WO2019026860A1 - Procédé de régulation d'expression génique et régulateur d'expression génique - Google Patents

Procédé de régulation d'expression génique et régulateur d'expression génique Download PDF

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WO2019026860A1
WO2019026860A1 PCT/JP2018/028511 JP2018028511W WO2019026860A1 WO 2019026860 A1 WO2019026860 A1 WO 2019026860A1 JP 2018028511 W JP2018028511 W JP 2018028511W WO 2019026860 A1 WO2019026860 A1 WO 2019026860A1
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mrna
gapmer
bases
aso
sequence
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PCT/JP2018/028511
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English (en)
Japanese (ja)
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忠士 和田
渡邉　肇
遼平 高田
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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Publication of WO2019026860A1 publication Critical patent/WO2019026860A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/13—Nucleic acids or derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the control of gene expression. More particularly, the present invention relates to a polynucleotide that suppresses the expression of mRNA and a method of using the same.
  • Patent Document 2 suppresses mRNA expression using two types of oligonucleotides, the antisense oligonucleotide of Patent Document 1 and the antisense oligonucleotide of the 5'-non-translated region of mRNA.
  • two types of oligonucleotides were required, and the dose amount was increased accordingly, and there was also a possibility of giving toxicity to tissues.
  • Nonpatent literature 1 when the target oligonucleotide is degraded using the conventional LNA-based antisense oligonucleotide (gapmer) to suppress gene expression (Patent Documents 3 and 4), it is used as a medicine because it has toxicity to the liver. There was a problem that it was difficult (nonpatent literature 1).
  • the present invention provides polynucleotides and methods for efficiently suppressing gene expression.
  • the following inventions are provided. (1) 5 to 9 bases (including the base of 3'-end of mRNA) from the base corresponding to the 3'-end of mRNA in the genome sequence, and 5 to 9 bases of 3'-end A polynucleotide capable of hybridizing to a continuous sequence (without the 3'-terminal base of mRNA). (2) 5 to 9 bases on the 5 'side (including the bases on the 3' end of the mRNA) from the bases corresponding to the 3 'end of the mRNA in the genome sequence, and 5 to 9 bases on the 3' side A polynucleotide having only a sequence capable of hybridizing to a continuous sequence (not including the 3'-terminal base of mRNA).
  • the nucleotide analogue is LNA, 2′-O-methylated RNA
  • a gene expression suppressing agent comprising the polynucleotide of any one of (1) to (7) as an active ingredient.
  • the gene expression inhibitor of (8) wherein the gene expression inhibitor is a translation inhibitor.
  • a medicine, a cosmetic and / or a food comprising the polynucleotide of any of (1) to (7) as an active ingredient.
  • a method for suppressing gene expression which comprises administering a polynucleotide of any one of (1) to (7).
  • the gene expression suppression method of (11), wherein the gene expression suppression method is a translation inhibition method.
  • a polynucleotide which can suppress gene expression efficiently with less side effects can be obtained.
  • FIG. 1 is a diagram showing the structural features of the gapmer and the mechanism of action.
  • FIG. 2 is an illustration of a reporter gene assay.
  • FIG. 3 shows the determination of the base sequence near the 3 'end of human RelA mRNA using the 3' RACE method.
  • FIG. 4 is a diagram showing processing at the 3 'end of mRNA.
  • FIG. 5 shows the nucleotide sequence of the 3 'end of human RelA mRNA and the design of the gapmer.
  • FIG. 6 shows the results of reporter gene assay in HeLa cells using each gapmer shown in FIG.
  • FIG. 7 is a diagram showing the nucleotide sequence of the 3 'end of mouse RelA mRNA and the design of gapmer.
  • FIG. 1 is a diagram showing the structural features of the gapmer and the mechanism of action.
  • FIG. 2 is an illustration of a reporter gene assay.
  • FIG. 3 shows the determination of the base sequence near the 3 'end of human RelA
  • FIG. 8 is a diagram showing reporter gene assay results in NIH 3T3 cells using each gapmer shown in FIG.
  • FIG. 9 is a diagram showing the nucleotide sequence of the 3 'end of human RelA mRNA and the design of gapmer.
  • FIG. 10 shows reporter gene assay results in HeLa cells using each gapmer shown in FIG.
  • FIG. 11 is a diagram showing the results of quantitative PCR for expression analysis of RelA in HeLa cells using each gapmer shown in FIG.
  • FIG. 12 shows a protocol for ASO evaluation using a mouse.
  • FIG. 13 is a diagram showing the value of each cholesterol in the blood of mice to which ASO was administered from the tail vein.
  • FIG. 14 shows the values of AST and ALT in the blood of mice to which ASO was administered from the tail vein.
  • FIG. 15 shows a protocol for ASO evaluation consisting of symmetrical gapmers using a mouse.
  • FIG. 16 is a diagram showing sequence information of symmetrical gapmers for mouse ApoB mRNA and the value of each cholesterol in the blood of mice to which they were administered.
  • FIG. 17 shows the values of AST and ALT in the blood of mice intraperitoneally administered gapmer.
  • FIG. 18 shows the analysis of the amount of ApoB mRNA in the liver of mice administered a symmetric gapmer to mouse ApoB mRNA.
  • FIG. 19 shows nucleotide sequence information of non-gapmer ASO.
  • FIG. 20 shows the results of a reporter gene assay in HeLa cells transfected with the ASO of FIG. FIG.
  • FIG. 21 is a diagram showing measurement of RelA mRNA amount in HeLa cells transfected with ASO of FIG. 19 by quantitative PCR.
  • FIG. 22 shows the procedure for transfecting ASO into HeLa cells and the protocol (A) and result (B) of the reporter gene assay.
  • Fig. 23 shows the localization of RelA mRNA in HeLa cells transfected with ASO-7 / + 7.
  • FIG. 24 shows the localization of RelA mRNA in HeLa cells transfected with ASO 1,2,8,13,14.
  • FIG. 25 shows the localization of RelA mRNA in HeLa cells transfected with gap P / C.
  • FIG. 26 shows localization of RelA mRNA in NTS-transfected HeLa cells.
  • FIG. 27 shows the localization of RelA mRNA in untreated HeLa cells.
  • FIG. 28 shows the relationship between the localization of RelA mRNA in ALa-7 / + 7-transfected HeLa cells and ASO-7 / + 7.
  • FIG. 29 is a diagram showing the amount of Acsl1 mRNA in the liver of mice to which any of 3'-gapAcsl1, 3'-non-gapAcsl1, SGgapAcsl1 and SGnon-gapAcsl1 was administered.
  • FIG. 29 is a diagram showing the amount of Acsl1 mRNA in the liver of mice to which any of 3'-gapAcsl1, 3'-non-gapAcsl1, SGgapAcsl1 and SGnon-gapAcsl1 was administered.
  • FIG. 30 shows changes in AST and ALT levels in serum of individual mice to which any of 3'-gapAcsl1, 3'-non-gapAcsl1, SGgapAcsl1 and SGnon-gapAcsl1 was administered.
  • FIG. 31 is a diagram showing the average transition of AST and ALT levels in the serum of mice to which any of 3'-gapAcsl1, 3'-non-gapAcsl1, SGgapAcsl1 and SGnon-gapAcsl1 was administered.
  • target mRNA is mRNA targeted for suppression of translation, which is transcribed from a gene by RNA polymerase II and is not cleaved to add poly A at the 3'-end.
  • target mRNA is mRNA targeted for suppression of translation, which is transcribed from a gene by RNA polymerase II and is not cleaved to add poly A at the 3'-end.
  • it is eukaryotic mRNA.
  • Eukaryotic organisms include, for example, humans, animals other than humans, plants, eukaryotic microbes (eg, yeast, fungi etc.), archaebacteria etc., but organisms having a poly A sequence at the 3'-end of mRNA If so, it can be the subject of the present invention.
  • cultured cells in vitro may also be the subject of the present invention. If necessary, all or part of these eukaryotes may be selected for application.
  • the "3'-end of mRNA” refers to the base of the 3'-end of mRNA which has been cleaved to add poly A after transcription. That is, after transcription, mRNA is cleaved at the 3'-untranslated region to add poly A, but it is cleaved, and the 3'-terminal base of the mRNA to which poly A is added is -It is called "end".
  • target mRNA is not particularly limited, and any mRNA to be translated can be targeted.
  • mRNA for translating a protein associated with a disease mRNA for translating a protein associated with a metabolism
  • mRNA for translating a protein involved in gene expression such as transcription or translation
  • mRNA for translating a protein involved in biosynthesis cells It includes, but is not limited to, mRNA that translates cyclically and proliferation related proteins.
  • it may be mRNA that translationally expresses a protein of unknown function.
  • anti-sense oligonucleotides which have a continuous sequence of 5 to 9 bases in both directions around the 3'-end of the target mRNA.
  • This sequence is also referred to as a sequence spanning the 3'-end. That is, according to the present invention, translation of a target mRNA can be efficiently suppressed by using a polynucleotide that hybridizes to a sequence spanning the 3'-end before the 3'-end cleavage of the mRNA.
  • hybridize means that the target mRNA and the polynucleotide of the present invention are bound by hydrogen bonding to form a double strand in a cell.
  • a polynucleotide that hybridizes to a sequence spanning the 3'-end of a target mRNA is used in the translational suppressor and the translational suppression method of the present invention.
  • 5 to 9 bases including the 3 'end of the cleavage point (including the "3'-end of mRNA" base) 5' to the 5 'side from the cleavage point of the 3' end, cleavage of the 3 'end 5 to 9 bases on the 3'-side of the point (containing the base at the 5'-end of the breakpoint but not the base at the 3'-end of the above breakpoint ("3'-end of mRNA")
  • the point is, as long as they can specifically bind to the target sequence to affect cleavage at the 3'-end of mRNA and suppress translation. More preferably, 6-8 bases on the 5'-side from the cleavage point at the 3'-end, 6-8 bases on the 3'-side of the cleavage point at the 3'-end, most preferably, cleavage at the 3'-end From the point of view, there are 7 bases on the 5'-side and 7 bases on the 3'-side of the cleavage point of the 3'-end, but not limited thereto.
  • a polynucleotide hybridizing not only to the sequence spanning the 3'-end but to a sequence including the sequence may be used.
  • a polynucleotide which does not induce degradation of mRNA by RNase H is preferable.
  • One embodiment of the translation inhibitor polynucleotide of the present invention uses a polynucleotide having a sequence that hybridizes to a sequence spanning the 3'-end of a target mRNA.
  • a polynucleotide that mechanically inhibits translation readily for any target mRNA can be designed, and a translation inhibitor can be produced.
  • ASO antisense nucleotides
  • the present invention solves this problem, and can easily design a sequence of antisense nucleotides having a translational inhibitory effect on any mRNA, and stably inhibit translation using antisense nucleotides having that sequence. That is, the invention of the present embodiment is mechanically applicable to any mRNA without particularly requiring screening experiments.
  • gapmers and non-gapmers can be used.
  • a gapmer is, as shown in FIG. 1, a nucleotide analog (eg, a sugar-modified nucleotide analog (eg, LNA)) having 1 to 3 bases at both ends (the number of bases at each end is different) ), And the remaining sequence is a polynucleotide consisting of DNA.
  • a nucleotide analog eg, a sugar-modified nucleotide analog (eg, LNA)
  • LNA sugar-modified nucleotide analog
  • a non-gapmer is, for example, a nucleotide analogue (eg, a sugar-modified nucleotide analogue (eg, LNA)), and the 1 to 3 bases at both ends (the number of bases at each end may be different) ) And a central portion of 1 to 3 bases, and the remaining sequence is a polynucleotide consisting of DNA.
  • the gapmer hybridizes with a target mRNA to form a duplex, and suppresses the expression of the target mRNA by degradation with RNase H.
  • RNase H a nucleotide analogue
  • both of the gapmer and nongapmer have the characteristic that almost no hepatotoxicity appears.
  • the effect of translational repression can be measured using the transcriptional activation mechanism of NF- ⁇ B in FIG. 2 using the plasmid described in FIG. That is, a plasmid having a region that binds NF-BB upstream of the promoter and a luciferase gene downstream of the promoter is used together with a polynucleotide that targets mRNA of RelA gene, a subunit of the NF- ⁇ B transcription factor.
  • the translation of RelA protein is suppressed in FIG. 2, so that the transcriptional activation of luciferase is suppressed and the phenomenon that the luciferase activity is lowered is utilized.
  • the polynucleotide that hybridizes to the sequence spanning the 3'-end of the target mRNA of the present invention to inhibit translation does not have to hybridize only to the sequence across all the 3'-end of the polynucleotide sequence. It may be hybridized to a sequence spanning the 3'-end. That is, translational inhibition of mRNA is considered to occur as long as it partially hybridizes to a sequence spanning the 3'-end of the target mRNA.
  • polynucleotide of the present invention may contain nucleotide analogues instead of natural nucleotides.
  • the polynucleotide of the present invention includes, for example, a polynucleotide including locked nucleic acids (LNA), a polynucleotide including a morpholino nucleic acid (eg, morpholino oligonucleotides (MO)), and analogs of nucleotides such as morpholino oligonucleotides (MO).
  • LNA locked nucleic acids
  • MO morpholino oligonucleotides
  • MO morpholino oligonucleotides
  • nucleotide may be a nucleotide, and the nucleotide (ribonucleotide or deoxyribonucleotide) may be a nucleotide analogue in which sugar, base and / or phosphate are chemically modified as long as the translation of the target mRNA can be suppressed. .
  • base-modified nucleotide analogues include 5-position modified uridine or cytidine (eg, 5-propynyl uridine, 5-propynyl cytidine, 5-methyl cytidine, 5-methyl uridine, 5- (2-amino) propyl) Uridine, 5-halocytidine, 5-halouridine, 5-methyloxyuridine etc.); 8-position-modified adenosine or guanosine (eg 8-bromognosine etc.); deazanucleotides (eg 7-deaza-adenosine etc.); O- and N And-alkylated nucleotides (eg, N6-methyl adenosine etc.) and the like.
  • 5-position modified uridine or cytidine eg, 5-propynyl uridine, 5-propynyl cytidine, 5-methyl cytidine, 5-methyl uridine, 5- (2-amino) propyl
  • LNA also referred to as 2 ′, 4′-BNA (also referred to as 2 ′, 4′-bridged nucleic acids)
  • an OH group at the 2 ′ position of ribonucleotide H, oR, R, OR'OR, halogen atom, SH, SR, NH 2, NHR, NR 2 or CN
  • R represents an alkyl group, an alkenyl group or an alkynyl group of 1-6 carbon atoms
  • R ′ is a 2′-position sugar modification substituted by — (CH 2 ) m — (m is an integer of 1 to 3) or the like, 5′-terminal phosphorylation modification with monophosphorylated 5 ′ end And morpholino nucleic acids containing morpholine rings instead of ribose or deoxyribose of nucleic acids.
  • O-methyl modified nucleic acid is also referred to as 2'-O-methylated RNA, 2'-O-methyl (Me)
  • O-methoxyethyl modified nucleic acid is 2) '-O-methoxyethylated RNA, also referred to as 2'-MOE
  • 2'-MOE O-methoxyethylated RNA
  • phosphate-modified nucleotide analogues include ones in which the phosphoester group linking adjacent ribonucleotides is substituted with a phosphothioate group.
  • nucleotide analogues can be introduced into the polynucleotide by known methods 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 response.
  • 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- ⁇ inflammatory cytokines
  • proinflammatory cytokine gene is transcriptionally activated by RelA and P50 by releasing I kappa B from the NF- kappa B complex, whereby mRNA is transcribed and translated into a cytokine protein.
  • the translation suppressor of the present invention may target the mRNA of these cytokines as a target to suppress translation
  • the cytokine expression of cytokines may also be achieved by suppressing the expression of all or part of transcription factors that promote cytokine gene expression. It can suppress the expression and suppress the inflammatory response.
  • transcription factors for such cytokine genes include NF- ⁇ B, AP-1, and STAT3. Therefore, a translational suppressor of the NF- ⁇ B gene can be used as an anti-inflammatory agent.
  • the translational inhibitors of the present invention may also be applied to diseases and conditions mediated by the inflammatory cytokine cascade.
  • Diseases and conditions 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 hemorrhage, 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 hemorrhage, stuttering, ionizing radiation exposure, acute pancreatitis, adult respiratory distress syndrome (ARDS).
  • ARDS adult respiratory distress syndrome
  • Reperfusion injury includes the following: post pump syndrome, ischemia reperfusion injury.
  • Cardiovascular disease includes the following: Cardiotonic twitch syndrome, myocardial infarction, congestive heart failure.
  • Infectious diseases include: HIV infection / HIV neuropathy, meningitis, hepatitis, septic arthritis, peritonitis, pneumonia epiglottitis, E. coli O 157: H7, hemolytic uremic syndrome / thrombolytic thrombocytopenic purpura Disease, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, leprosy, toxic shock syndrome, streptococcal myositis, gas gangrene, Mycobacterium tuberculosis (human tuberculosis), Mycobacterium aviun Intracellulare (avian tuberculosis bacterial intracellular substance infection), Pneumocystis Carinii (pneumocystis carinii) pneumonia, pelvic inflammatory disease, testicular inflammation / epididymitis, Legionella, Lyme disease, influenza A, Epstein-Barr virus, virus related hemophagocytic syndrome, viral encephalitis / As
  • Obstetrics / gynecology symptoms include: preterm birth, miscarriage, infertility.
  • Inflammatory diseases / autoimmune diseases include: rheumatoid arthritis / seronegative arthropathy, osteoarthritis, inflammatory bowel disease, systemic lupus erythematosus, iris ciliary body inflammation / uveitis optic neuritis, sporadic Pulmonary Fibrosis, Systemic Vasculitis / Wagener's Granulomatosis, Sarcoidosis Orchialitis / Vervectomy Reversal.
  • Allergic / atopic diseases include: asthma, allergic rhinitis, eczema, allergic contact dermatitis, allergic conjunctivitis, hypersensitivity pneumonia.
  • Malignant diseases include: ALL, AML, CML, CLL, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, colorectal cancer, nasopharyngeal cancer, malignant histiocytosis, paraneoplastic syndrome / high malignancy Calcemia.
  • Transplants include: organ graft rejection, graft versus host disease, cachexia.
  • cystic fibrosis familial blood phagocytic lymphocytosis, sickle cell anemia.
  • Dermatologically includes: psoriasis, alopecia.
  • Neurological disorders include the following: multiple sclerosis, migraine.
  • Renal disease includes 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, hemochromatitis, alpha-1 antitrypsin deficiency, diabetes, Hashimoto's thyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axis assessment, primary biliary cirrhosis .
  • the target mRNA whose translation is suppressed by the translational suppressor of the present invention may be mRNA that expresses a protein causing 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.
  • Subjects of the anticancer agent and / or cancer treatment method of the present invention may include these neoplastic disorders.
  • sarcoma generally refers to a tumor consisting of a substance such as embryonic connective tissue and generally consisting of densely packed cells embedded in a fibrous substance or homogeneous substance.
  • sarcomas that can be treated by the anti-cancer agent and / or cancer treatment method of the present invention include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanoma, sarcoma, osteosarcoma, abrenege ( Abmethy's) sarcoma, lip sarcoma, liposarcoma, alveolar soft tissue sarcoma, ameloblastoma sarcoma, grape sarcoma, green sarcoma, choriocarcinoma, fetal sarcoma, Wilms tumor sarcoma, endometrial sarcoma, interstitial sarcoma, Ewing sarcoma,
  • melanoma is taken to mean a tumor arising from melanocyte cell lines of the skin and other organs.
  • Melanomas that can be treated by the anticancer agent and / or cancer treatment method of the present invention include, but are not limited to, for example, terminal melon melanoma, melanin-deficient melanoma, benign juvenile melanoma, cloud man Melanoma, S91 melanoma, Harding-Pase melanoma, juvenile melanoma, malignant melanoma, melanoma, nodular melanoma, subungual melanoma, and superficial enlargement melanoma.
  • carcinoma refers to a malignant neoplasm consisting of epithelial cells that tend to invade surrounding tissues and cause metastases.
  • carcinoma refers to a malignant neoplasm consisting of epithelial cells that tend to invade surrounding tissues and cause metastases.
  • carcinomas that can be treated by the anticancer agent and / or cancer treatment method of the present invention include, but are not limited to, for example, acinar carcinoma, acinar cell carcinoma, adenocarcinoma sac, adenoid cyst carcinoma, adenomatous carcinoma
  • Adrenal cortex carcinoma alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basal cell carcinoma, basal cell carcinoma, basal squamous cell carcinoma, bronchoalveolar carcinoma, bronchiolocarcinoma Bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, choriocarcinoma, colloid carcinoma, comedone carcinoma, endometrial carcinoma, phetiform carcinoma, scaly carcinoma, skin carcinoma, columnar carcinoma, columnar cell carcinoma, glandular duct Carcinoma, Carcinoma, Fetal Carcinoma, Brain-like Carcinoma, Epidermoid Carcinoma, Adenoid Epithelial Carcinoma, Outward Growth 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, ret genes, etc. By suppressing the expression of these genes, it is possible to exert an anticancer effect.
  • diseases to which the medicament of the present invention can be applied include, for example, cancer, AIDS, Alzheimer's disease, amyotrophic lateral sclerosis, atherosclerosis, autoimmune disease, cerebellar degeneration, cancer, diabetes mellitus, Glomerulonephritis, heart failure, macular degeneration, multiple sclerosis, myelodysplastic syndrome, Parkinson's disease, prostate hyperplasia, psoriasis, asthma, retinal degeneration, retinitis pigmentosa, rheumatoid arthritis, atherosclerotic plaque rupture, systemic lupus erythematosus, ulcer
  • colitis colitis viral infection, ischemia reperfusion injury, spinal cord injury, nerve injury, cardiac hypertrophy, and diamond blackfan anemia.
  • any disease that can be treated by suppressing the expression of a specific gene can be used without particular limitation.
  • the cause gene may be known or unknown, and if the cause gene is found in the future, the translation inhibitor of the present invention is applied to the gene to treat the disease. There is a possibility that
  • factor IX blood coagulation factor IX
  • hemophilia aminolevulinic acid synthase 1 (aminolevulinic acid synthetase 1): acute intermittent porphyria
  • transthyretin transthyretin, TTR
  • familial amyloid cardiomyopathy TGF- ⁇ : Glaucoma
  • VLA-4 multiple sclerosis
  • insulin-like growth factor-1 insulin like growth factor 1
  • Grb2 acute myeloid leukemia
  • Androgen receptor (androgen receptor) prostate cancer
  • survivin Solid cancer
  • Hif-1 ⁇ solid cancer
  • insulin receptor substrate-1 insulin receptor substrate-1 (insulin receptor substrate-1): eye angiogenesis
  • psoriasis ICAM-1: pouchitis
  • huntintin Huntingtin's disease
  • SOD-1 amyotrophic side Cord sclerosis
  • cells into which a translation inhibitor has been introduced can be obtained.
  • the polynucleotide according to the present invention can be introduced into cells using a conventional gene transfer method, such as calcium phosphate method, liposome method, electroporation (electroporation), gene gun (gene gun), whisker method, microinjection Methods, laser injection method, protoplast method (plant, yeast), Agrobacterium method (plant), lithium chloride method (yeast), etc. may be used, but not limited thereto.
  • Introduction to an individual can also be administered to the affected area by injection into the tail or abdominal cavity or the like, gene cancer, external preparations and the like.
  • the translational suppressor of the present invention can be used in vitro or in vivo. That is, it is possible to inhibit translation in cells in a culture vessel such as in a test tube to examine the function of the gene or to regulate metabolism to use for production of a substance.
  • kits comprising a translation inhibitor.
  • the kit include kits containing the above-described gene transfer reagent, selection reagent (antibiotics etc.) and the polynucleotide of the present invention, and a kit obtained by adding a culture medium thereto.
  • a method of hybridizing a target mRNA with a polynucleotide capable of hybridizing to a sequence spanning the 3'-end and suppressing the translation of the target mRNA According to the method for suppressing translation of target mRNA of the present invention, it is not necessary to create multiple antisenses for each target mRNA, measure the suppression efficiency, and select the most suitable one, so that gene expression can be performed mechanically. It can be suppressed.
  • the polynucleotides of the invention can also be administered sequentially. Using DDS technology, it can be released gradually into cells and tissues to continuously suppress gene expression, and can also be used for the prevention and treatment of chronic diseases.
  • the in vitro translation system is a system for translating a protein in a cell-free extract or in a solution containing a translation enzyme, a substrate necessary for translation, and the like.
  • an in vitro translation system for example, a rabbit reticulocyte system (Rabbit Reticulocyte Lysate System), a system of wheat germ extract (Wheat Germ Extract), insect culture cell extract, human culture cell extract (see Example 8) And those using cell extracts such as E. coli extracts, and those using a mixture of those produced separately and purified from proteins involved in translation etc. Either system can be used in the present invention. .
  • a rabbit reticulocyte system Rabinit Reticulocyte Lysate System
  • Wheat Germ Extract wheat germ extract
  • insect culture cell extract human culture cell extract
  • human culture cell extract see Example 8
  • cell extracts such as E. coli extracts, and those using a mixture of those produced separately and purified from proteins involved in translation etc. Either system can be used in the present invention.
  • candidate substances having a translation inhibitory activity are introduced into the in vitro translation system together with the target mRNA or, if necessary, shifted in time of introduction.
  • the synthetic activity of the protein from mRNA may be measured.
  • the amount of protein synthesis can be measured by the enzyme activity.
  • Enzymatic activities include, for example, luminescence by luciferase, coloration by substrate cleavage by ⁇ -galactosidase, luminescence, measurement by radioisotope of phosphorylation by phosphorylation enzyme, measurement of substrate transfer activity by transferase, etc. It is not limited.
  • a protein When a protein does not have an enzyme activity, it can be quantified, for example, by electrophoresis such as SDS-PAGE (including Western blot), HPLC, MASS, quantification with antibody (ELISA, dot blot, etc.), etc. It is not restricted to these. Furthermore, the translational inhibitory activity may be measured by combining a plurality of these quantification methods.
  • oliconucleotides of the present invention may be used in combination. In that case, it is preferable to use a method of screening for a translation inhibitor by an in vitro translation system.
  • the translation inhibitor containing the siRNA of the present invention may be an antiinflammatory agent and / or an anticancer agent, and may be in the form of a kit containing the antiinflammatory agent and / or the anticancer agent.
  • cells containing the translation inhibitor are also included in the scope of the present invention.
  • FIG. 2 In order to analyze the gene expression suppression effect by ASO, a reporter gene assay was adopted (FIG. 2). As shown in FIG. 2, expression of firefly luciferase from plasmid pGL4.32 is dependent on a promoter with a transcription factor NF- ⁇ B response element. Since the firefly luciferase activity depends on the NF- ⁇ B activity, the firefly luciferase activity decreases when the expression of the subunit RelA protein of NF--B is suppressed. The expression of RelA gene can be evaluated at the protein level or the mRNA level by Western blotting or quantitative PCR.
  • Transfection efficiency is corrected by transfecting cultured cells together with plasmid pGL4.32 with plasmid pGL4.75 (which expresses Renilla luciferase) without the NF- ⁇ B response element, and intracellular NF- ⁇ B The activity can be assessed quantitatively.
  • siScrable (final concentration 40 nM) is a siRNA negative control
  • siRelA ⁇ 0 is a siRNA against the target sequence containing the polyA signal sequence of RelA mRNA (final concentration 40 nM), 19-mer gapmer to the same region (gapRelA ⁇ 0/19, final concentration 10 nM) or 10 mer gapmer (gapRelA ⁇ 0/10, final concentration 10 nM)
  • Untreated represents untreated cells, respectively.
  • FIG. 2 Two types of plasmids (pGL4.32 and pGL4.75, manufactured by Promega) shown in FIG. 2 were transfected into HeLa cells.
  • One of the targets of ASO in the present invention was human RelA mRNA.
  • ASO When the translation reaction of RelA protein, one of the subunits of transcription factor NF- ⁇ ⁇ B, is suppressed by ASO, NF- ⁇ B activity decreases and the expression of firefly luciferase gene from the reporter gene decreases (Fig. 2) .
  • the gene expression suppression effect by ASO can be analyzed by Western blotting (analysis of RelA protein) or quantitative PCR (analysis of RelA mRNA) (FIG. 2).
  • Fig. 2 shows the gapmer for RelA mRNA (referred to as the 19-mer gapR ⁇ 0.19 (or gap P / C) targeted to the sequence containing the polyA signal of RelA mRNA as a positive control) and the siRNA (siRelA for the same region).
  • gapRelA ⁇ 0/19 and siRelA ⁇ 0 show the expression suppression effect of RelA gene in any of the assays.
  • gapRelA ⁇ 0/10 is a negative control showing no antisense effect because it targets the same region as gapRel A ⁇ 0/19 but has a short length of 10 mer.
  • nucleotide sequence of the 3 'end of human RelA mRNA was used the nucleotide sequence information (NM_001145138.1) of human RelA mRNA registered in NCBI and the 3' end of RelA mRNA in HeLa cells in use. An experiment was conducted to compare the nucleotide sequences of Total RNA was extracted from HeLa cells, and the nucleotide sequence was determined by the 3 'RACE (Rapid Amplification of cDNA Ends) method.
  • FIG. 3A shows the sequence of each clone (a short sequence of 7 bases, a short sequence of 5 bases, and a short sequence of 4 bases) together with the nucleotide sequence of the 3 'end registered by NCBI.
  • FIG. 3B shows the ratio (proportion) of clones shorter by 7 bases, 5 bases or 4 bases than the 3 'end registered by NCBI.
  • the 3 'processing factor (CstF) binds, and cleavage (cleavage) of the 3' terminal sequence of mRNA occurs at the arrow portion (FIG. 4 (B)). From here, extension of the poly (A) chain starts with PAP (C). The inventors determined this arrow part by the 3 'RACE method (FIG. 5). From the cleavage site, poly (A) chain elongation enzyme (PAP) causes poly (A) chain elongation (FIG. 4 (C)). Incidentally, these series of 3 'processing reactions occur in the nucleus.
  • a gapmer that acts on the target mRNA at a stage before the poly (A) chain is added We designed an ASO that spans the mRNA cleavage site (FIG. 5).
  • the sequence downstream of the cleavage site can not be known from the sequence information of mRNA of the database such as NCBI because poly (A) chain is attached. Therefore, based on the sequence information derived from the genome, base sequence information downstream of the cleavage site (downstream from the arrow in FIG. 5) was obtained, and a gapmer consisting of four types of 13-mers was designed.
  • FIG. 5 shows the sequences of these four gapmers, as well as the sequences used for the design. The sequences of these four gapmers are also shown in Table 1 below.
  • the cleavage site was designated as the origin according to the positional information of each target sequence.
  • the upper case of the base sequence of ASO represents LNA, and the lower case represents DNA.
  • FIG. 6 shows the result of transfecting HeLa cells to a final concentration of 1, 3 or 10 nM with the gapmer of FIG. 5, and the vertical axis (Relative luciferase activity) represents the relative activity of luciferase after correction.
  • GapP / C gapRelA ⁇ 0/19, final concentration 10 nM
  • siRNA against RelA mRNA si P / C, final concentration 40 nM
  • NTS final concentration 10 nM
  • NIH 3T3 cells were transfected with a reporter plasmid and four 13-mer gapmers shown in FIG. 7, respectively, in order to confirm whether the same occurred in cultured cells derived from mice.
  • Arrows in FIG. 7 indicate cleavage portions of mRNA.
  • the upper row (Mouse RelA_NM_009045.4) is sequence information registered in NCBI, and originally the part after the arrow was a poly (A) chain.
  • the 3 'end sequence of mouse RelA mRNA was determined from the total RNA derived from liver by the 3' RACE method, it was identical to the information of NCBI.
  • the sequences after the arrows were extracted and described based on the genomic information.
  • Four types of gapmer type ASO were designed as shown in FIG. FIG.
  • the sequences of these four gapmers are also shown in Table 1 below.
  • the cleavage site was designated as the origin according to the positional information of each target sequence.
  • the upper case of the base sequence of ASO represents LNA, and the lower case represents DNA.
  • the gapmer in FIG. 7 was transfected into NIH3T3 cells to a final concentration of 1, 3 or 10 nM.
  • the reporter gene assay results in thus transfected NIH 3T3 cells are shown in FIG.
  • the vertical axis (Relative luciferase Activity) in FIG. 8 represents the relative activity of luciferase after correction.
  • gapmer (NTS, final concentration 10 nM) for nonspecific sequences was transfected.
  • expression suppression activity was able to be observed in all ASOs although there was a difference in strength (FIG. 8).
  • ASO may be hybridized to a target mRNA in a stage before poly (A) chain addition occurs in the cell nucleus to exert an antisense effect.
  • results of NIH 3T3 cells showed that -8 / + 5, ie, having the cleavage site of mRNA near the center of ASO, induces the strongest expression suppression activity (FIGS. 7 and 8). Therefore, in the subsequent experiments, we designed the cleavage site of the target mRNA to be located in the central part of ASO.
  • the symmetrical gapmer exerts a strong gene expression suppression effect With the cleavage site (arrow) at the center of human RelA mRNA, upstream and downstream, that is, nine bases each on both sides with the cleavage site at the center (-9 / +9), 8-base one (-8 / + 8), 7-base one (-7 / + 7), 6-base one (-6 / + 6), 5-base one (-5 / + 5) ) Prepared ASO ( Figure 9). Essentially, the part after the arrow is a poly (A) chain. Therefore, the sequence after the arrow was extracted and described based on the genome information. FIG. 9 shows the prepared gapmer sequence together with the sequence used for the design. The sequences of these gapmers are also shown in Table 1 below.
  • the upper case of the base sequence of ASO represents LNA, and the lower case represents DNA. -8 / + 5 has already been described in FIG.
  • the two bases at the 5 'and 3' ends of all of them are LNAs, and all other consist of DNA.
  • symmetric gapmer it is referred to as “symmetric gapmer” because of its structural feature that the cleavage site is located at the center to form a symmetric type (FIG. 9).
  • HeLa cells were transfected to a final concentration of 10 nM for the gapmer shown in FIG.
  • the reporter gene assay results in HeLa cells transfected with each gapmer shown in FIG. 9 are shown in FIG.
  • the vertical axis (Relative luciferase activity) in FIG. 10 represents the relative activity of luciferase after correction.
  • Gapmer gapP / C; gapRelA ⁇ 0/19, final concentration 10nM
  • siRNA against RelA mRNA siP / C, final concentration 40nM
  • NTS final concentration 10nM for nonspecific sequences
  • FIG. 10 HeLa cells were transfected with each gapmer of FIG. 9 or the gap control of positive control (gapP / C) to a final concentration of 10 nM or siRNA (siP / C) to a final concentration of 40 nM, and these transfections were performed. Total RNA was prepared from each of these cells.
  • Reverse transcription reaction is performed using oligo (dT) (hatched bar on the left side of each treatment group) or random primer (open bar on the right side of each treatment group), and the resulting cDNA is used as a material for quantitative PCR
  • the amount of RelA mRNA was analyzed (FIG. 11).
  • the vertical axis (Relative RelA mRNA level) in FIG. 11 indicates the relative amount of RelA mRNA after correction with the internal standard.
  • the numerical values in FIG. 11 represent the relative amounts of RelA mRNA after correction.
  • gap P / C and siP / C reduced the amount of mRNA to about 10% as compared with the untreated one (FIG. 11).
  • -7 / + 7 was reverse transcribed with oligo (dT)
  • RelA mRNA level seemed to be reduced at the same level as gap control P / C as positive control, but when reverse transcription was performed with random primer, The amount of RelA mRNA was reduced to only about half that of untreated (FIG. 11). This difference is that gapmer -7 / + 7 inhibits the reaction in which poly (A) is added to RelA mRNA.
  • the effect on the amount of -6 / + 6 RelA mRNA which has the ability to reduce luciferase activity by about 80% in reporter gene assay compared to control, is about 70% of the untreated one when reverse transcribed with random primer The degree was (FIG. 11). That is, it can be said that -6 / + 6 hardly reduces the amount of mRNA. From the above results, while the conventional gapmer degrades the target mRNA and exerts the expression suppression activity (US005962425A, US005955589A), the symmetric gapmer developed by the inventors hardly degrades the target mRNA It has been suggested that it may have shown the expression suppression activity.
  • apoB apolipoprotein B
  • Cholesterol is incorporated into protein components such as apoB, and lipid components are added to synthesize lipoproteins (Davidson NO, and Shelness, GS, Annu. Rev. Nutr. 20, 169-193, 2000.).
  • the lipid components are transformed into lipoproteins in the liver and then delivered systemically.
  • the world's first systemically available nucleic acid drug Kainamuro approved in the US has been reported to target apoB and specifically lower LDL-C levels (Wong, E., and Goldberg, T. Drug Forecast 39, 119-122, 2014.). Therefore, if ApoB is used as a candidate gene in an animal experiment using a mouse, an evaluation system has already been established, and the gene expression suppression effect can be easily confirmed. Since this method is generally adopted widely, it was decided to use it in this example.
  • ASO gapmer-type ASO (MM13 mer (sequences shown in Table 1 and FIG. 12 below)) against ApoB mRNA, which has been shown to have a strong inhibitory effect in previous studies (Nishina, K., et al., Nature Commun.) , 6: 79692015) to examine the conditions (FIG. 12).
  • ASO was administered via tail vein on Day 0. The dosage was 0.004 mg to 0.5 mg per animal in four steps, and the same amount was administered to each individual when administered a second time. One 0.5 mg dose is given for one, 0.1 to 0.004 mg doses for two animals, and the figures are mean values. Two PBSs were used and the average value was shown.
  • the mechanism of hepatotoxicity includes the type of nucleic acid (Krieg, M., et al., Nature Reviews Drug Discov., 6: 471-484, 2006) and its sequence (Burdick, AD, et al., Nucleic acid). Acid Res., 8: 488 2-4 891, 2014) etc. have been reported to be related. In the subsequent experiments, MM 13 mer was adopted as a positive control, and 0.02 mg per animal with little increase in AST value and ALT value was selected as a dose (FIG. 14).
  • the dotted line in the figure shows the cleavage site, and this is at the center to design a symmetrical gapmer.
  • the nucleotide sequence information of the 3 'end of mouse ApoB mRNA is determined by the present inventors after 3'RACE was performed using total RNA recovered from the liver as a material.
  • FIG. 16A shows the prepared gapmer sequences, along with the sequences used for the design. The sequences of these gapmers are also shown in Table 1 below.
  • the dotted line indicates the cleavage site. Essentially, the portion downstream of the dotted line is a poly (A) chain. Therefore, the sequences after the dotted line were extracted and described based on the genomic information.
  • the top row shows the sequence information of ApoB mRNA, the bars schematically show the mRNA, and the continuation of AAAA represents the poly (A) chain.
  • Light gray circles, upper case letters represent LNA, and black circles, lower case letters represent DNA.
  • Figures 16B-D were given intraperitoneally with ASO (dose 0.5 mg per animal). As a positive control, 0.02 mg of MM 13-mer was administered from the abdominal cavity. Blood was collected according to the protocol of FIG. 15, and LDL-cholesterol levels (LDL-C: FIG. 16B), HDL-cholesterol levels (HDL-C: FIG. 16C), and total cholesterol levels (T-CHO: FIG. 16D) in blood were determined. It was measured. MM 13 mer and PBS were administered in 2 mice, and -9 / + 9, -8 / + 8 and -7 / + 7 were administered in 4 animals. The numbers indicate the mean value.
  • LDL-cholesterol levels LDL-cholesterol levels
  • HDL-C HDL-C
  • T-CHO total cholesterol levels
  • the AST value in the symmetric gapmer-7 / + 7 developed by the inventors was 100 or less, and the ALT value was 45 or less, which was extremely weak as compared to the MM 13-mer (FIG. 17). From the above results, the symmetric gapmer designed to cross over the poly (A) chain addition site at the 3 'end of ApoB mRNA suppresses ApoB expression in mouse liver, and as a result, LDL-C level in blood It is thought that a decrease in
  • ASO composed of three symmetrical gapmers induced a decrease in the amount of ApoB mRNA in all individuals as compared to those treated with PBS (FIG. 18).
  • ASO-7 / + 7 a significant reduction in the amount of ApoB mRNA was observed in all four mice administered (FIG. 18). From the above results, it can be said that the symmetric gapmer acts on ApoB mRNA in the liver and suppresses the expression of ApoB, thereby causing a decrease in the LDL-C level in blood.
  • Non-gapmer-type ASO which breaks down the structure of the gapmer, has a gene expression suppression effect
  • gapmer-type ASO effectively suppresses the expression of target genes, it has been widely reported that it shows strong liver damage when administered to mice (Burel, SA, et. Al. Nucleic Acid Res., 44: 2093-2109, 2016., Kasuya, T., et. Al. Sci. Rep., 27; 6: 30377, 2016.).
  • the positive control conventional gapmer MM 13-mer
  • hepatotoxicity is reported to be derived from RNase H1 activity (Burel, SA, et. Al. Nucleic Acid Res., 44: 2093-2109, 2016., Kasuya, T., et. Al. Sci. Rep., 27; 6: 30377, 2016.). That is, the gapmer causes RNase H1-dependent mRNA degradation, which is considered to be the main cause of hepatotoxicity.
  • a symmetric gapmer spanning the poly (A) chain addition site of the target mRNA and a non-gapmer type having LNA near the central part of the ASO sequence were prepared.
  • the seven ASOs used in the present invention are shown in FIG. 19 (the sequences of these ASOs are also shown in Table 1). Arrows indicate the cleavage portion of human RelA mRNA. Essentially, the part after the arrow is a poly (A) chain. Therefore, the sequence after the arrow was extracted and described based on the genome information.
  • the cleavage site (arrow) was at the center, and 7 bases (-7 / + 7) were prepared upstream and downstream.
  • LNA is shown in light gray and DNA is shown in black. Each was named based on the relative position (represented by a number) where LNA was inserted. The activity of those ASOs was examined by reporter gene assay.
  • FIG. 20 The results of the reporter gene assay in HeLa cells transfected in this way are shown in FIG.
  • the vertical axis (Relative luciferase Activity) in FIG. 20 represents luciferase activity after correction.
  • Gapmer gapP / C; gapRelA ⁇ 0/19, final concentration 10nM
  • siRNA against RelA mRNA siP / C, final concentration 40nM
  • NTS final concentration 10nM for nonspecific sequences
  • Reverse transcription reaction is performed using oligo (dT) (hatched bar on the left side of each treatment group) or random primer (open bar on the right side of each treatment group), and the resulting cDNA is used as a material for quantitative PCR
  • the amount of RelA mRNA was analyzed (FIG. 21).
  • the vertical axis in FIG. 21 shows the amount of RelA mRNA after correction with the internal standard.
  • the numerical values in FIG. 21 indicate the relative amounts of RelA mRNA after correction.
  • the positive control gap P / C reduced the amount of RelA mRNA to 10% level as compared to the control NTS (FIG. 21).
  • 1.1.8.14 and 1.2.8.13.14 of the non-gapmer structure only halved RelA mRNA amount compared with NTS (FIG. 21).
  • FIG. 22B gapmer NTS against the nonspecific sequence of the negative control showed no suppressive effect
  • Figures 23 to 27 show the results of in situ hybridization.
  • the intracellular localization of RelA mRNA and cyclohilin B mRNA was analyzed using RNAscope.
  • Figure 23 non-gapmer 1, 2, 8, 13, 14 ( Figure 24) and NTS ( Figure 26) and untreated cells ( Figure 27)
  • Figure 27 numerous black spots in the nucleus and cytoplasm It could be detected (occurring in the presence of RelA mRNA).
  • the positive control gap P / C transfected cells Fig. 25
  • almost no spots could be detected in the whole cells.
  • HeLa cells or NIH3T3 cells were suspended in DMEM medium containing 10% non-mobilized FBS to a concentration of about 0.5 ⁇ 10 5 cells / mL, and each 0.5 mL was spread on a 24-well plate. After 24 hours, transfection was performed when the cells were at about 40-50% confluence.
  • the medium in which HeLa cells were cultured was aspirated at 22 hours after transfection for activation of NF- ⁇ B, and DMEM medium containing 500 ⁇ L of 10% non-mobilized FBS, 20 ng / mL TNF- ⁇ was used. Replaced it. After replacing the medium, it was returned to the incubator (37 ° C., 5% CO 2 ) and allowed to stand for 2 hours.
  • Luciferase activity was measured using Dual-Luciferase Reporter Assay System (Promega) as follows. The transfected HeLa cells were washed twice with 500 ⁇ L of ice-cold Phosphate-Buffered Saline (PBS) (pH 7.4), 150 ⁇ L of passive lysis buffer (Promega) was added, and 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 obtained supernatant was used for Luciferase assay. Luminoskan luminometer (Thermo Scientific) was used for the measurement of luminescence intensity.
  • Quantitative PCR The expression level of the target gene was measured by StepOnePlus real-time PCR (Life Technologies) using GoTaq qPCR Master Mix (Promega) or KAPA SYBR Fast qPCR Kit (Nippon Genetics). When GoTaq qPCR Master Mix was used, the cycle was started with the following profile by adding 10 ⁇ L of GoTaq qPCR Master Mi, 5 pmol of gene specific Forward primer and Reverse primer to cDNA.
  • the primer sequences used for amplification of the target gene are as follows.
  • Human RelA 5'-GCAGTTTGATGATGGAAGACC-3 '(forward, SEQ ID NO: 18), 5'- CTGTC ACTAGGCGAGTTA-3' (reverse, SEQ ID NO: 19)
  • Human ⁇ -actin (endogenous control): 5'-GATAGCATTGCTTTTTCGTGTA-3 '(forward, SEQ ID NO: 20), 5'- TTCAACTGGTCTCAAGTCAG-3' (reverse, SEQ ID NO: 21)
  • Mouse ApoB 5'-GCTCAACTCAGGTTACCGTGA-3 '(forward, SEQ ID NO: 22), 5'- AGGGTGTACTGGCAAGTTTGG-3' (reverse, SEQ ID NO: 23)
  • Mouse Spt5 (endogenous control): 5'-GGTCCTACTGAGCATTGATGGTGAG-3 '(forward, SEQ ID NO: 24), 5'- TCAGGCTTCCAGG
  • NF- ⁇ B p65 Antibody C-20: sc-372 (Santa Cruz Biotech), and Monoclonal Anti- ⁇ -Tubulin Antibody Produced in Mouse (Sigma) were used.
  • ECL Anti-Rabbit IgG HRP-Linked F (ab) 2 Fragment (GE healthcare), and polyclonal rabbit anti mouse immunoglobulins / hrp (DAKO) were used.
  • Luminata Classico Western HRP (Millipore) was used for detection and visualized with LPR-140EX (AISIN).
  • 3 'RACE 3 'RACE was performed as follows using GeneRacer Kit (Invitrogen). 90 ng GeneRacer Oligogo T, 1 ⁇ L dNTP Mixture (2.5 mM each) and water were added to 300 ng of total RNA prepared from each sample to make the total volume 7 ⁇ L. The mixture was allowed to stand at 65 ° C. for 5 minutes and then allowed to stand on ice for 5 minutes.
  • Nested PCR was performed using the 1st PCR product. Add 100 ⁇ l of 1 ⁇ PCR product to 5 ⁇ l of 10 ⁇ ExTaq buffer, 4 ⁇ l of dNTP Mix (2.5 mM each), 0.25 ⁇ l of ExTaq, 20 pmol of gene-specific Forward primer and Reverse primer, and make the total amount 50 ⁇ l. I cycled in my profile.
  • the PCR product was electrophoresed on a 1.2% agarose gel and cut out and purified.
  • FastGene Gel / PCR Extraction (Nippon Genetics) was used and purified according to the protocol.
  • the cut-out and purified product was subjected to A-tailing reaction using ExTaq.
  • ExTaq To 25 ⁇ L of the excised and purified PCR product, 5 ⁇ L of 10 ⁇ ExTaq buffer, 1 ⁇ L of 10 mM dATP (NEB), 0.25 ⁇ L of ExTaq, and water were added to make the total volume 50 ⁇ L, and left at 70 ° C. for 30 minutes.
  • the product subjected to the A-tailing reaction was purified according to the protocol using FastGene Gel / PCR Extraction (Nippon Genetics).
  • the product subjected to the A-tailing reaction was ligated to pGEM T-easy vector.
  • T4 ligation buffer To 1 ⁇ L of the product subjected to the A-tailing reaction, 5 ⁇ L of T4 ligation buffer, 1 ng of pGEM T-easy (Promega), 1 ⁇ L of T4 ligase (Promega), and water were added to make the total volume 10 ⁇ L, and left at room temperature for 60 minutes. After standing, the whole reaction solution was added to DH5a (Toyobo), and left standing in ice for 30 minutes. After standing, heat shock was applied for 45 seconds in a 42 ° C. water bath, and the plate was allowed to stand in ice for 2 minutes or more.
  • DH5a Toyobo
  • plasmids were prepared from the obtained colonies using a Qiaprep miniprep kit (Qiagen) according to the protocol. The prepared plasmid was subjected to sequencing analysis service from Eurofin Co., Ltd., and the nucleotide sequence of the cloning site was determined.
  • In situ Hybridization In situ hybridization was performed as follows using RNAscopeHD reagent-BROWN (Advanced cell diagnostics).
  • the samples were washed with wash buffer and treated sequentially with AMP1 reagent to AMP6 reagent. After AMP6 reagent treatment, it was washed with wash buffer, DAB reagent was dropped to the sample, and left to stand at room temperature for 10 minutes. The samples were washed with distilled water and then counterstained with Mayer's hematoxylin (Merck). After staining, it was dehydrated with ethanol and cleared with remozole (nacalai tesque). The sample was sealed with MountQuick (Daichi Sangyo Co., Ltd.) and observed with a microscope.
  • mice Female 5 to 6 week old Jc1: ICR mice were purchased from CLEA Japan, Inc. (hereinafter CLEA) and bred under conventional conditions. A conditioning period of one week or more was provided until the start of the experiment. The mice were housed at 23 ⁇ 2 ° C., 60% ⁇ 5% humidity, with a 12 hour light / dark cycle. Standard feed (CE-2, CLEA) and water were at free-fed conditions. All experiments were conducted in accordance with the guidelines for appropriate implementation of animal experiments formulated by the Japan Science Council in 2006. The protocol of this experiment has been approved by the Experimental Animal Committee of Osaka University, National University Corporation (approved No. 25-1-1).
  • ASO ASO was administered to mice in the amount and schedule shown in the figure and in the specification from the tail vein or the abdominal cavity.
  • the second administration was performed on Day 7 with the first administration day of nucleic acid as Day 0, and blood was collected on Day. It was carried out on 10 days and various cholesterol levels were measured. For Day 10, we also measured AST and ALT values.
  • the nucleic acid was injected from the tail vein to each animal at 0.5, 0.1, 0.02, 0.004 mg (two each). The second dose was not administered only for the 0.5 mg volume (1 individual).
  • RNA Extraction Cultured cells or excised mouse liver were treated with Sepazol RNA I super G (Nacalai Tesque) according to the instructions.
  • Sepazol RNA I super G Nacalai Tesque
  • extracting RNA from mouse liver tissue was performed 4,500 rpm, once shredded at 10 seconds using a bead-type cell crusher Micro Smash TM (TOMY). Three 3.0 sigma zirconia beads were added to the tube. After crushing, the supernatant was collected by centrifugation at top speed (17, 900 g) at 4 ° C. for 5 minutes to remove tissue fragments.
  • Purification of RNA was performed by Direct-zol RNA MiniPrep (Zymo Research) according to the instruction.
  • FIG. 28A shows the relationship between ReA mRNA localization in ASO -7 / + 7-transfected HeLa cells and ASO -7 / + 7, and FIG. 28B shows in gap P / C-transfected HeLa cells. It shows the localization of RelA mRNA and the intracellular behavior of gap P / C.
  • ASO that is, a double strand formed by hybridizing a single-stranded antisense nucleic acid to a target mRNA
  • ASO is very stable. Without heating, that is, a temperature exceeding the Tm of ASO, or treatment with a substance exhibiting strong alkalinity, the double strand does not break back to the single strand state.
  • helicase helicase having an activity to specifically dissociate the double strand formed between gapmer -7 / + 7 and RelA mRNA
  • RelA mRNA is Since gapmer -7 / + 7 can keep translation suppressed while remaining in the cytoplasm (Fig. 22), it is not considered that helicase has eliminated the double stranded state.
  • gap P / C once forms a double-stranded state with the target RelA mRNA, but RNaseH1 activity causes the degradation of RelA mRNA, so this double-stranded state disappears and eventually, ASO gap P / C C returns to single stranded state in the cell. Since gap P / C in a single strand state is not present in the cell as the original target RelA mRNA, it hybridizes to an off target mRNA having an antisense sequence similar to that of RelA mRNA, resulting in a double strand. Is formed and degradation is caused by RNase H1 (FIG. 28B). And again, ASO gap P / C returns to single stranded state. This chain reaction causes degradation of off target mRNA other than the target one after another.
  • the main cause of hepatotoxicity is the off target effect of the gapmer, ie RNase H1-dependent degradation of mRNA having a similar sequence to the target sequence of ASO in non-target mRNA (Burel, S. A., et. Al. Nucleic Acid Res., 44: 2093-2109, 2016., Kasuya, T., et. Al. Sci. Rep., 27: 6: 30377, 2016.).
  • the symmetric gapmer invented by the present inventors does not degrade the target, the duplex state between the gapmer and the target mRNA continues, and as a result, the gapmer becomes easily single stranded in the cell. There is no return. Therefore, the possibility of the off target effect is low, and as a result, it is considered that induction of hepatotoxicity is suppressed to a very low level. Therefore, as shown in FIG. 19, it can be considered that the symmetrical gapmer straddling the poly (A) chain addition site hardly induces the increase of the AST value or the ALT value in blood.
  • the gapmer-type ASO and non-gapmer against mRNA of mouse Acsl1 gene were preparedd type ASO. Specifically, ASO (Table 2, SGnon-gapAcsl1) in which a 2-residue LNA was introduced at the center of the location where the DNA of gapmer-type ASO (Table 2, SGgapAcsl) and the 12 residues of the central part of that ASO continue. Prepared.
  • ASO with the same structure as ASO designed by Ashiya et al., Changing only its target sequence. Specifically, we prepared gapmer type (3'-gapAcsl1) and non-gapmer type (3'-non-gapAcsl1) of symmetrical ASO across the portion to which the poly (A) chain of Acsl1 mRNA is added (Table) 2). Each ASO purchased Gene Design Co., Ltd. for synthesis. When C was LNAylated, methylcytosine was LNAylated. Phosphorothioation was performed between all bases.
  • nucleotide sequences of 3'-gapAcsl1 and 3'-non-gapAcsl1 are shown in SEQ ID NO: 33, and those of SGgapAcsl1 and SGnon-gapAcsl1 are shown in SEQ ID NO: 34 (gapmer type and non-gapmer type In the above, the base sequences are the same except that the LNAized bases are different).
  • LNA is in upper case and DNA in lower case.
  • the 10th base (“t” for 3'-gapAcsl1 and “T” for 3'-non-gapAcsl1) is cleaved in its target mRNA and poly A is It is a base at a position capable of binding to "3'-end of mRNA", which is the base of 3'-end of mRNA to be added.
  • the ICR mice were administered ASO at a dose of 0.5 mg / animal from the abdominal cavity.
  • PBS was administered as a negative control.
  • Three individuals were analyzed each.
  • FIG. 29 shows the results when each primer set was used (the result of the set of Primer-1 is represented by “Primer1”, and the result of Primer-2 is represented by “Primer2”).
  • each primer of the primer set used for quantitative PCR is as follows: Acsl1 Forward Primer-1: 5'-TGCCAGAGCTGATTGACATTC-3 '(SEQ ID NO: 35) Acsl1 Reverse Primer-1: 5'-GGCATACCAGAAGGTGGTGAG-3 '(SEQ ID NO: 36) Acsl1 Forward Primer-2: 5'-ACCAGCCCTATGAGTGGATT-3 '(SEQ ID NO: 37) Acsl1 Reverse Primer-2: 5'-CAAGGCTTGAACCCCTTCTG-3 '(SEQ ID NO: 38)
  • Standard deviations were calculated from the mean values of AST value, ALT value, and Acsl1 mRNA amount, and error bars were indicated (Fig. 29B, Fig. 31). Analysis of variance of ANOVA was performed using statistical analysis software R, and tests of statistical significance were performed by Tukey method (***, p ⁇ 0.005, *, p ⁇ 0.05).
  • FIG. 29 shows the amount of Acsl1 mRNA in the liver of mice administered with any of 3'-gapAcsl1, 3'-non-gapAcsl1, SGgapAcsl1 and SGnon-gapAcsl1, where A is the result for each treated mouse individual, And B shows the mean of each administration mouse population. A significant decrease in Ascl1 expression level was observed in all the ASO-administered populations compared to the PBS-administered individuals (FIG.
  • FIG. 30 shows the transition of AST value (A) and ALT value (B) in the serum of individual mice to which any of 3'-gapAcsl1, 3'-non-gapAcsl1, SGgapAcsl1 and SGnon-gapAcsl1 was administered.
  • FIG. 31 shows the average of the transition of AST value (A) and ALT value (B) in serum in each administration mouse population.
  • remarkable AST level and ALT level increase only in the group who received ASO (SGgapAcsl1), which is reported to show remarkable hepatotoxicity in the paper of Tsuchiya et al.
  • non-gapmer ASO does not induce hepatotoxicity. Therefore, the non-gapmer type ASO invented by the inventors is widely adopted in the field of nucleic acid drug development from now on, and it is expected that it will become the mainstream of future nucleic acid drugs.
  • the present invention is applicable to the pharmaceutical industry, the health food industry and the like.

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Abstract

La présente invention concerne un polynucléotide et un procédé de régulation efficace de l'expression génique. Sont décrits : un polynucléotide pouvant s'hybrider avec une séquence contiguë de 5 à 9 bases (comprenant la base de l'extrémité 3' de l'ARNm) du côté 5' depuis la base correspondant à l'extrémité 3' de l'ARNm et 5 à 9 bases (non comprise la base de l'extrémité 3' de l'ARNm) du côté 3' dans une séquence génomique; le polynucléotide précité, comprenant au moins un analogue nucléotidique; et un régulateur d'expression génique comportant le polynucléotide en tant que composant actif.
PCT/JP2018/028511 2017-07-31 2018-07-30 Procédé de régulation d'expression génique et régulateur d'expression génique WO2019026860A1 (fr)

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WO2013015152A1 (fr) * 2011-07-22 2013-01-31 公立大学法人横浜市立大学 TECHNIQUE POUR LE CLIVAGE D'UNE PARTIE D'UNE CHAÎNE poly(A) ET/OU D'UNE SÉQUENCE 3'-TERMINALE D'UN ARNm POUR INHIBER UNE RÉACTION DE TRADUCTION

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WO2013015152A1 (fr) * 2011-07-22 2013-01-31 公立大学法人横浜市立大学 TECHNIQUE POUR LE CLIVAGE D'UNE PARTIE D'UNE CHAÎNE poly(A) ET/OU D'UNE SÉQUENCE 3'-TERMINALE D'UN ARNm POUR INHIBER UNE RÉACTION DE TRADUCTION

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BINDER R ET AL.: "Degradation of Apolipoprotein II mRNA Occurs via Endonucleolytic Cleavage at 5' -AAU-3' /5' -UAA-3' Elements in Single-stranded Loop Domains of the 3'-Noncoding Region", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 264, no. 28, 5 October 1989 (1989-10-05), pages 16910 - 16918, XP055678548 *
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