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

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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
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이아름
이지윤
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의료법인 성광의료재단
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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.

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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 WO2023008918A1 (fr)

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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

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
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