[go: up one dir, main page]

WO2023008918A1 - Cellule souche génétiquement modifiée ayant une expression du gene b2m inhibée, et son procédé d'utilisation - Google Patents

Cellule souche génétiquement modifiée ayant une expression du gene b2m inhibée, et son procédé d'utilisation Download PDF

Info

Publication number
WO2023008918A1
WO2023008918A1 PCT/KR2022/011100 KR2022011100W WO2023008918A1 WO 2023008918 A1 WO2023008918 A1 WO 2023008918A1 KR 2022011100 W KR2022011100 W KR 2022011100W WO 2023008918 A1 WO2023008918 A1 WO 2023008918A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
cell
gene
vector
somatic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/KR2022/011100
Other languages
English (en)
Korean (ko)
Inventor
이아름
이지윤
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Industry Academic Cooperation Foundation of College of Medicine Pochon CHA University
Original Assignee
Industry Academic Cooperation Foundation of College of Medicine Pochon CHA University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Industry Academic Cooperation Foundation of College of Medicine Pochon CHA University filed Critical Industry Academic Cooperation Foundation of College of Medicine Pochon CHA University
Publication of WO2023008918A1 publication Critical patent/WO2023008918A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • 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
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • 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
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • 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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • 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
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • 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
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • NK cells do not require separate antigen presentation
  • research on NK cell-based immune anti-cancer drugs is increasing.
  • the injected NK cells are rejected by the patient's immune system, their lifespan in vivo is limited and there is the hassle of multiple injections, so attempts to extend the lifespan of NK cells used as cell therapy continues. .
  • Another aspect is to provide a method for preparing somatic cells into immune rejection evasion stem cells.
  • the major immunocompatibility antigens are six types of HLA (HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR) are cell surface proteins, and humans are derived from paternal genes. It is known that 6 species and 6 species derived from the maternal gene are expressed, that is, a total of 6 pairs. In more detail, normal somatic cells are known to express a total of three pairs of HLA-A, HLA-B, and HLA-C belonging to MHC class I.
  • activation may refer to the production of a gene that is not expressed at all or a protein that has no activity even if it is expressed.
  • depression may mean that the B2M gene is expressed at a lower level than in non-engineered somatic cells, or that the activity of the protein is low even if the protein is expressed.
  • the parental cell is a cell that has not been artificially manipulated, and refers to a cell freshly isolated from the human body and a cell cultured therefrom.
  • the Cas9 protein may include a nuclear localization sequence or signal (NLS) at the 5'- or 3'-, or both ends of the Cas9 protein to be located in the nucleus of a eukaryotic cell, the NLS It can be one or more.
  • NLS nuclear localization sequence or signal
  • RNA-guided CRISPR clustered regularly interspaced short palindrome repeats
  • CRISPR clustered regularly interspaced short palindrome repeats
  • Cas9 knocks out target genes, activates transcription and activates single guide RNA (sgRNA) (i.e., crRNA-tracrRNA fusion transcripts). ), which is known to target numerous genetic loci.
  • sgRNA single guide RNA
  • the B2M gene or genetic manipulation artificially performed to reduce the B2M protein expression or activity may be induced by the Cas9 protein or the Cpf1 protein.
  • the Cas9 protein or Cpf1 protein that can be used for the above gene manipulation includes Cas9 protein derived from Streptococcus pyogenes, Cas9 protein derived from Campylobacter jejuni, Streptococcus thermophiles It can be induced using at least one selected from the group consisting of a Cas9 protein derived from, a Cas9 protein derived from Streptococcus aureus, a Cas9 protein derived from Neisseria meningitidis, and a Cpf1 protein. .
  • Guide sequences can be selected to target any target sequence.
  • a target sequence is a sequence within the genome of a cell.
  • Exemplary target sequences include those that are unique in the target genome.
  • the vector includes expression control elements such as promoters, operators, initiation codons, stop codons, polyadenylation signals, and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and can be prepared in various ways according to purposes.
  • the vector's promoter may be constitutive or inducible.
  • the expression vector includes a selectable marker for selecting a host cell containing the vector and, in the case of a replicable expression vector, an origin of replication. Vectors can replicate autonomously or integrate into host DNA.
  • Stem cells of one aspect may further include a substance inducing differentiation into differentiated cells of ectoderm, mesoderm, or endoderm, and may be differentiated into cells of ectoderm, mesoderm, or endoderm by the differentiation-inducing substance.
  • stem cells of one aspect may be differentiated into teratomas including ectoderm, mesoderm, or endoderm.
  • the genetically engineered immune cells in which differentiation is induced from the SCNT-PSCs may have reduced expression or activity of the B2M gene or B2M protein, thereby exhibiting characteristics as immune rejection evasion immune cells.
  • One aspect provides a cell therapy agent containing the stem cells or immune cells prepared above.
  • the cell therapy agent may further include, in addition to the immune rejection evasion stem cells, a substance for inducing differentiation into differentiated cells of ectoderm, mesoderm, or endoderm, and may include all Lineage cells differentiated by the substance for inducing differentiation.
  • D-mannitol As an isotonizing agent, D-mannitol, sorbitol, etc. are mentioned, for example.
  • the term "individual” refers to a subject in need of treatment of a disease, more specifically an individual having a cancer disease or cancer cells, more specifically a human (cancer patient) or non-human primate, rodent ( It may refer to mammals such as rats, mice, guinea pigs, etc.), mice, dogs, cats, horses, cows, sheep, pigs, goats, camels, and antelopes.
  • Figure 2 is a diagram showing the results of knocking out the B2M gene of mouse fibroblasts and confirming whether knocking out actually occurred by PCR electrophoresis
  • Figure 2A is a diagram showing the results of confirming through 160/184 primers
  • Figure 2B is a diagram showing the results confirmed through 185/184 primers.
  • FIG. 6 is a view confirming the result of inducing teratoma formation by injecting the prepared somatic cell replicating pluripotent stem cell line into a testicular capsule.
  • FIG. 9a shows the frequency of CD3-NK1.1+ natural killer cells (CD-NK1.1+) and markers showing the killing ability (NKP46, Ly49 , KLRG1) is a confirmation diagram (control of normal mice).
  • Figure 9b compares the frequency (CD3-NK1.1+) of natural killer cells (CD3-NK1.1+) and markers (NKP46, Ly49, KLRG1) showing the killing ability in the spleen cells of mice in which the B2M gene was knocked out. it is one degree
  • 11a is a diagram showing the results of inducing differentiation of wild-type embryonic stem cells into NK cells, a type of blood cell, and confirming the morphology at days 1, 2, 3, 5, 6, 9, 10, 11, 12, and 13; am.
  • FIG. 12B is a diagram showing the results of confirming whether differentiation of induced gamma delta T cells was effectively induced into gamma delta cells on day 11 using a single marker, double expression, triple expression, and quadruple expression markers.
  • 12c is a diagram showing the results of confirming the expression of gamma delta T cell markers through immunostaining.
  • Example 1 Identification of pluripotent stem cells prepared through somatic cell nuclear transfer from fibroblasts knocked out of the B2M gene
  • genomic DNA was obtained from fibroblasts of wild-type mice in which the B2M gene was not knocked out and the prepared B2M KO fibroblasts. was extracted. From this, whether the B2M gene was actually knocked out was confirmed through electrophoresis using two types of 160/184 and 185/184 primers, respectively, and GAPDH was used as a control gene.
  • the results confirmed through the 160/184 primers are shown in FIG. 2A, and the results confirmed through the 185/184 primers are shown in FIG. 2B.
  • the primers used are as follows.
  • the differentiation into the endoderm was confirmed by confirming the differentiation pattern into the muscle fibers structure, which was heavily stained in the middle, through Masson's trichrome staining. Accordingly, it was confirmed that the somatic replicating pluripotent stem cells in which the B2M gene was knocked out can effectively differentiate into three germ layers. Therefore, it was confirmed that the somatic cell replicating pluripotent stem cells in which the B2M gene was knocked out had the unique ability of an undifferentiated pluripotent embryonic stem cell line capable of differentiating into any tissue thereafter.
  • the composition of the differentiation media for differentiation was as follows.
  • Mesoderm differentiation media contained Apel-2 media, 10ng/ml bFGF, 3nM CHIR, 100ng/ml ascorbic acid, 20ng/ml VEGFA, 50ng/ml SCF and 25ng/ml BMP4. To differentiate into mesoderm, the cells were treated with the differentiation media and differentiated for 2 days.
  • Media for inducing stabilization while inducing entry into lymphoid cells include Apel-2 media, 10 ng/ml of bFGF, 20 ng/ml of VEGFA, 250 ng/ml of SCF, 200 ng/ml of FLT3L, and 20 ng/ml of GCSF, 50ng/ml EGF, 50ng/ml IL-7, 100ng/ml IL-15, 50ng/ml IL-5, 40ng/ml IL-2, 25ng/ml DLL1 and 10nM EZH inhibitor The cells were treated and cultured for 3-5 days to differentiate them.
  • Mouse embryonic stem cell maintenance media is DMEM containing high glucose, NEAA (nonessential amino acid) 5ml, 20% SR, mercaptoethanol (500 ⁇ l/500ml), penicillin 5ml, LIF (1 ⁇ l/ml) , 1x) and separated into single cells using Matrigel. 200,000 cells in a 6-well culture dish and 50,000 cells in a 12-well culture dish were attached to the culture dish for 2 days and proliferated. At the point at which differentiation began, the cells used Apel2 media as a basis, and media compositions for differentiating blood Lineage cells were used according to each stage. The composition of the media used to differentiate into a type of blood lineage cell was as follows, and the processing steps were as follows.
  • Differentiation media for hemangioblastic progenitor cells were Apel-2 media, 10 ng/ml bFGF, 100 ng/ml VEGFA, 250 ng/ml SCF, 200 ng/ml FLT3L, 20 ng/ml GCSF, 100 ng/ml Cells were treated with media containing TPO, 20 ng/ml EPO, 50 ng/ml IL-6, and 50 ng/ml IGF-1, and the cells were differentiated for 2 days.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Immunology (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Reproductive Health (AREA)
  • Gynecology & Obstetrics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Virology (AREA)
  • Transplantation (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une cellule souche génétiquement modifiée ayant une expression ou une activité réduite du gène B2M ou de la protéine B2M, et un procédé d'utilisation de celle-ci. Un aspect concerne des cellules souches pluripotentes (PCNT-PSC) préparé par préparation de cellules somatiques, dont le gène B2M a été inactivé, et la soumission de celui-ci à un transfert nucléaire de cellule somatique, et des cellules immunitaires induites pour se différencier de celles-ci. Les cellules souches pluripotentes et les cellules immunitaires de l'aspect sont capables de se différencier en cellules NK et en lymphocytes T gamma delta, qui sont des cellules immunitaires normales, même si le gène B2M a été inactivé. En outre, étant donné que les molécules HLA de classe I sont induites pour être éliminées par ciblage du gène B2M, les cellules préparées peuvent survivre dans le corps pendant une longue durée et réduire au minimum le nombre d'injections par le biais d'une fuite de rejet immunitaire "hors du rayon" qui ne donne pas lieu à une réaction de lymphocytes T allogéniques, et peuvent ainsi être utilisés en tant que cellules à faible réponse immunitaire ou cellules souches d'échappement à rejet immunitaire et cellules immunitaires et ainsi largement utilisés en tant que produit de thérapie cellulaire.
PCT/KR2022/011100 2021-07-28 2022-07-28 Cellule souche génétiquement modifiée ayant une expression du gene b2m inhibée, et son procédé d'utilisation Ceased WO2023008918A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2021-0099492 2021-07-28
KR20210099492 2021-07-28

Publications (1)

Publication Number Publication Date
WO2023008918A1 true WO2023008918A1 (fr) 2023-02-02

Family

ID=85087962

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2022/011100 Ceased WO2023008918A1 (fr) 2021-07-28 2022-07-28 Cellule souche génétiquement modifiée ayant une expression du gene b2m inhibée, et son procédé d'utilisation

Country Status (2)

Country Link
KR (11) KR102847814B1 (fr)
WO (1) WO2023008918A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20160139045A (ko) * 2003-12-30 2016-12-06 주식회사 에이치바이온 배아 줄기 세포주 및 이의 제조방법
KR20190137010A (ko) * 2018-05-30 2019-12-10 주식회사 강스템바이오텍 Hla 유전자가 제거된 인간 유도만능줄기세포 유래 중간엽줄기세포 및 그의 제조 방법
KR20210032449A (ko) * 2018-07-17 2021-03-24 더 리젠츠 오브 더 유니버시티 오브 캘리포니아 면역조작된 만능성 줄기세포로부터 유래된 키메라 항원 수용체 t 세포

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106536721B (zh) * 2014-08-06 2020-12-04 车医科学大学校产学协力团 核酸酶介导的编辑编码hla的基因所产生的免疫相容性细胞
MX2019008413A (es) * 2017-01-13 2019-09-13 Univ California Celulas pluripotentes inmunodiseñadas.
US10724052B2 (en) * 2018-09-07 2020-07-28 Crispr Therapeutics Ag Universal donor cells

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20160139045A (ko) * 2003-12-30 2016-12-06 주식회사 에이치바이온 배아 줄기 세포주 및 이의 제조방법
KR20190137010A (ko) * 2018-05-30 2019-12-10 주식회사 강스템바이오텍 Hla 유전자가 제거된 인간 유도만능줄기세포 유래 중간엽줄기세포 및 그의 제조 방법
KR20210032449A (ko) * 2018-07-17 2021-03-24 더 리젠츠 오브 더 유니버시티 오브 캘리포니아 면역조작된 만능성 줄기세포로부터 유래된 키메라 항원 수용체 t 세포

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BOGOMIAKOVA M.E., SEKRETOVA E.K., EREMEEV A.V., SHUVALOVA L.D., BOBROVSKY P.A., ZERKALENKOVA E.A., LEBEDEVA O.S., LAGARKOVA M.A.: "Derivation of induced pluripotent stem cells line (RCPCMi007-A-1) with inactivation of the beta-2-microglobulin gene by CRISPR/Cas9 genome editing", STEM CELL RESEARCH, ELSEVIER, NL, vol. 55, 1 August 2021 (2021-08-01), NL , pages 102451, XP093029427, ISSN: 1873-5061, DOI: 10.1016/j.scr.2021.102451 *
ZHA SHIJUN, TAY JOHAN CHIN-KANG, ZHU SUMIN, LI ZHENDONG, DU ZHICHENG, WANG SHU: "Generation of Mesenchymal Stromal Cells with Low Immunogenicity from Human PBMC-Derived β2 Microglobulin Knockout Induced Pluripotent Stem Cells", CELL TRANSPLANTATION, SAGE, US, vol. 29, 1 January 2020 (2020-01-01), US , pages 096368972096552, XP093029428, ISSN: 0963-6897, DOI: 10.1177/0963689720965529 *

Also Published As

Publication number Publication date
KR102847814B1 (ko) 2025-08-21
KR20250128286A (ko) 2025-08-27
KR20250124799A (ko) 2025-08-20
KR20250127017A (ko) 2025-08-26
KR20250124798A (ko) 2025-08-20
KR20250128285A (ko) 2025-08-27
KR20250130539A (ko) 2025-09-02
KR20250127018A (ko) 2025-08-26
KR20250130538A (ko) 2025-09-02
KR20250124801A (ko) 2025-08-20
KR20250124800A (ko) 2025-08-20
KR20230020351A (ko) 2023-02-10

Similar Documents

Publication Publication Date Title
Takagi et al. Reversal of X-inactivation in female mouse somatic cells hybridized with murine teratocarcinoma stem cells in vitro
Laky et al. The role of IL-7 in thymic and extrathymic development of TCRγδ cells
AU2016278959A1 (en) CRISPR/Cas9 complex for introducing a functional polypeptide into cells of blood cell lineage
WO2019050071A1 (fr) Composition destinée à la prévention ou au traitement de la fibrose hépatique, contenant un exosome ou un acide ribonucléique dérivé d'exosome
WO2019231266A1 (fr) Cellule souche mésenchymateuse dérivée d'une cellule souche pluripotente induite humaine à gène hla supprimé et son procédé de préparation
WO2016048107A1 (fr) Composition pharmaceutique pour la prévention ou le traitement de maladies immunitaires ou de maladies inflammatoires, comprenant des cellules souches traitées par de l'interféron gamma ou de l'interleukine-1 beta, ou une culture de celles-ci
JP2024042096A (ja) 神経幹細胞組成物および神経変性障害を処置するための方法
WO2019198995A1 (fr) Procédé de conversion à base d'exosomes pour cellules immunitaires
CN107208055B (zh) 引起免疫原性反应降低的被修饰细胞
WO2023282688A1 (fr) Cellule souche mésenchymateuse ayant une résistance au stress oxydatif, son procédé de préparation et son utilisation
WO2022018884A1 (fr) Agent thérapeutique destiné à l'épidermolyse bulleuse dystrophique
WO2018030630A1 (fr) Vecteur non viral en minicercle portant un gène sox et procédé pour sa construction
WO2020209636A1 (fr) Procédé pour induire une reprogrammation directe d'une cellule urinaire dans une cellule progénitrice rénale et composition pharmaceutique comprenant ladite cellule reprogrammée par le même procédé pour prévenir ou traiter une maladie des lésions cellulaires rénales
WO2023008918A1 (fr) Cellule souche génétiquement modifiée ayant une expression du gene b2m inhibée, et son procédé d'utilisation
WO2023167575A1 (fr) Cellules souches faiblement immunogènes, cellules faiblement immunogènes différenciées ou dérivées de cellules souches, et leur procédé de production
Bernardino-Sgherri et al. Unusual chromosome cleavage dynamic in rodent neonatal germ cells
WO2025225866A1 (fr) Cellules souches pluripotentes induites universelles hypo-immunogènes
WO2021107234A1 (fr) Cellules souches hématopoïétiques à inactivation simultanée de gènes ccr5/cxcr4 personnalisées pour le traitement ou la prévention d'une infection par le vih, et leur procédé de préparation
WO2022010220A1 (fr) Nouvelles cellules de transplantation ayant une immunogénicité réduite
CN114929866B (zh) 基因改造巨核细胞、改造血小板和它们的制造方法
WO2019117454A1 (fr) Additif de milieu pour transformation cellulaire hautement efficace à l'aide d'un facteur de régulation de stress d'organite cellulaire
WO2022035287A1 (fr) Cellule ayant un gène corrigé ex vivo et utilisation associée
KR20200011817A (ko) 혈액 응고인자 ⅷ의 교정용 조성물 및 이를 이용한 방법
WO2019212123A1 (fr) Procédé pour augmenter la capacité de migration de cellules dendritiques, et son utilisation
WO2022211604A1 (fr) Cellules souches modifiées avec un gène mutant de fe-fviii, cellules endothéliales différenciées à partir de celles-ci, et composition pharmaceutique les contenant pour la prévention ou le traitement de l'hémophilie

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22849894

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22849894

Country of ref document: EP

Kind code of ref document: A1