WO2019026860A1 - Gene expression regulation method and gene expression regulator - Google Patents
Gene expression regulation method and gene expression regulator Download PDFInfo
- Publication number
- 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
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- mrna
- gapmer
- bases
- aso
- sequence
- Prior art date
Links
Images
Classifications
-
- 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.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Animal Behavior & Ethology (AREA)
- Biotechnology (AREA)
- Pharmacology & Pharmacy (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Epidemiology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Cosmetics (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention provides a polynucleotide and a method for efficiently regulating gene expression. Disclosed are: a polynucleotide capable of hybridizing with a contiguous sequence of 5-9 bases (including the base of the 3' end of the mRNA) of the 5' side from the base corresponding to the 3' end of the mRNA and 5-9 bases (not including the base of the 3' end of the mRNA) of the 3' side in a genomic sequence; the aforementioned polynucleotide, including at least one nucleotide analog; and a gene expression regulator having the polynucleotide as an active component.
Description
本発明は、遺伝子発現の制御に関する。より詳しくは、mRNAの発現を抑制するポリヌクレオチドおよびその使用方法に関する。
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.
従来、遺伝子発現を抑制する方法としては、アンチセンスRNA、リボザイム、RNAiなどの技術が知られている。
Conventionally, as methods for suppressing gene expression, techniques such as antisense RNA, ribozyme, RNAi and the like are known.
例えば、特許文献1に記載の発明は、mRNAのポリAより3’-側にハイブリダイズするアンチセンスオリゴヌクレオチドを用いてmRNAの発現を抑制するものであるが、投与量が多く必要であり、組織への毒性の問題もあった。
For example, in the invention described in Patent Document 1, although the expression of mRNA is suppressed using an antisense oligonucleotide that hybridizes to the 3'-side of polyA of mRNA, a large dose is required. There was also the issue of toxicity to tissues.
特許文献2に記載の発明は、特許文献1のアンチセンスオリゴヌクレオチドと、mRNAの5’-非翻訳領域のアンチセンスオリゴヌクレオチドの2種類のオリゴヌクレオチドを使用してmRNAの発現を抑制するものであるが、この場合、2種類のオリゴヌクレオチドが必要であり、投与量もその分多くなり、組織へ毒性を与えるおそれもあった。
The invention described in 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. However, in this case, two types of oligonucleotides were required, and the dose amount was increased accordingly, and there was also a possibility of giving toxicity to tissues.
さらに、従来のLNAを用いたアンチセンスオリゴヌクレオチド(gapmer)を用いて標的のmRNAを分解して遺伝子発現を抑制する場合(特許文献3および4)に、肝臓に対する毒性があるため、医薬として用いることが困難である、という問題があった(非特許文献1)。
Furthermore, 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).
そこで、投与量が少なく、人体や動物への毒性が低く、効率よく遺伝子発現を抑制する薬剤および方法が求められていた。
Therefore, there has been a demand for a drug and method for efficiently suppressing gene expression with a low dose, low toxicity to humans and animals.
本発明は、効率よく遺伝子発現を抑制するポリヌクレオチドおよび方法を提供する。
The present invention provides polynucleotides and methods for efficiently suppressing gene expression.
本発明によれば、以下の発明が提供される。
(1)ゲノム配列中、mRNAの3’-末端に対応する塩基から5’-側の5~9塩基(mRNAの3’-末端の塩基を含む)、および3’-側の5~9塩基(mRNAの3’-末端の塩基は含まない)の連続した配列にハイブリダイズし得るポリヌクレオチド。
(2)ゲノム配列中、mRNAの3’-末端に対応する塩基から5’-側の5~9塩基(mRNAの3’-末端の塩基を含む)、および3’-側の5~9塩基(mRNAの3’-末端の塩基は含まない)の連続した配列にハイブリダイズし得る配列のみを有するポリヌクレオチド。
(3)ゲノム配列中、mRNAの3’-末端に対応する塩基から5’-側の5~9塩基(mRNAの3’-末端の塩基を含む)、および3’-側の5~9塩基(mRNAの3’-末端の塩基は含まない)の連続した配列のアンチセンス配列を含むポリヌクレオチド。
(4)ゲノム配列中、mRNAの3’-末端に対応する塩基から5’-側の5~9塩基(mRNAの3’-末端の塩基を含む)、および3’-側の5~9塩基(mRNAの3’-末端の塩基は含まない)の連続した配列のアンチセンス配列からなるポリヌクレオチド。
(5)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)~(4)のいずれかのポリヌクレオチド
(6)前記ヌクレオチド類似体が、LNA、2’-O-メチル化RNA、2’-O-メトキシエチル化RNA、および/またはモルフォリノ核酸である、(5)のポリヌクレオチド。
(7)前記ポリヌクレオチドが、前記ヌクレオチド類似体を両末端および中央部に有する非ギャップマーである、(5)または(6)のポリヌクレオチド。
(8)(1)~(7)のいずれかのポリヌクレオチドを有効成分として含有する、遺伝子発現抑制剤。
(9)遺伝子発現抑制剤が、翻訳阻害剤である、(8)の遺伝子発現抑制剤。
(10)(1)~(7)のいずれかのポリヌクレオチドを有効成分として含有する、医薬、化粧品および/または食品。
(11)(1)~(7)のいずれかのポリヌクレオチドを投与することを包含する、遺伝子発現抑制方法。
(12)遺伝子発現抑制方法が、翻訳阻害方法である、(11)の遺伝子発現抑制方法。
(13)医薬を製造するための、(1)~(7)のいずれかのポリヌクレオチドの使用。 According to the present invention, 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).
(3) 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 comprising an antisense sequence of a continuous sequence (not including the 3'-terminal base of mRNA).
(4) 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 consisting of an antisense sequence of a continuous sequence (not including the 3'-terminal base of mRNA).
(5) The polynucleotide of any one of (1) to (4), wherein the polynucleotide comprises at least one nucleotide analogue (6) The nucleotide analogue is LNA, 2′-O-methylated RNA, The polynucleotide of (5), which is a 2'-O-methoxyethylated RNA and / or a morpholino nucleic acid.
(7) The polynucleotide of (5) or (6), wherein the polynucleotide is a non-gapmer having the nucleotide analogue at both ends and at the center.
(8) A gene expression suppressing agent comprising the polynucleotide of any one of (1) to (7) as an active ingredient.
(9) The gene expression inhibitor of (8), wherein the gene expression inhibitor is a translation inhibitor.
(10) A medicine, a cosmetic and / or a food comprising the polynucleotide of any of (1) to (7) as an active ingredient.
(11) A method for suppressing gene expression, which comprises administering a polynucleotide of any one of (1) to (7).
(12) The gene expression suppression method of (11), wherein the gene expression suppression method is a translation inhibition method.
(13) Use of the polynucleotide of any of (1) to (7) for producing a medicament.
(1)ゲノム配列中、mRNAの3’-末端に対応する塩基から5’-側の5~9塩基(mRNAの3’-末端の塩基を含む)、および3’-側の5~9塩基(mRNAの3’-末端の塩基は含まない)の連続した配列にハイブリダイズし得るポリヌクレオチド。
(2)ゲノム配列中、mRNAの3’-末端に対応する塩基から5’-側の5~9塩基(mRNAの3’-末端の塩基を含む)、および3’-側の5~9塩基(mRNAの3’-末端の塩基は含まない)の連続した配列にハイブリダイズし得る配列のみを有するポリヌクレオチド。
(3)ゲノム配列中、mRNAの3’-末端に対応する塩基から5’-側の5~9塩基(mRNAの3’-末端の塩基を含む)、および3’-側の5~9塩基(mRNAの3’-末端の塩基は含まない)の連続した配列のアンチセンス配列を含むポリヌクレオチド。
(4)ゲノム配列中、mRNAの3’-末端に対応する塩基から5’-側の5~9塩基(mRNAの3’-末端の塩基を含む)、および3’-側の5~9塩基(mRNAの3’-末端の塩基は含まない)の連続した配列のアンチセンス配列からなるポリヌクレオチド。
(5)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)~(4)のいずれかのポリヌクレオチド
(6)前記ヌクレオチド類似体が、LNA、2’-O-メチル化RNA、2’-O-メトキシエチル化RNA、および/またはモルフォリノ核酸である、(5)のポリヌクレオチド。
(7)前記ポリヌクレオチドが、前記ヌクレオチド類似体を両末端および中央部に有する非ギャップマーである、(5)または(6)のポリヌクレオチド。
(8)(1)~(7)のいずれかのポリヌクレオチドを有効成分として含有する、遺伝子発現抑制剤。
(9)遺伝子発現抑制剤が、翻訳阻害剤である、(8)の遺伝子発現抑制剤。
(10)(1)~(7)のいずれかのポリヌクレオチドを有効成分として含有する、医薬、化粧品および/または食品。
(11)(1)~(7)のいずれかのポリヌクレオチドを投与することを包含する、遺伝子発現抑制方法。
(12)遺伝子発現抑制方法が、翻訳阻害方法である、(11)の遺伝子発現抑制方法。
(13)医薬を製造するための、(1)~(7)のいずれかのポリヌクレオチドの使用。 According to the present invention, 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).
(3) 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 comprising an antisense sequence of a continuous sequence (not including the 3'-terminal base of mRNA).
(4) 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 consisting of an antisense sequence of a continuous sequence (not including the 3'-terminal base of mRNA).
(5) The polynucleotide of any one of (1) to (4), wherein the polynucleotide comprises at least one nucleotide analogue (6) The nucleotide analogue is LNA, 2′-O-methylated RNA, The polynucleotide of (5), which is a 2'-O-methoxyethylated RNA and / or a morpholino nucleic acid.
(7) The polynucleotide of (5) or (6), wherein the polynucleotide is a non-gapmer having the nucleotide analogue at both ends and at the center.
(8) A gene expression suppressing agent comprising the polynucleotide of any one of (1) to (7) as an active ingredient.
(9) The gene expression inhibitor of (8), wherein the gene expression inhibitor is a translation inhibitor.
(10) A medicine, a cosmetic and / or a food comprising the polynucleotide of any of (1) to (7) as an active ingredient.
(11) A method for suppressing gene expression, which comprises administering a polynucleotide of any one of (1) to (7).
(12) The gene expression suppression method of (11), wherein the gene expression suppression method is a translation inhibition method.
(13) Use of the polynucleotide of any of (1) to (7) for producing a medicament.
本発明によれば、副作用が少なく、効率よく、遺伝子発現を抑制できるポリヌクレオチドが得られる。
According to the present invention, a polynucleotide which can suppress gene expression efficiently with less side effects can be obtained.
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を付加されるために切断される前の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, a translation inhibitor and a translation suppression method are provided that suppress translation of a protein from a target mRNA. As used herein, 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. Say. Preferably, 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. In addition, 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.
本明細書において、「mRNAの3’-末端」とは、転写後、ポリAが付加されるために、切断されたmRNAの3’-末端の塩基をいう。すなわち、mRNAは転写後、ポリAが付加されるために3’-非翻訳領域で切断されるが、切断され、ポリAが付加されるmRNAの3’-末端の塩基を「mRNAの3’-末端」という。
As used herein, 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".
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、細胞周期、増殖関連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 to be translated can be targeted. For example, 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. In addition, it may be mRNA that translationally expresses a protein of unknown function.
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3’-末端を中心として、その両方向に5~9塩基の連続した配列のアンチセンスオリゴヌクレオチドを用いる。この配列を3’-末端を跨ぐ配列ともいう。すなわち、本発明は、mRNAの3’-末端切断前の3’-末端を跨ぐ配列にハイブリダイズするポリヌクレオチドを用いることにより効率よく標的mRNAの翻訳を抑制することができる。ここでハイブリダイズする、とは、細胞内において標的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 present invention, anti-sense oligonucleotides are used 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. Here, “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.
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3’-末端を跨ぐ配列にハイブリダイズするポリヌクレオチドを使用する。この場合、3’-末端の切断点から5’-側に5~9塩基(切断点の3’-末端(「mRNAの3’-末端」)の塩基を含む)、3’-末端の切断点の3’-側に5~9塩基(切断点の5’-末端の塩基を含むが、上記切断点の3’-末端の塩基(「mRNAの3’-末端」)を含まない)の合計、10~18塩基の標的mRNA配列にハイブリダイズするポリヌクレオチドを用いることが好ましい。要は、標的配列に特異的に結合してmRNAの3’-末端の切断に影響を与え、翻訳を抑制できればよい。より好ましくは、3’-末端の切断点から5’-側に6~8塩基、3’-末端の切断点の3’-側に6~8塩基、最も好ましくは、3’-末端の切断点から5’-側に7塩基、3’-末端の切断点の3’-側に7塩基であるが、これらに限られない。また、3’-末端を跨ぐ配列にハイブリダイズして翻訳を抑制できる限り、3’-末端を跨ぐ配列のみではなく、該配列を含む配列にハイブリダイズするポリヌクレオチドを用いてもよい。ただし、RNaseHによるmRNAの分解を誘導しないポリヌクレオチドであることが好ましい。
In a more preferred embodiment of the present invention, 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. In this case, 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")) It is preferred to use polynucleotides which hybridize to a total of 10-18 bases of the target mRNA sequence. 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. In addition, as long as hybridization can be performed to a sequence spanning the 3'-end to suppress translation, a polynucleotide hybridizing not only to the sequence spanning the 3'-end but to a sequence including the sequence may be used. However, a polynucleotide which does not induce degradation of mRNA by RNase H is preferable.
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3’-末端を跨ぐ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(ASO)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 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. In this case, there is an advantage that a polynucleotide that mechanically inhibits translation readily for any target mRNA can be designed, and a translation inhibitor can be produced. In the prior art, in order to inhibit gene expression by antisense technology, it was not sufficient to design a region to be used as an antisense strand, and it was necessary to confirm by experiment whether there is a translational inhibitory effect. Depending on the region, the introduction of antisense nucleotides (ASO) may not produce inhibitory activity. 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.
本発明においては、ギャップマー(gapmer)および非ギャップマー(nongapmer)を使用することができる。ギャップマー(gapmer)とは、図1に示すように、ヌクレオチド類似体(例えば、糖が修飾されたヌクレオチド類似体(例えばLNA))を両末端の1~3塩基(各末端の塩基数は異なっていてもよい)に有し、残りの配列がDNAからなるポリヌクレオチドをいう。非ギャップマー(nongapmer)とは、例えば、ヌクレオチド類似体(例えば、糖が修飾されたヌクレオチド類似体(例えばLNA))を両末端の1~3塩基(各末端の塩基数は異なっていてもよい)および中央部の1~3塩基に有し、残りの配列がDNAからなるポリヌクレオチドである。gapmerは図1に示すように、標的mRNAとハイブリダイズして二重鎖を形成し、RNaseHによる分解により標的mRNAの発現を抑制するものである。しかしながら、後に述べるように、従来のgapmerの場合は肝毒性が出るという問題がある。本発明のASOの設計方法により設計した場合は、gapmer、nongapmerとも肝毒性がほとんど出ないという特徴がある。
In the present invention, gapmers and non-gapmers can be used. A gapmer (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 non-gapmer (nongapmer) 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. As shown in FIG. 1, the gapmer hybridizes with a target mRNA to form a duplex, and suppresses the expression of the target mRNA by degradation with RNase H. However, as described later, in the case of the conventional gapmer, there is a problem that hepatotoxicity occurs. When designed according to the design method of ASO of the present invention, both of the gapmer and nongapmer have the characteristic that almost no hepatotoxicity appears.
翻訳抑制の効果は、図2に記載のプラスミドを用いて図2のNF-κB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NF-κB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、NF-κB転写因子のサブユニットであるRelA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、RelA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 repression can be measured using the transcriptional activation mechanism of NF-κB 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-κB transcription factor. When introduced into cells, 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.
本発明の標的mRNAの3’-末端を跨ぐ配列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3’-末端を跨ぐ配列のみにハイブリダイズする必要はなく、一部分でも3’-末端を跨ぐ配列にハイブリダイズすればよい。すなわち、標的mRNAの3’-末端を跨ぐ配列に一部でもハイブリダイズする限り、mRNAの翻訳阻害が起きると考えられる。
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.
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。本発明のポリヌクレオチドは、例えば、LNA(locked nucleic acids)を含むポリヌクレオチド、モルフォリノ核酸を含むポリヌクレオチド(例えば、モルフォリノオリゴヌクレオチド(MO:morpholino oligonucleotides))のようなヌクレオチドの類似体を含むポリヌクレオチドであってもよく、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。
Also, the 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). It 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. . Examples of 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.
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(2’,4’-BNA(2’,4’-Bridged nucleic acids)ともいう)、リボヌクレオチドの2’位のOH基が、H、OR、R、OR’OR、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し、R’は-(CH2)m-(mは、1~3の整数である)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、核酸のリボースまたはデオキシリボースの代わりにモルフォリン環を含むモルフォリノ核酸等が挙げられる。2’位糖修飾には、例えば、O-メチル(修飾核酸は2’-O-メチル化RNA、2’-O-メチル(Me)とも称される)またはO-メトキシエチル(修飾核酸は2’-O-メトキシエチル化RNA、2’-MOEとも称される)が好ましく用いられる。
In addition, as a sugar-modified nucleotide analogue, for example, 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 (where,, 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. For example, O-methyl (modified nucleic acid is also referred to as 2'-O-methylated RNA, 2'-O-methyl (Me)) or O-methoxyethyl (modified nucleic acid is 2) '-O-methoxyethylated RNA, also referred to as 2'-MOE) is preferably used.
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 phosphate-modified nucleotide analogues include ones in which the phosphoester group linking adjacent ribonucleotides is substituted with a phosphothioate group.
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 the polynucleotide by known methods 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、NF-κB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。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、NF-κB、AP-1、STAT3が挙げられる。したがって、NF-κB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 response. 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. Such 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. Therefore, while 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. Examples of transcription factors for such cytokine genes include NF-κB, AP-1, and STAT3. Therefore, a translational suppressor of the NF-κB 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 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:
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 hemorrhage, 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 the following: post pump 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 disease includes the following: Cardiotonic twitch 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***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、***/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 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 / 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 / gynecology symptoms include: preterm 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***/精管切除反転術。
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.
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。
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.
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。
Transplants 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, this includes: cystic fibrosis, familial blood phagocytic lymphocytosis, 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 disorders 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、***。
Renal disease includes 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, hemochromatitis, alpha-1 antitrypsin deficiency, diabetes, Hashimoto's thyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axis assessment, 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。
By applying the translation inhibitor of the present invention to target mRNAs encoding proteins that cause diseases and conditions mediated by these inflammatory cytokine cascades, inflammation can be reduced and / or treated.
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。
Furthermore, 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. 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. Subjects 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 consisting of a substance such as embryonic connective tissue and generally consisting of densely packed cells embedded in a fibrous substance or homogeneous substance. Examples of 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, muscle Membrane sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, B cell immunoblast sarcoma, lymphoma, T cell immunoblast sarcoma, Iensen sarcoma , Kaposi's sarcoma, Kupffer cell sarcoma, hemangiosarcoma, white blood sarcoma, malignant mesenchymal sarcoma, paraosseous sarcoma, reticular sarcoma, rous sarcoma, serous cystic sarcoma, synovial sarcoma, and capillary dilatation (telan giectaltic) including sarcomas.
「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 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.
「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 consisting of epithelial cells that tend to invade surrounding tissues and cause metastases. The term "carcinoma" refers to a malignant neoplasm consisting of epithelial cells that tend to invade surrounding tissues and cause metastases. The 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, Ulcer Carcinoma, Fibrosum Carcinoma, Geliniform Carcinoma, Gelatinous Carcinoma, Giant Cell Carcinoma, Giant Cell Gigantocellulare) Carcinoma, Adenocarcinoma, Granulosa Cell Carcinoma, Haemoroid Carcinoma, Hepatocarcinoma, Hepatocellular Carcinoma, Hultre Cell Carcinoma, Hyalin Carcinoma, Clear Cell Gland (Hypephephroid) Carcinoma, Childhood Fetal Carcinoma, Carcinoma in situ Intraepidermal carcinoma, Intraepithelial carcinoma, Krompecher carcinoma, Kuruchizky cell carcinoma, large cell carcinoma, lenticular carcinoma, lenticulare carcinoma, fatty carcinoma, lymphoepithelial carcinoma, carcinomatous medullare, marrow Carcinoma (medullary carcinoma), melanoma carcinoma, carcinogenic carcinoma (carcinoma mole), mucinous carcinoma, mucous carcinoma (carcinoma muciparum), mucous cell carcinoma, mucinous epidermoid carcinoma, mucosal carcinoma (cocinoco mucosum), mucosal carcinoma (mucous) carcinoma), myxoma carcinoma (carcinoma myxomatodes), nasopharyngeal carcinoma, oat cell, osteogenic carcinoma, osteocarcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, squamous cell carcinoma, squamous carcinoma pultaceous carcinoma), renal cell carcinoma, spare cell carcinoma, sarcomatoid carcinoma, schneider carcinoma, scleral carcinoma, scrotal carcinoma, sign ring cell carcinoma, simple carcinoma, small cell carcinoma, solanoid carcinoma, spheroid cell carcinoma, Spindle cell carcinoma, cavernous carcinoma, squamous cell carcinoma, squamous cell carcinoma, string carcinoma, vasodilation carcinoma, capillary dilatation carcinoma, transitional cell carcinoma, nodular carcinoma, nodular Includes tuberous carcinoma, wart-like carcinoma, and choriocarcinoma.
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, ret genes, etc. By suppressing the expression of these genes, it is possible to exert an anticancer effect.
また、本発明の医薬が適用し得る疾患としては、例えば、癌、エイズ、アルツハイマー病、筋萎縮性側索硬化症、アテローム性動脈硬化症、自己免疫疾患、小脳変性症、癌、真性糖尿病、糸球体腎炎、心不全、黄斑変性、多発性硬化症、骨髄異形成症候群、パーキンソン病、前立腺過形成、乾癬、喘息、網膜変性、網膜色素変性症、関節リウマチ、アテロームプラーク破裂、全身性エリテマトーデス、潰瘍性大腸炎、ウイルス感染、虚血再灌流損傷、脊髄損傷、神経損傷、心肥大、およびダイアモンドブラックファン貧血などが挙げられるがこれらに限られない。要は、特定の遺伝子の発現を抑制することにより治療できる疾患であれば特に制限されずに使用できる。現在原因遺伝子が判明しているものでも、未解明のものであってもよく、将来的に原因遺伝子が見つかったら、その遺伝子に対して本発明の翻訳阻害剤を適用して疾患を治療することができる場合があり得る。
In addition, 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 These include, but are not limited to: colitis colitis, viral infection, ischemia reperfusion injury, spinal cord injury, nerve injury, cardiac hypertrophy, and diamond blackfan anemia. The point is that 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
特に有望と考えられるものとしては、以下の遺伝子、疾患を標的にすることが好ましい(遺伝子名:疾患名の組み合わせで記載)。factor IX(血液凝固IX因子):血友病、aminolevulinic acid synthase 1(アミノレブリン酸合成酵素1):急性間欠性ポルフィリン症、transthyretin(トランスチレチン、TTR):家族性アミロイド心筋症、TGF-β:緑内障、VLA-4:多発性硬化症、insulin-like growth factor-1(インスリン様成長因子1):先端巨大症、Grb2:急性骨髄性白血病、Androgen receptor(アンドロゲンレセプター):前立腺がん、survivin:固形癌、Hif-1α:固形癌、insulin receptor substrate-1(インスリン受容体基質- 1):目血管新生、乾癬、ICAM-1:回腸嚢炎、huntintin:ハンチントン病、SOD-1:筋萎縮性側索硬化症、factor IX:凝固障害、Hsp27:癌、Bcl-2:黒色腫、白血病、骨髄腫、clusterin(クラステリン):前立腺癌、非小細胞肺癌、CTGF:肥厚性瘢痕。
Among the genes considered to be particularly promising, it is preferable to target the following genes and diseases (gene name: described by a combination of disease names). 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-β: Glaucoma, VLA-4: multiple sclerosis, insulin-like growth factor-1 (insulin like growth factor 1): acromegaly, Grb2: acute myeloid leukemia, Androgen receptor (androgen receptor): prostate cancer, survivin: Solid cancer, Hif-1α: solid cancer, insulin receptor substrate-1 (insulin receptor substrate-1): eye angiogenesis, psoriasis, ICAM-1: pouchitis, huntintin: Huntingtin's disease, SOD-1: amyotrophic side Cord sclerosis, factor IX: coagulation disorder, Hsp27: cancer, Bcl-2: melanoma, leukemia, myeloma, clusterin (clusterin): prostate cancer, non-small cell lung cancer, CTGF: hypertrophic scar.
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。個体への導入は尾部や腹腔内等への注射やジーンガン、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。
Furthermore, according to the present invention, 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.
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(抗生物質等)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。
Also, according to the present invention, there is provided a kit comprising a translation inhibitor. Examples of 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.
また、本発明によれば、3’-末端を跨ぐ配列にハイブリダイズし得るポリヌクレオチドを標的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。
Also, according to the present invention, there is provided 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.
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 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.
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 of screening for a substance having translation inhibitory activity using an in vitro translation system. 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. As 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. . Not only translation systems but systems capable of transcription and translation in the same system have also been developed, but 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(ウエスタンブロットを含む)、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 inhibitors by the in vitro translation system, 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. In the measurement of the synthesis activity of a protein, if the protein has an enzyme activity, 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 β-galactosidase, luminescence, measurement by radioisotope of phosphorylation by phosphorylation enzyme, measurement of substrate transfer activity by transferase, etc. It is not limited. 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.
本発明のオリコヌクレオチドに加えて、他の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、タンパク質などを組み合わせて用いてもよい。その場合、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を用いるのが好ましい。
In addition to the oliconucleotides of the present invention, other antisense strand polynucleotides, ribozymes, aptamers, antibiotics, antibodies, proteins and the like 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.
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 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. In addition, cells containing the translation inhibitor are also included in the scope of the present invention.
レポータージーンアッセイ
ASOによる遺伝子発現抑制効果を解析する目的で、レポータージーンアッセイを採用した(図2)。図2に示すように、プラスミドpGL4.32からのホタルルシフェラーゼの発現は、転写因子NF-κB応答配列を有するプロモーターに依存している。ホタルルシフェラーゼ活性は、NF-κB活性に依存するので、NF-κBのサブユニットRelAタンパク質の発現が抑制されるとホタルルシフェラーゼ活性は低下する。ウエスタンブロット法や定量PCRにより、RelA遺伝子の発現をタンパク質レベルあるいはmRNAレベルで評価することができる。NF-κB応答配列を有しないプラスミドpGL4.75(ウミシイタケルシフェラーゼを発現する)をプラスミドpGL4.32と一緒に培養細胞にトランスフェクションすることにより、トランスフェクション効率を補正し、細胞内のNF-κB活性を定量的に評価できる。siScrable(最終濃度40nM)はsiRNAのネガティブコントロール、siRelA ±0はRelA mRNAのポリAシグナル配列を含む標的配列に対するsiRNA(最終濃度40nM)、同領域に対する19merのgapmer(gapRelA ±0/19、最終濃度10nM)あるいは10merのgapmer(gapRelA ±0/10、最終濃度10nM)、Untreatedは未処理の細胞をそれぞれ表す。 Reporter Gene Assay 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-κB response element. Since the firefly luciferase activity depends on the NF-κB 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-κB response element, and intracellular NF-κB 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.
ASOによる遺伝子発現抑制効果を解析する目的で、レポータージーンアッセイを採用した(図2)。図2に示すように、プラスミドpGL4.32からのホタルルシフェラーゼの発現は、転写因子NF-κB応答配列を有するプロモーターに依存している。ホタルルシフェラーゼ活性は、NF-κB活性に依存するので、NF-κBのサブユニットRelAタンパク質の発現が抑制されるとホタルルシフェラーゼ活性は低下する。ウエスタンブロット法や定量PCRにより、RelA遺伝子の発現をタンパク質レベルあるいはmRNAレベルで評価することができる。NF-κB応答配列を有しないプラスミドpGL4.75(ウミシイタケルシフェラーゼを発現する)をプラスミドpGL4.32と一緒に培養細胞にトランスフェクションすることにより、トランスフェクション効率を補正し、細胞内のNF-κB活性を定量的に評価できる。siScrable(最終濃度40nM)はsiRNAのネガティブコントロール、siRelA ±0はRelA mRNAのポリAシグナル配列を含む標的配列に対するsiRNA(最終濃度40nM)、同領域に対する19merのgapmer(gapRelA ±0/19、最終濃度10nM)あるいは10merのgapmer(gapRelA ±0/10、最終濃度10nM)、Untreatedは未処理の細胞をそれぞれ表す。 Reporter Gene Assay 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-κB response element. Since the firefly luciferase activity depends on the NF-κB 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-κB response element, and intracellular NF-κB The activity can be assessed quantitatively. siScrable (
図2中にある2種類のプラスミド(pGL4.32とpGL4.75、プロメガ社製)をHeLa細胞にトランスフェクションした。本発明におけるASOの標的の1つは、ヒトRelA mRNAとした。転写因子NF-κBのサブユニットの1つであるRelAタンパク質の翻訳反応がASOにより抑制されると、NF-κB活性が低下し、レポーター遺伝子からのホタルルシフェラーゼ遺伝子の発現が低下する(図2)。その結果、ルシフェラーゼ活性は減少する。ASOによる遺伝子発現抑制効果は、ウエスタンブロット法(RelAタンパク質の解析)や定量PCR法(RelA mRNAの解析)で解析できる(図2)。また、NF-κB応答配列を有しないpGL4.75プラスミドは、レポータージーンアッセイにおける内部標準として用いた。図2は、RelA mRNAに対するgapmer(ポジティブコントロールでRelA mRNAのポリAシグナルを含む配列を標的とした19merからなるgapRelA±0/19(あるいはgap P/C)と呼ぶ)や同領域に対するsiRNA(siRelA±0)の遺伝子発現抑制効果を、レポータージーンアッセイ法、ウエスタンブロット法および定量PCRで観察した結果を示している。gapRelA±0/19とsiRelA±0は、どのアッセイにおいてもRelA遺伝子の発現抑制効果を示している。gapRelA ±0/10はgapRelA ±0/19と同じ領域を標的とするが長さが10merと短いため、アンチセンス効果を示さないネガティブコントロールである。
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. When the translation reaction of RelA protein, one of the subunits of transcription factor NF- 転 写 B, is suppressed by ASO, NF-κB activity decreases and the expression of firefly luciferase gene from the reporter gene decreases (Fig. 2) . As a result, the luciferase activity is reduced. 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). Also, the pGL4.75 plasmid without the NF-κB response element was used as an internal standard in the reporter gene assay. 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). The result of having observed the gene expression inhibitory effect of +/- 0) by the reporter gene assay method, a western blot method, and quantitative PCR is shown. 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.
ヒトRelA mRNAの3’末端の塩基配列の決定
発明者らは、NCBIに登録されているヒトRelA mRNAの塩基配列情報(NM_001145138.1)と使用しているHeLa細胞中のRelA mRNAの3’末端の塩基配列を比較するための実験を行った。HeLa細胞から全RNAを抽出し、3’RACE(Rapid Amplification of cDNA Ends)法により塩基配列を決定した。その結果、HeLa細胞内のRelA mRNAの3’末端の塩基配列は、NCBIに登録されていたもの(上段(RelA_NM_001145138.1))より約5塩基短いことがわかった(図3)。22個クローンを解析した結果、7塩基あるいは4塩基短いものも若干ながら存在することがわかった(図3)。図3Aに、NCBIで登録された3’末端の塩基配列と共に各クローンの配列(7塩基短い配列、5塩基短い配列、および4塩基短い配列)を示す。図3Bは、NCBIで登録された3’末端より、7塩基、5塩基、あるいは4塩基短いクローンの比率(割合)を示している。 Determination of the nucleotide sequence of the 3 'end of human RelA mRNA We 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. As a result, it was found that the nucleotide sequence of the 3 'end of RelA mRNA in HeLa cells was about 5 bases shorter than that registered in NCBI (upper row (RelA_NM_001145138.1)) (FIG. 3). As a result of analysis of 22 clones, it was found that some 7 bases or 4 bases shorter were still present (FIG. 3). 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.
発明者らは、NCBIに登録されているヒトRelA mRNAの塩基配列情報(NM_001145138.1)と使用しているHeLa細胞中のRelA mRNAの3’末端の塩基配列を比較するための実験を行った。HeLa細胞から全RNAを抽出し、3’RACE(Rapid Amplification of cDNA Ends)法により塩基配列を決定した。その結果、HeLa細胞内のRelA mRNAの3’末端の塩基配列は、NCBIに登録されていたもの(上段(RelA_NM_001145138.1))より約5塩基短いことがわかった(図3)。22個クローンを解析した結果、7塩基あるいは4塩基短いものも若干ながら存在することがわかった(図3)。図3Aに、NCBIで登録された3’末端の塩基配列と共に各クローンの配列(7塩基短い配列、5塩基短い配列、および4塩基短い配列)を示す。図3Bは、NCBIで登録された3’末端より、7塩基、5塩基、あるいは4塩基短いクローンの比率(割合)を示している。 Determination of the nucleotide sequence of the 3 'end of human RelA mRNA We 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. As a result, it was found that the nucleotide sequence of the 3 'end of RelA mRNA in HeLa cells was about 5 bases shorter than that registered in NCBI (upper row (RelA_NM_001145138.1)) (FIG. 3). As a result of analysis of 22 clones, it was found that some 7 bases or 4 bases shorter were still present (FIG. 3). 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.
mRNAの3’側プロセッシング
図4で示したように、転写伸長反応はmRNA重合酵素であるRNA polymerase II (Pol II)が仲介する(Proudfoot, N. J. Genes & Dev., 25:1770-1782, 2011.)。Pol IIは、ポリAシグナル(AAUAAA)を超え、下流にRNA鎖を伸長する(図4(A))。その後、図4(A)に示したように、AAUAAAに3’側プロセッシング因子CPSFが結合し、さらにpoly(A) polymerase(PAP)やmRNA切断因子(CF)、さらにはGU rich配列に結合する3’側プロセッシング因子(CstF)が結合して、mRNAの3’末端配列の開裂(切断)が矢印部分で生じる(図4(B))。ここからPAPによりpoly(A)鎖の伸長が始まる(C)。発明者らは3’RACE法により、この矢印部分を決定した(図5)。開裂部位からは、poly(A)鎖伸長酵素(PAP)によりpoly(A)鎖の伸長が生じる(図4(C))。ちなみにこれら一連の3’プロセッシング反応は、核内で生じる。 3. 3 'Processing of mRNA As shown in FIG. 4, the transcription and elongation reaction is mediated by mRNA polymerase enzyme RNA polymerase II (Pol II) (Proudfoot, NJ Genes & Dev., 25: 1770-1782, 2011. ). Pol II extends the RNA strand downstream beyond the poly A signal (AAUAAA) (FIG. 4 (A)). Thereafter, as shown in FIG. 4 (A), the 3 'processing factor CPSF binds to AAUAAA, and further binds to poly (A) polymerase (PAP), mRNA cleaving factor (CF), and GU rich sequences. 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.
図4で示したように、転写伸長反応はmRNA重合酵素であるRNA polymerase II (Pol II)が仲介する(Proudfoot, N. J. Genes & Dev., 25:1770-1782, 2011.)。Pol IIは、ポリAシグナル(AAUAAA)を超え、下流にRNA鎖を伸長する(図4(A))。その後、図4(A)に示したように、AAUAAAに3’側プロセッシング因子CPSFが結合し、さらにpoly(A) polymerase(PAP)やmRNA切断因子(CF)、さらにはGU rich配列に結合する3’側プロセッシング因子(CstF)が結合して、mRNAの3’末端配列の開裂(切断)が矢印部分で生じる(図4(B))。ここからPAPによりpoly(A)鎖の伸長が始まる(C)。発明者らは3’RACE法により、この矢印部分を決定した(図5)。開裂部位からは、poly(A)鎖伸長酵素(PAP)によりpoly(A)鎖の伸長が生じる(図4(C))。ちなみにこれら一連の3’プロセッシング反応は、核内で生じる。 3. 3 'Processing of mRNA As shown in FIG. 4, the transcription and elongation reaction is mediated by mRNA polymerase enzyme RNA polymerase II (Pol II) (Proudfoot, NJ Genes & Dev., 25: 1770-1782, 2011. ). Pol II extends the RNA strand downstream beyond the poly A signal (AAUAAA) (FIG. 4 (A)). Thereafter, as shown in FIG. 4 (A), the 3 'processing factor CPSF binds to AAUAAA, and further binds to poly (A) polymerase (PAP), mRNA cleaving factor (CF), and GU rich sequences. 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.
poly(A)鎖が付加される前の段階で標的mRNAに作用するgapmer
発明者らは、mRNA開裂部位を跨ぐようなASOを設計した(図5)。開裂部位より下流の配列はpoly(A)鎖が付くため、NCBI等のデータベースのmRNAの配列情報からでは知ることはできない。そのため、ゲノム由来の配列情報を基にして開裂部位より下流(図5の矢印より下流部分)の塩基配列情報を取得し、4種類の13merからなるgapmerを設計した。図5には、設計に用いた配列と共に、これらの4種のgapmerの配列を示す。これらの4種のgapmerの配列は、以下の表1にも示す。それぞれの標的配列の位置情報に従い、開裂部分を原点として命名した。ASOの塩基配列の大文字はLNA、小文字はDNAを表している。 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.
発明者らは、mRNA開裂部位を跨ぐようなASOを設計した(図5)。開裂部位より下流の配列はpoly(A)鎖が付くため、NCBI等のデータベースのmRNAの配列情報からでは知ることはできない。そのため、ゲノム由来の配列情報を基にして開裂部位より下流(図5の矢印より下流部分)の塩基配列情報を取得し、4種類の13merからなるgapmerを設計した。図5には、設計に用いた配列と共に、これらの4種のgapmerの配列を示す。これらの4種のgapmerの配列は、以下の表1にも示す。それぞれの標的配列の位置情報に従い、開裂部分を原点として命名した。ASOの塩基配列の大文字はLNA、小文字はDNAを表している。 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.
これらをHeLa細胞にトランスフェクションしてレポータージーンアッセイを行った。結果を図6に示す。図6は、図5のgapmerを終濃度1、3、あるいは10nMとなるようにHeLa細胞にトランスフェクションしたもので、縦軸(Relative luciferase activity)は、補正後のルシフェラーゼの相対活性を表している。ポジティブコントロールとしてgapP/C(gapRelA ±0/19、終濃度10nM)、RelA mRNA に対する市販のsiRNA(si P/C、終濃度40nM)、あるいは非特異的な配列に対するgapmer(NTS、終濃度10nM)をそれぞれトランスフェクションした。その結果、標的配列が下流にずれていくに従って発現抑制効果は減少した(図6)。しかし、いずれのgapmerにおいても強弱に差はあるが発現抑制効果を観察することができた(図6)。非特異的な配列に対するgapmer NTSは抑制効果を示さない(図6、Kakiuchi-Kiyota, S., et al., Toxicol. Sci., 138, 234-248, 2014.)。
These were transfected into HeLa cells for reporter gene assay. The results are shown in FIG. 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) as a positive control, commercially available siRNA against RelA mRNA (si P / C, final concentration 40 nM), or gapmer against nonspecific sequences (NTS, final concentration 10 nM) Were transfected respectively. As a result, the expression suppression effect decreased as the target sequence shifted downstream (FIG. 6). However, the expression suppression effect was able to be observed in all gapmers although there was a difference in strength (FIG. 6). The gapmer NTS on nonspecific sequences shows no suppressive effect (FIG. 6, Kakiuchi-Kiyota, S., et al., Toxicol. Sci., 138, 234-248, 2014.).
同様のことがマウス由来の培養細胞で生じるか確認する目的で、NIH3T3細胞にレポータープラスミドと図7に示した4種類の13merのgapmerをそれぞれトランスフェクションした。図7の矢印は、mRNAの開裂部分を示している。上段(Mouse RelA_NM_009045.4)は、NCBIに登録済みの配列情報で、本来は矢印以降の部分はpoly(A)鎖となっていた。3’RACE法で肝臓由来の全RNAを材料としてマウスRelA mRNAの3’末端配列を決定すると、NCBIの情報と同一であった。矢印以降の配列は、ゲノム情報を基にして抽出し、記載した。4種類のgapmer型ASOを図7のように設計した。図7には、設計に用いた配列と共に、これらの4種のgapmerの配列を示す。これらの4種のgapmerの配列は、以下の表1にも示す。それぞれの標的配列の位置情報に従い、開裂部分を原点として命名した。ASOの塩基配列の大文字はLNA、小文字はDNAを表している。
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. When 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. 7 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.
図7のgapmerを終濃度1、3、あるいは10nMとなるようにNIH3T3細胞にトランスフェクションした。このようにトランスフェクションしたNIH3T3細胞におけるレポータージーンアッセイ結果を図8に示す。図8の縦軸(Relative luciferase Activity)は、補正後のルシフェラーゼの相対活性を表している。ネガティブコントロールとして非特異的な配列に対するgapmer(NTS、終濃度10nM)をトランスフェクションした。その結果、HeLa細胞と同様に、すべてのASOにおいて強弱に差はあるものの発現抑制活性を観察することができた(図8)。
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. As a negative control, gapmer (NTS, final concentration 10 nM) for nonspecific sequences was transfected. As a result, as in HeLa cells, expression suppression activity was able to be observed in all ASOs although there was a difference in strength (FIG. 8).
この結果から、ASOは細胞の核内でpoly(A)鎖付加が生じる前の段階にある標的mRNAにハイブリダイズしてアンチセンス効果を発揮する可能性が強く示唆された。興味深いことにNIH3T3細胞の結果から、-8/+5、すなわちmRNAの開裂部位をASOの中央付近に有するものが一番強い発現抑制活性を誘導することがわかった(図7と8)。よって、本発明者らは以降の実験で、ASOの中央部分に標的mRNAの開裂部位がくるように設計した。
From this result, it is strongly suggested that 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. Interestingly, the 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.
対称型gapmerは強い遺伝子発現抑制効果を発揮する
ヒトRelA mRNAに対して開裂部位(矢印)を中央にして、上流・下流、すなわち開裂部位を中央にして両側にそれぞれ9塩基のもの(-9/+9)、8塩基のもの(-8/+8)、7塩基のもの(-7/+7)、6塩基のもの(-6/+6)、5塩基のもの(-5/+5)のASOを準備した(図9)。本来、矢印以降の部分はpoly(A)鎖である。よって、ゲノム情報を基にして矢印以降の配列を抽出し、記載した。図9には、設計に用いた配列と共に、準備したgapmerの配列を示す。これらのgapmerの配列は、以下の表1にも示す。ASOの塩基配列の大文字はLNA、小文字はDNAを表している。-8/+5は図5に記載済みである。それらすべての5’と3’端の2塩基はLNAで、それら以外は全てDNAから成る。以下、開裂部位を中央に配置した対称型をしているという構造上の特徴から、「対称型gapmer」と呼ぶ(図9)。 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. Hereinafter, 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).
ヒトRelA mRNAに対して開裂部位(矢印)を中央にして、上流・下流、すなわち開裂部位を中央にして両側にそれぞれ9塩基のもの(-9/+9)、8塩基のもの(-8/+8)、7塩基のもの(-7/+7)、6塩基のもの(-6/+6)、5塩基のもの(-5/+5)のASOを準備した(図9)。本来、矢印以降の部分はpoly(A)鎖である。よって、ゲノム情報を基にして矢印以降の配列を抽出し、記載した。図9には、設計に用いた配列と共に、準備したgapmerの配列を示す。これらのgapmerの配列は、以下の表1にも示す。ASOの塩基配列の大文字はLNA、小文字はDNAを表している。-8/+5は図5に記載済みである。それらすべての5’と3’端の2塩基はLNAで、それら以外は全てDNAから成る。以下、開裂部位を中央に配置した対称型をしているという構造上の特徴から、「対称型gapmer」と呼ぶ(図9)。 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. Hereinafter, 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).
図9のgapmerを終濃度10 nMとなるようにHeLa細胞にトランスフェクションした。図9に示す各gapmerでトランスフェクションしたHeLa細胞におけるレポータージーンアッセイ結果を図10に示す。図10の縦軸(Relative luciferase activity)は、補正後のルシフェラーゼの相対活性を表している。ポジティブコントロールとしてgapmer(gapP/C;gapRelA ±0/19、終濃度10nM)、RelA mRNAに対する市販のsiRNA(siP/C、終濃度40nM)、あるいは非特異的な配列に対するgapmer(NTS、終濃度10nM)をそれぞれトランスフェクションした。UTは、未処理の細胞を表している。図10中の数値は、補正後のルシフェラーゼ活性である。レポータージーンアッセイの結果、-9/+9、-8/+8と-7/+7は、未処理のものに比較して約5%までルシフェラーゼ活性を低下させた(図10)。ポジティブコントロールとして用いたgapRelA ±0/19(gap P/C)やRelA mRNAに対する市販のsiRNA(siP/C、Takaesu, G., et al., J. Mol. Biol. 326, 105-115, 2003.)の示すレポータージーンアッセイの値が約10%であることを考慮すると、-9/+9、-8/+8、-7/+7は非常に強い発現抑制効果を示すものと言える(図10)。-6/+6や-5/+5は抑制効果が若干弱くなった(図10)。
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) as a positive control, commercially available siRNA against RelA mRNA (siP / C, final concentration 40nM), or gapmer (NTS, final concentration 10nM for nonspecific sequences) Each was transfected. UT stands for untreated cells. The numerical values in FIG. 10 are the luciferase activity after correction. As a result of reporter gene assay, -9 / + 9, -8 / + 8 and -7 / + 7 reduced the luciferase activity to about 5% as compared to the untreated one (FIG. 10). Commercially available siRNA against gapRelA ± 0/19 (gap P / C) or RelA mRNA used as positive control (siP / C, Takaesu, G., et al., J. Mol. Biol. 326, 105-115, 2003 Considering that the value of the reporter gene assay indicated by.) Is about 10%, -9 / + 9, -8 / + 8, -7 / + 7 can be said to show a very strong expression suppression effect (Fig. 10). -6 / + 6 and -5 / + 5 had a slightly weaker inhibitory effect (Fig. 10).
次に定量PCRアッセイによりASOをトランスフェクションした細胞内のRelA mRNA量について検討した。この結果を図11に示す。図10と同様に図9の各gapmerまたはポジティブコントロールのgapmer(gapP/C)を終濃度10nMあるいはsiRNA(siP/C)を終濃度40nMとなるようにHeLa細胞にトランスフェクションし、これらのトランスフェクションしたそれぞれの細胞から、全RNAを調整した。逆転写反応をoligo(dT)(各処理群の左側のハッチングを施した棒)あるいはrandom primer(各処理群の右側の白抜きの棒)を用いて行い、できたcDNAを材料として定量PCRによりRelA mRNA量を解析した(図11)。図11の縦軸(Relative RelA mRNA level)は、内部標準で補正後のRelA mRNAの相対量を示している。図11中の数値は、補正後のRelA mRNAの相対量を表している。
Next, the amount of RelA mRNA in cells transfected with ASO was examined by quantitative PCR assay. The results are shown in FIG. As in 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.
その結果、ポジティブコントロールのgap P/CやsiP/Cは、未処理のものに比較してmRNA量を約10%程度まで減少させていた(図11)。-7/+7はoligo(dT)で逆転写した場合、RelA mRNA量はポジティブコントロールのgap P/Cと同じレベルで減じているように見えたが、randomプライマーで逆転写反応した場合は、RelA mRNA量を未処理の約半分までしか減少させていなかった(図11)。この違いは、RelA mRNAにpoly(A)が付加される反応をgapmer -7/+7は阻害している。レポータージーンアッセイでコントロールに比較して約80%までルシフェラーゼ活性を低下させる能力を有する-6/+6のRelA mRNA量への影響は、randomプライマーで逆転写した場合は、未処理のものの約70%程度であった(図11)。すなわち、-6/+6はほとんどmRNA量を減じていないといえる。以上の結果より、従来のgapmerが標的のmRNAを分解して発現抑制活性を発揮している(US005962425A、US005955589A)のに対し、発明者らの開発した対称型gapmerは、標的mRNAをほとんど分解しないで発現抑制活性を示している可能性が示唆された。
As a result, the positive controls gap P / C and siP / C reduced the amount of mRNA to about 10% as compared with the untreated one (FIG. 11). When -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.
マウスApoB遺伝子を標的としたASOのアンチセンス活性評価
apolipoproteinB(apoB)はリポタンパク質の主要な構成要素である。コレステロールはapoBなどのタンパク質成分に取り込まれ、脂質成分が付加されリポタンパク質が合成される(Davidson N.O., and Shelness, G.S., Annu. Rev. Nutr. 20, 169-193, 2000.)。脂質成分は肝臓においてリポタンパク質へと作り変えられてから全身へと送り届けられる。米国で承認されている世界初の全身投与可能な核酸医薬品カイナムロは、apoBを標的としLDL-C値を特異的に低下させることが報告されている(Wong, E., and Goldberg, T. Drug Forecast 39, 119-122, 2014.)。よって、マウスを用いた動物実験においてApoBを候補遺伝子とすれば評価系が既に確立しているため遺伝子発現抑制効果を簡便に確認することができる。本法は一般的に広く採用されているため、本実施例でも使用することにした。 Evaluation of antisense activity of ASO targeting mouse ApoB gene apolipoprotein B (apoB) is a major component of lipoproteins. 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.
apolipoproteinB(apoB)はリポタンパク質の主要な構成要素である。コレステロールはapoBなどのタンパク質成分に取り込まれ、脂質成分が付加されリポタンパク質が合成される(Davidson N.O., and Shelness, G.S., Annu. Rev. Nutr. 20, 169-193, 2000.)。脂質成分は肝臓においてリポタンパク質へと作り変えられてから全身へと送り届けられる。米国で承認されている世界初の全身投与可能な核酸医薬品カイナムロは、apoBを標的としLDL-C値を特異的に低下させることが報告されている(Wong, E., and Goldberg, T. Drug Forecast 39, 119-122, 2014.)。よって、マウスを用いた動物実験においてApoBを候補遺伝子とすれば評価系が既に確立しているため遺伝子発現抑制効果を簡便に確認することができる。本法は一般的に広く採用されているため、本実施例でも使用することにした。 Evaluation of antisense activity of ASO targeting mouse ApoB gene apolipoprotein B (apoB) is a major component of lipoproteins. 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.
まず、先行研究により強い抑制効果を持つことが示されているApoB mRNAに対するgapmer型のASO(MM13mer(配列を以下の表1および図12に示す);Nishina, K., et al., Nature Commun., 6:79692015)を用いて条件検討した(図12)。ASOを尾静脈よりDay.0で投与した。投与量は1匹あたり0.004mgから0.5mgまで4段階刻みで、2回目に投与する際も各個体に同量を投与した。0.5mg投与は1匹、0.1mgから0.004mgまでの投与は2匹で行い、図は平均値である。PBSは2匹で行い、平均値を示した。ASOを投与する前日(Day.-1)、投与してから3日後(Day.3)、7日後(Day.7)に採血した。7日目の採血後、再びマウス尾静脈にASOを投与した。投与してから10日後(Day.10)に再び採血した(図12)。採血した血清中の総コレステロール値、HDL-コレステロール値、LDL-コレステロール値をそれぞれ測定した。結果を図13に示す。
First, a 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. Blood was collected on the day before ASO administration (Day.-1), 3 days after administration (Day.3), and 7 days (Day.7). After blood collection on day 7, ASO was again administered to the mouse tail vein. Blood was collected again 10 days after administration (Day 10) (Figure 12). The total cholesterol, HDL-cholesterol and LDL-cholesterol levels in the collected blood serum were each measured. The results are shown in FIG.
その結果、いずれのコレステロール値においてもASOの投与量が増加するにしたがってDay.-1の値より顕著に低下した(図13)。同時にこれらのマウスのDay.10に測定した血液中のAST値及びALT値は、投与量の増加に伴い顕著に上昇した(図14)。特にMM 13merを1匹あたり0.1mgと0.5mgを投与したもののASTおよびALTの値は非常に高く、激しい肝障害を生じている可能性が示唆された。
As a result, as the dose of ASO increased at any cholesterol level, it decreased significantly from the value of Day.-1 (FIG. 13). At the same time, the AST and ALT values in blood measured on Day 10 of these mice significantly increased with the increase in dose (FIG. 14). The AST and ALT values were very high, especially with 0.1 mg and 0.5 mg of MM 13 mer per animal, suggesting that severe liver damage may have occurred.
肝毒性のメカニズムとしては、核酸の種類(Krieg, M., et al., Nature Reviews Drug Discov., 6:471-484, 2006)、その配列(Burdick, A. D., et al., Nucleic Acid Res., 8:4882-4891, 2014)等が関係することが報告されている。以降の実験ではMM 13merをポジティブコントロールとして採用し、AST値及びALT値がほとんど上昇しない1匹あたり0.02mgを投与量として選んだ(図14)。
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).
マウスを用いた対称型gapmerのアンチセンス活性評価
ApoB mRNAのpoly(A)鎖付加部位を跨ぐように設計した対称型gapmerからなるASOの活性を評価するために、図15に示したプロトコールを採用した。ASO投与日(Day.0)の5日前(Day.-5)、及び投与した日から3日後のDay.3に採血した。Day.3では、肝臓の摘出も実施した。MM 13merの投与量は、肝毒性の出にくい1匹あたり0.02 mg、対称型gapmerはその25倍量の0.5 mgを腹腔より投与した。実際に投与した対称型gapmerの塩基配列を、図16Aに示した。図中の点線は開裂部位を示しており、ここを中央にして対称型gapmerを設計している。ちなみにマウスApoB mRNAの3’末端の塩基配列情報は、肝臓より回収した全RNAを材料として3’RACEを実施し、本発明者らが決定したものである。 Evaluation of antisense activity of symmetrical gapmer using mouse To evaluate the activity of ASO consisting of symmetrical gapmer designed to cross over the poly (A) chain addition site of ApoB mRNA, the protocol shown in FIG. 15 is adopted did. Blood was collected 5 days before the day of ASO administration (Day. 0) (Day. -5), and on Day. 3 three days after the day of administration. OnDay 3 we also performed liver removal. The dose of MM 13-mer was 0.02 mg per animal, which is less likely to cause hepatotoxicity, and the symmetrical gapmer was dosed with 0.5 mg of its 25-fold amount from the abdominal cavity. The nucleotide sequence of the symmetric gapmer actually administered is shown in FIG. 16A. 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.
ApoB mRNAのpoly(A)鎖付加部位を跨ぐように設計した対称型gapmerからなるASOの活性を評価するために、図15に示したプロトコールを採用した。ASO投与日(Day.0)の5日前(Day.-5)、及び投与した日から3日後のDay.3に採血した。Day.3では、肝臓の摘出も実施した。MM 13merの投与量は、肝毒性の出にくい1匹あたり0.02 mg、対称型gapmerはその25倍量の0.5 mgを腹腔より投与した。実際に投与した対称型gapmerの塩基配列を、図16Aに示した。図中の点線は開裂部位を示しており、ここを中央にして対称型gapmerを設計している。ちなみにマウスApoB mRNAの3’末端の塩基配列情報は、肝臓より回収した全RNAを材料として3’RACEを実施し、本発明者らが決定したものである。 Evaluation of antisense activity of symmetrical gapmer using mouse To evaluate the activity of ASO consisting of symmetrical gapmer designed to cross over the poly (A) chain addition site of ApoB mRNA, the protocol shown in FIG. 15 is adopted did. Blood was collected 5 days before the day of ASO administration (Day. 0) (Day. -5), and on Day. 3 three days after the day of administration. On
図16Aに示すように、開裂部位を中央にして3種類の対称型gapmerを設計し、準備した。図16Aには、設計に用いた配列と共に、準備したgapmerの配列を示す。これらのgapmerの配列は、以下の表1にも示す。点線部分は開裂部位を示している。本来、点線より下流の部分はpoly(A)鎖である。よって、ゲノム情報を基にして点線以降の配列を抽出し、記載した。最上段はApoB mRNA の配列情報、棒はmRNAを模式的に示し、AAAAの連続はpoly(A)鎖を表している。薄いグレーの丸で大文字はLNA、黒丸で小文字はDNAをそれぞれ表している。図16BからDは、ASO(投与量は1匹あたり0.5mg)で腹腔より投与した。ポジティブコントロールとしてMM 13merを0.02mg、腹腔より投与した。図15のプロトコールに従って採血し、血液中のLDL-コレステロール値(LDL-C:図16B)、HDL-コレステロール値(HDL-C:図16C)、および総コレステロール値(T-CHO:図16D)を測定した。MM 13merとPBS投与は2匹で行い、-9/+9、-8/+8と-7/+7投与は4匹で行った。数値は、平均値を示している。
As shown in FIG. 16A, three symmetrical gapmers were designed and prepared with the cleavage site at the center. 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.
ASOを投与してから3日後の血清中のLDL-C値は、PBS以外のASOを投与した全てのマウスでDay.-5の値に比較して顕著に減少した。ASO -7/+7を投与したマウスのLDL-Cが顕著にDay.3で減少していた(図16B ApoB ASO-7/+7 p<0.01)。-9/+9や-8/+8に関しても減少していた(図16B)。HDL-CやT-CHOに関しては、ASO投与による変化はほとんどなかった(図16CとD)。ポジティブコントロールのMM 13merは、HDL-CやT-CHOの値を若干減少させているようである(図16CとD)。
Three days after ASO administration, LDL-C levels in the serum were significantly reduced in all mice receiving ASO other than PBS, as compared to the values on Day.-5. The LDL-C of the mice administered ASO-7 / + 7 was significantly decreased on Day 3 (FIG. 16B ApoB ASO-7 / + 7 p <0.01). It also decreased for -9 / + 9 and -8 / + 8 (FIG. 16B). As for HDL-C and T-CHO, there was almost no change by ASO administration (FIGS. 16C and D). The positive control MM 13 mer seems to slightly reduce the values of HDL-C and T-CHO (FIGS. 16C and D).
図15のプロトコールで実験を実施し、図16で回収したDay.3の血液を用いて、AST値とALT値を測定した。その結果、肝毒性の指標であるAST値やALT値については、ASO-7/+7で若干の上昇が観察された(図17、AST値p<0.05、ALT値p<0.01)。MM 13merの場合、1匹あたり0.5mg投与すると、AST値は400以上、ALT値は600以上となり、これは激しい肝障害を誘導したためである(図14)。一方、発明者らの開発した対称型gapmer-7/+7におけるAST値は100以下、ALT値も45以下となり、MM 13merと比較すると極めて弱いものであった(図17)。以上の結果より、ApoB mRNAの3’末端のpoly(A)鎖付加部位を跨ぐように設計した対称型gapmerは、マウスの肝臓でApoBを発現抑制し、その結果、血中のLDL-C値の低下が誘導されたと考えられる。
The experiment was carried out according to the protocol of FIG. 15, and the blood of Day 3 collected in FIG. 16 was used to measure the AST value and the ALT value. As a result, a slight increase was observed in ASO-7 / + 7 for the AST value and the ALT value, which are indicators of hepatotoxicity (FIG. 17, AST value p <0.05, ALT value p <0.01). In the case of MM 13 mer, when administered at 0.5 mg per animal, the AST value is 400 or more, and the ALT value is 600 or more, because the severe liver injury was induced (FIG. 14). On the other hand, 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
実際に腹腔から投与したASOが肝臓に達し、肝臓中のApoB mRNAに作用したか確かめるため、ASO投与3日後に摘出した肝臓より全RNAを抽出し定量PCRで解析した。Aはrandom primer、Bはoligo dT primerでそれぞれ逆転写反応を行った。縦軸は、内部標準で補正後のApoB mRNAの相対量を示している。その結果、3種類の対称型gapmerからなるASOは、PBS処理のものに比較してすべての個体でApoB mRNA量の低下を誘導した(図18)。特にASO-7/+7では、投与した4匹すべてのマウスで顕著なApoB mRNA量の低下が観察された(図18)。以上の結果から、対称型gapmerは肝臓のApoB mRNAに働きかけ、ApoBの発現を抑制し、その結果、血液中のLDL-C値の低下を引き起こしたと言える。
In order to confirm whether ASO actually administered from the peritoneal cavity reached the liver and affected ApoB mRNA in the liver, total RNA was extracted from the liver excised 3 days after ASO administration and analyzed by quantitative PCR. Reverse transcription was performed using A as a random primer and B as an oligo dT primer. The vertical axis shows the relative amount of ApoB mRNA corrected with the internal standard. As a result, 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). In particular, in the case of 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.
gapmer構造を壊すnon-gapmer型ASOは遺伝子発現抑制効果を有する
gapmer型ASOは標的遺伝子の発現を効率的に抑制するものの、マウスに投与した際に強い肝障害を示すことが多数報告されている(Burel, S. A., et. al. Nucleic Acid Res., 44:2093-2109, 2016., Kasuya, T., et. al. Sci. Rep., 27;6:30377, 2016.)。発明者らの実験においても図14の結果のように、ポジティブコントロールの従来型gapmer(MM 13mer)は、1匹あたり0.1 mg以上投与すると極めて強い肝毒性が惹起された。よって、gapmer型の副作用である肝障害を取り除くことは、核酸医薬品開発において必須の課題である。一方、肝毒性はRNaseH1活性に由来することが報告されている(Burel, S. A., et. al. Nucleic Acid Res., 44:2093-2109, 2016., Kasuya, T., et. al. Sci. Rep., 27;6:30377, 2016.)。すなわち、gapmerによりRNaseH1依存的なmRNA分解が生じ、これが肝毒性の主原因と考えられている。 Non-gapmer-type ASO, which breaks down the structure of the gapmer, has a gene expression suppression effect Although 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.). Also in the experiments of the inventors, as shown in FIG. 14, when the positive control conventional gapmer (MM 13-mer) was administered at 0.1 mg or more per animal, extremely strong hepatotoxicity was elicited. Therefore, it is an essential task in nucleic acid drug development to remove the liver disorder that is a side effect of gapmer type. On the other hand, 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.
gapmer型ASOは標的遺伝子の発現を効率的に抑制するものの、マウスに投与した際に強い肝障害を示すことが多数報告されている(Burel, S. A., et. al. Nucleic Acid Res., 44:2093-2109, 2016., Kasuya, T., et. al. Sci. Rep., 27;6:30377, 2016.)。発明者らの実験においても図14の結果のように、ポジティブコントロールの従来型gapmer(MM 13mer)は、1匹あたり0.1 mg以上投与すると極めて強い肝毒性が惹起された。よって、gapmer型の副作用である肝障害を取り除くことは、核酸医薬品開発において必須の課題である。一方、肝毒性はRNaseH1活性に由来することが報告されている(Burel, S. A., et. al. Nucleic Acid Res., 44:2093-2109, 2016., Kasuya, T., et. al. Sci. Rep., 27;6:30377, 2016.)。すなわち、gapmerによりRNaseH1依存的なmRNA分解が生じ、これが肝毒性の主原因と考えられている。 Non-gapmer-type ASO, which breaks down the structure of the gapmer, has a gene expression suppression effect Although 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.). Also in the experiments of the inventors, as shown in FIG. 14, when the positive control conventional gapmer (MM 13-mer) was administered at 0.1 mg or more per animal, extremely strong hepatotoxicity was elicited. Therefore, it is an essential task in nucleic acid drug development to remove the liver disorder that is a side effect of gapmer type. On the other hand, 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.
粕谷らの論文では、gapmer構造の中央部分にLNAを配したnon-gapmerは、肝毒性を全く示さないと報告している(Kasuya, T., et. al. Sci. Rep., 27;6:30377, 2016.)。
In a paper by Tatsuya et al., Non-gapmer having LNA in the central part of the gapmer structure has been reported to show no liver toxicity (Kasuya, T., et. Al. Sci. Rep., 27; 6) : 30377, 2016.).
そこで、標的mRNAのpoly(A)鎖付加部位を跨ぐ対称型gapmerとASOの配列の中央部付近にLNAを有したnon-gapmer型を準備した。本発明で用いた7種類のASOを図19に示した(これらのASOの配列を表1にも示す)。矢印は、ヒトRelA mRNAの開裂部分を示している。本来、矢印以降の部分はpoly(A)鎖である。よって、ゲノム情報を基にして矢印以降の配列を抽出し、記載した。開裂部位(矢印)を中央にして、上流・下流に7塩基のもの(-7/+7)を準備した。ASOの塩基配列中でLNAは薄いグレーで表し、DNAは黒で表した。LNAの挿入されている相対位置(番号で表示)に基づき、それぞれを命名した。それらASOの活性をレポータージーンアッセイで検討した。
Therefore, 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. In the base sequence of ASO, 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.
図19のASOを終濃度10nMとなるようにHeLa細胞にトランスフェクションした。このようにトランスフェクションしたHeLa細胞におけるレポータージーンアッセイの結果を図20に示す。図20の縦軸(Relative luciferase Activity)は、補正後のルシフェラーゼ活性を表している。ポジティブコントロールとしてgapmer(gapP/C;gapRelA ±0/19、終濃度10nM)、RelA mRNAに対する市販のsiRNA(siP/C、終濃度40nM)、あるいは非特異的な配列に対するgapmer(NTS、終濃度10nM)をそれぞれトランスフェクションした。図20中の数値は、補正後のルシフェラーゼの相対活性である。
HeLa cells were transfected with ASO of FIG. 19 to a final concentration of 10 nM. 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) as a positive control, commercially available siRNA against RelA mRNA (siP / C, final concentration 40nM), or gapmer (NTS, final concentration 10nM for nonspecific sequences) Each was transfected. Numerical values in FIG. 20 are relative activities of luciferase after correction.
その結果、14merからなるnon-gapmer構造の1.2.8.13.14が、最も強い発現抑制効果を示した(図20)。また、non-gapmer型の1.7.8.14においてもポジティブコントロールのgapmer(gap P/C)の示す値のレベルまで発現を抑制した(図20)。
As a result, 1.2.8.13.14 of the 14-mer non-gapmer structure showed the strongest expression suppression effect (FIG. 20). In addition, the expression was suppressed also in the non-gapmer type 1.7.8.14 to the level of the value indicated by the positive control gapmer (gap P / C) (FIG. 20).
次にそれぞれのASOをトランスフェクションしたHeLa細胞から全RNAを抽出し、定量PCRにより細胞中のRelA mRNAに関して検討を加えた。逆転写反応をoligo(dT)(各処理群の左側のハッチングを施した棒)あるいはrandom primer(各処理群の右側の白抜きの棒)を用いて行い、できたcDNAを材料として定量PCRによりRelA mRNA量を解析した(図21)。図21の縦軸は、内部標準で補正後のRelA mRNA量を示している。図21中の数値は、補正後のRelA mRNAの相対量を示している。
Next, total RNA was extracted from each ASO-transfected HeLa cell, and analysis was performed on RelA mRNA in the cell by quantitative PCR. 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.
その結果、ポジティブコントロールのgap P/Cは、コントロールのNTSに比較して10%レベルまでRelA mRNA量を減じていた(図21)。これに対してnon-gapmer構造の1.7.8.14や1.2.8.13.14はNTSと比較してRelA mRNA量をおよそ半減するにとどまった(図21)。以上の結果からpoly(A)鎖付加部位を跨ぐASOでnon-gapmer構造を有するものは、1)遺伝子発現抑制効果を有している、2)mRNAの分解をほとんど経由することなく発現抑制効果を発揮していることが示された。
As a result, the positive control gap P / C reduced the amount of RelA mRNA to 10% level as compared to the control NTS (FIG. 21). On the other hand, 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). From the above results, ASO straddling the poly (A) chain addition site and having a non-gapmer structure has 1) an effect of suppressing gene expression, and 2) an expression suppression effect with almost no degradation of mRNA It was shown that the
RelA mRNAの細胞内局在と肝毒性
RNAscopeによるin situハイブリダイゼーションを行い、HeLa細胞中に存在するRelA mRNAに関して検討を加えた。プロトコールを図22Aに示した。トランスフェクションの4時間後、細胞を2つに分け、レポータージーンアッセイとin situハイブリダイゼーション(ISH)を実施した(図22)。その結果、レポータージーンアッセイにおいて対称型gapmerの-7/+7とnon-gapmer構造のASO 1,2,8,13,14およびポジティブコントロールのgap P/Cで強い発現抑制活性を観察した。 Intracellular Localization of RelA mRNA and Hepatotoxicity In situ hybridization with RNAscope was performed to examine the RelA mRNA present in HeLa cells. The protocol is shown in FIG. 22A. Four hours after transfection, cells were split in two and reporter gene assay and in situ hybridization (ISH) were performed (FIG. 22). As a result, in the reporter gene assay, a strong expression inhibitory activity was observed in the symmetric gapmer -7 / + 7 and the non-gapmer ASO 1, 2, 8, 13, 14 and the positive control gap P / C.
RNAscopeによるin situハイブリダイゼーションを行い、HeLa細胞中に存在するRelA mRNAに関して検討を加えた。プロトコールを図22Aに示した。トランスフェクションの4時間後、細胞を2つに分け、レポータージーンアッセイとin situハイブリダイゼーション(ISH)を実施した(図22)。その結果、レポータージーンアッセイにおいて対称型gapmerの-7/+7とnon-gapmer構造のASO 1,2,8,13,14およびポジティブコントロールのgap P/Cで強い発現抑制活性を観察した。 Intracellular Localization of RelA mRNA and Hepatotoxicity In situ hybridization with RNAscope was performed to examine the RelA mRNA present in HeLa cells. The protocol is shown in FIG. 22A. Four hours after transfection, cells were split in two and reporter gene assay and in situ hybridization (ISH) were performed (FIG. 22). As a result, in the reporter gene assay, a strong expression inhibitory activity was observed in the symmetric gapmer -7 / + 7 and the
一方、ネガティブコントロールの非特異的配列に対するgapmer NTSは、抑制効果を全く示さなかった(図22B)。図23から図27は、in situハイブリダイゼーションの結果である。RNAscopeを用いてRelA mRNAとcyclohilin B mRNAの細胞内局在を解析した。-7/+7(図23)とnon-gapmer1,2,8,13,14(図24)とNTS(図26)および未処理の細胞(図27)では、核および細胞質に多数の黒いスポット(RelA mRNAの存在している場所で生じる)を検出することができた。一方、ポジティブコントロールのgap P/Cをトランスフェクションした細胞(図25)では、細胞全体でほとんどスポットを検出することができなかった。ハウスキーピング遺伝子(house keeping gene)のcyclophilin B mRNAは強く検出されているので、gap P/CのRelA mRNAに相当するスポットが無いのは実験上のミスではない。よって、gap P/Cは細胞内のRelA mRNAを効率的に分解したといえる。
On the other hand, gapmer NTS against the nonspecific sequence of the negative control showed no suppressive effect (FIG. 22B). 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. In -7 / + 7 (Figure 23), non-gapmer 1, 2, 8, 13, 14 (Figure 24) and NTS (Figure 26) and untreated cells (Figure 27), numerous black spots in the nucleus and cytoplasm It could be detected (occurring in the presence of RelA mRNA). On the other hand, in the positive control gap P / C transfected cells (Fig. 25), almost no spots could be detected in the whole cells. As the housekeeping gene cyclophilin B mRNA is strongly detected, it is not an experimental mistake that there is no spot corresponding to the gap P / C RelA mRNA. Thus, it can be said that gap P / C efficiently degraded intracellular RelA mRNA.
これに対して、発明者らの発明した対称型gapmerの-7/+7とnon-gapmerの1,2,8,13,14からなるASOは、細胞内のRelA mRNAをほとんど分解していないといえる。また、これらの結果は定量PCRの結果を強く反映していた(図21)。以上の結果より、標的mRNAのpoly(A)鎖付加部位を跨ぐように設計した対称型gapmerあるいはnon-gapmer型のASOは、標的mRNAの分解をほとんど経由せずに発現抑制効果、すなわちアンチセンス効果を発揮していることがわかった。
On the other hand, ASO consisting of -7 / + 7 of the symmetric gapmer invented by the inventors and 1, 2, 8, 13 and 14 of non-gapmer hardly decomposes RelA mRNA in cells. It can be said. In addition, these results strongly reflected the results of quantitative PCR (FIG. 21). From the above results, it is found that the symmetrical gapmer or non-gapmer ASO designed to cross over the poly (A) chain addition site of the target mRNA has an expression suppression effect, ie, antisense, with almost no degradation of the target mRNA. It turned out that the effect was exhibited.
材料と方法
核酸合成
全てのASOは株式会社ジーンデザインに合成を依頼し購入した。ASOの塩基配列は下記表1にまとめた。LNAを大文字で、DNAを小文字で記した。CがLNA化される場合はメチルシトシンをLNA化している。全ての塩基間はホスホロチオエート化されている。 Materials and Methods Nucleic Acid Synthesis All ASOs were purchased by request of Gene Design Inc. for synthesis. The nucleotide sequences of ASO are summarized in Table 1 below. LNA is in upper case and DNA in lower case. When C is LNA-ized, methyl cytosine is LNA-ized. All the bases are phosphorothioated.
核酸合成
全てのASOは株式会社ジーンデザインに合成を依頼し購入した。ASOの塩基配列は下記表1にまとめた。LNAを大文字で、DNAを小文字で記した。CがLNA化される場合はメチルシトシンをLNA化している。全ての塩基間はホスホロチオエート化されている。 Materials and Methods Nucleic Acid Synthesis All ASOs were purchased by request of Gene Design Inc. for synthesis. The nucleotide sequences of ASO are summarized in Table 1 below. LNA is in upper case and DNA in lower case. When C is LNA-ized, methyl cytosine is LNA-ized. All the bases are phosphorothioated.
細胞培養
HeLa細胞、及びNIH3T3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。 Cell culture HeLa cells and NIH3T3 cells were treated with 10% non-mobilized FBS (Gibco), penicillin-streptomycin (Life technologies) in DMEM (Dulbecco's-modified Eagle's medium; Sigma) at 37 ° C, 5% CO 2 Cultured.
HeLa細胞、及びNIH3T3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。 Cell culture HeLa cells and NIH3T3 cells were treated with 10% non-mobilized FBS (Gibco), penicillin-streptomycin (Life technologies) in DMEM (Dulbecco's-modified Eagle's medium; Sigma) at 37 ° C, 5% CO 2 Cultured.
レポータージーンアッセイ
約0.5×105 cells/mLとなるよう、HeLa細胞あるいはNIH3T3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。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。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 あるいは各種ASO, 1μL Lipofectamine2000(Ingitrogen)を100μL OptiMEM(Ingitrogen)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。 Reporter Gene Assay 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. 0.8 μg pGL4.32 [luc2P / NF-κB-RE / Hygro] plasmid, 20 ng pGL4.75 [hRluc / CMV] plasmid, siRNA or various ASO, 1 μL Lipofectamine 2000 (Ingitrogen) is mixed in 100 μL OptiMEM (Ingitrogen), and room temperature Let stand for 20 minutes. After standing, transfection was performed by adding the mixed solution to the culture solution.
約0.5×105 cells/mLとなるよう、HeLa細胞あるいはNIH3T3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。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。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 あるいは各種ASO, 1μL Lipofectamine2000(Ingitrogen)を100μL OptiMEM(Ingitrogen)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。 Reporter Gene Assay 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. 0.8 μg pGL4.32 [luc2P / NF-κB-RE / Hygro] plasmid, 20 ng pGL4.75 [hRluc / CMV] plasmid, siRNA or various ASO, 1 μL Lipofectamine 2000 (Ingitrogen) is mixed in 100 μL OptiMEM (Ingitrogen), and room temperature Let stand for 20 minutes. After standing, transfection was performed by adding the mixed solution to the culture solution.
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。
The medium in which HeLa cells were cultured was aspirated at 22 hours after transfection for activation of NF-κB, and DMEM medium containing 500 μL of 10% non-mobilized FBS, 20 ng / mL TNF-α 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.
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 activity was measured using Dual-Luciferase Reporter Assay System (Promega) as follows. The transfected HeLa cells were washed twice with 500 μL of ice-cold Phosphate-Buffered Saline (PBS) (pH 7.4), 150 μL 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 μL of the obtained supernatant was used for Luciferase assay. Luminoskan luminometer (Thermo Scientific) was used for the measurement of luminescence intensity.
定量PCR
標的遺伝子の発現量はGoTaq qPCR Master Mix(Promega)あるいは、KAPA SYBR Fast qPCR Kit(日本ジェネティクス)を用いて、StepOnePlus real-time PCR(Life Technologies)で測定した。GoTaq qPCR Master Mixを用いた場合、cDNAにGoTaq qPCR Master Mi×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。 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 μL of GoTaq qPCR Master Mi, 5 pmol of gene specific Forward primer and Reverse primer to cDNA.
標的遺伝子の発現量はGoTaq qPCR Master Mix(Promega)あるいは、KAPA SYBR Fast qPCR Kit(日本ジェネティクス)を用いて、StepOnePlus real-time PCR(Life Technologies)で測定した。GoTaq qPCR Master Mixを用いた場合、cDNAにGoTaq qPCR Master Mi×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。 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 μL of GoTaq qPCR Master Mi, 5 pmol of gene specific Forward primer and Reverse primer to cDNA.
初期熱変性 95℃ 5分
PCR反応 (50 cycle) 95℃ 30秒(熱変性)
55℃ 60秒(アニーリング)
72℃ 30秒(伸長) Initial heat denaturation 95 °C 5 minutes
PCR reaction (50 cycles) 95 °C 30 seconds (heat denaturation)
55 °C 60 seconds (annealing)
72 °C 30 seconds (stretching)
PCR反応 (50 cycle) 95℃ 30秒(熱変性)
55℃ 60秒(アニーリング)
72℃ 30秒(伸長) Initial heat denaturation 95 °
PCR reaction (50 cycles) 95 °
55 °
72 °
KAPA SYBR Fast qPCR Kitを用いた場合、cDNAにKAPA qPCR Master Mix 10μL, ROX High Reference Dye 0.4μL, 遺伝子特異的 Forward primer及びReverse primerを4pmol加え以下のプロファイルでサイクルを開始した。
When KAPA SYBR Fast qPCR Kit was used, 10 μL of KAPA qPCR Master Mix, 0.4 μL of ROX High Reference Dye, 4 pmol of gene-specific Forward primer and Reverse primer were added to the cDNA, and the cycle was started with the following profile.
初期熱変性 95℃ 30秒
PCR反応 (40 cycle) 95℃ 5秒(熱変性)
60℃ 30秒(アニーリング・伸長) Initial heat denaturation 95 °C 30 seconds
PCR reaction (40 cycles) 95 °C 5 seconds (heat denaturation)
60 °C 30 seconds (annealing / elongation)
PCR反応 (40 cycle) 95℃ 5秒(熱変性)
60℃ 30秒(アニーリング・伸長) Initial heat denaturation 95 °
PCR reaction (40 cycles) 95 °
60 °
標的遺伝子の増幅に用いたプライマー配列は以下の通りである。
ヒトRelA:5’-GCAGTTTGATGATGAAGACC-3’(forward、配列番号18),5’-CTGTCACTAGGCGAGTTA-3’(reverse、配列番号19)
ヒトβ-actin(内在性コントロール):5’-GATAGCATTGCTTTCGTGTA-3’(forward、配列番号20),5’-TTCAACTGGTCTCAAGTCAG-3’(reverse、配列番号21)
マウスApoB:5’-GCTCAACTCAGGTTACCGTGA-3’(forward、配列番号22),5’-AGGGTGTACTGGCAAGTTTGG-3’(reverse、配列番号23)
マウスSpt5(内在性コントロール):5’-GGTCCTACTGAGCATTGATGGTGAG-3’(forward、配列番号24),5’-TCAGGCTTCCAGGAGCTTCCCTAGG-3’(reverse、配列番号25) 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 β-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'- TCAGGCTTCCAGGAGCTTCCCTAGG-3' (reverse, SEQ ID NO: 25)
ヒトRelA:5’-GCAGTTTGATGATGAAGACC-3’(forward、配列番号18),5’-CTGTCACTAGGCGAGTTA-3’(reverse、配列番号19)
ヒトβ-actin(内在性コントロール):5’-GATAGCATTGCTTTCGTGTA-3’(forward、配列番号20),5’-TTCAACTGGTCTCAAGTCAG-3’(reverse、配列番号21)
マウスApoB:5’-GCTCAACTCAGGTTACCGTGA-3’(forward、配列番号22),5’-AGGGTGTACTGGCAAGTTTGG-3’(reverse、配列番号23)
マウスSpt5(内在性コントロール):5’-GGTCCTACTGAGCATTGATGGTGAG-3’(forward、配列番号24),5’-TCAGGCTTCCAGGAGCTTCCCTAGG-3’(reverse、配列番号25) 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 β-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'- TCAGGCTTCCAGGAGCTTCCCTAGG-3' (reverse, SEQ ID NO: 25)
ウエスタンブロット
HeLa 細胞を300μLのRIPA buffer(25mM Tris・HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS; Pierce)に1×Halt Protease Inhibitor Cocktail-EDTA-Free(Thermo Fisher Scientific)を加えたもので懸濁後、Handy Sonic UR-20P (TOMY)を用いてソニケーションを行った。その後、4℃,15,000gで10分間遠心分離し、上清を得た。得られた上清を用い、ウエスタンブロットを行った。一次抗体としてNF-κB p65 Antibody (C-20): sc-372(Santa Cruz Biotech)、及びMonoclonal Anti-γ-Tubulin antibody produced in mouse(Sigma)を用いた。二次抗体にはECL Anti-Rabbit IgG, HRP-Linked F(ab)2 Fragment (GE healthcare)、及びpolyclonal rabbit anti mouse immunoglobulins/hrp(DAKO)を用いた。検出にはLuminata Classico Western HRP(Millipore)を用い、LPR-140EX(AISIN)で可視化した。 Western Blot HeLa cells in 300 μL of RIPA buffer (25 mM Tris · HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS; Pierce) in 1 × Halt Protease Inhibitor Cocktail-EDTA-Free (Thermo) After suspension with the addition of Fisher Scientific), sonication was performed using Handy Sonic UR-20P (TOMY). Thereafter, it was centrifuged at 15,000 g at 4 ° C. for 10 minutes to obtain a supernatant. Western blot was performed using the obtained supernatant. As primary antibodies, NF-κB p65 Antibody (C-20): sc-372 (Santa Cruz Biotech), and Monoclonal Anti-γ-Tubulin Antibody Produced in Mouse (Sigma) were used. As secondary antibodies, 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).
HeLa 細胞を300μLのRIPA buffer(25mM Tris・HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS; Pierce)に1×Halt Protease Inhibitor Cocktail-EDTA-Free(Thermo Fisher Scientific)を加えたもので懸濁後、Handy Sonic UR-20P (TOMY)を用いてソニケーションを行った。その後、4℃,15,000gで10分間遠心分離し、上清を得た。得られた上清を用い、ウエスタンブロットを行った。一次抗体としてNF-κB p65 Antibody (C-20): sc-372(Santa Cruz Biotech)、及びMonoclonal Anti-γ-Tubulin antibody produced in mouse(Sigma)を用いた。二次抗体にはECL Anti-Rabbit IgG, HRP-Linked F(ab)2 Fragment (GE healthcare)、及びpolyclonal rabbit anti mouse immunoglobulins/hrp(DAKO)を用いた。検出にはLuminata Classico Western HRP(Millipore)を用い、LPR-140EX(AISIN)で可視化した。 Western Blot HeLa cells in 300 μL of RIPA buffer (25 mM Tris · HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS; Pierce) in 1 × Halt Protease Inhibitor Cocktail-EDTA-Free (Thermo) After suspension with the addition of Fisher Scientific), sonication was performed using Handy Sonic UR-20P (TOMY). Thereafter, it was centrifuged at 15,000 g at 4 ° C. for 10 minutes to obtain a supernatant. Western blot was performed using the obtained supernatant. As primary antibodies, NF-κB p65 Antibody (C-20): sc-372 (Santa Cruz Biotech), and Monoclonal Anti-γ-Tubulin Antibody Produced in Mouse (Sigma) were used. As secondary antibodies, 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はGeneRacer Kit(Invitrogen)を用いて以下のように行った。各サンプルから調製したtotal RNA 300ngに90ng GeneRacer OligodT, 1μL dNTP Mixture(2.5mM each)、水を加えて全量を7μLにした。混合液を65℃で5分間静置後、氷上で5分間静置した。氷冷したサンプルに、5×First strand buffer 4μL, 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。混合液を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。 3 'RACE
3 'RACE was performed as follows using GeneRacer Kit (Invitrogen). 90 ng GeneRacer Oligogo T, 1 μL dNTP Mixture (2.5 mM each) and water were added to 300 ng of total RNA prepared from each sample to make thetotal volume 7 μL. The mixture was allowed to stand at 65 ° C. for 5 minutes and then allowed to stand on ice for 5 minutes. To the ice-cold sample, 4 μL of 5 × First strand buffer, 1 mL of 0.1 M DTT, 1 μL of 10 mM dNTP, 0.5 μL of RNasin Plus RNase Inhibitor (Promega), 0.5 μL of SuperScript III Reverse Transcriptase, and water were added to a total volume of 20 μL. The mixture was incubated at 50 ° C. for 60 minutes and then at 70 ° C. for 30 minutes to synthesize cDNA.
3’RACEはGeneRacer Kit(Invitrogen)を用いて以下のように行った。各サンプルから調製したtotal RNA 300ngに90ng GeneRacer OligodT, 1μL dNTP Mixture(2.5mM each)、水を加えて全量を7μLにした。混合液を65℃で5分間静置後、氷上で5分間静置した。氷冷したサンプルに、5×First strand buffer 4μL, 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。混合液を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。 3 'RACE
3 'RACE was performed as follows using GeneRacer Kit (Invitrogen). 90 ng GeneRacer Oligogo T, 1 μL dNTP Mixture (2.5 mM each) and water were added to 300 ng of total RNA prepared from each sample to make the
合成したcDNA 1μLに10×PCR buffer for KOD plus 5μL, 2mM dNTPs 5μL, 25mM MgSO4 2μL, KOD plus(Toyobo)0.5μL, 遺伝子特異的Forward primer及びReverse primerを20pmolずつ、水を加えて全量を50μLにし、以下のプロファイルでサイクルを行った。
To 1 μL of the synthesized cDNA, add 5 μL of 10 × PCR buffer for KOD plus, 5 μL of 2 mM dNTPs, 2 μL of 25 mM MgSO 4 , 0.5 μL of KOD plus (Toyobo), 20 pmol of gene-specific Forward primer and Reverse primer, and add 50 μL of total water And cycled with the following profile:
初期熱変性 94℃ 5分
PCR反応 (30 cycle) 94℃ 30秒(熱変性)
60℃ 30秒(アニーリング)
68℃ 60秒(伸長) Initial heat denaturation 94 °C 5 minutes
PCR reaction (30 cycles) 94 °C 30 seconds (heat denaturation)
60 °C 30 seconds (annealing)
68 °C 60 seconds (stretching)
PCR反応 (30 cycle) 94℃ 30秒(熱変性)
60℃ 30秒(アニーリング)
68℃ 60秒(伸長) Initial heat denaturation 94 °
PCR reaction (30 cycles) 94 °
60 °
68 °
1st PCR産物を用いてNested PCRを行った。100倍希釈した1st PCR産物に10× ExTaq buffer 5μL, dNTP Mixture (2.5mM each) 4μL, ExTaq 0.25μL,遺伝子特異的Forward primer及びReverse primerを20pmolずつ、水を加えて全量を50μLにし、以下のプロファイルでサイクルを行った。
Nested PCR was performed using the 1st PCR product. Add 100 μl of 1 × PCR product to 5 μl of 10 × ExTaq buffer, 4 μl of dNTP Mix (2.5 mM each), 0.25 μl of ExTaq, 20 pmol of gene-specific Forward primer and Reverse primer, and make the total amount 50 μl. I cycled in my profile.
初期熱変性 98℃ 5分
PCR反応 (30 cycle) 98℃ 30秒(熱変性)
60℃ 30秒(アニーリング)
72℃ 60秒(伸長) Initial heat denaturation 98 °C 5 minutes
PCR reaction (30 cycles) 98 °C 30 seconds (heat denaturation)
60 °C 30 seconds (annealing)
72 °C 60 seconds (stretching)
PCR反応 (30 cycle) 98℃ 30秒(熱変性)
60℃ 30秒(アニーリング)
72℃ 60秒(伸長) Initial heat denaturation 98 °
PCR reaction (30 cycles) 98 °
60 °
72 °
PCR産物を1.2%アガロースゲルで電気泳動し、切出精製を行った。切出精製にはFastGene Gel/PCR Extraction(日本ジェネティクス)を用い、プロトコールに従って精製した。
The PCR product was electrophoresed on a 1.2% agarose gel and cut out and purified. For excision and purification, FastGene Gel / PCR Extraction (Nippon Genetics) was used and purified according to the protocol.
切出精製した産物にExTaqを用いてA-tailing反応を行った。切出精製したPCR産物25μLに10 x ExTaq buffer 5μL, 10mM dATP(NEB) 1μL, ExTaq 0.25μL, 水を加え全量を50μLにし70℃で30分間静置した。A-tailing反応を行った産物はFastGene Gel/PCR Extraction(日本ジェネティクス)を用い、プロトコールに従って精製した。
The cut-out and purified product was subjected to A-tailing reaction using ExTaq. To 25 μL of the excised and purified PCR product, 5 μL of 10 × ExTaq buffer, 1 μL of 10 mM dATP (NEB), 0.25 μL of ExTaq, and water were added to make the total volume 50 μL, 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).
A-tailing反応を行った産物をpGEM T-easy vectorにライゲーションした。A-tailing反応を行った産物1μLにT4 ligation buffer 5μL, pGEM T-easy(Promega)1ng, T4 ligase(Promega)1μL、水を加えて全量を10μLにし、室温で60分間静置した。静置後、反応液全量をDH5a(Toyobo)に加え、氷中で30分静置した。静置後、42℃の水浴で45秒間ヒートショックを与え氷中で2分以上静置した。あらかじめ、37℃に保温したSOC培地を1mL加え、37℃で60分間静置した。静置後、アンピシリン(50μg/mL)を含むLB培地に播種した。37℃で一晩静置後、得られたコロニーからQiaprep miniprep kit(Qiagen)を用い、プロトコールに従ってプラスミドを調製した。調製したプラスミドはユーロフィン株式会社にシークエンス解析サービス依頼し、クローニング部位の塩基配列を決定した。
The product subjected to the A-tailing reaction was ligated to pGEM T-easy vector. To 1 μL of the product subjected to the A-tailing reaction, 5 μL of T4 ligation buffer, 1 ng of pGEM T-easy (Promega), 1 μL of T4 ligase (Promega), and water were added to make the total volume 10 μL, 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. 1 mL of SOC medium previously kept at 37 ° C. was added, and left at 37 ° C. for 60 minutes. After standing, it was inoculated on LB medium containing ampicillin (50 μg / mL). After standing overnight at 37 ° C., 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 ハイブリダイゼーション
in situ ハイブリダイゼーションはRNAscopeHD reagent-BROWN(Advanced cell diagnostics)を使用し、以下のように行った。 In situ Hybridization In situ hybridization was performed as follows using RNAscopeHD reagent-BROWN (Advanced cell diagnostics).
in situ ハイブリダイゼーションはRNAscopeHD reagent-BROWN(Advanced cell diagnostics)を使用し、以下のように行った。 In situ Hybridization In situ hybridization was performed as follows using RNAscopeHD reagent-BROWN (Advanced cell diagnostics).
ミリセルEZスライド(millipore)上で培養した細胞をPBSで洗浄後10%中性緩衝ホルマリンで30分間、室温で固定した。その後、50%エタノール、70%エタノール、100%エタノールに段階的に浸漬し、脱水した。次に、70%エタノール、50%エタノール、PBSに段階的に浸漬することで再水和した。水和した試料の水分を軽く切り、試料に直接過酸化水素水を滴下し、室温で10分間静置した。静置した試料をPBSで洗浄した後、ProteasePlusを試料に直接滴下し、室温で10分間静置した。試料をPBSで洗浄後、遺伝子特異的プローブを滴下し、40℃で2時間静置した。試料をwash バッファーで洗浄し、AMP1試薬からAMP6試薬で順次処理した。AMP6試薬処理を行った後、washバッファーで洗浄し、DAB試薬を試料に滴下し、室温で10分間静置した。試料を蒸留水で洗浄後、マイヤーヘマトキシリン(Merck)でカウンター染色した。染色後、エタノール で脱水し、レモゾール(nacalai tesque)で透徹を行った。試料をマウントクイック(大道産業株式会社)で封入し、顕微鏡で観察した。
Cells cultured on Millicell EZ slides (millipore) were washed with PBS and fixed with 10% neutral buffered formalin for 30 minutes at room temperature. Thereafter, it was immersed stepwise in 50% ethanol, 70% ethanol, 100% ethanol and dehydrated. Next, it was rehydrated by stepwise immersion in 70% ethanol, 50% ethanol and PBS. The moisture of the hydrated sample was lightly removed, hydrogen peroxide solution was dropped directly to the sample, and the sample was allowed to stand at room temperature for 10 minutes. After washing the stationary sample with PBS, Protease Plus was dropped directly on the sample and allowed to stand at room temperature for 10 minutes. After washing the sample with PBS, a gene specific probe was dropped and allowed to stand at 40 ° C. for 2 hours. 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.
マウスの飼育
5~6週齢の雌Jc1:ICRマウスを日本クレア株式会社(以下、クレア)から購入し、コンベショナルな条件下で飼育した。実験開始まで1週間以上の順化期間を設けた。マウスは、23±2℃で、湿度60%±5%、12時間おきの明暗サイクルで飼育した。標準的な飼料(CE-2、クレア)と水は、自由に食する条件にした。すべての実験は、2006年に日本学術会議が策定した動物実験の適正な実施に向けたガイドラインに従って行った。本実験のプロトコールは、国立大学法人大阪大学の実験動物委員会の承認を得ている(承認No. 25-1-1)。 Breeding ofmice 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).
5~6週齢の雌Jc1:ICRマウスを日本クレア株式会社(以下、クレア)から購入し、コンベショナルな条件下で飼育した。実験開始まで1週間以上の順化期間を設けた。マウスは、23±2℃で、湿度60%±5%、12時間おきの明暗サイクルで飼育した。標準的な飼料(CE-2、クレア)と水は、自由に食する条件にした。すべての実験は、2006年に日本学術会議が策定した動物実験の適正な実施に向けたガイドラインに従って行った。本実験のプロトコールは、国立大学法人大阪大学の実験動物委員会の承認を得ている(承認No. 25-1-1)。 Breeding of
ASOの投与
ASOは尾静脈あるいは腹腔から図中および明細書中に示したそれぞれの量及び日程でマウスに投与した。図12~14に示すMM 13mer ASOの条件検討を行った実験では、最初の核酸投与日をDay.0として2回目の投与をDay.7に行い、採血はDay.-1、3、7、10日に行い各種コレステロール値を測定した。Day.10についてはAST値及びALT値の測定も行った。核酸は1匹あたり0.5、0.1、0.02、0.004mgになるように尾静脈より注射した(各2個体)。0.5mg容量のみ2回目の投与を行わなかった(1個体)。図15~18に示す本発明の核酸を投与した実験では、核酸を投与した日をDay.0とするとDay.-5及びDay.3に採血を行い、コレステロール値の測定を行った。Day.3に関してはAST値及びALT値の測定を行った。MM 13mer ASOのみ0.02mg/匹(2個体)、他の核酸は0.5mg/匹の容量で腹腔内投与した(各4個体)。全ての実験において、陰性対照としてPBSを投与した(2個体)。 Administration of 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. In the experiment in which the condition of MM 13mer ASO shown in FIGS. 12 to 14 was examined, the second administration was performed onDay 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). In the experiments in which the nucleic acid of the present invention shown in FIGS. 15-18 was administered, blood was collected on Day.-5 and Day.3 assuming that the day on which the nucleic acid was administered was Day. 0, and the cholesterol level was measured. The AST value and ALT value were measured for Day.3. MM 13 mer ASO alone was intraperitoneally administered at a volume of 0.02 mg / animal (2 individuals) and the other nucleic acid at a volume of 0.5 mg / animal (4 individuals each). PBS was administered as a negative control in all experiments (2 individuals).
ASOは尾静脈あるいは腹腔から図中および明細書中に示したそれぞれの量及び日程でマウスに投与した。図12~14に示すMM 13mer ASOの条件検討を行った実験では、最初の核酸投与日をDay.0として2回目の投与をDay.7に行い、採血はDay.-1、3、7、10日に行い各種コレステロール値を測定した。Day.10についてはAST値及びALT値の測定も行った。核酸は1匹あたり0.5、0.1、0.02、0.004mgになるように尾静脈より注射した(各2個体)。0.5mg容量のみ2回目の投与を行わなかった(1個体)。図15~18に示す本発明の核酸を投与した実験では、核酸を投与した日をDay.0とするとDay.-5及びDay.3に採血を行い、コレステロール値の測定を行った。Day.3に関してはAST値及びALT値の測定を行った。MM 13mer ASOのみ0.02mg/匹(2個体)、他の核酸は0.5mg/匹の容量で腹腔内投与した(各4個体)。全ての実験において、陰性対照としてPBSを投与した(2個体)。 Administration of 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. In the experiment in which the condition of MM 13mer ASO shown in FIGS. 12 to 14 was examined, the second administration was performed on
コレステロールやAST、ALT値の測定
Goldenrod ANIMAL LANCET(Bio Research Center)を用いてマウスの頬より採血を行い、BD Microtainer Tubes(Becton, Dickinson and Company)中に血液を回収後15分~30分以内の間に10,000rpm、室温で2分間遠心して血清を分離した。血清はオリエンタル酵母工業株式会社に送付し、総コレステロール、LDL-コレステロール、HDL-コレステロール、AST、ALTそれぞれの数値を測定した。 Measurement of cholesterol, AST and ALT levels Blood was collected from the cheeks of mice using Goldenrod ANIMAL LANCET (Bio Research Center), and within 15 to 30 minutes after the blood was collected in BD Microtainer Tubes (Becton, Dickinson and Company) The serum was separated by centrifugation at 10,000 rpm for 2 minutes at room temperature. The serum was sent to Oriental Yeast Co., Ltd., and the values of total cholesterol, LDL-cholesterol, HDL-cholesterol, AST and ALT were measured.
Goldenrod ANIMAL LANCET(Bio Research Center)を用いてマウスの頬より採血を行い、BD Microtainer Tubes(Becton, Dickinson and Company)中に血液を回収後15分~30分以内の間に10,000rpm、室温で2分間遠心して血清を分離した。血清はオリエンタル酵母工業株式会社に送付し、総コレステロール、LDL-コレステロール、HDL-コレステロール、AST、ALTそれぞれの数値を測定した。 Measurement of cholesterol, AST and ALT levels Blood was collected from the cheeks of mice using Goldenrod ANIMAL LANCET (Bio Research Center), and within 15 to 30 minutes after the blood was collected in BD Microtainer Tubes (Becton, Dickinson and Company) The serum was separated by centrifugation at 10,000 rpm for 2 minutes at room temperature. The serum was sent to Oriental Yeast Co., Ltd., and the values of total cholesterol, LDL-cholesterol, HDL-cholesterol, AST and ALT were measured.
RNA抽出
培養細胞あるいは摘出したマウスの肝臓を、セパゾールRNA IスーパーG(ナカライテスク)により説明書に従って処理した。マウス肝臓組織よりRNAを抽出する場合は、ビーズ式細胞破砕装置Micro SmashTM(TOMY)を用いて4,500rpm、10秒で1回破砕処理を行った。チューブには3.0σのジルコニアビーズを各3個加えた。破砕後トップスピード(17,900g)で4℃、5分間遠心して上清だけ回収し組織片を取り除いた。RNAの精製は、Direct-zol RNA MiniPrep (Zymo Research)により説明書に従って精製した。続いて、50~1000ngのRNAを用いて50~500ng oligo (dT)あるいはrandom primerとdNTP Mixture(最終濃度0.77mM)の混合液(13μL)を65℃で5分間静置後、氷上で5分静置した。氷冷したサンプルに、5×First strand buffer 4μL、0.1M DTT 1μL、RNasin Plus RNase Inhibitor(Promega)1μL、SuperScript III Reverse Transcriptase(逆転写酵素)1μLをそれぞれ加えて全量を20μLにした。混合液を50℃で30分から60分インキュベート後、70℃で30分あるいは80℃で10分インキュベートして逆転写酵素を失活させ、cDNAとした。 RNA Extraction Cultured cells or excised mouse liver were treated with Sepazol RNA I super G (Nacalai Tesque) according to the instructions. When 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. Then, 50-500 ng oligo (dT) or a mixture (13 μL) of random primer and dNTP Mixture (final concentration 0.77 mM) using 50 to 1000 ng of RNA for 5 minutes at 65 ° C, then on ice for 5 minutes Let stand. To the ice-cold sample, 4 μL of 5 × First strand buffer, 1 μL of 0.1 M DTT, 1 μL of RNasin Plus RNase Inhibitor (Promega), and 1 μL of SuperScript III Reverse Transcriptase (reverse transcriptase) were added to make the total volume to 20 μL. The mixture was incubated at 50 ° C. for 30 to 60 minutes, and then incubated at 70 ° C. for 30 minutes or at 80 ° C. for 10 minutes to inactivate the reverse transcriptase to obtain cDNA.
培養細胞あるいは摘出したマウスの肝臓を、セパゾールRNA IスーパーG(ナカライテスク)により説明書に従って処理した。マウス肝臓組織よりRNAを抽出する場合は、ビーズ式細胞破砕装置Micro SmashTM(TOMY)を用いて4,500rpm、10秒で1回破砕処理を行った。チューブには3.0σのジルコニアビーズを各3個加えた。破砕後トップスピード(17,900g)で4℃、5分間遠心して上清だけ回収し組織片を取り除いた。RNAの精製は、Direct-zol RNA MiniPrep (Zymo Research)により説明書に従って精製した。続いて、50~1000ngのRNAを用いて50~500ng oligo (dT)あるいはrandom primerとdNTP Mixture(最終濃度0.77mM)の混合液(13μL)を65℃で5分間静置後、氷上で5分静置した。氷冷したサンプルに、5×First strand buffer 4μL、0.1M DTT 1μL、RNasin Plus RNase Inhibitor(Promega)1μL、SuperScript III Reverse Transcriptase(逆転写酵素)1μLをそれぞれ加えて全量を20μLにした。混合液を50℃で30分から60分インキュベート後、70℃で30分あるいは80℃で10分インキュベートして逆転写酵素を失活させ、cDNAとした。 RNA Extraction Cultured cells or excised mouse liver were treated with Sepazol RNA I super G (Nacalai Tesque) according to the instructions. When 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. Then, 50-500 ng oligo (dT) or a mixture (13 μL) of random primer and dNTP Mixture (final concentration 0.77 mM) using 50 to 1000 ng of RNA for 5 minutes at 65 ° C, then on ice for 5 minutes Let stand. To the ice-cold sample, 4 μL of 5 × First strand buffer, 1 μL of 0.1 M DTT, 1 μL of RNasin Plus RNase Inhibitor (Promega), and 1 μL of SuperScript III Reverse Transcriptase (reverse transcriptase) were added to make the total volume to 20 μL. The mixture was incubated at 50 ° C. for 30 to 60 minutes, and then incubated at 70 ° C. for 30 minutes or at 80 ° C. for 10 minutes to inactivate the reverse transcriptase to obtain cDNA.
mRNAのpoly(A)鎖付加部位を跨ぐ対称型gapmerが、血清中のAST値やALT値の上昇をほとんど誘導しない理由
対称型gapmer-7/+7を従来のgapmer(MM13mer)と比較すると、gapmer-7/+7からなるASOをマウスに投与した場合、肝毒性の指標となる血清中のAST値およびALT値の上昇が顕著に抑えられていた(図14と17)。 The reason why the symmetric gapmer across the poly (A) chain addition site of mRNA hardly induces the elevation of the AST value or ALT value in serum When comparing the symmetric gapmer-7 / + 7 with the conventional gapmer (MM13mer), When ASO consisting of gapmer-7 / + 7 was administered to mice, the elevation of serum AST and ALT levels, which is an index of hepatotoxicity, was significantly suppressed (FIGS. 14 and 17).
対称型gapmer-7/+7を従来のgapmer(MM13mer)と比較すると、gapmer-7/+7からなるASOをマウスに投与した場合、肝毒性の指標となる血清中のAST値およびALT値の上昇が顕著に抑えられていた(図14と17)。 The reason why the symmetric gapmer across the poly (A) chain addition site of mRNA hardly induces the elevation of the AST value or ALT value in serum When comparing the symmetric gapmer-7 / + 7 with the conventional gapmer (MM13mer), When ASO consisting of gapmer-7 / + 7 was administered to mice, the elevation of serum AST and ALT levels, which is an index of hepatotoxicity, was significantly suppressed (FIGS. 14 and 17).
レポータージーンアッセイで対称型gapmer-7/+7と従来のgapmer(gap P/C)は強い発現抑制活性を示しているので、抑制効果は両者間で同等であるといえる。興味深いことにin situハイブリダイゼーションの結果から、細胞内のRelA mRNAの局在とその存在量に対する影響は両者で大きく異なっていることがわかった(図28)。図28Aは、ASO -7/+7をトランスフェクションしたHeLa細胞内のRelA mRNAの局在とASO -7/+7の関係を示し、図28Bはgap P/CをトランスフェクションしたHeLa細胞内のRelA mRNAの局在とgap P/Cの細胞内での振る舞いを示す。
In the reporter gene assay, the symmetric gapmer-7 / + 7 and the conventional gapmer (gap P / C) show strong expression suppression activity, so the suppression effect can be said to be equal between the two. Interestingly, the results of in situ hybridization revealed that the localization of RelA mRNA in cells and the influence on its abundance were significantly different between the two (FIG. 28). 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.
すなわち、-7/+7はRelA mRNAの局在や存在量にほとんど影響を与えていない(図23)。しかし、gap P/CはRelA mRNA量を顕著に減じていた(図25)。これらを考察すると、-7/+7をトランスフェクションした細胞内にはRelA mRNAが存在し続けているので、ASOである-7/+7はRelA mRNAと持続的にハイブリダイゼーションしている。また、RelA mRNAの翻訳反応が効率的に抑制されていることからも、ハイブリダイゼーションが細胞中で継続していることは明らかである。
That is, -7 / + 7 hardly affected the localization or abundance of RelA mRNA (Fig. 23). However, gap P / C significantly reduced the amount of RelA mRNA (FIG. 25). In consideration of these, since RelA mRNA continues to be present in cells transfected with -7 / + 7, ASO -7 / + 7 is continuously hybridized with RelA mRNA. In addition, it is also clear that hybridization continues in cells, because the translation reaction of RelA mRNA is efficiently suppressed.
一般にASO、すなわち一本鎖のアンチセンス核酸が標的mRNAにハイブリダイズして形成された2本鎖は、非常に安定である。加熱、すなわちASOのTmを上回る温度になるか、あるいは強いアルカリ性を示すような物質で処理しないと2本鎖が解け一本鎖状態に戻ることはない。
In general, ASO, that is, a double strand formed by hybridizing a single-stranded antisense nucleic acid to a target mRNA, 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.
また、gapmer -7/+7とRelA mRNA間で作られた2本鎖を特異的に解離させるような活性を有するヘリケース(helicase)の存在と関与が考えられるが、上述したようにRelA mRNAが細胞質に存在し続けているのにgapmer -7/+7は翻訳を抑制した状態を保つことができているので(図22)、ヘリケースが2本鎖状態を解消したとは考えられない。
In addition, although the presence and involvement of helicase (helicase) having an activity to specifically dissociate the double strand formed between gapmer -7 / + 7 and RelA mRNA may be considered, as described above, 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.
よって、gapmer -7/+7をトランスフェクションした細胞内にRelA mRNA が存在し続けている以上、両者間で形成される2本鎖状態は存在し続けているといえる(図28A)。一方、gap P/Cのin situハイブリダイゼーションの結果は、RNaseH1活性で標的のmRNA(本発明ではRelA mRNA)が効率的に分解したことを明確に示している。そうするとgap P/Cの標的のmRNAは細胞中に存在しない。要するにgap P/Cは一旦標的であるRelA mRNAと2本鎖状態を形成するが、RNaseH1活性によりRelA mRNAの分解が生じるため、この2本鎖状態は解消し、結局、ASOであるgap P/Cは細胞内で一本鎖の状態に戻る。一本鎖状態となったgap P/Cは本来の標的であるRelA mRNAが細胞内に存在しないので、RelA mRNAと類似のアンチセンス配列を有するoff target mRNAに対してハイブリダイズし、2本鎖が形成され、RNaseH1により分解が生じる(図28B)。そして、再びASO gap P/Cは1本鎖状態に戻る。この連鎖反応により、次々に標的以外のoff target mRNAの分解が生じてしまう。
Therefore, as long as RelA mRNA continues to be present in the gapmer-7 / + 7-transfected cells, it can be said that the double-stranded state formed between the two continues (FIG. 28A). On the other hand, the results of in situ hybridization of gap P / C clearly show that RNase H1 activity efficiently degraded the target mRNA (in the present invention, RelA mRNA). Then the gap P / C target mRNA is not present in the cell. In short, 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.
Burelらや粕谷らの論文では、肝毒性の主因はgapmerのoff target効果、すなわち標的以外のmRNAでASOの標的配列と似た配列を有したmRNAのRNaseH1依存的な分解だとしている(Burel, S. A., et. al. Nucleic Acid Res., 44:2093-2109, 2016., Kasuya, T., et. al. Sci. Rep., 27;6:30377, 2016.)。
In the article by Burel et al. And Tsuchiya et al., 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.).
然るに、本発明者らの発明した対称型gapmerは標的を分解しないという特性から、gapmer-標的mRNA間の2本鎖状態は継続し、その結果、細胞内で容易にgapmerが一本鎖状態に戻ることはない。そのためoff target効果の可能性は低く、結果として肝毒性の誘導がきわめて低いレベルに抑えられていると考えられる。よって、図19にあるようにpoly(A)鎖付加部位を跨ぐ対称型gapmerは、血液中のAST値やALT値の上昇をほとんど誘導しなかったものと考察することができる。
However, due to the property that 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.
さらに、マウスAcsl1遺伝子のmRNAに対して、上記粕谷らの論文(Kasuya, T., et. al. Sci. Rep., 27;6:30377, 2016.)の情報に従ってgapmer型ASOとnon-gapmer型ASOを準備した。具体的には、gapmer型ASO(表2、SGgapAcsl1)およびそのASOの中央部分の12残基のDNAが連続する箇所の中央に2残基のLNAを導入したASO(表2、SGnon-gapAcsl1)を準備した。さらに、我々は粕谷らの設計したASOと同じ構造を有するASOで、その標的配列のみを変えて準備した。具体的には、Acsl1 mRNAのpoly(A)鎖が付加される部分を跨ぐ対称型ASOのgapmer型(3’-gapAcsl1)およびnon-gapmer型(3’-non-gapAcsl1)を準備した(表2)。各ASOは、株式会社ジーンデザインに合成を依頼し購入した。CがLNA化される場合は、メチルシトシンをLNA化した。全ての塩基間をホスホロチオエート化した。
Furthermore, according to the information of Kasuya et al. (Kasuya, T., et. Al. Sci. Rep., 27; 6: 30377, 2016.), the gapmer-type ASO and non-gapmer against mRNA of mouse Acsl1 gene. Prepared 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. Furthermore, we 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.
表2に加えて、3’-gapAcsl1および3’-non-gapAcsl1の塩基配列を配列番号33に、そしてSGgapAcsl1およびSGnon-gapAcsl1の塩基配列を配列番号34に示す(gapmer型とnon-gapmer型とでは、LNA化した塩基が異なる以外は同じ塩基配列である)。以下の表2では、LNAを大文字で、DNAを小文字で記した。3’-gapAcsl1および3’-non-gapAcsl1では、10番目の塩基(3’-gapAcsl1の「t」および3’-non-gapAcsl1の「T」)が、その標的mRNAにおいて切断され、ポリAが付加されるmRNAの3’-末端の塩基である「mRNAの3’-末端」に対して結合し得る位置の塩基である。
In addition to Table 2, the 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). In Table 2 below, LNA is in upper case and DNA in lower case. For 3'-gapAcsl1 and 3'-non-gapAcsl1, 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.
手法:
ICRマウスに腹腔から0.5mg/匹の用量でASOを投与した。ネガティブコントロールとしてPBSを投与した。それぞれ3個体ずつ解析した。 Technique:
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.
ICRマウスに腹腔から0.5mg/匹の用量でASOを投与した。ネガティブコントロールとしてPBSを投与した。それぞれ3個体ずつ解析した。 Technique:
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.
1回目のASO投与をDay.0とすると、投与の2日前(Day.-2)と投与から4日後(Day.4)に採血を行い、肝毒性の指標であるAST値およびALT値を確認した(図30および31)。また、Day.4においてマウス肝臓組織を回収し、RNAを抽出して組織中に残されたAcsl1 RNA量を定量PCRにより測定した(図29)。Acsl1遺伝子の発現量はそれぞれの個体のSpt5量により補正し、1個体あたりに含まれるAcsl1 mRNA量の相対値を表示した(図29の縦軸「relative quantity/normalized by Spt5」)。Acsl1に対するプライマーセットは下記の2種類を用いた。図29に、各プライマーセットを用いた場合の結果を示す(Primer-1のセットの結果を「Primer1」、そしてPrimer-2の結果を「Primer2」で表した)。
Assuming that the first ASO administration is Day 0, blood is collected 2 days before (Day.-2) and 4 days after (Day 4) administration to confirm the AST value and ALT value, which are indicators of hepatotoxicity (Figures 30 and 31). In addition, mouse liver tissue was recovered on day 4, RNA was extracted, and the amount of Acsl1 RNA remaining in the tissue was measured by quantitative PCR (FIG. 29). The expression level of the Acsl1 gene was corrected based on the amount of Spt5 of each individual, and the relative value of the amount of Acsl1 mRNA contained per one individual was displayed (the vertical axis "relative quantity / normalized by Spt5" in FIG. 29). The following two types of primer sets for Acsl1 were used. 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”).
定量PCRに用いたプライマーセットの各プライマーの配列は以下に示した通りである:
Acsl1 Forward Primer-1:5’-TGCCAGAGCTGATTGACATTC-3’(配列番号35)
Acsl1 Reverse Primer-1:5’-GGCATACCAGAAGGTGGTGAG-3’(配列番号36)
Acsl1 Forward Primer-2:5’-ACCAGCCCTATGAGTGGATTT-3’(配列番号37)
Acsl1 Reverse Primer-2:5’-CAAGGCTTGAACCCCTTCTG-3’(配列番号38) The sequences of each primer of the primer set used for quantitative PCR are 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)
Acsl1 Forward Primer-1:5’-TGCCAGAGCTGATTGACATTC-3’(配列番号35)
Acsl1 Reverse Primer-1:5’-GGCATACCAGAAGGTGGTGAG-3’(配列番号36)
Acsl1 Forward Primer-2:5’-ACCAGCCCTATGAGTGGATTT-3’(配列番号37)
Acsl1 Reverse Primer-2:5’-CAAGGCTTGAACCCCTTCTG-3’(配列番号38) The sequences of each primer of the primer set used for quantitative PCR are 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)
AST値、ALT値、Acsl1 mRNA量の平均値から標準偏差を計算し、エラーバーを表記した (図29B、図31)。統計解析ソフトRを用いてANOVAの分散分析を行い、Tukey法により統計学的有意差の検定を行った(***,p<0.005、*,p<0.05)。
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).
結果:
図29は、3’-gapAcsl1、3’-non-gapAcsl1、SGgapAcsl1およびSGnon-gapAcsl1のいずれかを投与したマウスの肝臓中のAcsl1 mRNA量を示し、Aはそれぞれの投与マウス個体の結果であり、そしてBは、各投与マウス個体群の平均を示す。ASOを投与したすべての個体群において、PBSを投与した個体群と比較するとAscl1発現量の有意な低下が認められた(図29B、p=0.0000063(SGgapAcsl1)、p=0.0046(SGnon-gapAcsl1)、p=0.0000055(3’-gapAcsl1)、p=000043(3’-non-gapAcsl1))。上記粕谷らの論文を参考にして合成した核酸SGnon-gapAcsl1については、gapmer型SGgapAcsl1を投与した個体群と比較して有意な差が認められた(それぞれp=0.0011(Primer1の場合), p=00091(Primer2の場合))。これに対して、本発明を基にnon-gapmer型として設計したASOを投与した個体群については、gapmer型のASOを投与した個体群と比較すると若干分解は緩やかであるように見えるものの有意な差は認められなかった(それぞれp=0.31(Primer1の場合)、p=0.25(Primer2の場合))。また、non-gapmer型として合成したASOを投与した個体群同士についても有意な差が認められた(図29B、p=0.021(SGnon-gapAcsl1 vs. 3’-non-gapAcsl1)) result:
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. 29B, p = 0.00000063 (SGgapAcsl1), p = 0.0046 (SGnon-gapAcsl1), p = 0.0000055 (3'-gapAcsl1), p = 000043 (3'-non-gapAcsl1)). Regarding the nucleic acid SGnon-gapAcsl1 synthesized with reference to the above-mentioned article by Sugaya et al., A significant difference was observed in comparison with the group to which gapmer-type SGgapAcsl was administered (in the case of p = 0.0011 (in the case of Primer1), p = 00091 (for Primer2)). On the other hand, in the population administered ASO designed as non-gapmer type based on the present invention, although the degradation seems to be mild compared with the population administered the gapmer type ASO, it is significant There was no difference (p = 0.31 (for Primer1), p = 0.25 (for Primer2)). In addition, significant differences were also observed among the groups to which ASO synthesized as non-gapmer type was administered (Fig. 29B, p = 0.021 (SG non-gap Acsl1 vs. 3'-non-gap Acsl1)).
図29は、3’-gapAcsl1、3’-non-gapAcsl1、SGgapAcsl1およびSGnon-gapAcsl1のいずれかを投与したマウスの肝臓中のAcsl1 mRNA量を示し、Aはそれぞれの投与マウス個体の結果であり、そしてBは、各投与マウス個体群の平均を示す。ASOを投与したすべての個体群において、PBSを投与した個体群と比較するとAscl1発現量の有意な低下が認められた(図29B、p=0.0000063(SGgapAcsl1)、p=0.0046(SGnon-gapAcsl1)、p=0.0000055(3’-gapAcsl1)、p=000043(3’-non-gapAcsl1))。上記粕谷らの論文を参考にして合成した核酸SGnon-gapAcsl1については、gapmer型SGgapAcsl1を投与した個体群と比較して有意な差が認められた(それぞれp=0.0011(Primer1の場合), p=00091(Primer2の場合))。これに対して、本発明を基にnon-gapmer型として設計したASOを投与した個体群については、gapmer型のASOを投与した個体群と比較すると若干分解は緩やかであるように見えるものの有意な差は認められなかった(それぞれp=0.31(Primer1の場合)、p=0.25(Primer2の場合))。また、non-gapmer型として合成したASOを投与した個体群同士についても有意な差が認められた(図29B、p=0.021(SGnon-gapAcsl1 vs. 3’-non-gapAcsl1)) result:
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. 29B, p = 0.00000063 (SGgapAcsl1), p = 0.0046 (SGnon-gapAcsl1), p = 0.0000055 (3'-gapAcsl1), p = 000043 (3'-non-gapAcsl1)). Regarding the nucleic acid SGnon-gapAcsl1 synthesized with reference to the above-mentioned article by Sugaya et al., A significant difference was observed in comparison with the group to which gapmer-type SGgapAcsl was administered (in the case of p = 0.0011 (in the case of Primer1), p = 00091 (for Primer2)). On the other hand, in the population administered ASO designed as non-gapmer type based on the present invention, although the degradation seems to be mild compared with the population administered the gapmer type ASO, it is significant There was no difference (p = 0.31 (for Primer1), p = 0.25 (for Primer2)). In addition, significant differences were also observed among the groups to which ASO synthesized as non-gapmer type was administered (Fig. 29B, p = 0.021 (SG non-gap Acsl1 vs. 3'-non-gap Acsl1)).
図30は、3’-gapAcsl1、3’-non-gapAcsl1、SGgapAcsl1およびSGnon-gapAcsl1のいずれかを投与した個々のマウスの血清中のAST値(A)およびALT値(B)の推移を示し、図31は、各投与マウス個体群における血清中のAST値(A)およびALT値(B)の推移の平均を示す。血清中AST値及びALT値を測定した結果、粕谷らの論文において顕著な肝毒性を示すことが報告されているASO(SGgapAcsl1)を投与した個体群でのみ、顕著なAST値及びALT値の上昇が認められた(図30および図31、p<0.05)。本発明のgapmer型ASO(3’-gapAcsl1)を投与した個体群についてのみ、SGgapAcsl1を投与した個体群と有意な差が認められなかった(AST値,p=0.26,ALT値,p=0.066)。
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. As a result of measuring AST level and ALT level in serum, 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. Were observed (Figures 30 and 31, p <0.05). No significant difference was found between the group receiving the gapmer type ASO (3'-gapAcsl1) of the present invention and the group receiving SGgapAcsl1 (AST value, p = 0.26, ALT value, p = 0.066). .
総括:
粕谷らの論文における報告の通り、gapmer型の核酸では著しい遺伝子発現抑制効果が認められ、non-gapmer型では抑制効果は見られなかった。本発明の核酸を投与した個体群では、gapmer型のASOは粕谷らの論文のASOと同程度の遺伝子発現抑制活性を示したものの、肝毒性は生じなかった。さらに、non-gapmer型のASOでもgapmer型ほどではないものの強い遺伝子発現抑制効果を示し、その一方で顕著な肝毒性は認められなかった。以上の結果から、本発明のpoly(A)鎖付加部位を跨ぐASOは、緩やかに標的遺伝子のmRNAを分解、あるいは分解せず、標的遺伝子の発現を抑制している可能性が示された。 Summary:
As reported in the paper by Tsuchiya et al., A significant gene expression suppression effect was observed for the gapmer type nucleic acid, and no suppression effect was seen for the non-gapmer type. In the population to which the nucleic acid of the present invention was administered, although the gapmer type ASO showed the same gene expression inhibitory activity as that of ASO of Shizutani et al., No hepatotoxicity occurred. Furthermore, even non-gapmer-type ASO showed a strong gene expression suppression effect although it was not as high as gapmer-type, while no significant hepatotoxicity was observed. From the above results, it is shown that ASO straddling the poly (A) chain addition site of the present invention may moderately or not degrade the mRNA of the target gene and suppress the expression of the target gene.
粕谷らの論文における報告の通り、gapmer型の核酸では著しい遺伝子発現抑制効果が認められ、non-gapmer型では抑制効果は見られなかった。本発明の核酸を投与した個体群では、gapmer型のASOは粕谷らの論文のASOと同程度の遺伝子発現抑制活性を示したものの、肝毒性は生じなかった。さらに、non-gapmer型のASOでもgapmer型ほどではないものの強い遺伝子発現抑制効果を示し、その一方で顕著な肝毒性は認められなかった。以上の結果から、本発明のpoly(A)鎖付加部位を跨ぐASOは、緩やかに標的遺伝子のmRNAを分解、あるいは分解せず、標的遺伝子の発現を抑制している可能性が示された。 Summary:
As reported in the paper by Tsuchiya et al., A significant gene expression suppression effect was observed for the gapmer type nucleic acid, and no suppression effect was seen for the non-gapmer type. In the population to which the nucleic acid of the present invention was administered, although the gapmer type ASO showed the same gene expression inhibitory activity as that of ASO of Shizutani et al., No hepatotoxicity occurred. Furthermore, even non-gapmer-type ASO showed a strong gene expression suppression effect although it was not as high as gapmer-type, while no significant hepatotoxicity was observed. From the above results, it is shown that ASO straddling the poly (A) chain addition site of the present invention may moderately or not degrade the mRNA of the target gene and suppress the expression of the target gene.
本発明者らの開発したASOの有用性
2014年時点で、gapmer活性を利用した核酸医薬品47品目が臨床試験段階にある(Sharma, V.K., et. al., Med.Chem.Comm., 5, 1454-1471, 2014)。しかし、gapmerで特にLNAを採用している場合、甚大な副作用として肝毒性が知られている。これは解決しなければならない危急の課題であった。発明者らは、本発明によりマウスでほとんど肝毒性を示さないASOを創出した。そして、発明者らの開発したASOは、標的mRNAの分解を引き起こさないかほとんど分解しない。この特性により、細胞中におけるoff target効果が抑制されていることがわかった。さらに、non-gapmer構造でありながら、特異的に標的遺伝子の発現を抑制することができるASOの開発にも成功した。先行研究によりnon-gapmer型ASOは肝毒性を誘導しないことが報告されている。よって、発明者らの発明したnon-gapmer型のASOは、今後広範に核酸医薬品開発現場で採用され、今後の核酸医薬品の主流となることが期待される。 Usefulness of the ASO developed by the present inventors As of 2014, 47 items of nucleic acid drugs using gapmer activity are in clinical trials (Sharma, VK, et. Al., Med. Chem. Comm., 5, 1454-1471, 2014). However, hepatotoxicity is known as a serious side effect, particularly when LNA is employed in the gapmer. This was an urgent issue that needs to be resolved. The inventors have created an ASO that exhibits little hepatotoxicity in mice according to the present invention. And, our developed ASO causes little or no degradation of the target mRNA. It was found that this property suppresses the off target effect in cells. Furthermore, we succeeded in developing ASO that can suppress the expression of target gene specifically even though it has a non-gapmer structure. Previous studies have reported that 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.
2014年時点で、gapmer活性を利用した核酸医薬品47品目が臨床試験段階にある(Sharma, V.K., et. al., Med.Chem.Comm., 5, 1454-1471, 2014)。しかし、gapmerで特にLNAを採用している場合、甚大な副作用として肝毒性が知られている。これは解決しなければならない危急の課題であった。発明者らは、本発明によりマウスでほとんど肝毒性を示さないASOを創出した。そして、発明者らの開発したASOは、標的mRNAの分解を引き起こさないかほとんど分解しない。この特性により、細胞中におけるoff target効果が抑制されていることがわかった。さらに、non-gapmer構造でありながら、特異的に標的遺伝子の発現を抑制することができるASOの開発にも成功した。先行研究によりnon-gapmer型ASOは肝毒性を誘導しないことが報告されている。よって、発明者らの発明したnon-gapmer型のASOは、今後広範に核酸医薬品開発現場で採用され、今後の核酸医薬品の主流となることが期待される。 Usefulness of the ASO developed by the present inventors As of 2014, 47 items of nucleic acid drugs using gapmer activity are in clinical trials (Sharma, VK, et. Al., Med. Chem. Comm., 5, 1454-1471, 2014). However, hepatotoxicity is known as a serious side effect, particularly when LNA is employed in the gapmer. This was an urgent issue that needs to be resolved. The inventors have created an ASO that exhibits little hepatotoxicity in mice according to the present invention. And, our developed ASO causes little or no degradation of the target mRNA. It was found that this property suppresses the off target effect in cells. Furthermore, we succeeded in developing ASO that can suppress the expression of target gene specifically even though it has a non-gapmer structure. Previous studies have reported that 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.
参考文献
(1)Burdick, A. D., Sciabola, S., Mantena, S. R., Hollingshead, B. D., Stanton, R., Warneke, J. A., Zeng, M., Martsen, E., Medvedev, A., Makarov, S. S., Reed, L. A., Davis, J. W. 2nd., and Whiteley, L.O. Sequence motifs associated with hepatotoxicity of locked nucleic acid-modified antisense oligonucleotides. Nucleic Acids Res., 8:4882-4891. doi: 10.1093/nar/gku142. Epub 2014.
(2)Burel, S. A., Hart, C.E., Cauntay, P., Hsiao, J., Machemer, T., Katz, M., Watt, A., Bui, H. H., Younis, H., Sabripour, M., Freier, S. M., Hung, G., Dan, A., Prakash, T.P., Seth, P. P., Swayze, E. E., Bennett, C. F., Crooke, S. T., and Henry, S.P. Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts. Nucleic Acids Res., 44:2093-2109. doi: 10.1093/nar/gkv1210, 2016.
(3)Davidson, N. O and Shelness, G.S., APOLIPOPROTEIN B: mRNA editing, lipoprotein assembly, and presecretory degradation. Annu. Rev. Nutr. 20, 169-193, 2000.
(4)Kakiuchi-Kiyota, S., Koza-Taylor, P. H., Mantena, S. R., Nelms, L. F., Enayetallah, A. E., Hollingshead, B. D., Burdick, A. D., Reed, L. A., Warneke, J. A., Whiteley, L. O., Ryan, A. M., and Mathialagan N. Comparison of Hepatic Transcription Profiles of Locked Ribonucleic Acid Antisense Oligonucleotides: Evidence of Distinct Pathways Contributing to Non-target Mediated Toxicity in Mice. Toxicol. Sci., 138, 234-248, 2014.
(5)Kasuya, T., Hori, S., Watanabe, A., Nakajima, M., Gahara, Y., Rokushima, M., Yanagimoto, T., and Kugimiya, A. Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides. Sci. Rep., 27;6:30377. doi: 10.1038/srep30377, 2016.
(6)Krieg, A. M., Therapeutic potential of Toll-like receptor 9 activation. Nature Rev. Drug Discov., 6:471-484, 2006.
(7)Nishina, K., Piao, W., Yoshida-Tanaka, K., Sujino, Y., Nishina, T., Yamamoto, T., Nitta, K., Yoshioka, K., Kuwahara, H., Yasuhara, H., Baba, T., Ono, F., Miyata, K., Miyake, K., Seth. P. P., Low A., Yoshida, M., Bennett, C. F., Kataoka, K., Mizusawa, H., Obika, S., and Yokota, T. DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing. Nature Commun., 6:7969. doi: 10.1038/ncomms8969, 2015.
(8)Proudfoot, N. J., Ending the message: poly(A) signals then and now. Genes & Dev., 25:1770-1782, 2011.
(9)Sharma, V. K., Sharmab, R. K., and Singh, S. K. Antisense oligonucleotides: modifications and clinical trials. Med. Chem. Commun., 5, 1454-1471, 2014.
Takaesu, G., Surabhi1, R. M., Park, K-J., Ninomiya-Tsuji, J., Matsumoto, K., and Gaynor, R. B. TAK1 is Critical for IkB kinase-mediated activation of the NF-κB pathway. J. Mol. Biol. 326, 105-115, 2003.
(10)Wong, E., and Goldberg, T. Mipomersen (Kynamro). A novel antisense oligonucleotide inhibitor for the management of homozygous familial hypercholesterolemia. Drug Forecast 39, 119-122, 2014. References (1) Burdick, AD, Sciabola, S., Mantena, SR, Hollingshead, BD, Stanton, R., Warneke, JA, Zeng, M., Martsen, E., Medvedev, A., Makarov, SS, Reed, LA, Davis,JW 2 nd ., And Whiteley, LO Sequence motifs associated with hepatotoxicity of locked nucleic acid modified with antisense oligonucleotides. Nucleic Acids Res., 8: 4882 4 891. doi: 10. 1093 / nar / gku142. .
(2) Burel, SA, Hart, CE, Cauntay, P., Hsiao, J., Machhemer, T., Katz, M., Watt, A., Bui, HH, Younis, H., Sabripour, M., Freier, SM, Hung, G., Dan, A., Prakash, TP, Seth, PP, Swayze, EE, Bennett, CF, Crooke, ST, and Henry, SP Hepatotoxicity of high affinity gapmer anti-antisense oligonucleotides is mediated by RNase H1 Dependent promiscuous reduction of very long pre-mRNA transcripts. Nucleic Acids Res., 44: 2093-2109. doi: 10.1093 / nar / gkv1210, 2016.
(3) Davidson, N. O and Shelness, GS, APOLIPOPROTEIN B: mRNA editing, lipoprotein assembly, and predecretory degradation. Annu. Rev. Nutr. 20, 169-193, 2000.
(4) Kakiuchi-Kiyota, S., Koza-Taylor, PH, Mantena, SR, Nelms, LF, Enayetallah, AE, Hollingshead, BD, Burdick, AD, Reed, LA, Warneke, JA, Whiteley, LO, Ryan, AM, and Mathialagan N. Comparison of Hepatic Transcription Profiles of Locked Acidic Acid Antisense Oligonucleotides: Evidence of Distinct Pathways Contributing to Non-target Mediated Toxity in Mice. Toxicol. Sci., 138, 234-248, 2014.
(5) Kasuya, T., Hori, S., Watanabe, A., Nakajima, M., Gahara, Y., Rokushima, M., Yanagimoto, T., and Kugimiya, A. Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides. Sci. Rep., 27; 6: 30377. doi: 10.1038 / srep30377, 2016.
(6) Krieg, A. M., Therapeutic potential of Toll-like receptor 9 activation. Nature Rev. Drug Discov., 6: 471-484, 2006.
(7) Nishina, K., Piao, W., Yoshida-Tanaka, K., Sujino, Y., Nishina, T., Yamamoto, T., Nitta, K., Yoshioka, K., Kuwahara, H., Yasuhara, H., Baba, T., Ono, F., Miyata, K., Miyake, K., Seth. PP, Low A., Yoshida, M., Bennett, CF, Kataoka, K., Mizusawa, H , Obika, S., and Yokota, T. DNA / RNA heteroduplex oligonucleotidenucleotide for highly efficient gene silencing. Nature Commun., 6: 7969. Doi: 10.1038 / ncomms8969, 2015.
(8) Proudfoot, N.J., Ending the message: poly (A) signals then and now. Genes & Dev., 25: 1770-1782, 2011.
(9) Sharma, V. K., Sharmab, R. K., and Singh, S. K. Antisense oligonucleotides: modifications and clinical trials. Med. Chem. Commun., 5, 1454-1471, 2014.
Takaesu, G., Surabhi1, RM, Park, KJ., Ninomiya-Tsuji, J., Matsumoto, K., and Gaynor, RB TAK1 is Critical for IkB kinase-mediated activation of the NF-κB pathway. J. Mol. Biol. 326, 105-115, 2003.
(10) Wong, E., and Goldberg, T. Mipomersen (Kynamro). A novel antisense oligonucleotide inhibitor for the management of homozygous familial hypercholesterolemia. Drug Forecast 39, 119-122, 2014.
(1)Burdick, A. D., Sciabola, S., Mantena, S. R., Hollingshead, B. D., Stanton, R., Warneke, J. A., Zeng, M., Martsen, E., Medvedev, A., Makarov, S. S., Reed, L. A., Davis, J. W. 2nd., and Whiteley, L.O. Sequence motifs associated with hepatotoxicity of locked nucleic acid-modified antisense oligonucleotides. Nucleic Acids Res., 8:4882-4891. doi: 10.1093/nar/gku142. Epub 2014.
(2)Burel, S. A., Hart, C.E., Cauntay, P., Hsiao, J., Machemer, T., Katz, M., Watt, A., Bui, H. H., Younis, H., Sabripour, M., Freier, S. M., Hung, G., Dan, A., Prakash, T.P., Seth, P. P., Swayze, E. E., Bennett, C. F., Crooke, S. T., and Henry, S.P. Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts. Nucleic Acids Res., 44:2093-2109. doi: 10.1093/nar/gkv1210, 2016.
(3)Davidson, N. O and Shelness, G.S., APOLIPOPROTEIN B: mRNA editing, lipoprotein assembly, and presecretory degradation. Annu. Rev. Nutr. 20, 169-193, 2000.
(4)Kakiuchi-Kiyota, S., Koza-Taylor, P. H., Mantena, S. R., Nelms, L. F., Enayetallah, A. E., Hollingshead, B. D., Burdick, A. D., Reed, L. A., Warneke, J. A., Whiteley, L. O., Ryan, A. M., and Mathialagan N. Comparison of Hepatic Transcription Profiles of Locked Ribonucleic Acid Antisense Oligonucleotides: Evidence of Distinct Pathways Contributing to Non-target Mediated Toxicity in Mice. Toxicol. Sci., 138, 234-248, 2014.
(5)Kasuya, T., Hori, S., Watanabe, A., Nakajima, M., Gahara, Y., Rokushima, M., Yanagimoto, T., and Kugimiya, A. Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides. Sci. Rep., 27;6:30377. doi: 10.1038/srep30377, 2016.
(6)Krieg, A. M., Therapeutic potential of Toll-like receptor 9 activation. Nature Rev. Drug Discov., 6:471-484, 2006.
(7)Nishina, K., Piao, W., Yoshida-Tanaka, K., Sujino, Y., Nishina, T., Yamamoto, T., Nitta, K., Yoshioka, K., Kuwahara, H., Yasuhara, H., Baba, T., Ono, F., Miyata, K., Miyake, K., Seth. P. P., Low A., Yoshida, M., Bennett, C. F., Kataoka, K., Mizusawa, H., Obika, S., and Yokota, T. DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing. Nature Commun., 6:7969. doi: 10.1038/ncomms8969, 2015.
(8)Proudfoot, N. J., Ending the message: poly(A) signals then and now. Genes & Dev., 25:1770-1782, 2011.
(9)Sharma, V. K., Sharmab, R. K., and Singh, S. K. Antisense oligonucleotides: modifications and clinical trials. Med. Chem. Commun., 5, 1454-1471, 2014.
Takaesu, G., Surabhi1, R. M., Park, K-J., Ninomiya-Tsuji, J., Matsumoto, K., and Gaynor, R. B. TAK1 is Critical for IkB kinase-mediated activation of the NF-κB pathway. J. Mol. Biol. 326, 105-115, 2003.
(10)Wong, E., and Goldberg, T. Mipomersen (Kynamro). A novel antisense oligonucleotide inhibitor for the management of homozygous familial hypercholesterolemia. Drug Forecast 39, 119-122, 2014. References (1) Burdick, AD, Sciabola, S., Mantena, SR, Hollingshead, BD, Stanton, R., Warneke, JA, Zeng, M., Martsen, E., Medvedev, A., Makarov, SS, Reed, LA, Davis,
(2) Burel, SA, Hart, CE, Cauntay, P., Hsiao, J., Machhemer, T., Katz, M., Watt, A., Bui, HH, Younis, H., Sabripour, M., Freier, SM, Hung, G., Dan, A., Prakash, TP, Seth, PP, Swayze, EE, Bennett, CF, Crooke, ST, and Henry, SP Hepatotoxicity of high affinity gapmer anti-antisense oligonucleotides is mediated by RNase H1 Dependent promiscuous reduction of very long pre-mRNA transcripts. Nucleic Acids Res., 44: 2093-2109. doi: 10.1093 / nar / gkv1210, 2016.
(3) Davidson, N. O and Shelness, GS, APOLIPOPROTEIN B: mRNA editing, lipoprotein assembly, and predecretory degradation. Annu. Rev. Nutr. 20, 169-193, 2000.
(4) Kakiuchi-Kiyota, S., Koza-Taylor, PH, Mantena, SR, Nelms, LF, Enayetallah, AE, Hollingshead, BD, Burdick, AD, Reed, LA, Warneke, JA, Whiteley, LO, Ryan, AM, and Mathialagan N. Comparison of Hepatic Transcription Profiles of Locked Acidic Acid Antisense Oligonucleotides: Evidence of Distinct Pathways Contributing to Non-target Mediated Toxity in Mice. Toxicol. Sci., 138, 234-248, 2014.
(5) Kasuya, T., Hori, S., Watanabe, A., Nakajima, M., Gahara, Y., Rokushima, M., Yanagimoto, T., and Kugimiya, A. Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides. Sci. Rep., 27; 6: 30377. doi: 10.1038 / srep30377, 2016.
(6) Krieg, A. M., Therapeutic potential of Toll-
(7) Nishina, K., Piao, W., Yoshida-Tanaka, K., Sujino, Y., Nishina, T., Yamamoto, T., Nitta, K., Yoshioka, K., Kuwahara, H., Yasuhara, H., Baba, T., Ono, F., Miyata, K., Miyake, K., Seth. PP, Low A., Yoshida, M., Bennett, CF, Kataoka, K., Mizusawa, H , Obika, S., and Yokota, T. DNA / RNA heteroduplex oligonucleotidenucleotide for highly efficient gene silencing. Nature Commun., 6: 7969. Doi: 10.1038 / ncomms8969, 2015.
(8) Proudfoot, N.J., Ending the message: poly (A) signals then and now. Genes & Dev., 25: 1770-1782, 2011.
(9) Sharma, V. K., Sharmab, R. K., and Singh, S. K. Antisense oligonucleotides: modifications and clinical trials. Med. Chem. Commun., 5, 1454-1471, 2014.
Takaesu, G., Surabhi1, RM, Park, KJ., Ninomiya-Tsuji, J., Matsumoto, K., and Gaynor, RB TAK1 is Critical for IkB kinase-mediated activation of the NF-κB pathway. J. Mol. Biol. 326, 105-115, 2003.
(10) Wong, E., and Goldberg, T. Mipomersen (Kynamro). A novel antisense oligonucleotide inhibitor for the management of homozygous familial hypercholesterolemia. Drug Forecast 39, 119-122, 2014.
本発明は、医薬産業、健康食品産業等に利用可能である。
The present invention is applicable to the pharmaceutical industry, the health food industry and the like.
Claims (13)
- ゲノム配列中、mRNAの3’-末端に対応する塩基から5’-側の5~9塩基(mRNAの3’-末端の塩基を含む)、および3’-側の5~9塩基(mRNAの3’-末端の塩基は含まない)の連続した配列にハイブリダイズし得るポリヌクレオチド。 In the genome sequence, 5 to 9 bases on the 5 'side (including the bases on the 3' end of the mRNA) from the base corresponding to the 3'-end of the mRNA, and 5 to 9 bases on the 3 'side (of the mRNA A polynucleotide capable of hybridizing to a continuous sequence of 3'-terminal base).
- ゲノム配列中、mRNAの3’-末端に対応する塩基から5’-側の5~9塩基(mRNAの3’-末端の塩基を含む)、および3’-側の5~9塩基(mRNAの3’-末端の塩基は含まない)の連続した配列にハイブリダイズし得る配列のみを有するポリヌクレオチド。 In the genome sequence, 5 to 9 bases on the 5 'side (including the bases on the 3' end of the mRNA) from the base corresponding to the 3'-end of the mRNA, and 5 to 9 bases on the 3 'side (of the mRNA A polynucleotide having only a sequence capable of hybridizing to a continuous sequence at the 3'-end (not including the base).
- ゲノム配列中、mRNAの3’-末端に対応する塩基から5’-側の5~9塩基(mRNAの3’-末端の塩基を含む)、および3’-側の5~9塩基(mRNAの3’-末端の塩基は含まない)の連続した配列のアンチセンス配列を含むポリヌクレオチド。 In the genome sequence, 5 to 9 bases on the 5 'side (including the bases on the 3' end of the mRNA) from the base corresponding to the 3'-end of the mRNA, and 5 to 9 bases on the 3 'side (of the mRNA A polynucleotide comprising an antisense sequence of a continuous sequence of 3'-terminal bases).
- ゲノム配列中、mRNAの3’-末端に対応する塩基から5’-側の5~9塩基(mRNAの3’-末端の塩基を含む)、および3’-側の5~9塩基(mRNAの3’-末端の塩基は含まない)の連続した配列のアンチセンス配列からなるポリヌクレオチド。 In the genome sequence, 5 to 9 bases on the 5 'side (including the bases on the 3' end of the mRNA) from the base corresponding to the 3'-end of the mRNA, and 5 to 9 bases on the 3 'side (of the mRNA A polynucleotide consisting of an antisense sequence of a continuous sequence of 3'-terminal bases).
- 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1~4のいずれかに記載のポリヌクレオチド。 The polynucleotide according to any one of the preceding claims, wherein said polynucleotide comprises at least one nucleotide analogue.
- 前記ヌクレオチド類似体が、LNA、2’-O-メチル化RNA、2’-O-メトキシエチル化RNA、および/またはモルフォリノ核酸である、請求項5に記載のポリヌクレオチド。 The polynucleotide according to claim 5, wherein the nucleotide analogue is LNA, 2'-O-methylated RNA, 2'-O-methoxyethylated RNA, and / or morpholino nucleic acid.
- 前記ポリヌクレオチドが、前記ヌクレオチド類似体を両末端および中央部に有する非ギャップマーである、請求項5または6に記載のポリヌクレオチド。 The polynucleotide according to claim 5 or 6, wherein the polynucleotide is a non-gapmer having the nucleotide analogue at both ends and in the middle.
- 請求項1~7のいずれかに記載のポリヌクレオチドを有効成分として含有する、遺伝子発現抑制剤。 A gene expression inhibitor comprising the polynucleotide according to any one of claims 1 to 7 as an active ingredient.
- 遺伝子発現抑制剤が、翻訳阻害剤である、請求項8に記載の遺伝子発現抑制剤。 The gene expression inhibitor according to claim 8, wherein the gene expression inhibitor is a translation inhibitor.
- 請求項1~7のいずれかに記載のポリヌクレオチドを有効成分として含有する、医薬、化粧品および/または食品。 A pharmaceutical, cosmetic and / or food comprising the polynucleotide according to any one of claims 1 to 7 as an active ingredient.
- 請求項1~7のいずれかに記載のポリヌクレオチドを投与することを包含する、遺伝子発現抑制方法。 A method for suppressing gene expression, comprising administering the polynucleotide according to any one of claims 1 to 7.
- 遺伝子発現抑制方法が、翻訳阻害方法である、請求項11に記載の遺伝子発現抑制方法。 The gene expression suppression method according to claim 11, wherein the gene expression suppression method is a translation inhibition method.
- 医薬を製造するための、請求項1~7のいずれかに記載のポリヌクレオチドの使用。 Use of a polynucleotide according to any of claims 1 to 7 for the manufacture of a medicament.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2019534511A JPWO2019026860A1 (en) | 2017-07-31 | 2018-07-30 | Gene expression control method and gene expression control substance |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2017-148706 | 2017-07-31 | ||
JP2017148706 | 2017-07-31 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2019026860A1 true WO2019026860A1 (en) | 2019-02-07 |
Family
ID=65232714
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2018/028511 WO2019026860A1 (en) | 2017-07-31 | 2018-07-30 | Gene expression regulation method and gene expression regulator |
Country Status (2)
Country | Link |
---|---|
JP (1) | JPWO2019026860A1 (en) |
WO (1) | WO2019026860A1 (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013015152A1 (en) * | 2011-07-22 | 2013-01-31 | 公立大学法人横浜市立大学 | TECHNIQUE FOR CLEAVING OUT PART OF poly(A) CHAIN AND/OR 3'-TERMINAL SEQUENCE OF mRNA TO INHIBIT TRANSLATION REACTION |
-
2018
- 2018-07-30 WO PCT/JP2018/028511 patent/WO2019026860A1/en active Application Filing
- 2018-07-30 JP JP2019534511A patent/JPWO2019026860A1/en active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013015152A1 (en) * | 2011-07-22 | 2013-01-31 | 公立大学法人横浜市立大学 | TECHNIQUE FOR CLEAVING OUT PART OF poly(A) CHAIN AND/OR 3'-TERMINAL SEQUENCE OF mRNA TO INHIBIT TRANSLATION REACTION |
Non-Patent Citations (4)
Title |
---|
BI YAN-LI ET AL.: "Establishment and application of thistriplex RT-PCR for ARV, REV and ALV", CHIN J VET SCI, vol. 32, no. 3, 1 March 2012 (2012-03-01), pages 341 - 344, XP055678564, DOI: 10.16303/j.cnki.1005-4545.2012.03.007 * |
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 * |
WADA T ET AL.: "Antisense morpholino targeting just upstream from a poly(A) tail junction of maternal mRNA removes the tail and inhibits translation", NUCLEIC ACIDS RESEARCH, vol. 40, no. 22, 1 December 2012 (2012-12-01), pages 1 - 10, XP055141644, ISSN: 0305-1048, DOI: 10.1093/nar/gks765 * |
YI JIANG ET AL.: "Evaluation of Four Candidate VNTR Loci for Genotyping 225 Chinese Clinical Mycobacterium Tuberculosis Complex Strains", BIOMED ENVIRON SCI, vol. 25, no. 1, 1 January 2012 (2012-01-01), pages 82 - 90, XP055678557, ISSN: 0895-3988, DOI: 10.3967/0895‐3988.2012.01.012 * |
Also Published As
Publication number | Publication date |
---|---|
JPWO2019026860A1 (en) | 2020-12-03 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP3236976B1 (en) | Rna interference agents for p21 gene modulation | |
JP6577073B2 (en) | Organic composition for treating beta-ENaC-related diseases | |
US20080145313A1 (en) | Compositions and Methods for the Treatment and Prevention of Neoplastic Disorders | |
EP2316491B1 (en) | Cell proliferation inhibitor | |
KR101605932B1 (en) | Organic compositions to treat hsf1-related diseases | |
US20120115923A1 (en) | Sirna Compositions Promoting Scar-Free Wound Healing of Skin and Methods for Wound Treatment | |
JP2010537640A (en) | Compositions of asymmetric RNA duplexes as microRNA mimetics or inhibitors | |
EP3134526A1 (en) | Lncrnas for therapy and diagnosis of cardiac hypertrophy | |
JP2007530431A (en) | Compositions and methods for treating pancreatic cancer | |
EP3452593B1 (en) | The incrna meg3 for therapy and diagnosis of cardiac remodelling | |
WO2019026860A1 (en) | Gene expression regulation method and gene expression regulator | |
JP2022541212A (en) | SiRNA sequences targeting the expression of the human genes JAK1 or JAK3 for therapeutic use | |
WO2011074652A1 (en) | Nucleic acid capable of inhibiting expression of hif-2α | |
Drufuca | ROLE OF MICRORNA IN MACROPHAGE POLARIZED ACTIVATION | |
Mols et al. | TNF-α stimulation inhibits siRNA-mediated RNA interference through a mechanism involving poly-(A) tail stabilization | |
TW201629216A (en) | Method for inhibiting translation and inhibitors for translation | |
WO2007100091A1 (en) | Composition for suppressing the expression of fucosyltransferase | |
JP2019508379A (en) | Therapeutic method and therapeutic composition for malignant tumor | |
WO2004027061A1 (en) | Method of screening remedy for proliferative disease | |
WO2019232640A1 (en) | Method for the identification and design of rna interference agents | |
CN116769828A (en) | Method for regulating and controlling activity of NRF2 transcription factor | |
WO2008127193A1 (en) | Metalled sirnas for facilitated targeting of suboptimal mrna sequences | |
Musaro et al. | Involvement of miRNAs in the Regulation | |
TW201718854A (en) | RNA interference agents for p21 gene modulation | |
TW201717969A (en) | Methods and compositions for treating malignant tumors associated with KRAS mutation |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 18842318 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2019534511 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 18842318 Country of ref document: EP Kind code of ref document: A1 |