WO2016104612A1 - Translation inhibition method and translation inhibitor - Google Patents

Translation inhibition method and translation inhibitor Download PDF

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WO2016104612A1
WO2016104612A1 PCT/JP2015/086042 JP2015086042W WO2016104612A1 WO 2016104612 A1 WO2016104612 A1 WO 2016104612A1 JP 2015086042 W JP2015086042 W JP 2015086042W WO 2016104612 A1 WO2016104612 A1 WO 2016104612A1
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polynucleotide
translation
target mrna
mrna
region
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PCT/JP2015/086042
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French (fr)
Japanese (ja)
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忠士 和田
渡邉　肇
遼平 高田
剛毅 真門
彩佳 北村
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国立大学法人大阪大学
株式会社陽進堂
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Priority to JP2016566447A priority Critical patent/JPWO2016104612A1/en
Publication of WO2016104612A1 publication Critical patent/WO2016104612A1/en

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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
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to a method for suppressing gene expression and an inhibitor, and more particularly, to a method for suppressing translation of mRNA and a translation inhibitor.
  • Non-patent Document 1 The biopharmaceutical field is booming. Currently, it is mainly protein preparations. In the future, growth of nucleic acid drugs that can be chemically synthesized with relatively small molecules is expected (Non-patent Document 1).
  • Nucleic acid drugs consist of DNA or RNA, or their analogs. The size is smaller than the protein preparation and larger than the small molecule drug. Nucleic acid drugs or candidate substances thereof are mainly composed of single-stranded or double-stranded molecules having a length of about 10 to 20 bases (Non-patent Document 2). It exhibits a double-stranded state with the target nucleic acid, mainly mRNA, by hydrogen bonds formed between bases. When a double chain is partially generated in a specific region of mRNA, 1) the target mRNA is degraded, and 2) binding between the target mRNA and a specific functional protein is inhibited (Non-patent Document 2). By utilizing the properties 1) and 2), the expression of a specific gene can be suppressed. That is, it can be used for disease treatment by utilizing the gene expression inhibitory activity of nucleic acid drugs.
  • Non-patent document 2 As a method for suppressing gene expression, an antisense method (Non-patent document 2), a ribozyme method (Non-patent document 3), or an RNAi (small interfering RNA: using siRNA) method (Non-patent document 4) has been developed.
  • the antisense method DNA in a single-stranded state or an analog thereof, for example, a polynucleotide containing LNA (locked nucleic acids) or a nucleic acid analog consisting of MO (morpholino oligonucleotides) is converted into a region around the mRNA translation initiation codon, Designed to form a duplex in the region surrounding the splicing site.
  • MO morpholino oligonucleotides
  • the ribozyme method and RNAi method achieve gene expression suppression mainly by using RNA to induce mRNA cleavage and degradation of the target gene. Inhibition of gene expression by ribozymes is not always sufficient.
  • RNAi mechanism Triggered by the discovery of the RNAi mechanism, a short double-stranded RNA, microRNA (miRNA) that works under physiological conditions was discovered.
  • miRNAs form duplexes in the 3 'untranslated region (UTR) of mRNA. This activates deadenylase and shortens the poly (A) chain (Non-patent Document 5).
  • the activity of the translation initiation factor complex acting on the 5 'UTR of mRNA is inhibited (Non-patent Document 6).
  • the translation reaction is suppressed.
  • siRNA forms a double strand consisting of a base sequence that is completely complementary to the target mRNA, and induces degradation of mRNA.
  • miRNA a duplex containing a mismatch is formed between target mRNAs, and mRNA is not cleaved or degraded (Non-patent Document 5).
  • Patent Document 1 Non-Patent Document 7
  • This method is considered to belong to a novel antisense method different from the conventional antisense method, ribozyme method or RNAi method due to the difference in the position on the mRNA where the double chain is formed and the suppression mechanism.
  • its application range is limited, and an efficient translation suppression method for a wide range of target mRNAs has been demanded.
  • a translation inhibitor and a translation suppression method that efficiently and stably suppress translation from mRNA.
  • a target mRNA translation inhibitor A target mRNA translation inhibitor.
  • An anti-inflammatory agent and / or an anticancer agent comprising the target mRNA translation inhibitor of (10) or (11).
  • (13) A cell into which the target mRNA translation inhibitor according to any one of (1) to (11) or the anti-inflammatory and / or anticancer agent according to (12) has been introduced.
  • (14) A kit comprising the target mRNA translation inhibitor according to any one of (1) to (11) and / or the anti-inflammatory agent and / or anticancer agent according to claim 12.
  • a first polynucleotide capable of hybridizing to the 5′-untranslated region and / or the translated region of the target mRNA, and a second polynucleotide capable of hybridizing to the 3′-untranslated region of the target mRNA A method for suppressing translation of a target mRNA, wherein the target mRNA is hybridized.
  • the first polynucleotide is a polynucleotide capable of hybridizing to a region containing the 5′-untranslated region of the target mRNA.
  • the method according to (27), wherein the substance having translation inhibitory activity is a polynucleotide.
  • the method according to (28), wherein the polynucleotide is the first polynucleotide and / or the second polynucleotide according to (1).
  • the target mRNA translation inhibitor according to any one of (1) to (11), wherein the second polynucleotide is siRNA, (12) an anti-inflammatory agent and / or an anticancer agent, ) Cells and / or (14) an anti-inflammatory and / or anticancer agent.
  • (31) The method for suppressing translation according to (15) to (25), wherein the second polynucleotide is siRNA, and / or the method for treating inflammation and / or the method for treating cancer according to (26).
  • translation from the target mRNA can be stably suppressed.
  • target mRNA refers to mRNA that is a target for suppressing translation and is transcribed from a gene by RNA polymerase II and has poly A at the 3'-end.
  • eukaryotic mRNA include, for example, humans, animals other than humans, plants, eukaryotic microorganisms (eg, yeast, mold, etc.), archaea, etc., but are organisms having a polyA sequence at the 3′-end of mRNA. If it exists, it can become the object of the present invention. In vitro cultured cells can also be the subject of the present invention. If necessary, all or a part of these eukaryotes may be selected for application.
  • the type of target mRNA is not particularly limited, and any mRNA can be used as long as the protein is translated.
  • mRNA that translates proteins related to diseases mRNA that translates proteins related to metabolism
  • mRNA that translates proteins involved in gene expression such as transcription and translation
  • mRNA that translates proteins involved in biosynthesis etc.
  • it is not limited to these.
  • it may be mRNA that expresses a protein whose function is unknown by translation.
  • the first polynucleotide capable of hybridizing to the 5′-untranslated region and / or the translated region of the target mRNA and the 3′-untranslated region of the mRNA are hybridized.
  • a second polynucleotide capable of soy may be used.
  • the term “hybridize” as used herein means that the target mRNA and the polynucleotide of the present invention are combined by hydrogen bonding in a cell to form a double strand.
  • a region containing a 5′-untranslated region (5′-UTR) of a target mRNA and a 3′-untranslated region Use polynucleotides that hybridize to '-UTR).
  • the untranslated region refers to a region other than the region encoding the protein of the target mRNA (translation region). That is, the protein-encoding region in mRNA is a region encoding a protein from the start codon AUG to the codon immediately before the stop codon, and 5′-upstream of the start codon and 3 ′ of the stop codon. -Downstream and before the poly A region are referred to as 5'-untranslated region and 3'-untranslated region, respectively.
  • the first polynucleotide capable of hybridizing to the 5′-untranslated region and / or the translated region of the target mRNA and the second polynucleotide capable of hybridizing to the 3′-untranslated region of the mRNA can be used.
  • More preferable polynucleotide combinations include a polynucleotide capable of hybridizing to a region containing a 5′-untranslated region and a polynucleotide hybridizing to a 3′-untranslated region (3′-UTR), more preferably 5 A polynucleotide capable of hybridizing to a '-untranslated region and a polynucleotide capable of hybridizing to a 3'-untranslated region, particularly preferably a polynucleotide capable of hybridizing to a 5'-terminal region in a 5'-untranslated region And a polynucleotide capable of hybridizing to the 3′-untranslated region, most preferably a polynucleotide capable of hybridizing to the 5′-terminal region in the 5′-untranslated region and immediately before poly A in the 3′-untranslated region Polynucleotides that can hybridize to the region can be used.
  • One embodiment of the polynucleotide of the translation inhibitor of the present invention uses a polynucleotide having a 5′-terminal region of the target mRNA and a sequence that hybridizes to the sequence immediately before and upstream of the poly A at the 3′-terminal. To do.
  • a translation inhibitor can be prepared by designing a polynucleotide that mechanically easily inhibits any target mRNA.
  • it is not sufficient to design a region to be used as an antisense strand, and it was necessary to confirm whether it has a translation inhibitory effect by experiment.
  • the present invention solves this problem, and can easily design an antisense nucleotide sequence having a translation suppressing effect on any mRNA, and stably inhibit translation using the antisense nucleotide having the sequence. That is, the invention of this embodiment does not require a screening experiment and can be mechanically applied to any mRNA.
  • the range of the length of the first polynucleotide that hybridizes to the 5′-untranslated region and / or the region containing the translation region of the target mRNA of the present invention to inhibit translation is preferably 18 to 30 bases, Preferably it is 18-22 bases, more preferably 19-21 bases, most preferably 20 bases.
  • a polynucleotide that hybridizes to 1 to 50 bases from the 5′-end is preferably used, and more preferably 5′- A polynucleotide that hybridizes to 1 to 30 bases, more preferably 1 to 22 bases, most preferably 1 to 20 bases from the end is used.
  • the effect of translational suppression can be measured by using the transcription activation mechanism of NF ⁇ B in FIG. 2 using the plasmid shown in FIG. That is, when a plasmid having a NF ⁇ B binding region upstream of a promoter and a luciferase gene downstream of the promoter is used and introduced into a cell together with a polynucleotide targeting the Rel gene mRNA which is a subunit of the NF ⁇ B transcription factor In FIG. 2, since the translation of Rel protein is suppressed, the transcription activation of luciferase is suppressed and the phenomenon that luciferase activity is lowered is utilized.
  • the polynucleotide that inhibits translation by hybridizing to the region containing the 5′-untranslated region of the target mRNA of the present invention does not require that all polynucleotide sequences hybridize only to the 5′-untranslated region. However, it may be hybridized to the 5′-untranslated region. That is, as long as it partially hybridizes to the 5'-untranslated region of the target mRNA, it may also hybridize to the initiation codon that is the translation start site of the target mRNA. That is, it is not necessary for the entire region of the polynucleotide of the present invention to hybridize only to the untranslated region, and only a part may hybridize to the untranslated region. Moreover, a part may hybridize with the arrangement
  • the second polynucleotide capable of hybridizing to the 3'-untranslated region of the target mRNA of the present invention hybridizes to the sequence from the downstream of the stop codon to the front of the poly A chain.
  • the range of the length of the polynucleotide that hybridizes to the 3′-untranslated region and inhibits translation is preferably 10 to 50 bases, more preferably 10 to 30 bases, still more preferably 10 to 20 bases, and most preferably Is 10 to 19 bases. Of these, most preferably, among the 3′-untranslated regions of the target mRNA, the 3′-end (immediately before the poly A region; a sequence starting from the 5′-side of AAA... A polynucleotide that hybridizes to 10 to 30 bases from 3) is preferably used.
  • the first and second polynucleotides used in the translation inhibitor or translation suppression method of the present invention are nucleic acid, amino acid, PEG (polyethylene glycol), o-nitrobenzyl group, methylene group, terephthalamide skeleton, stilbene skeleton and honokiol. They may be linked via a spacer made of a low molecular weight compound having physiological activity.
  • the number of spacers connecting the first and second polynucleotides is at least one, and the first and second polynucleotides may be connected using a plurality of spacers (for example, via).
  • the length of the spacer is preferably 3 to 50 bases, more preferably 3 to 30 bases, still more preferably 3 to 20 bases, and most preferably 3 to 18 bases.
  • an amino acid is used as a spacer, it is preferably 2 to 30 amino acids, more preferably 3 to 20 amino acids, and most preferably 3 to 18 amino acids.
  • PEG it is preferably a 2-30 mer, more preferably a 3-20 mer, most preferably a 3-18 mer with [—C—C—O—] as a unit.
  • a spacer composed of a molecule other than these it is preferable to use a spacer having a repeating unit or length corresponding to these.
  • the spacer is designed in length and sequence so that the polynucleotide can hybridize to the 5'-untranslated region and / or the translated region and 3'-untranslated region of the target mRNA, respectively.
  • the polynucleotide of the present invention may contain nucleotide analogues instead of natural nucleotides.
  • it may be a nucleic acid analog consisting of a polynucleotide containing LNA (locked nucleic acids) or an MO (morpholino ⁇ oligonucleotides) nucleic acid, and its nucleotide (ribonucleotide or deoxyribonucleotide) may be used as long as translation of the target mRNA can be suppressed.
  • LNA locked nucleic acids
  • MO morpholino ⁇ oligonucleotides
  • ribonucleotide or deoxyribonucleotide may be used as long as translation of the target mRNA can be suppressed.
  • It may be a nucleotide analog in which sugar, base and / or phosphate are chemically modified.
  • nucleotide analogs with modified bases include 5-position-modified uridine or cytidine (eg, 5-propynyluridine, 5-propynylcytidine, 5-methylcytidine, 5-methyluridine, 5- (2-amino) propyl Uridine, 5-halocytidine, 5-halouridine, 5-methyloxyuridine, etc .; 8-position modified adenosine or guanosine (eg, 8-bromognosin, etc.); Deazanucleotide (eg, 7-deaza-adenosine, etc.); O- and N -Alkylated nucleotides (eg N6-methyladenosine etc.) and the like.
  • 5-position-modified uridine or cytidine eg, 5-propynyluridine, 5-propynylcytidine, 5-methylcytidine, 5-methyluridine, 5- (2-amino) propyl Uridine, 5-ha
  • nucleotide analogues modified with sugar include, for example, LNA, MO, 2′-OH of ribonucleotide is H, OR, R, halogen atom, SH, SR, NH 2 , NHR, NR 2 , or 2′-position sugar modification substituted by CN (wherein R represents an alkyl group, alkenyl group or alkynyl group having 1 to 6 carbon atoms) or the like, and 5′-terminal phosphorylation modification in which the 5 ′ end is monophosphorylated Can be mentioned.
  • nucleotide analogues modified with phosphate include those obtained by substituting phosphothioate groups for phosphoester groups that bind adjacent ribonucleotides.
  • nucleotide analogues can be introduced into a polynucleotide by a known method such as chemical synthesis.
  • the target mRNA whose translation is suppressed by the translation inhibitor of the present invention may be mRNA that expresses a protein that causes an inflammatory reaction.
  • proteins that cause an inflammatory response include, but are not limited to, inflammatory cytokines (IL-12, IL-6, TNF- ⁇ ) and the like.
  • IL-12, IL-6, TNF- ⁇ proteins that cause an inflammatory response
  • transcription is activated by RelA and P50 by releasing I ⁇ B from the NF ⁇ B complex, whereby mRNA is transcribed and translated into cytokine protein (see FIG. 2).
  • translation may be suppressed by the translation inhibitor of the present invention targeting the mRNA of these cytokines themselves, but also by suppressing the gene expression of all or part of transcription factors that promote cytokine gene expression. Expression can be suppressed and inflammatory reaction can be suppressed.
  • transcription factors for cytokine genes include NF ⁇ B, AP-1, and STAT3. Therefore, the translation inhibitor of the NF ⁇ B
  • the translation inhibitor of the present invention can also be applied to diseases and symptoms mediated by the inflammatory cytokine cascade.
  • Diseases and symptoms mediated by the inflammatory cytokine cascade include, but are not limited to:
  • Systemic inflammatory response syndromes include: sepsis syndrome, Gram positive sepsis, Gram negative sepsis, culture negative sepsis, fungal sepsis, neutropenic fever, urinary sepsis, meningococcal bacteremia Traumatic bleeding, stuttering, ionizing radiation exposure, acute pancreatitis, adult respiratory distress syndrome (ARDS).
  • sepsis syndrome Gram positive sepsis, Gram negative sepsis, culture negative sepsis, fungal sepsis, neutropenic fever, urinary sepsis, meningococcal bacteremia Traumatic bleeding, stuttering, ionizing radiation exposure, acute pancreatitis, adult respiratory distress syndrome (ARDS).
  • ARDS adult respiratory distress syndrome
  • ⁇ Reperfusion injury includes: Postpump syndrome, ischemia reperfusion injury.
  • Cardiovascular diseases include: cardiac fall syndrome, myocardial infarction, congestive heart failure.
  • Infectious diseases include: HIV infection / HIV neuropathy, meningitis, hepatitis, septic arthritis, peritonitis, pneumonia epiglottis, E. coli O157: H7, hemolytic uremic syndrome / thrombolytic thrombocytopenic purpura Disease, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome, streptococcal myositis, gas gangrene, Mycobacterium tuberculosis (human tuberculosis), Mycobacterium aviun Intracellulare Pneumocystis Carinii pneumonia, pelvic inflammatory disease, testicular inflammation / epididymis, Legionella, Lyme disease, influenza A, Epstein-Barr virus, virus-related hemophagocytic syndrome, viral encephalitis / Aseptic meningitis.
  • Obstetrics / gynecological symptoms include: premature birth, miscarriage, infertility.
  • Inflammatory / autoimmune diseases include: rheumatoid arthritis / seronegative arthropathy, osteoarthritis, inflammatory bowel disease, systemic lupus erythematosus, iridocyclitis / uveitis optic neuritis, idiopathic Pulmonary fibrosis, systemic vasculitis / Wegener's granulomatosis, sarcoidosis testisitis / vasectomy reversal surgery.
  • Allergic / atopic diseases include: asthma, allergic rhinitis, eczema, allergic contact dermatitis, allergic conjunctivitis, hypersensitivity pneumonia.
  • Malignancies include: ALL, AML, CML, CLL, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, colorectal cancer, nasopharyngeal cancer, malignant histiocytosis, paraneoplastic / high malignant disease Calcemia.
  • Grafts include: organ graft rejection, graft versus host disease, cachexia.
  • Congenital which includes: cystic fibrosis, familial blood phagocyte lymphohistiocytosis, sickle cell anemia.
  • Dermatologically includes: psoriasis, alopecia.
  • Neurological diseases include the following: multiple sclerosis, migraine.
  • Renal diseases include the following: nephrotic syndrome, hemodialysis, uremia.
  • Toxic ones include: OKT3 therapy, anti-CD3 therapy, cytokine therapy, chemotherapy, radiation therapy, chronic salicylate poisoning.
  • Metabolic / idiopathic diseases include: Wilson's disease, hemochromatosis, alpha-1 antitrypsin deficiency, diabetes, Hashimoto's thyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axis evaluation, primary biliary cirrhosis .
  • Inflammation can be reduced and / or treated by applying the translation inhibitor of the present invention targeting mRNAs encoding proteins that cause diseases and symptoms mediated by these inflammatory cytokine cascades.
  • the target mRNA whose translation is suppressed by the translation inhibitor of the present invention may be mRNA that expresses a protein that causes a neoplastic disorder.
  • neoplastic disorder is meant any type of cancer or neoplasm or malignant tumor found in humans including, but not limited to: leukemia, lymphoma, melanoma, carcinoma and sarcoma.
  • the subject of the anticancer agent and / or cancer treatment method of the present invention may include these neoplastic disorders.
  • sarcoma generally refers to a tumor composed of a material such as embryonic connective tissue and generally composed of closely packed cells embedded in a fibrous or homogeneous material.
  • sarcomas that can be treated by the anticancer agents and / or cancer treatment methods of the present invention include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abmethy's) sarcoma, liposarcoma, liposarcoma, alveolar soft tissue sarcoma, enamel epithelial sarcoma, grape sarcoma, green sarcoma, choriocarcinoma, fetal sarcoma, Wilms tumor sarcoma, endometrial sarcoma, interstitial sarcoma, Ewing sarcoma , Fascial sarcoma
  • melanoma is taken to mean a tumor arising from the melanocytic system of the skin and other organs.
  • Melanoma that can be treated by the anticancer agent and / or cancer treatment method of the present invention is not limited, and examples thereof include terminal melanoma type melanoma, melanin-deficient melanoma, benign juvenile melanoma, and Cloudman.
  • carcinoma refers to a malignant neoplasm composed of epithelial cells that tend to infiltrate the surrounding tissues and give rise to metastases.
  • carcinoma refers to a malignant neoplasm composed of epithelial cells that tend to infiltrate the surrounding tissues and give rise to metastases.
  • the carcinoma that can be treated by the anticancer agent and / or cancer treatment method of the present invention is not limited, and examples thereof include acinar carcinoma, acinar cell carcinoma, glandular cystic carcinoma, adenoid cystic carcinoma, adenomatous carcinoma.
  • cancer-related genes include myc, src, ras, abl, bcl, rb, p53, apc, brca1, brca2, akt2, braf, hras, kras, kit, msh2, cdk4, pten, egfr, erbb2, fgfr1, Examples include fgfr3, flt3, jak2, pdgfra, plk3ca, and ret genes. By suppressing the expression of these genes, an anticancer effect can be exerted.
  • cells into which a translation inhibitor is introduced can be obtained.
  • the introduction of the polynucleotide according to the present invention into cells can be carried out by a normal gene introduction method, such as calcium phosphate method, liposome method, electroporation method (electroporation method), gene gun (gene gun), whisker method, microinjection. Method, laser injection method, protoplast method (plant, yeast), Agrobacterium method (plant), lithium chloride method (yeast), etc. are used, but not limited thereto.
  • a normal gene introduction method such as calcium phosphate method, liposome method, electroporation method (electroporation method), gene gun (gene gun), whisker method, microinjection.
  • Method laser injection method, protoplast method (plant, yeast), Agrobacterium method (plant), lithium chloride method (yeast), etc. are used, but not limited thereto.
  • the translation inhibitor of the present invention can be used in vitro or in vivo. That is, it can be used for substance production by inhibiting translation in
  • kits containing a translation inhibitor are provided.
  • the kit include the above-described gene introduction reagent, a kit containing a selection reagent and the polynucleotide of the present invention, and a kit in which a medium is added to them.
  • the first polynucleotide capable of hybridizing to the 5′-untranslated region and / or the translated region of the target mRNA and the second polynucleotide capable of hybridizing to the 3′-untranslated region of the mRNA There is provided a method of hybridizing to a target mRNA and suppressing translation of the target mRNA. According to the method for suppressing the translation of target mRNA of the present invention, it is not necessary to create a plurality of antisenses for each target mRNA, measure the suppression efficiency, and select the optimum one, and mechanically express the gene expression. Can be suppressed.
  • the polynucleotide of the present invention can also be administered continuously. Using DDS technology, it can be gradually released into cells and tissues to continuously suppress gene expression, and can also be used for prevention and treatment of chronic diseases.
  • an in vitro translation system is a system that translates proteins in a cell-free extract or in a solution containing a translation enzyme and a substrate necessary for translation.
  • in vitro translation systems include rabbit reticulocyte system (Rabbit® Reticulocyte® Lysate® System), wheat germ extract system (Wheat® Germ® Extract), insect cell extract, and human culture cell extract (see Example 8).
  • Rabbit® Reticulocyte® Lysate® System rabbit reticulocyte system
  • Wheat® Germ® Extract wheat germ extract system
  • insect cell extract and human culture cell extract (see Example 8).
  • human culture cell extract see Example 8
  • Both systems can be used in the present invention.
  • a translation system not only a translation system but also a system capable of performing transcription and translation in the same system has been developed, and these can also be used in the present invention.
  • candidate substances that may have translation-inhibiting activity are introduced into the in vitro translation system together with the target mRNA or, if necessary, in the in vitro translation system. What is necessary is just to measure the synthetic activity of the protein from mRNA.
  • the measurement of protein synthesis activity if the protein has enzyme activity, the amount of protein synthesis can be measured by the enzyme activity. Examples of the enzyme activity include luminescence by luciferase, color development by substrate cleavage by ⁇ -galactosidase, luminescence, measurement of phosphorylation by phosphorylase by radioisotope, measurement of substrate transfer activity by transferase, etc. Not limited.
  • the protein does not have enzyme activity, it can be quantified by methods such as electrophoresis such as SDS-PAGE (including Western blot), HPLC, MASS, and antibody quantification (ELISA, dot blot, etc.). Not limited to.
  • electrophoresis such as SDS-PAGE (including Western blot), HPLC, MASS, and antibody quantification (ELISA, dot blot, etc.). Not limited to.
  • electrophoresis such as SDS-PAGE (including Western blot), HPLC, MASS, and antibody quantification (ELISA, dot blot, etc.).
  • ELISA enzyme quantification
  • dot blot etc.
  • translation inhibition activity may be measured by combining a plurality of these quantitative methods.
  • the translation inhibitor used is not particularly limited, and examples thereof include antisense strand polynucleotides, ribozymes, aptamers, antibiotics, antibodies, proteins, and the like. However, it is not limited to these.
  • the in vitro translation system can also be suitably used for measuring the activity of the translation inhibitor of the present invention and screening for a more active polynucleotide and / or spacer combination.
  • the translation inhibitor of the present invention does not have a constant rate of introduction into cells
  • introduction into cells is achieved by using an in vitro translation system that does not include a step of introduction into cells.
  • Translation inhibitors having a low rate can also be measured quickly, and have the advantage that comprehensive screening by high-throughput screening is possible.
  • the translation inhibitor of the present invention may contain siRNA.
  • the siRNA sequence is preferably siRNA targeting the 3'-end of mRNA.
  • the number of siRNA bases is 15 to 40, more preferably 19 to 30, further preferably 19 to 25 bases, particularly preferably 19 bases, and a thymine overhang attached to the 3′-end. Is preferably used.
  • the translation inhibitor containing the siRNA of the present invention may be an anti-inflammatory agent and / or an anticancer agent, and may be in the form of a kit containing the anti-inflammatory agent and / or the anticancer agent. Further, a cell containing the translation inhibitor is also included in the scope of the present invention.
  • siRNA may be used, and the siRNA that binds immediately before the 3'-end of the target mRNA is preferably used.
  • the method of inhibiting translation using siRNA is also used as a method for treating inflammation and / or treating cancer.
  • HeLa cells were cultured in DMEM (Sigma-Aldrich) containing 10% non-immobilized FBS (Gibco) and penicillin-streptomycin (Life technologies) at 37 ° C. and 5% CO 2 .
  • Antisense nucleic acid polynucleotide including LNA
  • binds to the 5′-untranslated region of RelA, the start codon region, the region immediately before poly A, etc., with the transcription factor RelA shown in FIG. was synthesized and introduced.
  • Transfection into HeLa cells was performed as follows. HeLa cells were suspended in DMEM medium containing 10% non-immobilized FBS, and 0.5 mL was seeded in a 24-well plate so that the concentration was about 1 ⁇ 10 6 cells / mL. After 24 hours, when the cells are confluent at about 40-50%, pGL4.32 (see FIG. 1) NF ⁇ B cis-element + promoter + Luciferase gene; Promega 1 ⁇ g, pGL4.75 (Promega) 20 ng, LNA ASO 1 ⁇ L Lipofectamine 2000 (Invitrogen) mixed with 100 ⁇ L OptiMEM (Invitrogen) was added. By measuring the change in the expression of the luciferase gene linked to the promoter downstream of RelA, the translation inhibitory activity of RelA was measured.
  • ASO antisense oligo
  • 5′-end ASO targeting the 5 ′ end 20mer of RelA mRNA (SEQ ID NO: 5′-TCGCGCGTCCGCGCCGGCCT-3 ′).
  • First AUG ASO targeting 20mer (SEQ ID NO: 5'-CTGGGGCCGGTACCTGCTTG-3 ') containing the start codon.
  • 3'-end / 19mer RelA mRNA ASO targeting 3 'end 19mer (SEQ ID NO: 5: 5'-GACAACGGTTCGACCGATC-3').
  • 3'-end / 10mer RelA mRNA ASO targeting the 3 'end 10mer (SEQ ID NO: 5: 5'-TCGACCGATC-3').
  • the sequence of ASO used is as follows. 5'-end: RelA mRNA 5 'end 20mer ASO (SEQ ID NO: 12: 5'-TCCGGCCGCGCCTGCGCGCT-3') First AUG: 20mer ASO including the start codon (SEQ ID NO: 13: 5'-GTTCGTCCATGGCCGGGGTC-3 ') 3'-end / 19mer: RelA mRNA 3'-end 19mer ASO (SEQ ID NO: 14: 5'-CTAGCCAGCTTGGCAACAG-3 ') 3'-end / 10mer: RelA mRNA 3'-end 10mer ASO (SEQ ID NO: 15: 5'-CTAGCCAGCT-3 ')
  • TNF- ⁇ Stimulation with TNF- ⁇ was performed as follows. At 22 hours after transfection, the medium in which the HeLa cells were cultured was replaced with a DMEM medium containing 10% non-immobilized FBS and 20 ng / mL TNF- ⁇ . Thereafter, HeLa cells were collected in 2 hr. Luciferase Assay was performed as follows. The collected cells were washed with 0.5 ml of ice-cold PBS and then lysed with 150 ⁇ L of Passive Lysis Buffer (Promega). Dual-Luciferase Reporter Assay System (Promega) was used for the assay.
  • the luciferase activity when no ASO is added is defined as 100, and the inhibitory activity is shown as a relative value (hereinafter the same). 5'-end (5'-end in the figure), start codon (First AUG), 3'-end 19mer (3'-end / 19mer), 10mer (3'-end / 10mer) Even when 10 nM was added, about 65% of the activity remained.
  • 5'-end ASO targeting the 5 'end 20mer of RelA mRNA.
  • 3'-end / 10mer RelA mRNA ASO targeting the 3 'end 10mer.
  • junB-AS ASO that targets 20mer of 5 'end of junB mRNA.
  • junB-S Sense strand targeting the 5 'end 20mer of junB mRNA. The results of the luciferase assay are shown in FIG. Even when junB-AS and junB-S are combined with 3'-end / 10mer, about 70% of the activity remains, indicating that it is not non-specific suppression.
  • Example 3 ASO targeting RelA mRNA 5'-end in order to prove that the inhibitory effect when acting in combination with ASO targeting 3'-end and 5'-end of RelA mRNA is specific And the inhibitory effect when combined with a mismatched ASO targeting 3'-end was measured using Luciferase assay. ASO alone was used at a concentration of 5, 10 nM, and 5 nM each was used in combination of the two.
  • 5'-end ASO targeting the 5 'end 20mer of RelA mRNA.
  • 3'-end / 10mer RelA mRNA ASO targeting the 3 'end 10mer.
  • 3'-end / 10mer 3MM RelA mRNA A 3 base mismatch in ASO targeting the 3 'end 10mer (SEQ ID NO: 6: 5'-CTCTCCATCT-3').
  • 3'-end / 10mer 4MM RelA mRNA A 4-base mismatch is added to ASO targeting the 3'-end 10mer (SEQ ID NO: 7: 5'-TTCTCCATCT-3 '). The results are shown in FIG. It can be seen that the suppression effect is reduced when there is a mismatch.
  • Example 4 The translational inhibitory effect of ASO designed at the 3'-end of RelA mRNA was shortened by shortening 8mer and 6mer using Luciferase assay. ASO alone was used at a concentration of 5, 10 nM, and 5 nM each was used in combination of the two.
  • Example 5 Inhibition of translation by introducing a polynucleotide consisting of ASO designed at 5'-end of RelA mRNA and ASO designed at 3'-end with PEG type spacer (Gen Design) into HeLa cells in the same manner as above. The activity was examined.
  • 5'-end ASO targeting the 5 'end 20mer of RelA mRNA.
  • 3'-end / 10mer RelA mRNA ASO targeting the 3 'end 10mer.
  • 5 '/ 20-Sp9-3' / 10 9 molecule spacer inserted 5 '/ 20-Sp18-3' / 10: 18 spacer inserted
  • FIG. 1 ASO targeting the 5 'end 20mer of RelA mRNA.
  • 3'-end / 10mer RelA mRNA
  • ASO targeting the 3 'end 10mer 5 '/ 20-Sp9-3' / 10: 9 molecule spacer inserted 5 '/ 20-Sp18-3' / 10: 18 spacer inserted
  • RNA was prepared from cells transfected with Full LNA ASO targeting RelA mRNA, and the amount of RelA mRNA was quantified using qReal-Time PCR.
  • Total RNA was extracted from the collected cells using Sepasol-RNA I super (Nacalai Tesque). RT-PCR was performed using SuperScript III transcriptase (Invitrogen). Oligo (dT) Primers (Invitrogen) was used as a primer for reverse transcription reaction.
  • qReal-Time PCR was performed as follows using StepOnePlus TM Real-Time PCR System (Life Technologies). To the cDNA, 10 ⁇ L of GoTaq qPCR Master Mix (Promega), 5 pmol of gene-specific Forward Primer and Reverse Primer were added, and the cycle was started with the following profile. The number of PCR reaction cycles was 50. Initial heat denaturation 95 ° C for 5 minutes PCR reaction (50 cycles) 95 ° C for 30 seconds (thermal denaturation) 55 ° C 60 seconds (annealing) 72 ° C 30 seconds (extension)
  • ASO alone was used at concentrations of 3.3, 5, and 10 nM, two combinations of 5 nM each, and three combinations of 3.3 nM each.
  • 5'-end ASO targeting the 5 'end 20mer of RelA mRNA.
  • First AUG ASO targeting 20mer containing the start codon.
  • 3'-end / 19mer RelA mRNA ASO targeting the 3 'end 19mer.
  • 3'-end / 10mer RelA mRNA ASO targeting the 3 'end 10mer.
  • the results are shown in FIG. The decrease in RelA mRNA due to the addition of ASO was not observed except for 3'-end / 10mer, but rather the mRNA increased.
  • RNA was prepared from cells into which ASO targeting RelA mRNA was introduced, and the amount of RelA mRNA was quantified using qReal-Time PCR in the same manner as in Example 6.
  • 5'-end ASO targeting the 5 'end 20mer of RelA mRNA.
  • 3'-end / 10mer RelA mRNA ASO targeting the 3 'end 10mer.
  • 5 '/ 20-Sp9-3' / 10 Insert 9 molecule spacer
  • the result of inserting a spacer of 5 ′ / 20-Sp18-3 ′ / 10: 18 molecule is shown in FIG. RelA mRNA levels were not suppressed by ASO, but rather the relative mRNA levels could increase.
  • Example 8 Preparation of cell lysate for in vitro translation
  • Cell lysate was prepared according to the method of Rakotondrafara & Hentze (see Nature protocol 6, 563-571 (2011) An efficient factor-depleted mammalian in vitro translation system, FIG. 11).
  • HeLa cells cultured in a 10 cm petri dish were removed by trypsin treatment, collected by centrifugation, suspended in a hypotonic solution having the same volume as the pellet (FIG. 11), and left at 4 ° C. for 45 minutes. After standing, the cells were disrupted by adding and removing the suspension several times with a 1 mL syringe equipped with a 27G injection needle. After centrifugation, the supernatant was used as a cell lysate.
  • the antisense oligo is a 20mer antisense oligo (SEQ ID NO: 12) from the 5′-end of RelA mRNA or a 20mer antisense oligo (SEQ ID NO: 16: 5′-TCAAGGTCCAGCCGCTCAGC-3 ′) from the JunB 5′-end.
  • a 10-mer antisense oligo (SEQ ID NO: 15) from the 3′-end was linked by a PEG spacer and used. Triethylene-glycol was used for the PEG type spacer.
  • the gene serving as a template for translation is the DNA fragment (RelA 5'-UTR-Luciferase ORF-RelA 3'-UTR: SEQ ID NO: 17) shown in FIG. 14 or the DNA fragment (JunB 5'-UTR-Luciferase shown in FIG. 15).
  • ORF-RelA 3′-UTR: SEQ ID NO: 18) was prepared by total synthesis of each vector linked between HindIII-EcoRI sites of vector pcDNA3.1 +.
  • Each synthesized vector is cleaved at the restriction enzyme EcoRI site downstream of RelA 3'-UTR, mRNA is transcribed with T7 RNA polymerase, CAP and polyA are added by a conventional method, and added to the in vitro translation system along with the antisense oligo Then, the translation activity was measured. The results are shown in FIG. 14 and FIG.
  • FIG. 14 shows the relative activity of luciferase. When no treatment was set to 1, 5'-LNA and 3'-LNA did not cause much translational inhibition, but 5 '+ 3' showed about 40% translational inhibition.
  • the antisense oligo of the present invention is effective even if the gene is changed. That is, by synthesizing 5′-terminal 20 bases and 3′-terminal 10 bases for various genes and adding them separately or linking them with a spacer, the translation of the genes can be inhibited. It has been shown.
  • Examples 10 to 12 below were carried out by the following method.
  • Cell Culture HeLa cells were cultured in DMEM (Dulbecco's-modified Eagle's medium; Sigma) containing 10% non-immobilized FBS (Gibco) and penicillin-streptomycin (Life technologies) at 37 ° C. and 5% CO 2 .
  • DMEM Dulbecco's-modified Eagle's medium
  • FBS non-immobilized FBS
  • penicillin-streptomycin Life technologies
  • Transfection HeLa cells were suspended in DMEM medium containing 10% non-immobilized FBS, and 0.5 mL each was plated on a 24-well plate so that the transfection was about 1 ⁇ 10 5 cells / mL. Transfection was performed 24 hours later when the cells were at about 40-50% confluence.
  • Luciferase assay Luciferase activity was measured as follows using Dual-Luciferase Reporter Assay System (Promega). The transfected HeLa cells were washed twice with 500 ⁇ L of ice-cooled Phosphate-Buffered Saline (PBS) (pH 7.4), 150 ⁇ L of passive lysis buffer (Promega) was added, and the mixture was gently stirred at room temperature for 15 minutes. The cell lysate was centrifuged at 10,000 g for 5 minutes at room temperature, and 30 ⁇ L of the resulting supernatant was used for Luciferase assay. Luminoskan luminometer (Thermo Scientific) was used for the measurement of luminescence intensity.
  • siRNA siRNA was purchased from Nippon Gene Materials for synthesis. SiNegative (Universal Negative Control siRNA) was purchased from Nippon Gene Materials. 3'-end siRelA / antisense strand; 5'-cuagccagcuuggcaacagTT-3 '(SEQ ID NO: 19) 3'-end siRelA / Sense strand: 5'-cuguugccaagcuggcuagTT-3 '(SEQ ID NO: 20) siPositive / antisense strand; 5'-ugacguaaagggauagggcTT-3 '(SEQ ID NO: 21) T is the overhang part. siRNA was introduced into cells as dsRNA with TT as an overhang.
  • gapmer and mixmer gapmer and mixmer were purchased from Gene Design Co., Ltd. for synthesis.
  • 3'-end gapmer 5'-CTagccagcttGG-3 '(SEQ ID NO: 22) 5'-end LNA 5'-TCCGGCCGCGCCTGCGCGCT-3 '(SEQ ID NO: 23) 5'-end gapmer 5'-TCCGgccgcgcctgcgCGCT-3 '(SEQ ID NO: 24) 5'-end Mixmer 1 5'-TccgGccgCgccTgcgCgct-3 '(SEQ ID NO: 25) 5'-end Mixmer 2 5'-tccGgccGCGCCTgcgCgct-3 '(SEQ ID NO: 26)
  • Capital letters indicate LNA.
  • Example 10 Combination of 5'-end LNA and 3'-end siRNA LNA antisense oligo (5'-end) designed to hybridize to the 5'-end of human RelAmRNA and hybridize to the 3'-UTR end
  • the designed LNA ASO (3'-end / 10-mer), siRNA (3'-siR), siPositive, and siNegative were used alone or in combination, and their translational inhibitory effects were measured.
  • use 10nM when using siRNA alone, use 40nM.
  • use 5'-LNA at 5nM and siRNA at 20nM. did. Luciferase activity was expressed as a relative value when the value when siNegative was transfected was 1.
  • Example 11 5'-end LNA and 3'-end gapmer combination 5'-end LNA (5'-L) designed to hybridize to the 5'-end of human RelA mRNA and 3'-UTR end
  • the LNA gapmer (3'-end gapmer / 13mer; 3'-G) designed as described above was used alone or in combination, and its translation inhibitory effect was measured.
  • 5′-L was used alone, it was used at 10 nM or 5 nM.
  • 5'-LNA was used between 5 nM and 3'-G between 0.1 and 10 nM. Luciferase activity was expressed as a relative value when the value of cells not treated with antisense oligo was taken as 1.
  • Example 12 Combination of 3'-end gapmer and 5'-end gapmer, 5'-mixmer1, 5'-mixmer2 gapmer (5'-G), mixmer1 (5 designed to hybridize to the 5'-end of human RelA mRNA '-M1), mixmer 2 (5'-M2) and LNA gapmer (3'-G) designed to hybridize to the 3'-UTR end can be used singly or in combination, and its translational inhibitory effect It was measured. When each was used alone, it was used at 5 nM. When the two were combined, the 5′-side antisense oligo was used at 1 nM, 3 nM, and 10 nM, and 3′-G was used at 5 nM. Luciferase activity was expressed as a relative value when the value of cells not treated with antisense oligo was taken as 1.
  • [result] 3'-end gapmer (3'-G) was transfected at a concentration of 5 nM. 3'-G alone showed 81% translational inhibition at 5 nM. 5'-end gapmer (5'-G), 5'-mixmer1 (5'-M1), 5'-mixmer2 (5'-M2) at concentrations of 1, 3, and 10 nM respectively. '-G) Transfected with 5 nM. When each concentration of 5′-G, 5′-M1, 5′-M2 and 5 nM of 3′-G was allowed to act in combination, the translation inhibitory activity could be confirmed (FIG. 18). Based on the above results, 3'-G and 5'-end gapmer (5'-G), 5'-mixmer1 (5'-M1), or 5'-mixmer2 (5'-M2) combinations also suppress translation It was suggested that it can be done.
  • Example 13 Application of antisense oligonucleic acid to the 5 'and 3' terminal regions of mouse RelA mRNA and suppression of inflammation-related gene expression Mouse BALB / c Transfection of both 7-week-old ears with 10 ⁇ g of the following nucleic acid analogs It was mixed with the reagent Lipofectamine 2000 (Thermo Fisher Scientific) and applied. One mouse was used per sample. 1. Nucleic acid analog with scrambled sequence (control, 5'-GtgtAacaCgtcTataCgccCA-3 ') 2. 5′-RelA (5′-GgtcCcgtTcccGgccCcgC-3 ′ (SEQ ID NO: 27) 3.
  • 3′-RelA (5′-CagcGtgaTaagAcatTtaT-3 ′ (SEQ ID NO: 28) 4.
  • 5'-RelA + 3'-RelA sequences hybridizing to the 5′-end and 3′-end of mouse RelA mRNA (antisense) were used. The capital letter of the sequence is LNA.
  • a mixmer consisting of LNA and DNA was used as an antisense oligonucleic acid.
  • Nucleic acids synthesized by Gene Design Co., Ltd. were used. Two days later, inflammation was induced by applying 10 ⁇ l of 0.15% DNFB to both auricles.
  • mice Two hours later, the mice were euthanized by cervical dislocation and the auricles were collected and stored at -80 ° C.
  • the auricle was cooled with liquid nitrogen and crushed by pressurization, and then 300 ⁇ l of RIPA buffer was added. Next, it is finely crushed with Hiscotron until there are no tissue clumps.
  • power control is set to 7 (strength that prevents the sample from jumping out of the Eppendorf tube). And sonication was performed 10 times at 15 second intervals. The solution was centrifuged to recover about 150 ⁇ l of a clear solution portion, and the protein concentration was measured by the BCA method.
  • the target mRNA translation inhibitor and translation inhibition method of the present invention can be used in the manufacturing industry for reagents and / or pharmaceuticals, agriculture, and the like.

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Abstract

Provided is a method for inhibiting, effectively and in a stable manner, the translation of mRNA into protein. Disclosed are: a target mRNA translation inhibitor that contains a first polynucleotide which may be hybridized to the 5'-untranslated region and/or translated region of target mRNA, and a second polynucleotide which can be hybridized to the 3'-untranslated region of said mRNA; and/or a method for inhibiting the translation from mRNA using said translation inhibitor. According to the present invention, it is possible to inhibit the translation from target mRNA in a stable manner.

Description

翻訳抑制方法および翻訳抑制剤Translation suppression method and translation inhibitor
　本発明は、遺伝子発現の抑制方法および抑制剤、より詳しくは、ｍＲＮＡの翻訳抑制方法および翻訳抑制剤に関する。 The present invention relates to a method for suppressing gene expression and an inhibitor, and more particularly, to a method for suppressing translation of mRNA and a translation inhibitor.
　バイオ医薬品分野が活況を呈している。現行は主にタンパク質製剤である。今後は比較的低分子で化学合成可能な核酸医薬品の伸びが期待されている（非特許文献１）。 The biopharmaceutical field is booming. Currently, it is mainly protein preparations. In the future, growth of nucleic acid drugs that can be chemically synthesized with relatively small molecules is expected (Non-patent Document 1).
　核酸医薬品は、DNAあるいはRNA、あるいはそれらの類縁体からなる。大きさはタンパク質製剤より小さく低分子医薬品より大きい。核酸医薬品あるいはその候補物質は、長さがおよそ10から20塩基程度の1本鎖あるいは2本鎖の分子からなるものが主流である（非特許文献２）。塩基間で形成される水素結合により標的核酸、主にmRNAと2重鎖状態を呈する。mRNAの特定領域で部分的に2重鎖が生じると、１）標的mRNAが分解される、２）標的mRNAと特定の機能性タンパク質間との結合が阻害される（非特許文献２）。１）や２）の性質を利用することにより、特定遺伝子の発現を抑制することができる。つまり、核酸医薬品の有する遺伝子発現抑制活性を利用し、疾病治療に用いることができる。 Nucleic acid drugs consist of DNA or RNA, or their analogs. The size is smaller than the protein preparation and larger than the small molecule drug. Nucleic acid drugs or candidate substances thereof are mainly composed of single-stranded or double-stranded molecules having a length of about 10 to 20 bases (Non-patent Document 2). It exhibits a double-stranded state with the target nucleic acid, mainly mRNA, by hydrogen bonds formed between bases. When a double chain is partially generated in a specific region of mRNA, 1) the target mRNA is degraded, and 2) binding between the target mRNA and a specific functional protein is inhibited (Non-patent Document 2). By utilizing the properties 1) and 2), the expression of a specific gene can be suppressed. That is, it can be used for disease treatment by utilizing the gene expression inhibitory activity of nucleic acid drugs.
　遺伝子発現を抑制する方法として、アンチセンス法（非特許文献２）、リボザイム法（非特許文献３）、あるいはRNAi（small interfering RNA : siRNAを用いる）法（非特許文献４）が開発されてきた。アンチセンス法では、主に1本鎖状態にあるDNAあるいはその類縁体、例えばLNA（locked nucleic acids）を含むポリヌクレオチドやMO（morpholino oligonucleotides）からなる核酸類縁体をmRNAの翻訳開始コドン周辺領域やスプライシング部位周辺領域で2重鎖を形成するように設計する。これにより、翻訳阻害や正常なスプライシング反応を阻害することにより遺伝子発現の抑制効果を得る。しかしながら、これらアンチセンス法によっては十分な抑制効果を安定的に得ることは困難であるという問題があった。 As a method for suppressing gene expression, an antisense method (Non-patent document 2), a ribozyme method (Non-patent document 3), or an RNAi (small interfering RNA: using siRNA) method (Non-patent document 4) has been developed. . In the antisense method, DNA in a single-stranded state or an analog thereof, for example, a polynucleotide containing LNA (locked nucleic acids) or a nucleic acid analog consisting of MO (morpholino oligonucleotides) is converted into a region around the mRNA translation initiation codon, Designed to form a duplex in the region surrounding the splicing site. Thereby, the inhibition effect of gene expression is obtained by inhibiting translation inhibition and normal splicing reaction. However, there is a problem that it is difficult to stably obtain a sufficient suppression effect by these antisense methods.
　リボザイム法とRNAi法は、主にRNAを用いて標的遺伝子のmRNAの切断と分解を誘導することにより遺伝子発現抑制を達成する。リボザイムによる遺伝子発現抑制も必ずしも十分な抑制効果が得られるものでは無かった。 The ribozyme method and RNAi method achieve gene expression suppression mainly by using RNA to induce mRNA cleavage and degradation of the target gene. Inhibition of gene expression by ribozymes is not always sufficient.
　RNAiメカニズムの発見をきっかけにして、生理的条件下で働く短鎖の2本鎖RNA、microRNA（miRNA）が見出された。miRNAはmRNAの3’非翻訳領域（UTR）で2重鎖を形成する。これによりdeadenylaseが活性化され、poly(A)鎖が短くなる（非特許文献５）。同時にmRNAの5’UTRに作用する翻訳開始因子複合体の活性が阻害される（非特許文献６）。結果として、翻訳反応が抑制される。siRNAは標的mRNAと完全に相補的な塩基配列からなる2重鎖を形成してmRNAの分解を誘導する。これに対してmiRNAではミスマッチを含む2重鎖を標的mRNA間と形成し、mRNAの切断や分解は生じない（非特許文献５）。 Triggered by the discovery of the RNAi mechanism, a short double-stranded RNA, microRNA (miRNA) that works under physiological conditions was discovered. miRNAs form duplexes in the 3 'untranslated region (UTR) of mRNA. This activates deadenylase and shortens the poly (A) chain (Non-patent Document 5). At the same time, the activity of the translation initiation factor complex acting on the 5 'UTR of mRNA is inhibited (Non-patent Document 6). As a result, the translation reaction is suppressed. siRNA forms a double strand consisting of a base sequence that is completely complementary to the target mRNA, and induces degradation of mRNA. On the other hand, in miRNA, a duplex containing a mismatch is formed between target mRNAs, and mRNA is not cleaved or degraded (Non-patent Document 5).
　我々は、最近、母性mRNAのpoly(A)鎖ジャンクションすぐ上流に設計した25塩基からなるMOをゼブラフィッシュ初期胚やイトマキヒトデ卵中にマイクロインジェクションすることにより、poly(A)鎖の短小化あるいは切断が生じ、その結果、標的遺伝子の翻訳反応を人為的に抑制できる方法の開発に成功した（特許文献１、非特許文献７）。この方法は2重鎖が形成されるmRNA上の位置と抑制メカニズムの相違から、従来のアンチセンス法やリボザイム法あるいはRNAi法と異なる新規のアンチセンス法に属すると考えられる。しかしながら、その適用範囲は限られており、幅広い標的ｍＲＮＡに対する効率のよい翻訳抑制方法が求められていた。 We recently shortened or cleaved the poly (A) strand by microinjecting a 25-base MO designed just upstream of the maternal mRNA poly (A) strand into early zebrafish embryos and starfish eggs. As a result, the inventors succeeded in developing a method capable of artificially suppressing the translation reaction of the target gene (Patent Document 1, Non-Patent Document 7). This method is considered to belong to a novel antisense method different from the conventional antisense method, ribozyme method or RNAi method due to the difference in the position on the mRNA where the double chain is formed and the suppression mechanism. However, its application range is limited, and an efficient translation suppression method for a wide range of target mRNAs has been demanded.
ＷＯ２０１３／０１５１５２WO2013 / 015152
　ｍＲＮＡからの翻訳を効率よく安定的に抑制する翻訳抑制剤および翻訳抑制方法を提供する。 Provided are a translation inhibitor and a translation suppression method that efficiently and stably suppress translation from mRNA.
　本発明によれば、以下の発明が提供される。
（１）標的ｍＲＮＡの５’－非翻訳領域および／または翻訳領域にハイブリダイズし得る第一のポリヌクレオチドと、該ｍＲＮＡの３’－非翻訳領域にハイブリダイズしうる第二のポリヌクレオチドとを含む標的ｍＲＮＡの翻訳抑制剤。
（２）前記第一のポリヌクレオチドが、前記標的ｍＲＮＡの５’－非翻訳領域を含む領域にハイブリダイズし得るポリヌクレオチドである（１）の標的ｍＲＮＡの翻訳抑制剤。
（３）前記第一のポリヌクレオチドが、前記標的ｍＲＮＡの５’－末端または翻訳開始コドンを含む領域にハイブリダイズすることを特徴とする（１）または（２）の標的ｍＲＮＡの翻訳抑制剤。
（４）前記第二のポリヌクレオチドが、前記標的ｍＲＮＡのポリＡ配列の直前にハイブリダイズすることを特徴とする（１）～（３）のいずれかに記載の標的ｍＲＮＡの翻訳抑制剤。
（５）前記第一のポリヌクレオチドが、１８～３０塩基からなるポリヌクレオチドである、（１）～（４）のいずれかに記載の標的ｍＲＮＡの翻訳抑制剤。
（６）前記第二のポリヌクレオチドが、６～３０塩基からなるポリヌクレオチドである、（１）～（５）のいずれかに記載の標的ｍＲＮＡの翻訳抑制剤。
（７）前記ポリヌクレオチドがＤＮＡ、ＲＮＡ、ＬＮＡを含むポリヌクレオチドまたはモルフォリノオリゴ核酸である（１）～（６）のいずれかに記載の標的ｍＲＮＡの翻訳抑制剤。
（８）前記ポリヌクレオチドが少なくとも１つのヌクレオチド類似体を含む、（１）～（７）のいずれかに記載の標的ｍＲＮＡの翻訳抑制剤。
（９）前記第一のポリヌクレオチドと前記第二のポリヌクレオチドを少なくとも１つのスペーサーを介して連結したポリヌクレオチドを、前記標的ｍＲＮＡの５’－非翻訳領域および／または翻訳領域ならびに３’－非翻訳領域にそれぞれハイブリダイズし得るようにしたものである、（１）～（８）のいずれかに記載の標的ｍＲＮＡの翻訳抑制剤。
（１０）前記標的ｍＲＮＡが炎症反応を引き起こすタンパク質のｍＲＮＡである、（１）～（９）のいずれかに記載の標的ｍＲＮＡの翻訳抑制剤。
（１１）前記標的ｍＲＮＡがＮＦκＢのｍＲＮＡである、（１）～（１０）のいずれかに記載の標的ｍＲＮＡの翻訳抑制剤。
（１２）（１０）または（１１）の標的ｍＲＮＡの翻訳抑制剤を含む抗炎症剤および／または抗がん剤。
（１３）（１）～（１１）のいずれかに記載の標的ｍＲＮＡの翻訳抑制剤または（１２）に記載の抗炎症剤および／または抗がん剤が導入された細胞。
（１４）（１）～（１１）のいずれかに記載の標的ｍＲＮＡの翻訳抑制剤および／または請求項１２に記載の抗炎症剤および／または抗がん剤を含むキット。
（１５）標的ｍＲＮＡの５’－非翻訳領域および／または翻訳領域にハイブリダイズし得る第一のポリヌクレオチドと、該標的ｍＲＮＡの３’－非翻訳領域にハイブリダイズしうる第二のポリヌクレオチドとを該標的ｍＲＮＡにハイブリダイズさせる、標的ｍＲＮＡの翻訳を抑制する方法。
（１６）前記第一のポリヌクレオチドが、前記標的ｍＲＮＡの５’－非翻訳領域を含む領域にハイブリダイズし得るポリヌクレオチドである、（１５）の標的ｍＲＮＡの翻訳を抑制する方法。
（１７）前記第一のポリヌクレオチドが、前記標的ｍＲＮＡの５’－末端または翻訳開始コドンを含む領域にハイブリダイズする、（１５）または（１６）の標的ｍＲＮＡの翻訳を抑制する方法。
（１８）前記第二のポリヌクレオチドが、前記標的ｍＲＮＡのポリＡ配列の直前にハイブリダイズすることを特徴とする（１５）～（１７）のいずれかに記載の標的ｍＲＮＡの翻訳を抑制する方法。
（１９）前記第一のポリヌクレオチドが、１８～３０塩基からなるポリヌクレオチドである、（１５）～（１８）のいずれかに記載の標的ｍＲＮＡの翻訳を抑制する方法。
（２０）前記第二のポリヌクレオチドが、６～３０塩基からなるポリヌクレオチドである、（１５）～（１９）のいずれかに記載の標的ｍＲＮＡの翻訳を抑制する方法。
（２１）前記ポリヌクレオチドがＤＮＡ、ＲＮＡ、ＬＮＡを含むポリヌクレオチドまたはモルフォリノオリゴ核酸である（１５）～（２０）のいずれかに記載の標的ｍＲＮＡの翻訳を抑制する方法。
（２２）前記ポリヌクレオチドが少なくとも１つのヌクレオチド類似体を含むポリヌクレオチドである、（１５）～（２１）のいずれかに記載の標的ｍＲＮＡの翻訳を抑制する方法。
（２３）前記第一のポリヌクレオチドと前記第二のポリヌクレオチドを少なくとも１つのスペーサーを介して連結したポリヌクレオチドを、標的ｍＲＮＡの５’－非翻訳領域および／または翻訳領域ならびに３’－非翻訳領域にそれぞれハイブリダイズし得るようにしたものを用い、（１５）～（２２）のいずれかに記載の標的ｍＲＮＡの翻訳を抑制する方法。
（２４）前記標的ｍＲＮＡが炎症反応を引き起こすタンパク質のｍＲＮＡである、（１５）～（２３）のいずれかに記載の標的ｍＲＮＡの翻訳を抑制する方法。
（２５）前記標的ｍＲＮＡがＮＦκＢのｍＲＮＡである、（１５）～（２４）のいずれかに記載の標的ｍＲＮＡの翻訳を抑制する方法。
（２６）（２４）または（２５）の標的ｍＲＮＡの翻訳抑制方法を用いる炎症治療方法および／またはがん治療方法。
（２７）ｉｎ　ｖｉｔｒｏ翻訳系を用いて、翻訳阻害活性を有する物質をスクリーニングする方法。
（２８）前記翻訳阻害活性を有する物質がポリヌクレオチドである（２７）に記載の方法。
（２９）前記ポリヌクレオチドが（１）に記載の第一のポリヌクレオチドおよび／または第二のポリヌクレオチドである（２８）に記載の方法。
（３０）第二のポリヌクレオチドがsiRNAである、（１）～（１１）のいずれかに記載の標的ｍＲＮＡの翻訳抑制剤、（１２）の抗炎症剤および／または抗がん剤、（１３）の細胞、ならびに／または（１４）の抗炎症剤および／または抗がん剤を含むキット。
（３１）第二のポリヌクレオチドがsiRNAである、（１５）～（２５）の翻訳を抑制する方法、ならびに／または（２６）の炎症治療方法および／またはがん治療方法。 According to the present invention, the following inventions are provided.
(1) A first polynucleotide capable of hybridizing to the 5′-untranslated region and / or the translated region of the target mRNA, and a second polynucleotide capable of hybridizing to the 3′-untranslated region of the mRNA. A target mRNA translation inhibitor.
(2) The target mRNA translation inhibitor of (1), wherein the first polynucleotide is a polynucleotide capable of hybridizing to a region containing the 5′-untranslated region of the target mRNA.
(3) The target mRNA translation inhibitor according to (1) or (2), wherein the first polynucleotide hybridizes to the 5′-end of the target mRNA or a region containing a translation initiation codon.
(4) The target mRNA translation inhibitor according to any one of (1) to (3), wherein the second polynucleotide hybridizes immediately before the poly A sequence of the target mRNA.
(5) The target mRNA translation inhibitor according to any one of (1) to (4), wherein the first polynucleotide is a polynucleotide comprising 18 to 30 bases.
(6) The target mRNA translation inhibitor according to any one of (1) to (5), wherein the second polynucleotide is a polynucleotide comprising 6 to 30 bases.
(7) The target mRNA translation inhibitor according to any one of (1) to (6), wherein the polynucleotide is a polynucleotide containing DNA, RNA, LNA, or a morpholino oligonucleic acid.
(8) The target mRNA translation inhibitor according to any one of (1) to (7), wherein the polynucleotide comprises at least one nucleotide analogue.
(9) A polynucleotide in which the first polynucleotide and the second polynucleotide are linked via at least one spacer, a 5′-untranslated region and / or a translated region and 3′-non-translated region of the target mRNA. The target mRNA translation inhibitor according to any one of (1) to (8), which is capable of hybridizing to a translation region.
(10) The target mRNA translation inhibitor according to any one of (1) to (9), wherein the target mRNA is mRNA of a protein that causes an inflammatory reaction.
(11) The target mRNA translation inhibitor according to any one of (1) to (10), wherein the target mRNA is NFκB mRNA.
(12) An anti-inflammatory agent and / or an anticancer agent comprising the target mRNA translation inhibitor of (10) or (11).
(13) A cell into which the target mRNA translation inhibitor according to any one of (1) to (11) or the anti-inflammatory and / or anticancer agent according to (12) has been introduced.
(14) A kit comprising the target mRNA translation inhibitor according to any one of (1) to (11) and / or the anti-inflammatory agent and / or anticancer agent according to claim 12.
(15) a first polynucleotide capable of hybridizing to the 5′-untranslated region and / or the translated region of the target mRNA, and a second polynucleotide capable of hybridizing to the 3′-untranslated region of the target mRNA A method for suppressing translation of a target mRNA, wherein the target mRNA is hybridized.
(16) The method for suppressing translation of a target mRNA according to (15), wherein the first polynucleotide is a polynucleotide capable of hybridizing to a region containing the 5′-untranslated region of the target mRNA.
(17) The method for suppressing translation of a target mRNA according to (15) or (16), wherein the first polynucleotide hybridizes to a 5′-end of the target mRNA or a region containing a translation initiation codon.
(18) The method for suppressing translation of a target mRNA according to any one of (15) to (17), wherein the second polynucleotide hybridizes immediately before the poly A sequence of the target mRNA. .
(19) The method for suppressing translation of a target mRNA according to any one of (15) to (18), wherein the first polynucleotide is a polynucleotide comprising 18 to 30 bases.
(20) The method for suppressing translation of a target mRNA according to any one of (15) to (19), wherein the second polynucleotide is a polynucleotide comprising 6 to 30 bases.
(21) The method for suppressing translation of a target mRNA according to any one of (15) to (20), wherein the polynucleotide is a polynucleotide containing DNA, RNA, LNA, or a morpholino oligonucleic acid.
(22) The method for suppressing translation of a target mRNA according to any one of (15) to (21), wherein the polynucleotide is a polynucleotide comprising at least one nucleotide analogue.
(23) A polynucleotide in which the first polynucleotide and the second polynucleotide are linked via at least one spacer, the 5′-untranslated region and / or the translated region and 3′-untranslated of the target mRNA. A method for suppressing translation of a target mRNA according to any one of (15) to (22), wherein a region that is capable of hybridizing to each region is used.
(24) The method for suppressing translation of a target mRNA according to any one of (15) to (23), wherein the target mRNA is mRNA of a protein that causes an inflammatory reaction.
(25) The method for suppressing translation of a target mRNA according to any one of (15) to (24), wherein the target mRNA is mRNA of NFκB.
(26) A method for treating inflammation and / or a method for treating cancer using the method for suppressing translation of a target mRNA according to (24) or (25).
(27) A method of screening a substance having translation inhibitory activity using an in vitro translation system.
(28) The method according to (27), wherein the substance having translation inhibitory activity is a polynucleotide.
(29) The method according to (28), wherein the polynucleotide is the first polynucleotide and / or the second polynucleotide according to (1).
(30) The target mRNA translation inhibitor according to any one of (1) to (11), wherein the second polynucleotide is siRNA, (12) an anti-inflammatory agent and / or an anticancer agent, ) Cells and / or (14) an anti-inflammatory and / or anticancer agent.
(31) The method for suppressing translation according to (15) to (25), wherein the second polynucleotide is siRNA, and / or the method for treating inflammation and / or the method for treating cancer according to (26).
　本発明によれば、標的ｍＲＮＡからの翻訳を安定的に抑制できる。 According to the present invention, translation from the target mRNA can be stably suppressed.
ＮＦκＢおよびルシフェラーゼを含むアッセイ用プラスミドを示す図である。It is a figure which shows the plasmid for an assay containing NF (kappa) B and luciferase. ＮＦκＢ複合体による転写制御メカニズムを示す図である。It is a figure which shows the transcription | transfer control mechanism by NF (kappa) B complex. アンチセンス核酸の標的を示す図である。It is a figure which shows the target of an antisense nucleic acid. 翻訳阻害ポリヌクレオチドの翻訳阻害活性を示す図である。It is a figure which shows the translation inhibitory activity of a translation inhibition polynucleotide. 翻訳阻害ポリヌクレオチドの特異性を示す図である。It is a figure which shows the specificity of a translation inhibition polynucleotide. 突然変異を有するポリヌクレオチドの翻訳阻害活性を示す図である。It is a figure which shows the translation inhibitory activity of the polynucleotide which has a mutation. ポリヌクレオチドの長さと阻害活性を示す図である。It is a figure which shows the length and inhibitory activity of a polynucleotide. スペーサーにより連結されたポリヌクレオチドの翻訳阻害活性を示す図である。It is a figure which shows the translation inhibitory activity of the polynucleotide connected by the spacer. 翻訳阻害剤存在下でのｍＲＮＡ量を示す図である。It is a figure which shows the amount of mRNA in the presence of a translation inhibitor. 翻訳阻害剤存在下でのｍＲＮＡ量を示す図である。It is a figure which shows the amount of mRNA in the presence of a translation inhibitor. ＨｅＬａ細胞破砕液の調製方法を示す図である。It is a figure which shows the preparation method of a HeLa cell disruption liquid. in vitro翻訳系を示す図である。It is a figure which shows an in-vitro translation system. スペーサーで連結したアンチセンスオリゴとその効果を示すグラフである。It is a graph which shows the antisense oligo connected with the spacer, and its effect. スペーサー型アンチセンスオリゴの翻訳抑制活性を示すグラフである。It is a graph which shows the translation inhibitory activity of spacer type | mold antisense oligo. スペーサー型アンチセンスオリゴの翻訳抑制活性を示すグラフである。It is a graph which shows the translation inhibitory activity of spacer type | mold antisense oligo. ポリヌクレオチドとsiRNAの組合わせで翻訳抑制活性を示すグラフである。It is a graph which shows translation inhibitory activity by the combination of polynucleotide and siRNA. ポリヌクレオチドとgapmerの組合わせで翻訳抑制活性を示すグラフである。It is a graph which shows translation inhibitory activity by the combination of a polynucleotide and gapmer. gapmerおよびmixmerの翻訳抑制活性を示すグラフである。It is a graph which shows the translation inhibitory activity of a gapmer and a mixmer. マウスRelAおよびJunBタンパク質のウエスタン解析を示す図である。It is a figure which shows the western analysis of mouse | mouth RelA and JunB protein. ウェスタンブロット後の膜のポンソーS染色を示す図である。It is a figure which shows the Ponceau S dyeing | staining of the film | membrane after a Western blot.
　本発明によれば、標的ｍＲＮＡからのタンパク質の翻訳を抑制する翻訳抑制剤および翻訳抑制方法が提供される。本明細書において、標的ｍＲＮＡとは、翻訳を抑制する標的となるｍＲＮＡであって、遺伝子からＲＮＡポリメラーゼＩＩにより転写され、３’－末端にポリＡを有するｍＲＮＡを言う。好ましくは真核生物のｍＲＮＡである。真核生物とは、例えば、ヒト、ヒトを除く動物、植物、真核微生物（例えば、酵母、カビなど）、古細菌などを含むが、ｍＲＮＡの３’－末端にポリＡ配列を有する生物であれば本発明の対象となり得る。また、インビトロにおける培養細胞も本発明の対象となり得る。必要に応じて、これらの真核生物の全部または一部を適用対象として選択してもよい。 According to the present invention, there are provided a translation inhibitor and a translation suppression method that suppress translation of a protein from a target mRNA. As used herein, target mRNA refers to mRNA that is a target for suppressing translation and is transcribed from a gene by RNA polymerase II and has poly A at the 3'-end. Preferred is eukaryotic mRNA. Eukaryotes include, for example, humans, animals other than humans, plants, eukaryotic microorganisms (eg, yeast, mold, etc.), archaea, etc., but are organisms having a polyA sequence at the 3′-end of mRNA. If it exists, it can become the object of the present invention. In vitro cultured cells can also be the subject of the present invention. If necessary, all or a part of these eukaryotes may be selected for application.
　標的ｍＲＮＡの種類は特に限定されず、タンパク質が翻訳されるｍＲＮＡであれば全て対象になりうる。例えば、疾患に関連するタンパク質を翻訳するｍＲＮＡ、代謝に関連するタンパク質を翻訳するｍＲＮＡ、転写・翻訳等の遺伝子発現に関与するタンパク質を翻訳するｍＲＮＡ、生合成に関与するタンパク質を翻訳するｍＲＮＡ、などが挙げられるがこれらに限られない。また、機能が未知のタンパク質を翻訳により発現するｍＲＮＡであってもよい。 The type of target mRNA is not particularly limited, and any mRNA can be used as long as the protein is translated. For example, mRNA that translates proteins related to diseases, mRNA that translates proteins related to metabolism, mRNA that translates proteins involved in gene expression such as transcription and translation, mRNA that translates proteins involved in biosynthesis, etc. However, it is not limited to these. Alternatively, it may be mRNA that expresses a protein whose function is unknown by translation.
　本発明の翻訳抑制剤および翻訳抑制方法においては、標的ｍＲＮＡの５’－非翻訳領域および／または翻訳領域にハイブリダイズし得る第一のポリヌクレオチドと、該ｍＲＮＡの３’－非翻訳領域にハイブリダイズしうる第二のポリヌクレオチドを使用し得る。ここでハイブリダイズする、とは、細胞内で標的ｍＲＮＡと本発明のポリヌクレオチドとが水素結合により結合して二本鎖を形成することをいう。 In the translation inhibitor and translation suppression method of the present invention, the first polynucleotide capable of hybridizing to the 5′-untranslated region and / or the translated region of the target mRNA and the 3′-untranslated region of the mRNA are hybridized. A second polynucleotide capable of soy may be used. The term “hybridize” as used herein means that the target mRNA and the polynucleotide of the present invention are combined by hydrogen bonding in a cell to form a double strand.
　本発明のより好ましい実施形態としては、本発明の翻訳抑制剤および翻訳抑制方法において、標的ｍＲＮＡの５’－非翻訳領域（５’－ＵＴＲ）を含む領域および、３’－非翻訳領域（３’－ＵＴＲ）にハイブリダイズするポリヌクレオチドを使用する。 As a more preferred embodiment of the present invention, in the translation inhibitor and translation suppression method of the present invention, a region containing a 5′-untranslated region (5′-UTR) of a target mRNA and a 3′-untranslated region (3 Use polynucleotides that hybridize to '-UTR).
　本明細書において、非翻訳領域とは、標的ｍＲＮＡのタンパク質をコードする領域（翻訳領域）以外の領域をいう。すなわち、ｍＲＮＡ中のタンパク質をコードする領域とは、開始コドンＡＵＧから終止コドンの一つ手前のコドンまでがタンパク質をコードする領域であり、その開始コドンの５’－上流側と終止コドンの３’－下流側であってポリＡ領域の手前までをそれぞれ、５’－非翻訳領域、３’－非翻訳領域という。本発明においては、標的ｍＲＮＡの５’－非翻訳領域および／または翻訳領域にハイブリダイズし得る第一のポリヌクレオチドと、該ｍＲＮＡの３’－非翻訳領域にハイブリダイズしうる第二のポリヌクレオチドを使用し得る。より好ましいポリヌクレオチドの組合せとしては、５’－非翻訳領域を含む領域にハイブリダイズし得るポリヌクレオチドおよび３’－非翻訳領域（３’－ＵＴＲ）にハイブリダイズするポリヌクレオチド、さらに好ましくは、５’－非翻訳領域にハイブリダイズし得るポリヌクレオチドおよび３’－非翻訳領域にハイブリダイズし得るポリヌクレオチド、特に好ましくは、５’－非翻訳領域中５’－末端領域にハイブリダイズし得るポリヌクレオチドおよび３’－非翻訳領域にハイブリダイズし得るポリヌクレオチド、最も好ましくは、５’－非翻訳領域中５’－末端領域にハイブリダイズし得るポリヌクレオチドおよび３’－非翻訳領域中ポリＡの直前領域にハイブリダイズし得るポリヌクレオチドを用いることができる。 In this specification, the untranslated region refers to a region other than the region encoding the protein of the target mRNA (translation region). That is, the protein-encoding region in mRNA is a region encoding a protein from the start codon AUG to the codon immediately before the stop codon, and 5′-upstream of the start codon and 3 ′ of the stop codon. -Downstream and before the poly A region are referred to as 5'-untranslated region and 3'-untranslated region, respectively. In the present invention, the first polynucleotide capable of hybridizing to the 5′-untranslated region and / or the translated region of the target mRNA and the second polynucleotide capable of hybridizing to the 3′-untranslated region of the mRNA Can be used. More preferable polynucleotide combinations include a polynucleotide capable of hybridizing to a region containing a 5′-untranslated region and a polynucleotide hybridizing to a 3′-untranslated region (3′-UTR), more preferably 5 A polynucleotide capable of hybridizing to a '-untranslated region and a polynucleotide capable of hybridizing to a 3'-untranslated region, particularly preferably a polynucleotide capable of hybridizing to a 5'-terminal region in a 5'-untranslated region And a polynucleotide capable of hybridizing to the 3′-untranslated region, most preferably a polynucleotide capable of hybridizing to the 5′-terminal region in the 5′-untranslated region and immediately before poly A in the 3′-untranslated region Polynucleotides that can hybridize to the region can be used.
　本発明の翻訳阻害剤のポリヌクレオチドの１つの実施態様は、標的ｍＲＮＡの５’－末端領域と、３’－末端のポリＡの直前から上流の配列にハイブリダイズする配列を有するポリヌクレオチドを使用するものである。この場合、あらゆる標的ｍＲＮＡに対して機械的に容易に翻訳を阻害するポリヌクレオチドを設計し、翻訳阻害剤を作成できるという利点がある。従来技術においては、アンチセンス技術により遺伝子発現を阻害するためには、アンチセンス鎖として使用する領域を設計するだけでは足りず、実験により翻訳阻害効果があるかどうかを確かめる必要があった。領域によってはアンチセンスヌクレオチドを導入しても阻害活性が出ない場合があるからである。本願発明はこの問題を解決し、あらゆるｍＲＮＡに対して翻訳抑制効果のあるアンチセンスヌクレオチドの配列を容易に設計し、その配列を有するアンチセンスヌクレオチドを用いて安定的に翻訳を阻害し得る。すなわち、本実施形態の発明は特にスクリーニング実験を必要とせず、あらゆるｍＲＮＡに対して機械的に適用可能である。 One embodiment of the polynucleotide of the translation inhibitor of the present invention uses a polynucleotide having a 5′-terminal region of the target mRNA and a sequence that hybridizes to the sequence immediately before and upstream of the poly A at the 3′-terminal. To do. In this case, there is an advantage that a translation inhibitor can be prepared by designing a polynucleotide that mechanically easily inhibits any target mRNA. In the prior art, in order to inhibit gene expression by antisense technology, it is not sufficient to design a region to be used as an antisense strand, and it was necessary to confirm whether it has a translation inhibitory effect by experiment. This is because, depending on the region, there may be no inhibitory activity even when antisense nucleotides are introduced. The present invention solves this problem, and can easily design an antisense nucleotide sequence having a translation suppressing effect on any mRNA, and stably inhibit translation using the antisense nucleotide having the sequence. That is, the invention of this embodiment does not require a screening experiment and can be mechanically applied to any mRNA.
　本発明の標的ｍＲＮＡの５’－非翻訳領域および／または翻訳領域を含む領域にハイブリダイズして翻訳を阻害する第一のポリヌクレオチドの長さの範囲としては、１８～３０塩基が好ましく、より好ましくは１８～２２塩基、さらに好ましくは１９～２１塩基、最も好ましくは２０塩基である。これらのうちでも、最も好ましくは、標的ｍＲＮＡの５’－非翻訳領域のうちでも、５’－末端からの１～５０塩基にハイブリダイズするポリヌクレオチドが好適に用いられ、より好ましくは５’－末端から１～３０塩基、さらに好ましくは１～２２塩基、最も好ましくは１～２０塩基、にハイブリダイズするポリヌクレオチドが用いられる。 The range of the length of the first polynucleotide that hybridizes to the 5′-untranslated region and / or the region containing the translation region of the target mRNA of the present invention to inhibit translation is preferably 18 to 30 bases, Preferably it is 18-22 bases, more preferably 19-21 bases, most preferably 20 bases. Among these, most preferably, among the 5′-untranslated regions of the target mRNA, a polynucleotide that hybridizes to 1 to 50 bases from the 5′-end is preferably used, and more preferably 5′- A polynucleotide that hybridizes to 1 to 30 bases, more preferably 1 to 22 bases, most preferably 1 to 20 bases from the end is used.
　翻訳抑制の効果は、図１に記載のプラスミドを用いて図２のＮＦκＢの転写活性化機構を利用して測定することができる。すなわち、プロモーターの上流にＮＦκＢと結合する領域を有し、プロモーターの下流にルシフェラーゼ遺伝子を有するプラスミドを用い、ＮＦκＢ転写因子のサブユニットであるＲｅｌ遺伝子のｍＲＮＡを標的とするポリヌクレオチドと共に細胞に導入すると、図２において、Ｒｅｌタンパク質の翻訳が抑制されることから、ルシフェラーゼの転写活性化が抑制され、ルシフェラーゼ活性が低くなる現象を利用する。 The effect of translational suppression can be measured by using the transcription activation mechanism of NFκB in FIG. 2 using the plasmid shown in FIG. That is, when a plasmid having a NFκB binding region upstream of a promoter and a luciferase gene downstream of the promoter is used and introduced into a cell together with a polynucleotide targeting the Rel gene mRNA which is a subunit of the NFκB transcription factor In FIG. 2, since the translation of Rel protein is suppressed, the transcription activation of luciferase is suppressed and the phenomenon that luciferase activity is lowered is utilized.
　本発明の標的ｍＲＮＡの５’－非翻訳領域を含む領域にハイブリダイズして翻訳を阻害するポリヌクレオチドは、全てのポリヌクレオチド配列が５’－非翻訳領域のみにハイブリダイズする必要はなく、一部分でも５’－非翻訳領域にハイブリダイズすればよい。すなわち、標的ｍＲＮＡの５’－非翻訳領域に一部でもハイブリダイズする限り、標的ｍＲＮＡの翻訳開始部位である開始コドンなどにもハイブリダイズしてもよい。すなわち、本発明のポリヌクレオチドの全領域が非翻訳領域のみにハイブリダイズする必要はなく、一部が非翻訳領域にハイブリダイズすればよい。また、一部はタンパク質をコードするコーディング領域の配列とハイブリダイズしてもよい（図３参照）。 The polynucleotide that inhibits translation by hybridizing to the region containing the 5′-untranslated region of the target mRNA of the present invention does not require that all polynucleotide sequences hybridize only to the 5′-untranslated region. However, it may be hybridized to the 5′-untranslated region. That is, as long as it partially hybridizes to the 5'-untranslated region of the target mRNA, it may also hybridize to the initiation codon that is the translation start site of the target mRNA. That is, it is not necessary for the entire region of the polynucleotide of the present invention to hybridize only to the untranslated region, and only a part may hybridize to the untranslated region. Moreover, a part may hybridize with the arrangement | sequence of the coding region which codes protein (refer FIG. 3).
　本発明の標的ｍＲＮＡの３’－非翻訳領域にハイブリダイズし得る第二のポリヌクレオチドは、終止コドンの下流からポリＡ鎖の手前までの配列にハイブリダイズする。３’－非翻訳領域にハイブリダイズして翻訳を阻害するポリヌクレオチドの長さの範囲としては、１０～５０塩基が好ましく、より好ましくは１０～３０塩基、さらに好ましくは１０～２０塩基、最も好ましくは１０～１９塩基である。これらのうちでも、最も好ましくは、標的ｍＲＮＡの３’－非翻訳領域のうちでも、３’－末端（ポリＡ領域の直前。ＡＡＡ・・・の１つ５’－側から開始する配列。図３参照）からの１０～３０塩基にハイブリダイズするポリヌクレオチドが好適に用いられる。 The second polynucleotide capable of hybridizing to the 3'-untranslated region of the target mRNA of the present invention hybridizes to the sequence from the downstream of the stop codon to the front of the poly A chain. The range of the length of the polynucleotide that hybridizes to the 3′-untranslated region and inhibits translation is preferably 10 to 50 bases, more preferably 10 to 30 bases, still more preferably 10 to 20 bases, and most preferably Is 10 to 19 bases. Of these, most preferably, among the 3′-untranslated regions of the target mRNA, the 3′-end (immediately before the poly A region; a sequence starting from the 5′-side of AAA... A polynucleotide that hybridizes to 10 to 30 bases from 3) is preferably used.
　本発明の翻訳抑制剤または翻訳抑制方法に用いられる第一および第二のポリヌクレオチドは、核酸、アミノ酸、ＰＥＧ（ポリエチレングリコール）、o-ニトロベンジル基、メチレン基、テレフタルアミド骨格、スチルベン骨格およびホーノキオールなど生理活性を有する低分子化合物などからなるスペーサーを介して連結していてもよい。第一および第二のポリヌクレオチドを連結するスペーサーの数は少なくとも１つであり、複数のスペーサーを用いて（例えば、介して）第一および第二のポリヌクレオチドを連結してもよい。スペーサーの長さは、核酸を用いる場合は、３～５０塩基が好ましく、より好ましくは３～３０塩基、さらに好ましくは３～２０塩基、最も好ましくは３～１８塩基である。スペーサーとしてアミノ酸を用いる場合は、２～３０アミノ酸が好ましく、より好ましくは、３～２０アミノ酸、最も好ましくは３～１８アミノ酸である。ＰＥＧの場合は、[-C-C-O-]を単位として２～３０量体が好ましく、より好ましくは、３～２０量体、最も好ましくは３～１８量体である。これら以外の分子からなるスペーサーを用いる場合はこれらに相当する繰り返し単位または長さを有するスペーサーを用いることが好ましい。スペーサーは、ポリヌクレオチドが標的ｍＲＮＡの５’－非翻訳領域および／または翻訳領域ならびに３’－非翻訳領域にそれぞれハイブリダイズし得るように長さと配列を設計する。 The first and second polynucleotides used in the translation inhibitor or translation suppression method of the present invention are nucleic acid, amino acid, PEG (polyethylene glycol), o-nitrobenzyl group, methylene group, terephthalamide skeleton, stilbene skeleton and honokiol. They may be linked via a spacer made of a low molecular weight compound having physiological activity. The number of spacers connecting the first and second polynucleotides is at least one, and the first and second polynucleotides may be connected using a plurality of spacers (for example, via). When using a nucleic acid, the length of the spacer is preferably 3 to 50 bases, more preferably 3 to 30 bases, still more preferably 3 to 20 bases, and most preferably 3 to 18 bases. When an amino acid is used as a spacer, it is preferably 2 to 30 amino acids, more preferably 3 to 20 amino acids, and most preferably 3 to 18 amino acids. In the case of PEG, it is preferably a 2-30 mer, more preferably a 3-20 mer, most preferably a 3-18 mer with [—C—C—O—] as a unit. When using a spacer composed of a molecule other than these, it is preferable to use a spacer having a repeating unit or length corresponding to these. The spacer is designed in length and sequence so that the polynucleotide can hybridize to the 5'-untranslated region and / or the translated region and 3'-untranslated region of the target mRNA, respectively.
　また、本発明のポリヌクレオチドは、天然のヌクレオチドの代わりにヌクレオチドの類似体を含んでいても良い。例えば、ＬＮＡ（locked nucleic acids）を含むポリヌクレオチドやＭＯ（morpholino oligonucleotides）核酸からなる核酸類縁体であってもよく、上記標的ｍＲＮＡの翻訳を抑制できる限り、そのヌクレオチド（リボヌクレオチドまたはデオキシリボヌクレオチド）が、糖、塩基および／またはリン酸塩が化学修飾されたヌクレオチド類似体であっても良い。塩基が修飾されたヌクレオチド類似体としては、例えば、５位修飾ウリジン又はシチジン（例えば、５－プロピニルウリジン、５－プロピニルシチジン、５－メチルシチジン、５－メチルウリジン、５－（２－アミノ）プロピルウリジン、５－ハロシチジン、５－ハロウリジン、５－メチルオキシウリジン等）；８位修飾アデノシン又はグアノシン（例えば、８－ブロモグノシン等）；デアザヌクレオチド（例えば７－デアザ－アデノシン等）；Ｏ－及びＮ－アルキル化ヌクレオチド（例えば、Ｎ６－メチルアデノシン等）等が挙げられる。 Further, the polynucleotide of the present invention may contain nucleotide analogues instead of natural nucleotides. For example, it may be a nucleic acid analog consisting of a polynucleotide containing LNA (locked nucleic acids) or an MO (morpholino 核酸 oligonucleotides) nucleic acid, and its nucleotide (ribonucleotide or deoxyribonucleotide) may be used as long as translation of the target mRNA can be suppressed. It may be a nucleotide analog in which sugar, base and / or phosphate are chemically modified. Examples of nucleotide analogs with modified bases include 5-position-modified uridine or cytidine (eg, 5-propynyluridine, 5-propynylcytidine, 5-methylcytidine, 5-methyluridine, 5- (2-amino) propyl Uridine, 5-halocytidine, 5-halouridine, 5-methyloxyuridine, etc .; 8-position modified adenosine or guanosine (eg, 8-bromognosin, etc.); Deazanucleotide (eg, 7-deaza-adenosine, etc.); O- and N -Alkylated nucleotides (eg N6-methyladenosine etc.) and the like.
　また、糖が修飾されたヌクレオチド類似体としては、例えば、ＬＮＡ、ＭＯ、リボヌクレオチドの２’－ＯＨが、Ｈ、ＯＲ、Ｒ、ハロゲン原子、ＳＨ、ＳＲ、ＮＨ2、ＮＨＲ、ＮＲ2、もしくはＣＮ（ここで、Ｒは炭素数１－６のアルキル基、アルケニル基又はアルキニル基を示す）等によって置換された２’位糖修飾、５’末端がモノリン酸化された５’末端リン酸化修飾が挙げられる。 Examples of nucleotide analogues modified with sugar include, for example, LNA, MO, 2′-OH of ribonucleotide is H, OR, R, halogen atom, SH, SR, NH 2 , NHR, NR 2 , or 2′-position sugar modification substituted by CN (wherein R represents an alkyl group, alkenyl group or alkynyl group having 1 to 6 carbon atoms) or the like, and 5′-terminal phosphorylation modification in which the 5 ′ end is monophosphorylated Can be mentioned.
　リン酸塩が修飾されたヌクレオチド類似体としては、隣接するリボヌクレオチドを結合するホスホエステル基を、ホスホチオエート基で置換したものが挙げられる。 Examples of nucleotide analogues modified with phosphate include those obtained by substituting phosphothioate groups for phosphoester groups that bind adjacent ribonucleotides.
　これらのヌクレオチド類似体は、化学合成など公知の方法によりポリヌクレオチドに導入することができる。 These nucleotide analogues can be introduced into a polynucleotide by a known method such as chemical synthesis.
　本発明の翻訳抑制剤により翻訳が抑制される標的ｍＲＮＡは、炎症反応を引き起こすタンパク質を発現するｍＲＮＡであってもよい。炎症反応を引き起こすタンパク質としては、例えば、炎症性サイトカイン（ＩＬ－１２、ＩＬ－６、ＴＮＦ－α）などが挙げられるがこれらに限られない。かかる炎症性サイトカイン遺伝子は、ＮＦκＢ複合体からＩκＢが遊離することによりＲｅｌＡとＰ５０により転写が活性化されることにより、ｍＲＮＡが転写され、サイトカインタンパクに翻訳される（図２参照）。従って、これらサイトカイン自身のｍＲＮＡを標的として本発明の翻訳抑制剤により翻訳を抑制してもよいが、サイトカイン遺伝子発現を促進する転写因子の全部または一部の遺伝子発現を抑制することによってもサイトカインの発現を抑制し、炎症反応を抑制することができる。かかるサイトカイン遺伝子の転写因子としては、例えば、ＮＦκＢ、AP-1、STAT3が挙げられる。したがって、ＮＦκＢ遺伝子の翻訳抑制剤は、抗炎症剤として使用できる。 The target mRNA whose translation is suppressed by the translation inhibitor of the present invention may be mRNA that expresses a protein that causes an inflammatory reaction. Examples of proteins that cause an inflammatory response include, but are not limited to, inflammatory cytokines (IL-12, IL-6, TNF-α) and the like. In such inflammatory cytokine genes, transcription is activated by RelA and P50 by releasing IκB from the NFκB complex, whereby mRNA is transcribed and translated into cytokine protein (see FIG. 2). Accordingly, translation may be suppressed by the translation inhibitor of the present invention targeting the mRNA of these cytokines themselves, but also by suppressing the gene expression of all or part of transcription factors that promote cytokine gene expression. Expression can be suppressed and inflammatory reaction can be suppressed. Examples of such transcription factors for cytokine genes include NFκB, AP-1, and STAT3. Therefore, the translation inhibitor of the NFκB gene can be used as an anti-inflammatory agent.
　本発明の翻訳抑制剤は炎症性サイトカインカスケードにより媒介される疾患および症状にも適用し得る。炎症性サイトカインカスケードにより媒介される疾患および症状としては以下のものが含まれるがこれらに限られない。 The translation inhibitor of the present invention can also be applied to diseases and symptoms mediated by the inflammatory cytokine cascade. Diseases and symptoms mediated by the inflammatory cytokine cascade include, but are not limited to:
　全身性炎症性応答症候群には以下が含まれる：敗血症症候群、グラム陽性敗血症、グラム陰性敗血症、培養陰性敗血症、真菌性敗血症、好中球減少性発熱、尿路性敗血症、髄膜炎菌血症、外傷性出血、唸音、電離放射線曝露、急性膵炎、成人呼吸窮迫症候群（ＡＲＤＳ）。 Systemic inflammatory response syndromes include: sepsis syndrome, Gram positive sepsis, Gram negative sepsis, culture negative sepsis, fungal sepsis, neutropenic fever, urinary sepsis, meningococcal bacteremia Traumatic bleeding, stuttering, ionizing radiation exposure, acute pancreatitis, adult respiratory distress syndrome (ARDS).
　再還流傷害には以下が含まれる：後ポンプ症候群、虚血再還流傷害。 ∙ Reperfusion injury includes: Postpump syndrome, ischemia reperfusion injury.
　心臓血管性疾患には以下が含まれる：心臓性昏倒症候群、心筋梗塞、うっ血性心不全。 Cardiovascular diseases include: cardiac fall syndrome, myocardial infarction, congestive heart failure.
　感染性疾患には以下が含まれる：ＨＩＶ感染／ＨＩＶニューロパシー、髄膜炎、肝炎、敗血症性関節炎、腹膜炎、肺炎喉頭蓋炎、大腸菌Ｏ１５７：Ｈ７、溶血性尿毒症症候群／血栓崩壊性血小板減少性紫斑病、マラリア、デング出血熱、リーシュマニア症、らい病、トキシックショック症候群、連鎖球菌性筋炎、ガス壊疸、Mycobacterium結核（ヒト型結核）、Mycobacterium aviun Intracellulare（鳥型結核菌細胞内物質感染）、Pneumocystis Carinii（ニューモシスティスカリニ）肺炎、骨盤炎症性疾患、睾丸炎／精巣上体炎、レジオネラ、ライム病、Ａ型インフルエンザ、エプスタイン－バーウイルス、ウイルス関連血球貪食(hemiaphagocytic)症候群、ウイルス性脳炎／無菌性髄膜炎。 Infectious diseases include: HIV infection / HIV neuropathy, meningitis, hepatitis, septic arthritis, peritonitis, pneumonia epiglottis, E. coli O157: H7, hemolytic uremic syndrome / thrombolytic thrombocytopenic purpura Disease, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome, streptococcal myositis, gas gangrene, Mycobacterium tuberculosis (human tuberculosis), Mycobacterium aviun Intracellulare Pneumocystis Carinii pneumonia, pelvic inflammatory disease, testicular inflammation / epididymis, Legionella, Lyme disease, influenza A, Epstein-Barr virus, virus-related hemophagocytic syndrome, viral encephalitis / Aseptic meningitis.
　産科学／婦人科学的症状には以下を含む：早産、流産、不妊症。 Obstetrics / gynecological symptoms include: premature birth, miscarriage, infertility.
　炎症性疾患／自己免疫疾患には以下が含まれる：リウマチ性関節炎／セロネガティブ関節症、変形性関節症、炎症性腸疾患、全身性エリトマトーデス、虹彩毛様体炎／ブドウ膜炎視神経炎、特発性肺繊維症、全身性脈管炎／ウェーゲナー肉芽腫症、サルコイドーシス
睾丸炎／精管切除反転術。 Inflammatory / autoimmune diseases include: rheumatoid arthritis / seronegative arthropathy, osteoarthritis, inflammatory bowel disease, systemic lupus erythematosus, iridocyclitis / uveitis optic neuritis, idiopathic Pulmonary fibrosis, systemic vasculitis / Wegener's granulomatosis, sarcoidosis testisitis / vasectomy reversal surgery.
　アレルギー性／アトピー性疾患には以下が含まれる：喘息、アレルギー性鼻炎、湿疹、アレルギー性接触性皮膚炎、アレルギー性結膜炎、過敏性肺炎。 Allergic / atopic diseases include: asthma, allergic rhinitis, eczema, allergic contact dermatitis, allergic conjunctivitis, hypersensitivity pneumonia.
　悪性疾患には以下が含まれる：ＡＬＬ、ＡＭＬ、ＣＭＬ、ＣＬＬ、ホジキン病、非ホジキンリンパ腫、カポジ肉腫、結腸直腸癌、鼻咽頭癌、悪性組織球増殖症、新生物随伴症候群／悪性疾患の高カルシウム血症。 Malignancies include: ALL, AML, CML, CLL, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, colorectal cancer, nasopharyngeal cancer, malignant histiocytosis, paraneoplastic / high malignant disease Calcemia.
　移植片には以下を含む：器官移植片拒絶、移植片対宿主疾患、悪液質。 Grafts include: organ graft rejection, graft versus host disease, cachexia.
　先天性、これは以下を含む：嚢胞性繊維症、家族性血液食細胞性リンパ組織球増殖症、鎌状赤血球貧血。 Congenital, which includes: cystic fibrosis, familial blood phagocyte lymphohistiocytosis, sickle cell anemia.
　皮膚科学的には以下を含む：乾癬、脱毛症。 Dermatologically includes: psoriasis, alopecia.
　神経学的な疾患には以下を含む：多発性硬化症、片頭痛。 Neurological diseases include the following: multiple sclerosis, migraine.
　腎臓性疾患には以下を含む：ネフローゼ症候群、血液透析、尿毒症。 Renal diseases include the following: nephrotic syndrome, hemodialysis, uremia.
　毒性のあるものには以下を含む：ＯＫＴ３療法、抗ＣＤ３療法、サイトカイン療法、化学療法、放射線療法、慢性サリチレート中毒。 Toxic ones include: OKT3 therapy, anti-CD3 therapy, cytokine therapy, chemotherapy, radiation therapy, chronic salicylate poisoning.
　代謝性／特発性疾患には以下を含む：ウィルソン病、血液色素症、α－１アンチトリプシン欠損症、糖尿病、橋本甲状腺炎、骨粗鬆症、視床下部－下垂体－副腎軸評価、原発性胆汁性肝硬変。 Metabolic / idiopathic diseases include: Wilson's disease, hemochromatosis, alpha-1 antitrypsin deficiency, diabetes, Hashimoto's thyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axis evaluation, primary biliary cirrhosis .
　これらの炎症性サイトカインカスケードにより媒介される疾患および症状を引き起こすタンパク質をコードするｍＲＮＡを標的として本発明の翻訳阻害剤を適用することで炎症を軽減し得、および／または治療し得る。 Inflammation can be reduced and / or treated by applying the translation inhibitor of the present invention targeting mRNAs encoding proteins that cause diseases and symptoms mediated by these inflammatory cytokine cascades.
　また、本発明の翻訳抑制剤により翻訳が抑制される標的ｍＲＮＡは、腫瘍性障害を引き起こすタンパク質を発現するｍＲＮＡであってもよい。「腫瘍性障害」とは、限定されるわけではないが：白血病、リンパ腫、黒色腫、癌腫および肉腫を含む、ヒトに見出されるあらゆる種類の癌または新生物または悪性腫瘍を意味する。本発明の抗がん剤および／またはがん治療方法の対象としてはこれらの腫瘍性障害を含み得る。 In addition, the target mRNA whose translation is suppressed by the translation inhibitor of the present invention may be mRNA that expresses a protein that causes a neoplastic disorder. By “neoplastic disorder” is meant any type of cancer or neoplasm or malignant tumor found in humans including, but not limited to: leukemia, lymphoma, melanoma, carcinoma and sarcoma. The subject of the anticancer agent and / or cancer treatment method of the present invention may include these neoplastic disorders.
　「肉腫」という用語は概して、胚性結合組織のような物質から成り、概して線維物質または均質物質に埋込まれた密に充填された細胞から構成される腫瘍を指す。本発明の抗がん剤および／またはがん治療方法によって処置できる肉腫の例は、限定されるわけではないが、軟骨肉腫、線維肉腫、リンパ肉腫、黒色肉腫、粘液肉腫、骨肉腫、アバネシー（Ａｂｍｅｔｈｙ’ｓ）肉腫、脂肪肉腫、リポ肉腫、胞状軟部肉腫、エナメル上皮肉腫、ブドウ状肉腫、緑色肉腫、絨毛癌腫、胎児性肉腫、ウィルムス腫瘍肉腫、子宮内膜肉腫、間質性肉腫、ユーイング肉腫、筋膜肉腫、線維芽細胞肉腫、巨細胞肉腫、顆粒球性肉腫、ホジキン肉腫、特発性多発性色素性出血性肉腫、Ｂ細胞の免疫芽細胞肉腫、リンパ腫、Ｔ細胞の免疫芽細胞肉腫、イエンセン肉腫、カポジ肉腫、クップファー細胞肉腫、血管肉腫、白血肉腫、悪性間葉肉腫、傍骨性肉腫、細網肉腫、ラウス肉腫、漿液嚢胞性肉腫、滑膜肉腫、および毛細管拡張性（ｔｅｌａｎｇｉｅｃｔａｌｔｉｃ）肉腫を含む。 The term “sarcoma” generally refers to a tumor composed of a material such as embryonic connective tissue and generally composed of closely packed cells embedded in a fibrous or homogeneous material. Examples of sarcomas that can be treated by the anticancer agents and / or cancer treatment methods of the present invention include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abmethy's) sarcoma, liposarcoma, liposarcoma, alveolar soft tissue sarcoma, enamel epithelial sarcoma, grape sarcoma, green sarcoma, choriocarcinoma, fetal sarcoma, Wilms tumor sarcoma, endometrial sarcoma, interstitial sarcoma, Ewing sarcoma , Fascial sarcoma, fibroblast sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, B cell immunoblast sarcoma, lymphoma, T cell immunoblast sarcoma, Jensen sarcoma, Kaposi sarcoma, Kupffer cell sarcoma, hemangiosarcoma, leukemia sarcoma, malignant mesenchymal sarcoma, paraskeletal sarcoma, reticulosarcoma, rous sarcoma, serous cystic sarcoma, synovial sarcoma, and capillary Including the tonicity (telangiectaltic) sarcoma.
　「黒色腫」という用語は、皮膚および他の器官のメラニン細胞系から生じる腫瘍を意味すると解釈される。本発明の抗がん剤および／またはがん治療方法によって処置できる黒色腫は、限定されるわけではないが、例えば末端黒子型黒色腫、メラニン欠乏性黒色腫、良性若年性黒色腫、クラウドマン黒色腫、Ｓ９１黒色腫、ハーディング－パッセー黒色腫、若年性黒色腫、悪性黒子型黒色腫、悪性黒色腫、結節性黒色腫、爪下黒色腫、および表在拡大型黒色腫を含む。 The term “melanoma” is taken to mean a tumor arising from the melanocytic system of the skin and other organs. Melanoma that can be treated by the anticancer agent and / or cancer treatment method of the present invention is not limited, and examples thereof include terminal melanoma type melanoma, melanin-deficient melanoma, benign juvenile melanoma, and Cloudman. Includes melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, malignant melanoma, malignant melanoma, nodular melanoma, subungual melanoma, and superficial enlarged melanoma.
　「癌腫」という用語は、周囲組織に浸潤して転移を生じる傾向がある上皮細胞から成る、悪性新生物を指す。「癌腫」という用語は、周囲組織に浸潤して転移を生じる傾向がある上皮細胞から成る、悪性新生物を指す。本発明の抗がん剤および／またはがん治療方法によって処置できる癌腫は、限定されるわけではないが、例えば腺房癌腫、腺房細胞癌腫、腺嚢癌腫、腺様嚢胞癌腫、腺腫性癌腫、副腎皮質の癌腫、肺胞癌腫、肺胞細胞癌腫、基底細胞（ｂａｓａｌ ｃｅｌｌ）癌腫、基底細胞（ｂａｓｏｃｅｌｌｕｌａｒｅ）癌腫、類基底細胞癌腫、基底有棘細胞癌腫、気管支肺胞上皮癌腫、細気管支癌腫、気管支原性癌腫、大脳様（ｃｅｒｅｂｒｉｆｏｒｍ）癌腫、胆管細胞癌腫、絨毛癌腫、コロイド癌腫、面皰癌腫、子宮体癌腫、篩状癌腫、鎧状癌腫、皮膚癌腫、円柱癌腫、円柱細胞癌腫、腺管癌腫、硬癌腫、胎児性癌腫、脳様癌腫、類表皮（ｅｐｉｅｒｍｏｉｄ）癌腫、腺様上皮癌腫、外向発育癌腫、潰瘍癌腫、線維（ｆｉｂｒｏｓｕｍ）癌腫、膠様（ｇｅｌａｔｉｎｉｆｏｒｍ）癌腫、膠様（ｇｅｌａｔｉｎｏｕｓ）癌腫、巨細胞（ｇｉａｎｔ ｃｅｌｌ）癌腫、巨細胞（ｇｉｇａｎｔｏｃｅｌｌｕｌａｒｅ）癌腫、腺癌腫、顆粒膜細胞癌腫、毛母癌腫、血性（ｈｅｍａｔｏｉｄ）癌腫、肝細胞癌腫、ヒュルトレ細胞癌腫、ヒアリン癌腫、明細胞腺（ｈｙｐｅｍｅｐｈｒｏｉｄ）癌腫、小児胎児性癌腫、上皮内癌腫、表皮内癌腫、上皮内癌腫、Ｋｒｏｍｐｅｃｈｅｒ癌腫、クルチツキー細胞癌腫、大細胞癌腫、レンズ状（ｌｅｎｔｉｃｕｌａｒ）癌腫、レンズ状（ｌｅｎｔｉｃｕｌａｒｅ）癌腫、脂肪性癌腫、リンパ上皮癌腫、髄様癌腫（ｃａｒｃｉｎｏｍａ ｍｅｄｕｌｌａｒｅ）、髄様癌腫（ｍｅｄｕｌｌａｒｙ ｃａｒｃｉｎｏｍａ）、黒色癌腫、軟癌腫（ｃａｒｃｉｎｏｍａ ｍｏｌｌｅ）、粘液性癌腫、粘液分泌癌腫（ｃａｒｃｉｎｏｍａ ｍｕｃｉｐａｒｕｍ）、粘液細胞癌腫、粘液性類表皮癌、粘膜癌腫（ｃａｒｃｉｎｏｍａ ｍｕｃｏｓｕｍ）、粘膜癌腫（ｍｕｃｏｕｓ ｃａｒｃｉｎｏｍａ）、粘液腫癌腫（ｃａｒｃｉｎｏｍａ ｍｙｘｏｍａｔｏｄｅｓ）、上咽頭癌、燕麦細胞、骨化性癌腫、類骨癌腫、乳頭状癌腫、門脈周囲癌腫、前浸潤癌腫、有棘細胞癌腫、粥状癌腫（ｐｕｌｔａｃｅｏｕｓ ｃａｒｃｉｎｏｍａ）、腎細胞癌腫、予備細胞癌腫、肉腫様癌腫、シュナイダー癌腫、硬性癌腫、陰嚢癌腫、印環細胞癌腫、単純癌腫、小細胞癌腫、ソラノイド（ｓｏｌａｎｏｉｄ）癌腫、回転楕円面細胞癌腫、紡錘体細胞癌腫、海綿様癌腫、扁平上皮癌腫、扁平上皮細胞癌腫、ストリング癌腫（ｓｔｒｉｎｇ ｃａｒｃｉｎｏｍａ）、血管拡張性癌腫、毛細管拡張症様癌腫、移行細胞癌腫、結節状癌腫（ｃａｒｃｉｎｏｍａ ｔｕｂｅｒｏｓｕｍ）、結節状癌腫（ｔｕｂｅｒｏｕｓ ｃａｒｃｉｎｏｍａ）、いぼ状癌腫、および絨毛癌腫を含む。 The term “carcinoma” refers to a malignant neoplasm composed of epithelial cells that tend to infiltrate the surrounding tissues and give rise to metastases. The term “carcinoma” refers to a malignant neoplasm composed of epithelial cells that tend to infiltrate the surrounding tissues and give rise to metastases. The carcinoma that can be treated by the anticancer agent and / or cancer treatment method of the present invention is not limited, and examples thereof include acinar carcinoma, acinar cell carcinoma, glandular cystic carcinoma, adenoid cystic carcinoma, adenomatous carcinoma. Adrenocortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basal cell carcinoma, basal cell carcinoma, basal squamous cell carcinoma, bronchoalveolar carcinoma, bronchiolocarcinoma , Bronchogenic carcinoma, cerebriaform carcinoma, cholangiocarcinoma, choriocarcinoma, colloid carcinoma, comedoma carcinoma, endometrial carcinoma, phloem carcinoma, armor carcinoma, skin carcinoma, columnar carcinoma, columnar cell carcinoma, gland duct Carcinoma, hard carcinoma, fetal carcinoma, brain-like carcinoma, epidermoid carcinoma, adenoid epithelial carcinoma, outward growth carcinoma, ulcer carcinoma, fibrosum carcinoma, glue-like ( elatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, giant cell carcinoma, adenocarcinoma, granulosa cell carcinoma, mammary carcinoma, hematoid carcinoma, hepatocellular carcinoma, hurtre cell Carcinoma, hyaline carcinoma, hypermephroid carcinoma, childhood fetal carcinoma, intraepithelial carcinoma, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher carcinoma, Kultzky cell carcinoma, large cell carcinoma, lenticular carcinoma, lenticular (Lenticulare) carcinoma, fatty carcinoma, lymphoepithelial carcinoma, medullary carcinoma, medullary carcinoma, melanoma, carcinoma moll ), Mucinous carcinoma, mucin-secreting carcinoma (carcinoma muciparum), mucinous cell carcinoma, mucinous epidermoid carcinoma, mucosal carcinoma (carcinoma ｕ mucosum), mucosal carcinoma (mucous carcinoma), myxoma carcinoma (carcinoma throat) Oat cell, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, squamous cell carcinoma, pulmonary carcinoma, renal cell carcinoma, reserve cell carcinoma, sarcomatoid carcinoma, Schneider carcinoma, rigid carcinoma, scrotal carcinoma, signet ring cell carcinoma, simple carcinoma, small cell carcinoma, solanoid carcinoma, spheroid carcinoma, spindle cell carcinoma, spongiform carcinoma, squamous cell carcinoma, squamous cell Carcinoma, string carcinoma noma), including vasodilation carcinoma, telangiectasia-like carcinoma, transitional cell carcinoma, nodular carcinomas (carcinoma tuberosum), nodular carcinomas (tuberous carcinoma), verrucous carcinoma, and choriocarcinoma carcinomas.
　癌関連遺伝子としては、例えば、myc、src、ras、abl、bcl、rb、p53, apc、brca1、brca2、akt2、braf、hras、kras、kit、msh2、cdk4、pten、egfr、erbb2、fgfr1、fgfr3、flt3、jak2、pdgfra、plk3ca、ret遺伝子等が挙げられ、これらの遺伝子発現を抑制することにより、抗癌作用を発揮できる。 Examples of cancer-related genes include myc, src, ras, abl, bcl, rb, p53, apc, brca1, brca2, akt2, braf, hras, kras, kit, msh2, cdk4, pten, egfr, erbb2, fgfr1, Examples include fgfr3, flt3, jak2, pdgfra, plk3ca, and ret genes. By suppressing the expression of these genes, an anticancer effect can be exerted.
　また、本発明によれば、翻訳抑制剤を導入した細胞が得られる。細胞への本発明に係るポリヌクレオチドの導入は、通常の遺伝子導入の方法が使用でき、リン酸カルシウム法、リポソーム法、電気穿孔法（エレクトロポレーション法）、遺伝子銃（ジーンガン）、ウイスカー法、マイクロインジェクション法、レーザーインジェクション法、プロトプラスト法（植物、酵母）、アグロバクテリウム法（植物）、塩化リチウム法（酵母）などが用いられるがこれらに限られない。個体への導入は注射やジーンガン、外用剤などにより患部に投与することも可能である。本発明の翻訳抑制剤は、インビトロでもインビボでも使用できる。すなわち、試験管内などの培養容器中の細胞における翻訳を阻害して当該遺伝子の機能を調べたり、代謝を調節して物質生産等に利用できる。 Moreover, according to the present invention, cells into which a translation inhibitor is introduced can be obtained. The introduction of the polynucleotide according to the present invention into cells can be carried out by a normal gene introduction method, such as calcium phosphate method, liposome method, electroporation method (electroporation method), gene gun (gene gun), whisker method, microinjection. Method, laser injection method, protoplast method (plant, yeast), Agrobacterium method (plant), lithium chloride method (yeast), etc. are used, but not limited thereto. For introduction into an individual, it can also be administered to the affected area by injection, gene gun, external preparation or the like. The translation inhibitor of the present invention can be used in vitro or in vivo. That is, it can be used for substance production by inhibiting translation in cells in a culture vessel such as a test tube to examine the function of the gene, or regulating metabolism.
　また、本発明によれば、翻訳抑制剤を含むキットが提供される。キットとしては、上述の遺伝子導入試薬、選択試薬と本発明のポリヌクレオチドを含むキット、それらに培地を加えたキットなどが挙げられる。 Moreover, according to the present invention, a kit containing a translation inhibitor is provided. Examples of the kit include the above-described gene introduction reagent, a kit containing a selection reagent and the polynucleotide of the present invention, and a kit in which a medium is added to them.
　また、本発明によれば、標的ｍＲＮＡの５’－非翻訳領域および／または翻訳領域にハイブリダイズし得る第一のポリヌクレオチドと、該ｍＲＮＡの３’－非翻訳領域にハイブリダイズしうる第二のポリヌクレオチドとを標的ｍＲＮＡにハイブリダイズさせ、標的ｍＲＮＡの翻訳を抑制する方法が提供される。本発明の標的ｍＲＮＡの翻訳を抑制する方法によれば、標的ｍＲＮＡ毎に複数のアンチセンスを作成して抑制効率を測定して最適なものを選択する手間が不要で、機械的に遺伝子発現を抑制することができる。 Further, according to the present invention, the first polynucleotide capable of hybridizing to the 5′-untranslated region and / or the translated region of the target mRNA and the second polynucleotide capable of hybridizing to the 3′-untranslated region of the mRNA. There is provided a method of hybridizing to a target mRNA and suppressing translation of the target mRNA. According to the method for suppressing the translation of target mRNA of the present invention, it is not necessary to create a plurality of antisenses for each target mRNA, measure the suppression efficiency, and select the optimum one, and mechanically express the gene expression. Can be suppressed.
　本発明のポリヌクレオチドは連続的に投与することも可能である。ＤＤＳ技術を用いて、細胞、組織に徐々に放出して継続的に遺伝子発現を抑制することもでき、慢性病の予防、治療にも用いることができる。 The polynucleotide of the present invention can also be administered continuously. Using DDS technology, it can be gradually released into cells and tissues to continuously suppress gene expression, and can also be used for prevention and treatment of chronic diseases.
　本発明によれば、ｉｎ　ｖｉｔｒｏ翻訳系（in vitro translation system）を用いて翻訳阻害活性を有する物質をスクリーニングする方法が提供される。ｉｎ　ｖｉｔｒｏ翻訳系とは、無細胞抽出液中または翻訳酵素および翻訳に必要な基質等を含む溶液中でタンパク質を翻訳するシステムである。ｉｎ　ｖｉｔｒｏ翻訳系としては、例えば、ウサギ網状赤血球の系（Rabbit Reticulocyte Lysate System）、小麦胚芽抽出物の系（Wheat Germ Extract）、昆虫培養細胞抽出物、ヒト培養細胞抽出液（実施例８参照）、および大腸菌抽出物などの細胞抽出液を用いるものと、翻訳に関わるタンパク質などを個別に生産し、精製したものの混合物を用いるものとがあるが、本発明にはどちらのシステムも用いることができる。また、翻訳系のみでなく、同一の系で転写、翻訳までできるシステムも開発されているが、これらも本発明に使用できる。 According to the present invention, there is provided a method for screening a substance having translation inhibitory activity using an in vitro translation system (in vitro translation system). An in vitro translation system is a system that translates proteins in a cell-free extract or in a solution containing a translation enzyme and a substrate necessary for translation. Examples of in vitro translation systems include rabbit reticulocyte system (Rabbit® Reticulocyte® Lysate® System), wheat germ extract system (Wheat® Germ® Extract), insect cell extract, and human culture cell extract (see Example 8). , And those using cell extracts such as Escherichia coli extract, and those using separately produced and purified proteins involved in translation, etc. Both systems can be used in the present invention. . Further, not only a translation system but also a system capable of performing transcription and translation in the same system has been developed, and these can also be used in the present invention.
　ｉｎ　ｖｉｔｒｏ翻訳系により翻訳阻害物質をスクリーニングするには、翻訳阻害活性を有する可能性のある候補物質を標的ｍＲＮＡとともに、あるいは必要に応じて導入時期をずらしてｉｎ　ｖｉｔｒｏ翻訳系内に導入し、標的ｍＲＮＡからのタンパク質の合成活性を測定すればよい。タンパク質の合成活性の測定は、タンパク質が酵素活性を持つものであれば、その酵素活性によりタンパク質の合成量を測定できる。酵素活性としては、例えば、ルシフェラーゼによる発光、β－ガラクトシダーゼによる基質切断による発色、発光、リン酸化酵素によるリン酸化の放射性同位元素による測定、転移酵素による基質転移活性の測定などが挙げられるがこれらに限られない。タンパク質が酵素活性を持たない場合は、例えば、ＳＤＳ－ＰＡＧＥ等の電気泳動（ウェスタンブロット含む）、ＨＰＬＣ、ＭＡＳＳ、抗体による定量（ＥＬＩＳＡ、ｄｏｔ　ｂｌｏｔ等）などの方法により定量することができるがこれらに限られない。また、これらの定量方法を複数組み合わせて翻訳阻害活性を測定してもよい。 In order to screen for translation-inhibiting substances using an in vitro translation system, candidate substances that may have translation-inhibiting activity are introduced into the in vitro translation system together with the target mRNA or, if necessary, in the in vitro translation system. What is necessary is just to measure the synthetic activity of the protein from mRNA. As for the measurement of protein synthesis activity, if the protein has enzyme activity, the amount of protein synthesis can be measured by the enzyme activity. Examples of the enzyme activity include luminescence by luciferase, color development by substrate cleavage by β-galactosidase, luminescence, measurement of phosphorylation by phosphorylase by radioisotope, measurement of substrate transfer activity by transferase, etc. Not limited. If the protein does not have enzyme activity, it can be quantified by methods such as electrophoresis such as SDS-PAGE (including Western blot), HPLC, MASS, and antibody quantification (ELISA, dot blot, etc.). Not limited to. In addition, translation inhibition activity may be measured by combining a plurality of these quantitative methods.
　本発明のｉｎ　ｖｉｔｒｏ翻訳系により翻訳阻害物質をスクリーニングする方法において、用いられる翻訳阻害物質としては特に制限はされず、例えば、アンチセンス鎖のポリヌクレオチド、リボザイム、アプタマー、抗生物質、抗体、タンパク質などが挙げられるがこれらに限られない。また、ｉｎ　ｖｉｔｒｏ翻訳系は本発明の翻訳阻害剤の活性を測定し、より活性の高いポリヌクレオチドおよび／またはスペーサーの組合せなどをスクリーニングするためにも好適に使用し得る。 In the method for screening a translation inhibitor using the in vitro translation system of the present invention, the translation inhibitor used is not particularly limited, and examples thereof include antisense strand polynucleotides, ribozymes, aptamers, antibiotics, antibodies, proteins, and the like. However, it is not limited to these. The in vitro translation system can also be suitably used for measuring the activity of the translation inhibitor of the present invention and screening for a more active polynucleotide and / or spacer combination.
　本発明の翻訳阻害剤、特にスペーサーを含むアンチセンスオリゴヌクレオチドにおいては、細胞への導入率が一定ではないため、細胞への導入工程を含まないｉｎ　ｖｉｔｒｏ翻訳系を用いることで、細胞への導入率の低い翻訳阻害剤についても迅速に翻訳阻害活性を測定でき、ハイスループットスクリーニングによる網羅的スクリーニングも可能になるという利点を有する。 Since the translation inhibitor of the present invention, particularly an antisense oligonucleotide containing a spacer, does not have a constant rate of introduction into cells, introduction into cells is achieved by using an in vitro translation system that does not include a step of introduction into cells. Translation inhibitors having a low rate can also be measured quickly, and have the advantage that comprehensive screening by high-throughput screening is possible.
　本発明の翻訳阻害剤は、siRNAを含んでいてもよい。その場合、siRNAの配列としては、ｍＲＮＡの３’―末端をターゲットとするsiRNAが好ましい。この場合、siRNAの塩基数としては、１５～４０、より好ましくは１９～３０、さらに好ましくは１９～２５塩基、特に好ましくは１９塩基であり、３’－末端にチミンのオーバーハングを付したものが好ましく用いられる。 The translation inhibitor of the present invention may contain siRNA. In that case, the siRNA sequence is preferably siRNA targeting the 3'-end of mRNA. In this case, the number of siRNA bases is 15 to 40, more preferably 19 to 30, further preferably 19 to 25 bases, particularly preferably 19 bases, and a thymine overhang attached to the 3′-end. Is preferably used.
　本発明のsiRNAを含む翻訳阻害剤は、抗炎症剤および／または抗がん剤であってもよく、また、該抗炎症剤および／または該抗がん剤を含むキットの形態でもよい。また、該翻訳阻害剤を含む細胞も本発明の範囲に含まれる。 The translation inhibitor containing the siRNA of the present invention may be an anti-inflammatory agent and / or an anticancer agent, and may be in the form of a kit containing the anti-inflammatory agent and / or the anticancer agent. Further, a cell containing the translation inhibitor is also included in the scope of the present invention.
　本発明の翻訳を阻害する方法には、siRNAを使用してもよく、該siRNAは標的ｍＲＮＡの３’-末端の直前に結合するものが好ましく用いられる。siRNAを使用する翻訳を阻害する方法は、炎症治療および／またはがん治療方法としても用いられる。 In the method for inhibiting translation of the present invention, siRNA may be used, and the siRNA that binds immediately before the 3'-end of the target mRNA is preferably used. The method of inhibiting translation using siRNA is also used as a method for treating inflammation and / or treating cancer.
＜実施例１＞
　HeLa細胞は10%非動化済FBS(Gibco)、penicillin-streptomycin(Life technologies) を含むDMEM(Sigma-Aldrich)で37℃、5%CO2の条件下で培養した。このHeLa細胞に、図１に示す、転写因子ＲｅｌＡを標的ｍＲＮＡとし、ＲｅｌＡの５’－非翻訳領域、開始コドン領域、ポリＡの直前領域等に結合するアンチセンス核酸（ＬＮＡを含むポリヌクレオチド）を合成して導入した。 <Example 1>
HeLa cells were cultured in DMEM (Sigma-Aldrich) containing 10% non-immobilized FBS (Gibco) and penicillin-streptomycin (Life technologies) at 37 ° C. and 5% CO 2 . Antisense nucleic acid (polynucleotide including LNA) that binds to the 5′-untranslated region of RelA, the start codon region, the region immediately before poly A, etc., with the transcription factor RelA shown in FIG. Was synthesized and introduced.
　HeLa細胞へのトランスフェクションは以下のようにして行った。約1x106cells/mLとなるよう、HeLa細胞を10%非動化済FBSを含むDMEM培地に懸濁させ、0.5mLずつ24穴プレートに撒いた。24hr後、細胞が40～50%程度の集密状態の時に、pGL4.32(図１参照。ＮＦκＢのcis-element+promoter+Luciferase gene; Promega)1μg, pGL4.75(Promega)　20ng, LNA ASO及び1μL Lipofectamine2000(Invitrogen)を100μL OptiMEM (Invitrogen)に混合したものを添加した。ＲｅｌＡの翻訳阻害の程度により、ＲｅｌＡ下流のプロモーターに連結したルシフェラーゼ遺伝子の発現の変化を測定することにより、ＲｅｌＡの翻訳阻害活性を測定した。 Transfection into HeLa cells was performed as follows. HeLa cells were suspended in DMEM medium containing 10% non-immobilized FBS, and 0.5 mL was seeded in a 24-well plate so that the concentration was about 1 × 10 6 cells / mL. After 24 hours, when the cells are confluent at about 40-50%, pGL4.32 (see FIG. 1) NFκB cis-element + promoter + Luciferase gene; Promega 1 μg, pGL4.75 (Promega) 20 ng, LNA ASO 1 μL Lipofectamine 2000 (Invitrogen) mixed with 100 μL OptiMEM (Invitrogen) was added. By measuring the change in the expression of the luciferase gene linked to the promoter downstream of RelA, the translation inhibitory activity of RelA was measured.
　ASO（アンチセンスオリゴ）は単独では3.3, 5, 10nMの濃度で使用し、二つの組み合わせでは各5nM, 三つの組合わせでは各3.3nMを使用した。
5’-end:RelA mRNAの5’端20mer（配列番号１：5’-TCGCGCGTCCGCGCCGGCCT-3’）を標的とするASO。
First AUG:開始コドンを含む20mer（配列番号２：5’-CTGGGGCCGGTACCTGCTTG-3’）を標的とするASO。
3’-end/19mer:RelA mRNA 3’端19mer（配列番号３：5’-GACAACGGTTCGACCGATC-3’）を標的とするASO。
3’-end/10mer:RelA mRNA 3’端10mer（配列番号４：5’-TCGACCGATC-3’）を標的とするASO。 ASO (antisense oligo) was used alone at a concentration of 3.3, 5, 10 nM, 5 nM each for the two combinations, and 3.3 nM each for the three combinations.
5′-end: ASO targeting the 5 ′ end 20mer of RelA mRNA (SEQ ID NO: 5′-TCGCGCGTCCGCGCCGGCCT-3 ′).
First AUG: ASO targeting 20mer (SEQ ID NO: 5'-CTGGGGCCGGTACCTGCTTG-3 ') containing the start codon.
3'-end / 19mer: RelA mRNA ASO targeting 3 'end 19mer (SEQ ID NO: 5: 5'-GACAACGGTTCGACCGATC-3').
3'-end / 10mer: RelA mRNA ASO targeting the 3 'end 10mer (SEQ ID NO: 5: 5'-TCGACCGATC-3').
　使用したASOの配列は以下のとおりである。
5’-end:RelA mRNAの5’端20merのASO（配列番号１２：5’-TCCGGCCGCGCCTGCGCGCT-3’）
First AUG:開始コドンを含む20merのASO（配列番号１３：5’-GTTCGTCCATGGCCGGGGTC-3’）
3’-end/19mer:RelA mRNA 3’端19merのASO（配列番号１４：5’-CTAGCCAGCTTGGCAACAG-3’）
3’-end/10mer:RelA mRNA 3’端10merのASO（配列番号１５：5’-CTAGCCAGCT-3’） The sequence of ASO used is as follows.
5'-end: RelA mRNA 5 'end 20mer ASO (SEQ ID NO: 12: 5'-TCCGGCCGCGCCTGCGCGCT-3')
First AUG: 20mer ASO including the start codon (SEQ ID NO: 13: 5'-GTTCGTCCATGGCCGGGGTC-3 ')
3'-end / 19mer: RelA mRNA 3'-end 19mer ASO (SEQ ID NO: 14: 5'-CTAGCCAGCTTGGCAACAG-3 ')
3'-end / 10mer: RelA mRNA 3'-end 10mer ASO (SEQ ID NO: 15: 5'-CTAGCCAGCT-3 ')
　TNF-αによる刺激は以下のようにして行った。トランスフェクション後 22hrの時点で、HeLa 細胞を培養していた培地を10% 非動化済 FBS, 20ng/mL TNF-αを含む DMEM 培地 に交換した。その後、2hrでHeLa細胞を回収した。Luciferase Assayは以下のようにして行った。回収した細胞は氷冷したPBS 0.5 mLで洗浄後、150μLのPassive Lysis Buffer(Promega)で溶解した。アッセイにはDual-Luciferase Reporter Assay System(Promega)を用いた。 Stimulation with TNF-α was performed as follows. At 22 hours after transfection, the medium in which the HeLa cells were cultured was replaced with a DMEM medium containing 10% non-immobilized FBS and 20 ng / mL TNF-α. Thereafter, HeLa cells were collected in 2 hr. Luciferase Assay was performed as follows. The collected cells were washed with 0.5 ml of ice-cold PBS and then lysed with 150 μL of Passive Lysis Buffer (Promega). Dual-Luciferase Reporter Assay System (Promega) was used for the assay.
　その結果を図４に示す。ASO無添加の場合のルシフェラーゼ活性を１００として、それに対する阻害活性を相対値で示す（以下同様）。５’－末端（図の5’-end）、開始コドン（First AUG）、３’－末端１９ｍｅｒ（3’-end/19mer），１０ｍｅｒ（3’-end/10mer）の１種類のヌクレオチドの場合、１０ｎＭ添加しても約６５％程度は活性が残存していた。しかしながら、５’＋Ｆ（5’-end＋First AUG）、５’＋１９（5’-end＋3’-end/19mer）、５’＋１０（5’-end＋3’-end/10mer）、Ｆ＋１９（First AUG＋3’-end/19mer）、Ｆ＋１０（First AUG＋3’-end/10mer）、５’＋Ｆ＋１９（5’-end＋First AUG＋3’-end/19mer）、５’＋Ｆ＋１０（5’-end＋First AUG＋3’-end/10mer）の組合せについては、３９～２３％のルシフェラーゼ活性で、７７～６１％の抑制効果が得られた。 The result is shown in FIG. The luciferase activity when no ASO is added is defined as 100, and the inhibitory activity is shown as a relative value (hereinafter the same). 5'-end (5'-end in the figure), start codon (First AUG), 3'-end 19mer (3'-end / 19mer), 10mer (3'-end / 10mer) Even when 10 nM was added, about 65% of the activity remained. However, 5 '+ F (5'-end + First AUG), 5' + 19 (5'-end + 3'-end / 19mer), 5 '+ 10 (5'-end + 3'-end / 10mer), F + 19 (First AUG + 3'-end / 19mer), F + 10 (First AUG + 3'-end / 10mer), 5 '+ F + 19 (5'-end + First AUG + 3'-end / 19mer), 5' + F + 10 (5'-end + First AUG + 3'-end / 10mer) With a luciferase activity of 39 to 23%, an inhibitory effect of 77 to 61% was obtained.
＜実施例２＞
　次にRelA mRNAの3’-endと5’-endを標的とするASOを組合せて作用させたときの抑制効果が特異的であることを証明するためにjunB mRNAの（配列番号５：5’-GCTGAGCGGCTGGACCTTGA-3’）を標的としたASO（配列番号１６：5’-TCAAGGTCCAGCCGCTCAGC-3’）とRelA mRNAの3’-endを標的とするASO（配列番号１５）を組合わせて作用させた時の抑制効果をLuciferase assayを用いて測定した。ASOは単独では5, 10nMの濃度で使用し、二つの組み合わせでは各5nMを使用した。 <Example 2>
Next, in order to prove that the inhibitory effect when ASO targeting 3′-end and 5′-end of RelA mRNA is combined and acted on is specific (SEQ ID NO: 5: 5 ′ When ASO (SEQ ID NO: 16: 5′-TCAAGGTCCAGCCGCTCAGC-3 ′) targeting A-GCTGAGCGGCTGGACCTTGA-3 ′) and ASO (SEQ ID NO: 15) targeting the 3′-end of RelA mRNA are used in combination. The inhibitory effect was measured using Luciferase assay. ASO alone was used at a concentration of 5, 10 nM, and 5 nM each was used in combination of the two.
　5’-end:RelA mRNAの5’端20merを標的とするASO。
　3’-end/10mer:RelA mRNA 3’端10merを標的とするASO。
　junB-AS:junB mRNAの5’端20merを標的とするASO。
　junB-S:junB mRNAの5’端20merを標的とするセンス鎖。
　ルシフェラーゼアッセイの結果を図５に示す。junB-ASおよびjunB-Sとも3’-end/10merと組合わせても約７０％の活性が残存しており非特異的な抑制ではないことがわかる。 5'-end: ASO targeting the 5 'end 20mer of RelA mRNA.
3'-end / 10mer: RelA mRNA ASO targeting the 3 'end 10mer.
junB-AS: ASO that targets 20mer of 5 'end of junB mRNA.
junB-S: Sense strand targeting the 5 'end 20mer of junB mRNA.
The results of the luciferase assay are shown in FIG. Even when junB-AS and junB-S are combined with 3'-end / 10mer, about 70% of the activity remains, indicating that it is not non-specific suppression.
＜実施例３＞
　RelA mRNAの3’-endと5’-endを標的とするASOを組合せて作用させたときの抑制効果が特異的であることを証明するためにRelA mRNAの5’-endを標的としたASOと3’-endを標的とするASOにミスマッチを入れたものを組合わせて作用させた時の抑制効果をLuciferase assayを用いて測定した。ASOは単独では5, 10nMの濃度で使用し、二つの組み合わせでは各5nMを使用した。 <Example 3>
ASO targeting RelA mRNA 5'-end in order to prove that the inhibitory effect when acting in combination with ASO targeting 3'-end and 5'-end of RelA mRNA is specific And the inhibitory effect when combined with a mismatched ASO targeting 3'-end was measured using Luciferase assay. ASO alone was used at a concentration of 5, 10 nM, and 5 nM each was used in combination of the two.
　5’-end:RelA mRNAの5’端20merを標的とするASO。
　3’-end/10mer:RelA mRNA 3’端10merを標的とするASO。
　3’-end/10mer 3MM:RelA mRNA 3’端10merを標的とするASOに3塩基ミスマッチを入れたもの（配列番号６：5’-CTCTCCATCT-3’）。
　3’-end/10mer 4MM:RelA mRNA 3’端10merを標的とするASOに4塩基ミスマッチを入れたもの（配列番号７：5’-TTCTCCATCT-3’）。
　結果を図６に示す。ミスマッチがあると抑制効果が低下することがわかる。 5'-end: ASO targeting the 5 'end 20mer of RelA mRNA.
3'-end / 10mer: RelA mRNA ASO targeting the 3 'end 10mer.
3'-end / 10mer 3MM: RelA mRNA A 3 base mismatch in ASO targeting the 3 'end 10mer (SEQ ID NO: 6: 5'-CTCTCCATCT-3').
3'-end / 10mer 4MM: RelA mRNA A 4-base mismatch is added to ASO targeting the 3'-end 10mer (SEQ ID NO: 7: 5'-TTCTCCATCT-3 ').
The results are shown in FIG. It can be seen that the suppression effect is reduced when there is a mismatch.
＜実施例４＞
　RelA mRNAの3’-endに設計したASOを8mer, 6merを短くしたときの翻訳抑制効果をLuciferase assayを用いて測定した。ASOは単独では5, 10nMの濃度で使用し、二つの組み合わせでは各5nMを使用した。 <Example 4>
The translational inhibitory effect of ASO designed at the 3'-end of RelA mRNA was shortened by shortening 8mer and 6mer using Luciferase assay. ASO alone was used at a concentration of 5, 10 nM, and 5 nM each was used in combination of the two.
　5’-end:RelA mRNAの5’端20merを標的とするASO。
　3’-end/10mer:RelA mRNA 3’端10merを標的とするASO。
　3’-end/8mer:RelA mRNA 3’端8merを標的とするASO。
　3’-end/6mer:RelA mRNA 3’端6merを標的とするASO。
　結果を図７に示す。3’-endの配列が短くなるほど翻訳抑制効果は低くなることがわかる。 5'-end: ASO targeting the 5 'end 20mer of RelA mRNA.
3'-end / 10mer: RelA mRNA ASO targeting the 3 'end 10mer.
3'-end / 8mer: ASO targeting RelA mRNA 3 'end 8mer.
3'-end / 6mer: ASO targeting RelA mRNA 3 'end 6mer.
The results are shown in FIG. It can be seen that the shorter the 3′-end sequence, the lower the translational suppression effect.
＜実施例５＞
　RelA mRNAの5’-endに設計したASOと3’-endに設計したASOをPEG型スペーサー（ジーンデザイン社製）で一分子化したポリヌクレオチドを上記と同様にしてＨｅＬａ細胞に導入し翻訳阻害活性を調べた。 <Example 5>
Inhibition of translation by introducing a polynucleotide consisting of ASO designed at 5'-end of RelA mRNA and ASO designed at 3'-end with PEG type spacer (Gen Design) into HeLa cells in the same manner as above. The activity was examined.
　5’-end:RelA mRNAの5’端20merを標的とするASO。
　3’-end/10mer:RelA mRNA 3’端10merを標的とするASO。
　5’/20-Sp9-3’/10:9分子のスペーサーを挿入
　5’/20-Sp18-3’/10:18分子のスペーサーを挿入
　結果を図８に示す。 5'-end: ASO targeting the 5 'end 20mer of RelA mRNA.
3'-end / 10mer: RelA mRNA ASO targeting the 3 'end 10mer.
5 '/ 20-Sp9-3' / 10: 9 molecule spacer inserted 5 '/ 20-Sp18-3' / 10: 18 spacer inserted The results are shown in FIG.
＜実施例６＞
　RelA mRNAを標的とするFull LNA ASOを導入した細胞からtotal RNAを調製し、qReal-Time PCR用いてRelA mRNAの量を定量した。全RNAは、回収した細胞からSepasol-RNA I super(Nacalai Tesque)を使用して抽出した。RT-PCRはSuperScript III transcriptase (Invitrogen)を用いて実施した。Oligo (dT) Primers(Invitrogen)を逆転写反応用のprimerとして用いた。 <Example 6>
Total RNA was prepared from cells transfected with Full LNA ASO targeting RelA mRNA, and the amount of RelA mRNA was quantified using qReal-Time PCR. Total RNA was extracted from the collected cells using Sepasol-RNA I super (Nacalai Tesque). RT-PCR was performed using SuperScript III transcriptase (Invitrogen). Oligo (dT) Primers (Invitrogen) was used as a primer for reverse transcription reaction.
　抽出した total RNA 100ngにOligo(dT) Primers 50ng,水を加え全量を7μLにした。混合液を65℃で5分間インキュベート後、氷上で5分静置した。氷冷後、5xFirst strand buffer 4 μL、0.1M DTT 1mL、10mM dNTP 1μL、RNasin Plus RNase Inhibitor (Promega) 0.5μL、Superscript III Reverse Transcriptase 0.5μL、水を加え全量を20μLにした。反応液を50℃で60分インキュベート後、70℃で30分インキュベートし、cDNAを合成した。qReal-Time PCRは、StepOnePlusTM Real-Time PCR System (Life Technologies)を使用して以下のようにして行った。cDNAにGoTaq qPCR Master Mix(Promega) 10μL、遺伝子特異的Forward Primer及びReverse Primerを5pmol加え以下のプロファイルでサイクルを開始した。PCR反応のサイクル数は50回とした。
初期熱変性　　　　　　　　　95℃5分
PCR反応(50cycle)　　　　　　95℃30秒(熱変性)
　　　　　　　　　　　　　　55℃60秒(アニーリング)
　　　　　　　　　　　　　　72℃30秒(伸長) To 100 ng of the extracted total RNA, 50 ng of Oligo (dT) Primers and water were added to make the total volume 7 μL. The mixture was incubated at 65 ° C. for 5 minutes and then allowed to stand on ice for 5 minutes. After ice cooling, 5 × First strand buffer 4 μL, 0.1 M DTT 1 mL, 10 mM dNTP 1 μL, RNasin Plus RNase Inhibitor (Promega) 0.5 μL, Superscript III Reverse Transcriptase 0.5 μL, and water were added to a total volume of 20 μL. The reaction solution was incubated at 50 ° C. for 60 minutes and then incubated at 70 ° C. for 30 minutes to synthesize cDNA. qReal-Time PCR was performed as follows using StepOnePlus ™ Real-Time PCR System (Life Technologies). To the cDNA, 10 μL of GoTaq qPCR Master Mix (Promega), 5 pmol of gene-specific Forward Primer and Reverse Primer were added, and the cycle was started with the following profile. The number of PCR reaction cycles was 50.
Initial heat denaturation 95 ° C for 5 minutes
PCR reaction (50 cycles) 95 ° C for 30 seconds (thermal denaturation)
55 ° C 60 seconds (annealing)
72 ° C 30 seconds (extension)
　qReal-Time PCRに用いた遺伝子特異的Primerを下に記す。
RelA Forward:5’-GCAGTTTGATGATGAAGACC-3’（配列番号８）
RelA Reverse:5’-CTGTCACTAGGCGAGTTA-3’（配列番号９）
b-Actin Forward:5’-GATAGCATTGCTTTCGTGTA-3’（配列番号１０）
b-Actin Reverse:5’-TTCAACTGGTCTCAAGTCAG-3’（配列番号１１） The gene-specific primer used for qReal-Time PCR is shown below.
RelA Forward: 5'-GCAGTTTGATGATGAAGACC-3 '(SEQ ID NO: 8)
RelA Reverse: 5'-CTGTCACTAGGCGAGTTA-3 '(SEQ ID NO: 9)
b-Actin Forward: 5'-GATAGCATTGCTTTCGTGTA-3 '(SEQ ID NO: 10)
b-Actin Reverse: 5'-TTCAACTGGTCTCAAGTCAG-3 '(SEQ ID NO: 11)
　ASOは単独では3.3、5、10nMの濃度で使用し、二つの組み合わせでは各5nM、三つの組合わせでは各3.3nMを使用した。
5’-end:RelA mRNAの5’端20merを標的とするASO。
First AUG:開始コドンを含む20merを標的とするASO。
3’-end/19mer:RelA mRNA 3’端19merを標的とするASO。
3’-end/10mer:RelA mRNA 3’端10merを標的とするASO。
　結果を図９に示す。ASO添加によるRelA mRNAの減少は3’-end/10merを除いて観察されずむしろmRNAが増加する場合が見られた。 ASO alone was used at concentrations of 3.3, 5, and 10 nM, two combinations of 5 nM each, and three combinations of 3.3 nM each.
5'-end: ASO targeting the 5 'end 20mer of RelA mRNA.
First AUG: ASO targeting 20mer containing the start codon.
3'-end / 19mer: RelA mRNA ASO targeting the 3 'end 19mer.
3'-end / 10mer: RelA mRNA ASO targeting the 3 'end 10mer.
The results are shown in FIG. The decrease in RelA mRNA due to the addition of ASO was not observed except for 3'-end / 10mer, but rather the mRNA increased.
＜実施例７＞
　RelA mRNAを標的とするASOを導入した細胞からtotal RNAを調製し、実施例６と同様にしてqReal-Time PCR用いてRelA mRNAの量を定量した。
5’-end:RelA mRNAの5’端20merを標的とするASO。
3’-end/10mer:RelA mRNA 3’端10merを標的とするASO。
5’/20-Sp9-3’/10:9分子のスペーサーを挿入
5’/20-Sp18-3’/10:18分子のスペーサーを挿入
　結果を図１０に示す。RelAのmRNAレベルはASOによっては抑制されず、むしろ相対mRNA量が増える場合もあった。 <Example 7>
Total RNA was prepared from cells into which ASO targeting RelA mRNA was introduced, and the amount of RelA mRNA was quantified using qReal-Time PCR in the same manner as in Example 6.
5'-end: ASO targeting the 5 'end 20mer of RelA mRNA.
3'-end / 10mer: RelA mRNA ASO targeting the 3 'end 10mer.
5 '/ 20-Sp9-3' / 10: Insert 9 molecule spacer
The result of inserting a spacer of 5 ′ / 20-Sp18-3 ′ / 10: 18 molecule is shown in FIG. RelA mRNA levels were not suppressed by ASO, but rather the relative mRNA levels could increase.
＜実施例８＞
in vitro translation用細胞破砕液の調製
　細胞破砕液の調製はRakotondrafara & Hentzeの方法に従って行った（Nature protocol 6, 563-571 (2011) An efficient factor-depleted mammalian in vitro translation system、図１１参照）。10cmシャーレで培養したHeLa細胞をトリプシン処理によりはがして遠心分離により回収後、ペレットと同体積の低張液（図１１）に懸濁し、4℃で45分間静置した。静置後、27Gの注射針を装着した1mLシリンジで懸濁液の出し入れを複数回行うことで細胞を破砕した。遠心後、上清を細胞破砕液とした。 <Example 8>
Preparation of cell lysate for in vitro translation Cell lysate was prepared according to the method of Rakotondrafara & Hentze (see Nature protocol 6, 563-571 (2011) An efficient factor-depleted mammalian in vitro translation system, FIG. 11). HeLa cells cultured in a 10 cm petri dish were removed by trypsin treatment, collected by centrifugation, suspended in a hypotonic solution having the same volume as the pellet (FIG. 11), and left at 4 ° C. for 45 minutes. After standing, the cells were disrupted by adding and removing the suspension several times with a 1 mL syringe equipped with a 27G injection needle. After centrifugation, the supernatant was used as a cell lysate.
＜実施例９＞
in vitro translation系によるアンチオリゴの翻訳阻害試験
　in vitro translationは、図１２に記載の方法で行った。4 μL 細胞破砕液 (20mg protein/mL)、1 μL of translation buffer (16 mM HEPES, pH 7.6、20 mM クレアチンホスフェイト、0.1 μg/μl クレアチンキナーゼ、0.1 mM スペルミジン、100 μM 各種アミノ酸混合液)、0.4 μL of 1 M KOAc, 0.2 mL of 100 mM Mg(OAc)2, 1 μL RNasin Plus RNase Inhibitor (Promega), 0.25 pmol mRNA テンプレート, アンチセンスオリゴ を含む10 μLの反応溶液を調製し、30℃で5時間翻訳反応を行った。翻訳反応終了後1×Passive Lysate Bufferを100μL加えることで反応を停止し、Luciferase assay用のサンプルとし、ルシフェラーゼ活性を測定した。 <Example 9>
Anti-oligo translation inhibition test by in vitro translation system In vitro translation was performed by the method shown in FIG. 4 μL cell lysate (20 mg protein / mL), 1 μL of translation buffer (16 mM HEPES, pH 7.6, 20 mM creatine phosphate, 0.1 μg / μl creatine kinase, 0.1 mM spermidine, 100 μM various amino acid mixtures), Prepare a 10 μL reaction solution containing 0.4 μL of 1 M KOAc, 0.2 mL of 100 mM Mg (OAc) 2, 1 μL RNasin Plus RNase Inhibitor (Promega), 0.25 pmol mRNA template, and antisense oligo at 30 ° C. The translation reaction was performed for 5 hours. After completion of the translation reaction, 100 μL of 1 × Passive Lysate Buffer was added to stop the reaction, and a luciferase activity was measured using a sample for Luciferase assay.
　アンチセンスオリゴはRelA mRNAの5’-末端からの20merのアンチセンスオリゴ（配列番号１２）またはJunB 5’-末端からの20merのアンチセンスオリゴ（配列番号１６：5’-TCAAGGTCCAGCCGCTCAGC-3’）と、3’-末端からの10merのアンチセンスオリゴ（配列番号１５）をPEG型スペーサーにより連結したものを作成して用いた。PEG型スペーサーはTriethylene glycolを用いた。 The antisense oligo is a 20mer antisense oligo (SEQ ID NO: 12) from the 5′-end of RelA mRNA or a 20mer antisense oligo (SEQ ID NO: 16: 5′-TCAAGGTCCAGCCGCTCAGC-3 ′) from the JunB 5′-end. A 10-mer antisense oligo (SEQ ID NO: 15) from the 3′-end was linked by a PEG spacer and used. Triethylene-glycol was used for the PEG type spacer.
　翻訳の鋳型となる遺伝子は図１４に記載のＤＮＡ断片（RelA 5’-UTR-Luciferase ORF-RelA 3’-UTR:配列番号１７）または図１５に記載のＤＮＡ断片（JunB 5’-UTR-Luciferase ORF-RelA 3’-UTR:配列番号１８）をベクターpcDNA3.1+のHindIII-EcoRIサイト間にそれぞれ連結したものを全合成して作成した。合成したベクターをそれぞれRelA 3’-UTRの下流の制限酵素EcoRIサイトで切断し、T7 RNA polymeraseによりmRNAを転写させ、CAPとpolyAを常法により付加してin vitro翻訳系にアンチセンスオリゴとともに添加して翻訳活性を測定した。その結果を図１４および図１５に示す。 The gene serving as a template for translation is the DNA fragment (RelA 5'-UTR-Luciferase ORF-RelA 3'-UTR: SEQ ID NO: 17) shown in FIG. 14 or the DNA fragment (JunB 5'-UTR-Luciferase shown in FIG. 15). ORF-RelA 3′-UTR: SEQ ID NO: 18) was prepared by total synthesis of each vector linked between HindIII-EcoRI sites of vector pcDNA3.1 +. Each synthesized vector is cleaved at the restriction enzyme EcoRI site downstream of RelA 3'-UTR, mRNA is transcribed with T7 RNA polymerase, CAP and polyA are added by a conventional method, and added to the in vitro translation system along with the antisense oligo Then, the translation activity was measured. The results are shown in FIG. 14 and FIG.
　図１４のグラフはルシフェラーゼの相対活性を示す。無処理を１とした場合、5’-LNA、3’-LNAではあまり翻訳阻害が起きないが、5’+3’の場合は約40%の翻訳阻害が観察された。 FIG. 14 shows the relative activity of luciferase. When no treatment was set to 1, 5'-LNA and 3'-LNA did not cause much translational inhibition, but 5 '+ 3' showed about 40% translational inhibition.
　また、5’-末端が同じ配列のアンチセンスオリゴを用いた場合は、量依存的に翻訳が阻害された。それに対し、5’-末端が異なるJunB-SP9-RelAを用いた場合には翻訳阻害は15%しか起こらなかった。従って翻訳阻害は配列依存性があると考えられた。 In addition, when antisense oligos having the same sequence at the 5'-end were used, translation was inhibited in a dose-dependent manner. On the other hand, when JunB-SP9-RelA having a different 5'-end was used, translation inhibition occurred only 15%. Therefore, translational inhibition was considered to be sequence-dependent.
　次に、翻訳鋳型の5’-末端をJunB 5’-UTRにした遺伝子を上記と同様の方法で作成し、翻訳の阻害活性を測定した。その結果、JunBのアンチセンスオリゴでは量依存的に翻訳阻害が見られたが、RelA-SP9-RelAを添加しても翻訳の低下は少なかった。この実験によってもアンチセンスオリゴの配列特異性が認められた。 Next, a gene having the 5'-end of the translation template as JunB 5'-UTR was prepared in the same manner as described above, and the translation inhibitory activity was measured. As a result, translation inhibition was observed in the JunB antisense oligo in a dose-dependent manner, but there was little decrease in translation even when RelA-SP9-RelA was added. This experiment also confirmed the sequence specificity of the antisense oligo.
　また、遺伝子を変えても本発明のアンチセンスオリゴは有効であることが示された。すなわち、種々の遺伝子に対して5’-末端の20塩基、3’-末端の10塩基を合成し、別々に添加するか、スペーサーにより連結して投与することで、該遺伝子の翻訳を阻害できることが示された。 Also, it was shown that the antisense oligo of the present invention is effective even if the gene is changed. That is, by synthesizing 5′-terminal 20 bases and 3′-terminal 10 bases for various genes and adding them separately or linking them with a spacer, the translation of the genes can be inhibited. It has been shown.
　以下の実施例１０～１２については以下の方法で行った。
細胞培養
　HeLa細胞は10%非動化済FBS(Gibco)、penicillin-streptomycin(Life technologies) を含むDMEM(Dulbecco’s-modified Eagle’s medium; Sigma)で37℃、5% CO2の条件下で培養した。 Examples 10 to 12 below were carried out by the following method.
Cell Culture HeLa cells were cultured in DMEM (Dulbecco's-modified Eagle's medium; Sigma) containing 10% non-immobilized FBS (Gibco) and penicillin-streptomycin (Life technologies) at 37 ° C. and 5% CO 2 .
　トランスフェクション
　約1 x 105 cells/mLとなるよう、HeLa細胞を10%非動化済FBSを含むDMEM培地に懸濁させ、0.5 mLずつ24 穴プレートに撒いた。24 時間後、細胞が 40～50%程度の集密状態の時にトランスフェクションを実施した。pGL4.32[luc2P/NF-κB-RE/Hygro]プラスミド、pGL4.75[hRluc/CMV]プラスミドを用いた場合、0.8μg pGL4.32[luc2P/NF-κB-RE/Hygro]プラスミド、20ng pGL4.75[hRluc/CMV]プラスミド、siRNAあるいはアンチセンスオリゴ、1μL Lipofectamine 2000 (Invitrogen)を100μL OptiMEM (Invitrogen)に混合し、室温で20分間静置した。静置後、混合液を培養液に添加することでトランスフェクションを行った。 Transfection HeLa cells were suspended in DMEM medium containing 10% non-immobilized FBS, and 0.5 mL each was plated on a 24-well plate so that the transfection was about 1 × 10 5 cells / mL. Transfection was performed 24 hours later when the cells were at about 40-50% confluence. When using pGL4.32 [luc2P / NF-κB-RE / Hygro] plasmid, pGL4.75 [hRluc / CMV] plasmid, 0.8 μg pGL4.32 [luc2P / NF-κB-RE / Hygro] plasmid, 20 ng pGL4 .75 [hRluc / CMV] plasmid, siRNA or antisense oligo, 1 μL Lipofectamine 2000 (Invitrogen) was mixed with 100 μL OptiMEM (Invitrogen) and allowed to stand at room temperature for 20 minutes. After standing, transfection was performed by adding the mixed solution to the culture solution.
　TNF-αによる刺激
　pGL4.32[luc2P/NF-κB-RE/Hygro]プラスミドをトランスフェクションした場合、NF-κB の活性化のため、トランスフェクション後22時間で、HeLa細胞を培養していた培地をアスピレーターで除去し、500μLの10%非動化済FBS、20ng/mL TNF-αを含むDMEM培地に交換した。培地を交換後、インキュベーター(37℃、5%CO2)に戻し、2時間静置した。 Stimulation with TNF-α When pGL4.32 [luc2P / NF-κB-RE / Hygro] plasmid was transfected, the medium in which HeLa cells were cultured for 22 hours after transfection to activate NF-κB Was removed with an aspirator and replaced with 500 μL of DMEM medium containing 10% non-immobilized FBS and 20 ng / mL TNF-α. After exchanging the medium, the medium was returned to the incubator (37 ° C., 5% CO 2 ) and left to stand for 2 hours.
　Luciferase assay
　ルシフェラーゼ活性はDual-Luciferase Reporter Assay System (Promega)を用いて以下のように測定した。トランスフェクションを行ったHeLa細胞を氷冷したPhosphate-Buffered Saline(PBS)(pH 7.4) 500μLで二回洗浄後、150μLのpassive lysis buffer (Promega)を加え室温で15 分間穏やかに撹拌した。細胞溶解液を室温、10,000gで5分間遠心分離し、得られた上清30μLをLuciferase assayに用いた。発光強度の測定にはLuminoskan luminometer (Thermo Scientific)を用いた。 Luciferase assay
Luciferase activity was measured as follows using Dual-Luciferase Reporter Assay System (Promega). The transfected HeLa cells were washed twice with 500 μL of ice-cooled Phosphate-Buffered Saline (PBS) (pH 7.4), 150 μL of passive lysis buffer (Promega) was added, and the mixture was gently stirred at room temperature for 15 minutes. The cell lysate was centrifuged at 10,000 g for 5 minutes at room temperature, and 30 μL of the resulting supernatant was used for Luciferase assay. Luminoskan luminometer (Thermo Scientific) was used for the measurement of luminescence intensity.
　siRNA
siRNAは、ニッポンジーンマテリアルに合成を依頼し購入した。また、siNegative(ユニバーサルネガティブコントロールsiRNA)はニッポンジーンマテリアルより既存の商品を購入した。
3’-end　siRelA/antisense strand;　5’-cuagccagcuuggcaacagTT-3’（配列番号１９）
3’-end　siRelA/Sense strand:　5’-cuguugccaagcuggcuagTT-3’（配列番号２０）
siPositive/antisense strand;　5’-ugacguaaagggauagggcTT-3’（配列番号２１）
Tはオーバーハング部分。siRNAはTTをオーバーハングとするdsRNAとして細胞に導入した。 siRNA
siRNA was purchased from Nippon Gene Materials for synthesis. SiNegative (Universal Negative Control siRNA) was purchased from Nippon Gene Materials.
3'-end siRelA / antisense strand; 5'-cuagccagcuuggcaacagTT-3 '(SEQ ID NO: 19)
3'-end siRelA / Sense strand: 5'-cuguugccaagcuggcuagTT-3 '(SEQ ID NO: 20)
siPositive / antisense strand; 5'-ugacguaaagggauagggcTT-3 '(SEQ ID NO: 21)
T is the overhang part. siRNA was introduced into cells as dsRNA with TT as an overhang.
　gapmer及びmixmer
gapmerとmixmer はジーンデザイン株式会社に合成を依頼し購入した。
3’-end gapmer　5’-CTagccagcttGG-3’（配列番号２２）
5’-end LNA　5’-TCCGGCCGCGCCTGCGCGCT-3’（配列番号２３）
5’-end gapmer　5’-TCCGgccgcgcctgcgCGCT-3’（配列番号２４）
5’-end Mixmer 1　5’-TccgGccgCgccTgcgCgct-3’（配列番号２５）
5’-end Mixmer 2　5’-tccGgccGCGCCTgcgCgct-3’（配列番号２６）
大文字はＬＮＡを示す。 gapmer and mixmer
gapmer and mixmer were purchased from Gene Design Co., Ltd. for synthesis.
3'-end gapmer 5'-CTagccagcttGG-3 '(SEQ ID NO: 22)
5'-end LNA 5'-TCCGGCCGCGCCTGCGCGCT-3 '(SEQ ID NO: 23)
5'-end gapmer 5'-TCCGgccgcgcctgcgCGCT-3 '(SEQ ID NO: 24)
5'-end Mixmer 1 5'-TccgGccgCgccTgcgCgct-3 '(SEQ ID NO: 25)
5'-end Mixmer 2 5'-tccGgccGCGCCTgcgCgct-3 '(SEQ ID NO: 26)
Capital letters indicate LNA.
＜実施例１０＞
5’-end LNAと3’-end siRNAの組み合わせ
　ヒトRelAmRNAの5’-末端にハイブリダイズするように設計したLNAアンチセンスオリゴ(5’-end)と3’-UTR末端にハイブリダイズするように設計したLNA ASO(3’-end/10-mer)、siRNA (3’-siR)、siPositive、siNegativeを単独、あるいは組み合わせて使用し、その翻訳抑制効果を測定した。5’-LNAを単独で使用する場合は10nM、siRNAを単独で使用する場合は40nMの濃度で使用し、二つを組合わせる場合は、5’-LNAは5nM、siRNAは20nMの濃度で使用した。ルシフェラーゼ活性はsiNegativeをトランスフェクションしたときの値を１としたときの相対値で示した。 <Example 10>
Combination of 5'-end LNA and 3'-end siRNA LNA antisense oligo (5'-end) designed to hybridize to the 5'-end of human RelAmRNA and hybridize to the 3'-UTR end The designed LNA ASO (3'-end / 10-mer), siRNA (3'-siR), siPositive, and siNegative were used alone or in combination, and their translational inhibitory effects were measured. When using 5'-LNA alone, use 10nM, when using siRNA alone, use 40nM. When combining the two, use 5'-LNA at 5nM and siRNA at 20nM. did. Luciferase activity was expressed as a relative value when the value when siNegative was transfected was 1.
[結果]
　5’-end LNA、3’-end siRelA、3’-end LNA、siPositiveを単独で作用させた場合、それぞれ32%、35%、40%、91% の抑制効果が得られた(図１６)。組合わせて使用したときは、5’-end + 3’-end siRelAでは91%、5-end + 3’-endでは92%、3’-end + siPositive では 91%、5’-end + siNegativeでは43%翻訳抑制効果が得られた(図１６)。このことから、5’-end LNAとの組合わせは3’-end LNAだけで無く、3’-end siRNAでも良いことが示唆された。以上の結果より、全てが天然核酸からなるsiRNAと全てが核酸類縁体からなるアンチセンスLNAの組み合わせでも翻訳抑制効果が得られることが明らかとなった。 [result]
When 5′-end LNA, 3′-end siRelA, 3′-end LNA, and siPositive were allowed to act alone, suppression effects of 32%, 35%, 40%, and 91% were obtained, respectively (FIG. 16). . When used in combination, 91% for 5'-end + 3'-end siRelA, 92% for 5-end + 3'-end, 91% for 3'-end + siPositive, 5'-end + siNegative Then, a 43% translation inhibitory effect was obtained (FIG. 16). This suggests that the combination with 5'-end LNA is not limited to 3'-end LNA but may be 3'-end siRNA. From the above results, it has been clarified that a translation suppressing effect can be obtained even by a combination of siRNA composed entirely of natural nucleic acids and antisense LNA composed entirely of nucleic acid analogues.
＜実施例１１＞
5’-end LNAと3’-end gapmerの組み合わせ
　ヒトRelA mRNAの5’-末端にハイブリダイズするように設計した5’-end LNA (5’-L)と3’-UTR末端にハイブリダイズするように設計したLNA gapmer (3’-end gapmer/13mer; 3’-G)を単独、あるいは組み合わせて使用し、その翻訳抑制効果を測定した。5’-Lを単独で使用する場合は10nM、あるいは5nMで使用した。二つを組合わせる場合は、5’-LNAは 5nM 3’-Gは 0.1-10nMの間で使用した。ルシフェラーゼ活性はアンチセンスオリゴで処理していない細胞の値を１としたときの相対値で示した。 <Example 11>
5'-end LNA and 3'-end gapmer combination 5'-end LNA (5'-L) designed to hybridize to the 5'-end of human RelA mRNA and 3'-UTR end The LNA gapmer (3'-end gapmer / 13mer; 3'-G) designed as described above was used alone or in combination, and its translation inhibitory effect was measured. When 5′-L was used alone, it was used at 10 nM or 5 nM. When combining the two, 5'-LNA was used between 5 nM and 3'-G between 0.1 and 10 nM. Luciferase activity was expressed as a relative value when the value of cells not treated with antisense oligo was taken as 1.
[結果]
　3’-G を単独で 0.1、1、5、10nMでトランスフェクションさせた場合、0.1nMでは翻訳抑制効果は見られず(107%)、1nM以上ではそれぞれ、63%、90%、94%の翻訳抑制効果を得た。5nMの5’-Lとそれぞれの濃度の3’-Gを組合わせて使用したとき、0.1nMとでは62%, 1nMとでは93%、5nM、10nMとでは96%の翻訳抑制効果を得た（図１７）。以上の結果から、5’-Lとの組合わせは3’-end LNAだけで無く、3’-G（gapmerタイプ）でも良いことが示唆された。さらに3’-Gを3’側アンチセンス核酸として使用することで、強力な翻訳抑制効果を低濃度（10nM以下で）で得ることができた。 [result]
When 3'-G alone was transfected at 0.1, 1, 5, 10 nM, no translational inhibition was observed at 0.1 nM (107%), and 63%, 90%, and 94% at 1 nM and above, respectively. The translation suppression effect was obtained. When 5nM 5'-L and 3'-G of each concentration were used in combination, 62% was achieved with 0.1nM, 93% with 1nM, and 96% with 5nM and 10nM. (FIG. 17). From the above results, it was suggested that the combination with 5′-L may be 3′-G (gapmer type) as well as 3′-end LNA. Furthermore, by using 3′-G as the 3′-side antisense nucleic acid, a strong translation suppressing effect could be obtained at a low concentration (less than 10 nM).
＜実施例１２＞
3’-end gapmerと5’-end gapmer、5’-mixmer1、5’-mixmer2の組み合わせ
　ヒトRelA mRNAの5’-末端にハイブリダイズするように設計した gapmer(5’-G)、mixmer1 (5’-M1)、mixmer 2 (5’-M2)と3’-UTR末端にハイブリダイズするように設計したLNA gapmer (3’-G)を単独、あるいは組合わせて使用し、その翻訳抑制効果を測定した。それぞれを単独で使用する場合は5nMで使用した。二つを組み合わせる場合は、5’-側のアンチセンスオリゴは1nM、3nM、10nMで使用し3’-Gは5nMで使用した。ルシフェラーゼ活性はアンチセンスオリゴで処理していない細胞の値を１としたときの相対値で示した。 <Example 12>
Combination of 3'-end gapmer and 5'-end gapmer, 5'-mixmer1, 5'-mixmer2 gapmer (5'-G), mixmer1 (5 designed to hybridize to the 5'-end of human RelA mRNA '-M1), mixmer 2 (5'-M2) and LNA gapmer (3'-G) designed to hybridize to the 3'-UTR end can be used singly or in combination, and its translational inhibitory effect It was measured. When each was used alone, it was used at 5 nM. When the two were combined, the 5′-side antisense oligo was used at 1 nM, 3 nM, and 10 nM, and 3′-G was used at 5 nM. Luciferase activity was expressed as a relative value when the value of cells not treated with antisense oligo was taken as 1.
[結果]
　3’-end gapmer (3’-G)を5nMの濃度で、トランスフェクションした。3’-G単独では5nMでは81%の翻訳抑制効果を示した。5’-end gapmer (5’-G)、5’-mixmer1 (5’-M1)、5’-mixmer2 (5’-M2)をそれぞれ1、3、10nMの濃度で3’-end gapmer (3’-G)5nMとともにトランスフェクションした。各濃度の5’-G、5’-M1、5’-M2と5nMの3’-Gを組合わせて作用させると、翻訳抑制活性を確認することができた（図１８）。以上の結果から、3’-Gと5’-end gapmer (5’-G)、5’-mixmer1 (5’-M1)、あるいは5’-mixmer2 (5’-M2)の組合せでも翻訳を抑制できることが示唆された。 [result]
3'-end gapmer (3'-G) was transfected at a concentration of 5 nM. 3'-G alone showed 81% translational inhibition at 5 nM. 5'-end gapmer (5'-G), 5'-mixmer1 (5'-M1), 5'-mixmer2 (5'-M2) at concentrations of 1, 3, and 10 nM respectively. '-G) Transfected with 5 nM. When each concentration of 5′-G, 5′-M1, 5′-M2 and 5 nM of 3′-G was allowed to act in combination, the translation inhibitory activity could be confirmed (FIG. 18). Based on the above results, 3'-G and 5'-end gapmer (5'-G), 5'-mixmer1 (5'-M1), or 5'-mixmer2 (5'-M2) combinations also suppress translation It was suggested that it can be done.
＜実施例１３＞
マウスRelA mRNAの5’および3’末端領域に対するアンチセンスオリゴ核酸塗布と炎症関連遺伝子の発現抑制
　マウスBALB/c 7週齢の両方の耳介部に、以下の核酸類縁体をトータル10μｇとトランスフェクション試薬Lipofectamine 2000（Thermo Fisher Scientific社）と混合して塗布した。1サンプル当たりマウス1匹を使用した。
１．スクランブル配列を有する核酸類縁体（コントロール、5’-GtgtAacaCgtcTataCgccCA-3’）
２．5’-RelA（5’-GgtcCcgtTcccGgccCcgC-3’（配列番号２７）
３．3’-RelA（5’-CagcGtgaTaagAcatTtaT-3’（配列番号２８）
４．5’-RelA+3’-RelA
　5’-RelAおよび3’-RelAはマウスRelA mRNAの5’-末端および3’-末端にハイブリダイズする配列（アンチセンス）を用いた。配列の大文字はLNAである。アンチセンスオリゴ核酸としてLNAとDNAからなるmixmerを使用した。核酸は、ジーンデザイン株式会社により合成されたものを用いた。
　2日後、両方の耳介部に10μlの0.15%DNFBを塗布して炎症を惹起した。2時間後、頸椎脱臼によりマウスを安楽死させ耳介部を回収し、-80℃で保存した。耳介部を液体窒素で冷却し、加圧により破砕してから300μlのRIPAバッファーを添加した。次にヒスコトロンで組織の塊がなくなるまで細かく破砕し、さらに超音波発生器ＵＲ－２０Ｐ（株式会社トミー精工製）を用いてpower controlを７（エッペンドルフチューブ内からサンプルが飛び出さない程度の強度）に設定し、１５秒間隔で１０回、超音波処理を実施した。溶液を遠心分離して、透明の溶液部分約150μlを回収し、BCA法によりタンパク質濃度を測定した。1レーン当たり15μｇのタンパク質になるように10% SDSポリアクリルアミドゲルで電気泳動し、メンブレンに転写し、抗RelAタンパク質抗体（Santa Cruz, NFκB p65 (c-20) sc-732）、抗チュブリンタンパク質抗体（SIGMA, T6557）、および抗JunBタンパク質抗体（Santa Cruz, JunB (210) sc-73）を用いたウエスタンブロット法によりそれぞれのタンパク質を検出した。また、それぞれのレーンで等量のタンパク質が泳動されているのかを確認する目的で、転写後のメンブレンをポンソーＳで染色した。 <Example 13>
Application of antisense oligonucleic acid to the 5 'and 3' terminal regions of mouse RelA mRNA and suppression of inflammation-related gene expression Mouse BALB / c Transfection of both 7-week-old ears with 10 µg of the following nucleic acid analogs It was mixed with the reagent Lipofectamine 2000 (Thermo Fisher Scientific) and applied. One mouse was used per sample.
1. Nucleic acid analog with scrambled sequence (control, 5'-GtgtAacaCgtcTataCgccCA-3 ')
2. 5′-RelA (5′-GgtcCcgtTcccGgccCcgC-3 ′ (SEQ ID NO: 27)
3. 3′-RelA (5′-CagcGtgaTaagAcatTtaT-3 ′ (SEQ ID NO: 28)
4. 5'-RelA + 3'-RelA
For 5′-RelA and 3′-RelA, sequences hybridizing to the 5′-end and 3′-end of mouse RelA mRNA (antisense) were used. The capital letter of the sequence is LNA. A mixmer consisting of LNA and DNA was used as an antisense oligonucleic acid. Nucleic acids synthesized by Gene Design Co., Ltd. were used.
Two days later, inflammation was induced by applying 10 μl of 0.15% DNFB to both auricles. Two hours later, the mice were euthanized by cervical dislocation and the auricles were collected and stored at -80 ° C. The auricle was cooled with liquid nitrogen and crushed by pressurization, and then 300 μl of RIPA buffer was added. Next, it is finely crushed with Hiscotron until there are no tissue clumps. Furthermore, using an ultrasonic generator UR-20P (manufactured by Tommy Seiko Co., Ltd.), power control is set to 7 (strength that prevents the sample from jumping out of the Eppendorf tube). And sonication was performed 10 times at 15 second intervals. The solution was centrifuged to recover about 150 μl of a clear solution portion, and the protein concentration was measured by the BCA method. Electrophoresis on 10% SDS polyacrylamide gel to obtain 15 μg of protein per lane, transfer to membrane, anti-RelA protein antibody (Santa Cruz, NFκB p65 (c-20) sc-732), anti-tubulin protein antibody Each protein was detected by Western blotting using (SIGMA, T6557) and an anti-JunB protein antibody (Santa Cruz, JunB (210) sc-73). Further, the membrane after the transfer was stained with Ponceau S for the purpose of confirming whether an equal amount of protein was migrated in each lane.
［結果］
　ウエスタンブロットの結果、アンチセンスオリゴ核酸5’-RelAおよび3’-RelA、あるいはそれらを混合して使用した場合はscrambleに比較してRelAタンパク質量が顕著に低下していることがわかった（図１９）。RelAをサブユニットに持つ転写因子NFκBは、JunB遺伝子のプロモーター領域のNFκB結合サイトに結合してJunB遺伝子を活性化する。その結果発現誘導されたJunBタンパク質は、転写因子AP-1として作用し、炎症反応の悪性化に寄与する。今回、マウス耳介部を用いた実験でRelA mRNAの5’および3’末端を標的とするアンチセンスオリゴ核酸を混合して塗布することにより、JunBタンパク質レベルも低下した（図１９）。このことは、RelA mRNAに対するアンチセンスオリゴ核酸が炎症治療薬となりうることを示唆している。すなわち、本発明の抗炎症アンチセンスオリゴ核酸はｉｎ　ｖｉｔｒｏ、細胞レベルのみでなく、ｉｎ　ｖｉｖｏ、個体レベルでも効果を有することが確認された。また、JunBタンパク質の発現を抑制できることから、抗癌剤としても効果があると考えられた。ウェスタンブロット後のポンソーS染色では各レーンでほぼ同じ濃度で染色され、各レーン当たりのタンパク質量が等量であることが確認された（図２０）。 [result]
As a result of Western blotting, it was found that antisense oligonucleic acid 5'-RelA and 3'-RelA, or a mixture of these, significantly reduced the amount of RelA protein compared to scramble (Fig. 19). The transcription factor NFκB having RelA as a subunit binds to the NFκB binding site in the promoter region of the JunB gene and activates the JunB gene. As a result, JunB protein whose expression is induced acts as a transcription factor AP-1 and contributes to malignant inflammation reaction. In this experiment using a mouse auricle, the level of JunB protein was also reduced by mixing and applying antisense oligonucleic acid targeting the 5 ′ and 3 ′ ends of RelA mRNA (FIG. 19). This suggests that antisense oligonucleic acid against RelA mRNA can be an anti-inflammatory drug. That is, it was confirmed that the anti-inflammatory antisense oligonucleic acid of the present invention is effective not only in vitro and at the cellular level, but also in vivo and at the individual level. Moreover, since the expression of JunB protein can be suppressed, it was considered to be effective as an anticancer agent. In Ponceau S staining after Western blotting, each lane was stained at almost the same concentration, and it was confirmed that the amount of protein per lane was equal (FIG. 20).
　本発明の標的ｍＲＮＡの翻訳抑制剤および翻訳抑制方法は、試薬および／または医薬等の製造業、農業などに利用できる。 The target mRNA translation inhibitor and translation inhibition method of the present invention can be used in the manufacturing industry for reagents and / or pharmaceuticals, agriculture, and the like.

Claims (31)

  1. 　標的ｍＲＮＡの５’－非翻訳領域および／または翻訳領域にハイブリダイズし得る第一のポリヌクレオチドと、該ｍＲＮＡの３’－非翻訳領域にハイブリダイズしうる第二のポリヌクレオチドとを含む標的ｍＲＮＡの翻訳抑制剤。 A target mRNA comprising a first polynucleotide capable of hybridizing to the 5'-untranslated region and / or the translated region of the target mRNA and a second polynucleotide capable of hybridizing to the 3'-untranslated region of the mRNA Translation inhibitor.
  2. 　前記第一のポリヌクレオチドが、前記標的ｍＲＮＡの５’－非翻訳領域を含む領域にハイブリダイズし得るポリヌクレオチドである請求項１の標的ｍＲＮＡの翻訳抑制剤。 The target mRNA translation inhibitor according to claim 1, wherein the first polynucleotide is a polynucleotide capable of hybridizing to a region containing a 5'-untranslated region of the target mRNA.
  3. 　前記第一のポリヌクレオチドが、前記標的ｍＲＮＡの５’－末端または翻訳開始コドンを含む領域にハイブリダイズすることを特徴とする請求項１または２の標的ｍＲＮＡの翻訳抑制剤。 3. The target mRNA translation inhibitor of claim 1 or 2, wherein the first polynucleotide hybridizes to the 5'-end of the target mRNA or a region containing a translation initiation codon.
  4. 　前記第二のポリヌクレオチドが、前記標的ｍＲＮＡのポリＡ配列の直前にハイブリダイズすることを特徴とする請求項１～３のいずれかに記載の標的ｍＲＮＡの翻訳抑制剤。 The target mRNA translation inhibitor according to any one of claims 1 to 3, wherein the second polynucleotide hybridizes immediately before the poly A sequence of the target mRNA.
  5. 　前記第一のポリヌクレオチドが、１８～３０塩基からなるポリヌクレオチドである、請求項１～４のいずれかに記載の標的ｍＲＮＡの翻訳抑制剤。 The target mRNA translation inhibitor according to any one of claims 1 to 4, wherein the first polynucleotide is a polynucleotide comprising 18 to 30 bases.
  6. 　前記第二のポリヌクレオチドが、６～３０塩基からなるポリヌクレオチドである、請求項１～５のいずれかに記載の標的ｍＲＮＡの翻訳抑制剤。 The target mRNA translation inhibitor according to any one of claims 1 to 5, wherein the second polynucleotide is a polynucleotide comprising 6 to 30 bases.
  7. 　前記ポリヌクレオチドがＤＮＡ、ＲＮＡ、ＬＮＡを含むポリヌクレオチドまたはモルフォリノオリゴ核酸である請求項１～６のいずれかに記載の標的ｍＲＮＡの翻訳抑制剤。 The target mRNA translation inhibitor according to any one of claims 1 to 6, wherein the polynucleotide is a polynucleotide containing DNA, RNA, LNA, or a morpholino oligonucleic acid.
  8. 　前記ポリヌクレオチドが少なくとも１つのヌクレオチド類似体を含む、請求項１～７のいずれかに記載の標的ｍＲＮＡの翻訳抑制剤。 The target mRNA translation inhibitor according to any one of claims 1 to 7, wherein the polynucleotide comprises at least one nucleotide analogue.
  9. 　前記第一のポリヌクレオチドと前記第二のポリヌクレオチドを少なくとも１つのスペーサーを介して連結したポリヌクレオチドを、標的ｍＲＮＡの５’－非翻訳領域および／または翻訳領域ならびに３’－非翻訳領域にそれぞれハイブリダイズし得るようにしたものである請求項１～８のいずれかに記載の標的ｍＲＮＡの翻訳抑制剤。 A polynucleotide in which the first polynucleotide and the second polynucleotide are linked via at least one spacer, respectively, in the 5′-untranslated region and / or the translated region and 3′-untranslated region of the target mRNA. The target mRNA translation inhibitor according to any one of claims 1 to 8, wherein the target mRNA is capable of hybridizing.
  10. 　前記標的ｍＲＮＡが炎症反応を引き起こすタンパク質のｍＲＮＡである、請求項１～９のいずれかに記載の標的ｍＲＮＡの翻訳抑制剤。 The target mRNA translation inhibitor according to any one of claims 1 to 9, wherein the target mRNA is mRNA of a protein that causes an inflammatory reaction.
  11. 　前記標的ｍＲＮＡがＮＦκＢのｍＲＮＡである、請求項１～１０のいずれかに記載の標的ｍＲＮＡの翻訳抑制剤。 The target mRNA translation inhibitor according to any one of claims 1 to 10, wherein the target mRNA is NFκB mRNA.
  12. 　請求項１０または１１の標的ｍＲＮＡの翻訳抑制剤を含む抗炎症剤および／または抗がん剤。 An anti-inflammatory agent and / or anticancer agent comprising the target mRNA translation inhibitor of claim 10 or 11.
  13. 　請求項１～１１のいずれかに記載の標的ｍＲＮＡの翻訳抑制剤または請求項１２に記載の抗炎症剤および／または抗がん剤が導入された細胞。 A cell into which the target mRNA translation inhibitor according to any one of claims 1 to 11 or the anti-inflammatory agent and / or anticancer agent according to claim 12 has been introduced.
  14. 　請求項１～１１のいずれかに記載の標的ｍＲＮＡの翻訳抑制剤および／または請求項１２に記載の抗炎症剤および／または抗がん剤を含むキット。 A kit comprising the target mRNA translation inhibitor according to any one of claims 1 to 11 and / or the anti-inflammatory agent and / or anticancer agent according to claim 12.
  15. 　標的ｍＲＮＡの５’－非翻訳領域および／または翻訳領域にハイブリダイズし得る第一のポリヌクレオチドと、該ｍＲＮＡの３’－非翻訳領域にハイブリダイズしうる第二のポリヌクレオチドとを標的ｍＲＮＡにハイブリダイズさせる、標的ｍＲＮＡの翻訳を抑制する方法。 A target polynucleotide comprising a first polynucleotide capable of hybridizing to the 5′-untranslated region and / or the translated region of the target mRNA and a second polynucleotide capable of hybridizing to the 3′-untranslated region of the mRNA. A method for suppressing the translation of a target mRNA to be hybridized.
  16. 　　前記第一のポリヌクレオチドが、前記標的ｍＲＮＡの５’－非翻訳領域を含む領域にハイブリダイズし得るポリヌクレオチドである、請求項１５の標的ｍＲＮＡの翻訳を抑制する方法。 The method for suppressing translation of a target mRNA according to claim 15, wherein the first polynucleotide is a polynucleotide capable of hybridizing to a region containing a 5'-untranslated region of the target mRNA.
  17. 　前記第一のポリヌクレオチドが、前記標的ｍＲＮＡの５’－末端または翻訳開始コドンを含む領域にハイブリダイズする、請求項１５または１６の標的ｍＲＮＡの翻訳を抑制する方法。 The method for suppressing translation of a target mRNA according to claim 15 or 16, wherein the first polynucleotide hybridizes to a 5'-end of the target mRNA or a region containing a translation initiation codon.
  18. 　前記第二のポリヌクレオチドが、前記標的ｍＲＮＡのポリＡ配列の直前にハイブリダイズすることを特徴とする請求項１５～１７のいずれかに記載の標的ｍＲＮＡの翻訳を抑制する方法。 The method for suppressing translation of a target mRNA according to any one of claims 15 to 17, wherein the second polynucleotide hybridizes immediately before the poly A sequence of the target mRNA.
  19. 　前記第一のポリヌクレオチドが、１８～３０塩基からなるポリヌクレオチドである、請求項１５～１８のいずれかに記載の標的ｍＲＮＡの翻訳を抑制する方法。 The method for suppressing translation of a target mRNA according to any one of claims 15 to 18, wherein the first polynucleotide is a polynucleotide comprising 18 to 30 bases.
  20. 　前記第二のポリヌクレオチドが、６～３０塩基からなるポリヌクレオチドである、請求項１５～１９のいずれかに記載の標的ｍＲＮＡの翻訳を抑制する方法。 The method for suppressing translation of a target mRNA according to any one of claims 15 to 19, wherein the second polynucleotide is a polynucleotide comprising 6 to 30 bases.
  21. 　前記ポリヌクレオチドがＤＮＡ、ＲＮＡ、ＬＮＡを含むポリヌクレオチドまたはモルフォリノオリゴ核酸である請求項１５～２０のいずれかに記載の標的ｍＲＮＡの翻訳を抑制する方法。 The method for suppressing translation of a target mRNA according to any one of claims 15 to 20, wherein the polynucleotide is a polynucleotide containing DNA, RNA, LNA, or a morpholino oligonucleic acid.
  22. 　前記ポリヌクレオチドが少なくとも１つのヌクレオチド類似体を含むポリヌクレオチドである、請求項１５～２１のいずれかに記載の標的ｍＲＮＡの翻訳を抑制する方法。 The method for suppressing translation of a target mRNA according to any one of claims 15 to 21, wherein the polynucleotide comprises a polynucleotide comprising at least one nucleotide analogue.
  23. 　前記第一のポリヌクレオチドと前記第二のポリヌクレオチドを少なくとも１つのスペーサーを介して連結したポリヌクレオチドを、標的ｍＲＮＡの５’－非翻訳領域および／または翻訳領域ならびに３’－非翻訳領域にそれぞれハイブリダイズし得るようにしたものを用い、請求項１５～２２のいずれかに記載の標的ｍＲＮＡの翻訳を抑制する方法。 A polynucleotide in which the first polynucleotide and the second polynucleotide are linked via at least one spacer, respectively, in the 5′-untranslated region and / or the translated region and 3′-untranslated region of the target mRNA. 23. The method for suppressing translation of a target mRNA according to any one of claims 15 to 22, using a hybridized one.
  24. 　前記標的ｍＲＮＡが炎症反応を引き起こすタンパク質のｍＲＮＡである、請求項１５～２３のいずれかに記載の標的ｍＲＮＡの翻訳を抑制する方法。 The method for suppressing translation of a target mRNA according to any one of claims 15 to 23, wherein the target mRNA is mRNA of a protein that causes an inflammatory reaction.
  25. 　前記標的ｍＲＮＡがＮＦκＢのｍＲＮＡである、請求項１５～２４のいずれかに記載の標的ｍＲＮＡの翻訳を抑制する方法。 The method for suppressing translation of a target mRNA according to any one of claims 15 to 24, wherein the target mRNA is NFκB mRNA.
  26. 　請求項２４または２５の標的ｍＲＮＡの翻訳抑制方法を用いる炎症治療方法および／またはがん治療方法。 A method for treating inflammation and / or a method for treating cancer using the method for suppressing translation of a target mRNA according to claim 24 or 25.
  27. 　ｉｎ　ｖｉｔｒｏ翻訳系を用いて、翻訳阻害活性を有する物質をスクリーニングする方法。 A method of screening a substance having translation inhibitory activity using an in vitro translation system.
  28. 　前記翻訳阻害活性を有する物質がポリヌクレオチドである、請求項２７に記載の方法。 28. The method according to claim 27, wherein the substance having translation inhibitory activity is a polynucleotide.
  29. 　前記ポリヌクレオチドが請求項１に記載の第一のポリヌクレオチドおよび／または第二のポリヌクレオチドである請求項２８に記載の方法。 The method according to claim 28, wherein the polynucleotide is the first polynucleotide and / or the second polynucleotide according to claim 1.
  30. 　第二のポリヌクレオチドがsiRNAである、請求項１～１１のいずれかに記載の標的ｍＲＮＡの翻訳抑制剤、請求項１２の抗炎症剤および／または抗がん剤、請求項１３の細胞、ならびに／または請求項１４の抗炎症剤および／または抗がん剤を含むキット。 The target mRNA translation inhibitor of any one of claims 1 to 11, wherein the second polynucleotide is siRNA, the anti-inflammatory and / or anticancer agent of claim 12, the cell of claim 13, and A kit comprising the anti-inflammatory agent and / or anti-cancer agent of claim 14.
  31. 　第二のポリヌクレオチドがsiRNAである、請求項１５～２５の翻訳を抑制する方法、ならびに／または請求項２６の炎症治療方法および／またはがん治療方法。 The method for suppressing translation according to claims 15 to 25 and / or the method for treating inflammation and / or the method for treating cancer according to claim 26, wherein the second polynucleotide is siRNA.
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