WO2021251271A1 - Cellule présentant une expression supprimée du complexe majeur d'histocompatibilité (cmh) de classe i - Google Patents

Cellule présentant une expression supprimée du complexe majeur d'histocompatibilité (cmh) de classe i Download PDF

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WO2021251271A1
WO2021251271A1 PCT/JP2021/021234 JP2021021234W WO2021251271A1 WO 2021251271 A1 WO2021251271 A1 WO 2021251271A1 JP 2021021234 W JP2021021234 W JP 2021021234W WO 2021251271 A1 WO2021251271 A1 WO 2021251271A1
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tapasin
tap1
tap2
cells
function
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拓也 稲垣
悠款 平井
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帝人株式会社
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    • 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
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • 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
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention is a cell in which the expression of major histocompatibility complex (hereinafter referred to as "MHC") class I, which is also used in regenerative medicine and cell therapy, is suppressed to avoid immune rejection, and the production thereof. Regarding the method.
  • MHC major histocompatibility complex
  • cell therapy that prevents, treats, treats or alleviates human disease or damage by administering self-derived, allogeneic, or heterologous cells that have been processed or modified in vitro, or age, disease, injury, Or new cells using various cells including pluripotent stem cells for regenerative medicine to create functional and living tissues for the purpose of repairing or replacing the functions lost in tissues and organs due to congenital disorders.
  • Medical technology has been devised and is being put to practical use.
  • autologous transplantation in which cells are collected from the patient himself and transplanted to the same patient, and cells collected from the same species such as a donor and sent to the patient.
  • allogeneic transplantation allogeneic transplantation
  • xenotransplantation in which cells are collected from a different species such as an animal and transplanted to a patient (Fig. 1).
  • Allogeneic transplantation and xenotransplantation are not invasive to patients and have the characteristic that the manufacturing unit price tends to be low because they can be mass-produced as opposed to autologous transplantation, which is an individual production.
  • allogeneic or heterologous cells often have immunogenicity mainly due to MHC inconsistency and are subject to immunorejection.
  • MHC molecules The main function of MHC molecules is the regulation of innate immunity and the presentation of antigens to T cells in acquired immunity. Due to its function, there are two types of MHC, class I and class II. Class I molecules are present in the cell membranes of all nucleated cells and platelets. These are present because cytotoxic T cells rapidly kill the cells when infected cells or the like present non-self amino acids. Class II molecules are mainly distributed on the surface of some cells such as B cells, antigen-presenting cells, macrophages, and dendritic cells. These present foreign peptides taken up by phagocytosis to helper T cells, and as a result, promote antibody production. Due to these functions, innumerable polymorphisms are present in the peptide bond portion of MHC, and it is unlikely that they will match even if they are of the same species.
  • Non-Patent Document 1 Immune rejection caused by MHC inconsistency is thought to diminish the effects of cell medicines by reducing engraftment and duration of action. That is, interferon- ⁇ (hereinafter, "IFN-g") by direct recognition of allogeneic MHC-class I by allogeneic responsive CD8-positive T cells present in a certain number in T cells acutely. ”) And other cytokine production results in inflammation formation and aggression. Furthermore, chronically, donor-derived MHC-class I protein and MHC-class II are presented as antigens, antibodies are produced in the recipient's body, and MHC-mismatched cells are eliminated (Fig. 2).
  • IFN-g interferon- ⁇
  • MHC molecules are found in almost all vertebrate cells, and their expression mechanism is well conserved among species (Non-Patent Document 2). MHC in humans is particularly called human leukocyte antigen (hereinafter referred to as "HLA”), and MHC-class I and MHC-class II in humans are called HLA-class I and HLA-class II, respectively. There is.
  • HLA human leukocyte antigen
  • HLA-class I is expressed in a wide range of cell types and is an immunogen, so the HLA-class I type is matched as an approach to avoid immune rejection seen in transplanted cell drugs. Attempts have been made to suppress the expression of HLA-class I.
  • a method of matching HLA-class I types a method of establishing a cell bank that covers HLA-class I found in the target population is envisioned.
  • iPSC induced pluripotent stem cells for regenerative medicine
  • HLA-class I there are only 22 types of HLA-class I stored by 2017, and 30% of all Japanese can actually use HLA-class I iPSCs manufactured based on proper manufacturing standards. Limited to. As racial diversification is expected in the future, it is expected that a great deal of labor will be required to prepare and store cells corresponding to all HLA-class I types.
  • HLA-class I As an attempt to suppress the expression of HLA-class I, it is conceivable to suppress the expression of HLA-class I at the gene level.
  • HLA-class I is composed of antigen peptide, HLA-class I heavy chain, and ⁇ 2-microglobulin (hereinafter referred to as "B2M").
  • HLA-class I heavy chains are first translated and synthesized in the endoplasmic reticulum cavity.
  • the HLA-class I heavy chain during synthesis binds to the endoplasmic reticulum chaperone calnexin, and the three-dimensional structure is assembled.
  • B2M then binds to the HLA-class I heavy chain, the HLA-class I heavy chain separates from calnexin and binds to another chaperone called carreticulin. Proteins in the cytoplasm undergo degradation by the ubiquitin proteasome system when normal function is lost due to disruption of the three-dimensional structure or the like.
  • the degraded peptide is a heterodimer of Transporter Associated with Antigen Processing 1 (hereinafter referred to as "TAP1”) and Transporter Associated with Antigen Processing 2 (hereinafter referred to as "TAP2"). It is carried into the endoplasmic reticulum lumen by a transporter consisting of.
  • TAP1 Transporter Associated with Antigen Processing 1
  • TAP2 Transporter Associated with Antigen Processing 2
  • Non-Patent Document 3 the peptide carried by the TAP1 / TAP2 complex binds to the peptide bond groove of the HLA-class I heavy chain, and the HLA-class I molecule completes the three-dimensional structure, separates from carreticulin, and becomes a transport vesicle. Go in. HLA-class I molecules that fail to bind peptides cannot leave the carreticulin and exit the cell surface (Non-Patent Document 3).
  • HLA-class I expression mechanisms mediated by the proteasome-TAP1 / TAP2 complex, one of HLA-class I heavy chain expression, B2M expression, TAP1 / TAP2 expression, and tapasin expression. It is assumed that suppression of one reduces HLA-class I expression, and there have been multiple reports so far.
  • Non-Patent Document 4 iPSCs in which the A and B loci of the HLA-class I heavy chain are knocked out (hereinafter referred to as "KO") by genome editing technology, respectively, and the immunogenicity of these cells is reduced.
  • Non-Patent Document 4 since the sequences that can be targeted by this technique are limited, it is difficult to edit the genome with high efficiency, and there may be a problem in adapting to cells other than cells having infinite proliferation.
  • Non-Patent Document 7 Reported that B2M is involved in various intracellular functions such as cytokine signaling pathway, apoptosis, and cell proliferation. Therefore, cells targeting HLA-class I expression deficiency due to B2M function inhibition are considered to have a risk of causing problems in manufacturability and safety.
  • Non-Patent Document 8 Gadola et al. Have reported that mutations and deletions of TAP1 or TAP2 are the cause in a disease called Bare Lymphocyte Syndrome, in which HLA-class I and class II are not detected on the leukocyte surface (Non-Patent Document 8). .. Therefore, TAP1 or TAP2 dysfunction is expected to lead to HLA class I expression deficiency.
  • Non-Patent Document 9 Cui et al. Found that when TAP1 or Tapasin was KOed alone by genome editing technology, HLA-class I expression deficiency was markedly caused and the immunogenicity of the cells was reduced. It has been found (Non-Patent Document 9). Therefore, inhibition of TAP1, TAP2, or Tapasin function by KO or the like is considered to be one of the effective means for producing cells with low immunogenicity due to HLA-class I expression deficiency.
  • the problem to be solved by the present invention is a mammalian cell in which immune rejection is suppressed, so that the engraftment and duration of action of transplanted cells and tissues in the body are improved, and the frequency of frequent administration is also improved, and the production thereof. To provide a method.
  • MHC-class I in the mammalian cells, but as described above, the expression capacity of the protein by KO for any one of TAP1, TAP2, or Tapasin. It was thought that the expression of MHC-class I could be completely suppressed by eliminating the protein or by eliminating the function of the protein by an inhibitory protein corresponding to the protein, and thus the above-mentioned problem could be solved. ..
  • the inventors of the present invention have unexpectedly found that the above-mentioned problems may not be solved by themselves. That is, in mammalian cells, even if one of the TAP1, TAP2, or Tapasin genes is KO'd to completely eliminate the expression capacity of the protein, MHC-class I expression may still remain. I found.
  • this residual MHC-class I expression will be referred to as "unexpected residual MHC-class I expression”. This leads to unexpected residual immunogenicity.
  • An object of the present invention is to provide a mammalian cell in which at least a part of unexpected residual MHC-class I expression is suppressed, and a method for producing the same, even on the premise of such an unexpected fact.
  • Another problem to be solved by the present invention is to provide a method for suppressing at least a part of unexpected residual MHC-class I expression in mammalian cells in order to suppress immune rejection at the time of transplantation.
  • the inventors of the present invention have obtained bone marrow-derived mesenchymal stem cells as a method for obtaining cells in which HLA-class I expression is suppressed and the risk of affecting other cell functions is low, based on previous studies and clinical findings.
  • stem cells hereinafter referred to as "BM-MSC”
  • B2M KO which is essential for HLA-class I expression, was performed as a positive control.
  • Tapasin as opposed to TAP1 and TAP2, is a structural protein on the HLA-Class I pathway and has no functional overlap.
  • HLA-class I expression-related genes were combined and KO of TAP1 and Tapasin, or TAP2 and Tapasin, and the HLA-class I expression level was increased. It was newly found that it can be suppressed to 1.0% or 1.2%, and there is no significant difference from the HLA-class I expression level when B2M is KO (Figs. 4 and 5). That is, as a result of repeated studies to solve the problem of residual HLA-class I found in KO of TAP1, TAP2, or Tapasin alone, the present inventors have conducted studies, for example, TAP1 and Tapasin, or TAP2 and Tapasin.
  • KD gene knockdown
  • the present invention is a mammalian-derived cell having the protein expression ability of TAP1, TAP2, and Tapasin (TAP1, / or TAP2, and Tapasin have a protein expression ability of 20% or less of the wild type). Cells that have been treated) and / or (treated to reduce the function of TAP1 and / or TAP2 and Tapasin to 20% or less of the wild type).
  • the "treatment to make it 20% or less of the wild type” means the treatment to make it 0-20% of the wild type, and the treatment to completely eliminate it is also included (the same applies hereinafter).
  • the "treatment to 20% or less of the wild type” is performed as long as the degree of treatment for individual cells is within this numerical range.
  • the degree of processing for each may be different or uniform (the same shall apply hereinafter).
  • the present invention expresses (step 1) any one of TAP1, TAP2, and Tapasin proteins or both TAP1 and TAP2 proteins in mammalian-derived cells capable of expressing TAP1, TAP2, and Tapasin proteins.
  • a treatment is performed to eliminate the function and / or their functions, and (step 2) cells in which the expression of MHC-class I and / or the function is not completely eliminated are selected from among them, and (step 2).
  • Step 3) Among the selected cells, the cells subjected to the treatment for eliminating the protein expression ability and / or the function of TAP1 in Step 1 (Tapasin only, or Tapasin and TAP2) have the protein expression ability and / or the same.
  • the cells are obtained by subjecting Tapasin to 20% or less of the protein expression capacity and / or its function of the wild type.
  • the present invention has a protein expression capacity of TAP1, TAP2, and tapasin that is 20% or less of that of wild-type cells (TAP1, / or TAP2, and Tapasin) with respect to mammalian-derived cells having the protein expression capacity of TAP1, TAP2, and tapasin.
  • a method for producing hypoimmunogenic cells which comprises (steps) and / or (steps in which the functions of TAP1 and / or TAP2 and Tapasin are all 20% or less of the wild type).
  • the present invention relates to mammalian-derived cells capable of expressing TAP1, TAP2, and Tapasin proteins in (Step 1) expression of any one of TAP1, TAP2, and Tapasin proteins or both TAP1 and TAP2 proteins. Treatments are performed to eliminate the function and / or their functions, and (step 2) cells in which the expression of the major histocompatibility gene complex class I and / or its function has not completely disappeared are selected. Selected cells (Step 3) Among the selected cells, those treated to eliminate the protein expression capacity and / or its function of TAP1 in Step 1 (Tapasin only, or Tapasin and TAP2) have a protein expression capacity.
  • TAP1 protein expression capacity and / or treatment to reduce their function to 20% or less of wild type, and cells treated to eliminate Tapasin protein expression capacity and / or their function in step 1.
  • the present invention has (TAP1 and / or TAP2, and Tapasin protein expression capacity of 20% or less of the wild type) with respect to mammalian-derived cells having TAP1, TAP2, and Tapasin protein expression capacity.
  • This is a method for suppressing MHC-class I expression in the cells, which comprises (steps) and / or (steps in which the functions of TAP1 and / or TAP2 and Tapasin are all 20% or less of the wild type).
  • the IFN-g protein concentration in the culture supernatant after 6 hours and 24 hours of co-culture when BM-MSC KO of each target gene and CD8-positive T cells primed for BM-MSC were co-cultured. It is a measured graph (N 3, error bar represents standard error). It is a graph which showed the KD efficiency of TAP1, TAP2, and Tapasin genes by siRNA 72 hours after introduction.
  • a si-negative control is a siRNA with a random sequence that does not inhibit any mRNA.
  • transplantation includes constructing a tissue from cells and implanting it in the body, and administering the cells.
  • wild-type for mammalian-derived cells means a normal state in which the cells have their original functions, but the expression ability and function of TAP1, TAP2, and Tapasin are all the same. As long as it has not changed, it also includes those that have undergone some treatment, such as static culture.
  • Immunogenicity means a substance that causes an immune reaction.
  • immunogen induces antibody production and cell-mediated immunity.
  • Immuno rejection means that transplanted cells, etc. are attacked by the recipient's immune system.
  • NK cells, cytotoxic T cells, macrophages, etc. mainly work as the mechanism.
  • “Frequent administration” means administration of drugs (including cell drugs) multiple times at regular intervals.
  • Extracellular vesicles are vesicles consisting of lipid membranes secreted by cells and may contain proteins and / or nucleic acids such as mRNA, ncRNA, miRNA, and / or DNA.
  • Disappearance means to eliminate within the detection limit.
  • Protein expression ability refers to the function of expressing wild-type proteins.
  • the present invention is a mammalian-derived cell having a protein expression capacity of TAP1, TAP2, and tapasin (TAP1 and / or TAP2, and Tapasin have a protein expression capacity of 20% or less of the wild type). (Treatment) and / or (treatment to reduce the function of TAP1 and / or TAP2 and Tapasin to 20% or less of the wild type).
  • TAP1 and / or TAP2, and Tapasin means three aspects of (TAP1 and Tapasin), (TAP2 and Tapasin), and (TAP1, TAP2, and Tapasin).
  • the protein expression ability of TAP1 and / or TAP2 and the protein expression ability of tapasin are both wild type, that is, 10% or less of the mammalian cells which have not been treated with any of the above. It is more preferably less than or equal to%, and most preferably it disappears. In this case, their functions are inevitably reduced in response to the decrease in the expression capacity of TAP1 and / or TAP2 and tapasin, but further treatment may be added to reduce or eliminate the functions of TAP1 and / or TAP2 and tapasin. can.
  • TAP1 and / or TAP2 and Tapasin are all preferably 10% or less of the wild type, more preferably 5% or less, and most preferably eliminated. These hypofunctions are the result of treatments that reduce or eliminate the function of TAP1, TAP2, and / or Tapasin, even though they are the result of treatments that reduce or eliminate the protein expression capacity of TAP1, TAP2, and / or Tapasin. However, it may be the result of a combination of these processes.
  • a case where a treatment in which the protein expression ability of TAP1 is set to 20% of the wild type and a treatment in which the protein expression ability of tapasin is set to 20% in the wild type has been described, but other treatments in the present invention have been described.
  • at least a part of the effect of the treatment includes the effect of suppressing the unexpected residual MHC-class I expression.
  • a method for such a protein expression-reducing treatment or a function-reducing treatment one or more methods selected from the group consisting of genome editing, gene KD, introduction of an inhibitory protein, and addition of an inhibitory drug can be used.
  • the cells of the present invention had a protein expression capacity of TAP1, TAP2, and tapasin in mammalian cells (TAP1 and / or TAP2, and Tapasin protein expression capacity of 20% or less of the wild type). Processing) and / or (treatment to reduce the functions of TAP1 and / or TAP2 and Tapasin to 20% or less of the wild type), but here we assume the cells to be compared. That is, TAP1, only, TAP2 only, tapasin only, or (TAP1 and TAP2) only protein expression abilities and / or (they) in mammalian cells having TAP1, TAP2, and Tapasin protein expression abilities.
  • the cells of the present invention are preferably cells in which the expression of MHC-class I is further reduced as compared with such comparative cells.
  • it is a mammalian cell in which at least a part of unexpected residual MHC-class I expression is suppressed.
  • mammalian-derived cells having TAP1, TAP2, and Tapasin protein-expressing abilities were treated to reduce the TAP1, and Tapasin protein-expressing abilities to 20% or less of the wild-type cells of the present invention.
  • the treatment conditions for TAP1 protein expression capacity of 20% or less of wild-type and tapasin protein expression capacity of 20% or less of wild-type at the time of preparation of comparative cells are the corresponding cell preparations of the present invention. It is the same as the processing conditions adopted at times. In such cells of the present invention, the expression of MHC-class I is reduced as compared with either of these two types of comparative cells.
  • the cells of the present invention are any one of the mammalian-derived cells having the protein expression ability of TAP1, TAP2, and Tapasin selected from the group consisting of TAP1, TAP2, Tapasin, and TAP1 and TAP2. It is preferable that the cells do not completely eliminate the expression of MHC-class I even if they are treated to eliminate the protein expression ability and / or their functions. That is, it is preferable to treat mammalian-derived cells having an unexpected residual MHC-class I expression potential.
  • the cell of the present invention can also be described by the manufacturing process as follows. That is, the cell of the present invention is a protein of any one of TAP1, TAP2, and Tapasin or both TAP1 and TAP2 with respect to a mammalian-derived cell having a protein-expressing ability of TAP1, TAP2, and Tapasin (Step 1). A treatment is performed to eliminate the protein expression ability and / or their functions, and (step 2), cells in which MHC-class I expression and / or their functions are not completely eliminated are selected.
  • Step 3 the cells subjected to the treatment for eliminating the protein expression ability and / or the function of TAP1 in step 1 (Tapasin only, or Tapasin and TAP2) have the protein expression ability and / Or for cells treated to reduce their function to 20% or less of the wild type, and to eliminate the protein expression capacity and / or the function of TAP2 in step 1 (Tapasin only, or Tapasin and For cells treated to reduce the protein expression capacity and / or their function of TAP1) to 20% or less of the wild type, and to eliminate the protein expression capacity and / or function of Tapasin in step 1.
  • the treated cells are preferably cells obtained by subjecting Tapasin to 20% or less of the protein expression capacity and / or its function of the wild type.
  • the treatment of 10% or less of the wild type is preferable, and the treatment of 5% or less of the wild type is more preferable and disappears. Treatment is most preferred.
  • the present invention reduces the protein expression capacity of TAP1, / or TAP2, and Tapasin to 20% or less of the wild type with respect to mammalian-derived cells having the protein expression capacity of TAP1, TAP2, and Tapasin.
  • Step) and / or a step of reducing the function of TAP1 and / or TAP2 and Tapasin to 20% or less of the wild type, which is a method for producing hypoimmunogenic cells.
  • Such a step of reducing the protein expression capacity of TAP1 and / or TAP2 and Tapasin to 20% or less of the wild type) and / or (the function of TAP1 and / or TAP2 and Tapasin to be 20% or less of the wild type).
  • a step of inactivating the gene of TAP1 and / or TAP2 and Tapasin by genome editing or gene KD is preferable.
  • the step of reducing the wild type to 10% or less is preferable, and the step of reducing the wild type to 5% or less is more preferable, and the process is eliminated.
  • the process is most preferred.
  • Step 1 For a mammalian-derived cell having a protein-expressing ability of TAP1, TAP2, and Tapasin, (Step 1) one of the proteins of TAP1, TAP2, and Tapasin or TAP1 and TAP1 and A treatment was performed to eliminate the expressive capacity and / or their functions of both TAP2 proteins, and (Step 2) the expression and / or its function of the major histocompatibility gene complex class I was completely eliminated.
  • Step 3 For cells that had not been selected (Step 3) and that had been treated to eliminate the protein expression ability and / or its function of TAP1 in Step 1 (Tapasin only, or Tapasin and TAP2).
  • Treatment to reduce the protein expression capacity and / or their function to 20% or less of the wild type, and for cells subjected to the treatment to eliminate the protein expression capacity and / or the function of TAP2 in step 1 Treatment to reduce the protein expression capacity and / or their function of Tapasin alone or Tapasin and TAP1) to 20% or less of the wild type, and treatment to eliminate the protein expression capacity and / or its function of Tapasin in step 1. Treatment to reduce the protein expression capacity and / or their function of TAP1 and / or TAP2 to 20% or less of the wild type, and the expression capacity of both TAP1 and TAP2 proteins and / or in step 1. For cells that have been treated to eliminate their functions, a method for producing hypoimmunogenic cells including a treatment in which Tapasin's protein expression ability and / or its function is reduced to 20% or less of that of the wild type is preferable.
  • the present invention also relates to mammalian-derived cells capable of expressing TAP1, TAP2, and Tapasin proteins (TAP1, / or TAP2, and Tapasin, for example, to suppress immune rejection during transplantation).
  • MHC in the cell comprising a step of reducing the protein expression capacity to 20% or less of the wild type) and / or (a step of reducing the functions of TAP1 and / or TAP2 and Tapasin to 20% or less of the wild type).
  • -A method of suppressing class I expression comprising a step of reducing the protein expression capacity to 20% or less of the wild type) and / or (a step of reducing the functions of TAP1 and / or TAP2 and Tapasin to 20% or less of the wild type).
  • the step of reducing the wild type to 10% or less is preferable, and the step of reducing the wild type to 5% or less is more preferable, and the process is eliminated.
  • the process is most preferred.
  • the method for producing the same, and the method for suppressing MHC-class I expression treatment to reduce the protein expression capacity of TAP1 and / or TAP2 and Tapasin to 20% or less of the wild type) and / or ( TAP1 and / or TAP2, and tapasin function to 20% or less of the wild type), among which (TAP1 and / or TAP2, and tapasin protein expression capacity at least one is eliminated) and / Or (a treatment that eliminates at least one of the functions of TAP1 and / or TAP2 and Tapasin), and (a treatment that eliminates both TAP1 and / or TAP2 and Tapasin's protein expression capacity) and / or. (Treatment that eliminates the functions of TAP1 and / or TAP2 and Tapasin) is more preferable.
  • the animal-derived cells used for the preparation of the cells of the present invention are not particularly limited as long as they are culturable animal-derived cells, but stem cells are preferable, and among them, mesenchymal stem cells. Is preferable, and BM-MSC is particularly preferable.
  • cells derived from mammals are preferable, and cells derived from humans are particularly preferable.
  • the scope of the present invention also includes pharmaceutical compositions containing any of the above-mentioned cells of the present invention or extracellular vesicles derived thereof as an active ingredient.
  • the present invention provides cells in which MHC-class I expression is suppressed and a method for producing the same.
  • the method comprises the steps of combining the antigenic peptide transporter-related genes TAP1, TAP2, Tapasin with TAP1 and / or TAP2, and Tapasin to reduce or eliminate protein expression by genome editing and / or gene KD.
  • a step of cell sorting cells in which MHC-class I expression is suppressed may be included. This method may be, but is not limited to, a process for producing a cell population containing cells in which MHC-class I expression is suppressed.
  • cells are collected from the same species such as a donor, appropriately cultured, and then donated for the production of this cell.
  • the gene KD is performed by RNA interference.
  • RNA interference is a phenomenon in which mRNA having a base sequence complementary to double-stranded RNA is degraded. Therefore, by artificially designing RNA for an arbitrary gene, the expression of the gene can be suppressed.
  • protein function inhibition of TAP1, TAP2, or Tapasin may be selected as a method for obtaining the above-mentioned cells in which MHC-class I expression is suppressed.
  • Inhibition of protein function can be performed by introducing a virus-derived protein such as ICP47, US6, US3 or Nanobody.
  • expression of these proteins in cells can also inhibit the function of TAP1, TAP2, or Tapasin proteins.
  • the object of suppressing MHC-class I expression in the present invention can also be achieved by expressing in cells a protein capable of inhibiting the function of TAP1, TAP2, or Tapasin in this way.
  • low molecular weight compounds that inhibit protein expression or function of TAP1, TAP2, or Tapasin, if any, can also be implemented by adding them during culture.
  • genome editing refers to gene deletion or modification, that is, introducing one or more copies of a gene, deleting a gene, regulating gene expression, or modifying a gene. It can include mutating, methylating / demethylating a gene, acetylating / deacetylating a gene, and introducing a point mutation into a gene.
  • the genome editing technique may use a target-specific nuclease.
  • target-specific nuclease means a nuclease capable of recognizing and cleaving a specific site of DNA on the genome of interest.
  • the nuclease include a nuclease in which a domain that recognizes a specific target sequence on the genome and a domain that cleaves the domain are fused. Examples include, but are not limited to, zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and RNA-induced gene manipulation nucleases (RGENs).
  • ZFNs zinc finger nucleases
  • TALENs transcriptional activator-like effector nucleases
  • RGENs RNA-induced gene manipulation nucleases
  • a target gene is selected using an artificial chimeric protein composed of a domain that specifically binds to DNA of a specific sequence, generally called a zinc finger motif, a nuclease domain, and FokI, which is a restriction enzyme as a typical example. Recognize and disconnect. When two artificial chimeric proteins bind to adjacent target sequences, the nuclease domain forms a dimer and cleaves the DNA.
  • the target gene is recognized using an artificial chimeric protein composed of the DNA-binding domain of the TALE protein secreted from the phytopathogenic bacterium Xanthomonas and the nuclease domain of FokI, which is a restriction enzyme as a typical example. Disconnect.
  • the RGEN method uses a protein consisting of a complex of Cas protein and guide RNA.
  • a guide RNA containing a complementary sequence of the target genomic sequence recognizes and binds to the target sequence.
  • the Cas protein cleaves the vicinity of the target sequence.
  • the guide RNA may consist of CRISPR RNA (crRNA) and transactive crRNA (tracrRNA).
  • crRNA CRISPR RNA
  • tracrRNA transactive crRNA
  • sgRNA single-guide RNA in which these are linked by an artificial linker can also be used.
  • genome editing can occur via the non-homologous end joining (NHEJ) pathway, including by homologous recombination (HR) and / or microhomology-mediated end binding (MMEJ).
  • NHEJ non-homologous end joining
  • HR homologous recombination
  • MMEJ microhomology-mediated end binding
  • the method of the invention comprises the technique of introducing a protein or nucleic acid into a cell. Any suitable method can be used as a method for introducing such a protein or nucleic acid.
  • electroporation is used to introduce nucleic acids or proteins into cells for the purpose of inactivating target genes.
  • the electroporation method is a method in which nucleic acids and proteins in a solution are taken up into the cytoplasm by applying an electric pulse to cells. You can also use Nucleofection.
  • the virus can be used to introduce the nucleic acid into the cell.
  • the virus used for that purpose may include retrovirus, lentivirus, adenovirus, adeno-related virus, integrase-deficient lentivirus and the like.
  • the techniques for introducing proteins and nucleic acids are microinjection, nucleic acid introduction by exosomes, introduction by liposomes, introduction by polymer micelles, sonoporation, jet injection, hydrodynamic injection, introduction by magnetic field, calcium phosphate. It can include methods, introductions using macromolecular complexes, methods using cell permeable peptides, and the like. Methods for delivering nucleic acids to these cells are described in the following literature (Kim et al., 2010, Analytical and Bioanalytical Chemistry, Volume 397, 3173-3178).
  • cell sorting may be performed to separate cells in which MHC-class I expression is suppressed.
  • cell sorting means labeling the target cells with a fluorescent dye-binding antibody, a magnetic nanoparticle-binding antibody, or expression of a fluorescent / photoprotein, and sorting the target cells. Specific examples thereof include, but are not limited to, the flow cytometric cell sorting (FACS) method and the magnetically activated cell sorting (MACS) method as long as the cells can be separated.
  • FACS flow cytometric cell sorting
  • MCS magnetically activated cell sorting
  • cells labeled with fluorescent dye-binding antibody or fluorescent protein expression are flowed through the flow path in the machine under isolated conditions to optically examine the fluorescence brightness, cell size, and internal structural complexity of each cell. Can be detected. In addition, by setting criteria for fluorescence brightness and cell size, it is possible to separate the target cell population.
  • the MACS method by labeling cells with a magnetic nanoparticle-binding antibody, it is possible to sort the target cell population according to the expression level of the surface protein.
  • the prepared cell or cell-derived material, or pharmaceutical composition containing them is a transplant alternative or implant-to-host disease, heart disease, bone / cartilage injury, spinal cord injury, respiratory organs.
  • Disorders, muscle atrophic sclerosis, disc hernia, cerebral infarction, traumatic brain injury, diabetes and its complications, multiple sclerosis, Crohn's disease, myocardial infarction, anemia, Parkinson's disease, osteoarthritis, cancer It can be used in patients for the treatment of liver cirrhosis, autoimmune disease, Alzheimer's disease, cardiovascular disease, kidney disease, bone disease, liver disease, neurological disease, rheumatoid arthritis, systemic erythematosus, but is not limited to these.
  • the routes of administration of the pharmaceutical composition of the present invention include intraarterial, intravenous, subcutaneous, intradermal, intramuscular, intraarterial, intraperitoneal, intratracheal, intrapulmonary, intracostal, intrathoracic, and intracardiac.
  • Multiple routes of administration are envisioned, including, but not limited to, intrauterine, inhalation, intracerebral, intraocular, local, intrathecal, epidural, intramedullary, intrathecal, and intraneuronal.
  • composition of the present invention can also be used in the therapeutic methods described in the following documents, but is not limited to this (Saeedi et al., 2019, Stem Cell Investigation, 6:34) (Ilic et al., 2017, STEMCELLS, Volume 35, Issue 1, 17-25).
  • MHC-class I and its expression mechanism are widely conserved among species, and the cells of the present invention are made from mammalian-derived cells such as mice, dogs, pigs, and horses in certain embodiments. ..
  • the MHC-class I expression suppression technique of the present invention can be utilized for immunorejection suppression in organ transplantation from a heterologous animal.
  • Human BM-MSCs (Cat # C-12974, Lot # 438Z012.1) used for MSC culture were purchased from PromoCell. According to the standard culture method of PromoCell, the mesenchymal stem cell growth medium 2 (Cat # C-28009) sold by PromoCell was used as the medium, and the medium was changed once every 2-3 days. .. In addition, 30 mM HEPES (Cat # C-40020) and AccutaseTM Solution (Cat # C-41310) sold by PromoCell were used for cell detachment. The cells after exfoliation were collected by precipitation by centrifuging at 220 xg for 3 minutes or 5 minutes using a centrifuge (Koki Holdings, Cat # CF6RN).
  • Tissue Culture Flask (Cat # 3313-150, etc.) manufactured by AGC Technoglass Co., Ltd. and Cell Culture Multiwell Plate (Cat # 353046, etc.) manufactured by Corning Inc. were used as culture substrates.
  • sgRNA SgRNA was used for the purpose of KOing the target gene.
  • the Cas9 protein recognizes and cleaves the complementary target sequence specified by the sgRNA by forming a complex with the sgRNA.
  • TrueguideTM sgRNA from Thermo Fisher Scientific was used for each target gene.
  • TrueGuideTM sgRNA Negative Control non-targeting 1 was used for the negative control.
  • TAP1 ID # CRISPR695357_SGM: target sequence ACTGCTACTTCTCGCCGACT
  • TAP2 ID # CRISPR629083_SGM: target sequence GGGGGCTGCTAAAGCTAAGA
  • Tapasin ID # CRISPR673728_SGM: target sequence GAACCAACACTCGATCACCG
  • B2M ID # CRISPR983707_SGM: target sequence AGTCACATGGTTCACACGGC
  • the Cas9 protein recognizes and cleaves the complementary target sequence specified by the sgRNA by forming a complex with the sgRNA.
  • CleanCap® Cas9 mRNA (modified) (Cat # L-7206-100) manufactured by TriLink was used.
  • 5 ⁇ g (5 ⁇ L) of Cas9 mRNA was added to a cell suspension of 1 ⁇ 10 6 cells / 100 ⁇ L.
  • Electroporation Lonza's 4D-NucleofectorTM or Thermo Fisher Scientific's NeonTM was used as the electroporation.
  • the buffer solution and cuvette during electroporation are P1 primary cells 4D-NucleofectorTM X kit L (V4XP-1012, V4XP-1024) or P1 primary cells 4D-NucleofectorTM X kit S (V4XP). I used the one that came with -1032). Electroporation was carried out by the following steps. That is, after exfoliating the cells and counting the number of viable cells, suspend and centrifuge in phosphate buffer (PBS) (Fuji Film Wako Pure Chemical Industries, Ltd., Cat # 166-23555) and centrifuge (220xg, 5 minutes). Cleaning was performed by doing.
  • PBS phosphate buffer
  • the cells resuspended to 1 ⁇ 10 6 cells / 100 ⁇ L using the buffer solution in the kit were dispensed into the cuvette.
  • 5 ⁇ g of Cas9 mRNA and 0.5 nmol of sgRNA were added to the cell suspension.
  • the manufacturer-configured program DT-104 was used as the introduction protocol. Incubated at room temperature in a clean bench for 10 minutes after electroporation. Then, the cells after electroporation were seeded and cultured on a 6-well plate containing the above-mentioned MSC culture medium in advance.
  • NeonTM When NeonTM is used as an electroporator, suspend it in a buffer solution in the NeonTM Transfection System (Thermo Fisher Scientific, Cat # MPK10096) kit to a size of 1 ⁇ 10 6 cells / 100 ⁇ L, and then use the nucleic acid to be introduced. Mixed. After filling the cell suspension into a dedicated pipette, electroporation was performed under the conditions of 1400 V, 20 ms, and 2 pulses. Then, the cells after electroporation were seeded and cultured on a 6-well plate containing a medium in advance. Genomic DNA Extraction The Genomic DNA Purification Kit (Cat # T3010S) from NEW ENGLAND BioLabs, Monarch®, was used to extract genomic DNA from cells.
  • Thermo Fisher Scientific, Cat # MPK10096 NeonTM Transfection System
  • Genomic PCR For use in sequence analysis, the purpose was to amplify genomic DNA containing the region targeted by each sgRNA. Genomic PCR was performed using Toyobo's KOD-Plus-Neo (Cat # KOD-401). The experimental method followed the standard protocol included with the kit. In addition, the primers shown in the following table were used for each target gene. In the table, Fw indicates that it is Forward Primer, and Rv indicates that it is Reverse Primer.
  • the amount of surface protein can be quantified for each individual cell or bead.
  • Each analysis was performed using LSR HortessaTM X-20 manufactured by Becton Dickinson, Japan as an experimental device.
  • Flow Cytometry Cell Sorting Using FACSAriaTM IIIu from Becton Dickinson, Japan, which has a mechanism to collect cell populations according to fluorescence intensity in addition to the mechanism of the above-mentioned flow cytometry method, HLA-is accompanied by KO of the target gene. A cell population with reduced class I expression was obtained.
  • PBMC Human peripheral blood-derived monocytes
  • PBMC Human peripheral blood-derived monocytes
  • MEM MEM medium
  • the HLA-class I type of BM-MSC used was analyzed by the next-generation sequencing method.
  • the information in the following table provided by the purchaser was used.
  • Negative Selection Using Magnet Beads Negative selection was performed using the EasySepTM Human CD8 + T Cell Isolation Kit (Cat # 17953) from STEM CELL TECHNOLOGY to isolate CD8-positive T cells from primed PBMCs. The experimental method followed the standard protocol of the kit. When the isolated cells are used for other experiments, they are stained with human CD3 antibody-APC (Cat # 561804) and human CD8 antibody-PE (Cat # 561949) manufactured by Becton Dickinson of Japan, and flow cytometry. It was used after confirming that the CD3 and CD8 positive cell population, that is, CD8 positive T cells were contained in 90% or more of the whole by the method.
  • mRNA recovery When mRNA was recovered from cells, Qiagen's RNeasy Kit (Cat # 74106) was used. The experimental method followed the standard protocol included with the kit.
  • Reverse Transcription Reaction A reverse transcription reaction was performed using the Applied biosystems High-Capacity RNA-to-cDNA Kit (Cat # 4387406) for the purpose of converting mRNA to cDNA.
  • the experimental method followed the standard protocol included with the kit.
  • the experimental equipment used was a 2720 Thermal cycler manufactured by Applied biosystems.
  • Quantitative PCR Quantitative PCR was performed using TaqMan® Gene Expression Master Mix (Cat # 4369514) from Applied biosystems for the purpose of quantifying the amount of cDNA.
  • the experimental method followed the standard protocol included with the kit.
  • the TaqMan Assay used for each quantitative PCR is as follows.
  • the experimental equipment used was Quant Studio 3 manufactured by Applied biosystems.
  • TaqMan Assay Information TaqMan Assays corresponding to the assay IDs in the following table were purchased from Applied biosystems for each gene.
  • siRNA For siRNA, Thermo Fisher Scientific's Silencer® Select was used. Cat # s13779 was used when targeting TAP1, Cat # s13781 was used when targeting TAP2, and Cat # s13785 was used when targeting Tapasin. In addition, Cat # AM4611 was used as a negative control. At the time of introduction by electroporation, siRNA was added to 500 nM in a cell suspension of 1 ⁇ 10 6 cells / 100 ⁇ L.
  • Example 1 Examination of inhibitory effect of TAP1, TAP2, and Tapasin KO on HLA-class I expression using genome editing in BM-MSC Targeting each gene of TAP1, TAP2, and Tapasin to BM-MSC SgRNA and Cas9 mRNA were introduced by electroporation. In addition, sgRNA that does not specify a target sequence was used as a negative control. An sgRNA targeting the B2M gene, which is known to be HLA-class I underexpression by KO, was used as a positive control. The expression status of HLA-class I in each of the prepared cells was confirmed.
  • BM-MSC was cultured, stripped and collected, washed with PBS, and replaced with the buffer solution in P1 primary cells 4D-NucleofectorTM X kit L to adjust the cell concentration to 1 ⁇ 10 6 cells /. It was set to 100 ⁇ L. Cas9 mRNA (5 ⁇ L) and sgRNA 0.5 nmol (2.5 ⁇ L) were added to the cuvette in the kit. A 100 ⁇ L cell suspension was mixed therein, and a pulse voltage was applied by the program DT-104. The cell suspension was maintained at room temperature for 10 minutes and then seeded on a 6-well culture plate.
  • Flow cytometric cell sorting was performed about 1 week after gene transfer, and HLA-class I low-expressing cell populations were collected by cell sorting.
  • the cells after cell sorting were expanded and cultured for about 1 week, genomic DNA was extracted from each cell, the genomic sequence was amplified by the PCR method using a primer that specifies the region containing the target region of sgRNA, and sequence analysis was performed.
  • the KO efficiency (Inde l introduction rate) shown in the following table was calculated by regression analysis of the sequence waveform data.
  • the KO efficiency of the target gene in each cell was 90% or more, and the cell population was extremely low in contamination with unKO cells.
  • the HLA-class I expression level was measured by the flow cytometry method together with each of the above-mentioned cells (FIG. 4).
  • the relative values of each target gene in KO cells when the HLA-class I expression level of the negative control MSC was set to 1 were calculated and graphed (Fig. 5).
  • the peak in cells KO with TAP1, TAP2, or tapasin alone is located on the low fluorescence side compared to the peak seen in the negative control (Fig. 4), and HLA.
  • -It was shown that the expression level of class I was decreased.
  • the peaks in cells KOed with TAP1 and Tapasin or a combination of TAP2 and Tapasin have moved to the lower fluorescence side, and HLA-class I expression is suppressed more efficiently in individual cells. was shown (Fig. 4).
  • HLA-Class I expression level in cells KO with TAP1, TAP2, or Tapasin alone decreased to 6.6-9.8% of the negative control.
  • HLA-class I expression levels in cells KO with TAP1 and Tapasin, or a combination of TAP2 and Tapasin decreased to 1.0% or 1.2%. This was a significantly lower value than the cells KOed with TAP1, TAP2, or Tapasin alone, and there was no significant difference in the expression level compared with the cells KOed with B2M (Fig. 5).
  • Example 2 Examination of the reactivity of CD8-positive T cells to BM-MSC KO with TAP1, TAP2, and tapasin CD8-positive T cells observed when CD8-positive T cells and BM-MSC of different donors were co-cultured.
  • PBMCs after co-culturing commercially available PBMCs with BM-MSC in MEM medium supplemented with human serum and IL-2 for 18-24 days, CD8-positive T cells were obtained by negative selection using magnet beads. Isolated.
  • the target gene was co-cultured with KO BM-MSC in Example 2, and the culture supernatant after 6 hours of co-culture and the mRNA derived from cells and the culture supernatant after 24 hours of co-culture were collected.
  • the expression levels of IFN-g and CD3 mRNA were measured by a quantitative PCR method.
  • the IFN-g mRNA expression level was corrected by the CD3 mRNA expression level expressed only in T cells, and the relative value when the IFN-g mRNA expression level in the negative control MSC was set to 1 was calculated (FIG. 6).
  • the IFN-g concentration in the culture supernatant was measured using Cytometric Bead ArrayTM (Fig. 7).
  • TAP1, TAP2, or tapasin was co-cultured with MSC KO KO in combination with TAP1 and Tapasin, or TAP2 and Tapasin, as compared with MSC KO KO with TAP1, TAP2, or Tapasin alone after 6 hours of co-culture.
  • the amount of IFN-g mRNA produced in CD8-positive T cells was significantly reduced.
  • the IFN-g mRNA expression level in CD8-positive T cells co-cultured with TAP1 and Tapasin or KO cells in combination with TAP2 and Tapasin showed the same level of expression as when cultured only with CD8-positive T cells. (Fig. 6).
  • the immunogenicity of TAP1, Tapasin, or BM-MSC KO of TAP2 and Tapasin is lower than that of BM-MSC KO of any one of TAP1, TAP2, and Tapasin alone.
  • Example 3 Examination of inhibitory effect of TAP and Tapasin gene KD on HLA-class I expression using siRNA in BM-MSC Suppresses the expression of TAP1, TAP2, and Tapasin mRNAs on BM-MSC.
  • siRNA was introduced by electroporation.
  • commercially available BM-MSC is cultured, peeled and collected, washed with PBS, and suspended in the buffer solution included in the NeonTM Transfection System kit so that the cell concentration becomes 1 ⁇ 10 6 cells / 100 ⁇ L. did.
  • SiRNA that suppresses the expression of TAP1, TAP2, and Tapasin mRNA per 100 ⁇ L of cell suspension or SilencerTM Negative Control No.1 siRNA (si negative control) as a negative control should be 500 nM at the final concentration.
  • SiRNA was introduced into cells by applying a pulse voltage with NeonTM. mRNA was recovered from cells 72 hours after siRNA introduction, and after reverse transcription reaction, quantitative PCR was performed. The gene KD efficiency was calculated from the amount of mRNA of the target gene of each target siRNA-introduced cell with respect to the si-negative control-introduced cell (Fig. 8).
  • the target gene KD efficiency by siRNA that suppresses mRNA expression of TAP1, TAP2, and Tapasin was 70%, 86%, and 92%, respectively, and all of them suppressed mRNA expression with high efficiency. confirmed.
  • the cells were stained with human HLA-ABC antibody-APC 96 hours after siRNA introduction, and the HLA-class I expression level was measured by flow cytometry. The relative value of the expression level of each target gene KD cell when the HLA-class I expression level in the si negative control-introduced cells was set to 1 was calculated and graphed (Fig. 9).
  • HLA-class I decreased in TAP1, TAP2, or tapasin-KD cells as compared with si-negative control-introduced cells. Furthermore, the HLA-class I expression level was further reduced in cells KD with TAP1 and Tapasin or a combination of TAP2 and Tapasin as compared with KD of each target gene alone.
  • hypoimmunogenic mammalian cells according to the present invention are used, for example, in the pharmaceutical industry that provides cell medicines.

Abstract

L'invention concerne une cellule qui est dérivée d'un mammifère, ledit mammifère ayant des capacités pour exprimer des protéines TAP1, TAP2 et tapasine et ayantété soumis à un traitement pour abaisser les capacités pour exprimer les protéines TAP1 et/ou TAP2 et tapasine chacune à 20 % ou moins par rapport au type sauvage et/ou un traitement pour abaisser les fonctions de TAP1 et/ou TAP2 et tapasine chacune à 20 % ou moins par rapport au type sauvage. La présente invention résout le problème selon lequel l'expression du CMH de classe I reste parfois même après élimination de la capacité d'expression de l'un des TAP1, TAP2 et tapasine dans des cellules de mammifère, ledit problème ayant été inconnu jusqu'à présent. En tant que résultat, la présente invention empêche les propriétés de prise de greffe et la période d'action d'être détériorées par le rejet immunitaire d'une cellule administrée et permet à un tissu ou à une cellule transplantée d'exercer un effet thérapeutique suffisant dans les domaines de la médecine régénérative et des produits pharmaceutiques cellulaires.
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