WO2023008918A1 - Genetically modified stem cell having inhibited b2m gene expression, and method for using same - Google Patents

Abstract

The present invention relates to a genetically modified stem cell having reduced expression or activity of the B2M gene or B2M protein, and a method for using same. One aspect relates to pluripotent stem cells (PCNT-PSCs) prepared by preparing somatic cells, of which the B2M gene has been knocked out, and subjecting same to somatic cell nuclear transfer, and immune cells induced to differentiate from same. The pluripotent stem cells and immune cells of the aspect are capable of differentiation into NK cells and gamma delta T cells, which are normal immune cells, even if the B2M gene has been knocked out. Further, since HLA class I molecules are induced to be knocked out by targeting the B2M gene, the prepared cells can survive in the body for a long time and minimize the number of injections through "off-the-shelf" immune rejection escape which does not give rise to a reaction of allogeneic T cells, and thus may be utilized as low-immune cells or immune rejection escape stem cells and immune cells and thus widely utilized as a cell therapy product.

Description

Genetically engineered stem cells in which the expression of the B2M gene is inhibited, and methods for using the same

It relates to a genetically engineered stem cell in which the expression or activity of a B2M gene or B2M protein is reduced, and a method of using the same.

In the past 10 years, the discovery of pluripotent stem cell-derived cell therapy raw materials and technology development have continued. In particular, in order to establish a high-quality cell line as a raw material for cell therapy while avoiding immune rejection, which is one of the biggest difficulties in cell therapy products, a new cell therapy raw material with a completely different concept from the conventional adult-derived or pluripotent stem cell line-derived cell line is sought. There is a need. However, there is currently no known cell therapy technology that recovers cell damage while eliminating immune rejection by genetic manipulation in principle.

In order to solve these problems, development of off-the-shelf cell therapy products using immune effector cells from healthy donors is being developed. Autologous peripheral blood-derived T cells using chimeric antigen receptor (CAR) have been used as immune cell therapy, but it is difficult to obtain a sufficient number of T cells, and there are drawbacks that can only be used as self-therapeutic agents. . On the other hand, NK cells (natural killer cells) do not respond to rejection of allografts due to innate immunity and do not induce GVHD (Graft-versus-host-disease) even when injected in large amounts to immunocompromised patients, so they do not match HLA. It can be used even if it is not, and its utilization is increasing. In addition, since NK cells do not require separate antigen presentation, research on NK cell-based immune anti-cancer drugs is increasing. However, since 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. .

In addition, gamma delta T cells, which are currently emerging as a new immune cell therapy, are non-conventional T cells with functions not limited to MHC-mediated antigen presentation. In general, it is known to have the ability to efficiently identify and kill cancer cells because it moves to peripheral tissues rather than lymphoid organs and performs functions independent of MHC-dependent antigen presentation and has abundant cytokine secretion ability. In particular, since tumor cells have the ability to evade the immune system through MHC heteroantigen and low antigen proliferation, the need for development of MHC-independent gamma delta T cells as a new immunotherapeutic agent has emerged.

Therefore, studies to develop an allogeneic immune cell therapy agent that regulates these major tissue complexes (MHC) have recently been actively conducted.

One aspect is to provide genetically engineered stem cells having reduced expression or activity of a B2M gene or B2M protein.

One aspect is to provide genetically engineered immune cells in which the expression or activity of the B2M gene or B2M protein is reduced, differentiated from the stem cells.

One aspect is to provide a cell therapy product containing the stem cells or immune cells prepared above.

Another aspect is to provide a method for preparing somatic cells into immune rejection evasion stem cells.

Another aspect is to provide a method for preparing immune rejection cells from somatic replicating pluripotent stem cells.

Other objects and advantages of this application will become more apparent from the following detailed description taken in conjunction with the appended claims and drawings. Any person skilled in the art of this application or a similar technical field can sufficiently recognize and infer the content not described in this specification, and thus the description thereof will be omitted.

One aspect is a gene editing complex that specifically binds to the B2M (β2-microglobulin) gene, an exogenous nucleic acid molecule, or a gene in which the expression or activity of the B2M gene or B2M protein is reduced, including a vector into which the nucleic acid molecule is introduced. Fully engineered stem cells are provided.

Beta-2 microglobulin, also known as “B2M”, is a light chain of major histocompatibility antigen complex (MHC) class I molecules, and is an essential part of the commonly known major histocompatibility complex. am. In humans, B2M is encoded by the B2m gene located on chromosome 15, as opposed to other MHC genes located as a gene cluster on chromosome 6. The human B2M protein is known to have a molecular weight of 11.800 Daltons with 119 amino acids. The B2M gene is a mouse B2M gene Mus musculus (house mouse), for example, NCBI Accession No. It may be NC_000068.8, and the human B2M gene (Homo sapiens (human)) is, for example, NCBI Accession No. It may be NC_000015.10, but is not limited thereto. A person skilled in the art can easily identify the location and sequence of the mutation using the registration number. The specific sequence corresponding to the number registered in NCBI, UCSC genome browser or GenBank may change somewhat over time. It will be apparent to those skilled in the art that the scope of the present invention also extends to the altered sequence.

The major immunocompatibility antigens (MHC) 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.

The expression or activity of the B2M gene or B2M and the protein is lower than the level of expression or activity of the B2M gene or B2M protein measured in somatic cells or parent cells of the same species, or there is no expression or activity. That is, the expression or activity of the B2M gene or B2M protein in somatic cells is about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 55% or more, about It may be reduced by at least 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%. Genetically engineered somatic cells with reduced expression or activity of the B2M gene or B2M protein can be identified using any method known in the art. The term “inactivation” 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. The term “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.

Reduction in the expression or activity of the B2M gene or B2M protein may be due to mutation, substitution, or deletion of a part or all of the gene encoding the B2M, or insertion of one or more bases into the gene, which is caused by correction of the B2M gene. by means or gene editing complexes.

The gene editing complex may be selected from the group consisting of zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), AAV, and RNA-guided engineered nuclease (RGEN), among which, RGEN is specific to the B2M gene It may be by a CRISPR system including a guide RNA and a Cas protein that specifically bind to a sequence and a vector including the same.

The RGEN may include guide RNA and Cas protein that specifically bind to a specific sequence of the B2M gene.

In general, the well-known gene editing tool “CRISPR system” collectively includes a sequence encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., a tracrRNA or active portion tracrRNA), and a tracr-mate sequence (endogenous In the context of a CRISPR system, "direct repeats" and tracrRNA-engineered parts, including direct repeats), guide sequences (also referred to as "spacers" in the context of an endogenous CRISPR system), guide RNAs, or other sequences and transcripts from the CRISPR locus. refers to transcripts and other elements involved in the expression of, or inducing the activity of, CRISPR-related (“Cas”) genes, including In some embodiments, one or more elements of the CRISPR system are from a type I, type II or type III CRISPR system. In some embodiments, one or more elements of the CRISPR system are from a particular organism comprising an endogenous CRISPR system, eg, Streptococcus pyogenes. In general, CRISPR systems are characterized by an element (also referred to as a protospacer in the context of an endogenous CRISPR system) that promotes the formation of a CRISPR complex at the site of a target sequence. In the context of formation of a CRISPR complex, "target sequence" or "target gene" refers to a sequence for which a guide sequence is designed to have complementarity, wherein hybridization between the target sequence and the guide sequence enhances the formation of a CRISPR complex. Essentially perfect complementarity is not required, but there is sufficient complementarity to cause hybridization and enhance formation of the CRISPR complex. A target sequence can include any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, a target sequence can be located in the nucleus or cytoplasm of a cell.

The Cas protein forms an active endonuclease or nickase when complexed with two RNAs called CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). Non-limiting examples of the Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologues thereof or modified versions thereof. These enzymes are known; For example, the amino acid sequence of the Streptococcus pyogenes Cas9 protein can be obtained from the SwissProt database under accession number Q99ZW2. In some embodiments, the unmodified CRISPR enzyme, eg, Cas9, has DNA cleavage activity. In some embodiments, the CRISPR enzyme is Cas9 and may be a Cas9 from Streptococcus pyogenes or Streptococcus pneumoniae. In some embodiments, the Cas protein is codon-optimized for expression in eukaryotic cells.

In some embodiments, 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.

As used herein, the term "nuclear localization sequence or signal (NLS)" refers to an amino acid sequence that plays a role in transporting a specific substance (eg, protein) into the cell nucleus, and is generally ) into the cell nucleus (Kalderon D, et al., Cell 39:499509 (1984); Dingwall C, et al., J Cell Biol. 107(3):8419 (1988)). Localization sequences are not required for CRISPR complex activity in eukaryotes, but the inclusion of such sequences is believed to enhance the activity of the system, specifically targeting nucleic acid molecules in the nucleus.

In addition, the RNA-guided CRISPR (clustered regularly interspaced short palindrome repeats)-associated nuclease 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.

The Cas9 (or Cpf1) protein refers to an essential protein element in the CRISPR/Cas9 system, and information on the Cas9 (or Cpf1) gene and protein can be obtained from GenBank of the National Center for Biotechnology Information (NCBI). However, it is not limited thereto. The CRISPR-associated gene encoding the Cas (or Cpf1) protein is known to exist in about 40 or more different Cas (or Cpf1) protein families, and there are 8 gene sequences according to specific combinations of cas genes and repeat structures. CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube) can be defined. Thus, each CRISPR subtype can form a repeating unit to form a polyribonucleotide-protein complex.

In order to produce a B2M knock-out somatic cell according to one aspect, as a genome editing technology, a technology using a rare-cutting endonuclease that cuts a rare gene sequence with a very low abundance in the genome can be employed. there is.

The gene knock-out may refer to regulation of gene activity, eg, inactivation, by deletion, substitution, and/or insertion of one or more nucleotides of all or part of the gene (eg, one or more nucleotides). The gene inactivation refers to suppression or downregulation of gene expression or modification to encode a protein that has lost its original function. In addition, gene regulation can be achieved by simultaneously targeting both intronic regions surrounding one or more exons of a target gene, resulting in protein structural modification resulting from exon deletion, dominant negative protein expression, and soluble secreted competitive inhibitors. It may mean a change in the function of a gene as a result of expression or the like.

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. . When the Cas9 (or Cpf1) is encoded in DNA and delivered to an individual or cell, the DNA may generally (but not necessarily) include a regulatory element (eg, a promoter) operable in the target cell. The promoter for Cas9 (or Cpf1) expression may be, for example, CMV, EF-1 a, EFS, MSCV, PGK, or CAG promoter. A promoter for gRNA expression can be, for example, the HI, EF-la, tRNA or U6 promoter. The Cas9 (or Cpf1) coding sequence may include a nuclear localization signal (NLS) (e.g., SV40 NLS). In one example, the promoter may have tissue specificity or cell specificity.

The genetic manipulation artificially performed to reduce the expression or activity of the B2M gene or B2M protein is carried out in the proto-spacer-adjacent motif (PAM) sequence in the nucleic acid sequence constituting the B2M gene or adjacent to its 5' end or 3' end. Deletion of all or one or more nucleotides of the contiguous nucleotide sequence region located in the nucleotide sequence region of 1 bp to 50 bp, substitution with a nucleotide different from that of the wild-type gene, each independently selected from A, T, C and G 1 to 23 nucleotides, or a combination of the above modifications.

The rare cutting endonuclease is from the group consisting of meganuclease, zinc finger nuclease, CRISPR/Cas9 (Cas9 protein), CRISPR-Cpf1 (Cpf1 protein) and TALE-nuclease. There may be more than one selected. In one embodiment, the rare cutting endonuclease may be a Cas9 protein or a Cpf1 protein.

The terms "chimeric RNA", "chimeric guide RNA", "guide RNA", "single guide RNA" and "synthetic guide RNA" are used interchangeably and include guide sequences, tracr sequences and/or tracr mate sequences. refers to a polynucleotide sequence that The term “guide sequence” refers to an approximately 20 bp sequence within a guide RNA that directs a target site, and may be used interchangeably with the terms “guide” or “spacer”. Also, the term “tracr mate sequence” may be used interchangeably with the term “direct repeat(s)”. The guide RNA may be composed of two RNAs, that is, CRISPR RNA (crRNA) and transactivating crRNA (transactivating crRNA, tracrRNA), or a single-stranded RNA comprising parts of crRNA and tracrRNA and hybridizing with the target DNA. (single-chain RNA, sgRNA).

Generally, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In addition, any nucleotide sequence that can be used for genetic manipulation to reduce the expression or activity of the B2M gene or B2M protein can be used as a guide RNA without limitation. For example, the nucleotide sequence may be a sequence capable of hybridizing with the B2M gene. In addition, a portion of the guide RNA nucleotide sequence may be modified in order to modify/enhance the function of the guide RNA. Also in some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using an appropriate alignment algorithm, is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more. Optimal alignment can be determined using any algorithm suitable for aligning sequences, non-limiting examples of which are the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, the Burroughs-Wunsch algorithm. Algorithms based on the Burrows-Wheeler Transform (e.g. Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies) , ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn) and Maq (available at maq.sourceforge.net) In some embodiments, a guide sequence is about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40 , 45, 50, 75 or more nucleotides in length. In some embodiments, the guide sequence is about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less nucleotides in length. The ability of the guide sequence to induce sequence-specific binding of the CRISPR complex of can be assessed by any suitable assay, for example, components of the CRISPR system sufficient to form a CRISPR complex comprising the guide sequence being tested. corresponding target sequence, e.g., after transfection with a vector encoding a component of a CRISPR sequence, e.g., by assessment of preferential cleavage within the target sequence by a SURVEYOR assay as described herein. Similarly, cleavage of the target polynucleotide sequence can be performed by CRISPR comprising a control guide sequence different from the target sequence, the guide sequence to be tested, and the test guide sequence. It can be evaluated in vitro by providing components of the complex and comparing binding or cleavage rates between test and control guide sequence reactions at the target sequence. Other assays are possible and will occur to those skilled in the art.

Guide sequences can be selected to target any target sequence. In some embodiments, a target sequence is a sequence within the genome of a cell. Exemplary target sequences include those that are unique in the target genome.

In the complex, crRNA may be linked to tracrRNA.

Alternatively, the reduction in the expression or activity of the B2M gene or B2M protein may be caused by an exogenous nucleic acid molecule or a vector into which the nucleic acid molecule is introduced.

Exogenous nucleic acid molecules can be antisense oligonucleotides, ribozymes or interfering RNA (RNAi) molecules.

The "vector" is an expression vector capable of expressing a target protein in a suitable host cell, and may refer to a gene delivery system including essential regulatory elements operably linked to express a gene insert.

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. In addition, 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.

In addition, the vector is a plasmid vector, cosmid vector, viral vector, lentiviral vector, retrovirus vector, HIV (Human immunodeficiency virus) vector, MLV (Murineleukemia virus) vector, ASLV (Avian sarcoma / leukosis) Vector, SNV (Spleen necrosis virus) vector, RSV (Rous sarcoma virus) vector, MMTV (Mouse mammary tumor virus) vector, Adenovirus vector, Adenoassociated virus vector, Herpes simplex virus ) vector and one or more vectors selected from the group consisting of episomal vectors.

The term "somatic cell" may refer to any cell other than reproductive cells, and may be, for example, derived from or isolated from mammals such as humans, horses, sheep, pigs, goats, camels, antelopes, dogs, and mice. there is. In addition, the somatic cells include fibroblasts, spleen cells, hepatocytes, epithelial cells, muscle cells, nerve cells, hair cells, hair follicle cells, hair follicle cells, oral epithelial cells, somatic cells extracted from urine, gastric mucosal cells, and embryonic cells. Cells, G cells, B cells, pericytes, astrocytes, hemangioblasts, blood cells, neural stem cells, and oligodendrocytes may be one or more selected from the group consisting of, but is not limited thereto. However, in a specific embodiment of the present invention, the somatic cells were used as fibroblasts, splenocytes or hepatocytes.

The term "stem cell" refers to master cells capable of regenerating without limitation to form specialized cells of tissues and organs. Stem cells are pluripotent or multipotent cells capable of development. Stem cells can divide into two daughter stem cells, or one daughter stem cell and one originating ('transit') cell, which then proliferates into the mature, complete cell form of the tissue. These stem cells can be classified in various ways. One of the most commonly used methods is based on the differentiation capacity of stem cells: pluripotent stem cells that can differentiate into three germ layers, and multipotent stem cells that are limited to differentiate into more than a specific germ layer. ) and unipotent stem cells capable of differentiating only into a specific germ layer. The term "pluripotent stem cells" refers to stem cells with totipotency capable of differentiating into all three germ layers constituting a living body, and in general, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). ) may correspond to this.

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. In one embodiment, stem cells of one aspect may be differentiated into teratomas including ectoderm, mesoderm, or endoderm.

In one aspect, the stem cells may be, for example, induced pluripotent stem cells (iPSC), embryonic stem cells, somatic cell nuclear transfer-PSC or adult stem cells. In one embodiment, somatic cell nuclear transfer embryonic stem cells with reduced expression or activity of the B2M gene or B2M protein are effectively produced from somatic cells, thereby reducing the expression or activity of the B2M gene or B2M protein that induces low immunity to immune rejection evasion cells. Genetically engineered stem cells were prepared.

As used herein, the term "immunorejection avoidance cells" refers to cells that do not respond to immune rejection by supplementing the therapeutic disadvantages of adult stem cells or immune cells. Double immune rejection evasion stem cells refer to cells that maintain evasion from immune rejection while stabilizing various pluripotency-related abilities. The immune rejection evasion cells are genetically engineered cells in which the B2M gene is knocked out, "expression or activity is reduced" or B2M is "inactivated", or B2M protein is "decreased in expression or activity" or B2M is "inactivated". can mean

One aspect provides a genetically engineered immune cell having reduced expression or activity of a B2M gene or B2M protein differentiated from the stem cells.

The immune cells may be based on a substance that induces immune rejection stem cells in which the expression or activity of the prepared B2M gene or B2M protein is reduced into immune cells, and the substance that induces immune cells is a stem cell in the art. Any material used as a material that induces immune cells can be used without limitation.

The immune cells may be lymphoid-derived cells differentiated through the basis of hemangioblasts, for example, CD8+ T cells, helper CD4+ T cells, helper CD4+ T cells, regulatory T cells, CD3 cells, intraepithelial lymphocytes (IEL), It may be gamma delta T cells or NK cells. In one embodiment, differentiation-induced gamma delta T cells or NK cells may be prepared from pluripotent stem cells (SCNT-PSC) through somatic-cell nuclear transfer (SCNT).

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 term “cell therapy product” refers to a therapy using autologous, allogenic, or xenogenic cells to restore tissue function, and refers to a therapy used for suppression of cancer. It may be a cell therapy agent containing the prepared stem cells or immune cells as an active ingredient.

Immune rejection evasion stem cells included in the cell therapy include embryonic stem cell lines derived from nuclear transfer technology prepared by injecting nuclei of somatic cells that do not express B2M into enucleated oocytes, and may further contain a pharmaceutically acceptable carrier. can The pharmaceutically acceptable carrier may include, for example, saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, human serum albumin (HSA), and a mixture of one or more of these components. And, if necessary, other conventional additives such as antioxidants, buffers and bacteriostats may be added.

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. can

In addition, if necessary according to the formulation, the cell therapy agent may include a suspending agent, a solubilizing agent, a stabilizing agent, an isotonic agent, a preservative, an adsorption preventing agent, a surfactant, a diluent, an excipient, a pH adjusting agent, a pain reliever, a buffer, and a sulfur-containing agent. A reducing agent, an antioxidant and the like can be added as appropriate. Examples of the suspending agent include methyl cellulose, polysorbate 80, hydroxyethyl cellulose, gum arabic, traganthal, carboxymethyl cellulose sodium, and polyoxyethylene sorbitan monolaurate.

Examples of the solution adjuvant include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinic acid amide, polyoxyethylene sorbitan monolaurate, mecrogol, and castor oil fatty acid ethyl ester. Dextran 40, methylcellulose, gelatin, sodium sulfite, sodium metasulfate, etc. are mentioned as a stabilizer.

As an isotonizing agent, D-mannitol, sorbitol, etc. are mentioned, for example.

Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, sorbic acid, phenol, cresol, and chlorocresol.

Examples of the anti-adsorption agent include human serum albumin, lecithin, dextran, ethylene oxide propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, polyoxyethylene hydrogenated castor oil, and polyethylene glycol.

Examples of the sulfur-containing reducing agent include N-acetylcysteine, N-acetylhomocysteine, thioctoic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione, carbon atom number Those having sulfhydryl groups, such as 1-7 thioalkanoic acid, etc. are mentioned.

Antioxidants include, for example, erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbic acid palmitate, L-ascorbic acid and chelating agents such as stearate, sodium hydrogensulfite, sodium sulfite, triamyl gallate, propyl gallate, sodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate.

In addition, when the cell therapy is based on an adult patient weighing 70 kg, for example, about 1,000 to 10,000 cells / time, 1,000 to 100,000 cells / time, 1,000 to 1000,000 cells / time, 1,000 to 10,000,000, 1,000 ~100,000,000 cells/time, 1,000~1,000,000,000 cells/time, 1,000~10,000,000,000 cells/circle, once or several times a day at regular time intervals.

The injection product according to the present invention may be prepared in the form of a filled injection by taking an amount commonly known in the art according to the patient's constitution and defect type.

The cell therapy agent can be used for the prevention or treatment of cancer and contains CD47, human leukocyte antigen-E (human leukocyte antigen-E), and human leukocyte antigen-G (human leukocyte antigen-G) to avoid NK attack. It may further include a nucleic acid molecule encoding or a vector into which the nucleic acid molecule is introduced.

The term "prevention" refers to all activities to prevent the disease by removing the etiology of cancer or detecting it early.

The term "treatment" refers to any action that ameliorates or beneficially alters symptoms caused by cancer.

The term 'cancer' refers to a group of diseases characterized by overproliferation of cells and infiltration into surrounding tissues when the normal cell death balance is disrupted. The cancer is, for example, lung cancer, laryngeal cancer, stomach cancer, colon/rectal cancer, liver cancer, gallbladder cancer, pancreatic cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, prostate cancer, kidney cancer, carcinoma derived from epithelial cells such as skin cancer, bone cancer, muscle cancer, fat cancer, sarcoma derived from connective tissue cells (e.g. fibro cell carcinoma), leukemia, lymphoma, hematological cancer derived from hematopoietic cells (e.g. multiple myeloma), nerve tissue It may be one or more selected from the group consisting of tumors occurring in, but is not limited thereto.

In addition, one aspect provides a method for preventing or treating cancer comprising administering a cell therapy product containing the prepared stem cells or immune cells to a subject in need thereof.

As used herein, 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.

The cell therapy product containing the prepared stem cells or immune cells may be administered to a subject by various methods known in the art, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, It may be administered by oral administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc., but is not limited thereto.

Another aspect is a method for producing somatic cells into immune rejection evasion stem cells, comprising the step of contacting or inserting a gene editing complex that specifically binds to a B2M gene, an exogenous nucleic acid molecule, and a vector into which the nucleic acid molecule has been introduced into somatic cells. to provide.

The method may further include injecting the nucleus of a somatic cell as a donor cell into an enucleated oocyte.

Another aspect provides a method for preparing immune rejection cells from somatic replicating pluripotent stem cells.

The method for preparing the immune cells may further include editing a gene that specifically binds to the B2M gene, and then injecting the nucleus of a somatic cell as a donor cell into an enucleated oocyte. In addition, the method may further include a step of treating cells with a substance that induces immune rejection stem cells into immune cells and a substance that differentiates immune cells. The material for differentiating immune cells may be used in the art to include all materials for differentiating stem cells into immune cells.

The method for producing the immune cells includes contacting or inserting a gene editing complex that specifically binds to a B2M (β2-microglobulin) gene, an exogenous nucleic acid molecule, and a vector into which the exogenous nucleic acid molecule is introduced into somatic cells; preparing somatic cell replicating pluripotent stem cells (SCNT-PSCs) by injecting the nuclei of the somatic cells as donor cells into enucleated oocytes; and inducing differentiation into immune cells from the prepared somatic replicating pluripotent stem cells.

The immune cells prepared in the above method include CD8+ T cells, helper CD4+ T cells, helper CD4+ T cells, regulatory T cells, intraepithelial lymphocytes (IEL), CD3 cells, gamma delta T cells, or NK. may be cells. Using the method of one aspect, it is possible to manufacture gamma delta T cells or NK cells differentiated from pluripotent stem cells (SCNT-PSC) through somatic-cell nuclear transfer (SCNT) from somatic cells with high efficiency. do.

One aspect relates to pluripotent stem cells (PCNT-PSCs) prepared from somatic cells in which the B2M gene is knocked out and through somatic cell nuclear transfer therefrom, and immune cells induced to differentiate therefrom, pluripotent stem cells of one aspect and immune cells can differentiate into NK cells and gamma delta T cells, which are normal immune cells, even if the B2M gene is knocked out. In addition, since the prepared cells target the B2M gene and induce knockout of HLA class I molecules, they can be used for a long period of time in vivo through avoidance of immune rejection of the 'off-the-shelf' concept that does not induce a response of allogeneic T cells. Because it is viable and the number of injections can be minimized, it can be used as low-immunity cells or immune rejection stem cells and immune cells and can be widely used as a cell therapy agent.

1 is a diagram illustrating knocking out the B2M gene of mouse fibroblasts, performing subculture for 7 days, and confirming the cell morphology.

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. 3 is a diagram showing the morphology of fibroblasts in which the B2M gene was knocked out, prepared into pluripotent stem cells through somatic-cell nuclear transfer (SCNT).

4 is a view showing the results of confirming the OCT4, SSEA, and NANOG markers of the prepared somatic cell replicating pluripotent stem cell line through immunofluorescence staining.

5 is a diagram illustrating the result of confirming whether the prepared somatic cell replicating pluripotent stem cell line was actually knocked out by PCR electrophoresis, and the result of confirming through 160/184 primers and 185/184 primers.

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.

Figure 7 is a diagram confirming the immunohistochemical staining of teratoma tissue of the prepared somatic cell replicating pluripotent stem cell line, Figure 7A is a diagram showing the H-E staining results of the PBS-injected group as a control group in the teratoma tissue, and Figure 7B is a diagram showing the results of neural rosettes Figure 7C is a diagram confirming the differentiation pattern into a neural rosette structure, Figure 7C is a diagram confirming the differentiation pattern into a gut epithelium structure in the part marked with *, and Figure 7D is a diagram confirming the differentiation pattern into a cartilage structure. Figure 7E is a diagram confirming the differentiation pattern into the muscle fiber structure.

8 is a diagram confirming the results of confirming the expression of B2M in liver and spleen cells in which the B2M gene was knocked out with 160/184 and 185/184 primers at the mRNA and gDNA levels.

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

9c is a diagram confirming the frequency of natural killer cells, which are CD3-NK1.1+ cells, using splenocytes of wild-type mice and mice in which the B2M gene was knocked out.

10A is a diagram showing markers (NKP46, Ly49, KLRG1) showing the frequency and killing ability of natural killer cells, which are CD3-NK1.1+ cells, using liver cells of wild-type mice.

10B is a diagram comparing the frequency of natural killer cells, which are CD3-NK1.1+ cells, and markers (NKP46, Ly49, KLRG1) showing the killing ability in hepatocytes of mice in which the B2M gene was knocked out.

10C is a diagram confirming the frequency of natural killer cells, which are CD3-NK1.1+ cells, using liver cells of wild-type mice and mice in which the B2M gene is knocked out.

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. 11B is a diagram confirming the degree of differentiation at 13 days after differentiation of wild-type embryonic stem cells into NK cells, a type of blood cell.

Figure 11c shows differentiation of B2M knocked-out somatic pluripotent stem cells into NK cells, a type of blood cell, at 1, 2, 3, 5, 6, 9, 10, 11, 12, and 13 days thereafter. It is a diagram showing the result of confirming the shape.

11D is a diagram confirming the degree of differentiation at 13 days after differentiation of B2M knocked-out somatic cell-replicating pluripotent stem cells into NK cells, a type of blood cell.

12a is a diagram showing the result of inducing differentiation into gamma delta T cells from prepared stem cells and confirming their morphology.

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.

Figure 12d is a diagram showing the quantitative results of confirming the differentiation results by FACS.

FIG. 13a is a diagram showing results of B2M knockout verification in wild-type cells without B2M knockout, fibroblast cells (skin) induced with B2M knockout, and somatic cell replicating pluripotent stem cells (ES) therefrom.

Figure 13b shows wild-type B2M knockout-free and B2M knockout-induced fibroblasts (skin), somatic cell replicating pluripotent stem cells (B2M-/-SCNT-PSCs) and cells differentiated into NK cells (B2M-/-SCNT-PSCs) therefrom. -Blood cells) is a diagram showing the results of B2M knockout verification.

13c shows somatic cell-replicating pluripotent stem cells prepared from wild blood cells without B2M knockout and fibroblasts induced with B2M knockout and differentiated into gammadelta T cells (B2M−/−Blood cells). It is a diagram showing the results of B2M knockout verification in

Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

Example 1. Identification of pluripotent stem cells prepared through somatic cell nuclear transfer from fibroblasts knocked out of the B2M gene

1.1 Production of fibroblasts knocked out of the B2M gene

An experiment was performed to knock out the B2M gene from mouse skin fibroblasts.

Specifically, B2M-deficient mice homozygous for the B2mtm1Unc target mutation were purchased. The purchased B2mtm1Unc mutant strain was prepared by target disruption of the B2M gene, and was prepared by inbreeding 11 times between B6 mice and the B2mtm1Unc target mutant mouse strain using the 129-derived E14TG2aES cell line. Tissues were collected from the tail and skin of the mice. The collected tissue was cultured in a medium containing DMEM (Dulbecco's modified Eagle's medium), 10% fetal bovine serum (FBS), and 1% penicillin and streptomycin (P/S) until cells were sufficiently produced. Tissue attached to the cultured cells was removed, and fibroblasts in which the B2M gene was knocked out were subcultured for more than 7 days, and cells cultured for 7 days were identified and shown in FIG. 1 .

Then, in order to confirm whether the B2M gene defect actually appeared in the subcultured fibroblasts, 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.

160 mutant forward: TCT GGA CGA AGA GCA TCA GGG (SEQ ID NO: 1),

184 consensus sequence: TAT CAG TCT CAG TGG GGG TG (SEQ ID NO: 2);

185 wildtype forward: CTG AGC TCT GTT TTC GTC TG (SEQ ID NO: 3)

As confirmed in FIG. 2A, in all fibroblasts in which the B2M gene was knocked out, unlike the wild type, a band appeared at 420 bp, confirming that B2M was missing. In addition, in FIG. 2B, unlike the wild type, it was confirmed that a band appeared near 1 kb, confirming that the B2M gene was actually knocked out in the prepared fibroblasts.

1.2 Production of pluripotent stem cells through somatic cell nuclear transfer from fibroblasts in which the B2M gene was knocked out and confirmation of the ability of the prepared stem cells

8-10 weeks old female B6 mice were administered 5 IU PMSG and 5 IU hCG hormone. After 14 hours of hCG administration, cumulus cells were separated using hyaluronidase from the over-ovulated mice and eggs were separated. The isolated oocytes were stored in the KSOM culture medium at 37°C incubator until the start of the experiment. Then, for the somatic cell cloning experiment, the nucleus was removed from the mature egg, and the fibroblasts in which the B2M gene was knocked out prepared in Example 1.1 were subjected to somatic-cell nuclear transfer (SCNT) to produce somatic-cell cloned blastocysts. Pluripotent stem cells were prepared by isolating the inner cell mass of the blastocyst. The manufactured B2M knockout somatic cell replicating pluripotent stem cells (KO SCNT-PSCs) were prepared in DMEM, 20% FBS, 1% P/S, 1% nonessential amino acids (NEAA), 0.1% beta mercaptoethanol (beta mercaptoethanol) was cultured in a stem cell culture solution containing, and the results of confirming the morphology of the prepared cells, which were cultured over the third passage, are shown in FIG. 3 .

In order to confirm whether the pluripotent stem cells prepared according to the above method have stem cell ability with differentiation potential as actual stem cells, the prepared somatic cell replicating pluripotent stem cell line was washed in PBS supplemented with 0.1% BSA and 4% paraformaldehyde. (paraformaldehyde) was treated at room temperature for 30 minutes to fix, treated with PBS containing 0.1% Triton X, and stored in a refrigerator for 24 hours. OCT4, SSEA, and NANOG markers were confirmed by immunofluorescence staining. The results of confirming this by confocal fluorescence microscopy are shown in FIG. 4 .

As confirmed in FIG. 4 , it was confirmed that the prepared somatic cell replicating pluripotent stem cell line had stem cell ability capable of differentiating into all cells, with all of OCT4, SSEA, and NANOG clearly appearing.

Accordingly, FIG. 5 shows the results of electrophoresis using two types of 160/184 and 185/184 primers, respectively, to determine whether the B2M gene was actually knocked out in the prepared stem cells.

As confirmed in FIG. 5, the somatic cell replicating pluripotent stem cell line (SCNT-PSCs) showed a band at 420 bp when using the 160/184 primer, and confirmed that a band appeared around 1 kb when using the 185/184 primer. It was specifically confirmed that the SCNT-PSCs actually knocked out the B2M gene and could be used as immune rejection evasion stem cells.

Example 2. Confirmation of Stem Cell Potency of B2M Knockout Somatic Replicating Pluripotent Stem Cells (SCNT-PSCs)

Using the method used in Example 1, an experiment was performed to confirm whether the somatic replicating pluripotent stem cells in which the B2M gene was knocked out can exhibit stem cell function. F-12, 20% FBS, 1% P/S, 1% nonessential amino acids (NEAA), 0.1% beta mercaptoethanol (beta mercaptoethanol) into a testis capsule of severe combined immunodeficient mice, one testis was injected with only PBS as a control group, and the other testis was a B2M-deficient somatic cloned embryo. Stem cell 1X10 ⁶ cells were mixed with PBS and then injected to induce teratoma formation. The transplanted mice were managed in a SPF (Specific Pathogen Free) facility for 5 weeks, and teratomas formed in the control and transplanted areas were confirmed 5 weeks after transplantation, and are shown in FIG. 6 . As confirmed in FIG. 6 , it was confirmed that teratomas were formed in all transplanted areas 5 weeks after transplantation. In addition, the differentiation pattern of the teratoma tissue formed at the transplanted site was analyzed through immunohistochemical staining (HE and special staining), which is shown in FIGS. 7A to 7E.

Figure 7A is a diagram confirming the PBS-treated group, which is a control group, through H-E staining, and as confirmed in Figures 7B to 7E, differentiation into teratoma markers, endoderm, mesoderm, and ectoderm, was made. could confirm that Specifically, as confirmed in FIG. 7B, the differentiation into the ectoderm was confirmed by confirming the differentiation pattern into the neural rosette structure. In FIG. 7C, the differentiation into endoderm was confirmed by confirming the differentiation pattern into the gut epithelium structure in the part marked with *, and as confirmed in FIG. 7D, the circular shape in the middle of the figure stained with Alcian blue The differentiation into mesoderm was confirmed by confirming the differentiation pattern into the cartilage structure. In FIG. 7E, 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.

Example 3. Identification of knock-out of B2M gene from liver and spleen cells

3.1 Knock-out confirmation of B2M gene in liver and spleen cells

An experiment was performed to confirm whether the B2M gene could be effectively knocked out when the method and reagents used in Example 1 were used in liver and spleen cells. That is, an experiment was performed to verify by PCR whether B2M was accurately knocked out using the stem cell line prepared after knocking out B2M.

The liver and spleen cells in which the B2M gene was knocked out were subcultured for more than 7 days, and in order to confirm whether the B2M gene was actually defective in the subcultured liver and spleen cells, the B2M gene was not knocked out. Genomic DNA was extracted from the liver and spleen cells of untreated wild-type mice and the prepared B2M KO liver and spleen cells. From this, it was confirmed through electrophoresis using two types of 160/184 and 185/184 primers, respectively, whether the B2M gene was actually knocked out, and GAPDH was used as a control gene. The results confirmed with the /184 and 185/184 primers are shown in FIG. 8 .

As confirmed in FIG. 8 , when mRNA expression was confirmed in liver and spleen cells, it was confirmed that B2M was not expressed. Also, in gDNA, when confirmed with 160/184 primers, a band appeared at 420 bp, and when confirmed with 185/184 primers, it was confirmed that a band appeared around 1 kb, confirming that B2M was not expressed even at the gDNA level, and actually prepared It was confirmed that the B2M gene was effectively deleted in all of the cells. Therefore, in the normal group in which B2M expression was not inhibited, PCR showed banding of the B2M product, and in the skin, liver, and spleen tissues of the other B2M knockout mice, no bands for the B2M product were found. confirmed by

3.2 Identification of frequency of natural killer cells, CD3-NK1.1+ cells in liver and spleen

In order to confirm the frequency of CD3-NK1.1+ natural killer cells in the liver and spleen of B2M knockout mice, flow cytometry analysis (Fluorescence-activated cell sorting: FACS) was performed. For flow cytometric analysis, livers and spleens were extracted from tissues of normal mice and B2M knockout mice, mashed using an eppendorf lid in a sterile culture dish, and single cells were extracted by filtering with a 0.4uM filter. After washing these cells twice with DPBS (1200 rpm, 5 minutes), elution was performed with less than 5X10 ⁵ cells for flow cytometry analysis, and CD3 antibody and nk1.1 antibody (NKP46, Ly49, KLRG1) were attached to each (1:100) 20 Incubate in a refrigerator at 4° C. for 1 minute. Cells were then washed once with DPBS and analyzed on a flow cytometer. In the case of gamma delta T cells, each antibody was applied to the cells at a concentration of 1:100, incubated in a refrigerator for 20 minutes, washed once with DPBS, and analyzed in a flow cytometer. Figure 9a shows the results of flow cytometry analysis of wild-type mice performed on the spleen, and Figure 9b shows the results of flow cytometry analysis of B2M knockout mice. The result of confirming the frequency of killer cells is shown in FIG. 9c. Figure 10a shows the results of flow cytometric analysis of wild-type mice performed on the liver, and Figure 10b shows the results of flow cytometry analysis of B2M knockout mice. CD3-NK1.1+ cells of wild-type and B2M knockout mice The result of confirming the frequency of killer cells is shown in FIG. 10c.

9a to 9c, in the spleen, the frequency of natural killer cells, which are CD3-NK1.1+ cells, was 3.3±0.1% in wild-type mice and 3.6±0.1% in B2M knockout mice. , it was confirmed that a significant difference appeared (*P-value<0.03). In addition, activity in NK cells was 39.6% in wild-type mice and 48.9% in B2M knockout mice for NKP46, 24.1% for wild-type mice and 30.9% for B2M knockout mice for Ly49, and 30.9% for KLRG1. It was confirmed that there was a frequency difference of 14.4% for mice and 10.1% for B2M knockout mice.

In addition, as confirmed in FIGS. 10a to 10c, in the case of wild-type mice, the frequency of natural killer cells, which are CD3-NK1.1+ cells, in the liver was 5.8±1.3%, and in the case of B2M knockout mice, the frequency difference was 2.4±0.1%. confirmed to appear. The activity in NK cells was 9.4% in wild-type mice and 14.3% in B2M knockout mice for NKP46, and 11.5% in wild-type mice and 5.2% in B2M knockout mice for Ly49. In this case, it was confirmed that wild-type mice had a frequency of 12.1% and B2M knockout mice had a frequency of about 9.1%.

Example 4. Confirmation of Differentiation of B2M Knockout Somatic Replicating Pluripotent Stem Cells (SCNT-PSCs) into Immune Cells

An experiment was performed to determine whether effective differentiation of the somatic cell-replicating pluripotent stem cells prepared in Example 1 into immune cells, a type of lymphoid cells, was induced. Through such experiments, it is possible to confirm whether differentiation into immune cells is achieved while maintaining the B2M gene deficiency in lymphoid cells, and whether the differentiation degree of differentiated cells and the differentiation rate of the prepared pluripotent stem cells are embryonic stem cells. It was possible to check whether there was a difference such as retardation when compared to .

Specifically, differentiation of B2M knocked-out somatic pluripotent stem cells and wild-type embryonic stem cells into lymphoid cells including NK cells, a type of blood cell, was induced. The composition of the differentiation media for differentiation was as follows.

Mouse embryonic stem cell maintenance media is DMEM containing high glucose, NEAA (nonessential amino acid) 5ml, 20% SR, mercaptoethanol (500μl/500ml), penicillin 5ml, LIF (1μl/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.

1. 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.

2. Differentiation media for hemangioblastic progenitor cells were Apel-2 media, 10ng/ml bFGF, 100ng/ml VEGFA, 250ng/ml SCF, 200ng/ml FLT3L, 20ng/ml GCSF, 100ng/ml TPO , 20 ng/ml of EPO, 50 ng/ml of IL-6, and 50 ng/ml of IGF-1, and treated with media having the above composition to cells and differentiated for 2 days.

3. Apel-2 media for stabilization of hemangioblasts, bFGF at 10 ng/ml, VEGFA at 100 ng/ml, SCF at 250 ng/ml, FLT3L at 200 ng/ml, GCSF at 20 ng/ml, TPO at 100 ng/ml , 50ng/ml of IL-6, 50ng/ml of IGF-1, 20ng/ml of IL-7, 20ng/ml of IL-15, 50ng/ml of IL-5 and 25ng/ml of DLL1, Cells were treated with the media and differentiated for 6 days to stabilize the cells.

4. 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.

After the last step 4, differentiation into lymphoid cells such as T cells, gamma delta T cells, etc. including differentiation into NK cells, starting with lymphoblasts, was confirmed. Accordingly, FIG. 11a shows the result of confirming the wild-type embryonic stem cells, which confirmed the cell morphology on days 1, 2, 3, 5, 6, 9, 10, 11, 12, and 13 inducing the differentiation, in FIG. 11a. Fig. 11c shows the result of confirming somatic cell-replicating pluripotent stem cells, Fig. 11b shows the result of confirming the degree of differentiation in wild-type embryonic stem cells 13 days after induction of differentiation, and Fig. 11b shows the result of confirming somatic cell-replicating pluripotent stem cells knocked out of B2M. It is shown in Figure 11d. In addition, since this cell line is a cell in which gene editing is induced to emit GFP fluorescence, GFP is expressed in all cells.

As confirmed in FIGS. 11a to 11d, when blood Lineage NK cells were differentiated between embryonic stem cells and B2M-deficient somatic cell replicating pluripotent stem cells, there was no significant difference in the degree of delay or frequency of differentiation morphologically. Confirmed.

In addition, when differentiation was induced up to the 13th day by applying a 4-step differentiation media from stem cells, the level of differentiation into natural killer cells was reduced to 6.6% in the wild-type ES cell line (C57/BL6 STRAIN) and 6.4% in the case of B2M KO. It was confirmed that indicated This is confirmed to be about twice as high as the efficiency of differentiation into NK cells in the spleen of a mouse. Thus, even if pluripotent stem cells are prepared from fibroblasts and produced into stem cells through somatic cell cloning, they have the same induction ability as wild-type stem cells. Therefore, it was confirmed that efficient differentiation was possible, and that the efficiency was superior to that of conventional differentiation from splenocytes.

In addition, compared to the expression of B2M at 7.2% in embryonic stem cells induced to terminal differentiation, it was confirmed that the produced SCNT-PSCs expressed B2M at a significantly lower rate of 0.4%. It was confirmed that even hypoimmune NK cells capable of avoiding immune rejection can be effectively produced because they can be produced without expression of B2M.

Example 5. Confirmation of Differentiation of B2M Knockout Somatic Replicating Pluripotent Stem Cells (SCNT-PSCs) into Gamma Delta T Cells

Experiments were performed to confirm whether effective differentiation was induced from the somatic cell-replicating pluripotent stem cells prepared in Example 1 to gammadelta T cells while maintaining the B2M gene-defective state. The composition of the differentiation media used for differentiation is the same as Ari.

Specifically, the differentiation of somatic cell replicating pluripotent stem cells knocked out of B2M into gamma delta T cells, which is a type of lymphoid cell, was induced. The composition of the differentiation media for differentiation was as follows.

Mouse embryonic stem cell maintenance media is DMEM containing high glucose, NEAA (nonessential amino acid) 5ml, 20% SR, mercaptoethanol (500μl/500ml), penicillin 5ml, LIF (1μl/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.

1. Mesoderm differentiation media contained Apel-2 media, bFGF at 10 ng/ml, 3 nM CHIR, ascorbic acid at 100 ng/ml, VEGFA at 20 ng/ml, SCF at 50 ng/ml and BMP4 at 25 ng/ml. do. To differentiate into mesoderm, the cells were treated with the differentiation media and differentiated for 2 days.

2. 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.

3. Apel-2 media for stabilization of hemangioblasts, bFGF at 10 ng/ml, VEGFA at 100 ng/ml, SCF at 250 ng/ml, FLT3L at 200 ng/ml, GCSF at 20 ng/ml, and TPO at 100 ng/ml , 50ng/ml of IL-6, 50ng/ml of IGF-1, 20ng/ml of IL-7, 20ng/ml of IL-15, 50ng/ml of IL-5 and 25ng/ml of DLL1, Cells were treated with the media and differentiated for 6 days to stabilize the cells.

4. 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.

FACS and immunostaining were performed after differentiation in order to confirm whether differentiation into gamma delta T cells among blood cells was performed while maintaining the B2M gene-defective state. After the last step 4, it was confirmed that differentiation and maturation into T cells and NK cells including differentiation into gamma delta T cells, starting with lymphoblasts, appeared. In particular, it was confirmed that gamma delta T cells enter differentiation most quickly within 2-3 days after differentiation.

Accordingly, differentiation of the prepared stem cells into gamma delta T cells was induced, and the results of confirming the morphology were shown in FIG. 12A. In addition, after inducing differentiation of B2M KO fibroblasts together with CD3, NKG2D, and NK1.1 using a gamma delta TCR antibody, expression of these proteins was confirmed to effectively induce gamma delta cells on day 11 of the differentiated gamma delta T cells. 12b shows the results of confirming whether differentiation was induced with a single marker, double expression, triple expression, and quadruple expression marker. In addition, the result of confirming the expression of the marker through immunostaining is shown in FIG. 12c, and the result of confirming the differentiation result through FACS is shown in FIG. 12d.

As confirmed in FIG. 12a, when the differentiation of the prepared somatic cell-replicating pluripotent stem cells into gamma delta T cells was induced, there was no significant difference in the degree of delay or frequency of differentiation, and after about 12 days, gamma delta T cells were effectively transformed into gamma delta T cells. It was confirmed that differentiation was induced. As confirmed in FIGS. 12B and 12D , it was confirmed that 11 days after differentiation showed differentiation into gamma delta T cells and CD3 T cells. It was confirmed that the frequency of CD3 identified as a single marker was 4.9±0.6% and NK1.1 was 10.1±0.6%. It was confirmed that the frequency of NKG2D was 22.1±1.9% and the gamma delta TCR was 29.0±3.1%. In addition, FACS was performed to more clearly identify the gamma delta T cell population through double expression, triple expression, and quadruple expression. As a result, the ratio of CD3+gd TCR+ cells was 3.7±0.6%, and NKG2D+gd TCR+ cells were It was confirmed that it appeared as 12.6±1.5%. In addition, it was confirmed that NKG2D+NK1.1+ cells appeared at 7.6±0.8%. In the case of triple expression, it was confirmed that the expression of NKG2D+gd TCR+ cells in CD3 cells was 11.1±1.3%, and the frequency of NKG2D+gd TCR+ cells in NK1.1 cells was 12.7±1.6%. In particular, since the differentiation of each lineage into mature cells was not completed, it was confirmed that the cells were in the form of differentiated lymphoid progenitor cells because no significant difference in frequency between groups was observed even when triple expression was investigated in any combination. It was confirmed that 10.6±0.7% of NKG2D+gd TCR+ cells appeared in the final NK1.1+CD3+ cells. In addition, as confirmed in FIG. 12c, it was confirmed that the SCNT-PSCs prepared through immunostaining were differentiated into gamma delta T cells by confirming the expression of gamma delta T cell markers.

Therefore, in cells 11 days after the induction of differentiation from somatic cell replicating pluripotent stem cells into gamma delta T cells, a type of lymphoid cell, the expression of blood gamma T cells, which are considered early lymphoid progenitor cells, was 30%, which was remarkably high. It was confirmed that the expression was expressed in gamma delta T cells at a ratio.

Example 6. Verification of B2M knockout of immune cells differentiated from prepared somatic cell replicating pluripotent stem cells (SCNT-PSCs)

NK cells and gamma delta T cells, which are immune cells finally differentiated from somatic cell replicating pluripotent stem cells (SCNT-PSCs) prepared in Examples 3 and 4, are also knocked out of B2M, resulting in hypoimmunity that can avoid immune rejection. An experiment was performed to confirm whether or not production with immune cells was possible, and this was specifically confirmed through PCR, and the results are shown in FIGS. 13a to 13c. The experimental method was performed through the method identified in the above examples through the cells prepared in the above examples.

Specifically, the results of B2M knockout verification in wild type without B2M knockout, fibroblasts (skin) induced with B2M knockout, and somatic cell replicating pluripotent stem cells (ES) therefrom are shown in FIG. 13a, and wild type without B2M knockout. and B2M knockout in fibroblasts (skin) from which B2M knockout was induced, somatic cell replicating pluripotent stem cells (B2M−/−SCNT-PSCs) and cells differentiated into NK cells (B2M−/−Blood cells) The verified results are shown in FIG. 13B, in which somatic cell-replicating pluripotent stem cells were prepared from wild blood cells without B2M knockout and fibroblasts induced with B2M knockout and differentiated into gammadelta T cells (B2M-/ -Blood cells), the results of B2M knockout verification are shown in FIG. 13c.

As confirmed in FIGS. 13 a, b and c, B2M deletion was confirmed at 410 bp for the 160/184 primers and at 261 bp for the 185/184 primers. Therefore, fibroblasts with B2M knockout induced, when prepared as somatic cell replicating pluripotent stem cells, and NK cells and gamma delta T cells derived from the prepared B2M-/-SCNT-PSCs also showed B2M knockout, Since the expression of B2M does not appear until the actual final differentiation, it was confirmed that it is possible to effectively manufacture NK cells with low immunity capable of avoiding immune rejection.

Claims (13)

1. Gene editing complexes that specifically bind to the B2M (β2-microglobulin) gene, exogenous nucleic acid molecules, or genetic manipulations with reduced expression or activity of the B2M gene or B2M protein, including vectors into which the exogenous nucleic acid molecule has been introduced. stem cells.
2. The stem cell according to claim 1, wherein the stem cell is induced pluripotent stem cell (iPSC), embryonic stem cell, somatic cell nuclear transfer-PSC or adult stem cell.
3. The method according to claim 2, wherein the somatic cells are fibroblasts, spleen cells, hepatocytes, epithelial cells, muscle cells, nerve cells, hair cells, hair follicle cells, hair follicle cells, oral epithelial cells, somatic cells extracted from urine, gastric mucosal cells, Stem cells that are at least one selected from the group consisting of goblet cells, G cells, B cells, pericyte cells, astrocytes, hemangioblasts, blood cells, neural stem cells, and oligodendrocyte precursor cells.
4. The stem cell according to claim 1, wherein the gene editing complex is selected from the group consisting of zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and RNA-guided engeineered nucelase (RGEN).
5. The stem cell according to claim 4, wherein the RGEN comprises guide RNA and Cas protein that specifically bind to a specific sequence of the B2M gene.
6. The stem cell according to claim 1, wherein the exogenous nucleic acid molecule is an antisense oligonucleotide, a ribozyme or an interfering RNA (RNAi) molecule.
7. The method according to claim 1, wherein the vector is a plasmid vector, cosmid vector, viral vector, lentiviral vector, retrovirus vector, HIV (Human immunodeficiency virus) vector, MLV (Murineleukemia virus) vector, ASLV (Avian sarcoma / leukosis) ) vector, SNV (Spleen necrosis virus) vector, RSV (Rous sarcoma virus) vector, MMTV (Mouse mammary tumor virus) vector, Adenovirus vector, Adenoassociated virus vector, Herpes simplex virus Stem cells that are one or more vectors selected from the group consisting of virus vectors and episomal vectors.
8. A genetically engineered immune cell with reduced expression or activity of the B2M gene or B2M protein differentiated from the stem cell of claim 1.
9. The immune cell of claim 8, wherein the immune cell is a CD8+ T cell, helper CD4+ T cell, helper CD4+ T cell, regulatory T cell, intraepithelial lymphocyte (IEL), gamma delta T cell or NK cell.
10. A cell therapy agent comprising the stem cell of claim 1 or the immune cell of claim 8.
11. A method for preparing somatic cells into immune rejection evasion stem cells, comprising contacting or inserting a gene editing complex that specifically binds to a B2M gene, an exogenous nucleic acid molecule, and a vector into which the exogenous nucleic acid molecule has been introduced into somatic cells.
12. The method of claim 11, further comprising injecting the nucleus of the somatic cell as a donor cell into an enucleated oocyte.
13. contacting or inserting a gene editing complex that specifically binds to a B2M (β2-microglobulin) gene, an exogenous nucleic acid molecule, and a vector into which the exogenous nucleic acid molecule is introduced;

    preparing somatic cell replicating pluripotent stem cells (SCNT-PSCs) by injecting the nuclei of the somatic cells as donor cells into enucleated oocytes; and

    A method for preparing immune rejection avoiding immune cells from somatic replicating pluripotent stem cells comprising the step of inducing differentiation into immune cells from the prepared somatic replicating pluripotent stem cells.

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