WO2021107097A1 - Molécule d'acide nucléique pour traitement de l'hépatite b - Google Patents

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

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WO2021107097A1
WO2021107097A1 PCT/JP2020/044231 JP2020044231W WO2021107097A1 WO 2021107097 A1 WO2021107097 A1 WO 2021107097A1 JP 2020044231 W JP2020044231 W JP 2020044231W WO 2021107097 A1 WO2021107097 A1 WO 2021107097A1
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nucleic acid
acid molecule
sequence
nucleotide sequence
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豊 惠口
順一 安岡
智佐登 江村
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株式会社ボナック
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • 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 comprises a nucleic acid molecule that effectively suppresses the amplification of hepatitis B virus (HBV) DNA, and a nucleic acid molecule for suppressing the growth of hepatitis B virus, for treating hepatitis B, liver cirrhosis and liver cancer.
  • HBV hepatitis B virus
  • Hepatitis B virus is said to infect 1.3 million to 1.5 million people in Japan and about 350 million people worldwide, and is a major cause of chronic hepatitis along with hepatitis C virus. ing.
  • the current treatment can be expected to completely eliminate the HCV virus at a fairly high rate, but the current treatment for chronic hepatitis B cannot completely eliminate the HBV virus.
  • Current treatments for chronic hepatitis B include interferon (IFN) therapy and nucleic acid analog drug therapy.
  • IFN interferon
  • nucleic acid analog preparation calms hepatitis and improves liver function, but it is obliged to take it for a long period of time, and hepatitis relapses in most cases when the drug is discontinued.
  • Non-Patent Documents 1 and 2 transcription from cccDNA (covalenty closed circular DNA, closed circular DNA or completely closed double-stranded DNA), which is present in a trace amount in the nucleus of hepatocytes even after treatment, occurs after the end of treatment. Therefore, the development of a new innovative therapeutic agent having a different mechanism of action from the above-mentioned therapeutic method is required (Non-Patent Documents 1 and 2).
  • Non-Patent Document 3 The mechanism of action of the candidate for the treatment of chronic hepatitis B is summarized in Non-Patent Document 3. Above all, suppression of translation 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, which cannot be targeted by conventional small molecule drugs and antibody drugs, and are highly expected as next-generation drugs. As a result, it is expected to create medicines for diseases that have been difficult to treat, and the current situation is that research is being actively conducted all over the world.
  • RNA interference is known as a technique for suppressing gene expression in nucleic acid drugs. Suppression of gene expression by RNA interference is generally carried out, for example, by administering a short double-stranded RNA molecule to cells or the like.
  • the double-stranded RNA molecule is usually called siRNA (small interfering RNA).
  • siRNA small interfering RNA
  • Patent Documents 1 and 2 more effective single-stranded nucleic acid molecules have been newly found to replace siRNA.
  • Patent Document 3 the single-stranded nucleic acid molecule for the treatment of hepatitis B has also been produced.
  • the present invention comprises a nucleic acid molecule that effectively suppresses the amplification of hepatitis B virus DNA, and a medicament for suppressing the growth of hepatitis B virus containing the nucleic acid molecule, and for treating hepatitis B, liver cirrhosis, and liver cancer. It is an object of the present invention to provide a composition.
  • the present inventors include a region containing a nucleotide sequence targeting a specific partial sequence in the hepatitis B virus genomic DNA, a human NCAPH gene, a human Sp1 gene, and a human SOCS7 gene, and a region containing the complementary strand sequence thereof.
  • a single-stranded nucleic acid molecule linked using a specific linker has been found to remarkably suppress the amplification of hepatitis B virus DNA, and has completed the present invention.
  • [1] A single-stranded nucleic acid molecule that suppresses the amplification of hepatitis B virus DNA. Consists of region (X), linker region (Lx) and region (Xc) only The linker region (Lx) has a non-nucleotide structure containing at least one of a pyrrolidine skeleton and a piperidine skeleton.
  • One of the region (X) and the region (Xc) has the following SEQ ID NOs: 1 and 2: (SEQ ID NO: 1) 5'-GAAGAUGAGAAGGCACAGACG -3' (SEQ ID NO: 2) 5'-UCCUGAACUGGAGCCACCAGC -3' A sequence complementary to a part of the hepatitis B virus gene represented by, and SEQ ID NOs: 3 to 5: (SEQ ID NO: 3) 5'-GUUUUCUGAUUGGGAAGGAGC -3' (SEQ ID NO: 4) 5'-CUAAUCCUUGGGCUUCUGGAG -3' (SEQ ID NO: 5) 5'-UAUACUUCACUGCUUCUGGCC -3' A sequence complementary to a part of the human NCAPH gene represented by, and SEQ ID NOs: 6 and 7: (SEQ ID NO: 6) 5'-CCUGUGUGUGUACGUUUGUGC -3' (SEQ ID NO: 7) 5'-UGUACGUUUGUGC
  • nucleic acid molecule containing a nucleotide sequence complementary to the expression-suppressing sequence.
  • the region (X), the linker region (Lx) and the region (Xc) are arranged in this order from the 3'side to the 5'side, and the number of bases (X) in the region (X) and the region (Xc).
  • the nucleic acid molecule according to [1], wherein the number of bases (Xc) in) satisfies the condition of the following formula (1) or formula (2).
  • X 1 and X 2 are independently H 2 , O, S or NH; Y 1 and Y 2 are independently single bonds, CH 2 , NH, O or S; R 3 is a hydrogen atom or substituent attached to C-3, C-4, C-5 or C-6 on ring A; L 1 is an alkylene chain consisting of n atoms, where the hydrogen atom on the alkylene carbon atom is replaced by OH, OR a , NH 2 , NHR a , NR a R b , SH, or SR a. It may or may not be replaced, or L 1 is a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with oxygen atoms.
  • L 2 is an alkylene chain consisting of m atoms, where the hydrogen atom on the alkylene carbon atom is replaced by OH, OR c , NH 2 , NHR c , NR c R d , SH or SR c. It does not have to be replaced, or L 2 is a polyether chain in which one or more carbon atoms of the alkylene chain are replaced with oxygen atoms.
  • Y 2 is NH, O or S
  • the atom of L 2 bonded to Y 2 is carbon
  • the atom of L 2 bonded to OR 2 is carbon
  • the oxygen atoms are not adjacent to each other
  • R a , R b , R c and R d are independent substituents or protecting groups
  • l is 1 or 2
  • m is an integer in the range 0-30
  • n is an integer in the range 0-30
  • one carbon atom other than C-2 on the ring A may be replaced with nitrogen, oxygen or sulfur.
  • a carbon-carbon double bond or a carbon-nitrogen double bond may be contained in the ring A, and the region (Xc) and the region (X) are via -OR 1- or -OR 2-, respectively.
  • R 1 and R 2 may or may not be present, and if present, R 1 and R 2 are independently nucleotide residues or the structure (I), respectively.
  • [6] The nucleic acid molecule according to any one of [1] to [5], wherein the linker region (Lx) is represented by the following formula (I-4a) or (I-6a).
  • nucleic acid molecule according to any one of [1] to [10] wherein the total number of bases is 38 bases or more.
  • nucleic acid molecule according to any one of [1] to [11], which comprises any of the base sequences represented by the following SEQ ID NOs: 19 to 27, 29, 36, 49, 53, 54.
  • SEQ ID NO: 19 5'- CGUCUGUGCCUUCUCAUCUUCCC-Lx-GGGAAGAUGAGAAGGCACAGACGGG -3'
  • SEQ ID NO: 20 5'- GCUGGUGGCUCCAGUUCAGGACC-Lx-GGUCCUGAACUGGAGCCACCAGCAG -3'
  • SEQ ID NO: 21 5'-GCUCCUUCCCAAUCAGAAAACCC-Lx-GGGUUUUCUGAUUGGGAAGGAGCAU -3'
  • SEQ ID NO: 22 5'- CUCCAGAAGCCCAAGGAUUAGCC-Lx-GGCUAAUCCUUGGGCUUCUGGAGUG -3'
  • SEQ ID NO: 23 5'- GGCCAGAAGCAGUGAAGUAUACC-Lx-GGUAUA
  • a therapeutic agent for hepatitis B which comprises the nucleic acid molecule according to any one of [1] to [12].
  • a therapeutic agent for liver cirrhosis or liver cancer which comprises the nucleic acid molecule according to any one of [1] to [12].
  • a nucleotide sequence complementary to a contiguous 15 or more nucleotide sequences in the target genomic DNA sequence is included as an expression-suppressing sequence of the hepatitis B virus gene. Consecutive containing the nucleotide sequence represented by nucleotide number: (3) 1427-1445 (4) 1878-1896 or (5) 3467-3485 of the nucleotide sequence encoding the mRNA of the human NCAPH gene represented by SEQ ID NO: 68.
  • a nucleotide sequence complementary to a contiguous 15 or more nucleotide sequences in a target DNA sequence of 25 nucleotides or less is included as an expression-suppressing sequence of the human NCAPH gene.
  • the nucleotide number of the nucleotide sequence of the mRNA-encoding nucleotide sequence of the human SOCS7 gene represented by SEQ ID NO: 70 which contains a nucleotide sequence complementary to a contiguous 15 or more nucleotide sequences as an expression-suppressing sequence of the human Sp1 gene.
  • nucleic acid molecule according to any one of [17] to [19], further comprising a nucleotide sequence complementary to the expression-suppressing sequence.
  • the complementary nucleotide sequence is (C) In the nucleotide sequence represented by SEQ ID NO: n + 9 (n is an integer selected from 71 to 79), the nucleotide sequence completely complementary to the nucleotide sequence of (a) above (provided that G and U are used). The pairing is considered to be complementary), or (d) a nucleotide sequence that is completely complementary to the nucleotide sequence of (b) above in the nucleotide sequence represented by SEQ ID NO: p (p is an integer selected from 1 to 9).
  • nucleic acid molecule according to [19].
  • nucleotide sequence represented by SEQ ID NO: n (n is an integer selected from 71 to 79) and the nucleotide sequence represented by SEQ ID NO: n + 9 are included, or SEQ ID NO: p (p is 1 to 1 to 1).
  • nucleic acid molecule according to any one of [20] to [22], which is siRNA against a hepatitis B virus gene, a human NCAPH gene, a human Sp1 gene, or a human SOCS7 gene.
  • siRNA against a hepatitis B virus gene, a human NCAPH gene, a human Sp1 gene, or a human SOCS7 gene.
  • Described in [24] which comprises a nucleotide sequence represented by SEQ ID NO: m (m is an integer selected from 89 to 98) and a nucleotide sequence represented by SEQ ID NO: m + 9, which is annealed to the sequence. Nucleic acid molecule.
  • the nucleic acid molecule of the present invention can effectively suppress the amplification of hepatitis B virus DNA.
  • the pharmaceutical composition containing the nucleic acid molecule of the present invention is useful for suppressing the growth of hepatitis B virus, for treating hepatitis B, cirrhosis, and liver cancer by effectively suppressing the amplification of hepatitis B virus DNA. Is.
  • FIG. 1 is a schematic diagram showing an example of the nucleic acid molecule of the present invention.
  • FIG. 2 is a graph showing a relative value of the amount of hepatitis B virus DNA in the culture medium in the examples of the present invention.
  • FIG. 3 is a graph showing the relative value of the intracellular hepatitis B virus cccDNA amount in the examples of the present invention.
  • FIG. 4 is a graph showing the relative value of the amount of hepatitis B virus DNA in the culture medium in the examples of the present invention.
  • FIG. 5 is a graph showing the relative value of the intracellular hepatitis B virus cccDNA amount in the examples of the present invention.
  • FIG. 6 is a graph showing the relative value of the amount of hepatitis B virus HBs antigen in the culture supernatant in the examples of the present invention.
  • FIG. 7 is a graph showing a relative value of the amount of hepatitis B virus DNA in the culture medium in the examples of the present invention.
  • FIG. 8 is a graph showing the relative value of the intracellular hepatitis B virus cccDNA amount in the examples of the present invention.
  • FIG. 9 is a graph showing the relative value of the amount of hepatitis B virus DNA in the culture medium in the examples of the present invention.
  • FIG. 10 is a graph showing a relative value of the amount of hepatitis B virus DNA in the culture medium in the examples of the present invention.
  • FIG. 11 is a graph showing the relative value of the intracellular hepatitis B virus cccDNA amount in the examples of the present invention.
  • FIG. 12 is a graph showing the relative value of the amount of hepatitis B virus HBs antigen in the culture supernatant in the examples of the present invention.
  • FIG. 13 is a graph showing the relative value of the amount of hepatitis B virus DNA in the culture medium in the examples of the present invention.
  • FIG. 14 is a graph showing the relative value of the intracellular hepatitis B virus cccDNA amount in the examples of the present invention.
  • FIG. 15 is a graph showing the relative value of the amount of hepatitis B virus DNA in the culture medium in the examples of the present invention.
  • FIG. 12 is a graph showing the relative value of the amount of hepatitis B virus HBs antigen in the culture supernatant in the examples of the present invention.
  • FIG. 13 is a graph showing the relative value of the amount of
  • FIG. 16 is a graph showing the relative value of the intracellular hepatitis B virus cccDNA amount in the examples of the present invention.
  • FIG. 17 is a graph showing the relative value of the amount of hepatitis B virus DNA in the culture medium in the examples of the present invention.
  • Nucleic acid molecule for suppressing amplification of hepatitis B virus DNA The present invention provides a nucleic acid molecule having an amplification suppressing activity of hepatitis B virus DNA.
  • Hepatitis B virus invades hepatocytes due to infection and proliferates.
  • the immune function works to eliminate it, but it is impossible to selectively attack only the virus in hepatocytes, and the hepatocytes themselves attack. It is received and destroyed, leading to the development of hepatitis.
  • hepatitis B becomes severe, cirrhosis and liver cancer are caused.
  • Hepatitis B virus has incomplete double-stranded DNA that stores genetic information, DNA polymerase located in the center, which are the core (HBc antigen), outer shell (HBe antigen), and outer membrane (HBs antigen). ) Has a structure surrounded by.
  • cccDNA covalenty closed circular DNA
  • mRNA 3.5 kb, 2.4 kb, 2.1 kb, 0.7 kb
  • structural proteins HBs antigen, HBc antigen, HBe antigen, and reverse transcriptase-active polymerase are transcribed from them.
  • X protein is translated (Molecular Therapy 2013; 21 (5) 973-985, Figure 3a).
  • the viral genome and the above four types of mRNA contain some or all of the four ORFs (open reading frames) (S ORFs, core ORFs, X ORFs, and polymerase ORFs) that can be translated as proteins (Molecular Therapy 2013; 21 (5) 973-985, Figure 3a).
  • SORF consists of three proteins that make up HBsAg, large S protein (including pre-S1, pre-S2 and S regions), Middle S protein (including pre-S2 and S regions), and Small S protein (including pre-S2 and S regions). (Consists of only the S region) is coded.
  • the core ORF encodes a core protein and a pre-core protein.
  • the core protein forms core particles, and the pre-core protein becomes the HBe antigen after cleaving 19 hydrophobic signal peptides and 34 amino acid residues at the C-terminal.
  • the X ORF encodes an X protein that is thought to be involved in viral growth and the development of hepatocellular carcinoma.
  • the polymerase ORF also encodes a DNA polymerase protein with reverse transcriptase activity.
  • a certain type of mRNA is incorporated into core particles as pregenomic RNA, minus-strand DNA is synthesized by the action of reverse transcriptase, and then plus-strand DNA is synthesized to become incomplete circular double-stranded DNA. Furthermore, it is wrapped in an envelope formed from HBs antigen and becomes virus particles (Dane particles), which are released into the blood.
  • HBs antigen translated by mRNA, hollow particles containing HBc antigen and p22cr antigen (particles without DNA nuclei), HBe antigen passing through the hepatocellular membrane, etc. are Dane particle blood. It is released and secreted in large quantities into the blood as a route different from the medium release.
  • Diagnosis of hepatitis B is made by detecting the HBsAg and / or HBeAg in the blood.
  • a positive HBsAg in the blood indicates that HBV is present in the liver, that HBV components are synthesized, and that hepatitis B is infected at the time of examination.
  • HBsAg in the blood grasps the viral growth in the liver and provides an index for judging the completion of treatment.
  • the HBe antigen is a protein that is overproduced when HBV proliferates, and indicates that it is highly infectious when HBV is actively proliferating in the liver.
  • the activity of the nucleic acid molecule to suppress the expression of the hepatitis B virus gene is, for example, in cells in which the nucleic acid molecule to be evaluated is infected with hepatitis B virus or cells into which the hepatitis B virus genome has been introduced (preferably human cells).
  • the amount of hepatitis B virus HBs antigen or hepatitis B virus HBe antigen that was introduced and released (transferred) to the outside of the cell was determined by whether the nucleic acid molecule to be evaluated was introduced or a negative control nucleic acid molecule.
  • the amount of hepatitis B virus HBsAg or HBeAg can be evaluated by detecting the antigen by a known immunological method using an antibody that specifically recognizes hepatitis B virus HBsAg or HBeg. it can. Immunological methods include flow cytometry analysis, radioimmunoassay immunoassay (RIA method), ELISA method (Methods in Enzymol. 70: 419-439 (1980)), Western blotting, immunohistochemical staining, etc. Can be done.
  • Nucleic acid molecule that suppresses the amplification of hepatitis B virus DNA The present invention is complementary to the nucleotide sequence in the mRNA of the hepatitis B virus gene, the mRNA of the human NCAPH gene, the mRNA of the human Sp1 gene or the mRNA of the human SOCS7 gene.
  • a nucleic acid molecule (hereinafter, may be referred to as “nucleic acid molecule of the present invention”) that suppresses the amplification of hepatitis B virus DNA, which contains such a sequence.
  • the nucleic acid molecule of the present invention can be used as a sequence that suppresses the amplification of hepatitis B virus DNA with a specific site of mRNA of hepatitis B virus gene, mRNA of human NCAPH gene, mRNA of human Sp1 gene or mRNA of human SOCS7 gene. Contains complementary sequences.
  • the nucleotide sequence of the genomic DNA of hepatitis B virus the nucleotide sequence of Hepatitis B virus isolate 31388 represented by SEQ ID NO: 67 (registered as GenBank Accession No. MG571368 in the NCBI database) or a variant thereof. An array can be mentioned.
  • the nucleotide sequence of the mRNA of the human NCAPH gene is the nucleotide sequence of Homo sapiens non-SMC condensin I complex subunit H (NCAPH), transcript variant 1, mRNA represented by SEQ ID NO: 68 (GenBank Accession No. NM_015341 in the NCBI database). However, in the nucleotide sequence, "t” shall be read as “u”) or the nucleotide sequence of a variant thereof.
  • nucleotide sequence of the mRNA of the human Sp1 gene As the nucleotide sequence of the mRNA of the human Sp1 gene, the nucleotide sequence of Homo sapiens Sp1 transcription factor (SP1), transcript variant 1, mRNA represented by SEQ ID NO: 69 (registered as GenBank Accession No. NM_138473 XM_028606 in the NCBI database). However, in the nucleotide sequence, “t” shall be read as "u”) or a variant of the nucleotide sequence thereof.
  • SP1 Homo sapiens Sp1 transcription factor
  • nucleotide sequence of the mRNA of the human SOCS7 gene As the nucleotide sequence of the mRNA of the human SOCS7 gene, the nucleotide sequence of Homo sapiens suppressor of cytokine signaling 7 (SOCS7) and mRNA represented by SEQ ID NO: 70 is registered as GenBank Accession No. NM_014598 XM_371052 in the NCBI database. However, in the nucleotide sequence, "t” shall be read as "u”) or a variant of the nucleotide sequence thereof.
  • genomic nucleotide sequence of the hepatitis B virus strain represented by SEQ ID NO: 67 the nucleotide sequence of the human NCAPH gene mRNA represented by SEQ ID NO: 68, and SEQ ID NO: 69.
  • the nucleotide sequence of the mRNA of the human Sp1 gene represented by, and the nucleotide sequence of the mRNA of the human SOCS7 gene represented by SEQ ID NO: 70 (however, "t” may be read as "u” in the nucleotide sequence).
  • the position of nucleotide, the range of the nucleotide sequence, and the like are described based on the above, and in that case, the corresponding nucleotide and nucleotide sequence in any variant are also included in the description.
  • a sequence that suppresses the amplification of hepatitis B virus DNA (an expression-suppressing sequence of the mRNA of the hepatitis B virus gene, the mRNA of the human NCAPH gene, the mRNA of the human Sp1 gene or the mRNA of the human SOCS7 gene, hereinafter also referred to as "expression-suppressing sequence" Is a sequence complementary to the nucleotide sequence of a specific site of the mRNA of the hepatitis B virus gene, the mRNA of the human NCAPH gene, the mRNA of the human Sp1 gene or the mRNA of the human SOCS7 gene.
  • the "complementary sequence” is not only a sequence that is completely complementary to the target sequence (that is, hybridizes without mismatch), but can also be hubridized with the above-mentioned mRNA under physiological conditions of mammalian cells.
  • a sequence containing a mismatch of 1 to several nucleotides preferably 1 or 2 nucleotides.
  • a sequence having 90% or more, preferably 95% or more, 97% or more, 98% or more, 99% or more identity with respect to the complementary strand sequence of the target nucleotide sequence in the mRNA can be mentioned.
  • complementarity in individual bases is not limited to forming Watson-Crick base pairs with target bases, but forms Hoogsteen base pairs and Wobble base pairs. Including to do.
  • the "complementary nucleotide sequence” is a nucleotide sequence that hybridizes with the target sequence under stringent conditions.
  • the “stringent condition” is, for example, the condition described in Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999, for example, 6 ⁇ SSC (sodium chloride / sodium citrate). ) / Hybridization at 45 ° C, followed by 0.2 ⁇ SSC / 0.1% SDS / one or more washings at 50-65 ° C. Hybridization conditions can be appropriately selected.
  • Nucleotide sequence of the above-mentioned mRNA targeted by the expression-suppressing sequence Nucleotide sequence of the hepatitis B virus genomic DNA represented by SEQ ID NO: 67 Nucleotide number: (1) 1550-1568 or (2) Nucleotide sequence represented by 59-77 The human NCAPH gene represented by SEQ ID NO: 68.
  • Nucleotide encoding nucleotide sequence Nucleotide number: (3) 1427-1445 (4) 1878-1896 or (5) Nucleotide sequence represented by 3467-3485 Nucleotide encoding human Sp1 gene represented by SEQ ID NO: 69 Nucleotide number: (6) Nucleotide number of the nucleotide sequence shown by (6) 2141-2159 or (7) 2133-2151, or the mRNA-encoding nucleotide sequence of the human SOCS7 gene represented by SEQ ID NO: 70: (8) The nucleotide sequence shown in 2707-2725 or (9) 1621-1639 can be mentioned.
  • the expression-suppressing sequence may be complementary to all of each of these target sequences, or may be complementary to a part of the target sequence, but the specificity for the mRNA should be taken into consideration. For example, it is preferably complementary to 15 or more contiguous sequences in each target sequence.
  • the expression-suppressing sequence can further contain a sequence complementary to the nucleotide sequence of the mRNA adjacent to the target sequence, in addition to the sequence of 15 consecutive nucleotides or more in each of the target sequences.
  • the upper limit of the length of the nucleotide sequence targeted by the expression-suppressing sequence is not particularly limited, but considering the ease of synthesis and the like, for example, 100 nucleotides or less, preferably 50 nucleotides or less, more preferably 30 nucleotides or less, and further. It is a contiguous partial nucleotide sequence of the above mRNA, preferably 25 nucleotides or less. Therefore, the length of the nucleotide sequence targeted by the expression-suppressing sequence can be preferably a continuous 15 to 30 nucleotides, more preferably a continuous 15 to 25 nucleotide partial nucleotide sequence in the nucleotide sequence of the mRNA. ..
  • the nucleic acid molecule of the present invention may be RNA, DNA, or DNA / RNA chimera as long as it can suppress the amplification of hepatitis B virus DNA. Further, the nucleic acid molecule of the present invention may be a double-stranded nucleic acid or a single-stranded nucleic acid as long as it can suppress the amplification of hepatitis B virus DNA. In the case of a double-stranded nucleic acid, it may be any of double-stranded DNA, double-stranded RNA, DNA: RNA hybrid, and a hybrid of DNA / RNA chimera and DNA, RNA or DNA / RNA chimera.
  • one strand is an expression-suppressing sequence of the above-mentioned mRNA, that is, a target sequence containing any one of the above-mentioned mRNAs (1) to (9) (preferably of the above-mentioned mRNA).
  • a chain containing a sequence complementary to a sequence of 15 or more consecutive nucleotides in a continuous subsequence of 25 nucleotides or less (hereinafter, a sequence that binds to a target RNA and suppresses gene expression) is also referred to as a "guide strand".
  • the other strand contains at least a sequence complementary to the expression-suppressing sequence (hereinafter, a chain containing a sequence complementary to the expression-suppressing sequence is also referred to as a "passenger chain").
  • a chain containing a sequence complementary to the expression-suppressing sequence is also referred to as a "passenger chain”
  • the "complementary sequence” is synonymous with the complementarity of the expression-suppressing sequence to the nucleotide sequence of the mRNA.
  • the nucleic acid molecule of the present invention is a single-stranded nucleic acid
  • it has only the above-mentioned guide strand, and a sequence in which the guide strand and the passenger strand are linked via an arbitrary linker to suppress gene expression in the molecule.
  • a sequence complementary thereto may hybridize to form a double strand.
  • Examples of the constituent unit of the nucleic acid molecule of the present invention include a ribonucleotide residue and a deoxyribonucleotide residue. These nucleotide residues may be modified or unmodified, for example. By including, for example, a modified nucleotide residue, the nucleic acid molecule of the present invention can improve nuclease resistance and stability. Further, the nucleic acid molecule of the present invention may further contain a non-nucleotide residue in addition to the nucleotide residue, for example.
  • the structural unit of the region (guide chain or passenger chain) other than the linker is preferably a nucleotide residue.
  • Each region is composed of, for example, the following residues (1) to (3).
  • the nucleic acid molecule of the present invention may be labeled with, for example, a labeling substance.
  • the labeling substance is not particularly limited, and examples thereof include fluorescent substances, dyes, and isotopes.
  • the labeling substance include fluorescent groups such as pyrene, TAMRA, fluorescein, Cy3 dye, and Cy5 dye, and examples of the dye include Alexa dye such as Alexa488.
  • Isotopes include, for example, stable isotopes and radioactive isotopes. Stable isotopes, for example, have a low risk of exposure and do not require a dedicated facility, so that they are easy to handle and can reduce costs.
  • the stable isotope does not change the physical properties of the labeled compound, for example, and has excellent properties as a tracer.
  • Stable isotopes include, for example, 2 H, 13 C, 15 N, 17 O, 18 O, 33 S, 34 S and 36 S.
  • Nucleotide residues include sugars, bases and phosphoric acid as components. Ribonucleotide residues have a ribose residue as a sugar and bases adenine (A), guanine (G), cytosine (C) and uracil (U) (which can also be replaced with thymine (T)). However, the deoxyribonucleotide residue has a deoxyribose residue as a sugar and can be replaced with adenine (dA), guanine (dG), cytosine (dC) and thymine (dT) (uracil (dU)) as bases. ).
  • the modified nucleotide residue may be any of the components of the nucleotide residue.
  • the "modification" can be, for example, substitution, addition and / or elimination of the component, substitution, addition and / or elimination of an atom and / or functional group in the component.
  • the modified nucleotide residue may be, for example, a naturally occurring modified nucleotide residue or an artificially modified nucleotide residue.
  • Naturally derived modified nucleotide residues for example, Limbach et al. (1994, Summary: the modified nucleosides of RNA, Nucleic Acids Res. 22: 2183 to 2196) can be referred to.
  • ribophosphate skeleton examples include modification of the ribose-phosphate skeleton (hereinafter referred to as ribophosphate skeleton).
  • a ribose residue can be modified.
  • the ribose residue can modify, for example, the 2'-carbon, and specifically, for example, a hydroxyl group bonded to the 2'-carbon can be a hydrogen atom, a halogen atom such as fluorine, or an -O-alkyl group (eg,).
  • -O-Me group -O-acyl group (eg, -O-COMe group) and amino group selected from the group consisting of an atom or group, preferably selected from the group consisting of hydrogen atom, methoxy group and fluorine atom.
  • the ribose residue can be replaced with deoxyribose.
  • the ribose residue can be replaced with, for example, a stereoisomer, and may be replaced with, for example, an arabinose residue.
  • the ribophosphate skeleton may be replaced with, for example, a non-ribophosphate skeleton having a non-ribose residue and / or a non-phosphate.
  • examples of the non-ribophosphate skeleton include uncharged bodies of the ribophosphate skeleton.
  • Alternatives to nucleotides substituted with a non-ribophosphate skeleton include, for example, morpholino, cyclobutyl, pyrrolidine and the like.
  • Other examples of the alternative include artificial nucleic acid monomer residues. Specific examples include, for example, PNA (peptide nucleic acid), LNA (Locked Nucleic Acid), ENA (2'-O, 4'-C-Ethylenebridged Nucleic Acid), and PNA is preferable.
  • the phosphate group can also be modified in the ribophosphate skeleton.
  • the phosphate group closest to the sugar residue is called the ⁇ -phosphate group.
  • the ⁇ -phosphate group is negatively charged, and the charge is uniformly distributed over the two oxygen atoms unbonded to the sugar residue.
  • the two oxygen atoms that are unbonded to the sugar residue in the phosphodiester bond between the nucleotide residues are hereinafter also referred to as “non-linking oxygen”. ..
  • linking oxygen the two oxygen atoms bound to the sugar residue are hereinafter referred to as "linking oxygen”.
  • the ⁇ -phosphate group is preferably modified to be uncharged or to have an asymmetric charge distribution in unbound oxygen, for example.
  • the phosphate group may replace, for example, unbound oxygen.
  • the unbonded oxygen is, for example, S (sulfur), Se (sulfur), B (boron), C (carbon), H (hydrogen), N (nitrogen) and OR (R is an alkyl group or an aryl group). It can be replaced with the atom, preferably with S.
  • R is an alkyl group or an aryl group.
  • Such modified phosphate groups include, for example, phosphorothioate, phosphorodithioate, phosphoroselenate, boranophosphate, boranophosphate ester, phosphonate hydrogen, phosphoromidate, alkyl or arylphosphonate, and phosphotriester.
  • phosphorodithioate in which both of the above two unbound oxygens are substituted with S is preferable.
  • the phosphate group may replace the bound oxygen, for example.
  • the bound oxygen can be replaced, for example, with any of the atoms S (sulfur), C (carbon) and N (nitrogen), and such modified phosphate groups include, for example, cross-linked phosphoramidates substituted with N. , S-substituted crosslinked phosphorothioate, C-substituted crosslinked methylenephosphonate, and the like.
  • Substitution of bound oxygen is preferably carried out, for example, at at least one of the 5'-terminal nucleotide residue and the 3'-terminal nucleotide residue of the nucleic acid molecule of the present invention, and in the case of the 5'side, substitution by C is preferable, and 3'is substituted. On the side, substitution by N is preferred.
  • the phosphoric acid group may be replaced with, for example, a phosphorus-free linker.
  • linker include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioform acetal, form acetal, oxime, methylene imino, methylene methyl imino, methylene hydrazo, and methylene dimethyl.
  • Examples thereof include hydrazo and methyleneoxymethylimino, and preferred examples include a methylenecarbonylamino group and a 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 is as described above, and is preferably performed on the terminal phosphate group.
  • the phosphate group may modify the whole or one or more atoms in the phosphate group. In the former case, for example, the entire phosphate group may be substituted or deleted.
  • Modification of the terminal nucleotide residue includes, for example, addition of another molecule.
  • other molecules include functional molecules such as labeling substances and protecting groups.
  • the protecting group include S (sulfur), Si (silicon), B (boron), and an ester-containing group.
  • Functional molecules such as the labeling substance can be used, for example, for detecting the nucleic acid molecule of the present invention.
  • Other molecules may be added to the phosphate group of the nucleotide residue, or may be added to the phosphate group or sugar residue via a spacer.
  • the terminal atom of the spacer can be added or substituted, for example, to the bound oxygen of the phosphate group or the sugar residue O, N, S or C.
  • 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 that binds to these.
  • the spacer can also be added or substituted, for example, to the terminal atom of the nucleotide substitute such as PNA.
  • the spacer is not particularly limited, for example,-(CH 2 ) n -,-(CH 2 ) n N-,-(CH 2 ) n O-,-(CH 2 ) n S-, O (CH 2 CH 2).
  • the molecules added to the ends include dyes, intercalating agents (eg, aclysine), cross-linking agents (eg, solarene, mitomycin C), porphyrin (TPPC4, texaphyrin, sapphirine), polycyclic aromatics.
  • intercalating agents eg, aclysine
  • cross-linking agents eg, solarene, mitomycin C
  • porphyrin TPPC4, texaphyrin, sapphirine
  • Group hydrocarbons eg phenazine, dihydrophenazine
  • artificial endonucleases eg EDTA
  • lipophilic carriers eg cholesterol, cholic acid, adamantanacetic 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- (oleoyl) lithocholic acid, O3- (oleoyl) col Acids, dimethoxytrityls, or phenoxazines) and peptide complexes (eg, antennapedia peptides, Tat peptides), alkylating agents, phosphates, aminos, mercaptos,
  • the nucleic acid molecule of the present invention may be modified at the 5'end with, for example, a phosphate group or a phosphate group analog.
  • Phosphate groups include, for example, 5'monophosphate ((HO) 2 (O) PO-5'), 5'diphosphate ((HO) 2 (O) POP (HO) (O) -O-5.
  • the base is not particularly limited in the nucleotide residue.
  • the base may be a natural base or a non-natural base.
  • a general base, a modified analog thereof, or the like 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, nubularine, isoguanisine, tubercidine and the like.
  • the base is, for example, an alkyl derivative such as 2-aminoadenine, 6-methylated purine; an alkyl derivative such as 2-propylated purine; 5-halouracil and 5-halocytosine; 5-propynyl uracil and 5-propynylcitosine; 6- Azouracil, 6-azocitosine and 6-azotimine; 5-uracil (psoid uracil), 4-thiouracil, 5-halouracil, 5- (2-aminopropyl) uracil, 5-aminoallyl uracil; 8-halogenation, amination, thiol Uracil, thioalkylation, hydroxylation and other 8-substituted purines; 5-trifluoromethylation and other 5-substituted pyrimidines; 7-methylguanine; 5-substituted pyrimidines; 6-azapyrimidines; N-2, N- 6, and O-6 substituted purines (including 2-
  • the modified nucleotide residue may include, for example, a residue lacking a base, that is, a base-free ribophosphate skeleton.
  • the modified nucleotide residues are, for example, US Provisional Application No. 60 / 465,665 (Filing Date: April 25, 2003) and US Provisional Application No. 60 / 465,665 (Filing Date: April 25, 2003).
  • Residues described in WO 2004/080406 International Publication No. WO2004 / 080406
  • these documents can be incorporated by the present invention.
  • the method for synthesizing the nucleic acid molecule of the present invention is not particularly limited, and a conventionally known method can be adopted.
  • Examples of the synthesis method include a synthesis method by a genetic engineering method, a chemical synthesis method, and the like.
  • Examples of the genetic engineering method include an in vitro transcription synthesis method, 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 as the chemical synthesis method.
  • the amidite is not particularly limited, and commercially available amidites include, for example, RNA Phosphoramidites (2'-O-TBDMSi, trade name, Sansenri Pharmaceutical), ACE amidite and TOM amidite, CEE amidite, CEM amidite, TEM amidite and the like. Can be given.
  • nucleic acid molecule of the present invention examples include siRNA, antisense nucleic acid and the like.
  • examples of the nucleic acid molecule of the present invention include a single-stranded nucleic acid molecule in which a guide strand and a passenger strand complementary thereto can form a double strand linked via a linker.
  • siRNA is a guide strand containing a sequence complementary to all or part of a target sequence containing any of the above (1) to (9) (preferably a continuous partial sequence of 25 nucleotides or less), and complement thereof.
  • a double-stranded oligo RNA consisting of a passenger strand containing a specific sequence, which is incorporated into a RISC complex, and the complementary sequence in the guide strand forms a duplex with the target sequence to cleave the RNA.
  • "complementary sequence” has the same meaning as described above.
  • the length of siRNA is not particularly limited as long as the guide strand contains a sequence complementary to all or part of the target sequence containing any of (1) to (9) above, but the nucleotide sequence targeted by siRNA is not particularly limited. In principle, it can be 15 to 50 nucleotides, preferably 19 to 30 nucleotides, more preferably 19 to 27 nucleotides, and particularly preferably 19 to 21 nucleotides.
  • the guide chain and the passenger chain may have an additional nucleotide at the 5'or 3'end. The length of the additional nucleotide is usually about 2 to 4 nucleotides, and the total length of the siRNA is 19 nucleotides or more.
  • the additional nucleotide may be DNA or RNA, but DNA may be used to improve the stability of the nucleic acid.
  • Such additional nucleotide sequences include, for example, ug-3', uu-3', tg-3', tt-3', ggg-3', guuu-3', gttt-3', ttttt-3. Sequences such as', uuuuuu-3'can be mentioned, but are not limited to these.
  • the siRNA contains, as a sequence that suppresses the expression of the gene, a sequence complementary to all or part of the target sequence containing any of (1) to (9) above in the guide strand, but in one preferred embodiment.
  • a guide strand containing one of the following nucleotide sequences (SEQ ID NO: n (n is an integer selected from 71 to 79; where U may be T in the sequence)).
  • SEQ ID NO: n nucleotide sequences
  • a nucleic acid molecule or the like consisting of a passenger chain complementary thereto preferably containing SEQ ID NO: n + 9 (n is an integer selected from 71 to 79; however, U may be T in the sequence)).
  • Expression-suppressing sequence complementary to the nucleotide sequence in the ORF of the above mRNA and its complementary strand sequence (SEQ ID NO: 71) 5'-AGAUGAGAAGGCACAGACG -3' (SEQ ID NO: 72) 5'-CUGAACUGGAGCCACCAGC -3' (SEQ ID NO: 73) 5'-UUUCUGAUUGGGAAGGAGC -3' (SEQ ID NO: 74) 5'-AAUCCUUGGGCUUCUGGAG -3' (SEQ ID NO: 75) 5'-UACUUCACUGCUUCUGGCC -3' (SEQ ID NO: 76) 5'-UGUGUGUGUGUACGUUUGUGC -3' (SEQ ID NO: 77) 5'-UACGUUUGUGCCUCUGUAG -3' (SEQ ID NO: 78) 5'-UUUCUCUCCUGCUCCUACA -3' (SEQ ID NO: 79) 5'-UUCAUCUCUGCAUCUUC
  • the siRNA may have a 3'-overhang on one or both strands.
  • the length of the overhang is not particularly limited, the lower limit is, for example, 1 base length, the upper limit is, for example, 4 base length, 3 base length, and the range is, for example, 1. It is up to 4 bases long, 1 to 3 bases long, and 1 to 2 bases long.
  • the arrangement of overhangs is not particularly limited and may be any of A, U, G, C and T.
  • the overhang arrangement can be exemplified by, for example, TT, UU, CU, GC, UA, AA, CC, UG, CG, AU, etc. from the 3'side.
  • TT time to RNA-degrading enzymes
  • one of the following nucleotide sequences (SEQ ID NO: SEQ ID NO: n (n is an integer selected from 71 to 79; however, in the sequence, U) Is a guide chain containing (may be T) and a passenger chain having a 3'-overhang complementary thereto (preferably SEQ ID NO: n + 9 (n is an integer selected from 71 to 79; however, said). In the sequence, U may be T), including)).
  • Expression-suppressing sequence complementary to the nucleotide sequence in the ORF of the above mRNA and its complementary strand sequence (SEQ ID NO: 89) 5'-AGAUGAGAAAGGCACAGACGtt -3' (SEQ ID NO: 90) 5'-CUGAACUGGAGCCACCAGCtt -3' (SEQ ID NO: 91) 5'-UUUCUGAUUGGGAAGGAGCtt -3' (SEQ ID NO: 92) 5'-AAUCCUUGGGCUUCUGGAGtt -3' (SEQ ID NO: 93) 5'-UACUUCACUGCUUCUGGCCtt -3' (SEQ ID NO: 94) 5'-UGUGUGUGUACGUUUGUGCtt -3' (SEQ ID NO: 95) 5'-UACGUUUGUGCCUCUGUAGtt -3' (SEQ ID NO: 96) 5'-UUUCUCUCCUGCUCCUACAtt -3' (SEQ ID NO
  • the method for synthesizing siRNA is not particularly limited, and a conventionally known method for producing nucleic acid can be adopted.
  • a synthesis method for example, a nucleic acid containing the complementary sequence and a nucleic acid having a sequence complementary thereto are synthesized by a DNA / RNA automatic synthesizer, respectively, and in an appropriate annealing buffer at about 90 to about 95 ° C. Examples thereof include a method of preparing by denaturing for about 1 minute and then annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. It can also be prepared by synthesizing shRNA, which is a precursor of siRNA, and cleaving it with a dicer.
  • Nucleotide residues constituting siRNA may also undergo the same modifications as described above in order to improve stability, specific activity and the like. However, in the case of siRNA, if all ribonucleotide residues in the native RNA are replaced with modified forms, RNAi activity may be lost, so introduction of the minimum modified nucleotide residues that can function the RISC complex is necessary. is necessary.
  • An antisense nucleic acid is all or part of a target sequence (preferably a continuous partial sequence of 25 nucleotides or less) containing any of (1) to (9), preferably the nucleotide.
  • "complementary sequence” has the same meaning as described above.
  • the length of the antisense nucleic acid is not particularly limited, but is, for example, 10 to 100 nucleotides, preferably 15 to 40 nucleotides, and more preferably 15 to 30 nucleotides. It can be a nucleotide.
  • the antisense nucleic acid has the above-mentioned SEQ ID NO: (SEQ ID NO: n (n is an integer selected from 71 to 79; where U may be T in the sequence)) as the expression-suppressing sequence.
  • SEQ ID NO: n is an integer selected from 71 to 79; where U may be T in the sequence.
  • n is an integer selected from 71 to 79; where U may be T in the sequence
  • the antisense nucleic acid may be of the gapmer type.
  • the gapmer-type antisense nucleic acid is a nucleic acid having DNA and nucleic acids having modifications or crosslinks introduced on both sides thereof.
  • RNA complementary to the main chain forms a heteroduplex nucleic acid, and the RNA is degraded by RNAase H.
  • O-methylation at the 2'position of the sugar moiety improves the stability of the antisense nucleic acid and increases its binding affinity to the target.
  • the nuclease resistance of the antisense nucleic acid is enhanced.
  • the method for synthesizing the antisense nucleic acid is not particularly limited, and a conventionally known method for producing a nucleic acid can be adopted.
  • Examples of the synthesis method include a method of preparing nucleic acids containing the complementary sequences by synthesizing them with an automatic DNA / RNA synthesizer.
  • antisense nucleic acids containing the above-mentioned various modifications can also be chemically synthesized by a conventionally known method.
  • the nucleic acid molecule of the present invention is a single-stranded nucleic acid molecule that suppresses the amplification of hepatitis B virus DNA as described above, and has the following sequence. Sequences complementary to a part of the hepatitis B virus gene represented by numbers 1 and 2, sequences complementary to a part of the human NCAPH gene represented by SEQ ID NOs: 3 to 5 below, SEQ ID NOs: 6 and 7 below.
  • At least consecutive nucleotide sequences selected from a sequence complementary to a part of the human Sp1 gene represented by and a sequence complementary to a part of the human SOCS7 gene represented by SEQ ID NOs: 8 and 9 below. It is characterized by containing a gene expression-suppressing sequence, which comprises a nucleotide sequence consisting of 18 nucleotides (referred to as "r-nucleotide sequence").
  • the expression-suppressing sequence may be, for example, a sequence consisting of only the r-nucleotide sequence or a sequence containing the r-nucleotide sequence.
  • the expression-suppressing sequence is a sequence containing an r-nucleotide sequence, one or more nucleotides are added to the 5'end and / or 3'-end of the r-nucleotide sequence. (Here, "complementary" refers to the definition of a complementary sequence described later).
  • nucleotides (sequences) other than the expression-suppressing sequence in the region (X or Xc) containing the expression-suppressing sequence in the nucleic acid molecule of the present invention do not need to be complementary to the target gene.
  • the length of the expression-suppressing sequence is not particularly limited, and is, for example, 18 to 32 nucleotides in length, preferably 19 to 30 nucleotides in length, and more preferably 19, 20, 21 nucleotides in length.
  • the numerical range of the number of bases discloses all positive integers belonging to the range, and for example, the description of "1 to 4 bases” is "1, 2, 3, 4 bases”. Means all disclosures (the same shall apply hereinafter).
  • the single-stranded nucleic acid molecule of the present invention further has a complementary sequence that can be annealed with the expression-suppressing sequence.
  • the complementary sequence does not necessarily have to be completely complementary as long as it can be annealed with the expression-suppressing sequence under physiological conditions in the target cell. That is, the complementary sequence may be a sequence having 100% complementarity in a region overlapping the expression-suppressing sequence, for example, 90% to 100%, 93% to 100%, 95% to 100%, It may be a sequence having complementarity such as 98% to 100% and 99% to 100%.
  • the complementary sequence is the nucleotide sequence represented by SEQ ID NO: n + 9 below. Includes a sequence complementary to the r nucleotide sequence (referred to as "s nucleotide sequence").
  • the complementary sequence may be, for example, a sequence consisting 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 nucleotides in length, preferably 19 to 30 nucleotides in length, and more preferably 19, 20, 21 nucleotides in length.
  • the expression-suppressing sequence and the complementary sequence may be, for example, an RNA molecule consisting only of a ribonucleotide residue, or an RNA molecule containing a deoxyribonucleotide residue in addition to the ribonucleotide residue.
  • uracil (U) residue When the uracil (U) residue is replaced with a deoxyribonucleotide residue, it may be replaced with either dT or dU.
  • a region containing the expression-suppressing sequence and a region containing the complementary sequence are indirectly linked via a linker region.
  • the order of linking the region containing the expression-suppressing sequence and the region containing the complementary sequence is not particularly limited.
  • the 5'-terminal side of the expression-suppressing sequence and the 3'-terminal side of the complementary sequence are linker regions.
  • the 3'end side of the expression-suppressing sequence and the 5'end side of the complementary sequence may be linked via a linker region. The former is preferable.
  • the linker region may be composed of, for example, nucleotide residues or may be composed of non-nucleotide residues. Alternatively, it may be composed of both nucleotide residues and non-nucleotide residues. Preferably, the linker region is composed of non-nucleotide residues.
  • the single-stranded nucleic acid molecule of the present invention is a molecule in which the 5'side region and the 3'side region are annealed to each other to form a double-stranded structure (stem structure).
  • stem structure This can be said to be a form of shRNA (small hairpin RNA or short hairpin RNA).
  • shRNA has a hairpin structure and generally has one stem region and one loop region.
  • the nucleic acid molecule of this embodiment consists only of a region (X), a linker region (Lx) and a region (Xc), and the region (X) and the region (Xc) having a complementary structure form the linker region (X). It has a structure connected via Lx). Specifically, since one of the region (X) and the region (Xc) contains the expression-suppressing sequence and the other contains the complementary sequence, intramolecular annealing can be performed to obtain the region (X). A stem structure can be formed with the region (Xc), and the linker region (Lx) becomes a loop structure.
  • the nucleic acid molecule may have the region (Xc), the linker region (Lx) and the region (X) in this order from the 5'side to the 3'side, or from the 3'side to 5'. It may be held in this order toward the side.
  • the former is preferable.
  • the expression-suppressing sequence may be arranged in either the region (X) or the region (Xc), but it is preferably arranged on the downstream side of the complementary sequence, that is, on the 3'side of the complementary sequence. ..
  • the expression-suppressing sequence is the region ( It is preferably placed within X).
  • FIG. 1 (A) is a schematic diagram showing an outline of the order of each region
  • FIG. 1 (B) is a schematic diagram showing a state in which the nucleic acid molecule forms a duplex in the molecule. is there.
  • FIG. 1 (B) in the nucleic acid molecule, a duplex is formed between the region (Xc) and the region (X), and the Lx region loops according to its length.
  • FIG. 1 merely shows the connection order of the regions and the positional relationship of each region forming the duplex. For example, the length of each region, the shape of the linker region (Lx), and the like are included in this. Not limited.
  • the number of nucleotides in the region (Xc) and the region (X) is not particularly limited.
  • the length of each region is illustrated below, but the present invention is not limited thereto.
  • the lower limit of the number of nucleotides in the region (X) is, for example, 19 nucleotides, preferably 20 bases.
  • the upper limit is, for example, 50 nucleotides, preferably 30 nucleotides, and more preferably 25 nucleotides.
  • Specific examples of the number of nucleotides in the region (X) are, for example, 19 to 50 nucleotides, preferably 19 to 30 nucleotides, and more preferably 19 to 25 nucleotides.
  • the lower limit of the number of nucleotides in the region (Xc) is, for example, 19 nucleotides, preferably 20 nucleotides.
  • the upper limit is, for example, 50 nucleotides, preferably 30 nucleotides, and more preferably 25 nucleotides.
  • Specific examples of the number of nucleotides in the region (Xc) are, for example, 19 to 50 nucleotides, preferably 19 to 30 nucleotides, and more preferably 19 to 25 nucleotides.
  • the region (X or Xc) containing the expression-suppressing sequence may be composed of only the expression-suppressing sequence, or may include the expression-suppressing sequence.
  • the number of nucleotides in the expression-suppressing sequence is as described above.
  • the region containing the expression-suppressing sequence may further have an additional sequence on the 5'side and / or 3'side of the expression-suppressing sequence.
  • the addition sequence is preferably added to the linker region (Lx) side.
  • the expression-suppressing sequence Is preferably located within the region (X), in which case the additional sequence is added to the 5'side of the expression-suppressing sequence.
  • the number of nucleotides in the additional sequence is, for example, 1-31 nucleotides, preferably 1-21 nucleotides, more preferably 1-11 nucleotides, and particularly preferably 1, 2, 3, 4, 5 or 6 nucleotides.
  • the region containing the expression-suppressing sequence (one of X or Xc) has an additional sequence on the linker region (Lx) side
  • the region containing the complementary sequence (the other of X or Xc) is also the linker region (Lx).
  • the side contains a sequence complementary to the additional sequence.
  • the relationship between the number of nucleotides (X) in the region (X) and the number of nucleotides (Xc) in the region (Xc) satisfies, for example, the following conditions (1) or (2), and in the former case, specifically. Meets the condition of (4) below, for example.
  • the nucleic acid molecule schematically shown in FIG. 1 (B) satisfies the condition of the following (1).
  • X-Xc 1-10, preferably 1, 2 or 3, More preferably 1 or 2 ... (4)
  • X Xc ⁇ ⁇ ⁇ (2)
  • the linker region (Lx) preferably has a structure that does not cause self-annealing inside its own region.
  • linker region (Lx) contains a nucleotide residue as described above, its length is not particularly limited.
  • the linker region (Lx) preferably has a length such that the region (X) and the region (Xc) can form a duplex.
  • the lower limit of the number of nucleotides 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 100 bases. It is 80 bases, more preferably 50 bases.
  • the total length of the nucleic acid molecule is not particularly limited.
  • the total number of nucleotides has a lower limit of, for example, 38 nucleotides, preferably 42 nucleotides, and more, when the linker region (Lx) contains nucleotide residues. It is preferably 50 nucleotides, more preferably 51 nucleotides, and particularly preferably 52 nucleotides.
  • the upper limit is, for example, 300 nucleotides, preferably 200 nucleotides, more preferably 150 nucleotides, still more preferably 100 nucleotides, and particularly preferably 80 nucleotides.
  • the total number of nucleotides excluding the linker region (Lx) has a lower limit of, for example, 36 nucleotides, preferably 38 nucleotides.
  • the upper limit is, for example, 100 nucleotides, preferably 80 nucleotides, more preferably 60 nucleotides, and even more preferably 50 nucleotides.
  • Specific examples of the total number of nucleotides are, for example, 36 to 100 nucleotides, preferably 38 to 80 nucleotides, more preferably 42 to 60 nucleotides, and even more preferably 42 to 50 nucleotides.
  • the single-stranded nucleic acid molecule of the present invention has the linker region (Lx) having a non-nucleotide structure.
  • 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, the skeleton of a pyrrolidine derivative in which one or more carbons constituting the 5-membered ring of pyrrolidine are substituted, and when substituted, for example, a carbon atom other than the carbon of C-2. Is preferable.
  • the carbon may be replaced 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 within the 5-membered ring of pyrrolidine.
  • the carbon and nitrogen constituting the 5-membered ring of pyrrolidine may be, for example, hydrogen-bonded or a substituent as described later may be bonded.
  • the linker region (Lx) may be bound to the region (X) and the region (Xc) via, for example, any group of the pyrrolidine skeleton, preferably any one of the 5-membered rings. It is a carbon atom and nitrogen, preferably carbon (C-2) and nitrogen at the 2-position of the 5-membered ring.
  • the pyrrolidine skeleton include a proline skeleton and a prolinol skeleton. Since the proline skeleton, prolinol skeleton, and the like are, for example, in vivo substances and their reduced forms, they are also excellent in safety.
  • the piperidine skeleton may be, for example, the skeleton of a piperidine derivative in which one or more carbons constituting the 6-membered ring of piperidine are substituted, and when substituted, for example, a carbon atom other than carbon of C-2. Is preferable.
  • the carbon may be replaced with, for example, nitrogen, oxygen or sulfur.
  • the piperidine backbone may contain, for example, a carbon-carbon double bond or a carbon-nitrogen double bond within the 6-membered ring of piperidine.
  • the carbon and nitrogen constituting the 6-membered ring of piperidine may be bonded with, for example, a hydrogen group or a substituent as described later.
  • the linker region (Lx) may be attached to the region (X) and the region (Xc) via, for example, any group of the piperidine skeleton, preferably any one of the 6-membered rings. It is carbon atoms and nitrogen, more preferably carbon (C-2) and nitrogen at the 2-position of the 6-membered ring.
  • the linker region may contain, for example, only a non-nucleotide residue having the non-nucleotide structure, or may contain a non-nucleotide residue having the non-nucleotide structure and a nucleotide residue.
  • the linker region is represented by, for example, the following formula (I).
  • X 1 and X 2 are independently H 2 , O, S or NH; Y 1 and Y 2 are independently single bonds, CH 2 , NH, O or S; R 3 is a hydrogen atom or substituent attached to C-3, C-4, C-5 or C-6 on ring A.
  • L 1 is an alkylene chain consisting of n atoms, where the hydrogen atom on the alkylene carbon atom is replaced with OH, OR a , NH 2 , NHR a , NR a R b , SH, or SR a.
  • L 1 is a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with oxygen atoms.
  • 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
  • the oxygen atoms are not adjacent to each other
  • L 2 is an alkylene chain consisting of m atoms, where the hydrogen atom on the alkylene carbon atom is replaced by OH, OR c , NH 2 , NHR c , NR c R d , SH or SR c.
  • L 2 is a polyether chain in which one or more carbon atoms of the alkylene chain are replaced with oxygen atoms.
  • Y 2 is NH, O or S
  • the atom of L 2 bonded to Y 2 is carbon
  • the atom of L 2 bonded to OR 2 is carbon
  • the oxygen atoms are not adjacent to each other
  • R a , R b , R c and R d are independent substituents or protecting groups
  • l is 1 or 2
  • m is an integer in the range 0-30
  • n is an integer in the range 0-30
  • one carbon atom other than C-2 on the ring A may be substituted with nitrogen, oxygen, or sulfur, and a carbon-carbon double bond or carbon-nitrogen bond may be formed in the ring A.
  • the region (Xc) and the region (X) are bound to the linker region (Lx) via -OR 1- or -OR 2-, respectively.
  • R 1 and R 2 may or may not be present, and if present, R 1 and R 2 are independently nucleotide residues or the structure (I), respectively.
  • X 1 and X 2 are, for example, H 2 , O, S or NH, respectively, independently of each other.
  • X 1 when X 1 is H 2 , it means that X 1 forms CH 2 (methylene group) together with the carbon atom to which X 1 is bonded. The same is true for X 2.
  • Y 1 and Y 2 are independently single bonds, CH 2 , NH, O or S, respectively.
  • l 1 or 2.
  • ring A is a 5-membered ring, eg, the pyrrolidine skeleton.
  • the pyrrolidine skeleton include a proline skeleton, a prolinol skeleton, and the like, and these divalent structures can be exemplified.
  • ring A is a 6-membered ring, eg, the piperidine skeleton.
  • one carbon atom other than C-2 on ring A may be replaced with nitrogen, oxygen or sulfur.
  • the ring A may contain a carbon-carbon double bond or a carbon-nitrogen double bond in the ring A.
  • Ring A may be, for example, either L-type or D-type.
  • R 3 is a hydrogen atom or substituent attached to C-3, C-4, C-5 or C-6 on ring A.
  • the substituent R 3 may be 1, a plurality, or not present, and when there are a plurality of substituents, the same or different groups may be used.
  • R 4 and R 5 are, for example, independent substituents or protecting groups, which may be the same or different.
  • the substituents include, for example, 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.
  • Substituent R 3 may be one of these listed substituents.
  • the protecting group is, for example, a functional group that inactivates a highly reactive functional group, and examples thereof include known protecting groups.
  • the protecting group for the protecting group, for example, the description in the literature (JF W. McOmie, "Protecting Groups in Organic Chemistry” Prenum Press, London and New York, 1973) can be incorporated.
  • the protecting group is not particularly limited, and is, for example, tert-butyldimethylsilyl group (TBDMS), bis (2-acetoxyethyloxy) methyl group (ACE), triisopropylsilyloxymethyl group (TOM), 1- (2).
  • 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 same applies hereinafter.
  • L 1 is an alkylene chain consisting of n atoms.
  • the hydrogen atom on the alkylene carbon atom may or may not 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 replaced with oxygen atoms.
  • the polyether chain is, for example, polyethylene glycol.
  • L 2 is an alkylene chain consisting of m atoms.
  • the hydrogen atom on the alkylene carbon atom may or may not be substituted with , for example, OH, OR c , NH 2 , NHR c , NR c R d , SH or SR c.
  • L 2 may be a polyether chain in which one or more carbon atoms of the alkylene chain are replaced with oxygen atoms.
  • Y 2 is NH, O or S
  • the atom of L 2 bonded to Y 2 is carbon
  • the atom of L 2 bonded to OR 2 is carbon
  • the oxygen atoms are not adjacent to each other. That is, for example, when Y 2 is O, the oxygen atom of L 2 and the oxygen atom of L 2 are not adjacent to each other, and the oxygen atom of OR 2 and the oxygen atom of L 2 are not adjacent to each other.
  • N of L 1 and m of 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, for example, according to the desired length of the linker region (Lx).
  • n and m are preferably 0 to 30, more preferably 0 to 20, and even more preferably 0 to 15, respectively, from the viewpoints of manufacturing cost, yield, and the like.
  • 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, independent substituents or protecting groups, respectively.
  • the substituent and the protecting group are, for example, the same as described above.
  • the hydrogen atom may be independently replaced with a halogen such as Cl, Br, F and I, for example.
  • the region (Xc) and the region (X) bind to the linker region (Lx) via -OR 1- or -OR 2-, respectively.
  • R 1 and R 2 may or may not be present.
  • R 1 and R 2 are independently nucleotide residues or structures of the formula (I) above.
  • the linker region (Lx) is, for example, the non-nucleotide residue having the structure of the formula (I) excluding the nucleotide residues R 1 and / or R 2. It is formed from a group and the nucleotide residue.
  • the linker region (Lx) is, for example, concatenated with two or more of the non-nucleotide residues having the structure of the formula (I). It becomes a structure.
  • the structure of the formula (I) may include, for example, one, two, three or four. As described above, when a plurality of the structures are included, it is preferable that the structures (I) are directly connected. On the other hand, in the absence of R 1 and R 2 , the linker region (Lx) is formed only from the non-nucleotide residue having the structure of the formula (I).
  • the combination of the combination of the region (Xc) and the region (X) and the -OR 1- and -OR 2- is not particularly limited, and for example, any of the following conditions can be mentioned.
  • Condition 1) The region (Xc) is coupled to the structure of formula (I) via -OR 2- and the region (X) via -OR 1-.
  • Condition (2) The region (Xc) is coupled to the structure of formula (I) via -OR 1 -and the region (X) via -OR 2-.
  • n, m and q are not particularly limited and are as described above.
  • the nucleic acid molecule of the present invention has any of the following structural formulas: (SEQ ID NO: 19) 5'- CGUCUGUGCCUUCUCAUCUUCCC-Lx-GGGAAGAUGAGAAGGCACAGACGGG -3' (SEQ ID NO: 20) 5'- GCUGGUGGCUCCAGUUCAGGACC-Lx-GGUCCUGAACUGGAGCCACCAGCAG -3' (SEQ ID NO: 21) 5'-GCUCCUUCCCAAUCAGAAAACCC-Lx-GGGUUUUCUGAUUGGGAAGGAGCAU -3' (SEQ ID NO: 22) 5'- CUCCAGAAGCCCAAGGAUUAGCC-Lx-GGCUAAUCCUUGGGCUUCUGGAGUG -3' (SEQ ID NO: 23) 5'- GGCCAGAAGCAGUGAAGUAUACC-Lx-GGUAUACUUCACUGCUUCUGGCCUA -3' (SEQ ID NO: 24) 5'-GC
  • Lx has a structure represented by the above formula (I), and more preferably, the above formulas (I-1) to (I-9). It has a structure represented by any of the above formulas, more preferably a structure represented by the above formula (I-4a) or (I-6a), and particularly preferably a structure represented by the above formula (I-6a). Has a structure.) Among them, single-stranded nucleic acid molecules consisting of the nucleotide sequences represented by SEQ ID NOs: 19 to 27, 29, 36, 49, 53 and 54 are particularly preferable, and are represented by SEQ ID NOs: 19, 27, 29, 36, 49, 53 and 54.
  • the single-stranded nucleic acid molecule consisting of the nucleotide sequences represented by SEQ ID NOs: 29 is even more preferable, and the single-stranded nucleic acid molecule consisting of the nucleotide sequences represented by SEQ ID NOs: 29, 36 and 49 is even more preferable, and the single-stranded nucleic acid molecule consisting of the nucleotide sequence represented by SEQ ID NO: 36 is composed.
  • Single-stranded nucleic acid molecules are most preferred.
  • 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 nucleotide residue and a modified modified nucleotide residue.
  • the nucleic acid molecule of the present invention can improve nuclease resistance and stability by containing, for example, the modified nucleotide residue. Further, the nucleic acid molecule of the present invention may further contain a non-nucleotide residue in addition to the nucleotide residue, for example.
  • the nucleotide residue is preferable as the structural unit of the region other than the linker.
  • Each of the regions is composed of, for example, the following residues (1) to (3).
  • 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, for example, the following residues (1) to (7).
  • nucleic acid molecule of the present invention examples include a molecule composed of only 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 modified nucleotide residue. It may be both nucleotide residues.
  • the number of the modified nucleotide residues is not particularly limited, and is, for example, "1 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 residues is not particularly limited and is, for example, "1 or several", specifically, for example, 1 to 1 to several. 8 pieces, 1 to 6 pieces, 1 to 4 pieces, 1, 2 or 3 pieces.
  • the number of the modified ribonucleotide residues is not particularly limited, and is, for example, "1 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 the ribose residue is replaced with a deoxyribose residue.
  • the number of the deoxyribonucleotide residues is not particularly limited, and is, for example, "1 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 may be labeled with the labeling substance.
  • the labeling substance is not particularly limited, and examples thereof include fluorescent substances, dyes, and isotopes.
  • the labeling substance include fluorescent groups such as pyrene, TAMRA, fluorescein, Cy3 dye, and Cy5 dye, and examples of the dye include Alexa dye such as Alexa 488.
  • the isotope include a stable isotope and a radioactive isotope, and a stable isotope is preferable.
  • 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, 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.
  • nucleotide residues constituting a single-stranded nucleic acid molecule include, for example, sugars, bases and phosphoric acids as components.
  • 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, adenine (A), guanine (G), cytosine (C) and uracil (U) as a base, and the deoxyribose residue has.
  • it has a deoxyribose residue as a sugar and adenine (A), guanine (G), cytosine (C) and timine (T) as bases.
  • nucleotide residues examples include unmodified nucleotide residues and modified nucleotide residues.
  • the unmodified nucleotide residue is such that each of the components is, for example, the same or substantially the same as naturally occurring, preferably the same or substantially the same as 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 may be modified, for example, by any of the components of the unmodified nucleotide residue.
  • “modification” is, for example, substitution, addition and / or deletion of the component, substitution, addition and / or deletion of an atom and / or functional group in the component, and is referred to as "modification". be able to.
  • modified nucleotide residue include naturally occurring nucleotide residues and artificially modified nucleotide residues. For the naturally occurring modified nucleotide residue, for example, Limbach et al.
  • modified nucleosides of RNA Nucleic Acids Res. 22: 2183 to 2196
  • the modified nucleotide residue may be, for example, a residue of a substitute for the nucleotide.
  • Modification of the nucleotide residue includes, for example, modification of the ribose-phosphate skeleton (hereinafter, ribophosphate skeleton).
  • a ribose residue can be modified.
  • the ribose residue can modify, for example, the 2'carbon, and specifically, for example, the hydroxyl group bonded to the 2'carbon can be replaced with a halogen such as hydrogen or fluoro. By substituting the hydroxyl group of the 2'carbon with hydrogen, the ribose residue can be replaced with deoxyribose.
  • the ribose residue can be replaced with, for example, a stereoisomer, and may be replaced with, for example, an arabinose residue.
  • the ribophosphate skeleton may be replaced with, for example, a non-ribophosphate skeleton having a non-ribose residue and / or a non-phosphate.
  • the non-ribophosphate skeleton include uncharged bodies of the ribophosphate skeleton.
  • Substitutes for the nucleotides substituted with the non-ribophosphate skeleton include, for example, morpholino, cyclobutyl, pyrrolidine and the like.
  • Other examples of the alternative include artificial nucleic acid monomer residues. Specific examples include, for example, PNA (peptide nucleic acid), LNA (Locked Nucleic Acid), ENA (2'-O, 4'-C-Ethylenebridged Nucleic Acid), and PNA is preferable.
  • a phosphoric acid 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 uniformly distributed over two oxygen atoms unbonded to sugar residues.
  • the two oxygen atoms that are unbonded to the sugar residue in the phosphodiester bond between the nucleotide residues are hereinafter also referred to as “non-linking oxygen”.
  • linking oxygen the two oxygen atoms bonded to the sugar residue are hereinafter referred to as "linking oxygen”. It is preferable that the ⁇ -phosphate group is modified so that it becomes uncharged or the charge distribution in the unbound oxygen becomes asymmetrical, for example.
  • the phosphoric acid group may replace, for example, the unbound oxygen.
  • the oxygen is, for example, any one of S (sulfur), Se (sulfur), B (boron), C (carbon), H (hydrogen), N (nitrogen) and OR (R is an alkyl group or an aryl group).
  • the unbound 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. Of these, phosphorodithioates in which the two unbound oxygens are both substituted with S are preferred.
  • the phosphoric acid group may replace, for example, the bound oxygen.
  • the oxygen can be replaced with, for example, any atom of S (sulfur), C (carbon) and N (nitrogen), and the modified phosphate group can be, for example, with a crosslinked phosphoramidate, S substituted with N. Examples thereof include a substituted crosslinked phosphorothioate and a C-substituted crosslinked methylenephosphonate.
  • the substitution of the bound oxygen is preferably carried out, for example, at at least one of the 5'-terminal nucleotide residue and the 3'-terminal nucleotide residue of the nucleic acid molecule of the present invention, and in the case of the 5'side, the substitution with C is preferable. In the case of the'side, substitution by N is preferable.
  • the phosphoric acid group may be replaced with, for example, the phosphorus-free linker.
  • the linkers include, for example, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioform acetal, form acetal, oxime, methylene imino, methylene methyl imino, methylene hydrazo, methylene dimethyl. It contains hydrazo, methyleneoxymethylimino and the like, and preferably contains a methylenecarbonylamino group and a 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 and the 5'end, or both.
  • the modification is, for example, as described above, and is preferably performed on the terminal phosphate group.
  • the phosphoric acid group may, for example, modify the whole, or may modify one or more atoms in the phosphoric acid group. In the former case, for example, the entire phosphate group may be substituted or deleted.
  • Modification of the terminal nucleotide residue includes, for example, addition of another molecule.
  • the other molecule include functional molecules such as the above-mentioned labeling substance and protecting group.
  • the protecting group include S (sulfur), Si (silicon), B (boron), and an ester-containing group.
  • Functional molecules such as the labeling substance can be used, for example, for detecting the nucleic acid molecule of the present invention.
  • the other molecule may be added to the phosphate group of the nucleotide residue, or may be added to the phosphate group or the sugar residue via a spacer, for example.
  • the terminal atom of the spacer can be added or substituted, for example, to the bound oxygen of the phosphate group or O, N, S or C of a 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 that binds to these.
  • the spacer can also be added or substituted, for example, to the terminal atom of a nucleotide substitute such as PNA.
  • the spacer is not particularly limited, and is, for example,-(CH 2 ) n -,-(CH 2 ) n N-,-(CH 2 ) n O-,-(CH 2 ) n S-, O (CH 2).
  • CH 2 O) n CH 2 CH 2 OH non-basic sugar, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, morpholino, etc., as well as biotin and fluorescein reagents, etc. Good.
  • the molecule added to the terminal includes, for example, a dye, an intercalating agent (for example, acrydin), a cross-linking agent (for example, solarene, mitomycin C), porphyrin (TPPC4, texaphyllin, sapphirine), and a polycyclic type.
  • an intercalating agent for example, acrydin
  • a cross-linking agent for example, solarene, mitomycin C
  • porphyrin texaphyllin, sapphirine
  • a polycyclic type for example, a dye, an intercalating agent (for example, acrydin), a cross-linking agent (for example, solarene, mitomycin C), porphyrin (TPPC4, texaphyllin, sapphirine), and a polycyclic type.
  • Aromatic hydrocarbons eg phenazine, dihydrophenazine
  • artificial endonucleases eg EDTA
  • lipophilic carriers eg cholesterol, cholic acid, adamantan acetic 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- (oleoyl) lithocholic acid, O3- (oleoyl) Cholic acid, dimethoxytrityl, or phenoxazine) and peptide complexes (eg, Antennapedia peptide, Tat peptide), alkylating agents, phosphoric acid, amino, mercapto,
  • the 5'end may be modified with, for example, a phosphate group or a phosphate group analog.
  • the phosphoric acid group is, for example, 5'monophosphoric acid ((HO) 2 (O) PO-5'), 5'diphosphoric acid ((HO) 2 (O) POP (HO) (O) -O- 5'), 5'triphosphate ((HO) 2 (O) PO- (HO) (O) POP (HO) (O) -O-5'), 5'-guanosine cap (7-methylated or Unmethylated, 7m-GO-5'-(HO) (O) PO- (HO) (O) POP (HO) (O) -O-5'), 5'-adenosine cap (Appp), optional Modified or unmodified nucleotide cap structure (NO-5'-(HO) (O) PO- (HO) (O) POP (HO) (O) -O-5'), 5'monothiophosphate
  • 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 synthetic.
  • As the base for example, a general base, a modified analog thereof, or the like 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, nubularine, isoguanisine, tubercidine and the like.
  • the base is, for example, an alkyl derivative such as 2-aminoadenine, 6-methylated purine; an alkyl derivative such as 2-propylated purine; 5-halouracil and 5-halocytosine; 5-propynyl uracil and 5-propynylcitosine; 6 -Azouracil, 6-azocitosine and 6-azotimine; 5-uracil (psoid uracil), 4-thiouracil, 5-halouracil, 5- (2-aminopropyl) uracil, 5-aminoallyl uracil; 8-halogenation, amination, Thiolization, thioalkylation, hydroxylation and other 8-substituted purines; 5-trifluoromethylation and other 5-substituted pyrimidines; 7-methylguanine; 5-substituted pyrimidines; 6-azapyrimidines; N-2, N -6, and O-6 substituted purines (including
  • the modified nucleotide residue may include, for example, a residue lacking a base, that is, a base-free 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: March 8, 2004). Sun; WO2004 / 080406) can be used, and the present invention can be incorporated by reference to these documents.
  • the method for synthesizing the nucleic acid molecule of the present invention is not particularly limited, and a conventionally known method can be adopted.
  • Examples of the synthesis method include a synthesis method by a genetic engineering method, a chemical synthesis method, and the like.
  • Examples of the genetic engineering method include an in vitro transcription synthesis method, 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 examples of commercially available amidites include RNA Phosphoramidites (2'-O-TBDMSi, trade name, Sansenri Pharmaceutical), ACE amidite and TOM amidite, CEE amidite, CEM amidite, TEM amidite and the like. Can be given.
  • a vector encoding the nucleic acid molecule in an expressible state as a precursor of the nucleic acid molecule (expression vector of the present invention). ) Can also be provided.
  • the expression vector of the present invention is characterized by containing the DNA encoding the nucleic acid molecule of the present invention under the control of a functional promoter in the target cell.
  • the expression vector of the present invention is characterized by containing a promoter functionally linked to the DNA, and other configurations are not limited in any way.
  • the vector into which the DNA is inserted is not particularly limited, and for example, a general vector can be used, and examples thereof include a viral vector and a non-viral vector.
  • examples of the non-viral vector include a plasmid vector.
  • Hepatitis B virus DNA amplification inhibitor is a preparation that suppresses the amplification of hepatitis B virus DNA, and is characterized by containing the nucleic acid molecule of the present invention as an active ingredient. ..
  • the hepatitis B virus DNA amplification inhibitor includes, for example, a step of administering the nucleic acid molecule alone or in combination with a pharmacologically acceptable carrier to a subject in which the hepatitis B virus DNA is present.
  • the administration step is performed, for example, by bringing the nucleic acid molecule into contact with the administration subject.
  • the administration target include cells, tissues or organs of humans and non-human animals such as non-human mammals other than humans.
  • the administration may be, for example, in vivo or in vitro.
  • nucleic acid molecule of the present invention When the amplification of hepatitis B virus DNA is suppressed by the nucleic acid molecule of the present invention, the growth of hepatitis B virus is reduced. Therefore, the drug containing the nucleic acid molecule of the present invention as an active ingredient is useful for suppressing 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 the hepatitis B virus in the human can be suppressed.
  • Hepatitis B can be treated by using the pharmaceutical composition for suppressing 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.
  • treatment is used to include prevention and delay of onset of disease, improvement of disease, and improvement of prognosis.
  • Hepatitis B causes liver cirrhosis and liver cancer when it becomes severe.
  • the pharmaceutical composition for treating hepatitis B of the present invention liver cirrhosis and liver cancer can be treated. Therefore, the pharmaceutical composition of the present invention is useful for the treatment of liver cirrhosis and liver cancer.
  • liver cirrhosis and liver cancer in the human can be treated.
  • the medicament of the present invention may use an effective amount of the nucleic acid molecule of the present invention alone, or may be formulated as a pharmaceutical composition together with an arbitrary carrier, for example, a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include, for example, excipients such as sucrose and starch, binders such as cellulose and methyl cellulose, disintegrants such as starch and carboxymethyl cellulose, lubricants such as magnesium stearate and aerodyl, citric acid, etc. Fragrances such as menthol, preservatives such as sodium benzoate and sodium hydrogen sulfite, stabilizers such as citric acid and sodium citrate, suspending agents such as methylcellulose and polyvinylpyrrolid, dispersants such as surfactants, water, Diluting agents such as physiological saline, base wax and the like can be mentioned, but the present invention is not limited thereto.
  • the medicament of the present invention may further contain a reagent for introducing a nucleic acid.
  • Reagents for introducing nucleic acids include atelocollagen; liposomes; nanoparticles; lipofectin, lipofectamine, DOGS (transfectum), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, or poly (ethyleneimine) (PEI). And the like, cationic lipids and the like can be used.
  • the medicament of the present invention may be a pharmaceutical composition in which the nucleic acid molecule of the present invention is encapsulated in liposomes.
  • Liposomes are microclosed vesicles having an internal phase surrounded by one or more lipid bilayers, which can usually retain water-soluble substances in the internal phase and fat-soluble substances in the lipid bilayer.
  • the nucleic acid molecule of the invention may be retained in the liposome internal phase or in the lipid bilayer.
  • the liposome used in the present invention may be a monolayer membrane or a multilayer membrane, and the particle size can be appropriately selected in the range of, for example, 10 to 1000 nm, preferably 50 to 300 nm. Considering the deliverability to the target tissue, the particle size can be, for example, 200 nm or less, preferably 100 nm or less.
  • Examples of the method for encapsulating a water-soluble compound such as nucleic acid in liposomes include a lipid film method (vortex method), a reverse phase evaporation method, a surfactant removal method, a freeze-thaw method, and a remote loading method. Without limitation, any known method can be appropriately selected.
  • the medicament 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 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 for efficient delivery to a specific organ, tissue or cell in the living body. It may be conjugated to a ligand.
  • the nucleic acid molecule of the present invention can be conjugated to a surface receptor ligand or the like characteristic of liver cells in order to improve the delivery efficiency to the liver.
  • ligands include cholesterol and N-acetylgalactosamine (GalNAc) clusters.
  • Examples of N-acetylgalactosamine (GalNAc) clusters include compounds having the following structural formulas.
  • a buffer for example, a buffer, an isotonic agent, a solubilizing agent, a preservative, a viscous base, a chelating agent, a cooling agent, a pH adjusting agent, an antioxidant and the like are appropriately selected and added to the medicament of the present invention.
  • the buffer include a phosphate buffer, a boric acid buffer, a citric acid buffer, a tartrate buffer, an acetate buffer, and an amino acid.
  • the tonicity agent include sugars such as sorbitol, glucose and mannitol, polyhydric alcohols such as glycerin and propylene glycol, salts such as sodium chloride, and boric acid.
  • solubilizing agent examples include polyoxyethylene sorbitan monoolate (for example, polysorbate 80), polyoxyethylene hydrogenated castor oil, nonionic surfactants such as tyroxapol and pluronic, and polyhydric alcohols such as glycerin and macrogol. Be done.
  • preservatives include quaternary ammonium salts such as benzalkonium chloride, benzethonium chloride and cetylpyridinium chloride, and paraoxybenzoic acid such as methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate and butyl paraoxybenzoate.
  • Examples thereof include esters, benzyl alcohol, sorbic acid and salts thereof (sodium salt, potassium salt, etc.), timerosal (trade name), chlorobutanol, sodium dehydroacetate and the like.
  • Examples of the viscous base include water-soluble polymers such as polyvinylpyrrolidone, polyethylene glycol and polyvinyl alcohol, and celluloses such as hydroxyethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose and sodium carboxymethyl cellulose.
  • Examples of the chelating agent include sodium edetate and citric acid.
  • Examples of the refreshing agent include l-menthol, borneol, camphor, eucalyptus oil and the like.
  • Examples of the pH adjuster include sodium hydroxide, potassium hydroxide, sodium carbonate, sodium hydrogencarbonate, boric acid or a salt thereof (hosand), hydrochloric acid, citric acid or a salt thereof (sodium citrate, sodium dihydrogen citrate). Etc.), phosphoric acid or a salt thereof (disodium hydrogen phosphate, potassium dihydrogen phosphate, etc.), acetic acid or a salt thereof (sodium acetate, ammonium acetate, etc.), citric acid or a salt thereof (sodium tartrate, etc.) and the like.
  • Examples of the antioxidant include sodium hydrogen sulfite, dry sodium sulfite, sodium pyrosulfite, concentrated mixed tocopherol and the like.
  • the content of the nucleic acid molecule of the present invention in the pharmaceutical composition of the present invention is, for example, about 0.1 to 100% by weight of the entire pharmaceutical composition.
  • the pharmaceutical composition of the present invention is a liposome preparation
  • the molar ratio of the nucleic acid molecule of the present invention to the liposome component is usually 1 / 100,000 to 1 / 10,000.
  • the amount of liposomes encapsulating the nucleic acid molecule of the present invention contained in the liposome preparation is not particularly limited as long as the liposome particles do not aggregate and the amount can exert a sufficient medicinal effect, and is usually 10 to 10. It is 100 mM.
  • the dose of the medicament of the present invention varies depending on the purpose of administration, the administration method, the type and size of ocular surface disease, and the situation (gender, age, weight, etc.) of the subject to be administered.
  • a single dose of the nucleic acid of the present invention it is usually preferably 0.01 to 1000 ⁇ g, preferably 0.05 to 100 ⁇ g, more preferably 0.1 to 50 ⁇ g, once to 10 times, preferably 5 to 10 times a day.
  • Example 1 Synthesis of single-stranded nucleic acid molecules
  • the single-stranded nucleic acid molecules shown below were synthesized by an ABI3900 nucleic acid synthesizer (trade name, Applied Biosystems) based on the phosphoramidite method.
  • EMM amidite International Publication No. 2013/027843
  • RNA amidite (hereinafter, the same applies).
  • the deprotection of the amidite was performed according to a conventional method.
  • the synthesized single-stranded nucleic acid molecules were purified by HPLC and then freeze-dried.
  • single-stranded nucleic acid molecules As single-stranded nucleic acid molecules, single-stranded nucleic acid molecules (PH-HB-0001, PH-HB-0002) having the hepatitis B virus gene expression-suppressing sequence represented by SEQ ID NOs: 1 and 2, from the above-mentioned SEQ ID NO: 3. Single-stranded nucleic acid molecules (PH-HB-0003, PH-HB-0004, PH-HB-0005) having a human NCAPH gene expression-suppressing sequence represented by 5, and human Sp1 gene expression represented by SEQ ID NOs: 6 and 7 above.
  • Single-stranded nucleic acid molecules having an inhibitory sequence (PH-Sp1-1, PH-Sp1-4) and single-stranded nucleic acid molecules having a human SOCS7 gene expression inhibitory sequence represented by SEQ ID NOs: 8 and 9 (PH-SOCS7-) 1, PH-SOCS7-4) were synthesized as described above.
  • Lx is a linker region Lx
  • L-proline diamide amidite was used to formulate the following structural formula.
  • PH-HB-0001 (SEQ ID NO: 19) 5'- CGUCUGUGCCUUCUCAUCUUCCC-Lx-GGGAAGAUGAGAAGGCACAGACGGG -3' PH-HB-0002 (SEQ ID NO: 20) 5'- GCUGGUGGCUCCAGUUCAGGACC-Lx-GGUCCUGAACUGGAGCCACCAGCAG -3' PH-HB-0003 (SEQ ID NO: 21) 5'-GCUCCUUCCCAAUCAGAAAACCC-Lx-GGGUUUUCUGAUUGGGAAGGAGCAU -3' PH-HB-0004 (SEQ ID NO: 22) 5'- CUCCAGAAGCCCAAGGAUUAGCC-Lx-GGCUAAUCCUUGGGCUUCUGGAGUG -3' PH-HB-0005 (SEQ ID NO: 23) 5'- GGCCAGAAGCAGUGAAGUAUACC-Lx-GGUAUACUUCACUGCUUCUGGCCUA -3' PH-
  • Hep38.7-Tet cells are cultured in DMEM / F12 medium (Life Technologies, 10565-018) with 10% fetal bovine serum (FBS), 100 U / mL penicillin (Thermo Fisher, 15140122), 100 ⁇ g / mL streptomycin. (Thermo Fisher, 15140122), 10 mM HEPES, 5 ⁇ g / mL Insulin, 400 ⁇ g / mL G418 (Nakaraitesku, 09380-086) was added to the medium. During normal passage, a medium supplemented with 400 ng / mL tetracycline was used, and when HBV replication was initiated, a medium without tetracycline was used.
  • FBS fetal bovine serum
  • Thermo Fisher, 15140122 100 U / mL penicillin
  • streptomycin 100 ⁇ g / mL streptomycin.
  • 10 mM HEPES 5 ⁇ g /
  • PshRNA The introduced single-stranded nucleic acid molecules (hereinafter, also referred to as PshRNA) are as follows. (a) PH-scramble1583 (negative control) (b) PH-HBV_264 (Positive Control) (c) PH-HB-0001 (d) PH-HB-0002
  • RNAs were introduced into Hep38.7 cells by reverse transfection using Lipofectamin-RNAiMAX and seeded on 96 well plates using 100 ⁇ L medium to a concentration of 3.0 x 10 4 cells / well. The RNA concentrations were adjusted to final concentrations of 0.5 nM, 10 nM, and 500 nM. Specifically, using a 96-well plate, 10 ⁇ L was prepared using OPTI-MEM so that the respective RNA concentrations were 5 nM, 100 nM, and 5000 nM, and separately prepared RNAiMAX 0.2 ⁇ L and OPTI-MEM 9.8 ⁇ L. Was mixed with the mixture of 1 and incubated at room temperature for 20 minutes.
  • HBV DNA 72 hours after changing to a tetracycline-free medium, the entire supernatant (about 200 ⁇ L) was collected, centrifuged to precipitate the contaminated cells, and then Smytest EX-R & D (MBL, Cat) from 50 ⁇ L of the supernatant. DNA was extracted using No. GS-J0201). The obtained DNA was lysed in 50 ⁇ L of DW (Ambion (R) Nuclease-Free Water, AM9937).
  • HBV DNA was quantified by real-time PCR using StepOnePlus (ThermoFisher / Applied Biosystems).
  • the Primer and Taqman Probe used for quantification are as follows. -Forward primer: 5'-CACATCAGGATTCCTAGGACC-3' (SEQ ID NO: 61) ⁇ Reverse primer: 5'-AGGTTGGTGAGTGATTGGAG-3' (SEQ ID NO: 62) -Taqman probe: 5'-FAM-CAGAGTCTAGACTCGTGGTGGACTTC-TAMRA-3'(SEQ ID NO: 63)
  • StepOnePlus PCR was performed with a final volume of 25 ⁇ L using 5 ⁇ L of each Primer 1 ⁇ M, Probe 0.25 ⁇ M and DNA extract stock solution.
  • the conditions for PCR are 50 °C 2min ⁇ 95 °C 10min ⁇ (95 °C 15sec ⁇ 60 °C 1min) ⁇ 53 cycles And said.
  • the copy number of HBV DNA in the sample was calculated from the obtained data and the calibration curve obtained using HBV-plasmid 10 1 to 10 7 copies (Fig. 2).
  • CccDNA was quantified by real-time PCR using StepOnePlus, referring to the method of Takkenberg et al. (J. Hepatol. 2018, 69, 301-307).
  • the Primer and Taqman Probe used for quantification are as follows. -Forward primer: 5'-CTCCCCGTCTGTGCCTTCT-3'(SEQ ID NO: 64) -Reverse primer: 5'-GCCCCAAAGCCACCCAAG-3'(SEQ ID NO: 65) -Taqman probe: 5'-FAM-CGTCGCATGGARACCACCGTGAACGCC-TAMRA-3' (SEQ ID NO: 66)
  • StepOnePlus PCR was performed with a final volume of 25 ⁇ L using 5 ⁇ L of each Primer 0.9 ⁇ M, Probe 0.4 ⁇ M and DNA extract stock solution.
  • the conditions for PCR are 50 °C 2min ⁇ 95 °C 10min ⁇ (95 °C 10sec ⁇ 58 °C 5sec ⁇ 63 °C 10sec ⁇ 72 °C 10sec) ⁇ 55 cycles And said.
  • the number of copies of HBV cccDNA in the sample was calculated from the obtained data and the calibration curve obtained using HBV-cccDNA plasmid 10 1 to 10 7 copies (Fig. 3).
  • Example 2 Evaluation of the activity of the product of the present invention with respect to the amount of HBV DNA in the culture medium and the amount of intracellular HBV cccDNA
  • the introduced PshRNA is as follows. (a) PH-7070 (negative control) (b) PH-HBV_264 (Positive Control) (c) PH-HB-0003 (d) PH-HB-0004 (e) PH-HB-0005
  • the HBV DNA ratio tended to decrease at an RNA concentration of 500 nM compared to PH-7070.
  • a decrease in the cccDNA ratio was observed at an RNA concentration of 10 nM as compared with PH-7070.
  • Example 3 Evaluation of activity of the product of the present invention with respect to the amount of HBs antigen in the culture solution
  • the introduced PshRNA is as follows. (a) PH-7070 (negative control) (b) PH-HBV_264 (Positive Control) (c) PH-HB-0003 (d) PH-HB-0004 (e) PH-HB-0005
  • RNA was introduced into Hep38.7 cells so that the final RNA concentrations were 0.5 nM, 10 nM, and 500 nM, respectively, in the same manner as in Example 1, and the supernatant was collected after 72 hours after changing to a tetracycline-free medium. did.
  • the amount of HBV HBs antigen present in the supernatant is determined by HBs S Antigen Quantitative ELISA Kit, Rapid-II (Beacle, Cat No. BCL-SHP-21) (detection limit 0.05). nUnit / mL) was used for measurement (Fig. 6).
  • HBsAg ratio was observed at RNA concentrations of 10 nM and 500 nM compared to PH-7070.
  • Example 4 Evaluation of the activity of the product of the present invention with respect to the amount of HBV DNA in the culture medium and the amount of intracellular HBV cccDNA
  • the introduced PshRNA is as follows. (a) PH-7070 (negative control) (b) PH-HBV_264 (Positive Control) (c) PH-HB-0003 (d) PH-HB-0004 (e) PH-HB-0005
  • PH-HB-0003, PH-HB-0004 and PH-HB-0005 did not show a decrease in the HBV DNA ratio compared to PH-7070.
  • PH-HB-0003, PH-HB-0004 and PH-HB-0005 showed a decrease in cccDNA ratio at RNA concentrations of 10 nM and 500 nM compared to PH-7070.
  • the copy number of HBV DNA in the culture medium (Fig. 9) was calculated at the respective RNA final concentrations of 0.5 nM, 10 nM, and 500 nM by the same method as in Example 1.
  • PH-HB-0001, PH-HB-0007, PH-HB-0014, PH-HB-0027, PH-HB-0031, and PH-HB-0032 had a lower HBV DNA ratio than PH-7070. It was.
  • Example 6 Evaluation of the activity of the product of the present invention with respect to the amount of HBV DNA in the culture medium and the amount of intracellular HBV cccDNA
  • the introduced PshRNA is as follows. (a) PH-7070 (negative control) (b) PH-HBV_264 (Positive Control) (c) PH-SP1-1 (d) PH-SP1-4
  • the HBV DNA ratio decreased at RNA concentrations of 10 nM and 500 nM compared to PH-7070.
  • the cccDNA ratio was decreased at RNA concentrations of 10 nM and 500 nM as compared with PH-7070.
  • Example 7 Evaluation of activity of the product of the present invention with respect to the amount of HBs antigen in the culture solution
  • the introduced PshRNA is as follows. (a) PH-7070 (negative control) (b) PH-HBV_264 (Positive Control) (c) PH-SP1-1 (d) PH-SP1-4
  • the amount of HBV HBs antigen in the culture solution was measured at the respective RNA final concentrations of 0.5 nM, 10 nM, and 500 nM by the same method as in Example 3 (Fig. 12).
  • HBsAg ratio was reduced at the corresponding RNA concentration as compared with PH-7070.
  • Example 8 Evaluation of the activity of the product of the present invention with respect to the amount of HBV DNA in the culture medium and the amount of intracellular HBV cccDNA
  • the introduced PshRNA is as follows. (a) PH-7070 (negative control) (b) PH-HBV_264 (Positive Control) (c) PH-SP1-1 (d) PH-SP1-4
  • the cccDNA ratio decreased at an RNA concentration of 500 nM compared to PH-7070.
  • Example 9 Evaluation of the activity of the product of the present invention with respect to the amount of HBV DNA in the culture medium and the amount of intracellular HBV cccDNA
  • the introduced PshRNA is as follows. (a) PH-7070 (negative control) (b) PH-HBV_264 (Positive Control) (c) PH-SOCS7-1 (d) PH-SOCS7-4
  • PH-SOCS7-1 the cccDNA ratio was reduced at RNA concentrations of 10 nM and 500 nM compared to PH-7070.
  • PH-SOCS7-4 had a reduced cccDNA ratio at the corresponding RNA concentration compared to PH-7070.
  • Example 10 Number of cultured cells, culture procedure, and outline of HBV induction method 3.5 x 10 4 cells / well (100 ⁇ L medium, 96 well plate) of cells were seeded per well using a tetracycline-containing medium. After 20 hours, the medium was changed to a tetracycline-free medium and HBV replication was started. After 72 hours, the supernatant or cells were collected.
  • siRNAs of the following siRNA and negative controls were scr-1583 dn: GAC GCG GUA CGG AGA Att (SEQ ID NO: 109) and scr-1583 up: UUC UCC GUA. It is an siRNA consisting of CCG CGU Ctt (SEQ ID NO: 110).
  • siRNAs were introduced into Hep38.7 cells by reverse transfection using Lipofectamin-RNAiMAX and seeded on 96 well plates using 100 ⁇ L medium to a concentration of 3.0 x 10 4 cells / well. The RNA concentrations were adjusted to final concentrations of 0.5 nM, 10 nM, and 500 nM. Specifically, using a 96-well plate, 10 ⁇ L was prepared using OPTI-MEM so that the respective RNA concentrations were 5 nM, 100 nM, and 5000 nM, and separately prepared RNAiMAX 0.2 ⁇ L and OPTI-MEM 9.8 ⁇ L. Was mixed with the mixture of 1 and incubated at room temperature for 20 minutes.
  • the copy number of HBV DNA in the culture medium (FIG. 17) was calculated at the respective RNA final concentrations of 0.5 nM, 10 nM, and 500 nM by the same method as in Example 1.
  • the HBV DNA ratio decreased at RNA concentrations of 0.5 nM, 10 nM, and 500 nM as compared with the negative control.
  • the present invention is effective as a therapeutic agent for diseases caused by the expression of hepatitis B virus gene, such as hepatitis B, liver cirrhosis and liver cancer.

Abstract

L'invention fournit une molécule d'acide nucléique permettant d'inhiber de manière remarquable l'amplification de l'ADN du virus de l'hépatite B. Plus spécifiquement, l'invention concerne une molécule d'acide nucléique monocaténaire qui inhibe l'amplification de l'ADN du virus du gène de l'hépatite B. Cette molécule d'acide nucléique est caractéristique en ce qu'elle est constituée uniquement d'une région (X), d'une région de liaison (Lx) et d'une région (Xc). Ladite région de liaison (Lx) présente une structure non-nucléotidique contenant un squelette de pyrrolidine et/ou un squelette de pipéridine. Ladite région (X) et/ou ladite région (Xc) contient une séquence de bases liée à 18 bases ou plus parmi les séquences inhibant l'expression du gène du virus de l'hépatite B représenté par SEQ ID N°1 et 2, du gène NCAPH humain représenté par SEQ ID N°3 à 5, du gène Sp1 humain représenté par SEQ ID N°6 et 7, et du gène SOCS7 humain représenté par SEQ ID N°8 et 9.
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