, siRNA 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 siRNA, siRNA 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 inhibin beta E (INHBE).
  • the present disclosure provides the aforementioned siRNA and/or pharmaceutical composition and/or siRNA conjugate for treating and/or preventing a disease in a subject related to inhibin ⁇ E (INHBE) gene expression; in some embodiments, the disease is selected from metabolic disorders; in some embodiments, the disease is selected from metabolic syndrome (MS), cardiovascular disease, obesity, hepatitis, and kidney disease.
  • a disease in a subject related to inhibin ⁇ E IDHBE gene expression
  • the disease is selected from metabolic disorders; in some embodiments, the disease is selected from metabolic syndrome (MS), cardiovascular disease, obesity, hepatitis, and kidney disease.
  • the present disclosure provides a method for treating and/or preventing a disease associated with inhibin ⁇ E (INHBE) gene expression in a subject, comprising administering to the subject an effective amount or an effective dose of the siRNA, siRNA conjugate or the pharmaceutical composition described in the present disclosure; in some embodiments, the disease is selected from metabolic disorders; in some embodiments, the disease is selected from metabolic syndrome (MS), cardiovascular disease, obesity, hepatitis, and kidney disease.
  • a disease associated with inhibin ⁇ E (INHBE) gene expression comprising administering to the subject an effective amount or an effective dose of the siRNA, siRNA conjugate or the pharmaceutical composition described in the present disclosure; in some embodiments, the disease is selected from metabolic disorders; in some embodiments, the disease is selected from metabolic syndrome (MS), cardiovascular disease, obesity, hepatitis, and kidney disease.
  • MS metabolic syndrome
  • the present disclosure provides the use of the aforementioned siRNA and/or pharmaceutical composition and/or siRNA conjugate in the preparation of a medicament for treating and/or preventing a disease associated with inhibin ⁇ E (INHBE) gene expression; in some embodiments, the disease is selected from metabolic disorders; in some embodiments, the disease is selected from metabolic syndrome (MS), cardiovascular disease, obesity, hepatitis, and kidney disease.
  • a disease associated with inhibin ⁇ E IDHBE
  • the disease is selected from metabolic disorders; in some embodiments, the disease is selected from metabolic syndrome (MS), cardiovascular disease, obesity, hepatitis, and kidney disease.
  • the present disclosure provides use of the aforementioned siRNA and/or pharmaceutical composition and/or siRNA conjugate in the preparation of a medicament for inhibiting the expression of INHBE.
  • the present disclosure provides a method for inhibiting INHBE expression, comprising administering an effective amount or effective dose of the aforementioned siRNA and/or pharmaceutical composition and/or siRNA conjugate to a subject.
  • the present disclosure provides the aforementioned siRNA and/or pharmaceutical composition and/or siRNA conjugate for use in treating and/or preventing a disease; in some embodiments, the disease is selected from metabolic disorders; in some embodiments, the disease is selected from metabolic syndrome (MS), cardiovascular disease, obesity, hepatitis, and kidney disease.
  • MS metabolic syndrome
  • 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 siRNA and/or pharmaceutical composition and/or siRNA conjugate; in some embodiments, the The disease is selected from metabolic disorders; in some embodiments, the disease is selected from metabolic syndrome (MS), cardiovascular disease, obesity, hepatitis, and kidney disease.
  • MS metabolic syndrome
  • the present disclosure provides the use of the aforementioned siRNA and/or pharmaceutical composition and/or siRNA conjugate in the preparation of a medicament for treating and/or preventing a disease; in some embodiments, the disease is selected from metabolic disorders; in some embodiments, the disease is selected from metabolic syndrome (MS), cardiovascular disease, obesity, hepatitis, and kidney disease.
  • MS metabolic syndrome
  • the present disclosure provides a method for delivering siRNA that inhibits INHBE expression and/or replication to the liver in vivo, the method comprising administering the aforementioned siRNA and/or pharmaceutical composition and/or siRNA conjugate to a subject.
  • the present disclosure also provides a method for preparing siRNA or siRNA conjugates, which comprises: synthesizing the siRNA or siRNA conjugates described in the present disclosure.
  • the present disclosure also provides a siRNA or siRNA conjugate, characterized in that one or more bases U of any siRNA or siRNA conjugate of the present disclosure are replaced with base T, for example, 1, 2, 3, 3, 5, 6, 7, 8, 9, 10 or all bases U.
  • 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 disclosed compounds may exist in specific geometric or stereoisomeric forms.
  • the 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 enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the 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 disclosure.
  • the disclosed compounds may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers are included, such as enantiomers and diastereomers.
  • the disclosed compounds containing asymmetric carbon atoms may be isolated in optically pure forms or in racemic forms. Optically pure forms may be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents.
  • Optically active (R)- and (S)-isomers as well as 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 resolved by conventional methods known in the art, and then the pure enantiomer is recovered.
  • the separation of enantiomers and diastereomers is typically accomplished by using chromatography, which employs a chiral stationary phase and is optionally combined with a chemical derivatization method (e.g., by Amines form carbamates).
  • the present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but in which one or more atoms are replaced by atoms having an atomic mass or mass number different from that normally found in nature.
  • isotopes that can be incorporated into compounds of the present disclosure 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 , respectively.
  • deuterium when a position is specifically designated as deuterium (D), the position is understood to have deuterium (i.e., at least 10% deuterium incorporation) at least 1000 times greater than the natural abundance of deuterium (which is 0.015%).
  • the natural abundance of the compound in the example may be at least 1000 times greater than deuterium, at least 2000 times greater than deuterium, at least 3000 times greater than deuterium, at least 4000 times greater than deuterium, at least 5000 times greater than deuterium, at least 6000 times greater than deuterium or more abundant deuterium.
  • the present disclosure also includes various deuterated forms of compounds of formula I and formula II. Each available hydrogen atom connected to a carbon atom may be independently replaced by a deuterium atom.
  • deuterated starting materials may be used in the preparation of deuterated forms of compounds of Formula I and Formula II, or they may be synthesized using conventional techniques using deuterated reagents, including but not limited to deuterated borane, trideuterated borane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated ethyl iodide, deuterated methyl iodide, and the like.
  • the bonds Indicates that the configuration is not specified, that is, if there are chiral isomers in the chemical structure, the bond Can be or include both Although all of the above structural formulas are drawn as certain isomers for simplicity, the present disclosure may include all isomers, such as tautomers, rotational isomers, geometric isomers, diastereomers, racemates and enantiomers.
  • the bonds No configuration is specified, i.e., the bond The configuration can be E-type or Z-type, or include both E and Z configurations.
  • INHBE includes but is not limited to human INHBE, cynomolgus monkey INHBE, mouse INHBE, and rat INHBE, and its amino acid and complete coding sequence and mRNA sequence are easily obtained using public databases, such as GenBank, UniProt, OMIM, and the Macaca genome project website.
  • INHBE also refers to naturally occurring DNA sequence variations of the INHBE gene, such as single nucleotide polymorphisms (SNPs) in the INHBE gene.
  • SNPs single nucleotide polymorphisms
  • Exemplary SNPs can be found in the dbSNP database.
  • target sequence refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during INHBE transcription, including mRNAs that are RNA processing products 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 INHBE.
  • the sense strand (also referred to as SS, SS strand or sense strand) refers to a strand comprising a sequence identical or substantially identical to a target mRNA sequence;
  • the antisense strand (also referred to as AS or AS strand) refers to a strand having a sequence complementary to a target mRNA sequence.
  • the term "differences from the nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 106 by no more than 3 nucleotides, and comprises at least 15 consecutive nucleotides" is intended to indicate that the siRNA sense strand described herein comprises at least 15 consecutive nucleotides of the sense strand of any one of SEQ ID NO: 1 to SEQ ID NO: 106, or differs from at least 15 consecutive nucleotides of the sense strand of any one of SEQ ID NO: 1 to SEQ ID NO: 106 by no more than 3 nucleotides (optionally, differs by no more than 2 nucleotides; optionally, differs by 1 nucleotide).
  • 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, respectively, which include the bases of guanine, cytosine, adenine, thymidine and uracil. It is well known to those skilled in the art that the replacement of bases T and U will not significantly affect the properties of the RNAi agent sequence. U in the sequence of the present disclosure can be arbitrarily replaced by T, and the sequence obtained after the replacement is also within the scope of protection of the present disclosure.
  • RNAi agent can all exist independently in the form of salt, mixed salt or non-salt (such as free acid or free base).
  • a salt or a mixed salt it can be a pharmaceutically acceptable salt.
  • pharmaceutically acceptable salt includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.
  • the salt forms of the following structures corresponding to each structure are also within the scope of protection of the present disclosure.
  • the 3' position of the first nucleotide at the 3' end of each chain is a hydroxyl group; the 5' position of the first nucleotide at the 5' end of each chain is a hydroxyl group.
  • 2'-fluoro modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is substituted with fluorine
  • non-2'-fluoro 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 substituted with a non-fluorine group.
  • 2'-methoxy modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.
  • 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.
  • the term “inhibit” can be used interchangeably with “reduce”, “silence”, “downregulate”, “suppress” and other similar terms, and includes any level of inhibition. Inhibition can be assessed by the reduction of one or more of these variables in absolute or relative levels compared to the control level.
  • the control level can be any type of control level used in the art, such as a baseline level before administration or a level determined from an untreated or controlled subject, cell, or sample (e.g., only a buffer control or an inert agent control).
  • the remaining expression of mRNA can be used to characterize the degree of inhibition of siRNA on target gene expression, such as the remaining expression of mRNA being no more than 99%, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10%.
  • the "compound”, “ligand”, “nucleic acid ligand conjugate”, “siRNA conjugate”, “nucleic acid”, “conjugate”, “chemical modification”, “targeting ligand”, “dsRNA”, “RNAi” of the present disclosure can independently exist in the form of salt, mixed salt or non-salt (such as free acid or free base). When it exists in the form of salt or mixed salt, it can 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 salts, sodium salts, potassium salts, calcium salts, and magnesium salts, preferably sodium salts.
  • 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 basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol,
  • the preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. These salts can be prepared by methods known in the art.
  • Effective amount refers to the amount of a drug, compound, or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results.
  • beneficial or desired results include eliminating or reducing the risk, reducing the severity, or delaying the onset of a condition, including the biochemical, histological, and/or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes presented during the development of the condition.
  • beneficial or desired results include clinical results, such as reducing the incidence of various target gene, target mRNA, or target protein-related conditions of the present invention or improving one or more symptoms of the condition, reducing the dose of other agents required to treat the condition, enhancing the efficacy of another agent, and/or delaying the progression of a target gene, target mRNA, or target protein-related condition of the present invention in a patient.
  • patient As used herein, “patient,” “subject,” or “individual” are used interchangeably and include humans or non-human animals, such as mammals, such as humans or monkeys.
  • the siRNA 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 already has commercial customization services.
  • the modified nucleotide groups can be introduced into the siRNA 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 siRNA are also well known to those skilled in the art.
  • 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.
  • blunt end or blunt end are used interchangeably and refer to the absence of unpaired nucleotides or nucleotide analogs at a given end of the siRNA, i.e., no nucleotide overhangs. In most cases, a siRNA with both ends blunt-ended will be double-stranded throughout its entire length.
  • the group In It can be replaced by 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 means that a first compound or group is 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.
  • the human INHBE gene (NM_031479.5) was used as the target gene to design 19/21nt siRNA to meet the general rules of active siRNA.
  • the unmodified sense and antisense strand sequences are shown in Table 1a and Table 1b.
  • the sequences of the sense and antisense strands modified with 2'-fluoro, 2'-methoxy, etc. are detailed in Tables 2a and 2b.
  • the sequence represents the direction from 5' to 3' from left to right
  • the lowercase letter m indicates that the nucleoside adjacent to the left side of the letter m is a 2'-methoxy-modified nucleoside
  • the lowercase letter f indicates that the nucleoside adjacent to the left side of the letter f is a 2'-fluoro-modified nucleoside
  • the lowercase letter s indicates that the connection between the two nucleosides adjacent to the left and right of the letter s or the connected nucleosides and the delivery group NAG0052' or L96' is a thiophosphate diester connection
  • two adjacent nucleosides are connected by a phosphodiester group
  • the 3' position of the first nucleotide at the 3' end of each chain is a hydroxyl group
  • the 5' position of the first nucleotide at the 5' end of each chain is a hydroxyl group.
  • siRNA or siRNA conjugate of the present invention is in the form of a salt, for example, in the form of a sodium salt, the structure of the salt form corresponding to the structure in Table 3 below is Also within the scope of protection of this disclosure:
  • Base represents a base.
  • siRNA is no different from the usual phosphoramidite solid phase synthesis method.
  • the synthesis process is briefly described as follows: On the Dr.Oligo48 synthesizer (Biolytic), starting with the universal CPG carrier, nucleoside phosphoramidite monomers are connected one by one according to the synthesis procedure. 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
  • the oligonucleotide was cleaved from the solid support and soaked in a 3:1 28% ammonia and ethanol solution at 50°C for 16 hours. Then centrifuged, the supernatant was transferred to another centrifuge tube, concentrated and evaporated to dryness, and purified by C18 reverse chromatography with a mobile phase of 0.1M TEAA and acetonitrile, and DMTr was removed using a 3% trifluoroacetic acid solution. The target oligonucleotide was collected and freeze-dried, and identified as the target by LC-MS. The product was then quantified by UV (260 nm).
  • the obtained single-stranded oligonucleotides were annealed according to complementary pairing in an equal molar ratio, and the double-stranded siRNA obtained was finally dissolved in 1 ⁇ PBS and adjusted to the required concentration for the experiment.
  • siRNA conjugate containing NAG0052' was prepared according to the same method as described in Example WO2023138663A1; the siRNA conjugate containing L96' was prepared according to the same method as described in Example WO2014025805A1.
  • the psi-CHECK plasmid used in this example was purchased from Sangon Biotechnology (Shanghai) Co., Ltd.
  • the on-target sequence corresponding to the siRNA was constructed with the INHBE gene and inserted into the psiCHECK-2 plasmid, i.e., the GSCM on-target plasmid.
  • the plasmid contains the Renilla luciferase gene and the firefly luciferase gene.
  • the target sequence of the siRNA is inserted into the 3’UTR region of the Renilla luciferase gene.
  • the activity of the siRNA against the target sequence can be reflected by the detection of the expression of the Renilla luciferase after calibration with the firefly luciferase.
  • the detection uses the Dual-Luciferase Reporter Assay System (Promega, E2940).
  • 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 siRNA and corresponding plasmids using Lipofectamine 2000 (ThermoFisher, 11668019), and 0.2 ⁇ L of Lipofectamine 2000 was used per well.
  • the amount of plasmid transfection was 20 ng per well.
  • 9 concentration points were set for siRNA, and the final concentration of the highest concentration point was 20 nM, 4-fold gradient dilution, 20 nM, 5 nM, 1.25 nM, 0.3125 nM, 0.0781 nM, 0.0195 nM, 0.0049 nM, 0.0012 nM, 0.0003 nM.
  • the Dual-Luciferase Reporter Assay System (Promega, E2940) was used to detect the target level.
  • Hep3B cells were seeded in 96-well plates at approximately 20,000 cells/well, with 100 ⁇ L of culture medium per well.
  • samples were transfected with Lipofectamine RNAi MAX (ThermoFisher, 13778150) according to the product manual, and the final concentration of the sample transfection gradient was 20, 4, 0.8, 0.16, 0.032, 0.0064 and 0.00128 nM.
  • FG0417-L/FG0418-XL magnetic bead method
  • RNA reverse transcription experiment Takara, RR037A
  • Real-time quantitative PCR Thermo, 4444557
  • results are expressed as the percentage of human INHBE mRNA expression remaining relative to the siRNA-treated cells, wherein the IC50 value corresponds to the concentration of siRNA used when the percentage of INHBE mRNA expression remaining is 50%.
  • Example 5 Inhibitory activity of INHBE at multiple concentrations in primary human hepatocytes (PHH)
  • PHH cells were seeded in 96-well plates at approximately 30,000 cells/well with 100 ⁇ L of culture medium per well.
  • samples were transfected with Lipofectamine RNAi MAX (ThermoFisher, 13778150) according to the product manual, and the final concentration of the sample transfection gradient was 20, 4, 0.8, 0.16, 0.032, 0.0064 and 0.00128 nM.
  • FG0417-L/FG0418-XL magnetic bead method
  • RNA reverse transcription experiment Takara, RR037A
  • Real-time quantitative PCR Thermo, 4444557
  • siRNA was screened for on-target activity at the molecular level using 9 concentration gradients.
  • the experimental process, experimental materials and instruments used, dilution process, and result analysis method were the same as those in Example 3.
  • the result >20 means that under the condition of the highest concentration of 20 nM in this example, the inhibition rate cannot reach 50%, and the siRNA disclosed in the present invention has a good inhibitory effect on INHBE.
  • Example 8 Inhibitory activity of INHBE at multiple concentrations in human hepatoma cell line (Huh7)
  • Huh7 cells were seeded in 96-well plates at approximately 15,000 cells/well with 100 ⁇ L of culture medium per well.
  • samples were transfected with Lipofectamine RNAi MAX (ThermoFisher, 13778150) according to the product manual, and the final concentration of the sample transfection gradient was 20, 4, 0.8, 0.16, 0.032, 0.0064 and 0.00128 nM.
  • FG0417-L/FG0418-XL magnetic bead method
  • RNA reverse transcription experiment Takara, RR037A
  • Real-time quantitative PCR Thermo, 4444557
  • Example 9 Inhibitory activity of INHBE at multiple concentrations in primary monkey hepatocytes (PCH)
  • PCH cells were seeded in a 96-well plate at approximately 30,000 cells/well, with 100 ⁇ L per well. Culture medium. During transfection, refer to the product manual and use Lipofectamine RNAi MAX (ThermoFisher, 13778150) to transfect the sample. The final concentration of the gradient transfection of the sample is 20, 4, 0.8, 0.16, 0.032, 0.0064 and 0.00128 nM.
  • RNA of the cells was extracted using a high-throughput cell RNA extraction kit (FG0417-L/FG0418-XL, magnetic bead method), RNA reverse transcription experiment (Takara, RR037A) and real-time quantitative PCR (Thermo, 4444557) to measure the mRNA level of monkey INHBE, and the mRNA level of monkey INHBE was corrected according to the level of the GAPDH internal reference gene.
  • siRNA was screened for on-target activity at the molecular level in vitro using 9 concentration gradients.
  • the experimental process, experimental materials and instruments used, and dilution process were the same as in Example 3.
  • the data results in Table 17 show that compared with the control sequence, the siRNA disclosed in the present invention has a higher level of on-target inhibitory activity.
  • siRNA was screened for on-target activity at the molecular level using 9 concentration gradients.
  • the experimental process, experimental materials and instruments used, dilution process, and result analysis method were the same as those in Example 3.
  • the data results in Table 18 show that the siRNA disclosed herein has a high level of on-target inhibitory activity.

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Abstract

涉及靶向抑制素βE的siRNA、siRNA缀合物及其医药用途；具体地，涉及靶向INHBE的siRNA、siRNA缀合物、组合物及其医药用途。还涉及包含该siRNA的药物组合物、细胞或试剂盒，以及该siRNA、siRNA缀合物用于治疗和/或预防患相关障碍的受试者的方法。

Description

靶向抑制素βE的siRNA、siRNA缀合物及其医药用途
本公开要求2023年3月2日提交的中国专利申请202310194029.7、2023年4月19日提交的中国专利申请202310420703.9、2023年9月28日提交的中国专利申请202311271773.9和2024年1月8日提交的中国专利申请202410023838.6的优先权，上述专利申请的全部内容通过引用并入本文。
技术领域
本公开属于生物医药领域，具体涉及靶向抑制素βE基因(INHBE)的siRNA、缀合物、组合物及其医药用途。
背景技术
抑制素βE基因(INHBE)是TGF-β(转化生长因子-β)超家族蛋白中的成员之一，参与调节许多细胞过程,包括细胞增殖、细胞凋亡、免疫反应和激素分泌。抑制素负责抑制垂体分泌促卵泡素，可以参与调节许多不同的功能，例如下丘脑和垂体激素分泌、性腺激素分泌、生殖细胞发育和成熟、红细胞分化、胰岛素分泌、神经细胞存活、胚胎轴向发育或骨骼生长等。此外,INHBE可能在内质网应激条件下被上调,从而该蛋白可能抑制胰腺和肝脏上的细胞增殖和生长。
体脂分布是心血管疾病和代谢疾病的一个重要判断依据，在各种体脂分布中，有一种特殊的体脂分布，很容易导致严重的心血管疾病或者代谢类疾病，其特征是腰部脂肪分布较多，和/或臀部脂肪堆积较少，从而导致腰臀比(WHR)较大。由于体脂分布异常导致的代谢类疾病包括：2型糖尿病、高脂血症、血脂异常(低密度脂蛋白胆固醇LDL-C、甘油三酯、极低密度脂蛋白胆固醇VLDL-C、载脂蛋白B或其他脂质部分的循环水平升高或改变)、肥胖症(特别是腹部肥胖)、脂肪代谢障碍、胰岛素抵抗、非酒精性脂肪性肝炎、高血压和/或高血压、高血糖或葡萄糖或高血糖症、代谢综合征、冠状动脉疾病和其他动脉粥样硬化病症等。
目前，治疗代谢类疾病的方法主要有改变生活习惯、调节饮食、运动健身、服用降脂药(例如他汀类药物)，但是这些方法效果较差，并且服用降脂药会产生一些副作用。干扰RNAi(siRNA)是一种有效的沉默基因表达的方式，通过转录后调控机制，可以特异性降解与代谢类疾病相关的靶基因mRNA，例如INHBE，从而有效的抑制该靶基因表达，达到有效治疗此类疾病的目的。
发明内容
WO2022028462A涉及一种防脱靶修饰的siRNA，本公开引用上述专利申请的全文。
WO2023274395A涉及一种核酸配体及其缀合物，本公开引用上述专利申请的全文。
本公开提供一种靶向INHBE的siRNA。
一些实施方案中，本公开提供了一种siRNA，其包含形成双链区的正义链与反义链；
所述正义链包含至少15个(例如16、17、18、19、20、21)连续核苷酸，且与SEQ ID NO：1至SEQ ID NO：9中任一的核苷酸序列相差不超过3个(例如0、1、2、3)核苷酸；
所述反义链包含至少15个(例如16、17、18、19、20、21、22、23)连续核苷酸，且与SEQ ID NO：10或SEQ ID NO：18中任一的核苷酸序列相差不超过3个(例如0、1、2、3)核苷酸。
一些实施方案中，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，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，反义链与靶序列完全反向互补。
一些实施方案中，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，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，正义链与反义链完全反向互补。
一些实施方案中，本公开siRNA包含一个或两个平端。
一些具体的实施方案中，siRNA的每条链各自独立地包含具有1至2个未配对的核苷酸。
一些实施方案中，本公开siRNA包含位于所述反义链3’端的突出端。
一些实施方案中，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(例如19、20、21、22、23个核苷酸)。
一些实施方案中，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，反义链的长度为19-26个核苷酸。本公开提供的siRNA的正义链和反义链的长度比可以是19/19、19/20、19/21、19/22、19/23、19/24、19/25、19/26、20/19、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。
在一些实施方式中，所述siRNA的正义链和反义链的长度比为19/21、21/21、21/23或23/25。在一些实施方式中，所述siRNA的正义链和反义链的长度比为 19/21、21/21或21/23。在一些实施方案中，所述siRNA的正义链和反义链的长度比为19/21。
一些实施方案中，所述正义链包含至少15个连续核苷酸，且与SEQ ID NO：1至SEQ ID NO：9、SEQ ID NO:19至SEQ ID NO:122中任一的核苷酸序列相差不超过2个核苷酸。一些实施方案中，核苷酸序列相差不超过1个核苷酸；一些实施方案中，相差1个核苷酸；
一些实施方案中，所述反义链包含至少15个连续核苷酸序列，且与SEQ ID NO：10至SEQ ID NO：18、SEQ ID NO:123至SEQ ID NO:226中任一的核苷酸序列相差不超过2个核苷酸；一些实施方案中，核苷酸序列相差不超过1个核苷酸；一些实施方案中，相差1个核苷酸。
一些实施方案中，正义链包含SEQ ID NO：1至SEQ ID NO:9、SEQ ID NO：19至SEQ ID NO：122任一的核苷酸序列中的至少15个连续核苷酸。一些实施方案中，正义链包含含SEQ ID NO：1至SEQ ID NO:9、SEQ ID NO：19至SEQ ID NO：122任一的核苷酸序列中的至少16个连续核苷酸。一些实施方案中，正义链包含含SEQ ID NO：1至SEQ ID NO:9、SEQ ID NO：19至SEQ ID NO：122任一的核苷酸序列中的至少17个连续核苷酸。一些实施方案中，正义链包含含SEQ ID NO：1至SEQ ID NO:9、SEQ ID NO：19至SEQ ID NO：122任一的核苷酸序列中的至少19个连续核苷酸。
一些实施方案中，反义链包含SEQ ID NO:10至SEQ ID NO:18、SEQ ID NO：123至SEQ ID NO：226任一的核苷酸序列中的至少15个连续核苷酸。一些实施方案中，反义链包含SEQ ID NO:10至SEQ ID NO:18、SEQ ID NO：123至SEQ ID NO：226任一的核苷酸序列中的至少17个连续核苷酸。一些实施方案中，反义链包含SEQ ID NO:10至SEQ ID NO:18、SEQ ID NO：123至SEQ ID NO：226任一的核苷酸序列中的至少19个连续核苷酸。一些实施方案中，反义链包含SEQ ID NO:10至SEQ ID NO:18、SEQ ID NO：123至SEQ ID NO：226任一的核苷酸序列中的至少20个连续核苷酸。一些实施方案中，反义链包含SEQ ID NO:10至SEQ ID NO:18、SEQ ID NO：123至SEQ ID NO：226任一的核苷酸序列中的至少21个连续核苷酸。
一些实施方案中，所述正义链包含或选自以下的任一核苷酸序列：SEQ ID NO：1至SEQ ID NO:9、SEQ ID NO：19至SEQ ID NO：122。
一些实施方案中，所述反义链包含或选自以下的任一核苷酸序列：SEQ ID NO:10至SEQ ID NO:18、SEQ ID NO：123至SEQ ID NO：226。
在一些实施方案中，本文提供了一种靶向抑制素βE的siRNA，其包含形成双链区的正义链与反义链，其中所述正义链和反义链包含或选自以下组合：
1)SEQ ID NO:1所示的正义链和SEQ ID NO:10所示的反义链；
2)SEQ ID NO:2所示的正义链和SEQ ID NO:11所示的反义链；
3)SEQ ID NO:3所示的正义链和SEQ ID NO:12所示的反义链；
4)SEQ ID NO:4所示的正义链和SEQ ID NO:13所示的反义链；
5)SEQ ID NO:5所示的正义链和SEQ ID NO:14所示的反义链；
6)SEQ ID NO:6所示的正义链和SEQ ID NO:15所示的反义链；
7)SEQ ID NO:7所示的正义链和SEQ ID NO:16所示的反义链；
8)SEQ ID NO:8所示的正义链和SEQ ID NO:17所示的反义链；
9)SEQ ID NO:9所示的正义链和SEQ ID NO:18所示的反义链；
10)SEQ ID NO:19所示的正义链和SEQ ID NO:123所示的反义链；
11)SEQ ID NO:20所示的正义链和SEQ ID NO:124所示的反义链；
12)SEQ ID NO:21所示的正义链和SEQ ID NO:125所示的反义链；
13)SEQ ID NO:22所示的正义链和SEQ ID NO:126所示的反义链；
14)SEQ ID NO:23所示的正义链和SEQ ID NO:127所示的反义链；
15)SEQ ID NO:24所示的正义链和SEQ ID NO:128所示的反义链；
16)SEQ ID NO:25所示的正义链和SEQ ID NO:129所示的反义链；
17)SEQ ID NO:26所示的正义链和SEQ ID NO:130所示的反义链；
18)SEQ ID NO:27所示的正义链和SEQ ID NO:131所示的反义链；
19)SEQ ID NO:28所示的正义链和SEQ ID NO:132所示的反义链；
20)SEQ ID NO:29所示的正义链和SEQ ID NO:133所示的反义链；
21)SEQ ID NO:30所示的正义链和SEQ ID NO:134所示的反义链；
22)SEQ ID NO:31所示的正义链和SEQ ID NO:135所示的反义链；
23)SEQ ID NO:32所示的正义链和SEQ ID NO:136所示的反义链；
24)SEQ ID NO:33所示的正义链和SEQ ID NO:137所示的反义链；
25)SEQ ID NO:34所示的正义链和SEQ ID NO:138所示的反义链；
26)SEQ ID NO:35所示的正义链和SEQ ID NO:139所示的反义链；
27)SEQ ID NO:36所示的正义链和SEQ ID NO:140所示的反义链；
28)SEQ ID NO:37所示的正义链和SEQ ID NO:141所示的反义链；
29)SEQ ID NO:38所示的正义链和SEQ ID NO:142所示的反义链；
30)SEQ ID NO:39所示的正义链和SEQ ID NO:143所示的反义链；
31)SEQ ID NO:40所示的正义链和SEQ ID NO:144所示的反义链；
32)SEQ ID NO:41所示的正义链和SEQ ID NO:145所示的反义链；
33)SEQ ID NO:42所示的正义链和SEQ ID NO:146所示的反义链；
34)SEQ ID NO:43所示的正义链和SEQ ID NO:147所示的反义链；
35)SEQ ID NO:44所示的正义链和SEQ ID NO:148所示的反义链；
36)SEQ ID NO:45所示的正义链和SEQ ID NO:149所示的反义链；
37)SEQ ID NO:46所示的正义链和SEQ ID NO:150所示的反义链；
38)SEQ ID NO:47所示的正义链和SEQ ID NO:151所示的反义链；
39)SEQ ID NO:48所示的正义链和SEQ ID NO:152所示的反义链；
40)SEQ ID NO:49所示的正义链和SEQ ID NO:153所示的反义链；
41)SEQ ID NO:50所示的正义链和SEQ ID NO:154所示的反义链；
42)SEQ ID NO:51所示的正义链和SEQ ID NO:155所示的反义链；
43)SEQ ID NO:52所示的正义链和SEQ ID NO:156所示的反义链；
44)SEQ ID NO:53所示的正义链和SEQ ID NO:157所示的反义链；
45)SEQ ID NO:54所示的正义链和SEQ ID NO:158所示的反义链；
46)SEQ ID NO:55所示的正义链和SEQ ID NO:159所示的反义链；
47)SEQ ID NO:56所示的正义链和SEQ ID NO:160所示的反义链；
48)SEQ ID NO:57所示的正义链和SEQ ID NO:161所示的反义链；
49)SEQ ID NO:58所示的正义链和SEQ ID NO:162所示的反义链；
50)SEQ ID NO:59所示的正义链和SEQ ID NO:163所示的反义链；
51)SEQ ID NO:60所示的正义链和SEQ ID NO:164所示的反义链；
52)SEQ ID NO:61所示的正义链和SEQ ID NO:165所示的反义链；
53)SEQ ID NO:62所示的正义链和SEQ ID NO:166所示的反义链；
54)SEQ ID NO:63所示的正义链和SEQ ID NO:167所示的反义链；
55)SEQ ID NO:64所示的正义链和SEQ ID NO:168所示的反义链；
56)SEQ ID NO:65所示的正义链和SEQ ID NO:169所示的反义链；
57)SEQ ID NO:66所示的正义链和SEQ ID NO:170所示的反义链；
58)SEQ ID NO:67所示的正义链和SEQ ID NO:171所示的反义链；
59)SEQ ID NO:68所示的正义链和SEQ ID NO:172所示的反义链；
60)SEQ ID NO:69所示的正义链和SEQ ID NO:173所示的反义链；
61)SEQ ID NO:70所示的正义链和SEQ ID NO:174所示的反义链；
62)SEQ ID NO:71所示的正义链和SEQ ID NO:175所示的反义链；
63)SEQ ID NO:72所示的正义链和SEQ ID NO:176所示的反义链；
64)SEQ ID NO:73所示的正义链和SEQ ID NO:177所示的反义链；
65)SEQ ID NO:74所示的正义链和SEQ ID NO:178所示的反义链；
66)SEQ ID NO:75所示的正义链和SEQ ID NO:179所示的反义链；
67)SEQ ID NO:76所示的正义链和SEQ ID NO:180所示的反义链；
68)SEQ ID NO:77所示的正义链和SEQ ID NO:181所示的反义链；
69)SEQ ID NO:78所示的正义链和SEQ ID NO:182所示的反义链；
70)SEQ ID NO:79所示的正义链和SEQ ID NO:183所示的反义链；
71)SEQ ID NO:80所示的正义链和SEQ ID NO:184所示的反义链；
72)SEQ ID NO:81所示的正义链和SEQ ID NO:185所示的反义链；
73)SEQ ID NO:82所示的正义链和SEQ ID NO:186所示的反义链；
74)SEQ ID NO:83所示的正义链和SEQ ID NO:187所示的反义链；
75)SEQ ID NO:84所示的正义链和SEQ ID NO:188所示的反义链；
76)SEQ ID NO:85所示的正义链和SEQ ID NO:189所示的反义链；
77)SEQ ID NO:86所示的正义链和SEQ ID NO:190所示的反义链；
78)SEQ ID NO:87所示的正义链和SEQ ID NO:191所示的反义链；
79)SEQ ID NO:88所示的正义链和SEQ ID NO:192所示的反义链；
80)SEQ ID NO:89所示的正义链和SEQ ID NO:193所示的反义链；
81)SEQ ID NO:90所示的正义链和SEQ ID NO:194所示的反义链；
82)SEQ ID NO:91所示的正义链和SEQ ID NO:195所示的反义链；
83)SEQ ID NO:92所示的正义链和SEQ ID NO:196所示的反义链；
84)SEQ ID NO:93所示的正义链和SEQ ID NO:197所示的反义链；
85)SEQ ID NO:94所示的正义链和SEQ ID NO:198所示的反义链；
86)SEQ ID NO:95所示的正义链和SEQ ID NO:199所示的反义链；
87)SEQ ID NO:96所示的正义链和SEQ ID NO:200所示的反义链；
88)SEQ ID NO:97所示的正义链和SEQ ID NO:201所示的反义链；
89)SEQ ID NO:98所示的正义链和SEQ ID NO:202所示的反义链；
90)SEQ ID NO:99所示的正义链和SEQ ID NO:203所示的反义链；
91)SEQ ID NO:100所示的正义链和SEQ ID NO:204所示的反义链；
92)SEQ ID NO:101所示的正义链和SEQ ID NO:205所示的反义链；
93)SEQ ID NO:102所示的正义链和SEQ ID NO:206所示的反义链；
94)SEQ ID NO:103所示的正义链和SEQ ID NO:207所示的反义链；
95)SEQ ID NO:104所示的正义链和SEQ ID NO:208所示的反义链；
96)SEQ ID NO:105所示的正义链和SEQ ID NO:209所示的反义链；
97)SEQ ID NO:106所示的正义链和SEQ ID NO:210所示的反义链；
98)SEQ ID NO:107所示的正义链和SEQ ID NO:211所示的反义链；
99)SEQ ID NO:108所示的正义链和SEQ ID NO:212所示的反义链；
100)SEQ ID NO:109所示的正义链和SEQ ID NO:213所示的反义链；
101)SEQ ID NO:110所示的正义链和SEQ ID NO:214所示的反义链；
102)SEQ ID NO:111所示的正义链和SEQ ID NO:215所示的反义链；
103)SEQ ID NO:112所示的正义链和SEQ ID NO:216所示的反义链；
104)SEQ ID NO:113所示的正义链和SEQ ID NO:217所示的反义链；
105)SEQ ID NO:114所示的正义链和SEQ ID NO:218所示的反义链；
106)SEQ ID NO:115所示的正义链和SEQ ID NO:219所示的反义链；
107)SEQ ID NO:116所示的正义链和SEQ ID NO:220所示的反义链；
108)SEQ ID NO:117所示的正义链和SEQ ID NO:221所示的反义链；
109)SEQ ID NO:118所示的正义链和SEQ ID NO:222所示的反义链；
110)SEQ ID NO:119所示的正义链和SEQ ID NO:223所示的反义链；
111)SEQ ID NO:120所示的正义链和SEQ ID NO:224所示的反义链；
112)SEQ ID NO:121所示的正义链和SEQ ID NO:225所示的反义链；和
113)SEQ ID NO:122所示的正义链和SEQ ID NO:226所示的反义链。
一些实施方案中，所述正义链和/或反义链中至少一个核苷酸为修饰的核苷酸。
在一些实施方案中，全部的核苷酸为修饰的核苷酸。
在一些实施方案中，所述修饰的核苷酸为2'-甲氧基修饰的核苷酸、或2'-氟代修饰的核苷酸。
在一些实施方案中，siRNA的正义链中三个连续的核苷酸为2'-氟代修饰的核苷酸。
在一些实施方案中，siRNA的正义链中，位于5’端第7-9位的三个连续的核苷酸为2'-氟代修饰的核苷酸。
在一些实施方案中，siRNA的正义链中位于5’端第7-9位的三个连续的核苷酸为2'-氟代修饰的核苷酸，正义链其余位置均为2'-甲氧基修饰的核苷酸。在一些实施方案中，所述正义链中位于5’端第5位和第7-9位的四个核苷酸各自独立地为2’-氟代修饰的核苷酸，正义链其余位置均为2’-甲氧基修饰的核苷酸。
在一些实施方案中，按照5'末端到3'末端的方向，反义链的第2、6、12、14和16位的核苷酸各自独立地为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。在一些实施方案中，按照5'末端到3'末端的方向，所述反义链的第2、4、6、10、12、14、16、18位的核苷酸各自独立地为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。在一些实施方案中，按照5'末端到3'末端的方向，反义链的第2、6、14和16位的核苷酸各自独立地为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。
在一些实施方案中，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。所述修饰基团使得所述siRNA在生物样品或环境中具有增加的稳定性。在一些实施方案中，所述具有修饰基团的磷酸二酯基为硫代磷酸二酯基。
在一些实施方案中，硫代磷酸二酯基存在于选自以下的位置中的至少一处：
所述正义链的5'末端端部第1个核苷酸和第2个核苷酸之间；
所述正义链的5'末端端部第2个核苷酸和第3个核苷酸之间；
所述正义链的3'末端端部第1个核苷酸和第2个核苷酸之间；
所述正义链的3'末端端部第2个核苷酸和第3个核苷酸之间；
所述反义链的5'末端端部第1个核苷酸和第2个核苷酸之间；
所述反义链的5'末端端部第2个核苷酸和第3个核苷酸之间；
所述反义链的3'末端端部第1个核苷酸和第2个核苷酸之间；以及
所述反义链的3'末端端部第2个核苷酸和第3个核苷酸之间。
在一些实施方案中，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'末端端部第1个核苷酸和第2个核苷酸之间；和
所述正义链的5'末端端部第2个核苷酸和第3个核苷酸之间；和
所述反义链的5'末端端部第1个核苷酸和第2个核苷酸之间；和
所述反义链的5'末端端部第2个核苷酸和第3个核苷酸之间；和
所述反义链的3'末端端部第1个核苷酸和第2个核苷酸之间；和
所述反义链的3'末端端部第2个核苷酸和第3个核苷酸之间。
本公开还提供了一种siRNA缀合物，其包含上述siRNA中的任意一种和连接至所述siRNA末端的靶向配体。
在一些实施方案中，siRNA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，所述靶向配体包含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-异丁酰基半乳糖胺中的一种或多种，优选地，所述靶向配体包含N-乙酰基-半乳糖胺。在一些实施方案中，所述靶向配体连接至所述siRNA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siRNA中正义链或反义链的3’末端或5’末端连接。
在一些实施方案中，所述靶向配体通过磷酸二酯基团与siRNA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siRNA末端间接连接。一些实施方案中，通过磷酸二酯基团与siRNA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siRNA末端直接连接；一些实施方案中，通过磷酸二酯基团与siRNA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siRNA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siRNA正义链3’末端直接连接。
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。在一些实施方式中，本文所公开的靶向配体可以包括1、2、3、4或超过4个通过L2连接至分支基团的靶向部分。
在一些实施方式中，各靶向部分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。
在一些实施方式中，本公开中的靶向配体包括N-乙酰半乳糖胺做为靶向部分，
在一些实施方式中，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。在一些实施方式中，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。
在一些实施方式中，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。在一些实施方式中，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(Ⅱ)或式(III)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，本公开的siRNA和/或siRNA缀合物具有如表1a、1b或表2a、2b中任意一对正义链和反义链所示的核苷酸序列。
在一些实施方案中，本公开的siRNA和/或siRNA缀合物包含或选自如SEQ ID NO:275至SEQ ID NO:383中任一项
在一些实施方案中，本公开的siRNA和/或siRNA缀合物中的正义链包含或选自如SEQ ID NO:227至SEQ ID NO:242、SEQ ID NO:243至SEQ ID NO:250、SEQ ID NO:275至SEQ ID NO:383中任一项所示的核苷酸序列；和/或，所述反义链包含或选自如SEQ ID NO:251至SEQ ID NO:266、SEQ ID NO:267至SEQ ID NO:274、SEQ ID NO:384至SEQ ID NO:492中任一项所示的核苷酸序列。
在一些实施方案中，其中所述正义链和反义链包含或选自以下组合：1)SEQ ID NO:227所示的正义链和SEQ ID NO:251所示的反义链；
2)SEQ ID NO:228所示的正义链和SEQ ID NO:252所示的反义链；
3)SEQ ID NO:229所示的正义链和SEQ ID NO:253所示的反义链；
4)SEQ ID NO:230所示的正义链和SEQ ID NO:254所示的反义链；
5)SEQ ID NO:231所示的正义链和SEQ ID NO:255所示的反义链；
6)SEQ ID NO:232所示的正义链和SEQ ID NO:256所示的反义链；
7)SEQ ID NO:233所示的正义链和SEQ ID NO:257所示的反义链；
8)SEQ ID NO:234所示的正义链和SEQ ID NO:258所示的反义链；
9)SEQ ID NO:235所示的正义链和SEQ ID NO:259所示的反义链；
10)SEQ ID NO:236所示的正义链和SEQ ID NO:260所示的反义链；
11)SEQ ID NO:237所示的正义链和SEQ ID NO:261所示的反义链；
12)SEQ ID NO:238所示的正义链和SEQ ID NO:262所示的反义链；
13)SEQ ID NO:239所示的正义链和SEQ ID NO:263所示的反义链；
14)SEQ ID NO:240所示的正义链和SEQ ID NO:264所示的反义链；
15)SEQ ID NO:241所示的正义链和SEQ ID NO:265所示的反义链；
16)SEQ ID NO:242所示的正义链和SEQ ID NO:266所示的反义链；
17)SEQ ID NO:243所示的正义链和SEQ ID NO:267所示的反义链；
18)SEQ ID NO:244所示的正义链和SEQ ID NO:268所示的反义链；
19)SEQ ID NO:245所示的正义链和SEQ ID NO:269所示的反义链；
20)SEQ ID NO:246所示的正义链和SEQ ID NO:270所示的反义链；
21)SEQ ID NO:247所示的正义链和SEQ ID NO:271所示的反义链；
22)SEQ ID NO:248所示的正义链和SEQ ID NO:272所示的反义链；
23)SEQ ID NO:249所示的正义链和SEQ ID NO:273所示的反义链；和
24)SEQ ID NO:250所示的正义链和SEQ ID NO:274所示的反义链。
另一方面，本公开提供一种组合物，其包含本公开所述的siRNA、siRNA缀合物，和一种或多种药学上可接受的赋形剂，例如载剂(vehicle)、运载体(carrier)、稀释剂、和/或递送聚合物。
各种递药系统是已知的并且可以用于本公开siRNA或siRNA缀合物，例如封装在脂质体中、微粒、微囊、能够表达siRNA或siRNA缀合物的重组细胞、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。
另一方面，本公开提供一种上述的siRNA、siRNA缀合物或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。
另一方面，本公开提供一种治疗受试者疾病的方法，包括向受试者给予上述的siRNA、siRNA缀合物或组合物。
另一方面，本公开提供一种抑制受试者体内mRNA表达的方法，该方法包括给予受试者上述siRNA、siRNA缀合物或组合物。
另一方面，本公开提供一种体内递送表达抑制性寡聚化合物至肝脏的方法，该方法包括给与受试者上述siRNA、siRNA缀合物或组合物。
本文所公开的siRNA、siRNA缀合物或组合物和方法可以在细胞、细胞群、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公开所述的siRNA、siRNA缀合物或组合物，所述siRNA与靶向配体连接，从而抑制靶mRNA在受试者中的表达。
在一些实施方式中，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病理性上调。
本公开中所述的受试者是指确诊患有(或疑似患有、或易感于)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siRNA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，本公开还提供了一种药物组合物，其包含本公开的siRNA或siRNA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，所述的药物组合物的单位剂量为0.001mg-1000mg。
在某些实施方案中，基于组合物的总重量，所述的药物组合物含有0.01-99.99％的前述化合物或其可药用盐或其同位素取代物。在某些实施方案中，所述的药物组合物含有0.1-99.9％的前述化合物或其可药用盐或其同位素取代物。在某些实施方案中，所述的药物组合物含有0.5％-99.5％的前述化合物或其可药用盐或其同位素取代物。在某些实施方案中，所述的药物组合物含有1％-99％的前述化合物或其可药用盐或其同位素取代物。在某些实施方案中，所述的药物组合物含有2％-98％的前述化合物或其可药用盐或其同位素取代物。
在某些实施方案中，基于组合物的总重量，所述的药物组合物含有0.01％-99.99％的药学上可接受的赋形剂。在某些实施方案中，所述的药物组合物含有0.1％-99.9％的药学上可接受的赋形剂。在某些实施方案中，所述的药物组合物含有0.5％-99.5％的药学上可接受的赋形剂。在某些实施方案中，所述的药物组合物含有1％-99％的药学上可接受的赋形剂。在某些实施方案中，所述的药物组合物含有2％-98％的药学上可接受的赋形剂。
在一些实施方案中，上述siRNA、siRNA缀合物或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siRNA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，上述siRNA、siRNA缀合物或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siRNA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，上述siRNA、siRNA缀合物或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，siRNA缀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，上述siRNA、siRNA缀合物或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，siRNA缀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，上述siRNA、siRNA缀合物或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，siRNA缀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％。
本公开还提供了一种细胞，其包含本公开的siRNA或siRNA缀合物。
本公开还提供了一种试剂盒，其包含本公开的siRNA、siRNA缀合物或药物组合物。
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siRNA、siRNA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siRNA、siRNA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siRNA、siRNA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，siRNA、siRNA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，靶基因是抑制素βE(INHBE)。
本公开提供了前述siRNA和/或药物组合物和/或siRNA缀合物，用于治疗和/或预防受试者与抑制素βE(INHBE)基因表达相关的疾病；在一些实施方案中，所述疾病选自代谢障碍类疾病；在一些实施方案中，所述疾病选自代谢综合征(MS)、心血管疾病、肥胖症、肝炎、肾疾病。
本公开提供一种治疗和/或预防受试者中与抑制素βE(INHBE)基因表达相关的疾病的方法，包括给予受试者有效量或有效剂量的本公开所述的siRNA、siRNA缀合物或所述的药物组合物；在一些实施方案中，所述疾病选自代谢障碍类疾病；在一些实施方案中，所述疾病选自代谢综合征(MS)、心血管疾病、肥胖症、肝炎、肾疾病。
本公开提供了前述siRNA和/或药物组合物和/或siRNA缀合物在制备治疗和/或预防与抑制素βE(INHBE)基因表达相关的疾病的药物中的用途；在一些实施方案中，所述疾病选自代谢障碍类疾病；在一些实施方案中，所述疾病选自代谢综合征(MS)、心血管疾病、肥胖症、肝炎、肾疾病。
本公开提供了前述siRNA和/或药物组合物和/或siRNA缀合物在制备用于抑制INHBE的表达的药物中的用途。
本公开提供了一种抑制INHBE表达的方法，包括给予受试者有效量或有效剂量的前述siRNA和/或药物组合物和/或siRNA缀合物。
本公开提供了前述siRNA和/或药物组合物和/或siRNA缀合物，用于治疗和/或预防疾病；在一些实施方案中，所述疾病选自代谢障碍类疾病；在一些实施方案中，所述疾病选自代谢综合征(MS)、心血管疾病、肥胖症、肝炎、肾疾病。
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siRNA和/或药物组合物和/或siRNA缀合物；在一些实施方案中，所 述疾病选自代谢障碍类疾病；在一些实施方案中，所述疾病选自代谢综合征(MS)、心血管疾病、肥胖症、肝炎、肾疾病。
本公开提供了前述siRNA和/或药物组合物和/或siRNA缀合物在制备用于治疗和/或预防疾病的药物中的用途；在一些实施方案中，所述疾病选自代谢障碍类疾病；在一些实施方案中，所述疾病选自代谢综合征(MS)、心血管疾病、肥胖症、肝炎、肾疾病。
本公开提供了一种体内递送抑制INHBE表达和/或复制的siRNA至肝脏的方法，该方法包括给与受试者前述siRNA和/或药物组合物和/或siRNA缀合物。
本公开还提供了一种制备siRNA或siRNA缀合物的方法，其包括：合成本公开所述的siRNA、siRNA缀合物。
本公开还提供了一种siRNA或siRNA缀合物，其特征在于以碱基T，替换本公开任一siRNA或siRNA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个或全部碱基U。
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。
另一方面，在不指明构型的情况下，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。
另外，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。
本公开化合物可以是不对称的，例如，具有一个或多个立体异构体。除非另有说明，所有立体异构体都包括，如对映异构体和非对映异构体。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。
除另有说明，当一个位置被特别地指定为氘(D)时，该位置应理解为具有大于氘的天然丰度(其为0.015％)至少1000倍的丰度的氘(即，至少10％的氘掺入)。示例中化合物的具有大于氘的天然丰度可以是至少1000倍的丰度的氘、至少2000倍的丰度的氘、至少3000倍的丰度的氘、至少4000倍的丰度的氘、至少5000倍的丰度的氘、至少6000倍的丰度的氘或更高丰度的氘。本公开还包括各种氘化形式的式I和式II的化合物。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。本领域技术人员能够参考相关文献合成氘化形式的式I和式II化合物。在制备氘代形式的式I和式II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。
在不指明构型的情况下，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，键可以为或者同时包含两种构型。虽然为简便起见将全部上述结构式画成某些异构体形式，但是本公开可以包括所有的异构体，如互变异构体、旋转异构体、几何异构体、非对映异构体、外消旋体和对映异构体。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。
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“抑制素βE”、“INHBE”在本公开中可互换使用。INHBE包括但不限于人类INHBE、食蟹猴INHBE、小鼠INHBE、大鼠INHBE，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“INHBE”亦指INHBE基因的天然存在的DNA序列变异，如INHBE基因中的单核苷酸多态性(SNP)。示例性SNP可在dbSNP数据库中发现。
术语“靶序列”指在INHBE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。 在一个实施方式中，该靶序列在INHBE的蛋白质编码区内。
如本文所使用的，在RNA介导的基因沉默的情形中，正义链(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；反义链(又称AS或AS链)是指具有与靶mRNA序列互补的序列的链。
在描述本文所述的siRNA正义链的上下文中，术语“与SEQ ID NO：1至SEQ ID NO：106任一的核苷酸序列相差不超过3个核苷酸序列，且包含至少15个连续核苷酸”旨在表示本文所述的siRNA正义链包含如SEQ ID NO：1至SEQ ID NO：106中任一正义链的至少15个连续核苷酸，或与SEQ ID NO：1至SEQ ID NO：106中任一正义链的至少15个连续核苷酸相差不超过3个核苷酸(任选地，相差不超过2个核苷酸；任选地，相差1个核苷酸)。
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，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。本领域技术人员所熟知的是，碱基T和U的替换不会对RNAi剂序列的性质产生显著影响，本公开的序列中U可以任意替换为T，替换后得到的序列也在本公开的保护范围。本公开的序列中，对于同一条核酸链，以从5’端至3’端的方向为从左至右的方向，小写字母m表示该字母m左侧相邻的一个核苷为2'-甲氧基修饰的核苷；小写字母f表示该字母f左侧相邻的一个核苷为2'-氟代修饰的核苷；小写字母s表示与该字母s相邻的两个核苷之间为硫代磷酸二酯基连接，如无特殊说明，本公开的“RNAi剂”、“核苷酸”、“化合物”、“化学修饰”、“寡核苷酸”、“双链RNAi抑制剂分子”、“siRNA”、“siRNA缀合物”“dsRNA”、“核酸”和“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。当其以盐形式存在时，部分基团可能离子化形成阴离子/阳离子，例如，磷酸二酯基、硫代膦酸二酯基可以以阴离子形式存在，下列各结构对应的盐形式的结构也在本公开的保护范围中。如无特别说明，每条链3’端的第一个核苷酸的3’位置为羟基；每条链5’端的第一个核苷酸的5’位置是羟基。
上述修饰及连接基团分别具有如下表所示结构，表中Base表示碱基：

如本公开所使用的，术语“2'-氟代修饰的核苷酸”指核苷酸的核糖基2'位的羟基被氟取代形成的核苷酸，“非2'-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。
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。
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siRNA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，分别读取萤火虫化学发光值(Fir)和海肾化学发光值(Ren)，计算相对值Ratio＝Ren/Fir；本公开中，剩余mRNA表达量比例(或剩余活性％)＝Ratio(siRNA处理组)/Ratio(无siRNA对照组)，抑制率(％)＝100％-剩余mRNA表达量(％)。
如无特殊说明，本公开的“化合物”、“配体”、“核酸配体缀合物”、“siRNA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”指获得任一种或多种有益的或所需的治疗结果所必需的药物、化合物或药物组合物的量。对于预防用途，有益的或所需的结果包括消除或降低风险、减轻严重性或延迟病症的发作，包括病症、其并发症和在病症的发展过程中呈现的中间病理表型的生物化学、组织学和/或行为症状。对于治疗应用，有益的或所需的结果包括临床结果，诸如减少各种本公开靶基因、靶mRNA或靶蛋白相关病症的发病率或改善所述病症的一个或更多个症状，减少治疗病症所需的其它药剂的剂量，增强另一种药剂的疗效，和/或延缓患者的本公开靶基因、靶mRNA或靶蛋白相关病症的进展。
如本文所使用的，“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。
本公开提供的siRNA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siRNA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siRNA的方法也是本领域技术人员所熟知的。
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“平端”或“平末端”可互换使用，是指在siRNA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siRNA将在其整个长度范围内是双链的。
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(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。
除另有说明，“任选地”、“任选”、“可选的”或“可选”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“任选地，R1和R2直接相连成环”是指R1和R2直接相连成环可以发生但不必须存在，该说明包括R1和R2直接相连成环的情形和R1和R2不成环的情形。
在本公开的化学结构式中，其可以根据本文所述发明范围连接一个或多个任何基团。
本公开上下文中，基团中的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。
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，如冷泉港的抗体技术实验手册，分子克隆手册；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，为市场购买的常规试剂。
实施例1.人INHBE siRNA的设计
以人INHBE基因(NM_031479.5)作为靶基因，以满足活性siRNA的一般规则设计19/21nt的siRNA。未经修饰的正义链及反义链序列见表1a和表1b，经过 2’-氟代、2’-甲氧基等修饰的正义链和反义链序列详见表2a和2b。
表1a.未修饰的正义链、反义链

表1b.未修饰的正义链、反义链


表2a.经过修饰的正义链和反义链

表2b.经过修饰的正义链和反义链





上表2中，序列从左至右表示5’端至3’端的方向，小写字母m表示该字母m左侧相邻的核苷为2'-甲氧基修饰的核苷；小写字母f表示该字母f左侧相邻的核苷为2'-氟代修饰的核苷；小写字母s表示与该字母s左右相邻的两个核苷或相连的核苷与递送基团NAG0052’或L96’之间的连接为硫代磷酸二酯基连接；无特别说明，两个相邻的核苷之间通过磷酸二酯基连接；如无特别说明，每条链3’端的第一个核苷酸的3’位为羟基；每条链5’端的第一个核苷酸的5’位是羟基。
所述2'-甲氧基修饰的核苷、2'-氟代修饰的核苷、硫代磷酸二酯基、磷酸二酯基、NAG0052’和L96’的结构如下表所示，当本公开的siRNA或siRNA缀合物以盐形式存在时，例如以钠盐形式存在时，下表3中结构对应的盐形式的结构 也在本公开的保护范围以内：
表3
其中，Base表示碱基。
实施例2.siRNA、siRNA缀合物的合成
siRNA的合成与通常的亚磷酰胺固相合成法无异。合成过程简要描述如下：于Dr.Oligo48合成器(Biolytic)上，以通用CPG载体为起始，根据合成程序逐个连接核苷亚磷酰胺单体。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，使用碘吡啶/水溶液(柯乐玛)作为氧化剂。
固相合成完成后，寡核糖核苷酸自该固体支撑物裂解，采用3:1的28％氨水和乙醇溶液在50℃条件下浸泡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，最后所得到的双链siRNA溶于1×PBS中，并调整至实验所需浓度备用。
按照和WO2023138663A1实施例记载相同的方法制备得到含有NAG0052’的siRNA缀合物；按照和WO2014025805A1实施例记载相同的方法制备获得含有L96’的siRNA缀合物。
实施例3.psiCHECK在靶水平验证
在HEK293A细胞中采用9个浓度梯度对表2siRNA进行体外分子水平模拟在靶活性筛选。
本实施例使用的psi-CHECK质粒购自于生工生物工程(上海)股份有限公司。以INHBE基因构建siRNA对应的在靶序列，插入到psiCHECK-2质粒中，即GSCM在靶质粒。该质粒包含海肾荧光素酶基因及萤火虫荧光素酶基因。作为双报告基因系统，siRNA的靶序列插入到海肾荧光素酶基因的3’UTR区域，siRNA对于靶标序列的活性可以通过经萤火虫荧光素酶校准后的海肾荧光素酶表达情况的检测来反映，检测使用Dual-Luciferase Reporter Assay System(Promega，E2940)。
将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siRNA及对应质粒，Lipofectamine2000每孔使用0.2μL。质粒转染量为20ng每孔对于在靶序列质粒，siRNA共设置9个浓度点，最高浓度点终浓度为20nM，4倍梯度稀释，20nM，5nM，1.25nM，0.3125nM，0.0781nM，0.0195nM，0.0049nM，0.0012nM，0.0003nM。转染后24h，采用Dual-Luciferase Reporter Assay System(Promega，E2940)检测在靶水平。
本实施例使用的实验材料和仪器详见表4和表5。本实施例使用的稀释方案见表6。实验结果如表7所示。
表4.psi-CHECK实验耗材和试剂
表5.psi-CHECK实验仪器

表6.样品多浓度稀释方案
表7.psi-CHECK在靶活性筛选结果

表7结果表明：本公开的siRNA对INHBE有很好的在靶抑制活性。
实施例4.对人肝癌细胞系(Hep3B)中INHBE的多浓度点抑制活性
转染前24h，将Hep3B细胞以约20,000细胞/孔接种在96孔板中，每孔100μL培养基。转染时，参照产品说明手册，使用Lipofectamine RNAi MAX(ThermoFisher，13778150)转染样品，样品转染的梯度终浓度为20、4、0.8、0.16、0.032、0.0064和0.00128nM。在处理24小时后，使用高通量细胞RNA提取试剂盒(FG0417-L/FG0418-XL,磁珠法)进行细胞总RNA提取、RNA逆转录实验(Takara，RR037A)和实时定量PCR(Thermo，4444557)检测，测定人INHBE的mRNA水平，根据GAPDH内参基因水平对人INHBE的mRNA水平进行校正。
该实验涉及的仪器如表8所示。
表8.实验仪器
其中，在实时定量PCR检测时，采用的是探针Q-PCR检测实验，其引物信息如表9所示。
表9.Taqman引物信息表
结果分析方法
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siRNA处理的细胞的人INHBE mRNA表达剩余百分比来表示。其中IC50值对应的是INHBE mRNA表达剩余百分比为50％时，使用的siRNA的浓度。
表10数据结果表明，本公开的siRNA在Hep3B细胞中针对INHBE基因具有高水平的在靶抑制活性。
表10.siRNA在Hep3B细胞中的INHBE抑制活性
实施例5.对人原代肝细胞(PHH)中INHBE的多浓度点抑制活性
转染前24h，将PHH细胞以约30,000细胞/孔接种在96孔板中，每孔100μL培养基。转染时，参照产品说明手册，使用Lipofectamine RNAi MAX(ThermoFisher，13778150)转染样品，样品转染的梯度终浓度为20、4、0.8、0.16、0.032、0.0064和0.00128nM。在处理24小时后，使用高通量细胞RNA提取试剂盒(FG0417-L/FG0418-XL,磁珠法)进行细胞总RNA提取、RNA逆转录实验(Takara，RR037A)和实时定量PCR(Thermo，4444557)检测，测定人INHBE的mRNA水平，根据GAPDH内参基因水平对人INHBE的mRNA水平进行校正。
该实验涉及的仪器、引物信息、结果分析方法和靶基因mRNA表达剩余百分比的计算方法与实施例4相同。
表11数据结果表明，本公开的siRNA在PHH细胞中针对INHBE基因具有高水平的在靶抑制活性。
表11.本公开的siRNA在PHH细胞中的INHBE抑制活性

实施例6.psiCHECK在靶水平验证
在HEK293A细胞中采用2个浓度梯度对siRNA进行体外分子水平模拟在靶活性筛选。实验过程、所使用的实验材料和仪器以及稀释过程、结果分析方法与实施例3相同。
由表12结果可见，本公开的siRNA具有良好的对INHBE的抑制效果。
表12.本公开的siRNA对INHBE抑制效果
实施例7.psiCHECK在靶水平验证
在HEK293A细胞中采用9个浓度梯度对siRNA进行体外分子水平模拟在靶活性筛选。实验过程、所使用的实验材料和仪器以及稀释过程、结果分析方法均与实施例3相同。
由表13结果可见，结果中＞20表示在本实施例最高浓度20nM的条件下，抑制率也无法达到50％，本公开的siRNA具有良好的对INHBE的抑制效果。
表13.本公开的siRNA对INHBE抑制效果
实施例8：对人肝癌细胞系(Huh7)中INHBE的多浓度点抑制活性
转染前24h，将Huh7细胞以约15,000细胞/孔接种在96孔板中，每孔100μL培养基。转染时，参照产品说明手册，使用Lipofectamine RNAi MAX(ThermoFisher，13778150)转染样品，样品转染的梯度终浓度为20、4、0.8、0.16、0.032、0.0064和0.00128nM。在处理24小时后，使用高通量细胞RNA提取试剂盒(FG0417-L/FG0418-XL,磁珠法)进行细胞总RNA提取、RNA逆转录实验(Takara，RR037A)和实时定量PCR(Thermo，4444557)检测，测定人INHBE的mRNA水平，根据GAPDH内参基因水平对人INHBE的mRNA水平进行校正。
该实验涉及的仪器、引物信息、结果分析方法参考实施例4
表14数据结果表明，所有siRNA及其缀合物在Huh7细胞中针对INHBE基因具有高水平的在靶抑制活性。
表14 siRNA缀合物在Huh7细胞中的INHBE抑制活性
实施例9：对猴原代肝细胞(PCH)中INHBE的多浓度点抑制活性
转染前24h，将PCH细胞以约30,000细胞/孔接种在96孔板中，每孔100μL 培养基。转染时，参照产品说明手册，使用Lipofectamine RNAi MAX(ThermoFisher，13778150)转染样品，样品转染的梯度终浓度为20、4、0.8、0.16、0.032、0.0064和0.00128nM。在处理24小时后，使用高通量细胞RNA提取试剂盒(FG0417-L/FG0418-XL,磁珠法)进行细胞总RNA提取、RNA逆转录实验(Takara，RR037A)和实时定量PCR(Thermo，4444557)检测，测定猴INHBE的mRNA水平，根据GAPDH内参基因水平对猴INHBE的mRNA水平进行校正。
该实验涉及的仪器实施例4表7所示。
其中，在实时定量PCR检测时，采用的是探针Q-PCR检测实验，其引物信息如表15所示。
表15.Taqman引物信息表
结果分析方法和靶mRNA表达剩余百分比的计算方法同实施例4。
表16数据结果表明，所有siRNA缀合物在PCH细胞中针对INHBE基因具有高水平的在靶抑制活性。
表16 siRNA缀合物在PCH细胞中的INHBE抑制活性
实施例10.psiCHECK在靶水平验证
在HEK293A细胞中采用9个浓度梯度对siRNA进行体外分子水平模拟在靶活性筛选。实验过程、所使用的实验材料和仪器以及稀释过程与实施例3相同。表17数据结果表明，与对照序列相比，本公开siRNA具更高水平的在靶抑制活性。
表17本公开的siRNA对INHBE抑制效果
实施例11.psiCHECK在靶水平验证
在HEK293A细胞中采用9个浓度梯度对siRNA进行体外分子水平模拟在靶活性筛选。实验过程、所使用的实验材料和仪器以及稀释过程、结果分析方法均与实施例3相同。表18数据结果表明，本公开siRNA具有高水平的在靶抑制活性。
表18.本公开的siRNA对INHBE抑制效果

Claims (17)

  1. 一种siRNA，其包含形成双链区的正义链与反义链；
    所述正义链包含至少15个连续核苷酸，且与SEQ ID NO：1、2、3、4、5、6、7、8和9中的任一核苷酸序列相差不超过3个核苷酸；和
    所述反义链包含至少15个连续核苷酸，且与SEQ ID NO：10、11、12、13、14、15、16、17和18中的任一核苷酸序列相差不超过3个核苷酸。
  2. 根据权利要求1所述的siRNA，其中所述正义链包含与SEQ ID NO：1至9中的任一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；所述反义链包含与SEQ ID NO：10至18中的任一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；
    优选地，所述正义链包含与SEQ ID NO：1至9中的任一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；优选地，相差不超过1个核苷酸，和/或，
    所述反义链包含与SEQ ID NO：10至18中的任一核苷酸序列相差不超过3个核苷酸的至少21个连续核苷酸；优选地，相差不超过1个核苷酸。
  3. 根据权利要求1或2所述的siRNA，其包含选自以下任一组的正义链和反义链：
    组1)，SEQ ID NO：1所示的正义链和SEQ ID NO：10所示的反义链；
    组2)，SEQ ID NO：2所示的正义链和SEQ ID NO：11所示的反义链；
    组3)，SEQ ID NO：3所示的正义链和SEQ ID NO：12所示的反义链；
    组4)，SEQ ID NO：4所示的正义链和SEQ ID NO：13所示的反义链；
    组5)，SEQ ID NO：5所示的正义链和SEQ ID NO：14所示的反义链；
    组6)，SEQ ID NO：6所示的正义链和SEQ ID NO：15所示的反义链；
    组7)，SEQ ID NO：7所示的正义链和SEQ ID NO：16所示的反义链；
    组8)，SEQ ID NO：8所示的正义链和SEQ ID NO：17所示的反义链；和
    组9)，SEQ ID NO：9所示的正义链和SEQ ID NO：18所示的反义链。
  4. 根据权利要求1-3中任一项所述的siRNA，其中所述正义链和/或反义链中至少一个核苷酸为修饰的核苷酸。
  5. 根据权利要求4所述的siRNA，其中所述正义链中的三个连续核苷酸为2'-氟代修饰的核苷酸，优选地，按照5'末端到3'末端的方向，所述正义链的第7、8和9位的核苷酸各自独立地为2'-氟代修饰的核苷酸，和按照5'末端到3'末端的方向，所述反义链的第2、6、12和14位的核苷酸各自独立地为2'-氟代修饰的核苷酸。
  6. 根据权利要求1-5中任一项所述的siRNA，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。
  7. 根据权利要求6所述的siRNA，其中所述硫代磷酸二酯基存在于选自以下的位置中的至少一处：
    所述正义链的5'末端端部第1个核苷酸和第2个核苷酸之间；
    所述正义链的5'末端端部第2个核苷酸和第3个核苷酸之间；
    所述正义链的3'末端端部第1个核苷酸和第2个核苷酸之间；
    所述正义链的3'末端端部第2个核苷酸和第3个核苷酸之间；
    所述反义链的5'末端端部第1个核苷酸和第2个核苷酸之间；
    所述反义链的5'末端端部第2个核苷酸和第3个核苷酸之间；
    所述反义链的3'末端端部第1个核苷酸和第2个核苷酸之间；
    所述反义链的3'末端端部第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。
  8. 根据权利要求1-7中任一项所述的siRNA，其中，所述正义链包含SEQ ID NO:227至SEQ ID NO:242中任一项所示的核苷酸序列；
    和/或，所述反义链包含SEQ ID NO:251至SEQ ID NO:266中任一项所示的核苷酸序列。
  9. 一种siRNA缀合物，其包含：权利要求1至8中任一项所述的siRNA，和连接至所述siRNA末端的靶向配体；
    优选的，所述靶向配体靶向肝脏；
    更优选的，所述靶向配体结合脱唾液酸糖蛋白受体(ASGPR)，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-异丁酰基半乳糖胺中的一种或多种；
    更优选地，所述靶向配体具有如下所示的结构：

  10. 根据权利要求9所述的siRNA缀合物，其中所述靶向配体通过硫代磷酸二酯基团或磷酸二酯基团与siRNA中正义链或反义链的3’末端或5’末端连接；优选的，所述靶向配体与siRNA的正义链3’末端连接。
  11. 根据权利要求10所述的siRNA缀合物，其中，所述正义链包含如SEQ ID NO:243至SEQ ID NO:250中任一项所示的核苷酸序列；和/或所述反义链包含如SEQ ID NO:267至SEQ ID NO:274中任一项所示的核苷酸序列。
  12. 一种药物组合物，其包含根据权利要求1-8中任一项所述的siRNA或根据权利要求9-11中任一项所述的siRNA缀合物，和药学上可接受的载体。
  13. 根据权利要求1-8中任一项所述的siRNA或根据权利要求9-11中任一项所述的siRNA缀合物在制备用于降低抑制素βE(INHBE)表达的药物中的用途。
  14. 一种在有需要的受试者中降低抑制素βE(INHBE)表达的方法，所述方法包括给予所述受试者有效量或有效剂量的根据权利要求1-8中任一项所述的siRNA或根据权利要求9-11中任一项所述的siRNA缀合物或根据权利要求12所述的药物组合物。
  15. 一种在有需要的受试者中治疗和/或预防疾病的方法，所述方法包括给予所述受试者有效量或有效剂量的根据权利要求1-8中任一项所述的siRNA或根据权利要求9-11中任一项所述的siRNA缀合物、或根据权利要求12所述的药物组合物；优选的，所述疾病选自代谢障碍类疾病；更优选的，所述疾病选自代谢综合征(MS)、心血管疾病、肥胖症、肝炎、肾疾病。
  16. 一种在有需要的受试者中体内递送抑制INHBE表达和/或复制的siRNA至肝脏的方法，其中所述方法包括给与所述受试者根据权利要求1-8中任一项所述的siRNA或根据权利要求9-11中任一项所述的siRNA缀合物、或根据权利要求12所述的药物组合物。
  17. 一种制备siRNA或siRNA缀合物的方法，其包括：合成权利要求1-8中 任一项所述的siRNA或根据权利要求9-11中任一项所述的siRNA缀合物、或根据权利要求12所述的药物组合物。
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