+

WO2018199338A1 - Molécule d'acide nucléique pour le traitement de l'hépatite b - Google Patents

Molécule d'acide nucléique pour le traitement de l'hépatite b Download PDF

Info

Publication number
WO2018199338A1
WO2018199338A1 PCT/JP2018/017347 JP2018017347W WO2018199338A1 WO 2018199338 A1 WO2018199338 A1 WO 2018199338A1 JP 2018017347 W JP2018017347 W JP 2018017347W WO 2018199338 A1 WO2018199338 A1 WO 2018199338A1
Authority
WO
WIPO (PCT)
Prior art keywords
region
bases
nucleic acid
acid molecule
nucleotide sequence
Prior art date
Application number
PCT/JP2018/017347
Other languages
English (en)
Japanese (ja)
Inventor
一彰 茶山
忠彦 吉間
隆之 水谷
Original Assignee
国立大学法人広島大学
大日本住友製薬株式会社
株式会社ボナック
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人広島大学, 大日本住友製薬株式会社, 株式会社ボナック filed Critical 国立大学法人広島大学
Priority to JP2019514692A priority Critical patent/JPWO2018199338A1/ja
Publication of WO2018199338A1 publication Critical patent/WO2018199338A1/fr

Links

Images

Classifications

    • 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
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/02—Suppositories; Bougies; Bases therefor; Ovules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present invention relates to a nucleic acid molecule that effectively suppresses expression of a hepatitis B virus (HBV) gene, and for the suppression of hepatitis B virus proliferation, the treatment of hepatitis B, cirrhosis, and liver cancer, including the nucleic acid molecule.
  • HBV hepatitis B virus
  • hepatitis B virus is infected between 1.3 million and 1.5 million people in Japan, and about 350 million people worldwide. Main causes of chronic hepatitis along with hepatitis C virus It has become.
  • Chronic hepatitis C can be expected to eliminate the HCV virus at a considerably high rate by the current therapy, but the current therapy for chronic hepatitis B cannot completely eliminate the HBV virus.
  • Current therapies for chronic hepatitis B include interferon (IFN) therapy and nucleic acid analog preparation therapy.
  • IFN interferon
  • nucleic acid analog preparation sedates hepatitis and improves liver function, but is forced to take for a long time, and hepatitis relapses in most cases due to discontinuation of medication.
  • Non-patent Documents 1 and 2 transcription from cccDNA (covalently closed circular DNA, closed circular DNA, or completely closed double-stranded DNA) present in a minute amount in the hepatocyte nucleus after the treatment occurs after the treatment is completed. Therefore, development of a new innovative therapeutic agent having a different mechanism of action from the above-described therapeutic methods is demanded (Non-patent Documents 1 and 2).
  • Non-patent document 3 summarizes the mechanism of action of chronic hepatitis B drug candidates.
  • translational suppression of all viral proteins by siRNA can be expected to completely block the function of HBV as a virus.
  • Nucleic acid drugs can target molecules such as mRNA and miRNA that cannot be targeted by conventional low-molecular-weight drugs and antibody drugs, and are highly expected as next-generation drugs. As a result, the creation of pharmaceuticals for diseases that have been difficult to treat is expected, and the current situation is that research is actively conducted all over the world.
  • RNA interference As a technique for suppressing gene expression in nucleic acid medicine, for example, RNA interference (RNAi) is known. Inhibition of gene expression by RNA interference is generally performed, for example, by administering a short double-stranded RNA molecule to a cell or the like.
  • the double-stranded RNA molecule is usually referred to as siRNA (small interfering RNA).
  • siRNA small interfering RNA
  • the present invention relates to a nucleic acid molecule that effectively suppresses the expression of hepatitis B virus gene, and a medicament for inhibiting the growth of hepatitis B virus containing the nucleic acid molecule, the treatment of hepatitis B, cirrhosis, and liver cancer.
  • An object is to provide a composition.
  • the present inventors designed and synthesized two types of novel siRNA sequences for HBV genotype C. They were introduced into cultured human cells Huh7 expressing HBV virus, and their ability to suppress virus production was evaluated using the secreted amounts of S antigen and e antigen of HBV virus as indicators. As a result, it was found that the two sequences (us1, us2) have high inhibitory activity. Furthermore, the present inventors have found that a single-stranded nucleic acid molecule carrying these two sequences has an activity equivalent to that of siRNA, thereby completing the present invention.
  • a nucleic acid molecule comprising the following nucleotide sequence (i) or (ii) as a hepatitis B virus gene expression suppression sequence (i) a nucleotide sequence represented by SEQ ID NO: 2; A nucleotide sequence in which one or two bases have been deleted, substituted, inserted or added in the nucleotide sequence represented by SEQ ID NO: 2; or 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 2 A nucleotide sequence represented by SEQ ID NO: 1; A nucleotide sequence in which one or two bases have been deleted, substituted, inserted, or added in the nucleotide sequence represented by SEQ ID NO: 1; or 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 Nucleotide sequence having [2] The nucleic acid molecule according to [1], wherein the total number of bases is 100 bases or less.
  • a double-stranded nucleic acid molecule One strand contains the nucleotide sequence of (i) and the other strand contains the nucleotide sequence of (iii) annealed to the nucleotide sequence of (i); or one strand contains the nucleotide sequence of (ii), the other
  • nucleotide sequences of (i), (ii), (iii), and (iv) are the nucleotide sequences represented by SEQ ID NOs: 2, 1, 4, 3, respectively [3] to [6]
  • the linker region (Lx) has a non-nucleotide structure containing at least one of a pyrrolidine skeleton and a piperidine skeleton; And at least one of the region (X) and the region (Xc) includes the expression suppressing sequence; (B) comprising region (Xc), linker region (Lx), region (X), region (Y), linker region (Ly) and region (Yc) in this order from 5 ′ side to 3
  • X 1 and X 2 are each independently H 2 , O, S or NH; Y 1 and Y 2 are each independently a single bond, CH 2 , NH, O or S; R 3 is a hydrogen atom or substituent bonded to C-3, C-4, C-5 or C-6 on ring A; L 1 is an alkylene chain consisting of n carbon atoms, where the hydrogen atom on the alkylene carbon atom is OH, OR a , NH 2 , NHR a , NR a R b , SH, or SR a May be substituted and / or L 1 is a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with an oxygen atom, However, when Y 1 is NH, O or S, the atom of L 1 bonded to Y 1 is carbon, the atom of L 1 bonded to OR 1 is carbon, and oxygen atoms are not adjacent to each other; L 2 is an alkylene chain consisting of
  • the ring A may contain a carbon-carbon double bond or a carbon-nitrogen double bond
  • the region (Xc) and the region (X) are each bonded to the linker region (Lx) via —OR 1 — or —OR 2 —;
  • the region (Yc) and the region (Y) are each bonded to the linker region (Ly) via —OR 1 — or —OR 2 —,
  • R 1 and R 2 may be present or absent, and when present, R 1 and R 2 are each independently a nucleotide residue or the structure (I).
  • nucleic acid molecule according to any one of [10] to [19], wherein in (B), the number of bases (Yc) in the region (Yc) is 1 to 11 bases. [21] The nucleic acid molecule according to [20], wherein the number of bases (Yc) in the region (Yc) is 1 to 7 bases. [22] The nucleic acid molecule according to [20], wherein the number of bases (Yc) in the region (Yc) is 1 to 3 bases. [23] The nucleic acid molecule according to any one of [10] to [13], wherein in (A), the number of bases (Xc) in the region (Xc) is 19 to 30 bases.
  • nucleic acid molecule according to any one of [10] to [23], wherein the total number of bases is 80 or less.
  • nucleic acid molecule according to [10] wherein in (B), the linker regions (Lx) and (Ly) are composed of nucleotide residues of 1 to 20 bases.
  • nucleotide sequence of (i) and the nucleotide sequence of (iii), or the nucleotide sequence of (ii) and the nucleotide sequence of (iv) are linked by a group represented by the following formula: [1] The nucleic acid molecule according to any one of to [4].
  • nucleic acid molecule according to any one of [1] to [4], [10] to [13] or [23], which is represented by any of the following. 5'-AGUCUAGACUCGUGGUGGAUUCC-Lx-GGAAUCCACCACGAGUCUAGACUUU-3 '(SEQ ID NO: 13) 5'-GCAAGAUUCCUAUGGGAGUUUCC-Lx-GGAAACUCCCAUAGGAAUCUUGCUU-3 '(SEQ ID NO: 14) (In the sequence, -Lx- represents a group represented by the following formula.)
  • nucleic acid molecule according to any one of [1] to [4] or [10] to [22], which is represented by any of the following. 5'-AAGUCUAGACUCGUGGUGGAUUCC-Lx-GGAAUCCACCACGAGUCUAGACUUUC-Ly-G-3 '(SEQ ID NO: 17) 5'-AGCAAGAUUCCUAUGGGAGUUUCC-Lx-GGAAACUCCCAUAGGAAUCUUGCUUC-Ly-G-3 '(SEQ ID NO: 18) (In the sequence, -Lx- and -Ly- represent a group represented by the following formula.)
  • nucleic acid molecule according to any one of [1] to [4], [10], [12] to [22] or [26] represented by any of the following. 5'-AAGUCUAGACUCGUGGUGGAUUCCCCACACCGGAAUCCACCACGAGUCUAGACUUUCUUCGG-3 '(SEQ ID NO: 15) 5'-AGCAAGAUUCCUAUGGGAGUUUCCCCACACCGGAAACUCCCAUAGGAAUCUUGCUUCUUCGG-3 '(SEQ ID NO: 16) [32] An expression vector for expressing the nucleic acid molecule according to [5] to [9], [26] or [31].
  • a pharmaceutical composition comprising the nucleic acid molecule according to any one of [1] to [31] or the expression vector according to [32].
  • the pharmaceutical composition according to [33] which is used for inhibiting hepatitis B virus growth.
  • the expression of the hepatitis B virus gene can be effectively suppressed by the nucleic acid molecule of the present invention.
  • the pharmaceutical composition containing the nucleic acid molecule of the present invention effectively suppresses the expression of hepatitis B virus gene, particularly surface antigen gene, thereby suppressing hepatitis B virus growth, hepatitis B, cirrhosis, liver cancer. It is useful for the treatment of
  • FIG. 1 is a schematic diagram showing an example of the nucleic acid molecule of the present invention.
  • FIG. 2 is a schematic diagram showing another example of the nucleic acid molecule of the present invention.
  • FIG. 3 is a schematic diagram showing another example of the nucleic acid molecule of the present invention.
  • FIG. 4 is a schematic diagram showing another example of the nucleic acid molecule of the present invention.
  • FIG. 5 is a graph showing the suppressive activity of hepatitis B virus surface antigen (HBs antigen) gene expression of each siRNA designed based on the hepatitis B virus genome sequence of the present invention.
  • HBs antigen hepatitis B virus surface antigen
  • FIG. 6 is a graph showing the concentration-dependent HBs antigen expression suppression activity and HBe antigen expression suppression activity of each siRNA designed based on the hepatitis B virus genome sequence of the present invention.
  • FIG. 7 is a graph showing the HBs antigen expression-suppressing activity of the siRNA and single-stranded nucleic acid molecule of the present invention.
  • FIG. 8 is a graph showing the concentration-dependent HBs antigen expression suppression activity of the siRNA and single-stranded nucleic acid molecule of the present invention.
  • the present invention provides a nucleic acid molecule having activity of suppressing the expression of hepatitis B virus gene.
  • Hepatitis B virus invades hepatocytes by infection and proliferates.
  • the immune function works to eliminate it, but it is impossible to selectively attack only the virus in the hepatocyte, and the hepatocyte itself attacks. When received, it is destroyed, leading to the development of hepatitis.
  • Hepatitis B virus has an incomplete double-stranded DNA in which genetic information is conserved, and a DNA polymerase is centrally located in the core (HBc antigen), outer shell (HBe antigen), and outer membrane (HBs antigen). ).
  • cccDNA covalent circular DNA
  • mRNA 3.5 kb, 2.4 kb, 2.1 kb, 0.7 kb
  • HBs antigen, HBc antigen, HBe antigen and reverse transcriptase are structural proteins.
  • An active polymerase, X protein is translated (Molecular Therapy 2013; 21 (5) 973-985, FIG. 3a).
  • S ORF open reading frames
  • core ORF X ORF
  • polymerase ORF polymerase ORF
  • S ORF consists of three types of proteins that make up the HBs antigen, large S protein (including pre-S1, pre-S2 and S region), Middle S protein (including pre-S2 and S region), and Small S protein ( (Consisting only of the S region).
  • the core ORF encodes a core protein and a precore protein.
  • the core protein forms a core particle, and the pre-core protein becomes HBe antigen after 19 hydrophobic signal peptides and 34 amino acid residues at the C-terminus are cleaved.
  • the X ORF codes for an X protein that is thought to be involved in virus growth and the development of hepatocellular carcinoma.
  • the polymerase ORF encodes a DNA polymerase protein having reverse transcriptase activity.
  • a certain type of mRNA is incorporated into the core particle as pregenomic RNA, a minus-strand DNA is synthesized by the action of reverse transcriptase, and then a plus-strand DNA is synthesized into an incomplete circular double-stranded DNA. Furthermore, it is wrapped in an envelope formed from HBs antigens to become virus particles (Dane particles) and released into the blood.
  • HBs antigen translated by mRNA, hollow particles containing HBc antigen and p22cr antigen (particles without DNA nucleus), HBe antigen passing through liver cell membrane, etc. are Dane particle blood A large amount is released into the blood and secreted as a route different from the medium release.
  • Diagnosis of hepatitis B is performed by detecting the HBs antigen and / or HBe antigen in blood.
  • a positive HBs antigen in the blood indicates that HBV is present in the liver, HBV components are synthesized, and hepatitis B is infected at the time of examination.
  • the HBs antigen in the blood grasps the virus growth in the liver and provides an index for judging the completion of treatment.
  • HBe antigen is a protein that is excessively produced when HBV proliferates, and indicates that infectivity is strong when HBV is actively proliferating in the liver.
  • the nucleic acid molecule suppresses the expression of hepatitis B virus gene by introducing the nucleic acid molecule to be evaluated into a cell infected with hepatitis B virus or a cell into which hepatitis B virus genome has been introduced (preferably a human cell).
  • a cell infected with hepatitis B virus or a cell into which hepatitis B virus genome has been introduced preferably a human cell.
  • the amount of hepatitis B virus HBs antigen released (translocated) or the amount of hepatitis B virus HBe antigen has not been introduced or a negative control nucleic acid molecule has been introduced.
  • the amount of hepatitis B virus HBs antigen released or transferred from a cell infected with hepatitis B virus or a cell (preferably a human cell) into which hepatitis B virus genome has been introduced, or hepatitis B virus HBe antigen It can be evaluated by comparing it with the amount of.
  • the amount of hepatitis B virus HBs antigen or HBe antigen can be evaluated by detecting the antigen by a known immunological technique using an antibody that specifically recognizes hepatitis B virus HBs antigen or HBe antigen. it can. Examples of immunological methods include flow cytometry analysis, radioisotope immunoassay (RIA method), ELISA method (Methods in Enzymol. 70: 419-439 (1980)), Western blotting, immunohistochemical staining, etc. Can do.
  • the present invention provides a nucleic acid molecule comprising the following nucleotide sequence (i) or (ii) as an expression suppressing sequence and having an activity of suppressing the expression of hepatitis B virus gene: (i) represented by SEQ ID NO: 2 Nucleotide sequence; A nucleotide sequence in which one or two bases have been deleted, substituted, inserted or added in the nucleotide sequence represented by SEQ ID NO: 2; or 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 2 A nucleotide sequence represented by SEQ ID NO: 1; A nucleotide sequence in which one or two bases have been deleted, substituted, inserted, or added in the nucleotide sequence represented by SEQ ID NO: 1; or 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 Nucleotide sequence having
  • nucleotide sequences represented by SEQ ID NOs: 1 and 2 are the following sequences. 5'-UCCACCACGAGUCUAGACU-3 '(SEQ ID NO: 1) 5'-ACUCCCAUAGGAAUCUUGC-3 '(SEQ ID NO: 2)
  • the nucleotide sequences represented by SEQ ID NOs: 1 and 2 are the hepatitis B virus genotype C complete genome (GenBank Accession No. AB0143481, No. AB113875, No. AB113876, No. AB113878, No. AB113879). No. AB246344), nucleotide numbers 245 to 263 (5′-AGUCUAGACUCGUGGUGGA-3 ′ (SEQ ID NO: 3)) and nucleotide numbers 629 to 647 (5′-GCAAGAUUCCUAUGGGAGU-3 ′ (SEQ ID NO: 4)) Each corresponds to a sequence that is completely complementary to the corresponding sequence.
  • sequences represented by nucleotide numbers 245 to 263 and nucleotide numbers 629 to 647 are both sequences in the coding region of S protein and polymerase protein of hepatitis B virus. Due to the structure of the hepatitis B virus genome, the S protein and the coding region of the polymerase overlap, but they are translated into separate proteins due to the different reading frames. As described above, a plurality of ORFs may exist in mRNA transcribed from the hepatitis B virus genome.
  • hepatitis B virus surface antigen gene protein when the function of mRNA encoding hepatitis B virus surface antigen gene protein is inhibited by the nucleic acid molecule having the activity of suppressing the expression of hepatitis B virus gene of the present invention, translation of other proteins encoded by the RNA May also be suppressed.
  • the expression of the hepatitis B virus surface antigen gene protein is suppressed by the nucleic acid molecule having the activity of suppressing the expression of hepatitis B virus gene of the present invention, the proliferation of hepatitis B virus is suppressed, and as a result, other B types Hepatitis virus gene expression may be suppressed.
  • the expression suppression activity means that gene expression is suppressed as a result of suppression of gene transcription, degradation of gene transcript, and / or inhibition of protein translation from gene transcript.
  • the number of nucleotides to be deleted, substituted, inserted or added is as long as the resulting nucleic acid molecule has activity of suppressing the expression of hepatitis B virus gene. Although it is not particularly limited, it is usually 1 or 2, preferably 1.
  • the position of the base to be substituted is usually within 8 bases from the 5 ′ end, preferably within 7, 6, 5, 4, 3, 2 or 1 base, Alternatively, it is within 8 bases from the 3 ′ end, preferably within 7, 6, 5, 4, 3, 2 or 1 base.
  • the nucleotide sequence (i) may be a nucleotide sequence represented by SEQ ID NO: 5 or SEQ ID NO: 7.
  • the nucleotide sequence represented by SEQ ID NO: 5 is the nucleotide sequence represented by SEQ ID NO: 2 (C type, Genotype C) of hepatitis B virus genotype A (Genotype A) and B type (Genotype B). Corresponding sequence.
  • SEQ ID NO: 5 the 8th base and the 5th base from the 3 ′ end of the nucleotide represented by SEQ ID NO: 2 were substituted from A to U and from C to U, respectively. Is an array.
  • a sequence completely complementary to the nucleotide sequence represented by SEQ ID NO: 5 is shown in SEQ ID NO: 6.
  • the nucleotide sequence represented by SEQ ID NO: 7 is a sequence corresponding to the nucleotide sequence represented by SEQ ID NO: 2 (C type, Genotype C) of the hepatitis B virus genotype D (Genotype D).
  • SEQ ID NO: 7 the 5th base and the 2nd base from the 3 ′ end of the nucleotide represented by SEQ ID NO: 2 were substituted from C to U and from G to C, respectively. Is an array.
  • a sequence completely complementary to the nucleotide sequence represented by SEQ ID NO: 7 is shown in SEQ ID NO: 8.
  • the position of the base to be deleted is usually within 5 bases from the 5 ′ end, preferably within 4, 3, 2 or 1 base, or from the 3 ′ end. Within 5 bases, preferably within 4, 3, 2 or 1 base.
  • the position of the base to be inserted is usually between 5 and 4 bases from the 5 ′ end, preferably between 4 and 3 bases, 3 bases and 2 bases. Between bases, or between 2 bases and 1 base, or between 5 bases and 4 bases from the 3 ′ end, preferably between 4 bases and 3 bases, between 3 bases and 2 bases, or between 2 bases and 1 base Between.
  • the position to which a base is added is 1 base from the 5 'end or 1 base from the 3' end.
  • the degree of sequence identity is not particularly limited as long as the resulting nucleic acid molecule has the activity of suppressing the expression of hepatitis B virus gene, but usually 85% Above, preferably 90% or more.
  • identity % is determined by any algorithm known in the art, such as Needleman et al. (1970) (J. Mol. Biol. 48: 444-453), Myers and Miller (CABIOS, 1988, 4: 11-17) or the like. The Needleman et al.
  • Algorithm is incorporated in the GAP program of the GCG software package, and the identity (%) is, for example, BLOSUM 62 matrix or PAM250 matrix, and gap weight: 16, 14, 12, 10, 8, 6 or 4 and length weight: 1, 2, 3, 4, 5 or 6 can be used.
  • the Myers and Miller algorithms are also incorporated into the ALIGN program which is part of the GCG sequence alignment software package.
  • the ALIGN program for example, PAM120 weight restable table, gap length penalty 12, and gap penalty 4 can be used.
  • a method showing the lowest value among the above methods may be adopted.
  • the length of the nucleotide sequence is 17 to 21 bases, preferably 18 to 20 bases, more preferably 19 bases.
  • the expression suppression sequence may be, for example, a sequence consisting of the nucleotide sequence or a sequence containing the nucleotide sequence.
  • the length of the expression suppression sequence is not particularly limited, and is, for example, 18 to 32 bases long, preferably 19 to 30 bases long, and more preferably 19, 20, or 21 bases long.
  • the numerical range of the number of bases discloses all positive integers belonging to the range.
  • the description “1 to 4 bases” includes “1, 2, 3, 4 bases”. "Means all disclosures (the same applies hereinafter).
  • the nucleic acid molecule of the present invention preferably further has, for example, a complementary sequence that can be annealed with the expression suppressing sequence.
  • the complementary sequence is, for example, in the same strand as the expression suppressing sequence and forms a single-stranded nucleic acid molecule composed of one single strand.
  • the complementary sequence only needs to be annealable with the expression suppression sequence, for example.
  • the complementary sequence may be, for example, a sequence exhibiting 100% complementarity with the expression suppression sequence, or a sequence exhibiting complementarity of less than 100% within a range that can be annealed.
  • the complementarity is not particularly limited, and examples thereof include 90% to 100%, 93% to 100%, 95% to 100%, 98% to 100%, and 99% to 100%.
  • the nucleic acid molecule of the present invention is a nucleotide sequence of (i) and a complementary sequence capable of annealing to the nucleotide sequence of (i), wherein the complementary sequence is (iii) below; or ( a nucleotide sequence of ii) and a complementary sequence which can be annealed to the nucleotide sequence of (ii), wherein the complementary sequence is (iv) below.
  • nucleotide sequence represented by SEQ ID NO: 4 A nucleotide sequence in which one or two bases have been deleted, substituted, inserted or added in the nucleotide sequence represented by SEQ ID NO: 4; or 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 4
  • a nucleotide sequence represented by SEQ ID NO: 3 A nucleotide sequence in which one or two bases have been deleted, substituted, inserted or added in the nucleotide sequence represented by SEQ ID NO: 3; or 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 3
  • the number of nucleotides to be deleted, substituted, inserted, or added is as long as the resulting nucleic acid molecule has the activity of suppressing the expression of hepatitis B virus gene. Although it is not particularly limited, it is usually 1 or 2, preferably 1.
  • the position of the base to be substituted is usually within 8 bases from the 5 ′ end, preferably within 7, 6, 5, 4, 3, 2 or 1 base, Alternatively, it is within 8 bases from the 3 ′ end, preferably within 7, 6, 5, 4, 3, 2 or 1 base.
  • the position of the base to be deleted is usually within 5 bases from the 5 ′ end, preferably within 4, 3, 2 or 1 base, or from the 3 ′ end. Within 5 bases, preferably within 4, 3, 2 or 1 base.
  • the position of the base to be inserted is usually between 5 and 4 bases from the 5 ′ end, preferably between 4 and 3 bases, 3 bases and 2 bases. Between bases, or between 2 bases and 1 base, or between 5 bases and 4 bases from the 3 ′ end, preferably between 4 bases and 3 bases, between 3 bases and 2 bases, or between 2 bases and 1 base Between.
  • the position to which a base is added is one base from the 5 'end or one base from the 3' end.
  • the degree of sequence identity is not particularly limited as long as the resulting nucleic acid molecule has the activity of suppressing the expression of hepatitis B virus gene, but usually 85% Above, preferably about 90% or more.
  • the nucleotide sequence of (iii) is preferably a sequence that is completely complementary to the nucleotide sequence of (i), and the nucleotide sequence of (iv) is preferably completely complementary to the nucleotide sequence of (ii) Is an array.
  • the nucleic acid molecule of the present invention can be a DNA molecule, an RNA molecule, a chimeric nucleic acid molecule (hereinafter referred to as a chimeric nucleic acid molecule) or a hybrid nucleic acid molecule.
  • a chimeric nucleic acid molecule refers to a single-stranded or double-stranded nucleic acid molecule containing RNA and DNA in a single nucleic acid molecule
  • a hybrid nucleic acid molecule refers to one strand of a double-stranded nucleic acid molecule. Is a RNA molecule or a chimeric nucleic acid molecule and the other strand is a DNA molecule or a chimeric nucleic acid molecule.
  • the nucleic acid molecule of the present invention is single-stranded or double-stranded.
  • Double stranded embodiments include double stranded RNA molecules, double stranded DNA molecules, RNA / DNA hybrid nucleic acid molecules, RNA / chimeric nucleic acid hybrid nucleic acid molecules, chimeric nucleic acid / chimeric nucleic acid hybrid nucleic acid molecules and chimeric nucleic acid / DNA hybrid nucleic acids Includes molecules.
  • nucleotide sequence is described as an RNA sequence unless otherwise specified.
  • polynucleotide is DNA
  • uracil (U) is appropriately read as thymine (T).
  • nucleic acid molecule capable of specifically suppressing the expression of the hepatitis B virus gene of the present invention examples include, for example, siRNA molecules, dsRDC (double-strand RNA DNA Chimera) molecules, and the following single-stranded nucleic acids of the present invention.
  • the length of the nucleic acid molecule of the present invention is not particularly limited as long as it has the activity of suppressing the expression of hepatitis B virus gene, but it is usually 17 bases or more, preferably 19 bases or more, more preferably 21 bases or more.
  • the length of the nucleic acid molecule of the present invention is usually 200 bases or less, preferably 150 bases or less, more preferably 100 bases or less (eg, 90 bases or less, 80 bases or less, 70 or less) because of ease of synthesis or antigenicity problems. Bases or less, 60 bases or less, 50 bases or less).
  • the length of the nucleic acid molecule of the present invention is generally 17 bases to 200 bases, preferably 19 bases to 150 bases, more preferably 21 bases to 100 bases (21 bases to 90 bases, 21 bases to 80 bases, about 21 bases to 70 bases, 21 bases to 60 bases, 21 bases to 50 bases).
  • the total number of bases of the nucleic acid molecule is not particularly limited as long as it has activity to suppress the expression of hepatitis B virus gene, but is usually 17 bases or more, preferably 19 bases or more, more preferably 21 bases or more.
  • the total number of bases of the nucleic acid molecule of the present invention is usually 400 bases or less, preferably 300 bases or less, more preferably 200 bases or less (eg, 180 bases or less, 160 bases or less) due to ease of synthesis or antigenicity problems. 140 bases or less, 120 bases or less, 100 bases or less, 90 bases or less, 80 bases or less, 70 bases or less, 60 bases or less, 50 bases or less).
  • the length of the nucleic acid molecule of the present invention is generally 17 bases to 400 bases, preferably 19 bases to 300 bases, more preferably 21 bases to 200 bases (21 bases to 180 bases, 21 bases to 160 bases, 21 bases to 140 bases, 21 bases to 120 bases, 21 bases to 100 bases, 21 bases to 90 bases, 21 bases to 80 bases, 21 bases to 70 bases, 21 bases to 60 bases, 21 bases to 50 bases) .
  • the nucleic acid molecules of the present invention are preferably isolated. “Isolated” means that an operation to remove factors other than the target component has been performed, and that the naturally occurring state has been removed.
  • the purity of the “isolated nucleic acid” (percentage of the target nucleic acid weight in the total weight of the evaluation target) is usually 70% or more, preferably 80% or more, more preferably 90% or more, and still more preferably 99%. % Or more. 2. Double-stranded nucleic acid molecule
  • the nucleic acid molecule of the present invention is a double-stranded nucleic acid molecule.
  • the double-stranded nucleic acid molecule comprises a nucleotide sequence of (i) on one strand and a sequence annealed to the nucleotide sequence of (i) on the other strand, or a nucleotide sequence of (ii) on one strand In the other strand is annealed to the nucleotide sequence of (ii).
  • the sequence annealed to the nucleotide sequence (i) is not particularly limited as long as it can be annealed, but is preferably the nucleotide sequence (iii).
  • the sequence annealed to the nucleotide sequence of (ii) is not particularly limited as long as it can be annealed, but is preferably the nucleotide sequence of (iv).
  • nucleotide sequences (i), (ii), (iii), and (iv) may be nucleotide sequences represented by SEQ ID NOs: 2, 1, 4, 3, respectively.
  • the length of the double-stranded nucleic acid molecule of the present invention is not particularly limited as long as it has the activity of suppressing the expression of hepatitis B virus gene, but is usually 17 bases or more, preferably 19 bases or more, more preferably 21 bases or more. is there.
  • the length of the double-stranded nucleic acid molecule of the present invention is usually 100 bases or less, preferably 75 bases or less, more preferably 50 bases or less (eg, 45 bases or less, 40 or less, for ease of synthesis or antigenicity problems). Bases, 35 bases, 30 bases, 25 bases, 24 bases, 23 bases, 22 bases).
  • the length of the nucleic acid molecule of the present invention is usually 17 to 100 bases, preferably 19 to 75 bases, more preferably 21 to 50 bases (21 to 45 bases, 21 to 40 bases, 21 bases to 35 bases, 21 bases to 30 bases, 21 bases to 25 bases, 21 bases to 24 bases, 21 bases to 23 bases, 21 bases to 22 bases).
  • the double-stranded nucleic acid molecule may have an additional base (overhang sequence) that does not form a base pair at the 5 'and / or 3' end.
  • the length of the overhang sequence is not particularly limited as long as siRNA can specifically suppress the expression of the target gene, but is usually 5 bases or less, for example, 2 to 4 bases.
  • the additional base may be DNA or RNA, but the use of DNA can improve the stability of the RNA molecule. Examples of such an additional base sequence include ug-3 ′, uu-3 ′, tg-3 ′, tt-3 ′, ggg-3 ′, guuu-3 ′, gttt-3 ′, and ttttt-3. Examples of the sequence include ', uuuu-3', but are not limited thereto.
  • the total number of bases of the double-stranded nucleic acid molecule is not particularly limited as long as it has the activity of suppressing the expression of hepatitis B virus gene, but is usually 34 bases or more, preferably 38 bases or more, more preferably 42 bases or more. is there.
  • the total number of bases of the double-stranded nucleic acid molecule of the present invention is usually 200 bases or less, preferably 150 bases or less, more preferably 100 bases or less (eg, 90 bases or less) from the viewpoint of ease of synthesis and antigenicity. 80 bases or less, 70 bases or less, 60 bases or less, 50 bases or less, 48 bases or less, 46 bases or less, 44 bases or less).
  • the length of the nucleic acid molecule of the present invention is usually 34 to 200 bases, preferably 38 to 150 bases, more preferably 42 to 100 bases (42 to 90 bases, 42 to 80 bases, 42 bases to 70 bases, 42 bases to 60 bases, 42 bases to 50 bases, 42 bases to 48 bases, 42 bases to 46 bases, 42 bases to 44 bases).
  • tt-3 ′ is added to the nucleotide sequence represented by SEQ ID NO: 1, 2, 3, 4 represented by SEQ ID NO: 9, 10, 11, 12.
  • SEQ ID NO: 1, 2, 3, 4 represented by SEQ ID NO: 9, 10, 11, 12.
  • the double-stranded nucleic acid molecule is preferably a nucleotide sequence represented by SEQ ID NO: 10 and a nucleotide sequence represented by SEQ ID NO: 12 annealed to the sequence shown below; or a nucleotide represented by SEQ ID NO: 9
  • a double-stranded nucleic acid molecule comprising a sequence and a nucleotide sequence represented by SEQ ID NO: 11 annealed to the sequence.
  • the double-stranded nucleic acid molecule is preferably an siRNA molecule shown below.
  • siRNA refers to the double-stranded nucleic acid molecule in which the base sequence other than the overhang sequence is composed only of ribonucleotide residues.
  • the nucleic acid molecule of the present invention is used for suppressing the expression of the hepatitis B virus gene, and as the expression suppressing sequence for the hepatitis B virus gene, the nucleic acid molecule of the above (i) or (ii) It is characterized by comprising a nucleotide sequence.
  • the expression suppression sequence may be, for example, a sequence consisting of the nucleotide sequence or a sequence containing the nucleotide sequence.
  • the length of the expression suppression sequence is not particularly limited, and is, for example, 18 to 32 bases long, preferably 19 to 30 bases long, and more preferably 19, 20, or 21 bases long.
  • the numerical range of the number of bases discloses all positive integers belonging to the range.
  • the description “1 to 4 bases” includes “1, 2, 3, 4 bases”. "Means all disclosures (the same applies hereinafter).
  • the single-stranded nucleic acid molecule of the present invention further has, for example, a complementary sequence that can be annealed with the expression suppressing sequence.
  • the complementary sequence is, for example, in the same strand as the expression suppressing sequence and forms a single-stranded nucleic acid molecule composed of one single strand.
  • the complementary sequence only needs to be annealable with the expression suppression sequence, for example.
  • the complementary sequence may be, for example, a sequence exhibiting 100% complementarity with the expression suppression sequence, or a sequence exhibiting complementarity of less than 100% within a range that can be annealed.
  • the complementarity is not particularly limited, and examples thereof include 90% to 100%, 93% to 100%, 95% to 100%, 98% to 100%, and 99% to 100%.
  • the complementary sequence includes, for example, the nucleotide sequence (iii) or (iv).
  • nucleotide sequence (iii) or (iv) is referred to as an s nucleotide sequence.
  • the complementary sequence may be, for example, a sequence composed of the s nucleotide sequence or a sequence containing the s nucleotide sequence.
  • the length of the complementary sequence is not particularly limited, and is, for example, 18 to 32 bases long, preferably 19 to 30 bases long, and more preferably 19, 20, or 21 bases long.
  • the expression suppression sequence and the complementary sequence may each be, for example, an RNA molecule consisting only of ribonucleotide residues, or an RNA molecule containing deoxyribonucleotide residues in addition to ribonucleotide residues.
  • nucleic acid molecule examples include a form in which the expression suppressing sequence and the complementary sequence are directly linked and a form in which they are indirectly linked.
  • Examples of the direct linking include linking by a phosphodiester bond.
  • Examples of the indirect linkage include linkage via a linker region.
  • the order in which the expression suppressing sequence and the complementary sequence are linked is not particularly limited, and for example, the 3 ′ end of the expression suppressing sequence and the 5 ′ end of the complementary sequence may be linked. The 5 ′ end may be linked to the 3 ′ end of the complementary sequence, preferably the latter.
  • the linker region may be composed of, for example, nucleotide residues, may be composed of non-nucleotide residues, or may be composed of the nucleotide residues and non-nucleotide residues.
  • nucleotide residues include a ribonucleotide residue and a deoxyribonucleotide residue.
  • a molecule in which a 5′-side region and a 3′-side region are annealed with each other to form a double-stranded structure can be mentioned.
  • This can also be said to be a form of shRNA (small hairpin RNA or short hairpin RNA).
  • shRNA small hairpin RNA or short hairpin RNA.
  • the shRNA has a hairpin structure and generally has one stem region and one loop region.
  • the nucleic acid molecule of this embodiment includes, for example, a region (X), a linker region (Lx), and a region (Xc), and the linker region (Lx) is between the region (X) and the region (Xc). Takes a linked structure.
  • the region (Xc) preferably has a structure complementary to the region (X). Specifically, one of the region (X) and the region (Xc) has the expression suppressing sequence.
  • the other includes the complementary sequence. Since the region (X) and the region (Xc) each have one of the expression suppression sequence and the complementary sequence, for example, a stem structure can be formed by intramolecular annealing, and the linker region (Lx) It becomes a loop structure.
  • the nucleic acid molecule may have, for example, the region (Xc), the linker region (Lx), and the region (X) in the order from 5 ′ side to 3 ′ side, or from the 3 ′ side. You may have the said area
  • the expression suppression sequence may be arranged, for example, in either the region (X) or the region (Xc), and may be arranged downstream of the complementary sequence, that is, 3 ′ side of the complementary sequence. preferable.
  • FIG. 1A is a schematic diagram showing an outline of the order of each region
  • FIG. 1B is a schematic diagram showing a state in which the nucleic acid molecule forms a double strand in the molecule. is there.
  • the nucleic acid molecule forms a double strand between the region (Xc) and the region (X), and the Lx region loops according to its length.
  • FIG. 1 merely shows the linking order of the regions and the positional relationship of each region forming a duplex. For example, the length of each region, the shape of the linker region (Lx), etc. Not limited.
  • the number of bases in the region (Xc) and the region (X) is not particularly limited. Although the length of each area
  • the relationship between the number of bases (X) in the region (X) and the number of bases (Xc) in the region (Xc) satisfies, for example, the following (3) or (5) In this case, specifically, for example, the following condition (11) is satisfied.
  • X ⁇ Xc 1 to 10, preferably 1, 2 or 3, More preferably 1 or 2 (11)
  • X Xc (5)
  • the region may be a region composed of only the expression suppression sequence or a region including the expression suppression sequence, for example.
  • the number of bases of the expression suppression sequence is, for example, as described above.
  • the region containing the expression suppression sequence may further have an additional sequence on the 5 'side and / or 3' side of the expression suppression sequence, for example.
  • the number of bases of the additional sequence is, for example, 1 to 31 bases, preferably 1 to 21 bases, and more preferably 1 to 11 bases.
  • the number of bases in the region (Xc) is not particularly limited.
  • the lower limit of Xc is, for example, 19 bases.
  • the upper limit is, for example, 50 bases, preferably 30 bases, and more preferably 25 bases.
  • Specific examples of the number of bases in the region (Xc) are, for example, 19 to 50 bases, preferably 19 to 30 bases, more preferably 19 to 25 bases.
  • the number of bases in the region (X) is not particularly limited.
  • the lower limit is, for example, 19 bases, preferably 20 bases, and more preferably 21 bases.
  • the upper limit is 50 bases, for example, More preferably, it is 40 bases, More preferably, it is 30 bases.
  • the linker region (Lx) preferably has a structure that does not cause self-annealing within its own region.
  • the linker region (Lx) includes a nucleotide residue as described above, the length is not particularly limited.
  • the linker region (Lx) preferably has a length that allows the region (X) and the region (Xc) to form a double chain.
  • the lower limit of the number of bases in the linker region (Lx) is, for example, 1 base, preferably 2 bases, more preferably 3 bases, and the upper limit thereof is, for example, 100 bases, preferably 80 bases, more preferably 50 bases.
  • the total length of the nucleic acid molecule is not particularly limited.
  • the lower limit of the total number of bases is, for example, 38 bases, preferably 40 bases, more preferably 42 bases, still more preferably 44 bases.
  • the base is particularly preferably 46 bases, and the upper limit thereof is, for example, 300 bases, preferably 200 bases, more preferably 150 bases, still more preferably 100 bases, and particularly preferably 80 bases.
  • the lower limit of the total number of bases excluding the linker region (Lx) is, for example, 38 bases, preferably 40 bases, more preferably 42 bases, still more preferably 44 bases. Yes, particularly preferably 46 bases, and the upper limit is, for example, 300 bases, preferably 200 bases, more preferably 150 bases, still more preferably 100 bases, particularly preferably 80 bases. .
  • a second form of the single-stranded nucleic acid molecule is a molecule in which the 5 ′ region and the 3 ′ region are separately annealed in the molecule to form two double-stranded structures (stem structures).
  • the nucleic acid molecule of the present embodiment includes, for example, a 5 ′ side region (Xc), an internal region (Z), and a 3 ′ side region (Yc) from the 5 ′ side to the 3 ′ side in the order described above.
  • Z) is formed by connecting an inner 5 ′ side region (X) and an inner 3 ′ side region (Y), and the 5 ′ side region (Xc) is complementary to the inner 5 ′ side region (X).
  • the 3 ′ side region (Yc) is preferably complementary to the inner 3 ′ side region (Y).
  • the 5 ′ region (Xc) when the internal 5 ′ region (X) of the internal region (Z) has the expression suppressing sequence, the 5 ′ region (Xc) preferably has the complementary sequence, When the internal 3 ′ side region (Y) of the region (Z) has the expression suppressing sequence, the 3 ′ side region (Yc) preferably has the complementary sequence.
  • the inner 5 ′ region (X) of the inner region (Z) when the 5 ′ region (Xc) has the expression suppressing sequence, the inner 5 ′ region (X) of the inner region (Z) preferably has the complementary sequence, and the 3 ′ region When (Yc) has the expression suppression sequence, the internal 3 ′ side region (Y) of the internal region (Z) preferably has the complementary sequence.
  • the 5′-side region (Xc) is complementary to the inner 5′-side region (X), and the 3′-side region (Yc) is the inner 3′-side region (Y).
  • the region (Xc) is folded toward the region (X), and the region (Xc) and the region (X) can form a double chain by self-annealing.
  • the region (Yc) is folded toward the region (Y), and the region (Yc) and the region (Y) can form a double chain by self-annealing.
  • the inner region (Z) is connected to the inner 5 'region (X) and the inner 3' region (Y).
  • the region (X) and the region (Y) are directly connected, for example, and do not have an intervening sequence therebetween.
  • the inner region (Z) is defined as “the inner 5 ′ side region (X) and the inner 3 ′ side in order to indicate the arrangement relationship between the 5 ′ side region (Xc) and the 3 ′ side region (Yc)”.
  • the region (Y) is connected to each other ”, and in the inner region (Z), the 5 ′ side region (Xc) and the 3 ′ side region (Yc) are, for example,
  • the use of nucleic acid molecules is not limited to being a separate and independent region. That is, for example, when the internal region (Z) has the expression suppression sequence, the expression suppression sequence is arranged across the region (X) and the region (Y) in the internal region (Z). Also good.
  • the 5 'side region (Xc) and the inner 5' side region (X) may be directly connected or indirectly connected, for example.
  • direct linkage includes, for example, linkage by a phosphodiester bond.
  • a linker region (Lx) is provided between the region (Xc) and the region (X), and the region (Xc) and the region ( And X) are linked together.
  • the 3'-side region (Yc) and the internal 3'-side region (Y) may be directly connected or indirectly connected, for example.
  • direct linkage includes, for example, linkage by a phosphodiester bond.
  • a linker region (Ly) is provided between the region (Yc) and the region (Y), and the region (Yc) and the region ( And Y) are linked.
  • the nucleic acid molecule may have, for example, both the linker region (Lx) and the linker region (Ly), or one of them.
  • the linker region (Lx) is provided between the 5 ′ side region (Xc) and the inner 5 ′ side region (X), and the 3 ′ side region (Yc) and the inner 3 'The linker region (Ly) is not present between the side region (Y), that is, the region (Yc) and the region (Y) are directly linked.
  • the linker region (Ly) is provided between the 3 ′ side region (Yc) and the inner 3 ′ side region (Y), and the 5 ′ side region (Xc) and the The linker region (Lx) is not provided between the internal 5′-side region (X), that is, the region (Xc) and the region (X) are directly linked.
  • the linker region (Lx) and the linker region (Ly) each preferably have a structure that does not cause self-annealing within its own region.
  • FIG. 2 (A) is a schematic diagram showing an outline of the order of each region from the 5 ′ side to the 3 ′ side of the nucleic acid molecule
  • FIG. 2 (B) shows that the nucleic acid molecule is the molecule. It is a schematic diagram which shows the state which forms the double chain
  • FIG. 2 merely shows the connection order of the regions and the positional relationship of the regions forming the double chain.
  • the length of each region is not limited to this.
  • FIG. 3A is a schematic diagram showing, as an example, an outline of the order of each region from the 5 ′ side to the 3 ′ side of the nucleic acid molecule
  • FIG. FIG. 2 is a schematic diagram showing a state in which a double chain is formed in the molecule.
  • the nucleic acid molecule is divided between the 5′-side region (Xc) and the inner 5′-side region (X), and between the inner 3′-side region (Y) and the 3′-side.
  • a double chain is formed with the side region (Yc), and the Lx region and the Ly region have a loop structure.
  • FIG. 3 merely shows the order of connection of the regions and the positional relationship of the regions forming the double chain.
  • the length of each region is not limited thereto.
  • the number of bases in the 5 ′ region (Xc), the internal 5 ′ region (X), the internal 3 ′ region (Y) and the 3 ′ region (Yc) is particularly limited. For example, it is as follows.
  • the 5′-side region (Xc) may be complementary to the entire region of the inner 5′-side region (X), for example.
  • the region (Xc) has the same base length as the region (X), and is composed of a base sequence complementary to the entire region from the 5 ′ end to the 3 ′ end of the region (X).
  • the region (Xc) has the same base length as the region (X), and all bases in the region (Xc) are complementary to all bases in the region (X). That is, for example, it is preferably completely complementary.
  • the present invention is not limited to this.
  • 1 to several (2, 3, 4 or 5) bases may be non-complementary.
  • the 5′-side region (Xc) may be complementary to a partial region of the inner 5′-side region (X), for example.
  • the region (Xc) has, for example, the same base length as the partial region of the region (X), that is, consists of a base sequence having a base length shorter by one base or more than the region (X). preferable. More preferably, the region (Xc) has the same base length as the partial region of the region (X), and all the bases of the region (Xc) are included in the partial region of the region (X). It is preferred that it is complementary to all bases, that is, for example, completely complementary.
  • the partial region of the region (X) is preferably, for example, a region (segment) having a base sequence continuous from the 5 ′ terminal base (first base) in the region (X).
  • the 3′-side region (Yc) may be complementary to the entire region of the inner 3′-side region (Y), for example.
  • the region (Yc) has, for example, the same base length as the region (Y) and is composed of a base sequence complementary to the entire region from the 5 ′ end to the 3 ′ end of the region (Y).
  • the region (Yc) has the same base length as the region (Y), and all bases in the region (Yc) are complementary to all bases in the region (Y). That is, for example, it is preferable to be completely complementary.
  • the present invention is not limited to this.
  • 1 to several (2, 3, 4 or 5) bases may be non-complementary.
  • the 3′-side region (Yc) may be complementary to a partial region of the inner 3′-side region (Y), for example.
  • the region (Yc) has, for example, the same base length as the partial region of the region (Y), that is, consists of a base sequence having a base length shorter by one base or more than the region (Y). preferable. More preferably, the region (Yc) has the same base length as the partial region of the region (Y), and all the bases of the region (Yc) are included in the partial region of the region (Y). It is preferred that it is complementary to all bases, that is, for example, completely complementary.
  • the partial region of the region (Y) is preferably, for example, a region (segment) having a base sequence continuous from the base at the 3 'end (first base) in the region (Y).
  • the number of bases (Z) in the internal region (Z), the number of bases (X) in the internal 5 ′ side region (X), and the number of bases (Y) in the internal 3 ′ side region (Y) Relationship between the number of bases (Z) in the internal region (Z), the number of bases (Xc) in the 5′-side region (Xc), and the number of bases (Yc) in the 3′-side region (Yc) Satisfies, for example, the conditions of the following formulas (1) and (2).
  • Z X + Y (1)
  • the relationship between the number of bases (X) in the inner 5 ′ region (X) and the number of bases (Y) in the inner 3 ′ region (Y) is not particularly limited, For example, any condition of the following formula may be satisfied.
  • X Y (19) X ⁇ Y (20) X> Y (21)
  • the number of bases (X) in the inner 5 ′ side region (X), the number of bases (Xc) in the 5 ′ side region (Xc), the number of bases in the inner 3 ′ side region (Y) (Y ) And the number of bases (Yc) in the 3′-side region (Yc) satisfy, for example, the following conditions (a) to (d).
  • Y Yc (4)
  • X Xc (5) Y> Yc (6) (C)
  • the conditions of the following formulas (7) and (8) are satisfied.
  • X> Xc (7) Y> Yc (8) (D)
  • the conditions of the following formulas (9) and (10) are satisfied.
  • X Xc (9)
  • Y Yc (10)
  • the difference between the number of bases (X) in the inner 5 ′ side region (X) and the number of bases (Xc) in the 5 ′ side region (Xc), the inner 3 ′ side region ( The difference between the number of bases (Y) of Y) and the number of bases (Yc) of the 3 ′ side region (Yc) preferably satisfies the following condition, for example.
  • A The conditions of the following formulas (11) and (12) are satisfied.
  • FIG. 4 is a nucleic acid molecule comprising the linker region (Lx) and the linker region (Ly), (A) is the nucleic acid molecule of (a), (B) is the nucleic acid molecule of (b), (C) is an example of the nucleic acid molecule of (c), and (D) is an example of the nucleic acid molecule of (d).
  • a dotted line shows the state which has formed the double chain
  • FIG. 4 has the number of bases (X) in the inner 5 ′ side region (X) and the number of bases (Y) in the inner 3 ′ side region (Y) as “X ⁇ Y” in the formula (20).
  • FIG. 4 is merely a relationship between the inner 5 ′ side region (X) and the 5 ′ side region (Xc), and the relationship between the inner 3 ′ side region (Y) and the 3 ′ side region (Yc).
  • the length and shape of each region are not limited thereto, and the presence or absence of the linker region (Lx) and the linker region (Ly) is not limited thereto.
  • the nucleic acid molecules (a) to (c) include, for example, the 5 ′ side region (Xc) and the internal 5 ′ side region (X), and the 3 ′ side region (Yc) and the internal 3 ′ side.
  • the region (Y) has a base that cannot be aligned with any of the 5 ′ side region (Xc) and the 3 ′ side region (Yc) in the internal region (Z) by forming a double chain, respectively. It can be said that the structure has a base that does not form a double chain.
  • the base that cannot be aligned also referred to as a base that does not form a double chain
  • free base In FIG.
  • the free base region is indicated by “F”.
  • the number of bases in the region (F) is not particularly limited.
  • the number of bases (F) in the region (F) is, for example, the number of bases “X-Xc” in the case of the nucleic acid molecule (a), and “Y—Yc” in the case of the nucleic acid molecule (b). In the case of the nucleic acid molecule (c), it is the total number of bases “X—Xc” and “Y—Yc”.
  • the nucleic acid molecule (d) has a structure in which, for example, the entire region of the internal region (Z) is aligned with the 5 ′ side region (Xc) and the 3 ′ side region (Yc), It can also be said that the entire region (Z) forms a double chain.
  • the nucleic acid molecule (d) the 5 'end of the 5' side region (Xc) and the 3 'end of the 3' side region (Yc) are unlinked.
  • each region is exemplified below for the nucleic acid molecule, but the present invention is not limited to this.
  • the total number of bases of the free base (F) in the 5 ′ side region (Xc), the 3 ′ side region (Yc), and the internal region (Z) is the number of bases in the internal region (Z). .
  • the lengths of the 5 ′ side region (Xc) and the 3 ′ side region (Yc) depend on, for example, the length of the internal region (Z), the number of free bases (F), and the position thereof. Can be determined as appropriate.
  • the number of bases in the internal region (Z) is, for example, 19 bases or more.
  • the lower limit of the number of bases is, for example, 19 bases, preferably 20 bases, and more preferably 21 bases.
  • the upper limit of the number of bases is, for example, 50 bases, preferably 40 bases, and more preferably 30 bases.
  • Specific examples of the number of bases in the internal region (Z) include, for example, 19 bases, 20 bases, 21 bases, 22 bases, 23 bases, 24 bases, 25 bases, 26 bases, 27 bases, 28 bases, 29 bases, or , 30 bases.
  • the internal region (Z) may be, for example, a region composed only of the expression suppression sequence or a region including the expression suppression sequence.
  • the number of bases of the expression suppression sequence is, for example, as described above.
  • the internal region (Z) contains the expression suppression sequence it may further have an additional sequence on the 5 'side and / or 3' side of the expression suppression sequence.
  • the number of bases of the additional sequence is, for example, 1 to 31 bases, preferably 1 to 21 bases, more preferably 1 to 11 bases, and further preferably 1 to 7 bases.
  • the number of bases in the 5 ′ side region (Xc) is, for example, 1 to 29 bases, preferably 1 to 11 bases, more preferably 1 to 7 bases, and further preferably 1 to 4 bases. Particularly preferred are 1 base, 2 bases and 3 bases.
  • the internal region (Z) or the 3 'side region (Yc) includes the expression suppression sequence, for example, such a base number is preferable.
  • the number of bases in the internal region (Z) is 19 to 30 bases (for example, 19 bases)
  • the number of bases in the 5 ′ side region (Xc) is, for example, 1 to 11 bases
  • the number is preferably 1 to 7 bases, more preferably 1 to 4 bases, and still more preferably 1 base, 2 bases, and 3 bases.
  • the 5′-side region (Xc) may be, for example, a region composed only of the expression suppression sequence, or a region including the expression suppression sequence But you can.
  • the length of the expression suppression sequence is, for example, as described above.
  • the 5 'region (Xc) contains the expression suppression sequence, it may further have an additional sequence on the 5' side and / or 3 'side of the expression suppression sequence.
  • the number of bases of the additional sequence is, for example, 1 to 11 bases, and preferably 1 to 7 bases.
  • the number of bases in the 3 ′ side region (Yc) is, for example, 1 to 29 bases, preferably 1 to 11 bases, more preferably 1 to 7 bases, and further preferably 1 to 4 bases. Particularly preferred are 1 base, 2 bases and 3 bases.
  • the internal region (Z) or the 5 'side region (Xc) includes the expression suppression sequence, for example, such a base number is preferable.
  • the number of bases in the internal region (Z) is 19 to 30 bases (for example, 19 bases)
  • the number of bases in the 3 ′ side region (Yc) is, for example, 1 to 11 bases
  • the number is preferably 1 to 7 bases, more preferably 1 to 4 bases, and still more preferably 1 base, 2 bases, and 3 bases.
  • the 3 ′ side region (Yc) may be, for example, a region composed only of the expression suppression sequence, or a region including the expression suppression sequence But you can.
  • the length of the expression suppression sequence is, for example, as described above.
  • the 3 'side region (Yc) includes the expression suppression sequence, it may further have an additional sequence on the 5' side and / or 3 'side of the expression suppression sequence.
  • the number of bases of the additional sequence is, for example, 1 to 11 bases, and preferably 1 to 7 bases.
  • the number of bases in the internal region (Z), the 5′-side region (Xc), and the 3′-side region (Yc) is expressed by, for example, “Z ⁇ Xc + Yc” in the formula (2). Can do.
  • the number of bases “Xc + Yc” is, for example, the same as or smaller than the inner region (Z).
  • “Z ⁇ (Xc + Yc)” is, for example, 1 to 10, preferably 1 to 4, more preferably 1, 2 or 3.
  • the “Z ⁇ (Xc + Yc)” corresponds to the number of bases (F) in the free base region (F) in the internal region (Z).
  • the linker region (Lx) preferably has, for example, a length that allows the internal 5 ′ side region (X) and the 5 ′ side region (Xc) to form a double chain, and the linker region (Ly) ) Is, for example, preferably a length such that the inner 3 ′ side region (Y) and the 3 ′ side region (Yc) can form a double chain.
  • the lengths of the linker region (Lx) and the linker region (Ly) may be the same or different, and the base sequences thereof may be the same or different.
  • the lower limit of the number of bases in the linker region (Lx) and the linker region (Ly) is, for example, 1 base, preferably 2 bases, more preferably 3 bases, and the upper limit thereof is, for example, 100 bases, preferably 80 bases, more preferably 50 bases.
  • Specific examples of the number of bases in each linker region include 1 to 50 bases, 1 to 30 bases, 1 to 20 bases, 1 to 10 bases, 1 to 7 bases, and 1 to 4 bases. This is not a limitation.
  • the total length of the nucleic acid molecule is not particularly limited.
  • the lower limit of the total number of bases is, for example, 38 bases, preferably 40 bases, more preferably 42 bases, still more preferably 44 bases.
  • the base is particularly preferably 46 bases, and the upper limit thereof is, for example, 300 bases, preferably 200 bases, more preferably 150 bases, still more preferably 100 bases, and particularly preferably 80 bases. .
  • the lower limit of the total number of bases excluding the linker region (Lx) and the linker region (Ly) is, for example, 38 bases, preferably 40 bases, more preferably 42 bases, More preferably, it is 44 bases, particularly preferably 46 bases, and the upper limit is, for example, 300 bases, preferably 200 bases, more preferably 150 bases, still more preferably 100 bases, Preferably, it is 80 bases.
  • the 5 'end and the 3' end may be bound or unbound.
  • the nucleic acid molecule of this form is a circular single-stranded nucleic acid molecule.
  • the nucleic acid molecule of the present embodiment is preferably a non-phosphate group at the 5 'end, for example, since it can maintain unbonded at both ends.
  • a third form of the single-stranded nucleic acid molecule is a molecule in which the linker region has a non-nucleotide structure.
  • This embodiment can use the above description except that the linker region (Lx) and / or the linker region (Ly) has a non-nucleotide structure in the nucleic acid molecules of the first and second forms.
  • the non-nucleotide structure is not particularly limited, and examples thereof include polyalkylene glycol, pyrrolidine skeleton and piperidine skeleton.
  • examples of the polyalkylene glycol include polyethylene glycol.
  • the pyrrolidine skeleton may be, for example, a skeleton of a pyrrolidine derivative in which one or more carbons constituting the 5-membered ring of pyrrolidine are substituted.
  • the carbon at the 2-position of the 5-membered ring C— It is preferable that it is carbon atoms other than carbon of 2).
  • the carbon may be substituted with, for example, nitrogen, oxygen or sulfur.
  • the pyrrolidine skeleton may contain, for example, a carbon-carbon double bond or a carbon-nitrogen double bond in the 5-membered ring of pyrrolidine.
  • the carbon and nitrogen constituting the 5-membered ring of pyrrolidine may be bonded, for example, to hydrogen or a substituent as described below.
  • the linker region (Lx) is substituted with the region (X) and the region (Xc), and the linker region (Ly) is substituted with the region (Y) and the region (Yc), for example, the pyrrolidine skeleton.
  • the preferred position at which the substituent is substituted is any one carbon and nitrogen of the 5-membered ring, preferably at the 2-position of the 5-membered ring.
  • Carbon (C-2) and nitrogen are examples of the main non-nucleotide structure containing the pyrrolidine skeleton.
  • the proline, prolinol, and the like are excellent in safety because they are, for example, in-vivo substances and their reduced forms.
  • the piperidine skeleton may be, for example, a skeleton of a piperidine derivative in which one or more carbons constituting the six-membered ring of piperidine are substituted, and when substituted, for example, the carbon at the 2-position of piperidine (C-2) It is preferably a carbon atom other than carbon.
  • the carbon may be substituted with, for example, nitrogen, oxygen or sulfur.
  • the piperidine skeleton may contain, for example, a carbon-carbon double bond or a carbon-nitrogen double bond in the 6-membered ring of piperidine.
  • the carbon and nitrogen constituting the piperidine 6-membered ring may be bonded to, for example, a hydrogen group or a substituent as described later.
  • the linker region (Lx) includes the region (X) and the region (Xc), the linker region (Ly) includes the region (Y) and the region (Yc), and any one of the piperidine skeleton, for example. It may be bonded via a group, preferably any one carbon atom of the six-membered ring and nitrogen, more preferably carbon (C-2) at the 2-position of the six-membered ring and nitrogen. It is.
  • the linker region may include, for example, only a non-nucleotide residue having the non-nucleotide structure, or may include a non-nucleotide residue having the non-nucleotide structure and a nucleotide residue.
  • the linker region is represented by the following formula (I), for example.
  • X 1 and X 2 are each independently H 2 , O, S or NH; Y 1 and Y 2 are each independently a single bond, CH 2 , NH, O or S; R 3 is a hydrogen atom or substituent bonded to C-3, C-4, C-5 or C-6 on ring A; L 1 is an alkylene chain consisting of n carbon atoms, where the hydrogen atom on the alkylene carbon atom is OH, OR a , NH 2 , NHR a , NR a R b , SH, or SR a May be substituted and / or L 1 is a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with an oxygen atom,
  • L 2 is an alkylene
  • the ring A may contain a carbon-carbon double bond or a carbon-nitrogen double bond
  • the region (Xc) and the region (X) are each bonded to the linker region (Lx) via —OR 1 — or —OR 2 —;
  • the region (Yc) and the region (Y) are each bonded to the linker region (Ly) via —OR 1 — or —OR 2 —,
  • R 1 and R 2 may be present or absent, and when present, R 1 and R 2 are each independently a nucleotide residue or the structure (I).
  • X 1 and X 2 are each independently, for example, H 2 , O, S or NH.
  • X 1 being H 2 means that X 1 together with the carbon atom to which X 1 is bonded forms CH 2 (methylene group). The same is true for X 2.
  • Y 1 and Y 2 are each independently a single bond, CH 2 , NH, O or S.
  • l 1 or 2.
  • ring A is a 5-membered ring, for example, the pyrrolidine skeleton.
  • main non-nucleotide structure containing the pyrrolidine skeleton include proline and prolinol.
  • ring A is a 6-membered ring, for example, the piperidine skeleton.
  • one carbon atom other than C-2 on ring A may be substituted with nitrogen, oxygen or sulfur.
  • Ring A may contain a carbon-carbon double bond or a carbon-nitrogen double bond in ring A.
  • any optical isomer may be used.
  • R 3 is a hydrogen atom or a substituent bonded to C-3, C-4, C-5 or C-6 on the ring A.
  • R 3 is the above-described substituent, the substituent R 3 may be one, plural, or absent, and when plural, it may be the same or different.
  • the substituent R 3 is, for example, halogen, OH, OR 4 , NH 2 , NHR 4 , NR 4 R 5 , SH, SR 4 or an oxo group ( ⁇ O).
  • R 4 and R 5 are, for example, each independently a substituent or a protecting group, and may be the same or different.
  • substituents include halogen, alkyl, alkenyl, alkynyl, haloalkyl, aryl, heteroaryl, arylalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cyclylalkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, heterocyclylalkenyl. , Heterocyclylalkyl, heteroarylalkyl, silyl, silyloxyalkyl and the like. The same applies hereinafter.
  • the substituent R 3 may be any of these listed substituents.
  • the protecting group is, for example, a functional group that converts a highly reactive functional group to inert, and examples thereof include known protecting groups.
  • the protecting group for example, the description in the literature (J.F.W. McOmie, “Protecting Groups in Organic Chemistry” Prenum Press, London and New York, 1973) can be cited.
  • the protective group is not particularly limited, and examples thereof include tert-butyldimethylsilyl group (TBDMS), bis (2-acetoxyethyloxy) methyl group (ACE), triisopropylsilyloxymethyl group (TOM), 1- (2 -Cyanoethoxy) ethyl group (CEE), 2-cyanoethoxymethyl group (CEM), tolylsulfonylethoxymethyl group (TEM), dimethoxytrityl group (DMTr) and the like.
  • R 3 is OR 4
  • the protecting group is not particularly limited, and examples thereof include a TBDMS group, an ACE group, a TOM group, a CEE group, a CEM group, and a TEM group.
  • the silyl-containing group of [Chemical Formula 5] of Japanese Patent No. 5555346 is also exemplified. The same applies hereinafter.
  • L 1 is an alkylene chain consisting of n carbon atoms.
  • the hydrogen atom on the alkylene carbon atom may be substituted with, for example, OH, OR a , NH 2 , NHR a , NR a R b , SH, or SR a .
  • L 1 may be a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with an oxygen atom.
  • the polyether chain is, for example, polyethylene glycol.
  • L 2 is an alkylene chain composed of m carbon atoms.
  • the hydrogen atom on the alkylene carbon atom may be substituted with, for example, OH, OR c , NH 2 , NHR c , NR c R d , SH or SR c , or may not be substituted.
  • L 2 may be a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with an oxygen atom.
  • Y 2 is NH, O or S
  • the L 2 atom bonded to Y 2 is carbon
  • the L 2 atom bonded to OR 2 is carbon
  • oxygen atoms are not adjacent to each other. That is, for example, when Y 2 is O, the oxygen atom and the oxygen atom of L 2 are not adjacent, and the oxygen atom of OR 2 and the oxygen atom of L 2 are not adjacent.
  • N in L 1 and m in L 2 are not particularly limited, and the lower limit is, for example, 0, and the upper limit is not particularly limited.
  • n and m can be appropriately set according to the desired length of the linker region (Lx) or (Ly), for example.
  • n and m are each preferably 0 to 30, more preferably 0 to 20, and still more preferably 0 to 15 from the viewpoint of production cost and yield.
  • n + m is, for example, 0 to 30, preferably 0 to 20, and more preferably 0 to 15.
  • R a , R b , R c and R d are, for example, each independently a substituent or a protecting group.
  • the substituent and the protecting group are the same as described above, for example.
  • hydrogen atoms may be independently substituted with halogens such as Cl, Br, F and I, for example.
  • the region (Xc) and the region (X) are in the linker region (Lx), the region (Yc) and the region (Y) are in the linker region (Ly), for example, —OR 1 — Alternatively, the bonds are made through —OR 2 —.
  • R 1 and R 2 may or may not exist.
  • R 1 and R 2 are each independently a nucleotide residue or the structure of formula (I) above.
  • the linker region (Lx) and the linker region (Ly) are, for example, of the formula (I) except for the nucleotide residue R 1 and / or R 2 It is formed from the non-nucleotide residue consisting of a structure and the nucleotide residue.
  • the linker region (Lx) and the linker region (Ly) are, for example, the non-nucleotide residue having the structure of the formula (I) Two or more structures are connected.
  • the structure of the formula (I) may include 1, 2, 3, or 4, for example.
  • the structure of (I) may be directly linked or may be bonded via the nucleotide residue, for example.
  • R 1 and R 2 are not present, the linker region (Lx) and the linker region (Ly) are formed only from the non-nucleotide residue having the structure of the formula (I), for example.
  • the linker region (Lx) and the linker region (Ly) are formed from the non-nucleotide residue and the nucleotide residue, the non-nucleotide in the linker region (Lx) and the linker region (Ly)
  • the lower limit of the total number of nucleotide residues and the nucleotide residues is, for example, 2, 3, or 4, and the upper limit is, for example, 100, 80, or 50.
  • Specific examples of the number of ring A in each linker region include, for example, 2 to 50, 2 to 30, 2 to 20, 2 to 10, 2 to 7, 2 to 4, 2 to Although 3 etc. can be illustrated, it is not restrict
  • the number of the non-nucleotide residues in the linker region (Lx) and the linker region (Ly) is The lower limit is, for example, 1, 2, or 3.
  • the upper limit is, for example, 100, 80, or 50.
  • Specific examples of the number of ring A in each linker region include 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 7, 1 to 4, 1 to Although 2 etc. can be illustrated, it is not restrict
  • the combination of the region (Xc) and the region (X), the region (Yc) and the region (Y), and the —OR 1 — and —OR 2 — is not particularly limited.
  • One of the following conditions can be given.
  • Condition (1) The region (Xc) is bonded to the structure of the formula (I) through —OR 2 —, and the region (X) is bonded through —OR 1 —.
  • the region (Yc) is bonded to the structure of the formula (I) through —OR 1 —, and the region (Y) is bonded through —OR 2 —.
  • Condition (2) The region (Xc) is bonded to the structure of the formula (I) through —OR 2 —, and the region (X) is bonded through —OR 1 —.
  • the region (Yc) is bonded to the structure of the formula (I) through —OR 2 —, and the region (Y) is bonded through —OR 1 —.
  • Condition (3) The region (Xc) is bonded to the structure of the formula (I) through —OR 1 —, and the region (X) is bonded through —OR 2 —.
  • the region (Yc) is bonded to the structure of the formula (I) through —OR 1 —, and the region (Y) is bonded through —OR 2 —.
  • Condition (4) The region (Xc) is bonded to the structure of the formula (I) through —OR 1 —, and the region (X) is bonded through —OR 2 —.
  • the region (Yc) is bonded to the structure of the formula (I) through —OR 2 —, and the region (Y) is bonded through —OR 1 —.
  • Examples of the structure of the formula (I) include the following formulas (I-1) to (I-9), in which n and m are the same as those in the formula (I).
  • q is an integer of 0 to 10.
  • n, m and q are not particularly limited and are as described above.
  • a preferred embodiment of the nucleic acid molecule of the present invention in which the linker region Lx has the structure represented by the above formula (I-6a) is a nucleic acid molecule consisting of the base sequence represented by SEQ ID NO: 13 or 14.
  • a preferred embodiment of the nucleic acid molecule of the present invention when the linker regions Lx and Ly include nucleotide residues is a nucleic acid molecule consisting of the base sequence represented by SEQ ID NO: 15 or 16.
  • a preferred embodiment of the nucleic acid molecule of the present invention in which the linker regions Lx and Ly have the structure shown in the above formula (I-6a) is a nucleic acid molecule consisting of the base sequence represented by SEQ ID NO: 17 or 18. it can.
  • the linker regions Lx and Ly are more preferably a group represented by the following formula.
  • the structural unit of the nucleic acid molecule of the present invention is not particularly limited, and examples thereof include nucleotide residues.
  • the nucleotide residue include a ribonucleotide residue and a deoxyribonucleotide residue.
  • the nucleotide residue include an unmodified unmodified nucleotide residue and a modified modified nucleotide residue.
  • the nucleic acid molecule of the present invention can improve nuclease resistance and stability, for example, by including the modified nucleotide residue.
  • the nucleic acid molecule of the present invention may further contain a non-nucleotide residue in addition to the nucleotide residue, for example.
  • each of the constituent units in the region other than the linker is preferably the nucleotide residue.
  • Each region is composed of the following residues (1) to (3), for example. (1) Unmodified nucleotide residue (2) Modified nucleotide residue (3) Unmodified nucleotide residue and modified nucleotide residue
  • the structural unit of the linker region is not particularly limited, and examples thereof include the nucleotide residue and the non-nucleotide residue.
  • the linker region may be composed of, for example, only the nucleotide residue, may be composed of only the non-nucleotide residue, or may be composed of the nucleotide residue and the non-nucleotide residue.
  • the linker region is composed of the following residues (1) to (7), for example.
  • both structural units may be the same or different.
  • Specific examples include, for example, a form in which the constituent units of both linker regions are the nucleotide residues, a form in which the constituent units of both linker regions are the non-nucleotide residues, and the constituent units of one region are the nucleotide residues.
  • the other linker region is a non-nucleotide residue.
  • nucleic acid molecule of the present invention examples include a molecule composed only of the nucleotide residue, a molecule containing the non-nucleotide residue in addition to the nucleotide residue, and the like.
  • the nucleotide residue may be, for example, only the unmodified nucleotide residue, only the modified nucleotide residue, or the unmodified nucleotide residue and the modification. Both nucleotide residues may be used.
  • the number of the modified nucleotide residue is not particularly limited, and is, for example, “one or several”, specifically For example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and most preferably 1 or 2.
  • the number of the non-nucleotide residue is not particularly limited, and is, for example, “one or several”, specifically, for example, 1 to Eight, one to six, one to four, one, two or three.
  • the number of the modified ribonucleotide residue is not particularly limited, and is, for example, “one or several”. Specifically, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and most preferably 1 or 2.
  • the modified ribonucleotide residue relative to the unmodified ribonucleotide residue may be, for example, the deoxyribonucleotide residue in which a ribose residue is replaced with a deoxyribose residue.
  • the number of the deoxyribonucleotide residue is not particularly limited, and is, for example, “one or several” Specifically, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and most preferably 1 or 2.
  • the number of the modified deoxyribonucleotide residue is not particularly limited, and is, for example, “one or several”. Specifically, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and most preferably 1 or 2.
  • the modified deoxyribonucleotide residue relative to the unmodified deoxyribonucleotide residue may be, for example, the ribonucleotide residue in which a deoxyribose residue is replaced with a ribose residue.
  • the number of the ribonucleotide residue is not particularly limited, and is, for example, “one or several”. Specifically, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and most preferably 1 or 2.
  • the nucleic acid molecule of the present invention may contain, for example, a labeling substance and be labeled with the labeling substance.
  • the labeling substance is not particularly limited, and examples thereof include fluorescent substances, dyes, isotopes and the like.
  • the labeling substance include fluorophores such as pyrene, TAMRA, fluorescein, Cy3 dye, and Cy5 dye, and examples of the dye include Alexa dye such as Alexa488.
  • the isotope include a stable isotope and a radioactive isotope, and preferably a stable isotope.
  • the stable isotope has a low risk of exposure and does not require a dedicated facility, so that it is easy to handle and the cost can be reduced.
  • the stable isotope does not change the physical properties of the labeled compound, for example, and is excellent in properties as a tracer.
  • the stable isotope is not particularly limited, and examples thereof include 2 H, 13 C, 15 N, 17 O, 18 O, 33 S, 34 S, and 36 S.
  • the nucleic acid molecule of the present invention can suppress the expression of the hepatitis B virus gene. For this reason, the nucleic acid molecule of the present invention can be used, for example, as a therapeutic agent for diseases caused by hepatitis B virus.
  • “treatment” includes, for example, the meanings of preventing the disease, improving the disease, and improving the prognosis. Examples of the disease include hepatitis B, cirrhosis, and liver cancer.
  • the method of using the nucleic acid molecule of the present invention is not particularly limited, and for example, the nucleic acid molecule may be administered to an administration subject having the hepatitis B virus.
  • the administration target examples include non-human animals such as humans and non-human mammals other than humans. Moreover, for example, it may be administered to cells, tissues or organs such as human or non-human animals, and the nucleic acid molecule of the present invention is preferably administered to hepatocytes, liver tissues or liver. The administration may be, for example, in vivo or in vitro.
  • the cells are not particularly limited.
  • various cultured cells such as Huh7, A549, HeLa, 293, and COS7
  • pluripotent stem cells such as ES cells and iPS cells
  • somatic stem cells such as hematopoietic stem cells
  • pluripotent Examples include various cultured cells derived from stem cells or somatic stem cells, cells isolated from living bodies such as primary cultured cells, and the like.
  • the nucleic acid molecule of the present invention When the nucleic acid molecule of the present invention is administered to an administration subject in vivo, the nucleic acid molecule binds to a cell surface receptor in order to efficiently deliver the organ to a specific organ, tissue or cell in the living body. It may be conjugated with a ligand.
  • the nucleic acid molecule of the present invention can be conjugated with a surface receptor ligand or the like characteristic of liver cells in order to improve delivery efficiency to the liver.
  • ligands include cholesterol and N-acetylgalactosamine (GalNAc) clusters. Examples of the N-acetylgalactosamine (GalNAc) cluster include compounds having the following structural formula.
  • nucleic acid molecule of the present invention refers to the description of the composition of the present invention, expression suppression method, treatment method and the like described later.
  • nucleic acid molecule of the present invention can suppress the expression of the hepatitis B virus gene as described above, it is useful, for example, as a research tool for pharmaceuticals, diagnostic agents, agricultural chemicals, agricultural chemicals, medicine, life sciences, etc. It is.
  • nucleotide residues include, for example, sugars, bases and phosphates as constituent elements.
  • examples of the nucleotide residue include a ribonucleotide residue and a deoxyribonucleotide residue as described above.
  • the ribonucleotide residue has, for example, a ribose residue as a sugar, and has adenine (A), guanine (G), cytosine (C), and uracil (U) as bases
  • the deoxyribose residue is For example, it has a deoxyribose residue as a sugar and has adenine (A), guanine (G), cytosine (C) and thymine (T) as bases.
  • the nucleotide residue includes an unmodified nucleotide residue and a modified nucleotide residue.
  • each of the constituent elements is, for example, the same or substantially the same as that existing in nature, and preferably the same or substantially the same as that naturally occurring in the human body. .
  • the modified nucleotide residue is, for example, a nucleotide residue obtained by modifying the unmodified nucleotide residue.
  • the modified nucleotide residue for example, any of the constituent elements of the unmodified nucleotide residue may be modified.
  • “modification” refers to, for example, substitution, addition and / or deletion of the component, substitution, addition and / or deletion of atoms and / or functional groups in the component, and is referred to as “modification”. be able to.
  • modified nucleotide residue include naturally occurring nucleotide residues, artificially modified nucleotide residues, and the like. For example, Limbac et al.
  • modified nucleosides of RNA Nucleic Acids Res. 22: 2183-2196
  • the modified nucleotide residue may be, for example, a residue of the nucleotide substitute.
  • ribophosphate skeleton examples include modification of a ribose-phosphate skeleton (hereinafter referred to as ribophosphate skeleton).
  • a ribose residue can be modified.
  • the ribose residue can be modified, for example, at the 2′-position carbon.
  • a hydroxyl group bonded to the 2′-position carbon can be replaced with hydrogen or a halogen such as fluoro.
  • the ribose residue can be replaced with deoxyribose.
  • the ribose residue can be substituted with, for example, a stereoisomer, and can be substituted with, for example, an arabinose residue.
  • the ribophosphate skeleton may be substituted with a non-ribophosphate skeleton having a non-ribose residue and / or non-phosphate, for example.
  • the non-ribophosphate skeleton include uncharged ribophosphate skeletons.
  • the substitute for the nucleotide substituted with the non-ribophosphate skeleton include morpholino, cyclobutyl, pyrrolidine and the like.
  • Other examples of the substitute include artificial nucleic acid monomer residues. Specific examples include PNA (peptide nucleic acid), LNA (Locked Nucleic Acid), ENA (2'-O, 4'-C-Ethylene-bridged Nucleic Acid), and PNA is preferable.
  • a phosphate group can be modified.
  • the phosphate group closest to the sugar residue is called an ⁇ -phosphate group.
  • the ⁇ -phosphate group is negatively charged, and the charge is evenly distributed over two oxygen atoms that are not bound to a sugar residue.
  • the four oxygen atoms in the ⁇ -phosphate group in the phosphodiester bond between nucleotide residues, the two oxygen atoms that are non-bonded to the sugar residue are hereinafter referred to as “non-linking oxygen”.
  • the two oxygen atoms bonded to the sugar residue are hereinafter referred to as “linking oxygen”.
  • the ⁇ -phosphate group is preferably subjected to, for example, a modification that makes it uncharged or a modification that makes the charge distribution in the unbound oxygen asymmetric.
  • the phosphate group may replace the non-bonded oxygen, for example.
  • the oxygen is, for example, one of S (sulfur), Se (selenium), B (boron), C (carbon), H (hydrogen), N (nitrogen), and OR (R is an alkyl group or an aryl group).
  • R is an alkyl group or an aryl group.
  • the non-bonded oxygen for example, both are preferably substituted, and more preferably, both are substituted with S.
  • the modified phosphate group include phosphorothioate, phosphorodithioate, phosphoroselenate, boranophosphate, boranophosphate ester, phosphonate hydrogen, phosphoramidate, alkyl or arylphosphonate, and phosphotriester. Among them, phosphorodithioate in which the two non-bonded oxygens are both substituted with S is preferable.
  • the phosphate group may substitute, for example, the bonded oxygen.
  • the oxygen can be substituted, for example, with any atom of S (sulfur), C (carbon) and N (nitrogen), and the modified phosphate group is, for example, a bridged phosphoramidate, S substituted with N Substituted bridged phosphorothioates, bridged methylene phosphonates substituted with C, and the like.
  • the binding oxygen substitution is preferably performed, for example, on at least one of the 5 ′ terminal nucleotide residue and the 3 ′ terminal nucleotide residue of the nucleic acid molecule of the present invention. For the 'side, substitution with N is preferred.
  • the phosphate group may be substituted with, for example, the phosphorus-free linker.
  • the linker include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioform acetal, form acetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethyl. Hydrazo, methyleneoxymethylimino and the like, preferably methylenecarbonylamino group and methylenemethylimino group.
  • nucleic acid molecule of the present invention for example, at least one nucleotide residue at the 3 'end and the 5' end may be modified.
  • the modification may be, for example, either the 3 'end or the 5' end, or both.
  • the modification is, for example, as described above, and is preferably performed on the terminal phosphate group.
  • the phosphate group may be modified entirely, or one or more atoms in the phosphate group may be modified. In the former case, for example, the entire phosphate group may be substituted or deleted.
  • Examples of the modification of the terminal nucleotide residue include addition of other molecules.
  • Examples of the other molecule include functional molecules such as a labeling substance and a protecting group as described above.
  • Examples of the protecting group include S (sulfur), Si (silicon), B (boron), ester-containing groups, and the like.
  • the functional molecule such as the labeling substance can be used for detecting the nucleic acid molecule of the present invention, for example.
  • the other molecule may be added to the phosphate group of the nucleotide residue, for example, or may be added to the phosphate group or the sugar residue via a spacer.
  • the terminal atom of the spacer can be added or substituted, for example, to the binding oxygen of the phosphate group or O, N, S or C of the sugar residue.
  • the binding site of the sugar residue is preferably, for example, C at the 3 'position or C at the 5' position, or an atom bonded thereto.
  • the spacer can be added or substituted at a terminal atom of a nucleotide substitute such as PNA.
  • the spacer is not particularly limited.
  • n is a positive integer
  • n 3 or 6 is preferable.
  • the molecule to be added to the terminal includes, for example, a dye, an intercalating agent (for example, acridine), a crosslinking agent (for example, psoralen, mitomycin C), a porphyrin (TPPC4, texaphyrin, suffirin), a polycyclic Aromatic hydrocarbons (eg phenazine, dihydrophenazine), artificial endonucleases (eg EDTA), lipophilic carriers (eg cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis- O (hexadecyl) glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3- (oleoy
  • the 5 ′ end may be modified with, for example, a phosphate group or a phosphate group analog.
  • the phosphate group include 5 ′ monophosphate ((HO) 2 (O) P—O-5 ′), 5 ′ diphosphate ((HO) 2 (O) P—O—P (HO)) (O) -O-5 ′), 5 ′ triphosphate ((HO) 2 (O) PO— (HO) (O) PO—P (HO) (O) —O-5 ′) 5'-guanosine cap (7-methylated or unmethylated, 7m-GO-5 '-(HO) (O) PO- (HO) (O) PO-OP (HO) ( O) —O-5 ′), 5′-adenosine cap (Appp), any modified or unmodified nucleotide cap structure (N—O-5 ′-(HO) (O) PO— (HO) (O ) P—O—P (HO) (O) (O) (O) (O) (O
  • the base is not particularly limited.
  • the base may be, for example, a natural base or a non-natural base.
  • the base may be, for example, naturally derived or a synthetic product.
  • As the base for example, a general base or a modified analog thereof can be used.
  • Examples of the base include purine bases such as adenine and guanine, and pyrimidine bases such as cytosine, uracil and thymine.
  • Other examples of the base include inosine, thymine, xanthine, hypoxanthine, nubalarine, isoguanisine, and tubercidine.
  • the base examples include alkyl derivatives such as 2-aminoadenine and 6-methylated purine; alkyl derivatives such as 2-propylated purine; 5-halouracil and 5-halocytosine; 5-propynyluracil and 5-propynylcytosine; -Azouracil, 6-azocytosine and 6-azothymine; 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5- (2-aminopropyl) uracil, 5-aminoallyluracil; 8-halogenated, aminated, Thiolated, thioalkylated, hydroxylated and other 8-substituted purines; 5-trifluoromethylated and other 5-substituted pyrimidines; 7-methylguanine; 5-substituted pyrimidines; 6-azapyrimidines; N-2, N -6 and O-6 substituted purines (2-aminopropyladenyl 5-
  • the modified nucleotide residue may include, for example, a residue lacking a base, that is, an abasic ribophosphate skeleton.
  • the modified nucleotide residues are, for example, US Provisional Application No. 60 / 465,665 (filing date: April 25, 2003) and International Application No. PCT / US04 / 07070 (filing date: 2004/3). The residues described on the 8th of May) can be used, and the present invention can incorporate these documents.
  • Method for synthesizing nucleic acid molecule of the present invention is not particularly limited, and conventionally known methods can be adopted.
  • Examples of the synthesis method include a synthesis method using a genetic engineering technique, a chemical synthesis method, and the like.
  • Examples of genetic engineering techniques include in vitro transcription synthesis, a method using a vector, and a method using a PCR cassette.
  • the vector is not particularly limited, and examples thereof include non-viral vectors such as plasmids and viral vectors.
  • the chemical synthesis method is not particularly limited, and examples thereof include a phosphoramidite method and an H-phosphonate method.
  • a commercially available automatic nucleic acid synthesizer can be used.
  • amidite is generally used.
  • the amidite is not particularly limited, and commercially available amidites include, for example, RNA Phosphoramidates (2′-O-TBDMSi, trade name, Michisato Pharmaceutical), ACE amidite, TOM amidite, CEE amidite, CEM amidite, TEM amidite, etc. Is given.
  • the expression vector of the present invention comprises a nucleic acid encoding the nucleic acid molecule of the present invention, and is characterized by expressing the nucleic acid molecule of the present invention. “Expression” of the nucleic acid molecule of the present invention includes not only the case where the nucleic acid molecule of the present invention is formed by transcription but also the case where the nucleic acid molecule of the present invention is formed by processing after transcription.
  • the expression vector of the present invention is characterized by containing the nucleic acid, and other configurations are not limited at all.
  • the nucleic acid is inserted so that the vector can be expressed.
  • the vector into which the nucleic acid is inserted is not particularly limited, and for example, a general vector can be used, and examples thereof include viral vectors and non-viral vectors. Examples of the non-viral vector include a plasmid vector.
  • the above-described nucleic acid molecule of the present invention or the nucleic acid encoding the same is functionally used as a promoter capable of exhibiting promoter activity in cells to be administered (for example, human hepatocytes). It is connected.
  • the promoter used is not particularly limited as long as it can function in human hepatocytes to be administered.
  • a pol I promoter, pol II promoter, pol III promoter, or the like can be used as the promoter.
  • SV40-derived early promoter, viral promoter such as cytomegalovirus LTR, mammalian constituent protein gene promoter such as ⁇ -actin gene promoter, and RNA promoter such as tRNA promoter are used.
  • RNA expression it is preferable to use a pol III promoter as a promoter.
  • a pol III promoter include U6 promoter, H1 promoter, tRNA promoter and the like.
  • the expression vector preferably contains a transcription termination signal, that is, a terminator region, downstream of the nucleic acid molecule of the present invention or the nucleic acid encoding it.
  • a selection marker gene for selecting transformed cells a gene that imparts resistance to drugs such as tetracycline, ampicillin, and kanamycin, a gene that complements an auxotrophic mutation, and the like.
  • examples of vectors suitable for human administration include retroviruses, adenoviruses, virus vectors such as adeno-associated viruses, plasmid vectors, and the like.
  • adenovirus has advantages such as extremely high gene transfer efficiency and can be introduced into non-dividing cells.
  • gene expression is transient and usually lasts only about 4 weeks.
  • use of an adeno-associated virus that has relatively high gene transfer efficiency can be introduced into non-dividing cells, and can be integrated into the chromosome via an inverted terminal repeat (ITR) Also preferred.
  • ITR inverted terminal repeat
  • composition of the present invention is a composition for suppressing the expression of hepatitis B virus gene, and is characterized by comprising the nucleic acid molecule or expression vector of the present invention.
  • the composition of the present invention is characterized by containing the nucleic acid molecule or expression vector of the present invention, and other configurations are not limited at all.
  • the composition of the present invention can also be referred to as, for example, an expression suppression reagent.
  • the expression of the hepatitis B virus gene can be suppressed by administration to a subject in which the hepatitis B virus is present.
  • composition of the present invention is characterized by including the nucleic acid molecule or expression vector of the present invention.
  • the composition of the present invention is characterized by containing the nucleic acid molecule or expression vector of the present invention, and other configurations are not limited at all.
  • the composition of the present invention can also be referred to as, for example, a pharmaceutical product or a pharmaceutical composition.
  • the expression of the gene can be suppressed and the disease can be treated.
  • the disease are as described above, and include hepatitis B, cirrhosis, liver cancer and the like.
  • treatment includes, for example, the meanings of prevention of the above-mentioned diseases, improvement of the diseases, and improvement of the prognosis.
  • the method of using the pharmaceutical composition of the present invention is not particularly limited.
  • the nucleic acid molecule or the expression vector may be administered to an administration subject having the hepatitis B virus.
  • Examples of the administration target include cells, tissues or organs.
  • Examples of the administration subject include non-human animals such as humans and non-human mammals other than humans.
  • the administration may be, for example, in vivo or in vitro.
  • the cells are not particularly limited, and examples thereof include the cells described above.
  • the administration method is not particularly limited, and can be appropriately determined according to the administration subject, for example.
  • the administration subject is a cultured cell
  • examples thereof include a method using a transfection reagent and an electroporation method.
  • the pharmaceutical composition of the present invention may contain, for example, only the nucleic acid molecule or expression vector of the present invention, or may contain other additives.
  • the additive is not particularly limited, and for example, a pharmaceutically acceptable additive is preferable.
  • the type of the additive is not particularly limited, and can be appropriately selected depending on, for example, the type of administration target.
  • the nucleic acid molecule or the expression vector may form a complex with the additive, for example.
  • the additive can also be referred to as a complexing agent, for example.
  • the complex formation for example, the nucleic acid molecule can be efficiently delivered.
  • the binding between the nucleic acid molecule and the complexing agent is not particularly limited, and examples thereof include non-covalent binding. Examples of the complex include an inclusion complex.
  • the complexing agent is not particularly limited, and examples thereof include a polymer, cyclodextrin, adamantine and the like.
  • examples of the cyclodextrin include a linear cyclodextrin copolymer and a linear oxidized cyclodextrin copolymer.
  • Examples of the additive include a carrier, a binding substance to a target cell, a condensing agent, a fusing agent, an excipient, and the like.
  • Carriers include, for example, excipients such as sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic , Binders such as polyethylene glycol, sucrose, starch, disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium-glycol starch, sodium bicarbonate, calcium phosphate, calcium citrate, magnesium stearate, aerosil, talc, Lubricants such as sodium lauryl sulfate, fragrances such as citric acid, menthol, glycyrrhizin / ammonium salt, glycine, orange powder, sodium benzoate, sulfite Preservatives such as sodium, methylparaben and propylparaben, stabilizers such as citric acid, sodium citrate and
  • the pharmaceutical composition of the present invention may further contain a reagent for nucleic acid introduction.
  • the nucleic acid introduction reagent include cationic lipids such as lipofectin, lipofectamine, DOGS (transfectam), DOPE, DOTAP, DDAB, DHDAB, HDEAB, polybrene, or poly (ethyleneimine) (PEI); Polysaccharides such as schizophyllan (SPG) can be used.
  • retronectin, fibronectin, polybrene, or the like can be used as an introduction reagent.
  • Examples of the dosage unit form of the pharmaceutical composition of the present invention include liquids, tablets, pills, drinking liquids, powders, suspensions, emulsions, granules, extracts, fine granules, syrups, soaking agents, decoctions, and eye drops. , Lozenges, poultices, liniments, lotions, ointments, plasters, capsules, suppositories, enemas, injections (solutions, suspensions, etc.), patches, ointments, jelly, pasta Examples include agents, inhalants, creams, sprays, nasal drops, aerosols and the like.
  • the content of the nucleic acid molecule or expression vector of the present invention in the pharmaceutical composition is not particularly limited and is appropriately selected within a wide range, and is, for example, about 0.01 to 100% by weight of the whole pharmaceutical composition.
  • the concentration of the nucleic acid molecule or expression vector of the present invention in the pharmaceutical composition is not particularly limited and is appropriately selected within a wide range.
  • the concentration is about 0.01 nM to 1 M of the entire pharmaceutical composition, preferably about 0. 1 nM to 10 mM, more preferably 1 nM to 100 nM.
  • the pharmaceutical composition of the present invention is administered by a method according to various forms when used. For example, it is administered orally in the case of tablets, pills, drinking liquids, suspensions, emulsions, granules and capsules, and in the case of injections intravenous, intramuscular, intradermal, subcutaneous, intraarticular cavity, It is administered intraperitoneally or in tumor tissue. In the case of a suppository, it is administered intrarectally.
  • the dosage of the pharmaceutical composition of the present invention includes the activity and type of the active ingredient, the mode of administration (eg, oral and parenteral), the severity of the disease, the animal species to be administered, the drug acceptability of the administration target, the body weight
  • the amount of active ingredient per day for an adult is usually about 0.001 mg to about 2.0 g, although it varies depending on the age and the like.
  • the pharmaceutical composition of the present invention is usually safely administered to humans so that the nucleic acid molecule or expression vector of the present invention is delivered to a target cell (eg, hepatocyte, liver cancer cell).
  • a target cell eg, hepatocyte, liver cancer cell.
  • the pharmaceutical composition of the present invention is useful as a pharmaceutical composition for inhibiting the growth of hepatitis B virus.
  • the nucleic acid molecule or expression vector of the present invention By administering an effective amount of the nucleic acid molecule or expression vector of the present invention to a target human infected with hepatitis B virus, the growth of hepatitis B virus in the human can be suppressed. If the growth of hepatitis B virus can be suppressed, the onset of hepatitis B can be prevented, and if hepatitis B has already developed, it can be recovered.
  • Hepatitis B can be treated by using the pharmaceutical composition for inhibiting the growth of hepatitis B virus of the present invention. Therefore, the pharmaceutical composition of the present invention is useful for the treatment of hepatitis B.
  • hepatitis B in the human can be treated.
  • hepatitis B When hepatitis B becomes severe, it causes cirrhosis and liver cancer.
  • the pharmaceutical composition for treating hepatitis B of the present invention cirrhosis and liver cancer can be treated. Therefore, the pharmaceutical composition of the present invention is useful for the treatment of cirrhosis and liver cancer.
  • cirrhosis or liver cancer in the human By administering an effective amount of the nucleic acid molecule or expression vector of the present invention to a target human, cirrhosis or liver cancer in the human can be treated.
  • the expression suppression method of the present invention is a method of suppressing the expression of hepatitis B virus gene or a method of suppressing hepatitis B virus growth, wherein the present invention Using a nucleic acid molecule, an expression vector and / or a pharmaceutical composition.
  • the expression suppression method or hepatitis B virus growth suppression method of the present invention is characterized by using the nucleic acid molecule, expression vector and / or pharmaceutical composition of the present invention, and other steps and conditions are not limited. Not.
  • the expression suppression method or hepatitis B virus growth suppression method of the present invention includes, for example, a step of administering the nucleic acid molecule to a subject in which hepatitis B virus is present.
  • the administration step for example, the nucleic acid molecule is brought into contact with the administration subject.
  • the administration subject include cells, tissues, and organs.
  • the administration subject include non-human animals such as humans and non-human mammals other than humans.
  • the administration may be, for example, in vivo or in vitro.
  • the nucleic acid molecule may be administered alone, or the composition of the present invention containing the nucleic acid molecule may be administered.
  • the administration method is not particularly limited, and can be appropriately selected depending on, for example, the type of administration target.
  • the therapeutic method of the disease of this invention is characterized by including the process of administering the nucleic acid molecule of the said this invention, an expression vector, and / or pharmaceutical composition to a patient as mentioned above.
  • the therapeutic method of the present invention is characterized by using the nucleic acid molecule of the present invention, and other steps and conditions are not limited at all.
  • the diseases targeted by the present invention are, for example, as described above, and include hepatitis B, cirrhosis, liver cancer and the like.
  • the expression suppression method of the present invention can be used.
  • the administration method is not particularly limited, and may be, for example, oral administration or parenteral administration.
  • the dosage of the nucleic acid molecule of the present invention in the treatment method of the present invention is not particularly limited as long as it is a therapeutically effective amount for the above-mentioned disease, and the type, severity, species of animal to be administered, age, weight, drug Usually, it is about 0.0001 to about 100 mg / kg per adult, for example about 0.001 to about 10 mg / kg, preferably about 0.005 to about 5 mg / kg, although it varies depending on the acceptability, administration route, etc. obtain.
  • the amount can be administered, for example, at intervals of 3 times a day to once every 2 weeks, preferably once a day to once a week.
  • nucleic acid molecule is the use of the nucleic acid molecule, expression vector and / or pharmaceutical composition of the present invention for the suppression of hepatitis B virus gene expression.
  • the present invention also provides the nucleic acid molecule, expression vector and / or the present invention for use in inhibiting the expression of hepatitis B virus gene, inhibiting the growth of hepatitis B virus, or treating hepatitis B, cirrhosis, liver cancer.
  • a pharmaceutical composition is provided.
  • the present invention also provides the nucleic acid molecule, expression vector and / or the present invention for the suppression of hepatitis B virus gene expression, hepatitis B virus growth suppression, or the manufacture of a therapeutic agent for hepatitis B, cirrhosis or liver cancer. Or use of a pharmaceutical composition is provided.
  • Example 1 SiRNA evaluation method for HBV-derived mRNA For evaluation, established human hepatoma cell Huh7 cells were used. Huh7 cells were cultured using penicillin / streptomycin, 10% FBS-containing DMEM (high glucose) medium. In experiments after the introduction of the plasmid, a medium not containing penicillin / streptomycin was used.
  • Huh7 cells are seeded in a 24-well plate at 3 ⁇ 10 4 cells / 500 ⁇ L / well and 24 hours later, a plasmid that expresses a 1.4-fold long HBV genome by lipofection and expression of GaussiaLuc as a secreted luciferase for correction of introduction efficiency Plasmid was co-introduced.
  • Lipofectamine 3000 was used as the lipofection reagent.
  • RNAiMAX was used as the lipofection reagent.
  • RNAiMAX 1.5 ⁇ L of RNAiMAX was added to a 50 nM siRNA solution diluted with OPTI-MEM in a serum-free medium so that the total volume became 50 ⁇ L, and after 5 minutes, to 24 well Huh7 cells (with a final siRNA concentration of 5 nM) ) Added.
  • siRNA_upstream1_P S (us1) 5'-AGUCUAGACUCGUGGUGGAtt-3 '(SEQ ID NO: 11) 3'-ttUCAGAUCUGAGCACCACCU -5 '(SEQ ID NO: 9)
  • siRNA_upstream2_P S (us2) 5'- GCAAGAUUCCUAUGGGAGUtt-3 '(SEQ ID NO: 12) 3'-ttCGUUCUAAGGAUACCCUCA -5 '(SEQ ID NO: 10)
  • Negative Control siRNA Negative) 5′-UACUAUUCGACACGCGAAGtt-3 ′ (SEQ ID NO: 19) 3'-ttAUGAUAAGCUGUGCGCUUC -5 '(SEQ ID NO: 20)
  • GaussiaLuc assay was performed to correct the plasmid introduction efficiency using a part of the culture solution. Specifically, 15 ⁇ L of a substrate solution (NEB, BioLux Gaussia Luciferase Assay Kit) was added to 5 ⁇ L of the culture supernatant, and luminescence was measured with a plate reader after 1 minute.
  • a substrate solution NEB, BioLux Gaussia Luciferase Assay Kit
  • RNAiMAX 1.5 ⁇ L of RNAiMAX was added to 50 ⁇ L each of 1 nM, 10 nM, and 100 nM solutions of us1 siRNA and us2 siRNA to be introduced in serum-free medium OPTI-MEM, and these were added to Huh7 cells in the medium.
  • a final concentration of 0.1 nM (us1_0.1, us2_0.1), 1 nM (us1_1, us2_1), or 10 nM (us1_10, us2_10) was added and tested in the same manner as described above after 96 hours of plasmid introduction. Antigen and HBe antigen production inhibitory effects were observed (FIG. 6, n 3).
  • the present invention provides a nucleic acid molecule that is less toxic to cells and that can effectively suppress the expression of a hepatitis B virus gene, and a pharmaceutical composition containing the nucleic acid molecule.
  • the pharmaceutical composition of the present invention is useful for suppressing the growth of hepatitis B virus with reduced side effects, and hence for treating hepatitis B, cirrhosis, and liver cancer. It is.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Dispersion Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Mycology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne : une molécule d'acide nucléique qui inhibe efficacement l'expression du gène du virus de l'hépatite B ; et une composition pharmaceutique qui comprend ladite molécule d'acide nucléique et qui est utilisée pour inhiber la croissance du virus de l'hépatite B et pour traiter l'hépatite B, la cirrhose hépatique ou le cancer hépatique. Plus spécifiquement, l'invention concerne une molécule d'acide nucléique qui comprend la séquence nucléotidique définie dans (i) ou (ii) en tant que séquence pour inhiber l'expression du gène du virus de l'hépatite B. (i) une séquence nucléotidique représentée par la SEQ ID NO:2 ; une séquence nucléotidique représentée par la SEQ ID NO:2, une ou deux bases étant supprimées, substituées, insérées ou ajoutées ; ou une séquence nucléotidique qui a 90 % ou plus d'identité vis-à-vis de la séquence nucléotidique représentée par la SEQ ID NO:2. (ii) une séquence nucléotidique représentée par la SEQ ID NO:1 ; une séquence nucléotidique représentée par la SEQ ID NO:1, une ou deux bases étant supprimées, substituées, insérées ou ajoutées ; ou une séquence nucléotidique qui a 90 % ou plus d'identité vis-à-vis de la séquence nucléotidique représentée par la SEQ ID NO:1.
PCT/JP2018/017347 2017-04-27 2018-04-27 Molécule d'acide nucléique pour le traitement de l'hépatite b WO2018199338A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2019514692A JPWO2018199338A1 (ja) 2017-04-27 2018-04-27 Ｂ型肝炎治療用核酸分子

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2017088960 2017-04-27
JP2017-088960 2017-04-27

Publications (1)

Publication Number Publication Date
WO2018199338A1 true WO2018199338A1 (fr) 2018-11-01

Family

ID=63918465

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2018/017347 WO2018199338A1 (fr) 2017-04-27 2018-04-27 Molécule d'acide nucléique pour le traitement de l'hépatite b

Country Status (2)

Country Link
JP (1) JPWO2018199338A1 (fr)
WO (1) WO2018199338A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20190064628A (ko) * 2016-10-14 2019-06-10 프리시젼 바이오사이언시스 인코포레이티드 B형 간염 바이러스 게놈 내의 인식 서열에 대해 특이적인 조작된 메가뉴클레아제
WO2020255062A1 (fr) * 2019-06-20 2020-12-24 Janssen Sciences Ireland Unlimited Company Administration de nanoparticules lipidiques ou de liposomes de vaccins contre le virus de l'hépatite b (vhb)
WO2021107097A1 (fr) * 2019-11-28 2021-06-03 株式会社ボナック Molécule d'acide nucléique pour traitement de l'hépatite b
WO2021117770A1 (fr) * 2019-12-10 2021-06-17 富士フイルム株式会社 Composition pharmaceutique et agent de traitement
US11725194B2 (en) 2017-12-19 2023-08-15 Janssen Sciences Ireland Unlimited Company Hepatitis B virus (HBV) vaccines and uses thereof
US11788077B2 (en) 2018-04-12 2023-10-17 Precision Biosciences, Inc. Polynucleotides encoding optimized engineered meganucleases having specificity for a recognition sequence in the Hepatitis B virus genome

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012005368A1 (fr) * 2010-07-08 2012-01-12 株式会社ボナック Molécule d'acide nucléique monocaténaire destinée à réguler l'expression génique
WO2012017919A1 (fr) * 2010-08-03 2012-02-09 株式会社ボナック Molécule d'acide nucléique simple brin ayant un squelette alicyclique contenant de l'azote
JP2013537423A (ja) * 2010-08-17 2013-10-03 メルク・シャープ・エンド・ドーム・コーポレイション 低分子干渉核酸（ｓｉＮＡ）を用いたＢ型肝炎ウイルス（ＨＢＶ）遺伝子発現のＲＮＡ干渉媒介性阻害

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012005368A1 (fr) * 2010-07-08 2012-01-12 株式会社ボナック Molécule d'acide nucléique monocaténaire destinée à réguler l'expression génique
WO2012017919A1 (fr) * 2010-08-03 2012-02-09 株式会社ボナック Molécule d'acide nucléique simple brin ayant un squelette alicyclique contenant de l'azote
JP2013537423A (ja) * 2010-08-17 2013-10-03 メルク・シャープ・エンド・ドーム・コーポレイション 低分子干渉核酸（ｓｉＮＡ）を用いたＢ型肝炎ウイルス（ＨＢＶ）遺伝子発現のＲＮＡ干渉媒介性阻害

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20190064628A (ko) * 2016-10-14 2019-06-10 프리시젼 바이오사이언시스 인코포레이티드 B형 간염 바이러스 게놈 내의 인식 서열에 대해 특이적인 조작된 메가뉴클레아제
KR102305215B1 (ko) 2016-10-14 2021-09-28 프리시젼 바이오사이언시스 인코포레이티드 B형 간염 바이러스 게놈 내의 인식 서열에 대해 특이적인 조작된 메가뉴클레아제
US11725194B2 (en) 2017-12-19 2023-08-15 Janssen Sciences Ireland Unlimited Company Hepatitis B virus (HBV) vaccines and uses thereof
US11788077B2 (en) 2018-04-12 2023-10-17 Precision Biosciences, Inc. Polynucleotides encoding optimized engineered meganucleases having specificity for a recognition sequence in the Hepatitis B virus genome
WO2020255062A1 (fr) * 2019-06-20 2020-12-24 Janssen Sciences Ireland Unlimited Company Administration de nanoparticules lipidiques ou de liposomes de vaccins contre le virus de l'hépatite b (vhb)
WO2021107097A1 (fr) * 2019-11-28 2021-06-03 株式会社ボナック Molécule d'acide nucléique pour traitement de l'hépatite b
WO2021117770A1 (fr) * 2019-12-10 2021-06-17 富士フイルム株式会社 Composition pharmaceutique et agent de traitement

Also Published As

Publication number Publication date
JPWO2018199338A1 (ja) 2020-03-12

Similar Documents

Publication Publication Date Title
WO2018199338A1 (fr) Molécule d'acide nucléique pour le traitement de l'hépatite b
WO2015093495A1 (fr) Molécule d'acide nucléique simple brin pour la régulation de l'expression du gène tgf-β1
TWI527901B (zh) A single stranded nucleic acid molecule used to control gene expression
TWI515294B (zh) A single stranded nucleic acid molecule having a nitrogen-containing lipid ring skeleton
US8785121B2 (en) Single-stranded nucleic acid molecule for controlling gene expression
JP6882741B2 (ja) プロレニン遺伝子またはプロレニン受容体遺伝子の発現を抑制する一本鎖核酸分子およびその用途
JP6283859B2 (ja) ペリオスチン遺伝子の発現抑制核酸分子、ペリオスチン遺伝子の発現抑制方法、およびその用途
KR20200033927A (ko) 섬유증 치료제
WO2013133393A1 (fr) Molécule d'acide nucléique à simple brin pour l'inhibition de l'expression du gène vegf
WO2021241040A1 (fr) Molécule d'acide nucléique inhibant l'expression de gène de sars-cov-2 et utilisation associée
WO2021107097A1 (fr) Molécule d'acide nucléique pour traitement de l'hépatite b
JP2017046596A (ja) ＴＧＦ−β１遺伝子発現制御のための一本鎖核酸分子
US10337009B2 (en) Single-stranded nucleic acid molecule for inhibiting TGF-β1 expression
JP2014140341A (ja) Ｒｐｎ２遺伝子の発現抑制用核酸分子、ｒｐｎ２遺伝子の発現抑制方法、およびその用途
JP2020198784A (ja) 免疫機能を調節する核酸分子およびその用途
JP2015182988A (ja) ＮＦ−κＢＲｅｌＡ遺伝子の発現抑制剤、アレルギー性皮膚炎用医薬、およびその用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18789909

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2019514692

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18789909

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载