WO2021246165A1 - Acide nucléique pour la déméthylation d'un gène oasis et procédé de déméthylation l'utilisant - Google Patents

Acide nucléique pour la déméthylation d'un gène oasis et procédé de déméthylation l'utilisant Download PDF

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WO2021246165A1
WO2021246165A1 PCT/JP2021/018886 JP2021018886W WO2021246165A1 WO 2021246165 A1 WO2021246165 A1 WO 2021246165A1 JP 2021018886 W JP2021018886 W JP 2021018886W WO 2021246165 A1 WO2021246165 A1 WO 2021246165A1
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oasis
nucleic acid
guide rna
sequence
demethylation
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和則 今泉
敦 齋藤
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国立大学法人広島大学
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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
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • 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
    • A61P35/00—Antineoplastic agents
    • 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
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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
    • 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
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • the present invention relates to a nucleic acid for demethylation of an OASIS gene and a demethylation method using the same, and in particular, a nucleic acid for demethylation of the OASIS gene using epigenome editing and a demethylation method using the same. Regarding.
  • OASIS old astrocyte specificly induced substance
  • Non-Patent Document 2 it has been known that the OASIS gene is methylated and its expression is reduced in certain cancer cells.
  • Non-Patent Document 3 an epigenome editing technique for artificially inducing demethylation of a specific gene has been developed.
  • the method shown in Non-Patent Document 3 applies the CRISPR-Cas9 system, which is a genome editing technique, and fuses a demethylase enzyme with dCas9, which is a Cas9 variant having no DNA cleavage activity.
  • a guide RNA to demethylate the vicinity of the target sequence.
  • appropriate conditions, particularly suitable target positions of guide RNA differ depending on the gene for demethylation, and a specific method suitable for demethylation of the OASIS gene has not yet been clarified. ..
  • the present invention has been made in view of the above problems, and an object thereof is to enable artificial demethylation of a methylated OASIS gene, and further to suppress cell proliferation by demethylation of the OASIS gene. To be able to do it.
  • the present inventors have completed the present invention by finding that the epigenome editing technique using a predetermined guide RNA enables demethylation of the OASIS gene as a result of diligent research. did.
  • the nucleic acid for demethylation of the OASIS gene encodes a base sequence and a guide RNA encoding a fusion protein of dCas9 and a demethylase, which is arranged under the control of a promoter sequence.
  • the guide RNA contains a base sequence that recognizes a part or all of the base sequences from -327th to +511th, when the first base of the translation start point of the OASIS gene is +1st. It is characterized by having.
  • the nucleic acid for demethylation of the OASIS gene when introduced into cells, a fusion protein of dCas9 and a demethylase and a guide RNA are expressed, and the fusion protein binds to dCas9.
  • the guide RNA produced will be guided to the target sequence of the guide RNA in the genome of the cell.
  • the guide RNA is designed to recognize the promoter region of the OASIS gene as a target sequence, and the demethylase fused to dCas9 is induced in the promoter region of the OASIS gene. Therefore, the demethylase can act on the methylated base existing in the promoter region of the OASIS gene to cause demethylation.
  • the OASIS gene is transcriptionally induced. Therefore, the promoter region of the OASIS gene can be demethylated by introducing the nucleic acid according to the present invention into cells such as cancer cells in which the expression of p21 is reduced and cell proliferation continues. By promoting the expression of OASIS and increasing the expression of p21, cell proliferation of cancer cells and the like can be suppressed.
  • the guide RNA has a base sequence from -327th to +36th, where the first base of the translation start point of the OASIS gene is +1. It is preferable to have a sequence that recognizes a part or all of, or a part or all of the base sequence from the +489th to the +511th. Further, the guide RNA is a part or all of the base sequence from the 327th to the -305th, or from the +14th to the +36th, when the first base of the translation start point of the OASIS gene is the +1st. It is more preferable to have a sequence that recognizes a part or all of the base sequence of.
  • the demethylase fused to dCas9 that binds to the guide RNA is induced to a position where it can act suitably on the methylation site of the OASIS gene. , The demethylation effect can be improved.
  • the demethylase is preferably TET1.
  • the promoter sequence includes the first promoter sequence for controlling the expression of the fusion protein of dCas9 and the demethylase, and the expression of the guide RNA. It can include a second promoter sequence for control.
  • a promoter suitable for controlling the expression of the fusion protein of dCas9 and the demethylase can be selected, and a promoter suitable for controlling the expression of the guide RNA can be selected.
  • the base sequences encoding each of dCas9, demethylase, and guide RNA arranged under the control of the promoter sequence are linear or cyclic. It is preferable that it is contained in the vector of. In this case, it is preferable to select a vector suitable for introducing the nucleic acid into cells.
  • the method for demethylating the OASIS gene according to the present invention is characterized by comprising the step of introducing the nucleic acid for demethylation of the OASIS gene according to the present invention into the cell.
  • the step of introducing the nucleic acid for demethylation of the OASIS gene according to the present invention into the cell is included.
  • a fusion protein with a demethylase and a guide RNA can be expressed.
  • Demethylation increases the expression of the OASIS gene and also increases p21 downstream thereof. Therefore, for cells such as cancer cells that do not suppress the cell cycle and continue to grow, the cell proliferation of cancer cells and the like is suppressed by increasing the expression of p21 by using the method according to the present invention. can do.
  • the pharmaceutical composition for treating cancer according to the present invention is characterized by containing a nucleic acid for demethylation of the OASIS gene according to the present invention.
  • the nucleic acid for demethylation of the OASIS gene according to the present invention is contained, as described above, the methylated OASIS gene can be demethylated. can. Therefore, for example, it is possible to suppress the proliferation of cancer cells that continue to grow without proper control of cell proliferation due to methylation of the OASIS gene.
  • the present invention also relates to the use of the nucleic acid for producing the pharmaceutical composition for treating cancer, and further, the present invention comprises administering the nucleic acid to a cancer patient for the treatment of cancer. It also concerns how to include it.
  • nucleic acid for demethylation of the OASIS gene according to the present invention it is possible to artificially demethylate the base of the promoter region of the methylated OASIS gene. This can promote the expression of the OASIS gene. Further, this can increase the expression of p21, and as a result, cell proliferation can be suppressed.
  • FIG. 1 is a graph comparing the rates of methylation in the OASIS promoter region in each cancer tissue.
  • FIG. 2 (a) is a graph comparing the methylation rates in the OASIS promoter region of each cancer tissue, and
  • FIG. 2 (b) shows the methylation rate and OASIS gene expression in the OASIS promoter region of each cancer tissue. It is a graph which shows the inverse correlation of a level.
  • FIG. 3 is a model diagram for explaining the OASIS gene demethylation technique according to the present invention.
  • FIG. 4 is a photograph showing the results of Western blotting to confirm the expression of OASIS and p21 in three glioblastoma cell lines (U87MG, U138MG, U251MG).
  • FIG. 5A is a model diagram showing the promoter region of the OASIS gene
  • FIG. 5B is a bisulfite-treated base sequence for the OASIS promoter region of three glioblastoma cell lines (U87MG, U138MG, U251MG). It is a graph which shows the result of having performed the determination method (bisulfite sequencing).
  • FIG. 6 (a) is a model diagram for explaining the plasmid for demethylation of the OASIS promoter region prepared in this example, and FIG. 6 (b) shows the sequence of the guide RNA prepared in this example. It is a figure which shows.
  • FIG. 6 (a) is a model diagram for explaining the plasmid for demethylation of the OASIS promoter region prepared in this example
  • FIG. 6 (b) shows the sequence of the guide RNA prepared in this example. It is a figure which shows.
  • FIG. 7 (a) is a fluorescence micrograph confirming the expression of p21 in U251 MG cells into which a plasmid for demethylation of the OASIS promoter region prepared in this example was introduced
  • FIG. 7 (b) is a fluorescence micrograph. It is a graph which shows the ratio of the number of p21 positive cells to the DAPI positive cells observed under the microscope.
  • FIG. 8 is a photograph showing the results of Western blotting to confirm the expression of TET1 and TET2 in three glioblastoma cell lines (U87MG, U138MG, U251MG).
  • FIG. 9A is a photograph showing the results of western blotting for confirming the expression of OASIS and p21 in U251 MG cells into which the plasmid for demethylation of the OASIS promoter region prepared in this example was introduced.
  • FIG. 9B is a graph showing the methylation ratio of the OASIS promoter in U251 MG cells into which the plasmid has been introduced, and
  • FIG. 9 (c) shows the results of staining SA- ⁇ gal with U251 MG cells into which the plasmid has been introduced. It is a graph which shows the result of quantifying the positive signal by a photograph and its staining.
  • FIG. 1 is a graph comparing methylation in the promoter region of the OASIS gene in each cancer tissue, the left side of the graph (dark gray color) is the result of human cancer tissue, and the right side of the graph (light gray color). Shows the results of normal tissue, and the higher the value, the higher the rate of methylation.
  • cancer tissues such as cervical cancer, colon adenocarcinoma, renal cell carcinoma, lung squamous cell carcinoma, rectal cancer, and melanoma
  • methylation is stronger than that in normal tissues. I was able to confirm what was happening.
  • FIG. 2A is a graph comparing methylation in the promoter region of the OASIS gene of each cancer tissue, the left side (gray color) of the graph is the result of normal tissue, and the right side (black) of the graph is the tumor. The results of the tissue are shown, and the higher the value, the higher the rate of methylation. As shown in FIG.
  • FIG. 2 (a) it is normal in cancer tissues such as bladder-urothelial cancer, epithelial esophageal cancer, head and neck squamous epithelial cancer, lung squamous epithelial cancer, and epithelial clear cell cancer. It was confirmed that the methylation was stronger than that of the tissue.
  • FIG. 2B is a graph showing the correlation between the rate of methylation in the OASIS promoter region of each of these cancer tissues and the expression level of the OASIS gene. As shown in FIG. 2 (b), it was confirmed that the expression level of the OASIS gene decreased as the methylation level in the OASIS promoter region increased.
  • the present inventors also revealed that the methylation of the OASIS gene is enhanced and its expression is abolished in a specific cancer cell line, as will be shown in detail later in Examples. From the above results, it is suggested that the expression of OASIS is reduced or eliminated by DNA methylation of the OASIS gene in cancer cells, which hinders the cell proliferation inhibitory function and causes the cancer cells to proliferate continuously. To.
  • the present inventors have conceived a method for artificially demethylating the promoter region of the OASIS gene to increase the expression of OASIS and effectively suppress cell proliferation, and have provided means for achieving this. Completed.
  • the means applies epigenome editing using a fusion protein of dCas9 and a demethylase (eg, TET).
  • one embodiment of the present invention is an OASIS gene containing a base sequence encoding a fusion protein of dCas9 and a demethylase and a base sequence encoding each of a guide RNA, which are arranged under the control of a promoter sequence. It is a nucleic acid for demethylation.
  • the guide RNA is a base that recognizes a part or all of the base sequence from the 327th to the +511th, when the first base of the translation start point of the OASIS gene is the +1th base. Has an sequence.
  • the nucleic acid according to the present embodiment relates to an epigenome editing technique for modifying a modified state of DNA by applying a CRISPR-Cas9-based genome editing technique well known in the present technology.
  • the nucleic acid according to this embodiment relates to an epigenome editing technique for demethylating methylated DNA. Similar to the genome editing technology of the CRISPR-Cas9 system, this technique utilizes a guide RNA that recognizes the target sequence, and uses a variant of Cas9 that is a nuclease that binds to the guide RNA and cleaves the target sequence.
  • the variant of Cas9 is dCas9 whose nuclease activity has been abolished by an amino acid mutation.
  • dCas9 retains the function of binding to the target sequence in the intracellular genome via the guide RNA. Since dCas9 is fused with the demethylase in the present embodiment, the demethylase fused to dCas9 binds to the target sequence via the guide RNA and dCas9, and undergoes methylation in the vicinity of the target sequence. It can act on the base to cause demethylation.
  • the first base of the translation start point of the OASIS gene is +1 as a guide RNA
  • a guide RNA having the above is used.
  • the present embodiment aims to demethylate the methylated promoter region of the OASIS gene, particularly the CpG island region, and by designing the guide RNA so as to recognize the range of the above sequence, it can be effectively demethylated. Methylation can be achieved.
  • the recognition sequence of the guide RNA is, in particular, a part or all of the base sequence from -327th to +36th, or a part or all of the base sequence from +489th to +511th within the above range. It is preferable to have a sequence that recognizes. Furthermore, among them, a part or all of the base sequence from -327th to -305th, a part or all of the base sequence from +14th to +36th, or a part or all of the base sequence from +489th to +511th. It is more preferable to have a sequence that recognizes all. In the present embodiment, a part of the base sequence is preferably at least 20 consecutive bases, more preferably at least 10 consecutive bases, and further preferably at least 5 consecutive bases.
  • the demethylating enzyme is not particularly limited as long as it can exert a demethylating action on the DNA being methylated, and for example, TET1, TET2, TET3 and the like can be used. can.
  • the promoter for expressing the fusion protein of dCas9 and the demethylase and the guide RNA is not particularly limited as long as they can normally express them, but the fusion of dCas9 and the demethylase is not particularly limited. It preferably contains a first promoter suitable for protein expression and a second promoter suitable for guide RNA expression.
  • a first promoter for example, a CAG promoter, an EF-1 ⁇ promoter, a TK promoter and the like can be used.
  • the second promoter for example, U6 promoter, T7 promoter, H1 promoter and the like can be used.
  • the nucleic acid according to the present embodiment preferably contains a nuclear localization signal (NLS) sequence in order to promote the translocation into the nucleus after being introduced into the cell.
  • NLS nuclear localization signal
  • the NLS is not particularly limited as long as it can transfer the nucleic acid according to the present embodiment into the nucleus.
  • the nucleic acid is preferably contained in the vector so as to be suitably introduced into the cell, and the vector may be linear or cyclic.
  • the vector is not limited to the following, but for example, a plasmid vector, a cosmid vector, a virus vector, an artificial chromosome vector and the like can be used.
  • Another embodiment according to the present invention is a method for demethylating an OASIS gene using the nucleic acid according to the present invention.
  • the method is characterized by comprising the step of introducing the nucleic acid according to the present invention into a cell.
  • various methods well known in the present technical field can be used as the method for introducing the nucleic acid into the cell.
  • a chemical method using a cationic lipid reagent or a cationic non-lipid reagent, a physical method such as electroporation, a biological method using a virus, or the like can be used.
  • Another embodiment according to the present invention is a pharmaceutical composition for cancer treatment containing the nucleic acid according to the present invention. Since the pharmaceutical composition according to the present embodiment contains the above nucleic acid, as described above, the methylated OASIS gene can be demethylated. Therefore, for example, the OASIS gene is methylated to appropriately control cell proliferation. It is possible to suppress the growth of cancer cells that continue to grow without being treated. Therefore, the nucleic acid according to the present invention can be used for producing a pharmaceutical composition for treating cancer, and can also be used for a method including administration of the nucleic acid to a cancer patient for the treatment of cancer. be.
  • the target cancer is not particularly limited as long as the OASIS gene is methylated in the cancer cells and the cell proliferation is not appropriately controlled and continues to grow.
  • Such cancers include cervical cancer, colon adenocarcinoma, renal cell cancer, lung, for example, in FIGS. 1 and 2 in which methylation of the OASIS gene is confirmed to occur more frequently than in normal cells.
  • the dose of the pharmaceutical composition according to the present invention may be set so as to secure the amount required by the application target.
  • the primary antibodies were anti-p21 mouse IgG monoclonal antibody (Santa Cruz Biotechnology; 1000-fold diluted), anti- ⁇ -actin mouse IgG monoclonal antibody (Sigma-Aldrich; 100,000-fold diluted), and anti-OASIS mouse IgG monoclonal antibody (Murakami T. et. Al., Nature Cell Biology 11, 1205-1211, 2009; 1000-fold diluted) was diluted with Can Get Signal solution (TOYOBO) and used. The result of Western blotting is shown in FIG.
  • a bisulfite-treated nucleotide sequence determination method (bisulfite sequencing) was performed on the CpG island region, which is considered to be mainly methylated in the promoter region of the OASIS gene, to perform methylation in the region.
  • the proportion was evaluated.
  • FIG. 5 (a) in the promoter region of the OASIS gene, since the CpG island exists at the base sequence position of -569 to +256 when the base of the translation start point is +1.
  • Bisulfite sequencing was performed on the nucleotide sequence positions of 704 to +367. In particular, here, the above region was divided into two, -704 to -165 and -164 to +367, and the methylation ratio of each was examined.
  • each of 2 ⁇ 106 cells (U87MG, U138MG, U251MG) was cultured for 24 hours, and then genomic DNA was purified and extracted from the recovered cells using NucleoSpin Tissue XS (Macherey-Nagel). The extraction method was based on the manual attached to NucleoSpin Tissue XS. The extracted 2 ⁇ g of genomic DNA was treated with bisulfite (hydrosulfite) using a Methylamp Whole Cell Bisulfite Modification kit (Epigentek). The bisulfite processing method was based on the manual attached to the Methylamp Whole Cell Bisulfite Modification kit.
  • a PCR reaction was performed using the DNA synthase Epitaq HS (TakaraBio).
  • a fragment of 1071 bp was extracted from an agarose gel using a Gel / PCR Extension kit (FastGene), and then incorporated into a p-GEM T-easy Vector (Promega) for transformation.
  • Five cloned colonies were picked up in each sample and sequence analysis was performed to evaluate the rate of methylation in the promoter region of the OASIS gene. The result is shown in FIG. 5 (b).
  • a method for preparing a plasmid for expressing a fusion protein of dCas9 and a demethylase and a guide RNA in the cell will be described.
  • the design of the plasmid was based on the design for artificially inducing demethylation disclosed in Non-Patent Document 3 above. Specifically, using the pCAG-Neo vector (Fuji Film Wako Pure Chemical Industries, Ltd.), they can be expressed downstream of the CAG promoter of the above vector so that fusion proteins with NLS, dCas9, demethylase TET1 and GFP can be expressed.
  • the gene sequence encoding the above was inserted.
  • the GFP sequence was inserted to confirm the intracellular introduction of this plasmid.
  • the U6 promoter sequence and the guide RNA sequence shown below were inserted downstream of the sequence encoding the fusion protein so that the guide RNA could be expressed.
  • a plasmid for expressing the fusion protein of dCas9 and the demethylase and the guide RNA in the cell was obtained (see FIG. 6 (a)).
  • FIG. 7 (a) shows a photograph taken by a fluorescence microscope
  • guide RNA targets 327 to -305 and +14 to +36 which are within or near the range of -704 to -165 suggested as suitable guide RNA target ranges in FIG. 5 (b). It is suggested that the position is particularly preferable. Further, although it is outside the above range, it is suggested that it is also preferable to set +489 to +511 as the target position of the guide RNA.
  • TET1 was used as the demethylase, but the effectiveness of other demethylases was also investigated.
  • the TET family of demethylases in humans are known as TET1, TET2 and TET3.
  • the involvement of TET1 and TET2 in dysregulation of methylation in tumor tissues has been reported (Ichimura N. et al., Cancer Prevention Research 8, 702-712, 2015, Murata A. et al., Oncotarget 6,23372-23382). , 2015).
  • TET3 and methylation abnormalities in tumor tissues were compared. The method and results will be described below.
  • TET1 As shown in FIG. 8, in U87MG cells and U138MG cells (see FIG. 4) in which OASIS expression is observed, the expression level of TET1 is normally high both after DOXO treatment, which causes DNA damage. On the other hand, almost no expression of TET2 is observed. Expression of TET2 is observed in U251 MG cells (see FIG. 4) where the expression level of OASIS is extremely low, but the expression level of TET1 is very low. This suggests that TET1 is involved in demethylation in the OASIS promoter. Therefore, it is suggested that TET1 should be selected as the demethylase in U251 MG cells.
  • the plasmid prepared above using the lipofection reagent Screen Fect A (Fuji Film Wako Pure Chemical Industries, Ltd.) (guide RNA is SEQ ID NO: 1 sequence) was introduced into U251 MG cells.
  • guide RNA is SEQ ID NO: 1 sequence
  • control cells transfected with a plasmid (non-targeting) that cannot target the OASIS gene were also separately provided.
  • the cells were treated with 1 ⁇ g / ml doxorubicin (DOXO), which induces DNA damage, for 1 hour in order to induce the expression of the OASIS gene.
  • DOXO 1 ⁇ g / ml doxorubicin
  • the primary antibodies were anti-p21 mouse IgG monoclonal antibody (Santa Cruz Biotechnology; 1000-fold diluted), anti- ⁇ -actin mouse IgG monoclonal antibody (Sigma-Aldrich; 100,000-fold diluted), and anti-OASIS mouse IgG monoclonal antibody (Murakami T. et. Al., Nature Cell Biology, 11, 1205-1211, 2009; 1000-fold diluted) was diluted with Can Get Signal solution (TOYOBO) and used. The result is shown in FIG. 9 (a).
  • a plasmid expressing the guide RNA of the sequence of SEQ ID NO: 1 or the control guide RNA that cannot target the OASIS gene and the fusion protein of dCas9 and TET1 is introduced into U251 MG cells, respectively.
  • the rate of methylation of the OASIS gene in the CpG island in the cell was compared. The method and results will be described below.
  • the OASIS gene promoter in the CpG island is compared with the case where the plasmid expressing the control guide RNA is introduced. Methylation was significantly reduced. That is, it was clarified that demethylation can be induced in the promoter of the OASIS gene by introducing a plasmid expressing the guide RNA of SEQ ID NO: 1 into U251 MG cells.
  • a plasmid expressing the guide RNA of the sequence of SEQ ID NO: 1 or the control guide RNA that cannot target the OASIS gene and the fusion protein of dCas9 and TET1 was introduced into U251 MG cells, respectively.
  • the cell proliferation of the cells was compared.
  • staining of SA- ⁇ -Gal which is a cell senescence marker, was used. The method and results will be described below.
  • the quantification method was based on the manual attached to Cellular Senescence Plate Assay kit SPIDER- ⁇ -Gal.
  • the fluorescence of SPiDER- ⁇ -Gal (contained in the kit reaction reagent) emitted by reacting with intracellular SA- ⁇ -Gal was measured with a microplate reader Varioskan Flash (Thermo Fisher Scientific) (Ex: 535 nm).
  • N 3, mean ⁇ SD, Student's t-test, * P ⁇ 0.05). The result is shown in FIG. 9 (c).
  • the methylated OASIS gene can be artificially demethylated. Therefore, it can be said that cell proliferation can be suppressed by demethylation of the OASIS gene. Therefore, it is suggested that it can be applied to a cancer therapeutic agent that exerts a growth inhibitory effect on cancer cells that proliferate in a disorderly manner.

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Abstract

Acide nucléique selon la présente invention pour la déméthylation du gène OASIS comprenant, située sous le contrôle d'une séquence de promoteur, une séquence de base qui code une protéine de fusion d'enzyme de déméthylation de dCas9/déméthylation et une séquence de base qui code un ARN de guidage. Désignant la position +1 comme première base au point de départ de traduction du gène OASIS, l'ARN guide a une séquence de base qui reconnaît tout ou partie de la séquence de base de la position -327 à la position +511.
PCT/JP2021/018886 2020-06-03 2021-05-19 Acide nucléique pour la déméthylation d'un gène oasis et procédé de déméthylation l'utilisant WO2021246165A1 (fr)

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JP2016512264A (ja) * 2013-03-15 2016-04-25 ザ ジェネラル ホスピタル コーポレイション 特定のゲノム遺伝子座へのゲノムおよびエピゲノム調節タンパク質のｒｎａ誘導型標的化
WO2017090724A1 (fr) * 2015-11-25 2017-06-01 国立大学法人 群馬大学 Kit d'édition de méthylation d'adn et procédé d'édition de méthylation d'adn

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JP2016512264A (ja) * 2013-03-15 2016-04-25 ザ ジェネラル ホスピタル コーポレイション 特定のゲノム遺伝子座へのゲノムおよびエピゲノム調節タンパク質のｒｎａ誘導型標的化
WO2017090724A1 (fr) * 2015-11-25 2017-06-01 国立大学法人 群馬大学 Kit d'édition de méthylation d'adn et procédé d'édition de méthylation d'adn

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ROSE MICHAEL, SCHUBERT CLAUDIA, DIERICHS LAURA, GAISA NADINE T, HEER MATTHIAS, HEIDENREICH AXEL, KNÜCHEL RUTH, DAHL EDGAR: "OASIS/CREB3L1 is epigenetically silenced in human bladder cancer facilitating tumor cell spreading and migration in vitro", EPIGENETICS, LANDES BIOSCIENCE, US, vol. 9, no. 12, 2 December 2014 (2014-12-02), US , pages 1626 - 1640, XP055880583, ISSN: 1559-2294, DOI: 10.4161/15592294.2014.988052 *
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