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WO2023013970A1 - Composition contenant un miarn dérivé de vésicules extracellulaires de lymphocytes t en tant que principe actif, et son utilisation - Google Patents

Composition contenant un miarn dérivé de vésicules extracellulaires de lymphocytes t en tant que principe actif, et son utilisation Download PDF

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WO2023013970A1
WO2023013970A1 PCT/KR2022/011094 KR2022011094W WO2023013970A1 WO 2023013970 A1 WO2023013970 A1 WO 2023013970A1 KR 2022011094 W KR2022011094 W KR 2022011094W WO 2023013970 A1 WO2023013970 A1 WO 2023013970A1
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mir
cancer
cells
seq
nucleotide sequence
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Korean (ko)
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백문창
예경무
정도경
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Daegu Gyeongbuk Institute of Science and Technology
Industry Academic Cooperation Foundation of KNU
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Daegu Gyeongbuk Institute of Science and Technology
Industry Academic Cooperation Foundation of KNU
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    • 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
    • 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
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes

Definitions

  • the present invention relates to a composition comprising miRNA derived from extracellular endoplasmic reticulum of T cells as an active ingredient and a use thereof.
  • Cancer is a product of uncontrolled and disorderly cell proliferation caused by an excess of abnormal cells, and can be said to be a disease caused by genetic mutations from a molecular biological point of view.
  • small extracellular vesicles are membrane-structured small vesicles secreted from most cells.
  • the diameter of sEVs is approximately 30-10 nm, and sEVs contain various types of proteins, genetic materials (DNA, RNA, miRNA), lipids, etc. derived from the cell.
  • sEVs are not directly released from the plasma membrane, but originate from specific intracellular compartments called multivesicular bodies (MVBs) and are released and secreted outside the cell. That is, when the fusion of the polycystic body and the plasma membrane occurs, the vesicles are released into the extracellular environment, which is called sEV.
  • MVBs multivesicular bodies
  • MicroRNA one of the substances derived from extracellular endoplasmic reticulum, is a non-coding RNA with 22 base sequences or less, which degrades target mRNA or inhibits the transcription of target mRNA. to regulate gene expression. Its gene expression regulation is related to cell differentiation and cell proliferation. In particular, during embryogenesis, miRNAs are expressed at elusive times, which play an important role in each stage of embryonic development. However, although the important function of regulating miRNA differentiation continues to be discovered, the precise mechanism regulating miRNA expression remains unknown. Recently, the possibility of epigenetic expression of miRNAs with anticancer functions in human cancer cells has been suggested, and various evidences for the epigenetic regulation of miRNA clusters have been reported.
  • the anticancer miRNA encoding various DNA regions is abnormally hypermethylated in human breast cancer cell lines, so the gene is not activated, and DNMT (DNA methylatransferase) 5-Asa in AGS gastric cancer cell lines.
  • DNMT DNA methylatransferase 5-Asa in AGS gastric cancer cell lines.
  • An object of the present invention is to provide an anticancer composition comprising miRNAs exhibiting anticancer activity among miRNAs derived from extracellular endoplasmic reticulum of T cells as active ingredients.
  • Another object of the present invention is to provide a composition for enhancing immunity comprising, as an active ingredient, miRNAs that enhance the proliferation and activity of immune cells among miRNAs derived from the extracellular endoplasmic reticulum of T cells.
  • the present invention is any one selected from the group consisting of miR-101-3p, miR-181a-3p, miR-223-3p, miR-619-5p, miR-1246, miR-3182, miR-4787-5p and miR-5787.
  • an anti-cancer composition comprising at least one miRNA as an active ingredient.
  • the present invention comprises the steps of transfecting T cells with a vector containing IL-2, a linker and a transmembrane protein; isolating extracellular vesicles expressing IL-2 on the surface from the transfected T cells; Collecting miRNA from the extracellular vesicles; and selecting a miRNA that inhibits the expression of PD-L1 in cancer cells from among the collected miRNAs.
  • the present invention provides any one or more selected from the group consisting of miR-17-5p, miR-29a-3p, miR-92a-1-5p, miR-125a-5p, miR-181a-3p and miR-223-3p.
  • a composition for enhancing immunity comprising miRNA as an active ingredient.
  • the present invention comprises the steps of transfecting T cells with a vector containing IL-2, a linker and a transmembrane protein; isolating extracellular vesicles expressing IL-2 on the surface from the transfected T cells; Collecting miRNA from the extracellular vesicles; and selecting miRNAs that promote the expression of Ki-67, IFN- ⁇ and Granzyme B genes in T cells from among the collected miRNAs.
  • T cells transfected with a vector containing IL-2, a linker, and a transmembrane protein produce extracellular vesicles expressing IL-2 on their surface, and extracellular vesicles expressing IL-2 on their surface.
  • miRNAs contained in miR-101-3p, miR-181a-3p, miR-223-3p, miR-619-5p, miR-1246, miR-3182, miR-4787-5p and miR-5787 are cancer cells.
  • miR-17-5p, miR-29a-3p, miR-92a-1-5p, miR-125a-5p, miR-181a-3p and miR -223-3p showed an immune enhancing effect by increasing the expression levels of Ki-67, IFN- ⁇ and Granzyme B genes in T cells, so that the composition containing the miRNAs could be used as an anti-cancer composition and an immune enhancing composition. can be provided.
  • Figure 1 shows the results of RNA-Seq data analysis of the activity of miRNAs isolated from the extracellular vesicles of T cells expressing IL-2 on the surface.
  • FIG. 2 shows the results of classifying miRNAs that regulate PD-L1 expression in cancer cells among miRNAs isolated from the extracellular vesicles of T cells expressing IL-2 on their surface.
  • 3 is a result of classifying miRNAs that increase immune cell activity among miRNAs isolated from extracellular endoplasmic reticulum of T cells expressing IL-2 on the surface.
  • 4 is a result of evaluating the expression changes of PD-L1 and Rab 27a genes by transfecting cancer cells with miRNA that regulates PD-L1 expression in cancer cells.
  • 5 is a result of evaluating the expression level of PD-L1 protein by transfecting cancer cells with miRNA that regulates PD-L1 expression in cancer cells.
  • the present invention is any one selected from the group consisting of miR-101-3p, miR-181a-3p, miR-223-3p, miR-619-5p, miR-1246, miR-3182, miR-4787-5p and miR-5787.
  • an anti-cancer composition comprising at least one miRNA as an active ingredient.
  • the miR-101-3p consists of the nucleotide sequence represented by SEQ ID NO: 7
  • the miR-181a-3p consists of the nucleotide sequence represented by SEQ ID NO: 8
  • the miR-223-3p consists of the nucleotide sequence represented by SEQ ID NO: 9
  • the miR-619-5p consists of the nucleotide sequence represented by SEQ ID NO: 10
  • the miR-1246 consists of the nucleotide sequence represented by SEQ ID NO: 11
  • the miR-3182 consists of the nucleotide sequence represented by SEQ ID NO: 10 12
  • the miR-4787-5p may consist of the nucleotide sequence represented by SEQ ID NO: 13
  • the miR-5787 may consist of the nucleotide sequence represented by SEQ ID NO: 14.
  • the miRNA has the effect of suppressing the expression of PD-L1 and Rab 27a genes in cancer cells, and thus can exhibit anticancer activity.
  • the miRNA is derived from the extracellular endoplasmic reticulum of T cells, and the extracellular endoplasmic reticulum can express IL-2 on its surface.
  • the anticancer composition is melanoma, colon cancer, lung cancer, skin cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, perianal cancer, breast cancer, fallopian tube carcinoma, endometrium Carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hawkins' disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma , Bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, and pituitary gland adenoma to prevent or treat any one or more cancer diseases selected from the group consisting of may be for
  • the present invention comprises the steps of transfecting T cells with a vector containing IL-2, a linker and a transmembrane protein; isolating extracellular vesicles expressing IL-2 on the surface from the transfected T cells; Collecting miRNA from the extracellular vesicles; and selecting a miRNA that inhibits the expression of PD-L1 in cancer cells from among the collected miRNAs.
  • the extracellular vesicles are small extracellular vesicles (sEVs) having a diameter of 30 to 100 nm, and may include exosomes, microvesicles, and the like.
  • sEVs small extracellular vesicles
  • the linker a linker sequence commonly used in the art may be used, and specifically, a linker having an amino acid sequence represented by SEQ ID NO: 27 (GSTSGSGKPGSGEGSTKG) may be used.
  • the transmembrane protein is epidermal growth factor receptor, insulin receptor, platelet-derived growth factor (PDGF) receptor, vascular endothelial growth factor receptor, fibroblast growth factor receptor, cholecystokinin (CCK) receptor, neurotrophic factor Any one selected from the group consisting of (Neurotrophic factor; NGF) receptor, Hepatocyte growth factor (HGF) receptor, Ephrin (Eph) receptor, angiopoietin receptor, and RTK (Related to receptor tyrosine kinase) receptor It may be the transmembrane domain of one or more receptors.
  • NGF Neurotrophic factor
  • HGF Hepatocyte growth factor
  • Ephrin Ephrin
  • RTK Related to receptor tyrosine kinase
  • the present invention provides any one or more selected from the group consisting of miR-17-5p, miR-29a-3p, miR-92a-1-5p, miR-125a-5p, miR-181a-3p and miR-223-3p.
  • a composition for enhancing immunity comprising miRNA as an active ingredient.
  • the miR-17-5p consists of the nucleotide sequence represented by SEQ ID NO: 21, the miR-29a-3p consists of the nucleotide sequence represented by SEQ ID NO: 22, and the miR-92a-1-5p consists of the nucleotide sequence represented by SEQ ID NO: 23
  • the miR-125a-5p consists of the nucleotide sequence represented by SEQ ID NO: 24, the miR-181a-3p consists of the nucleotide sequence represented by SEQ ID NO: 25, and the miR-181a-3p consists of the nucleotide sequence represented by SEQ ID NO: 25.
  • 223-3p may consist of the nucleotide sequence represented by SEQ ID NO: 26.
  • the miRNA can increase the expression levels of Ki-67, IFN- ⁇ and Granzyme B genes in T cells.
  • the miRNA is derived from the extracellular endoplasmic reticulum of T cells, and the extracellular endoplasmic reticulum can express IL-2 on its surface.
  • the present invention comprises the steps of transfecting T cells with a vector containing IL-2, a linker and a transmembrane protein; isolating extracellular vesicles expressing IL-2 on the surface from the transfected T cells; Collecting miRNA from the extracellular vesicles; and selecting miRNAs that promote the expression of Ki-67, IFN- ⁇ and Granzyme B genes in T cells from among the collected miRNAs.
  • HEK-293FT cells human embryonic kidney
  • DMEM medium Hyclone
  • Jurkat cells human T lymphocytes
  • RPMI 1640 medium Hyclone
  • HEK-293FT cells were seeded in a 6-well plate at a density of approximately 1 ⁇ 10 6 cells/well and cultured in Lipofectamine 2000 Reagent (ref. 11668-027) under Opti-MEM (ref. 31985-070; Gibco) medium.
  • Example 3 the lentivirus used Jurkat transduction into cells
  • Lentiviruses expressing 10 ⁇ g/mL polybrene and 500 ⁇ g/mL IL-2 were added to 0.3 mL RPMI medium and treated with Jurkat cells (1 ⁇ 10 6 cells/ml). Spinoculation was performed by centrifuging the lentivirus and cell mixture at 30° C. at 1,200 ⁇ g for 90 minutes. Cells were then incubated overnight at 37°C with lentivirus. Excess virus was removed and fresh medium supplemented with 10% fetal bovine serum was added the next day.
  • Control Jukat cells or IL-2-expressing Jukat cells were seeded at a density of about 2 ⁇ 10 6 cells/well, and cultured for 24 hours in RPMI medium without addition of fetal calf serum. Subsequently, in order to isolate small extracellular vesicles (sEV) or sEV IL -2 (IL-2 surface-expressed extracellular vesicles), each supernatant obtained from each cell was centrifuged at 300xg, 2500xg, and 10,000xg consecutively. . The supernatant was then filtered through a 0.2 ⁇ m syringe filter and centrifuged at 120,000 ⁇ g. The sEV pellet was resuspended in PBS and centrifuged again at 120,000xg. The purified sEV pellet was resuspended in PBS or 1X cell lysis buffer for the next experiment.
  • sEV small extracellular vesicles
  • IL-2 IL-2 surface-expressed extracellular vesicles
  • Example 5 IL-2 surface expression extracellular Small RNA sequencing of the endoplasmic reticulum
  • RNA libraries were generated by the NextSeq 500 system using single-end 75 sequencing (Illumina, San Diego, CA., USA).
  • Sequence reads were mapped with the bowtie2 software tool to obtain Bam files (alignment files). Mature miRNA sequences were used as references for mapping. The number of reads mapped to mature miRNA sequences were extracted from alignment files using bedtools (v2.25.0) and Bioconductor (R development Core Team, 2011) using the R (version 3.2.2) statistical programming language. Read counts were used to determine the expression levels of miRNAs. Quantile normalization method was used for comparison between samples.
  • FIG. 1 RNA-seq data analysis of differentially expressed genes (FIG. 1), miRNAs regulating PD-L1 expression in cancer cells (FIG. 2) and miRNAs increasing immune cell activity (FIG. 3) were identified.
  • Example 6 human origin melanoma cells microRNA mimic oligo transfection
  • hsa-miRNA mimic oligos (hsa-miR-10a - 5p, hsa-miR-92a-1-5p, hsa-miR-101-3p, hsa-miR- 146a-5p, hsa-miR-150-5p, hsa-miR-181a-3p, hsa-miR-200c-3p, hsa-miR-hsa-miR-223-3p, hsa-miR-619-5p, hsa- miR-1246p, hsa-miR-1285-5p, hsa-miR-1908-5p, hsa-miR-1972, hsa-miR-3138, hsa-miR-3182, hsa-miR-3195, hsa-miR-3654, hsa-miR
  • Example 7 hsa - miRNAs mimic oligos After transfection human origin PD-L1 in melanoma cells and RAB 27a gene expression analysis
  • mRNA (Cat; R2062, ZYMO RESEARCH) was extracted from the cells, and then nanodrop (DS-11 Series Spectrophotometer; DeNovix) was used. to measure the concentration of mRNA.
  • nanodrop DS-11 Series Spectrophotometer; DeNovix
  • Real-time polymerase chain reaction was performed using the Step One Plus Real-Time PCR System (Applied Biosystems).
  • the relative mRNA level of the sample was calculated by Ct (comparative threshold cycle) analysis after normalization for the amount of Gapdh in the same sample, and was expressed as a 2 - ⁇ Ct value modified from the initial Ct value.
  • Example 8 western blot selected using hsa - miRNAs mimic oligos 8 types of transfection
  • ECL enhanced chemiluminescence
  • FIG. 5 it was confirmed that PD-L1 expression in human melanoma cells transfected with hsa-miRNA mimic oligos was significantly reduced compared to the control group.
  • miR-101-3p and miR-181a-3p are key miRNAs that significantly suppress PD-L1 expression in human-derived melanoma cells.
  • the genetic sequence of key miRNAs that inhibit melanoma PD-L1 expression selected in this experiment is as follows.
  • hsa-miR-101-3p UACAGUACUGUGAUAACUGAA (SEQ ID NO: 7)
  • hsa-miR-1246 AAUGGAUUUUUGGAGCAGG (SEQ ID NO: 11)
  • hsa-miR-4787-5p GCGGGGGUGGCGGCGGCAUCCC (SEQ ID NO: 13)
  • Example 9 In isolated primary CD8+ T cells microRNA mimic oligo transfection
  • hsa-miRNA mimic oligos (hsa-miR-17-5p, hsa-miR-29a-3p, hsa-miR-92a-1-5p, hsa-miR-125a-5p, hsa-miR-125a-5p, hsa-miR-125a-5p, hsa-miR-29a-3p, miR-181a-3p and hsa-miR-223-3p) were dispensed at 1 ⁇ l each.
  • each hsa-microRNA mimic oligo was added to a 100 ⁇ l Nucleocuvette Vessel (Cat; PCK-2005, Lonza), and 4D Nucleofector Core unit (Serial no: 510B0263, Lonza) and 4D Nucleofector X unit (Serial no: 510 X 0441 , Lonza) for transfection.
  • 500 ⁇ l of RPMI 1640 medium supplemented with 10% fetal bovine serum to the corresponding Nucleocuvette, suck it up with single use pipettes, and place 12 well plate ( REF; 3513, Corning) and cultured for 24 hours in a 5% CO 2 incubator at 37°C.
  • Example 10 hsa - miRNAs mimic oligos Evaluation of cytotoxic ability of primary CD8+ T cells after transfection
  • hsa-miRNA mimic oligos (hsa-miR-17-5p, hsa-miR-29a-3p, hsa-miR-92a-1-5p, hsa-miR-125a-5p, hsa-miR-181a-3p and hsa-miR-223-3p) into primary CD8+ T cells, and then mRNA (Cat; R2062, ZYMO RESEARCH) was extracted from the cells and mRNA was measured using nanodrop (DS-11 Series Spectrophotometer; DeNovix). The concentration of was measured.
  • Real-time polymerase chain reaction was performed using the Step One Plus Real-Time PCR System (Applied Biosystems).
  • the relative mRNA level of the sample was calculated by Ct (comparative threshold cycle) analysis after normalization for the amount of Gapdh in the same sample, and was expressed as a 2 - ⁇ Ct value modified from the initial Ct value.
  • hsa-miR-181a-3p ACCAUCGACCGUUGAUUGUACC (SEQ ID NO: 25)

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Abstract

La présente invention concerne : une composition contenant, en tant que principe actif, un miARN dérivé de vésicules extracellulaires de lymphocytes T ; et son utilisation. Il a été confirmé que des vésicules extracellulaires exprimant IL-2 sur la surface sont produites dans des lymphocytes T transfectés avec un vecteur contenant IL-2, un lieur et une protéine transmembranaire, miR-101-3p, miR-181a-3p, miR-223-3p, miR-619-5p, miR-1246, miR-3182, miR-4787-5p et miR-5787 parmi les miARN contenus dans les vésicules extracellulaires exprimant IL-2 sur la surface inhibent l'expression de gènes PD-L1 et Rab 27a dans des cellules cancéreuses, présentant ainsi une activité anticancéreuse, et miR-17-5p, miR-29a-3p, miR-92a-1-5p, miR-125a-5p, miR-181a-3p et miR-223-3p présentent un effet d'augmentation de l'immunité consistant à augmenter les niveaux d'expression de gènes Ki-67, IFN-γ et granzyme B dans les lymphocytes T. Par conséquent, une composition contenant les miARN est fournie en tant que composition anticancéreuse et une composition pour améliorer l'immunité.
PCT/KR2022/011094 2021-08-05 2022-07-28 Composition contenant un miarn dérivé de vésicules extracellulaires de lymphocytes t en tant que principe actif, et son utilisation Ceased WO2023013970A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108403712A (zh) * 2018-02-26 2018-08-17 中国人民解放军总医院 miR-223-3p抑制骨肉瘤生长和转移
WO2020157288A1 (fr) * 2019-02-01 2020-08-06 Universität Basel Cellules immunitaires résistantes aux inhibiteurs de la calcineurine destinées à être utilisées dans une thérapie par transfert cellulaire adoptive
KR20210027080A (ko) * 2019-09-02 2021-03-10 경북대학교 산학협력단 Il-2 표면 발현 세포외 소포체를 유효성분으로 포함하는 암 질환 예방 또는 치료용 조성물

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011122889A2 (fr) 2010-03-31 2011-10-06 주식회사 강스템홀딩스 Procédé d'inhibition de la sénescence de cellules souches adultes en inhibant l'expression de miarn
KR101820264B1 (ko) 2017-01-18 2018-01-18 건국대학교 산학협력단 줄기세포 유래 엑소좀을 포함하는 암의 예방 또는 치료용 조성물

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108403712A (zh) * 2018-02-26 2018-08-17 中国人民解放军总医院 miR-223-3p抑制骨肉瘤生长和转移
WO2020157288A1 (fr) * 2019-02-01 2020-08-06 Universität Basel Cellules immunitaires résistantes aux inhibiteurs de la calcineurine destinées à être utilisées dans une thérapie par transfert cellulaire adoptive
KR20210027080A (ko) * 2019-09-02 2021-03-10 경북대학교 산학협력단 Il-2 표면 발현 세포외 소포체를 유효성분으로 포함하는 암 질환 예방 또는 치료용 조성물

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DIAZ-SALAZAR CARLOS, SUN JOSEPH C.: "Coordinated Viral Control by Cytotoxic Lymphocytes Ensures Optimal Adaptive NK Cell Responses", CELL REPORTS, ELSEVIER INC, US, vol. 32, no. 12, 22 September 2020 (2020-09-22), US , pages 108186, XP093032822, ISSN: 2211-1247, DOI: 10.1016/j.celrep.2020.108186 *
RODRÍGUEZ-GALÁN ANA, FERNÁNDEZ-MESSINA LOLA, SÁNCHEZ-MADRID FRANCISCO: "Control of Immunoregulatory Molecules by miRNAs in T Cell Activation", FRONTIERS IN IMMUNOLOGY, vol. 9, 25 September 2018 (2018-09-25), XP093032579, DOI: 10.3389/fimmu.2018.02148 *
YANGYANG XU, BING WAN, XI CHEN, PING ZHAN, YUAN ZHAO, TIANLI ZHANG, HONGBING LIU, MUHAMMAD ZUBAIR AFZAL, SAID DERMIME, STEVEN N. H: "The association of PD-L1 expression with the efficacy of anti-PD-1/PD-L1 immunotherapy and survival of non-small cell lung cancer patients: a meta-analysis of randomized controlled trials", TRANSLATIONAL LUNG CANCER RESEARCH, SOCIETY FOR TRANSLATIONAL CANCER RESEARCH (STCR), HONG KONG, vol. 8, no. 4, 1 August 2019 (2019-08-01), Hong Kong , pages 413 - 428, XP055752050, ISSN: 2218-6751, DOI: 10.21037/tlcr.2019.08.09 *

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