WO2019093308A1 - miRNAを含むがん治療用医薬組成物 - Google Patents
miRNAを含むがん治療用医薬組成物 Download PDFInfo
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- 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
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- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- 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
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
- C12N2310/141—MicroRNAs, miRNAs
Definitions
- the present invention relates to a pharmaceutical composition for treating cancer, which comprises a transcript of a gene encoding a specific miRNA or a processed product thereof.
- a microRNA (hereinafter referred to as miRNA) is a functional nucleic acid that is encoded on the genome and undergoes a multistep production process to finally become a microRNA of about 20-25 bases in length. miRNAs are classified into functional ncRNAs (non-coding RNAs) and are being elucidated to play an important role in various biological phenomena (eg, gene expression regulation, etc.). Various miRNAs known to date, including human miRNAs, are registered in miRBase (http://www.mirbase.org/reference).
- miRNAs are involved in the onset and progression of cancer, cardiovascular disease, neurodegenerative disease, psychiatric disease, chronic inflammatory disease, and the like. Particularly in recent years, it has been pointed out that miRNAs are deeply involved in cell carcinogenesis and cell senescence.
- Patent Document 1 describes that miR-22 promotes cell senescence and suppresses cancer invasion and / or metastasis.
- Patent Document 2 also describes that a composition containing miR-34 can be used for the treatment of cancer.
- the present inventors keenly searched for miRNAs having a therapeutic effect on cancer among a large number of miRNAs.
- the present inventors found a specific miRNA having a cancer therapeutic effect significantly higher than the miRNAs described in the prior art, and came to complete the present invention.
- the present invention is a pharmaceutical composition for cancer treatment, which comprises a transcript of a gene encoding miRNA or a processed product thereof,
- the miRNA is one or more miRNAs selected from the group consisting of miR-3140, miR-137, miR-631, and miR-657, It relates to a pharmaceutical composition.
- the cancer is a solid cancer.
- the solid cancer is characterized by being colorectal cancer, pancreatic cancer, tongue cancer, mesothelioma, uterine sarcoma, osteosarcoma, breast cancer, lung cancer, or head and neck cancer. I assume.
- the transcript of the gene encoding the miRNA or the processed product thereof is expressed from the 5 'end side of pri-miRNA, pre-miRNA, double-stranded mature-miRNA, pre-miRNA It is characterized in that it is a single-stranded mature-miRNA or a single-stranded mature-miRNA expressed from the 3 'end side of a pre-miRNA.
- said miRNA is (I) mature-miRNA consisting of the sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO: 11; (Ii) 1 to 5 bases of substitution, addition, and / or to mature-miRNA consisting of the sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO: 11 MiRNA, which has a deletion and has a cancer therapeutic effect; or (Iii) has 80% or more sequence homology to mature-miRNA consisting of the sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO: 11, Cancer treatment effect, miRNA; It is characterized by being.
- the miRNA is characterized in that it is chemically modified.
- said chemical modification is LNA formation, BNA formation, ENA formation, 2'-OMe modification, phosphorothioation, S-TuD formation, morpholino modification, peptide addition, glycosylation, aptamer addition It is characterized in that it is one or more chemical modifications selected from the group consisting of hydrophobic molecule addition, polymer addition and unmodified DNA addition.
- the pharmaceutical composition further comprises a nucleic acid transfection agent.
- the transfection agent is a lipid based transfection agent, a polymer based transfection agent, a magnetic particle based transfection agent, an exosome for nucleic acid delivery, or a viral protein for nucleic acid delivery. It features.
- the transfection agent is GGGGDD (G4D2), GGGGGGDD (G6D2), GGGGGGGGDD (G8D2), GGGGGGGGGGDD (G10D2), AAAAAAD (A6D), AAAAAADD (A6D2), AAAAAAK (A6K), AAAAAAKK (A6K2), VVVVVVD (V6D), VVVVVVDD (V6D2), VVVVVVVK (V6K), VVVVVKK (V6K2), LLLLLLD (L6D), LLLL L L D (L 6 D 2), L L L L L L L L L It is characterized in that it is a transfection agent containing the peptide shown.
- the pharmaceutical composition of the invention is characterized in that it is for topical administration.
- the pharmaceutical composition of the present invention is characterized in that it is used in combination with another anticancer agent.
- the other anticancer agent is an alkylating agent, a platinum preparation, an antimetabolite, a topoisomerase inhibitor, a microtubule inhibitor, an anticancer antibiotic, a molecular targeting agent, a hormone It is characterized in that it is one or more anti-cancer agents selected from the group consisting of a preparation, an immunomodulator, an interferon, an interleukin, a plant-derived anti-cancer agent, and a BRM preparation.
- Another embodiment of the present invention is the use of a transcript of a gene encoding miRNA or a processed product thereof for producing a pharmaceutical composition for treating cancer,
- the miRNA is one or more miRNAs selected from the group consisting of miR-3140, miR-137, miR-631, and miR-657, For use.
- Another embodiment of the present invention is a method of treating cancer, comprising Applying the therapeutically effective amount of an anticancer pharmaceutical composition containing a transcript of a gene encoding miRNA or a processed product thereof to a cancer patient, Including
- the miRNA is one or more miRNAs selected from the group consisting of miR-3140, miR-137, miR-631, and miR-657, It relates to the treatment method.
- FIG. 1 illustrates the flow of screening for miRNA in this example.
- FIG. 2 illustrates the flow of screening of miRNA in this example.
- FIG. 3 shows four miRNAs showing growth inhibitory effect on many types of cancer cells selected in the screening of miRNAs.
- FIG. 4 shows the effect of the miRNA of the present invention on tongue cancer cell lines (HSC-4 cells, OSC19 cells).
- FIG. 5 shows the effect of the miRNA of the present invention on tongue cancer cell lines (SAS cells).
- FIG. 6 shows the effect of the miRNA of the present invention on mesothelioma cell lines (MSTO-211H cells).
- FIG. 7 shows the effect of the miRNA of the present invention on mesothelioma cell line (EHMES-10 cells).
- FIG. 1 illustrates the flow of screening for miRNA in this example.
- FIG. 2 illustrates the flow of screening of miRNA in this example.
- FIG. 3 shows four miRNAs showing growth inhibitory effect on many types of cancer cells selected in the screening of mi
- FIG. 8 shows the effect of miRNA of the present invention on uterine sarcoma cell line (MES-SA cells).
- FIG. 9 shows the effect of the miRNA of the present invention on an osteosarcoma cell line (U2-OS cells).
- FIG. 10 shows the effect of the miRNA of the present invention on cancer stem cell lines (MDA-MB231-luc-D3H2LN cells).
- FIG. 11 shows the effect of the miRNA of the present invention on lung cancer cell lines (A549 cells).
- FIG. 12 shows the effect of miRNA of the present invention on colon cancer cell lines (SW620 cells, SW480 cells, HT29 cells).
- FIG. 13 shows the cancer therapeutic effect of the miRNA of the present invention in vivo.
- FIG. 14 shows the results of imaging analysis of tumor tissue at 12, 19, 26, and 33 days after tumor cell transplantation.
- FIG. 15 shows a graph that quantifies the imaging analysis result in FIG.
- FIG. 16 shows the results of comparing tumor weights at the endpoints.
- FIG. 17 shows the results of imaging analysis of tumor tissue after tumor cell transplantation.
- FIG. 18 shows the results of comparing tumor weights at the endpoints.
- FIG. 19 shows the schedule of Experiment 7.
- FIG. 20 shows the imaging results when miR-3140-3p was administered once.
- FIG. 21 shows the imaging results when miR-3140-3p was administered once.
- FIG. 22 shows the survival rate of mice when miR-3140-3p was administered once.
- FIG. 23 shows the imaging results when miR-3140-3p was administered twice.
- FIG. 24 shows the imaging results when miR-3140-3p was administered twice.
- FIG. 25 shows mouse survival rates when miR-3140-3p was administered twice.
- FIG. 26 shows the imaging results when miR-3140-3p was administered three times.
- FIG. 27 shows the imaging results when miR-3140-3p was administered 3 times.
- FIG. 28 shows the survival rate of mice when miR-3140-3p was administered 3 times.
- FIG. 29 shows the results of Experiment 8.
- FIG. 30 shows a comparison of the tumor suppressor effects of miR-3140-3p and cisplatin.
- FIG. 31 shows a comparison of the tumor suppressor effects of miR-3140-3p and cisplatin.
- FIG. 32 shows a comparison of the tumor suppression effect of miR-3140-3p and cisplatin.
- FIG. 33 shows a comparison of the survival rates of the miR-3140-3p administration group and the cisplatin administration group.
- the present invention relates to a pharmaceutical composition for cancer treatment comprising a transcript of a gene encoding miRNA (micro-RNA) or a processed product thereof.
- miRNA miRNA
- micro-RNA miRNA
- Common miRNAs are biosynthesized through a series of processes.
- the primary transcript of the gene encoding miRNA is called Primary miRNA transcript (pri-miRNA) and generally has a stem loop hairpin structure.
- the pri-miRNA is cleaved by the microprocessor complex, takes a hairpin form by the RNase III enzyme Drosha, and produces a precursor miRNA (pre-miRNA) which is an intermediate precursor of about 70 bases.
- pre-miRNA is then transported from the nucleus to the cytoplasm.
- cytoplasm In the cytoplasm, it is further cleaved by another RNase III enzyme, Dicer, to produce double-stranded mature miRNA.
- Dicer another RNase III enzyme
- -5p is added to the one expressed from the 5 'end side of the precursor among the two strands
- 3-p is added to the one expressed from the 3' end side, “hsa-miR-21” It is written as "-5 p", "hsa-miR-21-3 p”.
- known miRNAs are registered in principle at miRBase (http://www.mirbase.org/).
- only one strand of mature miRNA may exhibit a desired effect, each strand may exhibit a desired effect, and a desired effect may be exhibited in a double stranded state.
- desired effects may be exhibited in the pri-miRNA state or the pre-miRNA state.
- the nucleic acid contained in the composition of the present invention is a transcript of a gene encoding one or more miRNAs selected from the group consisting of miR-3140, miR-137, miR-631, and miR-657, or the processing thereof It may be a product, or it may be a variant or variant that retains the function of the nucleic acid.
- the sequence of the transcript of the gene encoding miR-3140 or its processed product used in one embodiment of the present invention is as follows.
- the sequence of the Intron region encoding miR-3140 pri-miRNA is shown in SEQ ID NO: 4.
- the sequence of the transcript of the gene encoding miR-137 or its processed product used in one embodiment of the present invention is as follows.
- the sequence of the Intron region encoding miR-137 pri-miRNA is shown in SEQ ID NO: 7.
- the sequence of the transcript of the gene encoding miR-631 or its processed product used in one embodiment of the present invention is as follows.
- the sequence of the Intron region encoding miR-631 pri-miRNA is shown in SEQ ID NO: 10.
- the sequence of the transcript of the gene encoding miR-657 or its processed product used in one embodiment of the present invention is as follows.
- the sequence of the Intron region encoding miR-657 pri-miRNA is shown in SEQ ID NO: 13.
- the nucleic acid contained in the composition of the present invention is, for example, 1 against mature-miRNA consisting of the sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO: 11. It may be a nucleic acid having 2, 3, 4 or 5 bases of substitution, addition, and / or deletion and having an effect of inhibiting the growth of cancer cells.
- the substitution of bases for mature-miRNA may be, for example, conservative substitution of RNA known in the art.
- nucleic acid contained in the composition of the present invention is, for example, a mature-miRNA consisting of the sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO: 11 It may be a nucleic acid having sequence homology (or sequence identity) of 80% or more (preferably 85% or more, 90% or more, 95% or more) and having an effect of inhibiting the growth of cancer cells .
- a mature-miRNA consisting of the sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO: 11 is easily produced by an RNA synthesis apparatus generally used in the art. Nucleic acids substituted, added and / or deleted of specific bases can be easily produced as well. In addition, many companies conduct contract synthesis of nucleic acids, and it is easy to obtain RNA of the desired sequence from such companies. Therefore, those skilled in the art can routinely, without undue burden, mature—from the sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 11 The nature and function of miRNA variants and variants can be investigated.
- nucleic acid contained in the composition of the present invention may be subjected to chemical modification known in the art for the purpose of improving the stability, specificity and the like of RNA.
- Chemical modifications that can be used in the present invention include, for example, LNA (Locked Nucleic Acid), BNA (Bridged Nucleic Acid), ENA (2′-O, 4′-C-Ethylene-bridged Nucleic Acid), 2 ′.
- the present invention can be used for treatment of various cancers, but can be suitably used, for example, for solid cancer. More preferably, the subject of the present invention may be colon cancer, pancreatic cancer, tongue cancer, mesothelioma, uterine sarcoma, osteosarcoma, breast cancer, lung cancer or head and neck cancer.
- the composition of the present invention may further comprise a nucleic acid transfection agent.
- nucleic acid transfection agents that may be used in the present invention include lipid based transfection agents, polymer based transfection agents, magnetic particle based transfection agents, exosomes for nucleic acid delivery, or viral proteins for nucleic acid delivery.
- lipid based transfection agents examples include cationic lipids.
- cationic lipids nucleic acid-cationic lipid complexes are taken up into cells via endocytosis and released into the cytoplasm, whereby nucleic acids are introduced into cells (lipofection).
- lipofection Specifically, for example, various commercially available lipofection reagents may be used.
- polymer-based transfection agents include, for example, cationic polymers.
- the cationic polymer forms a nucleic acid-polymer complex when in contact with the nucleic acid, and the complex is attached to the cell membrane via electrostatic interaction and taken up into the cell via endocytosis.
- cationic peptides and their derivatives eg, polylysine, polyornithine
- linear or branched synthetic polymers eg, polybrene, polyethyleneimine
- polysaccharide-based transfer molecules eg, cyclodextrin, chitosan
- Natural polymers eg, histone, collagen
- transfection agents used in so-called nano-DDS such as transfection agents using block copolymers that form micelled nanoparticles, transfection agents using carbon nanohorns, etc. can also be used in the present invention.
- magnétique particle-based transfection agents include transfection agents using magnetic particles coated with cationic molecules.
- the magnetic particle-based transfection agent performs transfection by attaching a nucleic acid to the surface of the magnetic particle and then magnetically introducing the magnetic particle into cells.
- various commercially available magnetic particles for transfection may be used.
- transfection agents for nucleic acids using generally available exosomes and transfection agents using virus proteins such as adenovirus can also be used in the present invention.
- GGGGDD G4D2
- GGGGGGDD G6D2
- GGGGGGGGDD G6D2
- GGGGGGGGDD G10D2
- AAAAAAD A6D
- AAAAAADD A6D2
- AAAAAAK A6K
- AAAAAAKK A6K2
- the pharmaceutical composition for treating cancer of the present invention can be used in combination with other anticancer agents known in the art.
- Other anticancer agents to be used in combination include, but are not limited to, for example, alkylating agents, platinum preparations, antimetabolites, topoisomerase inhibitors, microtubule inhibitors, anticancer antibiotics, molecular targeting agents, hormone preparations,
- One or more anti-cancer agents selected from the group consisting of immunomodulators, interferons, interleukins, plant-derived anti-cancer agents, BRM formulations can be used.
- the mode of combined use of the pharmaceutical composition for cancer treatment of the present invention and other anticancer agents known in the art is not limited, and one skilled in the art can be selected depending on the type and treatment stage of the target cancer. For example, a doctor can perform in various ways.
- the pharmaceutical composition for treating cancer and the other anticancer agent of the present invention may be administered to a subject simultaneously or at different times.
- the pharmaceutical composition for treating cancer and the other anticancer agent of the present invention may be prepared as a combination containing each and may be administered to a subject.
- the pharmaceutical composition for cancer treatment of the present invention and the other anticancer agent may be prepared as a kit separately provided.
- a combination drug comprising the pharmaceutical composition for cancer treatment of the present invention and the other anticancer agent. May be administered to the subject.
- the pharmaceutical composition for cancer treatment of the present invention and the other anticancer agent are administered at different times
- the pharmaceutical composition for cancer treatment of the present invention and the other anticancer agent May be administered at intervals of time
- the pharmaceutical composition for treating cancer according to the present invention and the other anticancer agent may be administered from different administration routes.
- the administration route of the pharmaceutical composition for cancer treatment of the present invention is not limited, and may be systemic administration or local administration.
- the administration route is, for example, oral administration including sublingual administration, inhalation administration, direct administration to a target tissue by a catheter or injection, intravenous administration including infusion, transdermal administration by patch etc, suppository, or naso-gastric Parenteral administration such as administration by forced enteral feeding using a tube, a transnasal intestine, a gastric fistula tube, or an intestinal fistula tube can be mentioned.
- the dosage form of the pharmaceutical composition for cancer treatment of the present invention may be appropriately determined depending on the administration route, and is not limited, but injections, drops, tablets, capsules, fine granules, powders, solutions And aqueous solutions dissolved in syrup, etc., patches, suppositories and the like.
- the subject to which the pharmaceutical composition of the present invention is administered is not limited.
- the present invention can be used for mammals (human, pig, bovine, monkey, baboon, dog, cat, rat, mouse, etc.).
- mammals human, pig, bovine, monkey, baboon, dog, cat, rat, mouse, etc.
- humans can be excluded from the subject.
- the administration method (administration route, dose, frequency of administration per day, timing of administration, etc.) of the pharmaceutical composition of the present invention to a subject is not limited, and the health condition of the subject, the degree of disease, type of drug used in combination A person skilled in the art (for example, a doctor) can make an appropriate decision depending on the situation.
- TIG-3 cells human fibroblasts
- Automata automatic dispenser Bravo
- RNAiMAX 0.35 ⁇ L RNAiMAX (Invitrogen) was added to 70 ⁇ L serum free medium (Serum Free Medium (SFM)). The 2: 1 solution was added to a 96 well plate ( ⁇ -plate; ibidi) using Bravo. At 3: 2, 0.7 ⁇ L of miRNA (Stock Conc. 2 ⁇ M) was added using Bravo and mixed by pipetting. 4: Incubated for 20 minutes at room temperature. The cell suspension diluted to 5: 3.5 ⁇ 10 4 cells / mL was dispensed in 70 ⁇ L portions using Bravo. 6: 37 ° C., 5% CO 2 incubation.
- the SA- ⁇ -galactosidase assay was performed on day 7 with day 0 on which transfection was performed.
- the operation procedure is shown below.
- the value of the positive control was subtracted from the value of 1: negative control in each plate.
- 2 The value of each miRNA was subtracted from the value of negative control.
- the value of 3: 2 was divided by the value of 1 to obtain a score value.
- 4 The scatter value was drawn by taking the score value of cell number on the horizontal axis and the score value of cell size on the vertical axis.
- 6 Among the 579 types of candidates, 349 types of miRNAs that induced activation of the cellular senescence marker ⁇ -galactosidase were identified as senescence-induced miRNAs.
- 349 types of senescence-induced miRNAs obtained by screening, using an automatic dispenser Bravo (Agilent), various cancer cell lines (colorectal cancer cell line HCT116 (p53 wild type and p53 deficient) , Pancreatic cancer cell lines BxPC-3 and CFPAC-1, tongue cancer cell line HSC-4). Transfection was performed according to the following procedure.
- RNAiMAX 0.35 ⁇ L RNAiMAX (Invitrogen) was added to 70 ⁇ L serum free medium (Serum Free Medium (SFM)). The 2: 1 solution was added to a 96 well plate ( ⁇ -plate; ibidi) using Bravo. At 3: 2, 0.7 ⁇ L of miRNA (Stock Conc. 200 nM) was added using Bravo and mixed by pipetting. 4: Incubated for 20 minutes at room temperature. The cell suspension diluted with 5: 3.5 ⁇ 10 4 cells / mL was dispensed in 70 ⁇ L portions using Bravo. 6: 37 ° C., 5% CO 2 incubation.
- the cell viability was evaluated using PrestoBlue (Invitrogen) on day 5, with day 0 of transfection being performed.
- PrestoBlue Invitrogen
- the operating procedure is shown below.
- the medium was replaced with a medium containing PrestoBlue diluted 1:20 and incubated at 37 ° C. for 1 hour. 2.
- cancer cell line colonal cancer cell lines HCT116 (p53 wild type and p53 deficient), pancreatic cancer cell lines BxPC-3 and CFPAC-1, tongue cancer cell line HSC-4) Selected miRNAs that suppress cell proliferation more significantly than miR-34a-5p.
- a tongue cancer cell line (SAS) was transfected with four miRNAs (miR-137-3p, miR-631-5p, miR-657-3p, miR-3140-3p), and cell proliferation was observed. Transfection was performed according to the following procedure.
- miR-3140-3p which had a particularly high cell growth inhibitory effect, was selected from malignant pleural mesothelioma cell lines (MSTO-211H and EHMES-10), uterine sarcoma cell line (MES-SA), bone Each was transfected into a sarcoma cell line (U2-OS), and cell proliferation was observed.
- miR-22-3p and miR-34a-5p which have been shown to have cytostatic effects in previous studies, were used. Transfection was performed according to the following procedure.
- miR-3140-3p shows extremely high cell growth inhibitory effect as compared with miR-22-3p and miR-34a-5p which are prior art.
- MiR-3140-3p was transfected into a highly metastatic cancer cell line of breast cancer (MDA-MB231-luc-D3H2LN cells), and cell proliferation was observed.
- MDA-MB231-luc-D3H2LN cells highly metastatic cancer cell line of breast cancer
- miR-22-3p and miR-34a-5p which have been shown to have cytostatic effects in previous studies, were used. Transfection was performed according to the following procedure.
- RNAiMAX Invitrogen, 500 ⁇ L of serum free medium (Serum Free Medium (SFM)) and 5 ⁇ L of RNAiMAX (Invitrogen) were added.
- SFM serum Free Medium
- RNAiMAX RNAiMAX
- 2: 1 solution 12.5 ⁇ L each of miR-Control or miR-3140-3p, miR-22-3p, and miR-34a-5p (stock conc. 2 ⁇ M) were added.
- (Final concentration 12.5 nM) 3 Incubated for 20 minutes at room temperature.
- the expression of the apoptosis marker Annexin V in breast cancer cells transfected with miR-3140-3p was analyzed by the following procedure. 1: Transfection of miR-3140-3p was performed in the same manner as above. 2: 6 days after transfection, cells were collected together with the supernatant. 3: The sample for FACS was prepared according to the protocol of Annexin V assay kit. 4: Cells stained with Annexin V-FITC were stained using an antibody against cancer stem cell marker CD44 (eBioScience). 5: The prepared sample was analyzed using a cell sorter (SONY).
- miR-3140-3p showed a very high cell growth inhibitory effect on a breast cancer stem cell line (MDA-MB231-luc-D3H2LN cells).
- MiR-3140-3p was transfected into a lung cancer cell line (A549) and cell proliferation was observed.
- A549 a lung cancer cell line
- miR-22-3p and miR-34a-5p which have been shown to have cytostatic effects in previous studies, were used. Transfection was performed according to the following procedure.
- miR-3140-3p showed a very high cytostatic effect on lung cancer cell lines (A549).
- MiR-3140-3p was transfected into a colon cancer cell line (SW620, SW480, HT29) and cell proliferation was observed. Transfection was performed according to the following procedure.
- experiments were performed using experimental animals to which malignant pleural mesothelioma cells were transplanted.
- miRNA administration A negative control which is a control sequence and miR-3140-3p which is a miRNA of the present invention were used.
- A6K (3D Matrix) was used as a nucleic acid delivery reagent.
- nucleic acid 100 ⁇ M nucleic acid (miRNA) was diluted to 71.4 ⁇ M with 10% saline and sterile water. The 2% 1% A6K solution was sonicated for 5 minutes prior to use. 3: The diluted nucleic acid and 1% A6K solution were mixed at a ratio of 1: 1 to obtain a administered nucleic acid. 4: 50 ⁇ L each of the administered nucleic acid was administered subcutaneously to the SCID mouse (tumor site).
- the experimental results were evaluated in the following procedure. 1: The nucleic acid was administered every 1 or 2 days from 4 days after transplantation. 2: A total of 13 doses were administered, and 34 days after transplantation was used as an endpoint. 3: The mouse was dissected, a tumor located under the skin was removed, and its weight was measured.
- mice in which miR-3140-3p was introduced to the tumor site the weight of the tumor was significantly lower than that in the control group.
- EHMES-10 cells were used. 1: The cells on the dish were washed twice with PBS ( ⁇ ). 2: The cells were detached using trypsin. 3: suspended in culture medium and subjected to cell counting. 4: Centrifugated under conditions of 1000 rpm 3 min and pelleted down. 5: The cells were resuspended in PBS ( ⁇ ) to 2.0 ⁇ 10 7 cells / mL.
- miRNA administration A negative control which is a control sequence and miR-3140-3p which is a miRNA of the present invention were used.
- A6K (3D Matrix) was used as a nucleic acid delivery reagent.
- nucleic acid 100 ⁇ M nucleic acid (miRNA) was diluted to 71.4 ⁇ M with 10% saline and sterile water. The 2% 1% A6K solution was sonicated for 5 minutes prior to use. 3: The diluted nucleic acid and 1% A6K solution were mixed at a ratio of 1: 1 to obtain a administered nucleic acid. 4: 50 ⁇ L each of the administered nucleic acid was administered subcutaneously to the SCID mouse (tumor site).
- the experimental results were evaluated in the following procedure. 1: The nucleic acid was administered every one or two days from 2 days after transplantation. 2: A total of 13 doses were administered, and 33 days after transplantation was used as an endpoint. 3: The tumor size was traced by administering luciferin intraperitoneally at 12, 19, 26, 33 days after transplantation and performing imaging. 4: The mouse was dissected at the end point, the tumor located under the skin was removed, and its weight was measured.
- FIG. 14 and FIG. 14 The imaging analysis results of the tumor tissue at 12, 19, 26, 33 days after tumor cell transplantation are shown in FIG. 14 and FIG. As shown in FIGS. 14 and 15, in the mice into which miR-3140-3p was introduced in the tumor site, the increase in tumor was suppressed as compared to the control group. Similar results were also shown in FIG. 16, which compares tumor weights at the endpoints.
- miRNA administration A negative control which is a control sequence, and miR-3140-3p, miR-137, miR-631 and miR-657 which are miRNA of the present invention were used.
- A6K (3D Matrix) was used as a nucleic acid delivery reagent.
- Administration of miRNA to mice was performed according to the following procedure.
- nucleic acid 100 ⁇ M nucleic acid (miRNA) was diluted to 71.4 ⁇ M with 10% saline and sterile water. The 2% 1% A6K solution was sonicated for 5 minutes prior to use. 3: The diluted nucleic acid and 1% A6K solution were mixed at a ratio of 1: 1 to obtain a administered nucleic acid. 4: 50 ⁇ L each of the administered nucleic acid was administered subcutaneously to the SCID mouse (tumor site).
- the experimental results were evaluated in the following procedure. 1: The nucleic acid was administered every 1 or 2 days from 3 days after transplantation. 2: A total of 11 doses were administered, and 28 days after transplantation was used as an endpoint. 3: Tumor size was traced by administering luciferin intraperitoneally 7, 14, 21 and 28 days after transplantation and performing imaging. 4: The mouse was dissected at the end point, the tumor located under the skin was removed, and its weight was measured.
- FIG. 17 The imaging analysis result of the tumor tissue after tumor cell transplantation is shown in FIG. As shown in FIG. 17, in the mice into which the miRNA of the present invention was introduced into the tumor site, the increase in tumor was suppressed as compared to the control group. Similar results were also shown in FIG. 18 comparing tumor weights at the endpoints.
- the miRNA of the present invention exerts an extremely high antitumor effect also in vivo.
- the tumor suppressor effect of miR-3140-3p in vivo was examined using an intrathoracic orthotopic transplant model mouse.
- a 6-week-old male mouse (C-B-17 / Icr-scid / scidJcl) was used.
- the tumor cells used the malignant pleural mesothelioma cell line EHMES-10 expressing a luciferase gene.
- a mouse was intraperitoneally administrated with a mixed anesthetic of 0.1 mL of medetomidine hydrochloride, midazolam hydrochloride and butorphanol tartrate per 10 g of body weight intraperitoneally to the mouse.
- 7 After anesthesia, 100 ⁇ L of the miRNA / A6K mixture was administered into the mouse thoracic cavity.
- 8 Imaging was performed every other week from the first imaging, and tumor growth was observed.
- 9 The date of death of the mouse was recorded, and the mouse survival rate was calculated.
- the dosing and imaging schedule is shown in FIG.
- a mixed anesthetic of medetomidine hydrochloride, midazolam and butorphanol tartrate was prepared as shown in Table 4 below.
- the miRNA / A6K mixture was prepared as in Table 5.
- RNA shown in Table 6 was administered instead of the miRNA of the present invention.
- miRNA was administered as a double strand in combination with a complementary strand containing a partial mismatch.
- the miR-3140-3p was also shown to significantly suppress malignant pleural mesothelioma in the groups to which miR-3140-3p was administered twice and three times, as in the group to which it was administered once (FIG. 23) ⁇ 28).
- mice The survival rate of mice was further improved in the group to which miR-3140-3p was administered three times (FIG. 28).
- the calculation of the IC 50 value of miR-3140-3p was performed using malignant pleural mesothelioma cell EHMES-10.
- RNAiMAX (Invitrogen) was added to 25 ⁇ L serum free medium (Serum Free Medium (SFM)).
- SFM serum Free Medium
- 2 Serially dilute miR-Control and miR-3140-3p to final concentrations of 40 nM, 20 nM, 10 nM, 5 nM, 1 nM, 500 nM, 100 pM, 50 pM, 10 pM, 5 pM, 1 pM and SFM / RNAiMAX complex Mixed.
- 3 Incubated for 20 minutes at room temperature. 75 ⁇ L of cells at 4: 6.7 ⁇ 10 4 cells / mL were added to each well. 5: 37 ° C., 5% CO 2 and incubated.
- Cell viability was examined 5 days after transfection using Cell Counting Kit 8 (DOJINDO). The procedure is shown below: (i) The Cell Counting Kit was diluted 10-fold with culture medium. (Ii) A Cell Counting Kit diluted 200 ⁇ l was added to each well. (Iii) incubated at 37 ° C., 5% CO 2 for 1 hour. (Iv) The value of 450 nm / 600 nm was measured using a plate reader.
- FIG. 29 it was revealed that miR-3140-3p exhibits a growth inhibitory effect on malignant pleural mesothelioma cell line EHMES-10 even at a very low concentration.
- the IC 50 value was calculated to be 0.57 nM.
- the tumor suppressor effect of cisplatin which is the first choice drug for malignant pleural mesothelioma
- the tumor suppressor effect of miR-3140-3p in vivo We compared at.
- a 6-week-old male mouse (C-B-17 / Icr-scid / scidJcl) was used.
- the tumor cells used the malignant pleural mesothelioma cell line EHMES-10 expressing the luciferase gene.
- a mouse was intraperitoneally administrated with a mixed anesthetic of 0.1 mL of medetomidine hydrochloride, midazolam hydrochloride and butorphanol tartrate per 10 g of body weight intraperitoneally to the mouse.
- 7 After anesthesia, 100 ⁇ L of the miRNA / A6K mixture was injected into the thoracic cavity for the miRNA administration group, and cisplatin (6 mg / kg) was administered intraperitoneally for the cisplatin administration group.
- Imaging was performed every other week from the first imaging, and tumor growth was observed.
- a mixed anesthetic was prepared as shown in Table 4 above.
- the miRNA / A6K mixture was prepared as shown in Tables 5 and 6 above.
- Cisplatin was prepared as shown in Table 7 below.
- FIGS. The experimental results are shown in FIGS. As shown in each figure, it was shown that miR-3140-3p can exert an anti-tumor effect equal to or greater than that of cisplatin, which is also a first-line drug for malignant pleural mesothelioma.
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Abstract
Description
前記miRNAが、miR-3140、miR-137、miR-631、およびmiR-657からなる群から選択される1または複数のmiRNAであることを特徴とする、
医薬組成物に関する。
(i)配列番号1、配列番号2、配列番号5、配列番号8、または、配列番号11で表される配列からなるmature-miRNA;
(ii)配列番号1、配列番号2、配列番号5、配列番号8、または、配列番号11で表される配列からなるmature-miRNAに対して、1~5塩基の置換、付加、および/または欠失を有し、がん治療効果を有する、miRNA;または、
(iii)配列番号1、配列番号2、配列番号5、配列番号8、または、配列番号11で表される配列からなるmature-miRNAに対して、80%以上の配列相同性を有し、がん治療効果を有する、miRNA;
であることを特徴とする。
前記miRNAが、miR-3140、miR-137、miR-631、およびmiR-657からなる群から選択される1または複数のmiRNAであることを特徴とする、
使用に関する。
前記方法は、がん患者に対して、治療上有効量の、miRNAをコードする遺伝子の転写産物またはそのプロセシング産物を含む抗がん用医薬組成物を適用するステップ、
を含み、
前記miRNAが、miR-3140、miR-137、miR-631、およびmiR-657からなる群から選択される1または複数のmiRNAであることを特徴とする、
治療方法に関する。
2:1の溶液を、Bravoを用いて96well plate(μ-plate;ibidi)に加えた。
3:2にmiRNA(Stock Conc. 2μM)を、Bravoを用いて0.7μL加え、ピペッティングにより混和した。
4:室温で20分間インキュベートした。
5:3.5×104cells/mLに希釈した細胞懸濁液を、Bravoを用いて70μLずつ分注した。
6:37℃,5%CO2条件下でインキュベートした。
2:3.7%ホルマリン溶液を加え、室温で10分間インキュベートし細胞を固定した。
3:PBS(-)を用いて2回洗浄を行った。
4:Stain Solutionを加え、室温で30分間インキュベートし、染色した。
5:PBS(-)を用いて3回洗浄を行った。
6:染色し終わったプレートは、自動写真撮影装置Operetta(Perkin Elmer)を用いて全視野撮影を行った。
7:撮影した写真は、画像解析ソフトColumbus(Perkin Elmer)を用いて定量解析を行った。
2:2%ホルマリン溶液を加え、室温で5分間インキュベートし細胞を固定した。
3:PBS(-)を用いて2回洗浄した。
4:β-Gal stain solutionを用事調製し、ウェルに加えた。
5:37℃で12-16時間インキュベートした。
6:染色し終わったプレートは、自動写真撮影装置Opera(Perkin Elmer)を用いて撮影した。
得られた細胞数、細胞の大きさの数値をそれぞれ、ネガティブコントロールであるランダム配列miRNAの数値およびポジティブコントロールであるmiR-34a-5pの数値を用いてスコア化した。スコア化の方法を以下に示した。
1:ネガティブコントロールの値からポジティブコントロールの値を引いた。
2:ネガティブコントロールの値からそれぞれのmiRNAの値を引いた。
3:2の値を1の値で割り、スコア値とした。
4:横軸に細胞数のスコア値を縦軸に細胞の大きさのスコア値を取り散布図を描いた。
5:miR-22-3pおよびmiR-22-5pのスコア値よりも少なくとも一方が高いスコア値を示したmiRNAを老化誘導miRNA候補として579種類同定した。
6:579種類の候補中、細胞老化マーカーであるβ-ガラクトシダーゼの活性化を誘導したmiRNAを老化誘導miRNAとして349種類同定した。
トランスフェクションは以下の手順で実施した。
2:1の溶液を、Bravoを用いて96well plate(μ-plate;ibidi)に加えた。
3:2にmiRNA(Stock Conc. 200nM)をBravoを用いて0.7μL加え、ピペッティングにより混和した。
4:室温で20分間インキュベートした。
5:3.5×104cells/mL希釈した細胞懸濁液をBravoを用いて70μLずつ分注した。
6:37℃,5%CO2条件下でインキュベートした。
2.Enspire(Perkin Elmer)を用いて蛍光値(Ex/Em=560nm/590nm)を測定した。
3.試薬のみのウェルから得られる蛍光値をバックグラウンドとして、細胞生存率を求めた。
2:1の溶液に核酸(Control,miR-137-3p,miR-631-5p,miR-657-3p,miR-3140-3p,4つのmiRNAの等量混合物)をそれぞれ1μLずつ加えた。(終濃度10nM)
3:室温で20分間インキュベートした。
4:6.7×104cells/mLに希釈した細胞懸濁液を1.5mLずつ35mm dishに加えた。
5:37℃,5%CO2条件下でインキュベートした。
6:トランスフェクション後5日後に細胞計数を行った。
2:1の溶液にmiRNA溶液をそれぞれ4μLずつ加えた(表3参照)。(終濃度10nM)
3:室温で20分間インキュベートした。
4:6.7×104cells/mLに希釈した細胞懸濁液を1.5mLずつ35mmdishに加えた。
5:37℃,5%CO2条件下でインキュベートした。
6:トランスフェクション後2日後に再び1-3の操作を行った。
7:6により調製した溶液を5で培養中のdishにそれぞれ添加した。
8:最初のトランスフェクション後7日後に細胞計数を行った。
2:1の溶液にmiR-ControlまたはmiR-3140-3p,miR-22-3p,miR-34a-5p(stock conc. 20μM)をそれぞれ1μL加えた。(終濃度10nM)
3:室温で20分間インキュベートした。
4:6.7×104cells/mLに希釈した細胞懸濁液を1.5mLずつ35mmdishに加えた。
5:37℃,5%CO2条件下でインキュベートした。
6:トランスフェクション後4日後に細胞計数を行った。
2:1の溶液にmiR-ControlまたはmiR-3140-3p,miR-22-3p,miR-34a-5p(stock conc. 2μM)をそれぞれ12.5μL加えた。(終濃度12.5nM)
3:室温で20分間インキュベートした。
4:6.7×104cells/mLに希釈した細胞懸濁液を1.5mLずつ35mmdishに加えた。
5:37℃,5%CO2条件下でインキュベートした。
6:トランスフェクション後4日後に細胞計数を行った。
1:上記と同様の手順で、miR-3140-3pのトランスフェクションを行なった。
2:トランスフェクション6日後に細胞を上清ごと回収した。
3:Annexin V assay kitのプロトコルに従い、FACS用サンプルを調製した。
4:Annexin V-FITCにより染色した細胞を、がん幹細胞マーカーであるCD44に対する抗体(eBioScience)を用いて染色した。
5:調製したサンプルを、セルソーター(SONY)を用いて解析した。
2:1の溶液にmiR-ControlまたはmiR-3140-3p,miR-22-3p,miR-34a-5p(stock conc. 20μM)をそれぞれ1μL加えた。(終濃度10.0nM)
3:室温で20分間インキュベートした。
4:6.7×104cells/mLに希釈した細胞懸濁液を1.5mLずつ35mmdishに加えた。
5:37℃,5%CO2条件下でインキュベートした。
6:トランスフェクション後6日後に細胞計数を行った。
2:1の溶液にmiR-ControlまたはmiR-3140-3p,miR-22-3p,miR-34a-5p(stock conc. 2μM)をそれぞれ12.5μL加えた。(終濃度10.0nM)
3:室温で20分間インキュベートした。
4:6.7×104cells/mLに希釈した細胞懸濁液を1.5mLずつ35mmdishに加えた。
5:37℃,5%CO2条件下でインキュベートした。
6:トランスフェクション後7日後に細胞計数を行った。
悪性胸膜中皮腫細胞MSTO-211H細胞を使用した。
1:ディッシュ上の細胞をPBS(-)で2回ウォッシュした。
2:トリプシンを用いて細胞を剥がした。
3:培地で懸濁し、細胞計数を行った。
4:1000rpm 3minの条件で遠心し、ペレットダウンした。
5:細胞を2.0×107cells/mLとなるようPBS(-)で再懸濁した。
マウスは6週齢のC-B-17/Icr-scid/scid Jcl(SCID マウス)を使用した。SCIDマウスの皮下に調製した細胞懸濁液100μLを投与し、細胞を定着させた。
コントロール配列であるネガティブコントロールと、本発明のmiRNAであるmiR-3140-3pを用いた。核酸デリバリー試薬として、A6K(3Dマトリックス社)を用いた。マウスへのmiRNAの投与は以下の手順で実施した。
2:1%A6K溶液を使用前に5分間超音波処理を行った。
3:希釈した核酸と1%A6K溶液を1:1の割合で混合し、投与核酸とした。
4:SCIDマウス皮下(腫瘍部)に投与核酸を50μLずつ投与した。
以下の手順で実験結果の評価を行った。
1:移植後4日後から、1日または2日おきに核酸を投与した。
2:計13回投与し、移植後34日後をエンドポイントとした。
3:マウスを解剖し、皮下にある腫瘍を摘出し、その重量を測定した。
悪性胸膜中皮腫細胞EHMES-10細胞を使用した。
1:ディッシュ上の細胞をPBS(-)で2回ウォッシュした。
2:トリプシンを用いて細胞を剥がした。
3:培地で懸濁し、細胞計数を行った。
4:1000rpm 3minの条件で遠心し、ペレットダウンした。
5:細胞を2.0×107cells/mLとなるようPBS(-)で再懸濁した。
マウスは6週齢のC-B-17/Icr-scid/scid Jcl(SCID マウス)を使用した。SCIDマウスの皮下に調製した細胞懸濁液100μLを投与し、細胞を定着させた。
コントロール配列であるネガティブコントロールと、本発明のmiRNAであるmiR-3140-3pを用いた。核酸デリバリー試薬として、A6K(3Dマトリックス社)を用いた。マウスへのmiRNAの投与は以下の手順で実施した。
2:1%A6K溶液を使用前に5分間超音波処理を行った。
3:希釈した核酸と1%A6K溶液を1:1の割合で混合し、投与核酸とした。
4:SCIDマウス皮下(腫瘍部)に投与核酸を50μLずつ投与した。
以下の手順で実験結果の評価を行った。
1:移植後2日後から、1日または2日おきに核酸を投与した。
2:計13回投与し、移植後33日後をエンドポイントとした。
3:移植後12、19、26、33日後にルシフェリンを腹腔内に投与し、イメージングを行うことで腫瘍の大きさをトレースした。
4:エンドポイントでマウスを解剖し、皮下にある腫瘍を摘出し、その重量を測定した。
舌がん細胞株HSC-4細胞を使用した。
1:ディッシュ上の細胞をPBS(-)で2回ウォッシュした。
2:トリプシンを用いて細胞を剥がした。
3:培地で懸濁し、細胞計数を行った。
4:1000rpm 3minの条件で遠心し、ペレットダウンした。
5:細胞を2.0×107cells/mLとなるようPBS(-)で再懸濁した。
マウスは6週齢のC-B-17/Icr-scid/scid Jcl(SCID マウス)を使用した。SCIDマウスの皮下に調製した細胞懸濁液100μLを投与し、細胞を定着させた。
コントロール配列であるネガティブコントロールと、本発明のmiRNAであるmiR-3140-3p、miR-137、miR-631およびmiR-657を用いた。核酸デリバリー試薬として、A6K(3Dマトリックス社)を用いた。マウスへのmiRNAの投与は以下の手順で実施した。
2:1%A6K溶液を使用前に5分間超音波処理を行った。
3:希釈した核酸と1%A6K溶液を1:1の割合で混合し、投与核酸とした。
4:SCIDマウス皮下(腫瘍部)に投与核酸を50μLずつ投与した。
以下の手順で実験結果の評価を行った。
1:移植後3日後から、1日または2日おきに核酸を投与した。
2:計11回投与し、移植後28日後をエンドポイントとした。
3:移植後7、14、21、28日後にルシフェリンを腹腔内に投与し、イメージングを行うことで腫瘍の大きさをトレースした。
4:エンドポイントでマウスを解剖し、皮下にある腫瘍を摘出し、その重量を測定した。
1:マウス腹腔内に体重10gあたり0.1mLの塩酸メデトミジン、ミダゾラム、酒石酸ブトルファノールの混合麻酔薬を投与した。
2:麻酔後マウス胸部の毛を剃り、ハサミで表皮を切開した。
3:マウス胸腔内に27Gのインスリン用シリンジを用いて、腫瘍細胞(3×107cells/mL)を100μL移植した。
4:移植後3日後、IVIS SpectrumCT In Vivo Imaging Systemを用いて、腫瘍細胞のイメージングを行なった。
5:イメージング後、移植に成功したマウスを用いて、群分けを行なった。
6:マウス腹腔内に体重10gあたり0.1mLの塩酸メデトミジン、ミダゾラム、酒石酸ブトルファノールの混合麻酔薬を投与し、マウスを麻酔した。
7:麻酔後、マウス胸腔内にmiRNA/A6K混合物を100μL投与した。
8:最初のイメージングから1週間おきにイメージングを行い、腫瘍増大を観察した。
9:マウスの死亡日時を記録し、マウス生存率を算出した。
1:25μLの無血清培地(Serum Free Medium(SFM))に対し0.25μLのRNAiMAX(Invitrogen)を加えた。
2:miR-ControlおよびmiR-3140-3pを終濃度が40nM、20nM、10nM、5nM、1nM、500pM、100pM、50pM、10pM、5pM、1pMとなるように段階希釈し、SFM/RNAiMAX複合体と混合した。
3:室温で20分間インキュベートした。
4:6.7×104cells/mLの細胞を75μLずつ各wellに加えた。
5:37℃、5%CO2条件下でインキュベートした。
6:トランスフェクション後5日後にCell Counting Kit 8(DOJINDO)を用いて細胞の生存率を検討した。以下にその手順を示す
(i)Cell Counting Kit を培地で10倍希釈した。
(ii)各wellに200μLずつ希釈したCell Counting Kitを添加した。
(iii)37℃、5%CO2条件下で1時間インキュベートした。
(iv)プレートリーダーを用いて450nm/600nmの値を測定した。
1:マウス腹腔内に体重10gあたり0.1mLの塩酸メデトミジン、ミダゾラム、酒石酸ブトルファノールの混合麻酔薬を投与した。
2:麻酔後マウス胸部の毛を剃り、ハサミで表皮を切開した。
3:マウス胸腔内に27Gのインスリン用シリンジを用いて、腫瘍細胞(3×107cells/mL)を100μL移植した。
4:移植後4日後にIVIS SpectrumCT In Vivo Imaging Systemを用いて腫瘍細胞のイメージングを行なった。
5:イメージング後、移植に成功したマウスを用いて、群分けを行なった。
6:マウス腹腔内に体重10gあたり0.1mLの塩酸メデトミジン、ミダゾラム、酒石酸ブトルファノールの混合麻酔薬を投与し、マウスを麻酔した。
7:麻酔後、miRNA投与群にはmiRNA/A6K混合物を胸腔内に100μL、シスプラチン投与群にはシスプラチン(6mg/kg)を腹腔内に投与した。
8:最初のイメージングから1週間おきにイメージングを行い、腫瘍増大を観察した。
Claims (15)
- miRNAをコードする遺伝子の転写産物またはそのプロセシング産物を含むがん治療用医薬組成物であって、
前記miRNAが、miR-3140、miR-137、miR-631、およびmiR-657からなる群から選択される1または複数のmiRNAであることを特徴とする、
医薬組成物。 - 請求項1に記載の医薬組成物であって、
前記がんが、固形がんであることを特徴とする、
医薬組成物。 - 請求項2に記載の医薬組成物であって、
前記固形がんが、大腸がん、膵臓がん、舌がん、中皮腫、子宮肉腫、骨肉腫、乳がん、肺がん、または頭頸部がんであることを特徴とする、
医薬組成物。 - 請求項1に記載の医薬組成物であって、
前記miRNAをコードする遺伝子の転写産物またはそのプロセシング産物は、pri-miRNA、pre-miRNA、2本鎖mature-miRNA、pre-miRNAの5’末端側から発現する1本鎖mature-miRNA、または、pre-miRNAの3’末端側から発現する1本鎖mature-miRNAであることを特徴とする、
医薬組成物。 - 請求項4に記載の医薬組成物であって、
前記miRNAが、
(i)配列番号1、配列番号2、配列番号5、配列番号8、または、配列番号11で表される配列からなるmature-miRNA;
(ii)配列番号1、配列番号2、配列番号5、配列番号8、または、配列番号11で表される配列からなるmature-miRNAに対して、1~5塩基の置換、付加、および/または欠失を有し、がん治療効果を有する、miRNA;または、
(iii)配列番号1、配列番号2、配列番号5、配列番号8、または、配列番号11で表される配列からなるmature-miRNAに対して、80%以上の配列相同性を有し、がん治療効果を有する、miRNA;
であることを特徴とする、
医薬組成物。 - 請求項1~5のいずれか1項に記載の医薬組成物であって、
前記miRNAが、化学的に修飾されていることを特徴とする、
医薬組成物。 - 請求項6に記載の医薬組成物であって、
前記化学的修飾が、LNA化、BNA化、ENA化、2’-OMe修飾、ホスホロチオエート化、S-TuD化、モルフォリノ修飾、ペプチド付加、糖鎖付加、アプタマー付加、疎水性分子付加、高分子付加、および、非修飾DNA付加からなる群から選択される、1または複数の化学的修飾であることを特徴とする、
医薬組成物。 - 請求項1~7のいずれか1項に記載の医薬組成物であって、
前記医薬組成物が、核酸トランスフェクション剤をさらに含むことを特徴とする、
医薬組成物。 - 請求項8に記載の医薬組成物であって、
前記トランスフェクション剤が、脂質系トランスフェクション剤、ポリマー系トランスフェクション剤、磁気粒子系トランスフェクション剤、核酸デリバリー用エクソソーム、または、核酸デリバリー用ウイルスタンパク質であることを特徴とする、
医薬組成物。 - 請求項8に記載の医薬組成物であって、
前記トランスフェクション剤は、GGGGDD(G4D2)、GGGGGGDD(G6D2)、GGGGGGGGDD(G8D2)、GGGGGGGGGGDD(G10D2)、AAAAAAD(A6D)、AAAAAADD(A6D2)、AAAAAAK(A6K)、AAAAAAKK(A6K2)、VVVVVVD(V6D)、VVVVVVDD(V6D2)、VVVVVVK(V6K)、VVVVVVKK(V6K2)、LLLLLLD(L6D)、LLLLLLDD(L6D2)、LLLLLLK(L6K)、または、LLLLLLKK(L6K2)のアミノ酸配列で表されるペプチドを含むトランスフェクション剤であることを特徴とする、
医薬組成物。 - 請求項1~10のいずれか1項に記載の医薬組成物であって、
前記医薬組成物は、局所投与用であることを特徴とする、
医薬組成物。 - 請求項1~11のいずれか1項に記載の医薬組成物であって、
他の抗がん剤と併用されることを特徴とする、
医薬組成物。 - 請求項12に記載の医薬組成物であって、
前記他の抗がん剤が、アルキル化剤、白金製剤、代謝拮抗剤、トポイソメラーゼ阻害剤、微小管阻害剤、抗がん性抗生物質、分子標的薬、ホルモン製剤、免疫調節薬、インターフェロン、インターロイキン、植物由来抗がん剤、BRM製剤からなる群から選択される、1または複数の抗がん剤であることを特徴とする、
医薬組成物。 - miRNAをコードする遺伝子の転写産物またはそのプロセシング産物の、がん治療用医薬組成物の製造のための使用であって、
前記miRNAが、miR-3140、miR-137、miR-631、およびmiR-657からなる群から選択される1または複数のmiRNAであることを特徴とする、
使用。 - がんの治療方法であって、
前記方法は、がん患者に対して、治療上有効量の、miRNAをコードする遺伝子の転写産物またはそのプロセシング産物を含む抗がん用医薬組成物を適用するステップ、
を含み、
前記miRNAが、miR-3140、miR-137、miR-631、およびmiR-657からなる群から選択される1または複数のmiRNAであることを特徴とする、
治療方法。
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AU2018364916A AU2018364916B2 (en) | 2017-11-09 | 2018-11-06 | Therapeutic pharmaceutical composition for cancer including miRNA |
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CN201880071699.7A CN111405901A (zh) | 2017-11-09 | 2018-11-06 | 含有miRNA的癌症治疗用医药组合物 |
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