WO2024002279A1 - Immunocompatible human pluripotent stem cell, method for preparing same, and use thereof - Google Patents

Immunocompatible human pluripotent stem cell, method for preparing same, and use thereof Download PDF

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WO2024002279A1
WO2024002279A1 PCT/CN2023/104109 CN2023104109W WO2024002279A1 WO 2024002279 A1 WO2024002279 A1 WO 2024002279A1 CN 2023104109 W CN2023104109 W CN 2023104109W WO 2024002279 A1 WO2024002279 A1 WO 2024002279A1
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cells
hla
seq
pluripotent stem
acid sequence
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PCT/CN2023/104109
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French (fr)
Chinese (zh)
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杨黄恬
张鹏
饶森乐
章小清
刘玲
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中国科学院上海营养与健康研究所
上海市东方医院（同济大学附属东方医院）
同济大学
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Publication of WO2024002279A1 publication Critical patent/WO2024002279A1/en

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Definitions

  • the present invention belongs to the field of biotechnology. More specifically, the present invention relates to an immune-compatible human pluripotent stem cell that can escape immune rejection of a transplant recipient, its preparation method and application.
  • Cell transplantation therapy is the use of bioengineering methods to obtain the characteristics of certain cells with specific functions and/or through in vitro amplification, special culture, etc., to generate cells with specific and powerful functions and transplant them into the patient's body to repair, Replenish, replace, condition or remove damaged or diseased cells/tissues to cure disease. Therefore, the application of cell technology can treat various diseases, such as nervous system, skeletal diseases, diabetes, cardiovascular and cerebrovascular diseases and other degenerative and damaging diseases, and many other diseases that are refractory to traditional therapies or complement traditional therapies and have broad applications. prospect. However, transplant recipients will develop immune rejection of non-self cells, making it difficult for transplanted cells to survive in the recipient's body for a long time.
  • HLA human leukocyte antigen
  • immune rejection is an immune response induced by allogeneic antigens on the cell surface, which is mainly mediated by HLA in the human body.
  • HLA is extremely polymorphic, and HLA matching can reduce the immune rejection of transplanted cells by transplant recipients. Based on the immune rejection mechanism, the following methods are available to reduce immune rejection:
  • HLA matching It is mainly used in organ and hematopoietic stem cell transplantation to reduce rejection through donor/recipient HLA matching.
  • HLA high polymorphism
  • iPSCs Autologous induced pluripotent stem cells
  • iPSCs are derived from reprogrammed autologous somatic cells and have the same HLA genes as the donor. In theory, iPSCs and their derived cells can also escape autoimmune rejection.
  • autologous iPSCs and their derived cells have a long preparation cycle, difficult quality control, and high cost. They are difficult to apply in diseases that are time-sensitive and have a narrow treatment time window, and the user population is very limited.
  • the cells derived from iPSCs may have functional abnormalities, which may lead to safety risks.
  • many degenerative and damaging diseases tend to occur in older age groups, such as myocardial infarction, Parkinson's disease, etc., and it is more difficult to establish iPSCs in such groups.
  • the technical routes for obtaining universal cells include the following: (1) By knocking out a key HLA molecule on a chromosome, pseudo-HLA homozygous cells are constructed to increase the number of all-purpose cells.
  • the co-inventors of the present invention constructed the B2Mm/sHLAG embryonic stem cells (hESCs) line by simultaneously overexpressing membrane-localized HLA-G1 and soluble HLA-G5, thereby reducing the immunogenicity of human embryonic stem cells.
  • hESCs B2Mm/sHLAG embryonic stem cells
  • the invention provides an immune-compatible human pluripotent stem cell, its preparation method and application, which has a wider range of applications.
  • a method for preparing immune-compatible human pluripotent stem cells which includes transforming human pluripotent stem cells to:
  • the genome of human pluripotent stem cells is modified to fuse the polynucleotide encoding HLA-G1 with the endogenous B2M gene in human pluripotent stem cells, thereby expressing B2M-HLA-G1 fusion protein does not express free B2M protein; preferably, the HLA-G1 coding gene is introduced into the endogenous B2M exon 3 at the position before the stop codon, or the HLA-G1 coding gene is Replace the stop codon located in exon 3 of the endogenous B2M gene; more preferably, the transformation is performed by gene editing methods.
  • the B2M-HLA-G1 and B2M-HLA-G5 are expressed using recombinant vectors; preferably, the recombinant vectors include non-viral vectors or viral vectors.
  • the non-viral vector includes: a site-directed knock-in system or a transposon system.
  • the viral vectors include: lentiviral vectors.
  • the B2M-HLA-G5 fusion protein also includes a flexible connecting peptide located between B2M and HLA-G5, preferably a flexible connecting peptide as shown in SEQ ID NO: 3; And/or, the B2M-HLA-G5 fusion protein includes: B2M and HLA-G5 in sequence from the N-terminus to the C-terminus; preferably, the amino acid sequence of the B2M-HLA-G5 fusion protein is as SEQ ID NO: 8 shown; more preferably, the nucleic acid sequence of the B2M-HLA-G5 fusion protein is shown in SEQ ID NO: 10.
  • the preparation method includes one or more of the following A-D:
  • the HLA-G1 includes: a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1; or a polypeptide with an amino acid sequence having more than 90% sequence identity with SEQ ID NO: 1 and having a polypeptide as shown in SEQ ID NO: 1 Functionally derived polypeptides (including active fragments and active variants);
  • the CIITA protein includes: a polypeptide with an amino acid sequence as shown in SEQ ID NO: 5; or a polypeptide with an amino acid sequence having more than 90% sequence identity with SEQ ID NO: 5 and having a polypeptide as shown in SEQ ID NO: 5 Functional derived polypeptides (including active fragments and active variants);
  • the HLA-G5 includes: a polypeptide with an amino acid sequence as shown in SEQ ID NO: 6; or a polypeptide with an amino acid sequence having more than 90% sequence identity with SEQ ID NO: 6 and having a polypeptide as shown in SEQ ID NO: 6 Functional peptide fragments.
  • the "more than 90% sequence identity” includes: more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, More than 98% or more than 99% sequence identity.
  • an immune-compatible human pluripotent stem cell which (a) does not express free B2M protein but expresses HLA-G1 and secreted HLA-G5; and (b) does not express CIITA protein.
  • the human pluripotent stem cells fuse endogenous B2M genes in the genome with polynucleotides encoding HLA-G1; contain exogenous fusions encoding B2M-HLA-G5 The polynucleotide of the protein; and, the CIITA gene in the genome was knocked out.
  • an application of immune-compatible human pluripotent stem cells for producing cells through induced differentiation.
  • Prepare cells suitable for transplantation preferably, the cells suitable for transplantation are tissue or organ cells; more preferably, the tissue or organ cells include (but are not limited to): cardiovascular precursor cells, cardiomyocytes, Endothelial cells, smooth muscle cells, nerve cells, hematopoietic stem cells, myeloid cells (including granulocytes, monocytes, macrophages, red blood cells, platelets), lymphoid cells (including natural killer cells, T cells, B cells), retina Pigment epithelial cells, pancreatic islet B cells, liver cells (including hepatocytes, bile duct cells, liver endothelial cells, hepatic stellate cells, Kupffer cells, mesothelial cells), keratinocytes, skeletal muscle cells, adipocytes, osteocytes, cartilage cells, mesenchymal stem cells.
  • a method for preparing cells suitable for transplantation including: (a) preparing immune-compatible human pluripotent stem cells by the method of preparing immune-compatible human pluripotent stem cells according to the present invention ; (b) further induce differentiation of the cells of (a) to obtain cells suitable for transplantation; preferably, the cells suitable for transplantation are tissue or organ cells; more preferably, the tissue or organ cells include (But not limited to): cardiovascular precursor cells, cardiomyocytes, endothelial cells, smooth muscle cells, nerve cells, hematopoietic stem cells, myeloid cells (including granulocytes, monocytes, macrophages, red blood cells, platelets), lymphoid cells Cells (including natural killer cells, T cells, B cells), retinal pigment epithelial cells, pancreatic islet B cells, liver cells (including hepatocytes, bile duct cells, liver endothelial cells, hepatic stellate cells, Kupffer cells, me
  • the tissue or organ cells are cardiomyocytes, prepared by a method of inducing CHIR99021 and IWR-1 successively; or the tissue or The organ cells are endothelial cells, which are prepared by inducing CHIR99021, bFGF, and VEGF+BMP4.
  • the CHIR99021, IWR-1, bFGF, VEGF or BMP4 also includes their homofunctional molecules.
  • the CHIR99021 has a final concentration of 6 ⁇ M, and the final concentration can fluctuate within 50%, preferably within 40%, and preferably within 30%. It can float, preferably within 20%, and preferably within 10%.
  • the CHIR99021 treatment is for 2 ⁇ 0.5 days.
  • the IWR-1 has a final concentration of 5 ⁇ M, and the final concentration can fluctuate within 50%, preferably within 40%, and preferably within 30%. It can fluctuate up and down within 20%, preferably within 10%.
  • the IWR-1 treatment is for 2 ⁇ 0.5 days.
  • the bFGF has a final concentration of 50 ng/ml, and the final concentration can fluctuate within 50%, preferably within 40%, and preferably within 30% It can fluctuate up and down within 20%, preferably within 10%.
  • the bFGF treatment is for 1 ⁇ 0.25 days.
  • the VEGF has a final concentration of 50 ng/ml, and the final concentration can fluctuate within 50%, preferably within 40%, and preferably within 30% It can fluctuate up and down within 20%, preferably within 10%.
  • the BMP4 has a final concentration of 50 ng/ml, and the final concentration can fluctuate within 50%, preferably within 40%, and preferably within 30% It can fluctuate up and down within 20%, preferably within 10%.
  • the VEGF+BMP4 treatment is 2 ⁇ 0.5 days.
  • a cardiomyocyte or endothelial cell is provided, the immune-compatible human pluripotent stem cells prepared by the method of preparing immune-compatible human pluripotent stem cells of the present invention or the immune-compatible human pluripotent stem cells of the present invention. derived from human pluripotent stem cells.
  • Figure 2 Construction and acquisition strategy of immune-compatible cells.
  • A. Design gRNA at the Exon2 position of B2M and the Exon3 position of CIITA, co-transfect the gRNA expression plasmid and Cas9 expression plasmid into hPSCs, then select single clones and perform PCR sequencing verification.
  • the clones with frameshift mutations are B2M and CIITA double knockout clones.
  • B, B2Mm/sHLAG hPSCs were first constructed (see CN113528448A), and then gRNA and Cas9 plasmid were co-transfected. Single clones were selected and verified by PCR sequencing. Clones with frameshift mutations were CIITA -/- B2Mm/sHLAG-hPSCs.
  • FIG. 1 Immunofluorescence identification of the expression of pluripotency markers OCT4 and SOX2 in wt-hPSCs, DKO-hPSCs and CIITA -/- B2Mm/sHLAG-hPSCs cells. Scale bar: 20 ⁇ m.
  • FIG. 5 Molecular expression identification of HLA family.
  • A Flow cytometry identifies the expression of HLA I representative molecules HLA-A/B/C.
  • B Flow cytometry identification of HLA-G1 molecular expression.
  • D Western blot identifies expression of secreted HLA-G5 in cell supernatants. *p ⁇ 0.05.
  • FIG. 6 Identification of differentiated endothelial cells and cardiomyocytes.
  • FIG. 7 Evaluation of the recognition and killing effect of immune cells on cardiomyocytes derived from CIITA -/- B2Mm/sHLAG-hPSCs.
  • FIG. 8 Evaluation of cell retention after transplantation of hPSCs-derived cardiomyocytes in humanized mice after myocardial infarction.
  • A Study schematic.
  • B Immunofluorescence image of heart sections on day 28 after transplantation.
  • ⁇ -ACTININ myofilament structural protein.
  • FIG. 9 Evaluation of cardiac remuscularization in Hu-mice myocardial infarction.
  • FIG. 10 Other construction methods of immune-compatible human induced pluripotent stem cells (iPSC-IC). A, inserting foreign genes into the genome safe harbor site (AAVS1 site); B, using the transposon system to integrate foreign genes into the genome; C, two construction methods of expression cassettes.
  • iPSC-IC immune-compatible human induced pluripotent stem cells
  • FIG. 11 Detection of exogenous gene expression in human iPSCs monoclonal cell-derived cardiomyocytes constructed using a strategy of inserting exogenous genes into the genome using non-viral vectors.
  • A Flow cytometry was used to detect the expression level of HLAG1 on the cardiomyocyte membrane differentiated from each line. The right side is the statistical average fluorescence intensity;
  • B Western blot was used to detect HLAG5 secreted by cardiomyocytes differentiated from each line;
  • C Enzyme-linked immunoassay Adsorption and detection of HLAG5 secreted by cardiomyocytes differentiated from various lines.
  • B2Mm/sHLAG hESCs work better when they are mainly used in cell therapy for degenerative diseases. But when it is in some other transplant environments, there is still activation of immune cells (including antigen-presenting cells). For example, when the inventors applied it to cell therapy for acute injury, such as infarction, the level of inflammation in the injured part of the heart is high. Clinically, transplanted exogenous cells need to be implanted into the injury or adjacent to the injury site to play a therapeutic role. Injured tissue is often accompanied by excessive activation of the inflammatory response at the lesion, resulting in higher levels of inflammatory factors in tissue cells at the lesion.
  • the immune rejection response to such cells is enhanced.
  • the inventors identified targets for further modification. Based on this, the present invention also reveals a further modified immune-compatible cell, which has a wider application environment.
  • Additional exemplary stem cell lines include those available through the National Institutes of Health Human Embryonic Stem Cell Registry and the Howard Hughes Medical Institute HUES collection. Those obtained (as described in Cowan, CA et al., New England J. Med. 350: 13. (2004)).
  • pluripotent stem cells have the potential to differentiate into any of the following three germ layers: endoderm (e.g., gastric junction, gastrointestinal tract, lung, etc.), mesoderm (e.g., muscle, bone, blood, urogenital tissue) etc.) or ectoderm (such as epidermal tissue and nervous system tissue).
  • endoderm e.g., gastric junction, gastrointestinal tract, lung, etc.
  • mesoderm e.g., muscle, bone, blood, urogenital tissue
  • ectoderm such as epidermal tissue and nervous system tissue.
  • the term “pluripotent stem cells” as used herein also includes “induced pluripotent stem cells,” “iPS,” “iPSCs” or “iPSCs,” a type of pluripotent stem cell derived from non-pluripotent cells.
  • parental cells include somatic cells that have been reprogrammed by various means to induce a pluripotent, undifferentiated phenotype.
  • Such "iPS”, “iPSC” or “iPSCs” cells can be generated by inducing the expression of certain regulatory genes or by the exogenous application of certain proteins. Methods of inducing iPS cells are known in the art.
  • hiPSC”, “hiPSCs”, “hPSC” or “hPSCs” are human induced pluripotent stem cells
  • miPSC”, “miPSCs”, “mPSC” or “mPSCs” are murine induced pluripotent stem cells.
  • pluripotent stem cell characteristics refers to cellular characteristics that distinguish pluripotent stem cells from other cells.
  • the ability to produce progeny that can differentiate under appropriate conditions into cell types that collectively display characteristics associated with cell lineages from all three germinal layers (endoderm, mesoderm, and ectoderm) is a pluripotent stem cell characteristic.
  • the expression or non-expression of certain combinations of molecular markers is also characteristic of pluripotent stem cells.
  • human pluripotent stem cells express at least several, and in some embodiments all, markers from the following non-limiting list: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2 -49/6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rex1 and Nanog.
  • Cell morphologies associated with pluripotent stem cells are also characteristic of pluripotent stem cells. As described herein, cells do not need to pass pluripotency to be reprogrammed into endodermal progenitors and/or hepatocytes.
  • multipotent or “multipotent cell” refers to a cell type that can give rise to a limited number of other specific cell types. For example, induced pluripotent cells can form endoderm cells.
  • pluripotent blood stem cells can differentiate themselves into several types of blood cells, including lymphocytes, monocytes, neutrophils, etc.
  • oligopotent refers to the ability of adult stem cells to differentiate into only a few different cell types. For example, lymphoid or myeloid stem cells can form cells of the lymphoid or myeloid lineage, respectively.
  • unipotent refers to the ability of a cell to form a single cell type. For example, spermatogonial stem cells can only form sperm cells.
  • non-pluripotent cell refers to a mammalian cell that is not a pluripotent cell.
  • Examples of such cells include differentiated cells as well as progenitor cells.
  • Examples of differentiated cells include, but are not limited to, cells from tissues selected from bone marrow, skin, skeletal muscle, adipose tissue, and peripheral blood.
  • Exemplary cell types include, but are not limited to, fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T cells.
  • Starting cells for generating induced pluripotent cells, endodermal progenitor cells and hepatocytes can be non-pluripotent cells.
  • Differentiated cells include, but are not limited to, pluripotent cells, oligopotent cells, unipotent cells, progenitor cells and terminally differentiated cells. In certain embodiments, lower energy cells are considered “differentiated" relative to more energy cells.
  • the term "somatic cell” refers to the cells that form an organism. Somatic cells include cells that make up the organs, skin, blood, bones, and connective tissues of an organism, but do not include reproductive cells.
  • the cells may be from, for example, humans or non-human mammals. Exemplary non-human mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, cattle, and non-human primates.
  • the cells are from an adult human or non-human mammal. In some embodiments, the cells are from neonates, adults, or non-human mammals.
  • the term "subject” or “patient” refers to any animal, such as a domestic animal, a zoo animal, or a human.
  • a “subject” or “patient” may be a mammal, such as a dog, cat, bird, livestock, or a human.
  • "subject” and “patient” Specific examples of “persons” include, but are not limited to, individuals (especially humans) having diseases or conditions associated with the liver, heart, lungs, kidneys, pancreas, brain, nervous tissue, blood, bones, bone marrow, etc.
  • Mammalian cells may be derived from Human or non-human mammals.
  • non-human mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, cattle and non-human primates (e.g. , chimpanzees, macaques and apes).
  • HLA human leukocyte antigen
  • MHC human major histocompatibility complex
  • HLA I consists of at least three proteins, HLA-A, HLA-B and HLA-C, which present peptides from inside the cell.
  • HLA II includes at least five proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ and HLA-DR, which present antigens from extracellular sources to T lymphocytes.
  • MHC human or mouse (MHC) origin. Therefore, these terms are used interchangeably herein when referring to mammalian cells.
  • gene knockout refers to the process of rendering a specific gene inactive in the host cell in which it is found, resulting in no production or an inactive form of the protein of interest. As understood by those skilled in the art and described further below, this can be achieved in a number of different ways, including removing all or part of the nucleic acid sequence from the gene, or interrupting the sequence with other sequences, removing or altering regulatory components (e.g. promoter ) prevents genes from being transcribed, changes the reading frame, prevents translation by binding to mRNA, or changes the regulatory components of nucleic acids, etc.
  • regulatory components e.g. promoter
  • all or part of the coding region of the target gene can be removed or replaced with "nonsense" sequences, all or part of the regulatory sequences (such as promoters) can be removed or replaced, the translation initiation sequence can be removed or replaced, etc.
  • knockouts are performed at the level of genomic DNA, so that future generations of cells also permanently carry the knockout.
  • knock-in refers to the process of adding genetic functionality to a host cell. This results in increased levels of the encoded protein. As understood by those skilled in the art, this can be accomplished in several ways, including adding one or more additional gene copies to the host cell or altering the regulatory components of the endogenous gene, thereby increasing protein expression. This can be accomplished by modifying the promoter, adding a different promoter, adding enhancers, or modifying other gene expression sequences. Typically, knock-in techniques result in the integration of additional copies of the transgene into the host cell.
  • ⁇ -2 microglobulin or " ⁇ 2M” or “B2M” protein may refer to having the amino acid sequence shown in SEQ ID NO: 2 below, or having more than 90% of the amino acid sequence shown in SEQ ID NO: 2 Human ⁇ 2M protein with sequence identity and similar biological activity.
  • CIITA protein may refer to humans having the amino acid sequence shown in SEQ ID NO: 5 below, or having more than 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 5 and having similar biological activities. CIITA protein.
  • wild type refers to cells found in nature.
  • pluripotent stem cells as used herein, it also means iPSCs that may contain nucleic acid changes that lead to pluripotency but do not undergo the gene editing procedures of the invention to achieve low immunogenicity.
  • allogeneic refers to the genetic differences between the host organism and the transplanted cells in which the immune response is generated.
  • B2M -/- in this article means that diploid cells have inactive B2M genes in both chromosomes, which can be done in various ways.
  • CIITA -/- in this article refers to diploid cells with inactive CIITA genes in both chromosomes. This can be done in various ways, as described in this article.
  • m/sHLAG is an abbreviation for membrane (m) localized HLA-G1 and soluble (s) HLA-G5.
  • B2Mm/sHLAG hPSCs cell lines that overexpress both membrane-localized HLA-G1 and soluble HLA-G5.
  • no expression is a relative term, and also encompasses “low expression” and “extremely low expression”. For example, compared with the expression level in the wild type, the expression of the modified cells is reduced to that of the wild type. Type 15% or less, 10% or less, 8% or less, 5% or less, 3% or less, 2% or less, 1% or less or less.
  • the term “comprising” means that the various ingredients can be used together in the mixture or composition of the present invention. Therefore, the terms “consisting essentially of” and “consisting of” are included in the term “comprising”.
  • the term “effective amount” or “effective dose” refers to an amount that produces function or activity in a subject and is acceptable to humans and/or animals.
  • a "pharmaceutically acceptable” ingredient is one that is suitable for administration to a subject without undue adverse side effects (such as toxicity, irritation, and allergic reactions), that is, a substance that has a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents.
  • Every maximum numerical limitation given throughout this specification will include every lower numerical limitation as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation as if such higher numerical limitation were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were expressly written herein.
  • the present invention provides human pluripotent stem cells that avoid host immune responses due to the genetic manipulation of the present invention.
  • the genome of the human pluripotent stem cells has been modified to: not express free B2M protein, not express CIITA protein, but express (exogenous) HLA-G1 and secreted HLA-G5.
  • human pluripotent stem cells constructed through the construction method of the present invention are also within the protection scope of the present invention.
  • the human pluripotent stem cells allow the derivation of "off-the-shelf" cell products for the generation of specific tissues and organs. Being able to use derivatives of the human pluripotent stem cells in human patients yields significant benefits, including the ability to avoid long-term adjuvant immunosuppressive treatments and drug use typically seen in transplantation, for example, cardiomyocytes derived from the cells can It avoids recognition and killing by human immune cells and has better retention effect in cardiac ischemia/reperfusion injury model. It also offers significant cost savings because cell therapy can be used without the need for individual treatments for each patient. Therefore, the human pluripotent stem cells are immunocompatible (avoid host immune response), can be used in a wider patient population, and can serve as a universal cell source to generate generally accepted derivatives.
  • the present invention provides human pluripotent stem cells that avoid host immune responses due to several genetic manipulations described in the present invention.
  • the cells lack the major immune antigens that trigger immune responses and are engineered to evade recognition and killing by immune cells.
  • the human pluripotent stem cells of the present invention do not express free B2M protein and CIITA protein, but, as a preferred method, they can successfully express secreted B2M-HLA-G5 fusion protein.
  • the human pluripotent stem cells of the present invention are realized by integrating recombinant nucleic acids into the genome of the human pluripotent stem cells.
  • Exemplary techniques for genetic manipulation include homologous recombination, knock-in, ZFN (zinc finger nuclease), TALEN (transcription activator-like effector nuclease), CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9, and others. Site-specific nuclease technology. There are a number of CRISPR/Cas9 based technologies, see for example Doudna and Charpentier, Science doi: 10.1126/science.1258096, which is incorporated herein by reference. These techniques enable double-stranded DNA breaks at desired locus sites. These controlled double-strand breaks promote homologous recombination at specific loci.
  • This process focuses on targeting specific sequences of nucleic acid molecules, such as chromosomes, with endonucleases, which recognize and bind the sequences and induce double-stranded breaks in the nucleic acid molecules. Double-strand breaks are repaired by error-prone nonhomologous end joining (NHEJ) or by homologous recombination (HR).
  • NHEJ nonhomologous end joining
  • HR homologous recombination
  • pluripotent stem cells of the invention many different techniques can be used to engineer the pluripotent stem cells of the invention, as well as to render human pluripotent stem cells deficient in major immune antigens that trigger an immune response and engineered to evade immunity as described herein Cell recognition and killing.
  • CRISPR can be used to reduce the expression of active B2M and/or CIITA proteins in the modified cells, and use viral technology (such as lentivirus) to achieve stable gene transduction.
  • viral technology such as lentivirus
  • immune-compatible human pluripotent stem cells constructed by lentiviral infection are used.
  • lentiviral vectors usually require steps such as plasmid extraction, virus packaging and quality control, and are randomly integrated into the genome.
  • non-viral plasmid transfection construction methods are used to integrate the target gene into the target cell genome using site-directed knock-in or transposon systems (such as Piggybac, Sleeping beauty, etc.).
  • site-directed knock-in or transposon systems such as Piggybac, Sleeping beauty, etc.
  • a variety of methods can be used to screen monoclonal cell lines, such as drug screening, PCR screening, flow cytometry screening, ELISA screening, and immunoblotting.
  • a recombinant nucleic acid encoding a desired polypeptide such as a B2M-HLA-G1 fusion protein or a CIITA protein
  • a desired polypeptide such as a B2M-HLA-G1 fusion protein or a CIITA protein
  • the regulatory nucleotide sequence is generally suitable for the host cell and the subject to be treated.
  • suitable expression vectors and suitable regulatory sequences are known in the art for use in a variety of host cells.
  • the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancers or activation subsequence. Constitutive or inducible promoters known in the art are also contemplated.
  • the promoter may be a naturally occurring promoter, or a hybrid promoter combining elements of more than one promoter.
  • the expression construct may be present in the cell on an episome (eg, a plasmid), or the expression construct may be inserted into the chromosome.
  • the expression vector includes a selectable marker gene to allow selection of transformed host cells.
  • expression vectors comprising a nucleotide sequence encoding a variant polypeptide operably linked to at least one regulatory sequence. Regulatory sequences as used herein include promoters, enhancers and other expression control elements. In certain embodiments, expression vectors are designed for selection of the host cell to be transformed, the specific variant polypeptide desired to be expressed, the copy number of the vector, the ability to control the copy number, or any other protein encoded by the vector such as an antibiotic marker expression.
  • the present invention also provides cardiomyocytes derived from the human pluripotent stem cells, which are then transplanted into patients in need, thereby avoiding recognition and killing by human immune cells, and the derived cardiomyocytes are also provided. Use of cells in the preparation of compositions or medicaments for the treatment of heart-related diseases or conditions.
  • the invention also provides a human pluripotent stem cell that has good immune compatibility (avoidance of host immune response) due to several genetic manipulations described in the invention.
  • the cells lack the major immune antigens that trigger immune responses and are engineered to be more immune compatible (avoid host immune responses).
  • human pluripotent stem cells constructed through the construction method of the present invention are also within the protection scope of the present invention.
  • cardiomyocytes derived from the human pluripotent stem cells and then transplant them into patients in need, thereby avoiding recognition and killing by human immune cells, and also provide the preparation of the derived cardiomyocytes for treatment Use in compositions or medicines for heart-related diseases or disorders.
  • the heart-related diseases or disorders are myocardial injury, myocardial infarction, myocardial ischemia, ischemia-reperfusion injury or other cardiac injuries; more preferably , the heart-related disease or condition is ischemia/reperfusion injury.
  • the present invention provides the application of cardiomyocytes derived from human pluripotent stem cells in compositions or medicines that promote myocardial repair after myocardial infarction, improve cardiac function after myocardial infarction, and protect myocardial ischemic damage.
  • the present invention provides cardiomyocytes derived from the human pluripotent stem cells, which are then transplanted into patients in need, thereby avoiding recognition and killing by human immune cells, and also provides the human pluripotent stem cells.
  • the heart-related disease or disorder is myocardial injury, myocardial infarction, myocardial ischemia, ischemia-reperfusion injury or other cardiac injuries.
  • the heart-related disease or disorder is ischemia/reperfusion injury.
  • the present invention provides the use of the human pluripotent stem cell-derived cardiomyocytes in the preparation of compositions or medicines for promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction, and protecting myocardial ischemic damage. use.
  • the present invention provides the cardiomyocytes derived from human pluripotent stem cells for the treatment of heart-related diseases or conditions. For example, it is used to treat myocardial injury, myocardial infarction, myocardial ischemia, ischemia-reperfusion injury and other diseases or conditions.
  • the present invention provides the human pluripotent stem cell-derived cardiomyocytes for improving myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction, and protecting myocardial ischemic damage.
  • the present invention also provides a composition, which contains an effective amount of the derived cardiomyocytes and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • pharmaceutical preparations should match the mode of administration.
  • the pharmaceutical composition of the present invention can be made into an injection form, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition is preferably manufactured under sterile conditions.
  • the active ingredients are administered in amounts that are therapeutically effective.
  • composition of the present invention can be directly used to promote myocardial repair after myocardial infarction, improve cardiac function after myocardial infarction, and protect myocardial ischemic damage.
  • it can also be used in combination with other therapeutic agents or adjuvants.
  • these materials may be formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, typically at a pH of about 5-8, preferably at a pH of about 6-8.
  • the effective amount of the derived cardiomyocytes of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (eg, through clinical trials). The factors include but are not limited to: pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc.
  • the administration mode of the derived cardiomyocytes of the present invention is not particularly limited and can be systemic or local.
  • the derived cardiomyocytes of the present invention can be administered by local tissue injection, preferably myocardial injection.
  • other methods of injection are also possible, such as, but not limited to, intraperitoneal injection, intravenous injection, oral administration, subcutaneous injection, spinal intrathecal injection, intradermal injection, etc. administered to the subject.
  • the invention also provides a method for constructing the human pluripotent stem cells, which includes: modifying the human pluripotent stem cells so that they: do not express free B2M protein, express HLA-G1 and secreted HLA-G5; and do not express CIITA protein.
  • the coding gene of the HLA-G1 fragment may include the coding sequence of the heavy chain reading frame of HLA-G1, and the HLA-G1 fragment may include:
  • HLA-G1 heavy chain sequence HLA-G1 fragment
  • the amino acid sequence in b) specifically refers to: the amino acid sequence shown in SEQ ID NO: 1 through substitution, deletion or addition of one or more (specifically, it can be 1-50, 1-30, 1- 20 pieces, 1-10 pieces, 1-5 pieces, 1-3 pieces, 1 piece, 2 pieces or 3) amino acids, or add one or more (specifically, 1-50, 1-30, 1-20, 1-10) amino acids at the N-terminus and/or C-terminus , 1-5, 1-3, 1, 2, or 3) amino acids, and has the function of the polypeptide fragment whose amino acids are as shown in SEQ ID NO: 1.
  • the HLA-G1 fragment usually has a complete alpha heavy chain structure, which is different from the classic HLA I molecule.
  • the amino acid sequence in b) may have an identity of more than 90%, 93%, 95%, 97%, or 99% with SEQ ID NO: 1.
  • the HLA-G1 fragment is usually of human origin.
  • the first B2M fragment may include: c) a polypeptide fragment with an amino acid sequence as shown in SEQ ID NO: 2; or, d) an amino acid sequence that has more than 90% similarity with SEQ ID NO: 2 A polypeptide fragment having sequence identity and having the functions of the polypeptide fragment defined in c).
  • the amino acid sequence in d) specifically refers to: the amino acid sequence shown in SEQ ID NO: 2 through substitution, deletion or addition of one or more (specifically, it can be 1-50, 1-30, 1- 20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or one or more ( Specifically, it can be obtained from 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, and has
  • the amino acid is a functional polypeptide fragment of the polypeptide fragment shown in SEQ ID NO: 2.
  • the first B2M fragment usually has a ⁇ -pleated sheet structure, which mainly has the function of binding to the major histocompatibility complex class I heavy chain through non-covalent bonds.
  • the amino acid sequence in d) may have more than 90%, 93%, 95%, 97%, or 99% identity with SEQ ID NO: 2.
  • the first B2M fragment is typically of human origin.
  • the amino acid sequence of the first flexible connecting peptide segment may include (GS) n , (GGS) n , (GGSG) n , (GGGS) n A, (GGGGS) n A, (GGGGA) n A, (GGGGGG) n A and other sequences, where n is selected from an integer between 1 and 10.
  • the length of the amino acid sequence of the first flexible connecting peptide segment may be 5-26.
  • the first flexible connecting peptide segment may include a polypeptide fragment with an amino acid sequence as shown in SEQ ID NO: 3.
  • the B2M-HLA-G1 fusion protein includes the first B2M fragment and the HLA-G1 fragment in sequence from the N-terminus to the C-terminus, and the B2M-HLA-G1 fusion protein includes an amino acid sequence such as The polypeptide fragment shown in SEQ ID NO: 4.
  • Nucleic acid sequence encoding B2M-HLA-G1 fusion protein in which the exon DNA sequence of the endogenous B2M gene is bolded and the intron DNA sequence is underlined:
  • the method of integrating exogenous nucleic acid encoding B2M-HLA-G1 fusion protein into the genome of human pluripotent stem cells may specifically include: integrating the encoding gene of the HLA-G1 fragment with the human pluripotent stem cell genome. Endogenous B2M gene fusion.
  • the stop codon located in exon 3 of the endogenous B2M gene can be replaced by the gene encoding the HLA-G1 fragment.
  • the amino acid sequence in f) specifically refers to: the amino acid sequence shown in SEQ ID NO: 5 through substitution, deletion or addition of one or more (specifically, it can be 1-50, 1-30, 1- 20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or one or more ( Specifically, it can be obtained from 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, and has
  • the amino acid is a functional polypeptide fragment of the polypeptide fragment shown in SEQ ID NO: 5.
  • the CIITA fragment usually has a bi- or multifunctional domain, which mainly acts as a transcriptional activator and plays a key role in the expression of major histocompatibility complex (MHC) class II genes.
  • MHC major histocompatibility complex
  • the amino acid sequence in f) may have more than 90%, 93%, 95%, 97%, or 99% identity with SEQ ID NO: 5.
  • the CIITA fragment is usually of human origin.
  • the method of not expressing CIITA may specifically include: knocking out exon 3 of the CIITA gene in the human pluripotent stem cell genome; preferably, knocking out by a gene editing method; More preferably, the gRNA (GGGAGGCTTATGCCAATAT) with the nucleotide sequence shown in SEQ ID NO: 11 is used for gene editing.
  • the HLA-G5 fragment includes: i) a polypeptide fragment with an amino acid sequence as shown in SEQ ID NO: 6; or, j) an amino acid sequence with more than 90% of the sequence as SEQ ID NO: 6 A polypeptide fragment that is identical and has the functions of the polypeptide fragment defined in i).
  • the amino acid sequence in j) specifically refers to: the amino acid sequence shown in SEQ ID NO: 6 through substitution, deletion or addition of one or more (specifically, it can be 1-50, 1-30, 1- 20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or one or more ( Specifically, it can be obtained from 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, and has The amino acid is a functional polypeptide fragment of the polypeptide fragment shown in SEQ ID NO: 6.
  • the HLA-G5 fragment usually has a complete extracellular structure of the ⁇ heavy chain, which is different from the classic HLA I molecule and mainly has immunosuppressive functions (for example, it can bind to inhibitory receptors and thereby regulate B cells , T cells, NK cells and APC cell-mediated immune responses, etc.
  • These inhibitory receptors mainly include ILT2/CD85j/LILRB1, ILT4/CD85d/LILRB2, and KIR2DL4/CD158d, etc.).
  • the amino acid sequence in j) may have an identity (Sequence identity) of more than 90%, 93%, 95%, 97%, or 99% with SEQ ID NO: 6.
  • the HLA-G5 fragment is usually of human origin.
  • the B2M-HLA-G5 fusion protein may also include a second flexible connecting peptide segment, and the second flexible connecting peptide segment is usually located between the HLA-G5 fragment and the second B2M fragment.
  • the second flexible connecting peptide segment can usually be a flexible polypeptide of appropriate length consisting of glycine (G), serine (S) and/or alanine (A), so that adjacent protein domains can be relative to each other. freely move with each other.
  • the amino acid sequence of the second flexible connecting peptide segment may include (GS)n, (GGS)n, (GGSG)n, (GGGS)nA, (GGGGS)nA, (GGGGA)nA, Sequences such as (GGGGGG)nA, where n is selected from an integer between 1 and 10.
  • the length of the amino acid sequence of the second flexible connecting peptide segment may be 5-26.
  • the second flexible connecting peptide segment may include a polypeptide fragment with an amino acid sequence as shown in SEQ ID NO: 5.
  • the B2M-HLA-G5 fusion protein includes the second B2M fragment and the HLA-G5 fragment in sequence from the N-terminus to the C-terminus.
  • the B2M-HLA-G5 fusion protein It includes a polypeptide fragment with an amino acid sequence as shown in SEQ ID NO: 8.
  • the method of integrating exogenous nucleic acid encoding B2M-HLA-G5 fusion protein into the genome of human pluripotent stem cells can specifically include: integrating the encoding B2M-HLA-G5 fusion protein through a lentiviral vector.
  • the nucleic acid is integrated into the genome of human pluripotent stem cells, allowing them to express B2M-HLA-G5 fusion protein.
  • the construction method of human pluripotent stem cells can successfully integrate the HLA-G1 fragment sequence into two endogenous B2M sites of human pluripotent stem cells, knock out the CIITA fragment sequence, and encode the human pluripotent stem cell through a lentiviral vector.
  • the sequence of the B2M-HLA-G5 fusion protein is integrated into the genome of human pluripotent stem cells.
  • the endogenous HLA-A, -B, and -C proteins of the constructed human pluripotent stem cell line cannot reach the cell membrane surface.
  • CIITA was also knocked out to obtain pluripotent stem cells with excellent immune compatibility and unaffected pluripotency and proliferation ability; further, the pluripotent stem cells can be used as a source of target cells for cell transplantation.
  • interfering RNA molecules can be prepared according to the CIITA or B2M sequence information provided in the present invention.
  • the interfering RNA can be delivered into cells by using appropriate transfection reagents, or various methods known in the art can be used. Technology is delivered into cells.
  • siRNAs are typically approximately 21 nucleotides in length (eg, 21-23 nucleotides).
  • the hPSCs used are derived from human embryonic stem cells and are H9 strains.
  • B2M/CIITA double knockout hPSCs were constructed as controls.
  • gene editing methods CRISPR/Cas9 system is used in this example
  • the B2M and CIITA genes are knocked out simultaneously.
  • the inventor optimized the sequence suitable for the gene editing position, and based on this, designed the gRNA targeting the genomic position.
  • the gRNA sequence is shown in Table 1.
  • the gRNA was constructed into the plasmid initiated by the U6 promoter respectively, and the gRNA expression plasmids gRNA-B2M and gRNA-CIITA were obtained.
  • DKO-hPSCs the gRNA-B2M plasmid and Cas9-T2A-GFP (Addgene#44719) plasmid are first transferred into hPSCs. After 4 days of culture, low-density single-cell plating is performed. After single clone growth, the genomic DNA is manually picked and analyzed. PCR and sequencing analysis were performed to select clones with non-three-fold base deletions or insertions as B2M knockout hPSCs.
  • CIITA knockout hPSCs were further constructed using the same method as above, but the gRNA plasmid was replaced with gRNA-CIITA plasmid, and CIITA knockout single clones were further selected.
  • Knockout hPSCs were constructed by the above method. Clones with non-3-fold base frameshift mutations were obtained as B2M/CIITA double knockout hPSCs (DKO-hPSCs) ( Figure 2A).
  • the method of constructing CIITA -/- B2Mm/sHLAG-hPSCs is to use the gene editing method (CRISPR/Cas9 in this example) to knock out the CIITA gene based on the cells constructed in the patent application CN113528448A, specifically (Figure 1):
  • the CRISPR/Cas9 gene editing method was used to insert the HLA-G1 coding gene (recombinant expression of B2M-HLA-G1 fusion protein) before the stop codon of the B2M gene coding frame, which encodes a flexible (G4S) 4-linked peptide segment.
  • the constructed cell line only expresses the HLA-G1/B2M complex.
  • Other HLA I family molecules cannot be expressed normally on the cell membrane due to the lack of B2M subunits.
  • B2M-(G4S)4-HLA-G5 coding frame into the above-mentioned cell genome: combine the B2M CDS sequence without stop codon (SEQ ID NO: 2), (Gly4Ser)4 flexible linker peptide coding sequence and HLA -The G5 heavy chain coding sequence was integrated into BamHI and MluI of the puromycin-resistant lentiviral vector pLVX-CAG-Puro, and the obtained recombinant lentiviral vector was introduced into the cells to obtain B2Mm/sHLAG hPSCs (see CN113528448A Corresponding explanation).
  • the CIITA gene was knocked out using a gene editing method (the CRISPR/Cas9 system was used in this example), and the gRNA-CIITA plasmid (the gRNA sequence is shown in Table 1) and Cas9-T2A-GFP plasmid were transfected through LONZA nucleofection, and the Single clones were taken for DNA PCR sequencing, and clones with non-3-fold base frameshift mutations were selected to obtain CIITA-knockout B2Mm/sHLAG hPSCs and construct immune-compatible hPSCs, named CIITA -/- B2Mm/sHLAG-hPSCs ( Figure 2B).
  • the cells can be further differentiated into immune-compatible functional cells such as cardiomyocytes, endothelial cells, neural cells, NK cells, etc. using tissue-specific cell lines.
  • immune-compatible functional cells such as cardiomyocytes, endothelial cells, neural cells, NK cells, etc. using tissue-specific cell lines.
  • wild-type hPSCs wt-hPSCs
  • Flow cytometry and immunofluorescence were used to identify the expression of pluripotency markers in wt-hPSCs, DKO-hPSCs and CIITA -/- B2Mm/sHLAG-hPSCs.
  • the cultured wt-hPSCs, DKO-hPSCs and CIITA-/-B2Mm/sHLAG-hPSCs were grown to 70% confluence, digested with Accutase into single cells, collected by centrifugation, fixed, ruptured, and used with hPSCs pluripotent cell markers OCT4 and The cells were incubated with SSEA4 antibodies, then stained with APC fluorophore-conjugated secondary antibodies, and analyzed by flow cytometry. Isotype control antibodies were used in the control group. The test results are shown in Figure 3A-B.
  • the cultured wt-hPSCs, DKO-hPSCs and CIITA-/-B2Mm/sHLAG-hPSCs clones were grown to a suitable size, the culture medium was aspirated, fixed, ruptured and incubated with pluripotent cell markers OCT4 and SOX2 antibodies, and then used Alexa 488 fluorophore-coupled secondary antibody staining, and image collection using laser confocal microscopy.
  • the test results are shown in Figure 4.
  • the inventors In order to identify whether the cells constructed using the above method express HLA molecules, the inventors first used flow cytometry for identification.
  • Interferon gamma IFN- ⁇ was added to the culture system of wt-hPSCs, DKO-hPSCs and CIITA -/- B2Mm/sHLAG-hPSCs at a final concentration of 100ng/ml. After 48 hours of culture, Accutase was used to digest them into single cells. , cells were collected by centrifugation for detection of HLA molecule expression. Since there are many members of the HLA I and HLA II families, the representative HLA-A, -B, -C (representatives of the HLA I family) and HLA-DR (representatives of the HLA II family) are used to characterize wt-hPSCs and DKO-hPSCs.
  • IFN- ⁇ Interferon gamma
  • HLA-A/B/C antibodies are coupled to FITC fluorophore
  • HLA-DR antibodies are coupled to PE fluorophore.
  • Single cells were collected and directly incubated with corresponding antibodies, and flow cytometry was used to detect the expression of HLA I and HLA II molecules.
  • HLA-G1 identification also uses the flow cytometry method described above.
  • HLA-A, -B, and -C are members of the classic HLA I family. Comparing the expression of HLA-A, -B, and -C in wt-hPSCs, DKO-hPSCs, and CIITA -/- B2Mm/sHLAG-hPSCs, we found that HLA -A, -B, -C are only expressed in wt-hPSCs, and their expression is not detected in DKO-hPSCs and CIITA -/- B2Mm/sHLAG-hPSCs (Figure 5A); while HLA-G1 has immunosuppressive effects The molecule was expressed only in CIITA ⁇ / ⁇ B2Mm/sHLAG-hPSCs (Fig.
  • hPSCs were treated with interferon gamma (IFN- ⁇ ) for 48 hours, and the expression of HLA-DR was detected by flow cytometry, which showed that the fluorescence signal value in wt was significantly higher than that in DKO-hPSCs and CIITA -/- B2Mm/sHLAG-hPSCs (Figure 5C ).
  • IFN- ⁇ interferon gamma
  • the expression of secreted HLA-G5 molecules was identified using immunoblotting method.
  • wt-hPSCs, DKO-hPSCs and CIITA -/- B2Mm/sHLAG-hPSCs cells were cultured in hPSCs medium to 50% confluence, then replaced with DMEM/F12 basal medium and cultured for 24 hours, and the culture supernatant was collected, 0.25 ⁇ m The cell debris was removed by filtration through a membrane, and the supernatant was concentrated using a 10kd ultrafiltration tube. Before performing immunoblot identification, add loading buffer (containing ⁇ -mercaptoethanol) to a final concentration of 1x, denature the protein in a 95°C water bath for 10 minutes, and use 10% SDS-PAGE gel electrophoresis to separate the protein.
  • the detection antibody is 5A6G7.
  • hPSCs can be obtained from various tissue types through in vitro induction methods.
  • the inventor differentiated hPSCs into endothelial and cardiomyocytes.
  • Differentiated cells endothelial cells, day 6; cardiomyocytes, day 14
  • endothelial cells were collected, digested with Accutase into single cells, and filtered with a 70 ⁇ m filter to remove cell clumps.
  • Endothelial cells were detected by incubating the APC-conjugated CD144 antibody directly with the cells; cardiomyocytes were fixed and ruptured, and the cardiomyocyte marker cTNT antibody was used as the primary antibody, and the APC-conjugated anti-mouse IgG antibody was used as the secondary antibody for detection.
  • wt-hPSCs, DKO-hPSCs and CIITA -/- B2Mm/sHLAG-hPSCs cells grow to 100% confluence, they are replaced with differentiation medium RPMI1640+0.2%BSA+6 ⁇ M CHIR99021 for 2 days, and then replaced with RPMI1640+0.2%BSA +5 ⁇ M IWR-1 for 2 days, then replaced with RPMI1640 + 0.2% BSA every 2 days.
  • Beating cardiomyocytes began to appear on day 7, and were named wt-CMs, DKO-CMs and CIITA-/-B2Mm/sHLAG-hPSCs according to the different types of starting hPSCs.
  • the cell immunofluorescence identification method of cardiomyocytes is the same as the hPSCs pluripotency marker detection method described in Example 3, except that the primary antibody is replaced with the cardiomyocyte marker cTNT antibody and the gap junction Connexin 43 antibody.
  • cardiomyocytes derived from three hPSCs all have a good myofilament structure, and good gap junctions (Connexin-43) are formed between cells (Figure 6B).
  • Immunofluorescence identification of the expression of sarcomeric protein ⁇ -ACTININ and gap protein Connexin-43 in differentiated cardiomyocytes is shown in Figure 6C.
  • immune-compatible cardiomyocytes (CIITA -/- B2Mm/sHLAG-CMs) were obtained through differentiation.
  • CIITA -/- B2Mm/sHLAG-hPSCs and their derived cells have the ability to escape immune cell recognition and killing
  • the inventors used in vitro immune cells (PBMCs or NK92) and CIITA -/- B2Mm/sHLAG-hPSCs-derived myocardium.
  • PBMCs or NK92 vitro immune cells
  • CIITA -/- B2Mm/sHLAG-hPSCs-derived myocardium Cell co-culture method for identification. T cells were tested for activation markers, proliferation, killing effect on target cells, and secreted IFN- ⁇ .
  • NK cells perform target cell recognition and killing detection and secreted IFN- ⁇ detection.
  • T cells are activated by mismatched HLA molecules, so wt-CMs are used as controls and Comparative activation effect of CIITA -/- B2Mm/sHLAG-hPSCs on T cells.
  • Wt-CMs and CIITA -/- B2Mm/sHLAG-hPSCs were spread in 96-well plates, and IFN- ⁇ at a final concentration of 100ng/ml was added for 24 hours and incubated for 48 hours to stimulate the expression of HLA molecules. Afterwards, IFN- ⁇ was removed, and primary peripheral blood mononuclear cells (PBMCs, which are rich in T cells) were added according to the ratio of cardiomyocytes to human peripheral blood mononuclear cells (PMBCs) of 1:3.
  • PBMCs primary peripheral blood mononuclear cells
  • T cell marker CD3 antibody After co-culture for 48 hours, a total of Cultured PBMCs were incubated with T cell marker CD3 antibody and T cell early activation marker CD69 antibody, and then flow cytometry was performed.
  • the T cell activation abilities of wt-CMs and CIITA -/- B2Mm/sHLAG-hPSCs were compared based on the proportion of CD69-positive cells in CD3-positive cells.
  • the preliminary treatment is the same as "T cell activation marker detection”.
  • PBMCs are labeled with CFSE fluorescent label and then co-cultured with cardiomyocytes.
  • the culture time is 7 days.
  • PBMCs are collected, incubated with T cell marker CD3 antibody, and then flowed cell detection.
  • CFSE does not leak from cells in labeled cells but is diluted as cells divide, so the CFSE fluorescence signal decreases in proliferating T cells. Compare wt-CMs and Effect of CIITA -/- B2Mm/sHLAG-hPSCs on T cell proliferation.
  • the preliminary treatment is the same as "T cell activation marker detection”.
  • PBMCs are co-cultured with wt-CMs or CIITA -/- B2Mm/sHLAG-hPSCs for 3-4 days. Then the co-culture supernatant is collected and centrifuged at 300g for 3 minutes to remove the cells and Cell debris was then used to detect the lactate dehydrogenase activity in the supernatant using the Beyotime LDH detection kit to determine the degree of cell damage.
  • the preliminary treatment is the same as "T cell activation marker detection”.
  • PBMCs are co-cultured with wt-CMs or CIITA -/- B2Mm/sHLAG-hPSCs for 3-4 days. Then the co-culture supernatant is collected and centrifuged at 300g for 3 minutes to remove the cells and The cells were fragmented, and then the IFN- ⁇ content in the supernatant was detected using an IFN- ⁇ detection kit.
  • NK cells can recognize cells without HLA molecule expression, so DKO-CMs were used as a control in this example.
  • the preliminary treatment of cells is the same as "NK cell identification and killing detection of target cells”.
  • After co-culture for 3-4 days collect the co-culture supernatant, centrifuge at 300g for 3 minutes to remove cells and cell debris, and then use the IFN- ⁇ detection kit to detect the supernatant IFN- ⁇ content.
  • PBMCs Human PBMCs were co-cultured with cardiomyocytes, and expression of the early T cell activation marker CD69 was identified.
  • the results showed that compared with wt cardiomyocytes (wt-CMs), immune-compatible cardiomyocytes (CIITA -/- B2Mm/sHLAG-CMs) significantly reduced the activation of T cells; and CIITA -/- B2Mm/sHLAG-CMs The effect of cell proliferation was also significantly lower than that of wt-CMs.
  • CIITA -/- B2Mm/sHLAG-CMs used DKO-CMs as a control, co-cultured differentiated cardiomyocytes with NK-92 cells, and found that NK-92 had an effect on CIITA -/-
  • the killing effect of B2Mm/sHLAG-CMs cells was significantly lower than that of DKO-CMs, and the ability of CIITA -/- B2Mm/sHLAG-CMs to stimulate NK-92 to secrete IFN- ⁇ was also significantly lower than that of DKO-CMs, as shown in Figure 7C-F.
  • cardiomyocytes derived from CIITA -/- B2Mm/sHLAG-hPSCs are capable of evading recognition and killing by T cells and NK cells.
  • the inventors used immune system humanized mice (Hu-mice) to construct a heart injury model (this example An ischemia/reperfusion injury model was used in the model), and cardiomyocytes derived from different hPSCs were transplanted into the myocardium after modeling.
  • the specific steps are as follows: At the 10th week of immune reconstitution, the Hu-mice heart, where human immune cells have occupied a considerable proportion in the peripheral blood of mice, was subjected to ligation of the left anterior descending coronary artery and ischemia for 60 minutes, followed by reperfusion to construct myocardial ischemia.
  • I/R Blood/reperfusion
  • mice were randomly divided into I/R group, I/R+wt-CMs group, I/R+DKO-CMs group and I/R+CIITA -/- B2Mm/sHLAG-CMs group; cell transplantation was performed during reperfusion.
  • each mouse was injected with 5x10 5 amounts of wt-CMs, DKO-CMs and CIITA -/- B2Mm/sHLAG-CMs into the myocardium in the border area of myocardial infarction.
  • mice hearts were collected and embedded in OCT.
  • the mouse heart was divided into 12 layers at a certain distance from the apex to the ligation point, and the cells in each layer were frozen and sectioned. Immunofluorescence was then used to identify the transplanted cells, and Masson staining was used to count the proportion of the transplanted cell area in the scar area.
  • Immunofluorescence After frozen sectioning, the tissue was fixed, permeabilized and blocked, and then co-incubated with sarcomeric protein ⁇ -ACTININ antibody and human KU80 antibody (C48E7, CST). Use a fluorescence microscope to collect images and perform statistics.
  • qRT-PCR Hu-mice heart samples were collected 28 days after cell transplantation. RNA was extracted and converted into cDNA. Human cTNT primers were used to identify cell residence through fluorescence quantitative PCR.
  • the inventors used Masson staining to explore the repair effect of transplanted cells on the injured cardiac area in mice.
  • Remuscularization ratio remuscle area/scar area
  • Example 9 Establishment of immune-compatible human induced pluripotent stem cells and their expression using non-viral constructs
  • iPSC-IC immune-compatible human induced pluripotent stem cells
  • human iPSCs with deleted HLA-I and HLA-II molecules i.e., hPSCs (DKO-hPSCs)
  • hPSCs DKO-hPSCs
  • Fig. 10A gene site-directed knock-in
  • Fig. 10B transposon system
  • FIG. 10C The structure of the exogenous inserted fragment is shown in Figure 10C, in which the front and back parts of the gene expression box are connected through the 2A sequence, so that the protein in the front and back parts is broken into two parts during translation.
  • the result is that one expression box can express two independent proteins.
  • two forms of expression cassettes HLAG1-B2M-2A-HLAG5-B2M and HLAG5-B2M-2A-HLAG1-B2M, were designed to screen for optimal expression efficiency. Monoclonal cell lines.
  • HLAG1 and HLAG5 in constructed monoclonal immune-compatible human induced pluripotent stem cell-derived cardiomyocytes (iPSC-IC-CMs)
  • iPSC wild-type human iPSCs
  • iPSC-DKO human iPSCs derived from HLA-I and HLA-II molecules
  • iPSC-IC gene-knocked-in human iPSCs
  • iPSC-IC-AAVS-derived cardiomyocytes and iPSC-IC-Pbx-derived cardiomyocytes significantly higher expressed HLAG1.
  • HLAG1 in cardiomyocytes derived from iPSC monoclones constructed with the Piggybac transposon system and the HLAG5-B2M-2A-HLAG1-B2M structural expression cassette was the highest ( Figure 11A (right)).
  • ELISA and Western blotting were used to detect the secretion level of HLAG5 in the cardiomyocyte culture medium. It was found that monoclonal iPSC-derived cardiomyocytes inserted into the expression cassette of the HLAG5-B2M-2A-HLAG1-B2M structure had higher HLAG5 secretion levels, while those inserted into the HLAG1- No expression of HLAG5 was detected in the culture supernatant of monoclonal iPSC-derived cardiomyocytes containing the expression cassette of the B2M-2A-HLAG5-B2M structure (Fig. 11B).
  • the inserted fragment sequence is as follows:

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Abstract

Provided are an immunocompatible human pluripotent stem cell, a method for preparing same, and use thereof. The modified human pluripotent stem cell can be induced and differentiated into various tissues/organ cells, has very ideal immunocompatibility, and has a wide application environment.

Description

免疫兼容型人多能干细胞、其制备方法及应用Immune-compatible human pluripotent stem cells, preparation methods and applications thereof
本申请要求2022年6月29日提交、发明名称为“免疫兼容型人多能干细胞、其制备方法及应用”、申请号为CN202210752354.6的优先权，其全部内容并入本文。This application claims priority for the invention submitted on June 29, 2022, with the invention title "Immune-compatible human pluripotent stem cells, preparation methods and applications thereof" and application number CN202210752354.6, the entire content of which is incorporated into this article.
技术领域Technical field
本发明属于生物技术领域，更具体地，本发明涉及一种可逃逸移植受体免疫排斥的免疫兼容型人多能干细胞、其制备方法及应用。The present invention belongs to the field of biotechnology. More specifically, the present invention relates to an immune-compatible human pluripotent stem cell that can escape immune rejection of a transplant recipient, its preparation method and application.
背景技术Background technique
细胞移植治疗是把某些具有特定功能的细胞的特性，采用生物工程方法获取和/或通过体外扩增、特殊培养等处理后，产生的特异性功能强大的细胞移植到患者体内，以修复、补充、替换、调节或去除受损或病变细胞/组织，从而达到治愈疾病的目的。因此细胞技术的应用可以治疗各种疾病，如神经***、骨骼疾病、糖尿病、心脑血管疾病等退行性和损伤性疾病等许多传统疗法难治性病变或与传统疗法形成互补，具有广阔的应用前景。然而，移植受体会对非自身细胞产生免疫排斥，使得移植细胞难以长期在移植受体体内存活。即便是接收经过人白细胞抗原(human leukocyte antigen，HLA)匹配的合适供体组织器官，由于无法做到全部HLA的匹配，受体患者依然需长期服用免疫排斥药物，加重患者负担，同时也带来感染/癌症风险。Cell transplantation therapy is the use of bioengineering methods to obtain the characteristics of certain cells with specific functions and/or through in vitro amplification, special culture, etc., to generate cells with specific and powerful functions and transplant them into the patient's body to repair, Replenish, replace, condition or remove damaged or diseased cells/tissues to cure disease. Therefore, the application of cell technology can treat various diseases, such as nervous system, skeletal diseases, diabetes, cardiovascular and cerebrovascular diseases and other degenerative and damaging diseases, and many other diseases that are refractory to traditional therapies or complement traditional therapies and have broad applications. prospect. However, transplant recipients will develop immune rejection of non-self cells, making it difficult for transplanted cells to survive in the recipient's body for a long time. Even if suitable donor tissues and organs are received through human leukocyte antigen (HLA) matching, since all HLA matches cannot be achieved, the recipient patient still needs to take immune rejection drugs for a long time, which increases the burden on the patient and also brings about Infection/cancer risk.
免疫排斥的本质是细胞表面的同种异型抗原诱导的一种免疫应答，在人体中主要由HLA所介导。HLA具有极高的多态性，HLA匹配可降低移植受体对移植细胞的免疫排斥作用。基于免疫排斥机制，现有如下方法减轻免疫排斥：The essence of immune rejection is an immune response induced by allogeneic antigens on the cell surface, which is mainly mediated by HLA in the human body. HLA is extremely polymorphic, and HLA matching can reduce the immune rejection of transplanted cells by transplant recipients. Based on the immune rejection mechanism, the following methods are available to reduce immune rejection:
1、HLA配型。主要用于器官及造血干细胞移植中，通过供体/受体的HLA配型降低排斥作用。然而，由于合适供体的稀少及HLA存在极高的多态性，寻找合适供体难度很大，难以满足器官移植病人的需求的。1. HLA matching. It is mainly used in organ and hematopoietic stem cell transplantation to reduce rejection through donor/recipient HLA matching. However, due to the scarcity of suitable donors and the extremely high polymorphism of HLA, it is very difficult to find suitable donors and meet the needs of organ transplant patients.
2、自体诱导多能干细胞(induced pluripotent stem cells，iPSCs)。iPSCs来源于自身体细胞重编程，具备和供体完全一致的HLA基因，在理论上iPSCs及其衍生细胞也可逃逸自体免疫排斥。然而，自体iPSCs及其衍生细胞制备周期长、质控难、成本高，在时效性强、治疗时间窗口窄的疾病中应用难度较大，使用人群非常有限。其次，对于遗传疾病病人，其iPSCs源细胞存在功能异常，进而带来安全隐患。并且，不少退行性和损伤性疾病好发于高年龄段人群，如心肌梗死、帕金森氏症等，此类人群建立iPSCs难度较高。2. Autologous induced pluripotent stem cells (iPSCs). iPSCs are derived from reprogrammed autologous somatic cells and have the same HLA genes as the donor. In theory, iPSCs and their derived cells can also escape autoimmune rejection. However, autologous iPSCs and their derived cells have a long preparation cycle, difficult quality control, and high cost. They are difficult to apply in diseases that are time-sensitive and have a narrow treatment time window, and the user population is very limited. Secondly, for patients with genetic diseases, the cells derived from iPSCs may have functional abnormalities, which may lead to safety risks. Moreover, many degenerative and damaging diseases tend to occur in older age groups, such as myocardial infarction, Parkinson's disease, etc., and it is more difficult to establish iPSCs in such groups.
3、建立可覆盖大部分人群的HLA配型iPSCs库。由于HLA的极高多态性，建立和维持iPSCs配型库的成本极高，周期很长，短时间内仍难以应用。而且不同iPSCs细胞株来源的组织细胞，如心肌细胞之间也存在差异，使用非均一化的细胞可能对后续治疗产生不利影响。3. Establish an HLA-matched iPSCs library that can cover most of the population. Due to the extremely high polymorphism of HLA, the cost of establishing and maintaining an iPSCs matching library is extremely high, the cycle is long, and it is still difficult to apply it in a short time. Moreover, there are differences between tissue cells derived from different iPSCs cell lines, such as cardiomyocytes, and the use of non-uniformized cells may have adverse effects on subsequent treatments.
以上方法均存在获取难度大、成本高、难以质控的缺点，因此，建立通用型低免疫原性 的供体细胞是本领域的迫切需求。The above methods all have the disadvantages of difficulty in acquisition, high cost, and difficulty in quality control. Therefore, it is necessary to establish a universal low immunogenicity method. Donor cells are an urgent need in this field.
通过对目前已发表及公布的文献和专利的检索，已获得通用型细胞的技术路线有如下几种：(1)通过敲除一条染色体上的关键HLA分子，构建伪HLA纯合子细胞，增加所获细胞的普适性；(2)通过敲除HLA I家族HLA-A，-B，-C和HLA II家族分子，并辅以过表达“don’t eat me”信号CD47分子，膜定位HLAG分子及T细胞抑制因子PD-L1，逃逸免疫细胞的识别与杀伤作用；(3)通过完全敲除HLA I家族和HLA II家族分子，并辅以“don’t eat me”信号CD47分子的过表达，逃逸T细胞和巨噬细胞的识别与杀伤作用；(4)通过敲除介导抗原呈递的经典HLA I分子并过表达具有免疫抑制作用的HLA-G1与分泌型HLA-G5分子，逃逸细胞毒性T细胞及自然杀伤细胞(NK细胞)的识别与杀伤作用，如本发明中共同发明人章小清等申请的国家发明专利申请(CN113528448A)。对比上述4种技术路线，第(1)种不仅难度极高，需要多个HLA分子的单个等位基因进行精确操控，而且增加了基因组发生突变的风险；第(2)种需要引起大范围(大于13kb)的基因组缺失才能达到敲除-A，-B，-C的结果，对基因组带来潜在的不稳定性；第(3)种选择的免疫抑制信号单一，主要作用靶标为巨噬细胞，相比之下HLAG具备广谱免疫抑制作用，可能是更好的选择。不同的免疫抑制因子的作用对象、作用效果均存在差异，因此这种免疫抑制因子的选择是不同技术路线的关键。本发明的共同发明人采用同时过表达膜定位HLA-G1与可溶性HLA-G5构建了B2Mm/sHLAG胚胎干细胞(human embryonic stem cells，hESCs)系，降低了人胚胎干细胞的免疫原性，其中探讨了胚胎干细胞而未涉及hPSCs、且还有待进一步扩展其应用环境。Through a search of published literature and patents, the technical routes for obtaining universal cells include the following: (1) By knocking out a key HLA molecule on a chromosome, pseudo-HLA homozygous cells are constructed to increase the number of all-purpose cells. Obtain cell universality; (2) By knocking out HLA I family HLA-A, -B, -C and HLA II family molecules, supplemented by overexpression of the "don't eat me" signal CD47 molecule, membrane localization of HLAG molecules and the T cell inhibitory factor PD-L1 to escape the recognition and killing effect of immune cells; (3) by completely knocking out HLA I family and HLA II family molecules, supplemented by the "don't eat me" signal CD47 molecule Express, escaping the recognition and killing of T cells and macrophages; (4) By knocking out the classic HLA I molecules that mediate antigen presentation and overexpressing the immunosuppressive HLA-G1 and secretory HLA-G5 molecules, escaping The recognition and killing effect of cytotoxic T cells and natural killer cells (NK cells), such as the national invention patent application (CN113528448A) filed by the co-inventor Zhang Xiaoqing et al. Comparing the above four technical routes, the (1) method is not only extremely difficult, requiring precise control of a single allele of multiple HLA molecules, but also increases the risk of genome mutation; the (2) method requires causing a large range of ( Only genome deletions greater than 13kb can achieve the result of knocking out -A, -B, and -C, which will bring potential instability to the genome; the (3) selection has a single immunosuppressive signal and the main target is macrophages. , in comparison, HLAG has broad-spectrum immunosuppressive effects and may be a better choice. Different immunosuppressive factors have different targets and effects. Therefore, the selection of such immunosuppressive factors is the key to different technical routes. The co-inventors of the present invention constructed the B2Mm/sHLAG embryonic stem cells (hESCs) line by simultaneously overexpressing membrane-localized HLA-G1 and soluble HLA-G5, thereby reducing the immunogenicity of human embryonic stem cells. Among them, they discussed Embryonic stem cells do not involve hPSCs, and their application environment needs to be further expanded.
发明内容Contents of the invention
本发明提供了一种免疫兼容型人多能干细胞、其制备方法及应用，其具有更为广泛的应用。The invention provides an immune-compatible human pluripotent stem cell, its preparation method and application, which has a wider range of applications.
在本发明的第一方面，提供一种制备免疫兼容型人多能干细胞的方法，包括改造人多能干细胞，使之：In a first aspect of the present invention, a method for preparing immune-compatible human pluripotent stem cells is provided, which includes transforming human pluripotent stem cells to:
(a)不表达游离B2M蛋白，表达(包括过表达)HLA-G1与分泌型HLA-G5；以及(b)不表达CIITA蛋白。(a) does not express free B2M protein, expresses (including overexpression) HLA-G1 and secreted HLA-G5; and (b) does not express CIITA protein.
在一种或多种实施方式中，所述(a)中，改造人多能干细胞的基因组，将编码HLA-G1的多核苷酸与人多能干细胞中内源性的B2M基因融合，从而表达B2M-HLA-G1融合蛋白且不表达游离B2M蛋白；较佳地，将HLA-G1的编码基因引入到内源B2M外显子3中终止密码子前的位置，或以HLA-G1的编码基因替换位于内源B2M基因外显子3中的终止密码子；更佳地，通过基因编辑方法进行所述改造。In one or more embodiments, in (a), the genome of human pluripotent stem cells is modified to fuse the polynucleotide encoding HLA-G1 with the endogenous B2M gene in human pluripotent stem cells, thereby expressing B2M-HLA-G1 fusion protein does not express free B2M protein; preferably, the HLA-G1 coding gene is introduced into the endogenous B2M exon 3 at the position before the stop codon, or the HLA-G1 coding gene is Replace the stop codon located in exon 3 of the endogenous B2M gene; more preferably, the transformation is performed by gene editing methods.
在一种或多种实施方式中，所述(a)中，在人多能干细胞中引入外源的编码B2M-HLA-G5融合蛋白的多核苷酸，所述B2M-HLA-G5融合蛋白包括B2M和HLA-G5。In one or more embodiments, in (a), an exogenous polynucleotide encoding a B2M-HLA-G5 fusion protein is introduced into human pluripotent stem cells, and the B2M-HLA-G5 fusion protein includes B2M and HLA-G5.
在一种或多种实施方式中，利用重组载体表达所述B2M-HLA-G1和B2M-HLA-G5；较佳地，所述重组载体包括非病毒载体或病毒载体。In one or more embodiments, the B2M-HLA-G1 and B2M-HLA-G5 are expressed using recombinant vectors; preferably, the recombinant vectors include non-viral vectors or viral vectors.
在一种或多种实施方式中，所述非病毒载体包括：定点敲入***或转座子***。 In one or more embodiments, the non-viral vector includes: a site-directed knock-in system or a transposon system.
在一种或多种实施方式中，所述病毒载体包括：慢病毒载体。In one or more embodiments, the viral vectors include: lentiviral vectors.
在一种或多种实施方式中，所述B2M-HLA-G1融合蛋白中，还包括位于B2M和HLA-G1之间的柔性连接肽，优选如SEQ ID NO：3所示的柔性连接肽；和/或，所述B2M-HLA-G1融合蛋白自N端至C端依次包括：B2M和HLA-G1；较佳地，所述B2M-HLA-G1融合蛋白的氨基酸序列如SEQ ID NO：4所示；更佳地，所述B2M-HLA-G1融合蛋白的核酸序列如SEQ ID NO：9所示。In one or more embodiments, the B2M-HLA-G1 fusion protein also includes a flexible connecting peptide located between B2M and HLA-G1, preferably a flexible connecting peptide as shown in SEQ ID NO: 3; And/or, the B2M-HLA-G1 fusion protein includes: B2M and HLA-G1 in sequence from the N-terminus to the C-terminus; preferably, the amino acid sequence of the B2M-HLA-G1 fusion protein is as SEQ ID NO: 4 as shown; more preferably, the nucleic acid sequence of the B2M-HLA-G1 fusion protein is as shown in SEQ ID NO: 9.
在一种或多种实施方式中，所述B2M-HLA-G5融合蛋白中，还包括位于B2M和HLA-G5之间的柔性连接肽，优选如SEQ ID NO：3所示的柔性连接肽；和/或，所述B2M-HLA-G5融合蛋白自N端至C端依次包括：B2M和HLA-G5；较佳地，所述B2M-HLA-G5融合蛋白的氨基酸序列如SEQ ID NO：8所示；更佳地，所述B2M-HLA-G5融合蛋白的核酸序列如SEQ ID NO：10所示。In one or more embodiments, the B2M-HLA-G5 fusion protein also includes a flexible connecting peptide located between B2M and HLA-G5, preferably a flexible connecting peptide as shown in SEQ ID NO: 3; And/or, the B2M-HLA-G5 fusion protein includes: B2M and HLA-G5 in sequence from the N-terminus to the C-terminus; preferably, the amino acid sequence of the B2M-HLA-G5 fusion protein is as SEQ ID NO: 8 shown; more preferably, the nucleic acid sequence of the B2M-HLA-G5 fusion protein is shown in SEQ ID NO: 10.
在一种或多种实施方式中，所述(b)中，针对人多能干细胞基因组中CIITA基因的外显子3进行敲除；较佳地，通过基因编辑方法进行敲除；更佳地，以SEQ ID NO：11所示核苷酸序列的gRNA(GGGAGGCTTATGCCAATAT)进行基因编辑。In one or more embodiments, in (b), exon 3 of the CIITA gene in the human pluripotent stem cell genome is deleted; preferably, the knockout is performed by gene editing methods; more preferably , use the gRNA (GGGAGGCTTATGCCAATAT) with the nucleotide sequence shown in SEQ ID NO: 11 for gene editing.
在一种或多种实施方式中，所述制备方法包括如下的A-D中的一个或多个：In one or more embodiments, the preparation method includes one or more of the following A-D:
A、所述HLA-G1包括：氨基酸序列如SEQ ID NO：1所示的多肽；或，氨基酸序列与SEQ ID NO：1具有90％以上序列同一性且具有SEQ ID NO：1所示的多肽的功能的衍生多肽(包括活性片段、活性变体)；A. The HLA-G1 includes: a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1; or a polypeptide with an amino acid sequence having more than 90% sequence identity with SEQ ID NO: 1 and having a polypeptide as shown in SEQ ID NO: 1 Functionally derived polypeptides (including active fragments and active variants);
B、所述B2M包括：氨基酸序列如SEQ ID NO：2所示的多肽；或，氨基酸序列与SEQ ID NO：2具有90％以上序列同一性且具有SEQ ID NO：2所示的多肽的功能的衍生多肽(包括活性片段、活性变体)；B. The B2M includes: a polypeptide with an amino acid sequence as shown in SEQ ID NO: 2; or an amino acid sequence with more than 90% sequence identity as SEQ ID NO: 2 and having the function of the polypeptide shown in SEQ ID NO: 2 Derived polypeptides (including active fragments and active variants);
C、所述CIITA蛋白包括：氨基酸序列如SEQ ID NO：5所示的多肽；或，氨基酸序列与SEQ ID NO：5具有90％以上序列同一性且具有SEQ ID NO：5所示的多肽的功能的衍生多肽(包括活性片段、活性变体)；C. The CIITA protein includes: a polypeptide with an amino acid sequence as shown in SEQ ID NO: 5; or a polypeptide with an amino acid sequence having more than 90% sequence identity with SEQ ID NO: 5 and having a polypeptide as shown in SEQ ID NO: 5 Functional derived polypeptides (including active fragments and active variants);
D、所述HLA-G5包括：氨基酸序列如SEQ ID NO：6所示的多肽；或，氨基酸序列与SEQ ID NO：6具有90％以上序列同一性且具有SEQ ID NO：6所示的多肽的功能的多肽片段。D. The HLA-G5 includes: a polypeptide with an amino acid sequence as shown in SEQ ID NO: 6; or a polypeptide with an amino acid sequence having more than 90% sequence identity with SEQ ID NO: 6 and having a polypeptide as shown in SEQ ID NO: 6 Functional peptide fragments.
在一种或多种实施方式中，所述“90％以上序列同一性”包括：91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上或99％以上的序列同一性。In one or more embodiments, the "more than 90% sequence identity" includes: more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, More than 98% or more than 99% sequence identity.
在本发明的第二方面，提供一种免疫兼容型人多能干细胞，其(a)不表达游离B2M蛋白，表达HLA-G1与分泌型HLA-G5；以及(b)不表达CIITA蛋白。作为本发明优选的一种或多种实施方式，所述人多能干细胞：基因组中内源性的B2M基因与编码HLA-G1的多核苷酸融合；包含外源的编码B2M-HLA-G5融合蛋白的多核苷酸；且，基因组中CIITA基因被敲除。In a second aspect of the present invention, an immune-compatible human pluripotent stem cell is provided, which (a) does not express free B2M protein but expresses HLA-G1 and secreted HLA-G5; and (b) does not express CIITA protein. As one or more preferred embodiments of the present invention, the human pluripotent stem cells: fuse endogenous B2M genes in the genome with polynucleotides encoding HLA-G1; contain exogenous fusions encoding B2M-HLA-G5 The polynucleotide of the protein; and, the CIITA gene in the genome was knocked out.
在一种或多种实施方式中，所述免疫兼容型人多能干细胞，由本发明所述的制备免疫兼容型人多能干细胞的方法构建获得。In one or more embodiments, the immune-compatible human pluripotent stem cells are constructed and obtained by the method of preparing immune-compatible human pluripotent stem cells according to the present invention.
在本发明的第三方面，提供一种免疫兼容型人多能干细胞的应用，用于通过诱导分化制 备适于移植的细胞；较佳地，所述适于移植的细胞为组织或器官细胞；更佳地，所述组织或器官细胞包括(但不限于)：心血管前体细胞、心肌细胞、内皮细胞、平滑肌细胞、神经细胞、造血干细胞、髓系细胞(包括粒细胞、单核细胞、巨噬细胞、红细胞、血小板)、淋系细胞(包括自然杀伤细胞、T细胞、B细胞)、视网膜色素上皮细胞、胰岛B细胞、肝脏细胞(包括肝细胞，胆管细胞，肝内皮细胞，肝星状细胞，Kupffer细胞，间皮细胞)、角质形成细胞、骨骼肌细胞、脂肪细胞、骨细胞、软骨细胞、间充质干细胞。In a third aspect of the present invention, an application of immune-compatible human pluripotent stem cells is provided for producing cells through induced differentiation. Prepare cells suitable for transplantation; preferably, the cells suitable for transplantation are tissue or organ cells; more preferably, the tissue or organ cells include (but are not limited to): cardiovascular precursor cells, cardiomyocytes, Endothelial cells, smooth muscle cells, nerve cells, hematopoietic stem cells, myeloid cells (including granulocytes, monocytes, macrophages, red blood cells, platelets), lymphoid cells (including natural killer cells, T cells, B cells), retina Pigment epithelial cells, pancreatic islet B cells, liver cells (including hepatocytes, bile duct cells, liver endothelial cells, hepatic stellate cells, Kupffer cells, mesothelial cells), keratinocytes, skeletal muscle cells, adipocytes, osteocytes, cartilage cells, mesenchymal stem cells.
在本发明的第四方面，提供一种制备适于移植的细胞的方法，包括：(a)以本发明所述的制备免疫兼容型人多能干细胞的方法制备获得免疫兼容型人多能干细胞；(b)将(a)的细胞进一步进行诱导分化，获得适于移植的细胞；较佳地，所述适于移植的细胞为组织或器官细胞；更佳地，所述组织或器官细胞包括(但不限于)：心血管前体细胞、心肌细胞、内皮细胞、平滑肌细胞、神经细胞、造血干细胞、髓系细胞(包括粒细胞、单核细胞、巨噬细胞、红细胞、血小板)、淋系细胞(包括自然杀伤细胞、T细胞、B细胞)、视网膜色素上皮细胞、胰岛B细胞、肝脏细胞(包括肝细胞，胆管细胞，肝内皮细胞，肝星状细胞，Kupffer细胞，间皮细胞)、角质形成细胞、骨骼肌细胞、脂肪细胞、骨细胞、软骨细胞、间充质干细胞。In a fourth aspect of the present invention, a method for preparing cells suitable for transplantation is provided, including: (a) preparing immune-compatible human pluripotent stem cells by the method of preparing immune-compatible human pluripotent stem cells according to the present invention ; (b) further induce differentiation of the cells of (a) to obtain cells suitable for transplantation; preferably, the cells suitable for transplantation are tissue or organ cells; more preferably, the tissue or organ cells include (But not limited to): cardiovascular precursor cells, cardiomyocytes, endothelial cells, smooth muscle cells, nerve cells, hematopoietic stem cells, myeloid cells (including granulocytes, monocytes, macrophages, red blood cells, platelets), lymphoid cells Cells (including natural killer cells, T cells, B cells), retinal pigment epithelial cells, pancreatic islet B cells, liver cells (including hepatocytes, bile duct cells, liver endothelial cells, hepatic stellate cells, Kupffer cells, mesothelial cells), Keratinocytes, skeletal muscle cells, adipocytes, osteocytes, chondrocytes, mesenchymal stem cells.
在一种或多种实施方式中，所述制备适于移植的细胞的方法中，所述组织或器官细胞为心肌细胞，采用先后以CHIR99021、IWR-1诱导的方法制备；或所述组织或器官细胞为内皮细胞，采用先后以CHIR99021、bFGF、VEGF+BMP4诱导的方法制备。In one or more embodiments, in the method of preparing cells suitable for transplantation, the tissue or organ cells are cardiomyocytes, prepared by a method of inducing CHIR99021 and IWR-1 successively; or the tissue or The organ cells are endothelial cells, which are prepared by inducing CHIR99021, bFGF, and VEGF+BMP4.
在一种或多种实施方式中，所述CHIR99021、IWR-1、bFGF、VEGF或BMP4还包括它们的同功能分子。In one or more embodiments, the CHIR99021, IWR-1, bFGF, VEGF or BMP4 also includes their homofunctional molecules.
在一种或多种实施方式中，所述CHIR99021为终浓度6μM，所述终浓度可在50％内上下浮动，较佳地可在40％内上下浮动，较佳地可在30％内上下浮动，较佳地可在20％内上下浮动，较佳地可在10％内上下浮动。较佳地，所述CHIR99021处理2±0.5天。In one or more embodiments, the CHIR99021 has a final concentration of 6 μM, and the final concentration can fluctuate within 50%, preferably within 40%, and preferably within 30%. It can float, preferably within 20%, and preferably within 10%. Preferably, the CHIR99021 treatment is for 2±0.5 days.
在一种或多种实施方式中，所述IWR-1为终浓度5μM，所述终浓度可在50％内上下浮动，较佳地可在40％内上下浮动，较佳地可在30％内上下浮动，较佳地可在20％内上下浮动，较佳地可在10％内上下浮动。较佳地，所述IWR-1处理2±0.5天。In one or more embodiments, the IWR-1 has a final concentration of 5 μM, and the final concentration can fluctuate within 50%, preferably within 40%, and preferably within 30%. It can fluctuate up and down within 20%, preferably within 10%. Preferably, the IWR-1 treatment is for 2±0.5 days.
在一种或多种实施方式中，所述bFGF为终浓度50ng/ml，所述终浓度可在50％内上下浮动，较佳地可在40％内上下浮动，较佳地可在30％内上下浮动，较佳地可在20％内上下浮动，较佳地可在10％内上下浮动。较佳地，所述bFGF处理1±0.25天。In one or more embodiments, the bFGF has a final concentration of 50 ng/ml, and the final concentration can fluctuate within 50%, preferably within 40%, and preferably within 30% It can fluctuate up and down within 20%, preferably within 10%. Preferably, the bFGF treatment is for 1±0.25 days.
在一种或多种实施方式中，所述VEGF为终浓度50ng/ml，所述终浓度可在50％内上下浮动，较佳地可在40％内上下浮动，较佳地可在30％内上下浮动，较佳地可在20％内上下浮动，较佳地可在10％内上下浮动。In one or more embodiments, the VEGF has a final concentration of 50 ng/ml, and the final concentration can fluctuate within 50%, preferably within 40%, and preferably within 30% It can fluctuate up and down within 20%, preferably within 10%.
在一种或多种实施方式中，所述BMP4为终浓度50ng/ml，所述终浓度可在50％内上下浮动，较佳地可在40％内上下浮动，较佳地可在30％内上下浮动，较佳地可在20％内上下浮动，较佳地可在10％内上下浮动。In one or more embodiments, the BMP4 has a final concentration of 50 ng/ml, and the final concentration can fluctuate within 50%, preferably within 40%, and preferably within 30% It can fluctuate up and down within 20%, preferably within 10%.
在一种或多种实施方式中，所述VEGF+BMP4处理2±0.5天。 In one or more embodiments, the VEGF+BMP4 treatment is 2 ± 0.5 days.
在本发明的第五方面，提供一种心肌细胞或内皮细胞，由本发明所述的制备免疫兼容型人多能干细胞的方法制备获得的免疫兼容型人多能干细胞或由本发明所述的免疫兼容型人多能干细胞衍生获得。In the fifth aspect of the present invention, a cardiomyocyte or endothelial cell is provided, the immune-compatible human pluripotent stem cells prepared by the method of preparing immune-compatible human pluripotent stem cells of the present invention or the immune-compatible human pluripotent stem cells of the present invention. derived from human pluripotent stem cells.
在本发明的第六方面，提供所述的心肌细胞在制备用于治疗心脏相关的疾病或病症的组合物或药物中的用途；较佳地，所述心脏相关的疾病或病症是心肌损伤、心肌梗死、心肌缺血、缺血再灌注损伤或其他心脏损伤；更佳地，所述心脏相关的疾病或病症是缺血/再灌注损伤。In the sixth aspect of the present invention, the use of the cardiomyocytes in preparing compositions or medicines for treating heart-related diseases or disorders is provided; preferably, the heart-related diseases or disorders are myocardial damage, Myocardial infarction, myocardial ischemia, ischemia-reperfusion injury or other heart injuries; more preferably, the heart-related disease or disorder is ischemia/reperfusion injury.
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.
附图说明Description of drawings
图1、免疫兼容型细胞的构建及获取策略。Figure 1. Construction and acquisition strategy of immune-compatible cells.
图2、免疫兼容型细胞的构建及获取策略。A，在B2M的Exon2位置及CIITA的Exon3位置设计gRNA，将gRNA表达质粒与Cas9表达质粒共转染到hPSCs中，之后挑取单克隆，并进行PCR测序验证。存在移码突变的克隆为B2M与CIITA双敲除克隆。B，首先构建B2Mm/sHLAG hPSCs(参见CN113528448A)，然后共转染gRNA与Cas9质粒，挑取单克隆后进行PCR测序验证，存在移码突变的克隆为CIITA-/-B2Mm/sHLAG-hPSCs。Figure 2. Construction and acquisition strategy of immune-compatible cells. A. Design gRNA at the Exon2 position of B2M and the Exon3 position of CIITA, co-transfect the gRNA expression plasmid and Cas9 expression plasmid into hPSCs, then select single clones and perform PCR sequencing verification. The clones with frameshift mutations are B2M and CIITA double knockout clones. B, B2Mm/sHLAG hPSCs were first constructed (see CN113528448A), and then gRNA and Cas9 plasmid were co-transfected. Single clones were selected and verified by PCR sequencing. Clones with frameshift mutations were CIITA -/- B2Mm/sHLAG-hPSCs.
图3、流式细胞术鉴定wt-hPSCs，DKO-hPSCs和CIITA-/-B2Mm/sHLAG-hPSCs细胞的多能性标志物OCT4与SSEA4的表达。A，流式细胞术鉴定OCT4表达(左)及OCT4阳性率百分比(右)。B，流式细胞术鉴定SSEA4表达(左)及OCT4阳性率百分比(右)。n＝3。Figure 3. Flow cytometry identification of the expression of pluripotency markers OCT4 and SSEA4 in wt-hPSCs, DKO-hPSCs and CIITA -/- B2Mm/sHLAG-hPSCs cells. A, Flow cytometry identification of OCT4 expression (left) and OCT4 positive rate percentage (right). B, Flow cytometry identification of SSEA4 expression (left) and OCT4 positive rate percentage (right). n=3.
图4、免疫荧光鉴定wt-hPSCs，DKO-hPSCs和CIITA-/-B2Mm/sHLAG-hPSCs细胞的多能性标志物OCT4与SOX2的表达。标尺：20μm。Figure 4. Immunofluorescence identification of the expression of pluripotency markers OCT4 and SOX2 in wt-hPSCs, DKO-hPSCs and CIITA -/- B2Mm/sHLAG-hPSCs cells. Scale bar: 20 μm.
图5、HLA家族分子表达鉴定。A，流式鉴定HLA I代表性分子HLA-A/B/C的表达。B，流式鉴定HLA-G1分子表达。C，流式鉴定HLA-DR在细胞膜上富集程度，n＝3。D，免疫印迹鉴定分泌型HLA-G5在细胞上清中对的表达。*p＜0.05。Figure 5. Molecular expression identification of HLA family. A, Flow cytometry identifies the expression of HLA I representative molecules HLA-A/B/C. B, Flow cytometry identification of HLA-G1 molecular expression. C, Flow cytometry analysis of the enrichment degree of HLA-DR on the cell membrane, n=3. D, Western blot identifies expression of secreted HLA-G5 in cell supernatants. *p<0.05.
图6、分化的内皮细胞及心肌细胞鉴定。A，分化的内皮细胞形态及内皮细胞纯度鉴定。纯度鉴定通过流式细胞术鉴定内皮标志物CD144表达比例。比例尺＝100μm。n＝3。B，分化的心肌细胞形态及纯度鉴定。纯度鉴定通过流式细胞术鉴定心肌细胞标志物cTNT表达比例。比例尺＝100μm。n＝3。C，免疫荧光鉴定肌节蛋白α-ACTININ和间隙蛋白Connexin-43在分化的心肌细胞中的表达。比例尺＝10μm。Figure 6. Identification of differentiated endothelial cells and cardiomyocytes. A, Identification of differentiated endothelial cell morphology and endothelial cell purity. Purity identification was performed by flow cytometry to identify the expression ratio of endothelial marker CD144. Scale bar = 100 μm. n=3. B, Identification of morphology and purity of differentiated cardiomyocytes. Purity identification: Flow cytometry was used to identify the expression ratio of cardiomyocyte marker cTNT. Scale bar = 100 μm. n=3. C, Immunofluorescence identifies the expression of sarcomeric protein α-ACTININ and gap protein Connexin-43 in differentiated cardiomyocytes. Scale bar = 10 μm.
图7、免疫细胞对CIITA-/-B2Mm/sHLAG-hPSCs源心肌细胞的识别与杀伤作用评价。流式细胞术分析wt-CMs、CIITA-/-B2Mm/sHLAG-CMs分别与hPBMCs共培养对hPBMCs中T细胞早期激活的作用(A)，对T细胞增殖的激活作用(B)，hPBMCs对共培养的心肌细胞的杀伤作用(C)和IFN-γ的分泌量(D)。NK-92与DKO-CMs或CIITA-/-B2Mm/sHLAG-CMs共培养对心肌细胞的杀伤作用(E)及NK-92细胞的IFN-γ的分泌量(F)。n＝5(A)；n＝4(B)；n＝5(C)；n＝5(D)；n＝6(E)；n＝5(F)。*p＜0.05，***p＜0.001。 Figure 7. Evaluation of the recognition and killing effect of immune cells on cardiomyocytes derived from CIITA -/- B2Mm/sHLAG-hPSCs. Flow cytometry analysis of the effects of wt-CMs and CIITA -/- B2Mm/sHLAG-CMs co-cultured with hPBMCs on the early activation of T cells in hPBMCs (A), the activation of T cell proliferation (B), and the effect of hPBMCs on co-culture The killing effect of cultured cardiomyocytes (C) and the secretion of IFN-γ (D). The killing effect of NK-92 and DKO-CMs or CIITA -/- B2Mm/sHLAG-CMs on cardiomyocytes (E) and the amount of IFN-γ secreted by NK-92 cells (F). n=5(A); n=4(B); n=5(C); n=5(D); n=6(E); n=5(F). *p<0.05, ***p<0.001.
图8、人源化小鼠心梗后移植hPSCs源心肌细胞后细胞驻留评价。A，研究示意图。B，移植后第28天心脏切片免疫荧光图。α-ACTININ：肌丝结构蛋白。hu-KU80：人KU80蛋白，此处所用抗体只对人KU80蛋白有结合，对小鼠KU80没有交叉反应。比例尺＝500μm。C，细胞驻留统计结果(以面积计算)。雌鼠，n＝3-7；雄鼠，n＝3。D，细胞驻留统计结果(以qRT-PCR计算)，雌鼠，n＝4-5；雄鼠，n＝3。*p＜0.05，**p＜0.01，***p＜0.001。Figure 8. Evaluation of cell retention after transplantation of hPSCs-derived cardiomyocytes in humanized mice after myocardial infarction. A, Study schematic. B, Immunofluorescence image of heart sections on day 28 after transplantation. α-ACTININ: myofilament structural protein. hu-KU80: human KU80 protein. The antibody used here only binds to human KU80 protein and has no cross-reactivity to mouse KU80. Scale bar = 500 μm. C, Cell residency statistics (calculated as area). Female mice, n=3-7; male mice, n=3. D, statistical results of cell residence (calculated by qRT-PCR), female mice, n=4-5; male mice, n=3. *p<0.05, **p<0.01, ***p<0.001.
图9、Hu-mice心梗心脏再肌肉化评价。A，移植后第28天小鼠心脏切片Masson染色。比例尺＝1mm。B，小鼠心脏再肌肉化比例统计。雌鼠，n＝3-7；雄鼠，n＝3。*p＜0.05，**p＜0.01，***p＜0.001。Figure 9. Evaluation of cardiac remuscularization in Hu-mice myocardial infarction. A, Masson staining of mouse heart sections on day 28 after transplantation. Scale bar = 1 mm. B, Statistics on the remuscularization ratio of mouse hearts. Female mice, n=3-7; male mice, n=3. *p<0.05, **p<0.01, ***p<0.001.
图10、免疫兼容型人诱导多能干细胞(iPSC-IC)的其他构建方法。A，在基因组安全港(safe harbor)位点(AAVS1位点)***外源基因；B，利用转座子***将外源基因整合到基因组中；C，两种表达框的构建方式。Figure 10. Other construction methods of immune-compatible human induced pluripotent stem cells (iPSC-IC). A, inserting foreign genes into the genome safe harbor site (AAVS1 site); B, using the transposon system to integrate foreign genes into the genome; C, two construction methods of expression cassettes.
图11、检测利用非病毒载体的外源基因***基因组的策略构建的人iPSCs单克隆细胞源心肌细胞中外源基因的表达。A，流式细胞术检测各个系分化获得的心肌细胞膜上HLAG1的表达水平，右侧为统计的平均荧光强度；B，免疫印迹检测各个系分化获得的心肌细胞分泌的HLAG5；C，酶联免疫吸附检测各个系分化获得的心肌细胞分泌的HLAG5。Figure 11. Detection of exogenous gene expression in human iPSCs monoclonal cell-derived cardiomyocytes constructed using a strategy of inserting exogenous genes into the genome using non-viral vectors. A, Flow cytometry was used to detect the expression level of HLAG1 on the cardiomyocyte membrane differentiated from each line. The right side is the statistical average fluorescence intensity; B, Western blot was used to detect HLAG5 secreted by cardiomyocytes differentiated from each line; C, Enzyme-linked immunoassay Adsorption and detection of HLAG5 secreted by cardiomyocytes differentiated from various lines.
具体实施方式Detailed ways
本发明人在应用B2Mm/sHLAG hESCs的过程中发现，其在免疫兼容性方面还需改进。当将B2Mm/sHLAG hESCs主要应用于退行性疾病的细胞治疗，此时运行较好。但当其在某些其它移植环境中时，仍有免疫细胞(包括抗原呈递细胞)激活的现象。例如，当本发明人将其应用于急性损伤的细胞治疗中，如梗死心脏损伤部位的炎症水平高。在临床上，移植的外源细胞需要植入损伤或临近损伤部位发挥治疗作用，而损伤组织常伴随病变处炎症反应的过度激活，使得病变处组织细胞炎症因子水平较高。当移植hESC诱导分化的组织/器官细胞时，对此类细胞的免疫排斥反应增强。经过广泛的实验研究，分析了大量与此类现象可能相关的分子后，本发明人确定了进一步改造的靶点。基于此，本发明也揭示了一种进一步改造的免疫兼容型细胞，其具有更为广泛的应用环境。In the process of applying B2Mm/sHLAG hESCs, the inventor found that its immune compatibility needs to be improved. B2Mm/sHLAG hESCs work better when they are mainly used in cell therapy for degenerative diseases. But when it is in some other transplant environments, there is still activation of immune cells (including antigen-presenting cells). For example, when the inventors applied it to cell therapy for acute injury, such as infarction, the level of inflammation in the injured part of the heart is high. Clinically, transplanted exogenous cells need to be implanted into the injury or adjacent to the injury site to play a therapeutic role. Injured tissue is often accompanied by excessive activation of the inflammatory response at the lesion, resulting in higher levels of inflammatory factors in tissue cells at the lesion. When hESC-induced differentiated tissue/organ cells are transplanted, the immune rejection response to such cells is enhanced. After extensive experimental research and analysis of a large number of molecules potentially related to such phenomena, the inventors identified targets for further modification. Based on this, the present invention also reveals a further modified immune-compatible cell, which has a wider application environment.
术语the term
术语“多能干细胞(pluripotent stem cells，PSCs)”是指能够在保持未分化状态的同时自我更新和增殖并且可以在适当条件下被诱导分化成特化细胞类型的细胞。如本文所用，“多能干细胞”包括多能干细胞和其他类型的干细胞，包括胚胎、羊膜或体干细胞。如本文所用，人多能干细胞(hPSCs)可以是人胚胎干细胞(human embryonic stem cells，hESCs)，其是利用未经过体内发育的受精14天以内的人类胚胎分离或者获取的。示例性人干细胞系包括H9人多能干细胞系，例如，可以是商用的H9hESC细胞系。另外的示例性干细胞系包括可通过National Institutes of Health Human Embryonic Stem Cell Registry和Howard Hughes Medical Institute HUES集合获 得的那些(如Cowan，C.A.等人，New England J.Med.350：13.(2004))所述。The term "pluripotent stem cells (PSCs)" refers to cells that are capable of self-renewal and proliferation while remaining in an undifferentiated state and that can be induced to differentiate into specialized cell types under appropriate conditions. As used herein, "pluripotent stem cells" includes pluripotent stem cells and other types of stem cells, including embryonic, amniotic, or somatic stem cells. As used herein, human pluripotent stem cells (hPSCs) may be human embryonic stem cells (hESCs), which are isolated or obtained from human embryos within 14 days of fertilization that have not undergone in vivo development. Exemplary human stem cell lines include the H9 human pluripotent stem cell line, which may be, for example, the commercially available H9hESC cell line. Additional exemplary stem cell lines include those available through the National Institutes of Health Human Embryonic Stem Cell Registry and the Howard Hughes Medical Institute HUES collection. Those obtained (as described in Cowan, CA et al., New England J. Med. 350: 13. (2004)).
如本文所用，“多能干细胞”具有分化成如下三个胚层中任何一个的潜力：内胚层(例如胃连接、胃肠道、肺等)、中胚层(例如肌肉、骨骼、血液、泌尿生殖组织等)或外胚层(例如表皮组织和神经***组织)。如本文所用的术语“多能干细胞”还包括“诱导性多能干细胞”、“iPS”、“iPSC”或“iPSCs”，一种衍生自非多能细胞的多能干细胞。亲本细胞的实例包括已通过各种方式重编程以诱导性多能性、未分化表型的体细胞。这种“iPS”、“iPSC”或“iPSCs”细胞可以通过诱导某些调节基因的表达或通过某些蛋白质的外源应用来产生。诱导iPS细胞的方法是本领域已知的。如本文所用，“hiPSC”、“hiPSCs”、“hPSC”或“hPSCs”是人诱导性多能干细胞，“miPSC”、“miPSCs”、“mPSC”或“mPSCs”是鼠诱导性多能干细胞。As used herein, "pluripotent stem cells" have the potential to differentiate into any of the following three germ layers: endoderm (e.g., gastric junction, gastrointestinal tract, lung, etc.), mesoderm (e.g., muscle, bone, blood, urogenital tissue) etc.) or ectoderm (such as epidermal tissue and nervous system tissue). The term "pluripotent stem cells" as used herein also includes "induced pluripotent stem cells," "iPS," "iPSCs" or "iPSCs," a type of pluripotent stem cell derived from non-pluripotent cells. Examples of parental cells include somatic cells that have been reprogrammed by various means to induce a pluripotent, undifferentiated phenotype. Such "iPS", "iPSC" or "iPSCs" cells can be generated by inducing the expression of certain regulatory genes or by the exogenous application of certain proteins. Methods of inducing iPS cells are known in the art. As used herein, "hiPSC", "hiPSCs", "hPSC" or "hPSCs" are human induced pluripotent stem cells, and "miPSC", "miPSCs", "mPSC" or "mPSCs" are murine induced pluripotent stem cells.
术语“多能干细胞特征”是指将多能干细胞与其他细胞区分开的细胞特征。产生能够在适当条件下分化成共同显示与来自所有三个生发层(内胚层、中胚层和外胚层)的细胞谱系相关的特征的细胞类型的后代的能力是多能干细胞特征。分子标志物的某些组合的表达或非表达也是多能干细胞特征。例如，人多能干细胞表达来自以下非限制性列表的至少几种和在一些实施方案中所有标志物：SSEA-3、SSEA-4、TRA-1-60、TRA-1-81、TRA-2-49/6E、ALP、Sox2、E-钙粘蛋白、UTF-1、Oct4、Rex1和Nanog。与多能干细胞相关的细胞形态也是多能干细胞特征。如本文所述，细胞不需要通过多能性以被重编程为内胚层祖细胞和/或肝细胞。The term "pluripotent stem cell characteristics" refers to cellular characteristics that distinguish pluripotent stem cells from other cells. The ability to produce progeny that can differentiate under appropriate conditions into cell types that collectively display characteristics associated with cell lineages from all three germinal layers (endoderm, mesoderm, and ectoderm) is a pluripotent stem cell characteristic. The expression or non-expression of certain combinations of molecular markers is also characteristic of pluripotent stem cells. For example, human pluripotent stem cells express at least several, and in some embodiments all, markers from the following non-limiting list: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2 -49/6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rex1 and Nanog. Cell morphologies associated with pluripotent stem cells are also characteristic of pluripotent stem cells. As described herein, cells do not need to pass pluripotency to be reprogrammed into endodermal progenitors and/or hepatocytes.
术语“多能(multipotent)”或“多能细胞(multipotent cell)”是指可以产生有限数量的其他特定细胞类型的细胞类型。例如，诱导性多能细胞能够形成内胚层细胞。此外，多能血液干细胞可以将自身分化为几种类型的血细胞，包括淋巴细胞、单核细胞、中性粒细胞等。术语“寡能”是指成体干细胞分化成仅少数不同细胞类型的能力。例如，淋巴或骨髓干细胞能够分别形成淋巴或骨髓谱系的细胞。术语“单能”是指细胞形成单细胞类型的能力。例如，精原干细胞只能形成***细胞。The term "multipotent" or "multipotent cell" refers to a cell type that can give rise to a limited number of other specific cell types. For example, induced pluripotent cells can form endoderm cells. In addition, pluripotent blood stem cells can differentiate themselves into several types of blood cells, including lymphocytes, monocytes, neutrophils, etc. The term "oligopotent" refers to the ability of adult stem cells to differentiate into only a few different cell types. For example, lymphoid or myeloid stem cells can form cells of the lymphoid or myeloid lineage, respectively. The term "unipotent" refers to the ability of a cell to form a single cell type. For example, spermatogonial stem cells can only form sperm cells.
术语“非多能细胞”是指不是多能细胞的哺乳动物细胞。此类细胞的实例包括分化细胞以及祖细胞。分化细胞的实例包括但不限于来自选自骨髓、皮肤、骨骼肌、脂肪组织和外周血的组织的细胞。示例性细胞类型包括但不限于成纤维细胞、肝细胞、成肌细胞、神经元、成骨细胞、破骨细胞和T细胞。用于产生诱导性多能细胞、内胚层祖细胞和肝细胞的起始细胞可以是非多能细胞。分化的细胞包括但不限于多能细胞、寡能细胞、单能细胞、祖细胞和终末分化细胞。在特定的实施方案中，相对于更多能的细胞，较低能的细胞被认为是“分化的”。The term "non-pluripotent cell" refers to a mammalian cell that is not a pluripotent cell. Examples of such cells include differentiated cells as well as progenitor cells. Examples of differentiated cells include, but are not limited to, cells from tissues selected from bone marrow, skin, skeletal muscle, adipose tissue, and peripheral blood. Exemplary cell types include, but are not limited to, fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T cells. Starting cells for generating induced pluripotent cells, endodermal progenitor cells and hepatocytes can be non-pluripotent cells. Differentiated cells include, but are not limited to, pluripotent cells, oligopotent cells, unipotent cells, progenitor cells and terminally differentiated cells. In certain embodiments, lower energy cells are considered "differentiated" relative to more energy cells.
术语“体细胞”是形成生物体的细胞。体细胞包括构成生物体内器官、皮肤、血液、骨骼和***的细胞，但不包括生殖细胞。细胞可以来自例如人或非人哺乳动物。示例性的非人哺乳动物包括但不限于小鼠、大鼠、猫、狗、兔、豚鼠、仓鼠、羊、猪、马、牛和非人灵长类动物。在一些实施方案中，细胞来自成年人或非人哺乳动物。在一些实施方案中，细胞来自新生儿、成年人或非人哺乳动物。The term "somatic cell" refers to the cells that form an organism. Somatic cells include cells that make up the organs, skin, blood, bones, and connective tissues of an organism, but do not include reproductive cells. The cells may be from, for example, humans or non-human mammals. Exemplary non-human mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, cattle, and non-human primates. In some embodiments, the cells are from an adult human or non-human mammal. In some embodiments, the cells are from neonates, adults, or non-human mammals.
如本文所用，术语“受试者”或“患者”是指任何动物，例如驯养动物、动物园动物或人。“受试者”或“患者”可以是哺乳动物，如狗、猫、鸟、牲畜，也包括人。“受试者”和“患 者”的具体实例包括但不限于具有与肝脏、心脏、肺、肾、胰腺、脑、神经组织、血液、骨骼、骨髓等相关的疾病或病症的个体(特别是人)。哺乳动物细胞可以来自人或非人哺乳动物。示例性的非人哺乳动物包括但不限于小鼠、大鼠、猫、狗、兔、豚鼠、仓鼠、羊、猪、马、牛和非人灵长类动物(例如，黑猩猩、猕猴和猿类)。As used herein, the term "subject" or "patient" refers to any animal, such as a domestic animal, a zoo animal, or a human. A "subject" or "patient" may be a mammal, such as a dog, cat, bird, livestock, or a human. "subject" and "patient" Specific examples of "persons" include, but are not limited to, individuals (especially humans) having diseases or conditions associated with the liver, heart, lungs, kidneys, pancreas, brain, nervous tissue, blood, bones, bone marrow, etc. Mammalian cells may be derived from Human or non-human mammals. Exemplary non-human mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, cattle and non-human primates (e.g. , chimpanzees, macaques and apes).
术语“HLA”或“人白细胞抗原”复合物是编码人主要组织相容性复合物(MHC)蛋白质的基因复合物。构成HLA复合物的这些细胞表面蛋白质负责调节对抗原的免疫应答。在人中，存在两种HLA：I类和II类，“HLA I”和“HLA II”。HLA I至少包括三种蛋白，HLA-A、HLA-B和HLA-C，它们从细胞内部呈递肽。HLA II至少包括五种蛋白，HLA-DP、HLA-DM、HLA-DOB、HLA-DQ和HLA-DR，其将来自细胞外的抗原呈递给T淋巴细胞。应当理解，“MHC”或“HLA”的使用并不意味着限制，因为它取决于基因是来自人(HLA)还是鼠(MHC)。因此，当涉及哺乳动物细胞时，这些术语在本文中可互换使用。The term "HLA" or "human leukocyte antigen" complex is a complex of genes encoding human major histocompatibility complex (MHC) proteins. These cell surface proteins that make up the HLA complex are responsible for regulating the immune response to antigens. In humans, there are two types of HLA: Class I and Class II, "HLA I" and "HLA II." HLA I consists of at least three proteins, HLA-A, HLA-B and HLA-C, which present peptides from inside the cell. HLA II includes at least five proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ and HLA-DR, which present antigens from extracellular sources to T lymphocytes. It should be understood that the use of "MHC" or "HLA" is not meant to be limiting, as it depends on whether the gene is of human (HLA) or mouse (MHC) origin. Therefore, these terms are used interchangeably herein when referring to mammalian cells.
术语“基因敲除”是指使特定基因在其所在的宿主细胞中无活性的过程，其导致不产生目的蛋白质或无活性形式。如本领域技术人员所理解和下文进一步描述的，这可以通过多种不同方式实现，包括从基因中去除全部或部分核酸序列，或用其他序列中断序列，去除或改变调节组分(例如启动子)使得基因不被转录，改变阅读框，通过与mRNA的结合阻止翻译，或改变核酸的调节成分等。例如，可以去除或用“无义”序列替换目的基因的全部或部分编码区，可以去除或替换全部或部分调节序列(例如启动子)，可以去除或替换翻译起始序列等。通常，敲除在基因组DNA水平上进行，使得细胞的后代也永久地携带敲除。The term "gene knockout" refers to the process of rendering a specific gene inactive in the host cell in which it is found, resulting in no production or an inactive form of the protein of interest. As understood by those skilled in the art and described further below, this can be achieved in a number of different ways, including removing all or part of the nucleic acid sequence from the gene, or interrupting the sequence with other sequences, removing or altering regulatory components (e.g. promoter ) prevents genes from being transcribed, changes the reading frame, prevents translation by binding to mRNA, or changes the regulatory components of nucleic acids, etc. For example, all or part of the coding region of the target gene can be removed or replaced with "nonsense" sequences, all or part of the regulatory sequences (such as promoters) can be removed or replaced, the translation initiation sequence can be removed or replaced, etc. Typically, knockouts are performed at the level of genomic DNA, so that future generations of cells also permanently carry the knockout.
术语“基因敲入/引入”是指向宿主细胞添加遗传功能的过程。这导致编码蛋白质水平增加。如本领域技术人员所理解的，这可以通过几种方式实现，包括将一种或多种额外的基因拷贝添加到宿主细胞中或改变内源基因的调节组分，从而增加蛋白质的表达。这可以通过修饰启动子、添加不同的启动子、添加增强子或修饰其他基因表达序列来实现通常，敲入技术导致转基因的额外拷贝整合到宿主细胞中。The term "knock-in" refers to the process of adding genetic functionality to a host cell. This results in increased levels of the encoded protein. As understood by those skilled in the art, this can be accomplished in several ways, including adding one or more additional gene copies to the host cell or altering the regulatory components of the endogenous gene, thereby increasing protein expression. This can be accomplished by modifying the promoter, adding a different promoter, adding enhancers, or modifying other gene expression sequences. Typically, knock-in techniques result in the integration of additional copies of the transgene into the host cell.
术语“β-2微球蛋白”或“β2M”或“B2M”蛋白可以是指具有下文SEQ ID NO：2所示的氨基酸序列，或与SEQ ID NO：2所示的氨基酸序列具有90％以上的序列同一性且具有与其类似的生物活性的人β2M蛋白。术语“CIITA”蛋白可以是指具有下文SEQ ID NO：5所示的氨基酸序列，或与SEQ ID NO：5所示的氨基酸序列具有90％以上的序列同一性且具有与其类似的生物活性的人CIITA蛋白质。The term "β-2 microglobulin" or "β2M" or "B2M" protein may refer to having the amino acid sequence shown in SEQ ID NO: 2 below, or having more than 90% of the amino acid sequence shown in SEQ ID NO: 2 Human β2M protein with sequence identity and similar biological activity. The term "CIITA" protein may refer to humans having the amino acid sequence shown in SEQ ID NO: 5 below, or having more than 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 5 and having similar biological activities. CIITA protein.
在细胞的上下文中，“野生型”是指在自然界中发现的细胞。然而，在多能干细胞的背景下，如本文所用，它还意指可能含有导致多能性的核酸变化但不经历本发明的基因编辑程序以实现低免疫原性的iPSCs。In the context of cells, "wild type" refers to cells found in nature. However, in the context of pluripotent stem cells, as used herein, it also means iPSCs that may contain nucleic acid changes that lead to pluripotency but do not undergo the gene editing procedures of the invention to achieve low immunogenicity.
术语“同种异体”是指宿主生物和细胞移植的遗传差异，其中产生免疫应答。The term "allogeneic" refers to the genetic differences between the host organism and the transplanted cells in which the immune response is generated.
本文中的“B2M-/-”是指二倍体细胞在两条染色体中都具有失活的B2M基因，这可以以各种方式完成。类似的，本文中的“CIITA-/-”是指二倍体细胞在两条染色体中都具有失活的CIITA基因。如本文所述，这可以以各种方式完成。 "B2M -/- " in this article means that diploid cells have inactive B2M genes in both chromosomes, which can be done in various ways. Similarly, "CIITA -/- " in this article refers to diploid cells with inactive CIITA genes in both chromosomes. This can be done in various ways, as described in this article.
如本文所用，“m/sHLAG”为膜(m)定位HLA-G1与可溶性(s)HLA-G5的简称。As used herein, "m/sHLAG" is an abbreviation for membrane (m) localized HLA-G1 and soluble (s) HLA-G5.
如本文所用，“B2Mm/sHLAG hPSCs”细胞系为同时过表达膜定位HLA-G1与可溶性HLA-G5的细胞系。As used herein, "B2Mm/sHLAG hPSCs" cell lines are cell lines that overexpress both membrane-localized HLA-G1 and soluble HLA-G5.
如本文所用，所述的“表达”包括了“过表达”、“重组表达”。所述“过表达”例如与野生型中表达量相比，表达量发生显著性增加，如增加到野生型的1.2、1.5、2、3、5、8、10、15、20、30、50倍以上或更高。As used herein, the "expression" includes "overexpression" and "recombinant expression". Said "overexpression" means, for example, that the expression level is significantly increased compared to the expression level in the wild type, such as to 1.2, 1.5, 2, 3, 5, 8, 10, 15, 20, 30, 50 of the wild type. times or higher.
如本文所用，所述的“不表达”是相对而言的，也涵盖“低表达”、“极低表达”，例如与野生型中表达量相比，经改造的细胞的表达被降低至野生型的15％以下、10％以下、8％以下、5％以下、3％以下、2％以下、1％以下或更低。As used herein, "no expression" is a relative term, and also encompasses "low expression" and "extremely low expression". For example, compared with the expression level in the wild type, the expression of the modified cells is reduced to that of the wild type. Type 15% or less, 10% or less, 8% or less, 5% or less, 3% or less, 2% or less, 1% or less or less.
如本文所用，术语“含有”表示各种成分可一起应用于本发明的混合物或组合物中。因此，术语“主要由...组成”和“由...组成”包含在术语“含有”中。如本文所用，术语“有效量”或“有效剂量”是指可对受试者产生功能或活性的且可被人和/或动物所接受的量。As used herein, the term "comprising" means that the various ingredients can be used together in the mixture or composition of the present invention. Therefore, the terms "consisting essentially of" and "consisting of" are included in the term "comprising". As used herein, the term "effective amount" or "effective dose" refers to an amount that produces function or activity in a subject and is acceptable to humans and/or animals.
如本文所用，“药学上可接受的”的成分是适用于受试者而无过度不良副反应(如毒性、刺激和***反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。As used herein, a "pharmaceutically acceptable" ingredient is one that is suitable for administration to a subject without undue adverse side effects (such as toxicity, irritation, and allergic reactions), that is, a substance that has a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents.
本说明书中给出的每个最大数值限制都包括每个较低的数值限制，如同这些较低的数值限制在本文中明确写出一样。在整个说明书中给出的每个最小数值限制将包括每个更高的数值限制，如同在此明确写出这样的更高数值限制一样。本说明书中给出的每个数值范围将包括落入这样更宽的数值范围内的每个较窄的数值范围，如同这些较窄的数值范围都在本文中明确写出。Every maximum numerical limitation given throughout this specification will include every lower numerical limitation as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation as if such higher numerical limitation were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were expressly written herein.
本发明的细胞及其用途Cells of the present invention and uses thereof
本发明提供了一种人多能干细胞，其由于本发明的遗传操作从而避免宿主免疫应答。具体而言，所述人多能干细胞的基因组经由改造而：不表达游离B2M蛋白，不表达CIITA蛋白，但表达(外源的)HLA-G1与分泌型HLA-G5。本领域技术人员可以理解，通过本发明所述的构建方法构建获得的人多能干细胞也在本发明的保护范围以内。The present invention provides human pluripotent stem cells that avoid host immune responses due to the genetic manipulation of the present invention. Specifically, the genome of the human pluripotent stem cells has been modified to: not express free B2M protein, not express CIITA protein, but express (exogenous) HLA-G1 and secreted HLA-G5. Those skilled in the art can understand that human pluripotent stem cells constructed through the construction method of the present invention are also within the protection scope of the present invention.
所述人多能干细胞允许衍生用于产生特定的组织和器官的“现成的”细胞产品。能够在人患者中使用所述人多能干细胞的衍生物从而产生显著益处，包括避免通常在移植中看到的长期辅助免疫抑制治疗和药物使用的能力，比如，所述细胞衍生的心肌细胞可避免人免疫细胞对其的识别与杀伤，在心脏缺血/再灌注损伤模型中具备更好的驻留效果。它还可以显著节省成本，因为可以使用细胞疗法而无需为每位患者进行单独治疗。因此，所述人多能干细胞具有免疫兼容性(避免宿主免疫应答)，能用于更广的患者群体，可以作为产生普遍接受的衍生物的通用细胞来源。The human pluripotent stem cells allow the derivation of "off-the-shelf" cell products for the generation of specific tissues and organs. Being able to use derivatives of the human pluripotent stem cells in human patients yields significant benefits, including the ability to avoid long-term adjuvant immunosuppressive treatments and drug use typically seen in transplantation, for example, cardiomyocytes derived from the cells can It avoids recognition and killing by human immune cells and has better retention effect in cardiac ischemia/reperfusion injury model. It also offers significant cost savings because cell therapy can be used without the need for individual treatments for each patient. Therefore, the human pluripotent stem cells are immunocompatible (avoid host immune response), can be used in a wider patient population, and can serve as a universal cell source to generate generally accepted derivatives.
本发明提供了一种人多能干细胞，其由于本发明中所述的几种遗传操作从而避免宿主免疫应答。所述细胞缺乏引发免疫应答的主要免疫抗原，并且经过改造以逃逸免疫细胞的识别与杀伤作用。 The present invention provides human pluripotent stem cells that avoid host immune responses due to several genetic manipulations described in the present invention. The cells lack the major immune antigens that trigger immune responses and are engineered to evade recognition and killing by immune cells.
具体的，本发明所述的人多能干细胞不表达游离B2M蛋白和CIITA蛋白，但是，作为优选的一种方式，其可以成功表达分泌型B2M-HLA-G5融合蛋白。在一些实施方案中，本发明所述的人多能干细胞通过在所述人多能干细胞的基因组中整合重组核酸的方式实现。Specifically, the human pluripotent stem cells of the present invention do not express free B2M protein and CIITA protein, but, as a preferred method, they can successfully express secreted B2M-HLA-G5 fusion protein. In some embodiments, the human pluripotent stem cells of the present invention are realized by integrating recombinant nucleic acids into the genome of the human pluripotent stem cells.
示例性遗传操作的技术包括同源重组、敲入、ZFN(锌指核酸酶)、TALEN(转录激活因子样效应核酸酶)、CRISPR(成簇规律间隔短回文重复序列)/Cas9以及其他位点特异性核酸酶技术。存在大量基于CRISPR/Cas9的技术，参见例如Doudna and Charpentier，Science doi：10.1126/science.1258096，其通过引用并入本文。这些技术使得能够在所需的基因座位点处进行双链DNA断裂。这些受控的双链断裂促进特定基因座位点的同源重组。该过程集中于用核酸内切酶靶向核酸分子的特定序列，例如染色体，所述核酸内切酶识别并结合序列并在核酸分子中诱导双链断裂。通过易错的非同源末端连接(NHEJ)或通过同源重组(HR)修复双链断裂。Exemplary techniques for genetic manipulation include homologous recombination, knock-in, ZFN (zinc finger nuclease), TALEN (transcription activator-like effector nuclease), CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9, and others. Site-specific nuclease technology. There are a number of CRISPR/Cas9 based technologies, see for example Doudna and Charpentier, Science doi: 10.1126/science.1258096, which is incorporated herein by reference. These techniques enable double-stranded DNA breaks at desired locus sites. These controlled double-strand breaks promote homologous recombination at specific loci. This process focuses on targeting specific sequences of nucleic acid molecules, such as chromosomes, with endonucleases, which recognize and bind the sequences and induce double-stranded breaks in the nucleic acid molecules. Double-strand breaks are repaired by error-prone nonhomologous end joining (NHEJ) or by homologous recombination (HR).
如本领域技术人员所理解的，可以使用许多不同的技术来改造本发明的多能干细胞，以及如本文所述的使人多能干细胞缺乏引发免疫应答的主要免疫抗原，并且经过改造以逃逸免疫细胞的识别与杀伤作用。As will be appreciated by those skilled in the art, many different techniques can be used to engineer the pluripotent stem cells of the invention, as well as to render human pluripotent stem cells deficient in major immune antigens that trigger an immune response and engineered to evade immunity as described herein Cell recognition and killing.
通常，这些技术可以单独使用或组合使用。例如，在人多能干细胞的构建中，CRISPR可用于降低改造细胞中活性B2M和/或CIITA蛋白的表达，并用病毒技术(例如慢病毒)实现基因的稳定转导。此外，如本领域技术人员所理解的，尽管一个实施方案顺序地利用CRISPR/Cas9技术敲除B2M，然后通过CRISPR/Cas9技术敲除CIITA，但这些基因可以使用不同的技术以不同的顺序操作。Typically, these techniques can be used individually or in combination. For example, in the construction of human pluripotent stem cells, CRISPR can be used to reduce the expression of active B2M and/or CIITA proteins in the modified cells, and use viral technology (such as lentivirus) to achieve stable gene transduction. Additionally, as will be understood by those skilled in the art, although one embodiment sequentially knocks out B2M using CRISPR/Cas9 technology and then knocks out CIITA using CRISPR/Cas9 technology, these genes can be operated in a different order using different technologies.
作为一种优选的实施方式，采用慢病毒感染的方式构建的免疫兼容型人多能干细胞。在操作中，通常慢病毒载体需要进行质粒抽提、病毒包装与质控等步骤，而且是随机整合到基因组上。As a preferred embodiment, immune-compatible human pluripotent stem cells constructed by lentiviral infection are used. In operation, lentiviral vectors usually require steps such as plasmid extraction, virus packaging and quality control, and are randomly integrated into the genome.
作为一种优选的实施方式，采用非病毒的质粒转染的构建方式，以定点敲入或转座子***(如Piggybac、Sleeping beauty等)将目的基因整合到目标细胞基因组，相较于慢病毒体系在生产、制备流程管理及安全性方面具备更好的优势。As a preferred embodiment, non-viral plasmid transfection construction methods are used to integrate the target gene into the target cell genome using site-directed knock-in or transposon systems (such as Piggybac, Sleeping beauty, etc.). Compared with lentivirus The system has better advantages in production, preparation process management and safety.
本发明中，可采用多种方法来筛选单克隆细胞系，例如可包括药物筛选、PCR筛选、流式细胞术筛选、ELISA筛选及免疫印迹等。In the present invention, a variety of methods can be used to screen monoclonal cell lines, such as drug screening, PCR screening, flow cytometry screening, ELISA screening, and immunoblotting.
对于所有这些技术，使用公知的重组技术来产生如本文所述的重组核酸。在某些实施方案中，重组核酸(编码所需多肽例如B2M-HLA-G1融合蛋白或CIITA蛋白)可以与表达构建体中的一个或多个调节核苷酸序列可操作地连接。调节核苷酸序列通常适合宿主细胞和待治疗的受试者。本领域已知多种类型的合适表达载体和合适的调节序列用于多种宿主细胞。通常，一种或多种调节核苷酸序列可包括但不限于启动子序列、前导序列或信号序列、核糖体结合位点、转录起始和终止序列、翻译起始和终止序列、以及增强子或激活子序列。还考虑了本领域已知的组成型或诱导型启动子。启动子可以是天然存在的启动子，或组合多于一种启动子的元件的杂合启动子。表达构建体可以在细胞中存在于附加体(例如质粒)上，或者表达构建体可以***染色体中。在一个具体实施方案中，表达载体包括选择标记基因以允许选择转化的宿主细胞。某 些实施方案包括表达载体，其包含与至少一种调节序列可操作地连接的编码变体多肽的核苷酸序列。用于本文的调节序列包括启动子、增强子和其他表达控制元件。在某些实施方案中，设计表达载体用于选择待转化的宿主细胞、期望表达的特定变体多肽、载体的拷贝数、控制该拷贝数的能力或由载体编码的任何其他蛋白质如抗生素标志物的表达。For all of these techniques, well-known recombinant techniques are used to produce recombinant nucleic acids as described herein. In certain embodiments, a recombinant nucleic acid encoding a desired polypeptide, such as a B2M-HLA-G1 fusion protein or a CIITA protein, can be operably linked to one or more regulatory nucleotide sequences in an expression construct. The regulatory nucleotide sequence is generally suitable for the host cell and the subject to be treated. Various types of suitable expression vectors and suitable regulatory sequences are known in the art for use in a variety of host cells. Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancers or activation subsequence. Constitutive or inducible promoters known in the art are also contemplated. The promoter may be a naturally occurring promoter, or a hybrid promoter combining elements of more than one promoter. The expression construct may be present in the cell on an episome (eg, a plasmid), or the expression construct may be inserted into the chromosome. In a specific embodiment, the expression vector includes a selectable marker gene to allow selection of transformed host cells. certain Some embodiments include expression vectors comprising a nucleotide sequence encoding a variant polypeptide operably linked to at least one regulatory sequence. Regulatory sequences as used herein include promoters, enhancers and other expression control elements. In certain embodiments, expression vectors are designed for selection of the host cell to be transformed, the specific variant polypeptide desired to be expressed, the copy number of the vector, the ability to control the copy number, or any other protein encoded by the vector such as an antibiotic marker expression.
本发明中，也提供了从所述人多能干细胞衍生的心肌细胞，然后将其移植到需要的患者中，从而避免人免疫细胞对其的识别与杀伤，还提供了所述的衍生的心肌细胞在制备用于治疗心脏相关的疾病或病症的组合物或药物中的用途。The present invention also provides cardiomyocytes derived from the human pluripotent stem cells, which are then transplanted into patients in need, thereby avoiding recognition and killing by human immune cells, and the derived cardiomyocytes are also provided. Use of cells in the preparation of compositions or medicaments for the treatment of heart-related diseases or conditions.
如本领域技术人员所理解的，人多能干细胞允许衍生用于产生特定的组织和器官的“现成的”细胞产品。能够在人患者中使用所述人多能干细胞的衍生物从而产生显著益处，包括避免通常在移植中看到的长期辅助免疫抑制治疗和药物使用的能力，比如，所述细胞衍生的心肌细胞可避免人免疫细胞对其的识别与杀伤，在心脏缺血/再灌注损伤模型中具备更好的驻留效果。它还可以显著节省成本，因为可以使用细胞疗法而无需为每位患者进行单独治疗。因此，所述人多能干细胞具有免疫兼容性，能用于更广的患者群体，可以作为产生普遍接受的衍生物的通用细胞来源。As understood by those skilled in the art, human pluripotent stem cells allow the derivation of "off-the-shelf" cell products for the generation of specific tissues and organs. Being able to use derivatives of the human pluripotent stem cells in human patients yields significant benefits, including the ability to avoid long-term adjuvant immunosuppressive treatments and drug use typically seen in transplantation, for example, cardiomyocytes derived from the cells can It avoids recognition and killing by human immune cells and has better retention effect in cardiac ischemia/reperfusion injury model. It also offers significant cost savings because cell therapy can be used without the need for individual treatments for each patient. Therefore, the human pluripotent stem cells are immune compatible, can be used in a wider patient population, and can serve as a universal cell source to generate generally accepted derivatives.
本发明也提供一种人多能干细胞，其由于本发明中所述的几种遗传操作从而具有良好的免疫兼容性(避免宿主免疫应答)。所述细胞缺乏引发免疫应答的主要免疫抗原，并且经过改造而具有更好的免疫兼容性(避免宿主免疫应答)。本领域技术人员可以理解，通过本发明所述的构建方法构建获得的人多能干细胞也在本发明的保护范围以内。The invention also provides a human pluripotent stem cell that has good immune compatibility (avoidance of host immune response) due to several genetic manipulations described in the invention. The cells lack the major immune antigens that trigger immune responses and are engineered to be more immune compatible (avoid host immune responses). Those skilled in the art can understand that human pluripotent stem cells constructed through the construction method of the present invention are also within the protection scope of the present invention.
提供从所述人多能干细胞衍生的心肌细胞，然后将其移植到需要的患者中，从而避免人免疫细胞对其的识别与杀伤，还提供了所述的衍生的心肌细胞在制备用于治疗心脏相关的疾病或病症的组合物或药物中的用途，优选的，所述心脏相关的疾病或病症是心肌损伤、心肌梗死、心肌缺血、缺血再灌注损伤或其他心脏损伤；更优选的，所述心脏相关的疾病或病症是缺血/再灌注损伤。也可以说，本发明提供了所述人多能干细胞衍生的心肌细胞在促进心肌梗死后的心肌修复、改善心肌梗死后的心功能、保护心肌缺血损伤的组合物或药物中的应用。Provide cardiomyocytes derived from the human pluripotent stem cells, and then transplant them into patients in need, thereby avoiding recognition and killing by human immune cells, and also provide the preparation of the derived cardiomyocytes for treatment Use in compositions or medicines for heart-related diseases or disorders. Preferably, the heart-related diseases or disorders are myocardial injury, myocardial infarction, myocardial ischemia, ischemia-reperfusion injury or other cardiac injuries; more preferably , the heart-related disease or condition is ischemia/reperfusion injury. It can also be said that the present invention provides the application of cardiomyocytes derived from human pluripotent stem cells in compositions or medicines that promote myocardial repair after myocardial infarction, improve cardiac function after myocardial infarction, and protect myocardial ischemic damage.
在优选的实施方式中，所述心脏相关的疾病或病症是心肌损伤、心肌梗死、心肌缺血、缺血再灌注损伤或其他心脏损伤；更优选的，所述心脏相关的疾病或病症是缺血/再灌注损伤。也可以说，本发明提供了所述人多能干细胞衍生的心肌细胞在促进心肌梗死后的心肌修复、改善心肌梗死后的心功能、保护心肌缺血损伤的组合物或药物中的应用。In a preferred embodiment, the heart-related disease or disorder is myocardial injury, myocardial infarction, myocardial ischemia, ischemia-reperfusion injury or other cardiac injury; more preferably, the heart-related disease or disorder is deficiency. Blood/reperfusion injury. It can also be said that the present invention provides the application of cardiomyocytes derived from human pluripotent stem cells in compositions or medicines that promote myocardial repair after myocardial infarction, improve cardiac function after myocardial infarction, and protect myocardial ischemic damage.
从另一角度讲，本发明提供了从所述人多能干细胞衍生的心肌细胞，然后将其移植到需要的患者中，从而避免人免疫细胞对其的识别与杀伤，还提供了所述人多能干细胞衍生的心肌细胞在制备用于治疗心脏相关的疾病或病症的组合物或药物中的用途。作为本发明优选的一种方式，所述心脏相关的疾病或病症是心肌损伤、心肌梗死、心肌缺血、缺血再灌注损伤或其他心脏损伤。作为本发明更优选的一种方式，所述心脏相关的疾病或病症是缺血/再灌注损伤。也可以说，本发明提供了所述人多能干细胞衍生的心肌细胞在制备用于促进心肌梗死后的心肌修复、改善心肌梗死后的心功能、保护心肌缺血损伤的组合物或药物中的用途。 From another perspective, the present invention provides cardiomyocytes derived from the human pluripotent stem cells, which are then transplanted into patients in need, thereby avoiding recognition and killing by human immune cells, and also provides the human pluripotent stem cells. Use of pluripotent stem cell-derived cardiomyocytes in the preparation of compositions or medicaments for the treatment of heart-related diseases or conditions. As a preferred embodiment of the present invention, the heart-related disease or disorder is myocardial injury, myocardial infarction, myocardial ischemia, ischemia-reperfusion injury or other cardiac injuries. As a more preferred aspect of the present invention, the heart-related disease or disorder is ischemia/reperfusion injury. It can also be said that the present invention provides the use of the human pluripotent stem cell-derived cardiomyocytes in the preparation of compositions or medicines for promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction, and protecting myocardial ischemic damage. use.
从治疗的角度，也可以说，本发明提供了所述人多能干细胞衍生的心肌细胞用于治疗心脏相关的疾病或病症。例如，用于治疗心肌损伤、心肌梗死、心肌缺血、缺血再灌注损伤等疾病或病症。作为本发明优选的一种方式，本发明提供了所述人多能干细胞衍生的心肌细胞用于改善心肌梗死后的心肌修复、改善心肌梗死后的心功能、保护心肌缺血损伤。From a therapeutic perspective, it can also be said that the present invention provides the cardiomyocytes derived from human pluripotent stem cells for the treatment of heart-related diseases or conditions. For example, it is used to treat myocardial injury, myocardial infarction, myocardial ischemia, ischemia-reperfusion injury and other diseases or conditions. As a preferred embodiment of the present invention, the present invention provides the human pluripotent stem cell-derived cardiomyocytes for improving myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction, and protecting myocardial ischemic damage.
本发明还提供了一种组合物，它含有有效量的所述的衍生的心肌细胞，以及药学上可接受的载体。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。活性成分的给药量是治疗有效量。本发明的组合物可直接用于促进心肌梗死后的心肌修复、改善心肌梗死后的心功能、保护心肌缺血损伤。此外，还可同时与其它治疗剂或辅剂联合使用。The present invention also provides a composition, which contains an effective amount of the derived cardiomyocytes and a pharmaceutically acceptable carrier. Such carriers include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, pharmaceutical preparations should match the mode of administration. The pharmaceutical composition of the present invention can be made into an injection form, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The active ingredients are administered in amounts that are therapeutically effective. The composition of the present invention can be directly used to promote myocardial repair after myocardial infarction, improve cardiac function after myocardial infarction, and protect myocardial ischemic damage. In addition, it can also be used in combination with other therapeutic agents or adjuvants.
通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地，pH约为6-8。Typically, these materials may be formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, typically at a pH of about 5-8, preferably at a pH of about 6-8.
本发明所述的衍生的心肌细胞的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。本发明的衍生的心肌细胞的给药方式没有特别的限制，可以是全身的或局部的。例如，本发明的衍生的心肌细胞可通过局部组织注射的方式给予，优选地为心肌注射。此外，其它方式的注射也是可以的，例如包括但不限于腹腔注射、静脉注射、口服、皮下注射、脊髓鞘内注射、皮内注射等的方式给予受试者。The effective amount of the derived cardiomyocytes of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (eg, through clinical trials). The factors include but are not limited to: pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. The administration mode of the derived cardiomyocytes of the present invention is not particularly limited and can be systemic or local. For example, the derived cardiomyocytes of the present invention can be administered by local tissue injection, preferably myocardial injection. In addition, other methods of injection are also possible, such as, but not limited to, intraperitoneal injection, intravenous injection, oral administration, subcutaneous injection, spinal intrathecal injection, intradermal injection, etc. administered to the subject.
本发明的方法Method of the present invention
本发明也提供一种所述人多能干细胞的构建方法，包括：改造人多能干细胞，使之：不表达游离B2M蛋白，表达HLA-G1与分泌型HLA-G5；以及不表达CIITA蛋白。The invention also provides a method for constructing the human pluripotent stem cells, which includes: modifying the human pluripotent stem cells so that they: do not express free B2M protein, express HLA-G1 and secreted HLA-G5; and do not express CIITA protein.
本发明所提供的构建方法中，所述HLA-G1片段的编码基因可以包括HLA-G1的重链阅读框的编码序列，所述HLA-G1片段可以包括：In the construction method provided by the invention, the coding gene of the HLA-G1 fragment may include the coding sequence of the heavy chain reading frame of HLA-G1, and the HLA-G1 fragment may include:
a)氨基酸序列如SEQ ID NO：1所示的多肽片段；或，a) A polypeptide fragment with an amino acid sequence as shown in SEQ ID NO: 1; or,
b)氨基酸序列与SEQ ID NO：1具有90％以上序列同一性且具有a)限定的多肽片段的功能的多肽片段。b) A polypeptide fragment whose amino acid sequence has more than 90% sequence identity with SEQ ID NO: 1 and has the function of the polypeptide fragment defined in a).
HLA-G1重链序列(HLA-G1片段)的氨基酸序列：
Amino acid sequence of HLA-G1 heavy chain sequence (HLA-G1 fragment):
具体的，所述b)中的氨基酸序列具体指：如SEQ ID NO：1所示的氨基酸序列经过取代、缺失或者添加一个或多个(具体可以是1-50、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2 个、或3个)氨基酸而得到的，或者在N-末端和/或C-末端添加一个或多个(具体可以是1-50个、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，且具有氨基酸如SEQ ID NO：1所示的多肽片段的功能的多肽片段。例如，所述HLA-G1片段通常具有完整的α重链结构，与经典型HLA I分子不同，其主要具有免疫抑制的功能(例如，可以与抑制性受体相结合从而可以调控B细胞、T细胞、NK细胞和APC细胞介导的免疫反应等，这些抑制性受体主要包括ILT2/CD85j/LILRB1，ILT4/CD85d/LILRB2，和KIR2DL4/CD158d等)。所述b)中的氨基酸序列可与SEQ ID NO：1具有90％、93％、95％、97％、或99％以上的同一性。所述HLA-G1片段通常来源于人。Specifically, the amino acid sequence in b) specifically refers to: the amino acid sequence shown in SEQ ID NO: 1 through substitution, deletion or addition of one or more (specifically, it can be 1-50, 1-30, 1- 20 pieces, 1-10 pieces, 1-5 pieces, 1-3 pieces, 1 piece, 2 pieces or 3) amino acids, or add one or more (specifically, 1-50, 1-30, 1-20, 1-10) amino acids at the N-terminus and/or C-terminus , 1-5, 1-3, 1, 2, or 3) amino acids, and has the function of the polypeptide fragment whose amino acids are as shown in SEQ ID NO: 1. For example, the HLA-G1 fragment usually has a complete alpha heavy chain structure, which is different from the classic HLA I molecule. It mainly has immunosuppressive functions (for example, it can bind to inhibitory receptors to regulate B cells, T immune responses mediated by cells, NK cells and APC cells, etc. These inhibitory receptors mainly include ILT2/CD85j/LILRB1, ILT4/CD85d/LILRB2, and KIR2DL4/CD158d, etc.). The amino acid sequence in b) may have an identity of more than 90%, 93%, 95%, 97%, or 99% with SEQ ID NO: 1. The HLA-G1 fragment is usually of human origin.
本发明所提供的构建方法中，所述第一B2M片段可以包括：c)氨基酸序列如SEQ ID NO：2所示的多肽片段；或，d)氨基酸序列与SEQ ID NO：2具有90％以上序列同一性且具有c)限定的多肽片段的功能的多肽片段。In the construction method provided by the present invention, the first B2M fragment may include: c) a polypeptide fragment with an amino acid sequence as shown in SEQ ID NO: 2; or, d) an amino acid sequence that has more than 90% similarity with SEQ ID NO: 2 A polypeptide fragment having sequence identity and having the functions of the polypeptide fragment defined in c).
内源性B2M蛋白质(第一B2M片段)的氨基酸序列：
Amino acid sequence of endogenous B2M protein (first B2M fragment):
具体的，所述d)中的氨基酸序列具体指：如SEQ ID NO：2所示的氨基酸序列经过取代、缺失或者添加一个或多个(具体可以是1-50、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，或者在N-末端和/或C-末端添加一个或多个(具体可以是1-50个、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，且具有氨基酸如SEQ ID NO：2所示的多肽片段的功能的多肽片段。例如，所述第一B2M片段通常具有β折叠的片状结构，其主要具有与主要组织相容性复合物I类重链通过非共价键结合的功能。所述d)中的氨基酸序列可与SEQ ID NO：2具有90％、93％、95％、97％、或99％以上的同一性。所述第一B2M片段通常来源于人。Specifically, the amino acid sequence in d) specifically refers to: the amino acid sequence shown in SEQ ID NO: 2 through substitution, deletion or addition of one or more (specifically, it can be 1-50, 1-30, 1- 20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or one or more ( Specifically, it can be obtained from 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, and has The amino acid is a functional polypeptide fragment of the polypeptide fragment shown in SEQ ID NO: 2. For example, the first B2M fragment usually has a β-pleated sheet structure, which mainly has the function of binding to the major histocompatibility complex class I heavy chain through non-covalent bonds. The amino acid sequence in d) may have more than 90%, 93%, 95%, 97%, or 99% identity with SEQ ID NO: 2. The first B2M fragment is typically of human origin.
本发明所提供的构建方法中，所述B2M-HLA-G1融合蛋白还可以包括第一柔性连接肽段，所述第一柔性连接肽段通常位于HLA-G1片段和第一B2M片段之间。所述第一柔性连接肽段通常可以为一段长度合适的由甘氨酸(G)、丝氨酸(S)和/或丙氨酸(A)构成的柔性多肽，从而使相邻的蛋白质结构域可相对于彼此自由移动，例如，所述第一柔性连接肽段的氨基酸序列可以包括如(GS)n、(GGS)n、(GGSG)n、(GGGS)nA、(GGGGS)nA、(GGGGA)nA、(GGGGG)nA等序列，其中，n选自1-10之间的整数。在本发明的一些具体实施例中，所述第一柔性连接肽段的氨基酸序列的长度可以为5-26。在本发明的一些具体实施例中，所述第一柔性连接肽段可以包括氨基酸序列如SEQ ID NO：3所示的多肽片段。在本发明另一些具体实施例中，所述B2M-HLA-G1融合蛋白自N端至C端依次包括第一B2M片段和HLA-G1片段，所述B2M-HLA-G1融合蛋白包括氨基酸序列如SEQ ID NO：4所示的多肽片段。In the construction method provided by the present invention, the B2M-HLA-G1 fusion protein may also include a first flexible connecting peptide segment, and the first flexible connecting peptide segment is usually located between the HLA-G1 fragment and the first B2M fragment. The first flexible connecting peptide segment can usually be a flexible polypeptide of appropriate length consisting of glycine (G), serine (S) and/or alanine (A), so that adjacent protein domains can be relative to each other. freely move with each other. For example, the amino acid sequence of the first flexible connecting peptide segment may include (GS) n , (GGS) n , (GGSG) n , (GGGS) n A, (GGGGS) n A, (GGGGA) n A, (GGGGGG) n A and other sequences, where n is selected from an integer between 1 and 10. In some specific embodiments of the present invention, the length of the amino acid sequence of the first flexible connecting peptide segment may be 5-26. In some specific embodiments of the present invention, the first flexible connecting peptide segment may include a polypeptide fragment with an amino acid sequence as shown in SEQ ID NO: 3. In other specific embodiments of the present invention, the B2M-HLA-G1 fusion protein includes the first B2M fragment and the HLA-G1 fragment in sequence from the N-terminus to the C-terminus, and the B2M-HLA-G1 fusion protein includes an amino acid sequence such as The polypeptide fragment shown in SEQ ID NO: 4.
柔性(G4S)4连接肽段(第一柔性连接肽段)的氨基酸序列：
The amino acid sequence of the flexible (G 4 S) 4- linked peptide segment (the first flexible linking peptide segment):
B2M-HLA-G1融合蛋白的氨基酸序列：

Amino acid sequence of B2M-HLA-G1 fusion protein:

编码B2M-HLA-G1融合蛋白的核酸序列，其中，内源性B2M基因外显子DNA序列加粗，内含子DNA序列加下划线：

Nucleic acid sequence encoding B2M-HLA-G1 fusion protein, in which the exon DNA sequence of the endogenous B2M gene is bolded and the intron DNA sequence is underlined:

本发明所提供的构建方法中，将人多能干细胞的基因组中整合外源的编码B2M-HLA-G1融合蛋白的核酸的方法具体可以包括：将HLA-G1片段的编码基因与人多能干细胞中内源性的B2M基因融合。在本发明的一些具体实施例中，可以将HLA-G1片段的编码基因替换位于内源B2M基因外显子3中的终止密码子。In the construction method provided by the present invention, the method of integrating exogenous nucleic acid encoding B2M-HLA-G1 fusion protein into the genome of human pluripotent stem cells may specifically include: integrating the encoding gene of the HLA-G1 fragment with the human pluripotent stem cell genome. Endogenous B2M gene fusion. In some specific embodiments of the present invention, the stop codon located in exon 3 of the endogenous B2M gene can be replaced by the gene encoding the HLA-G1 fragment.
本发明所提供的构建方法中，所述CIITA片段可以包括：e)氨基酸序列如SEQ ID NO：5所示的多肽片段；或，f)氨基酸序列与SEQ ID NO：5具有90％以上序列同一性且具有e)限定的多肽片段的功能的多肽片段。In the construction method provided by the invention, the CIITA fragment may include: e) a polypeptide fragment with an amino acid sequence as shown in SEQ ID NO: 5; or, f) an amino acid sequence with more than 90% sequence identity as SEQ ID NO: 5 and having the functions of the polypeptide fragment defined in e).
具体的，所述f)中的氨基酸序列具体指：如SEQ ID NO：5所示的氨基酸序列经过取代、缺失或者添加一个或多个(具体可以是1-50、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，或者在N-末端和/或C-末端添加一个或多个(具体可以是1-50个、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，且具有氨基酸如SEQ ID NO：5所示的多肽片段的功能的多肽片段。例如，所述CIITA片段通常具有双或多功能结构域，其主要充当转录激活物，并在主要组织相容性复合物(MHC)II类基因的表达中起关键作用。所述f)中的氨基酸序列可与SEQ ID NO：5具有90％、93％、95％、97％、或99％以上的同一性。所述CIITA片段通常来源于人。Specifically, the amino acid sequence in f) specifically refers to: the amino acid sequence shown in SEQ ID NO: 5 through substitution, deletion or addition of one or more (specifically, it can be 1-50, 1-30, 1- 20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or one or more ( Specifically, it can be obtained from 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, and has The amino acid is a functional polypeptide fragment of the polypeptide fragment shown in SEQ ID NO: 5. For example, the CIITA fragment usually has a bi- or multifunctional domain, which mainly acts as a transcriptional activator and plays a key role in the expression of major histocompatibility complex (MHC) class II genes. The amino acid sequence in f) may have more than 90%, 93%, 95%, 97%, or 99% identity with SEQ ID NO: 5. The CIITA fragment is usually of human origin.
CIITA蛋白的氨基酸序列：
Amino acid sequence of CIITA protein:
本发明所提供的构建方法中，所述不表达CIITA的方法具体可以包括：针对人多能干细胞基因组中CIITA基因的外显子3进行敲除；较佳地，通过基因编辑方法进行敲除；更佳地，以SEQ ID NO：11所示核苷酸序列的gRNA(GGGAGGCTTATGCCAATAT)进行基因编辑。In the construction method provided by the present invention, the method of not expressing CIITA may specifically include: knocking out exon 3 of the CIITA gene in the human pluripotent stem cell genome; preferably, knocking out by a gene editing method; More preferably, the gRNA (GGGAGGCTTATGCCAATAT) with the nucleotide sequence shown in SEQ ID NO: 11 is used for gene editing.
本发明所提供的构建方法中，所述第二B2M片段可以包括：g)氨基酸序列如SEQ ID NO：2所示的多肽片段；或，h)氨基酸序列与SEQ ID NO：2具有90％以上序列同一性且具有g)限定的多肽片段的功能的多肽片段。In the construction method provided by the present invention, the second B2M fragment may include: g) a polypeptide fragment with an amino acid sequence as shown in SEQ ID NO: 2; or, h) an amino acid sequence that has more than 90% similarity with SEQ ID NO: 2 A polypeptide fragment having sequence identity and having the functions of the polypeptide fragment defined in g).
第二B2M片段的氨基酸序列：

Amino acid sequence of the second B2M fragment:

具体的，所述h)中的氨基酸序列具体指：如SEQ ID NO：2所示的氨基酸序列经过取代、缺失或者添加一个或多个(具体可以是1-50、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，或者在N-末端和/或C-末端添加一个或多个(具体可以是1-50个、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，且具有氨基酸如SEQ ID NO：2所示的多肽片段的功能的多肽片段。例如，所述第二B2M片段通常具有β折叠的片状结构，其主要具有与主要组织相容性复合物I类重链通过非共价键结合的功能。所述h)中的氨基酸序列可与SEQ ID NO：2具有90％、93％、95％、97％、或99％以上的同一性。所述第二B2M片段通常来源于人。Specifically, the amino acid sequence in h) specifically refers to: the amino acid sequence shown in SEQ ID NO: 2 through substitution, deletion or addition of one or more (specifically, it can be 1-50, 1-30, 1- 20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or one or more ( Specifically, it can be obtained from 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, and has The amino acid is a functional polypeptide fragment of the polypeptide fragment shown in SEQ ID NO: 2. For example, the second B2M fragment usually has a β-pleated sheet structure, which mainly has the function of binding to the major histocompatibility complex class I heavy chain through non-covalent bonds. The amino acid sequence in h) may have more than 90%, 93%, 95%, 97%, or 99% identity with SEQ ID NO: 2. The second B2M fragment is typically of human origin.
本发明所提供的构建方法中，所述HLA-G5片段包括：i)氨基酸序列如SEQ ID NO：6所示的多肽片段；或，j)氨基酸序列与SEQ ID NO：6具有90％以上序列同一性且具有i)限定的多肽片段的功能的多肽片段。In the construction method provided by the invention, the HLA-G5 fragment includes: i) a polypeptide fragment with an amino acid sequence as shown in SEQ ID NO: 6; or, j) an amino acid sequence with more than 90% of the sequence as SEQ ID NO: 6 A polypeptide fragment that is identical and has the functions of the polypeptide fragment defined in i).
HLA-G5重链序列的氨基酸序列：
Amino acid sequence of HLA-G5 heavy chain sequence:
具体的，所述j)中的氨基酸序列具体指：如SEQ ID NO：6所示的氨基酸序列经过取代、缺失或者添加一个或多个(具体可以是1-50、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，或者在N-末端和/或C-末端添加一个或多个(具体可以是1-50个、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，且具有氨基酸如SEQ ID NO：6所示的多肽片段的功能的多肽片段。例如，所述HLA-G5片段通常具有完整的α重链胞外结构，与经典型HLA I分子不同，其主要具有免疫抑制的功能(例如，可以与抑制性受体相结合从而可以调控B细胞、T细胞、NK细胞和APC细胞介导的免疫反应等，这些抑制性受体主要包括ILT2/CD85j/LILRB1，ILT4/CD85d/LILRB2，和KIR2DL4/CD158d等)。所述j)中的氨基酸序列可与SEQ ID NO：6具有90％、93％、95％、97％、或99％以上的同一性(Sequence identity)。所述HLA-G5片段通常来源于人。Specifically, the amino acid sequence in j) specifically refers to: the amino acid sequence shown in SEQ ID NO: 6 through substitution, deletion or addition of one or more (specifically, it can be 1-50, 1-30, 1- 20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or one or more ( Specifically, it can be obtained from 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, and has The amino acid is a functional polypeptide fragment of the polypeptide fragment shown in SEQ ID NO: 6. For example, the HLA-G5 fragment usually has a complete extracellular structure of the α heavy chain, which is different from the classic HLA I molecule and mainly has immunosuppressive functions (for example, it can bind to inhibitory receptors and thereby regulate B cells , T cells, NK cells and APC cell-mediated immune responses, etc. These inhibitory receptors mainly include ILT2/CD85j/LILRB1, ILT4/CD85d/LILRB2, and KIR2DL4/CD158d, etc.). The amino acid sequence in j) may have an identity (Sequence identity) of more than 90%, 93%, 95%, 97%, or 99% with SEQ ID NO: 6. The HLA-G5 fragment is usually of human origin.
本发明所提供的构建方法中，所述B2M-HLA-G5融合蛋白还可以包括第二柔性连接肽段，所述第二柔性连接肽段通常位于HLA-G5片段和第二B2M片段之间。所述第二柔性连接肽段通常可以为一段长度合适的由甘氨酸(G)、丝氨酸(S)和/或丙氨酸(A)构成的柔性多肽，从而使相邻的蛋白质结构域可相对于彼此自由移动，例如，所述第二柔性连接肽段的氨基酸序列可以包括如(GS)n、(GGS)n、(GGSG)n、(GGGS)nA、(GGGGS)nA、(GGGGA)nA、(GGGGG)nA等序列，其中，n选自1-10之间的整数。在本发明的一些具体实施例中，所述第二柔性连接肽段的氨基酸序列的长度可以为5-26。在本发明的一些具体实施例中，所述第二柔性连接肽段可以包括氨基酸序列如SEQ ID NO：5所示的多肽片段。在本发明另一具体实施例中，所述B2M-HLA-G5融合蛋白自N端至C端依次包括第二B2M片段和HLA-G5片段，所述B2M-HLA-G5融合蛋白 包括氨基酸序列如SEQ ID NO：8所示的多肽片段。In the construction method provided by the present invention, the B2M-HLA-G5 fusion protein may also include a second flexible connecting peptide segment, and the second flexible connecting peptide segment is usually located between the HLA-G5 fragment and the second B2M fragment. The second flexible connecting peptide segment can usually be a flexible polypeptide of appropriate length consisting of glycine (G), serine (S) and/or alanine (A), so that adjacent protein domains can be relative to each other. freely move with each other. For example, the amino acid sequence of the second flexible connecting peptide segment may include (GS)n, (GGS)n, (GGSG)n, (GGGS)nA, (GGGGS)nA, (GGGGA)nA, Sequences such as (GGGGGG)nA, where n is selected from an integer between 1 and 10. In some specific embodiments of the present invention, the length of the amino acid sequence of the second flexible connecting peptide segment may be 5-26. In some specific embodiments of the present invention, the second flexible connecting peptide segment may include a polypeptide fragment with an amino acid sequence as shown in SEQ ID NO: 5. In another specific embodiment of the present invention, the B2M-HLA-G5 fusion protein includes the second B2M fragment and the HLA-G5 fragment in sequence from the N-terminus to the C-terminus. The B2M-HLA-G5 fusion protein It includes a polypeptide fragment with an amino acid sequence as shown in SEQ ID NO: 8.
柔性(G4S)4连接肽段(第二柔性连接肽段)的氨基酸序列：
The amino acid sequence of the flexible (G4S) 4-linked peptide segment (the second flexible linking peptide segment):
B2M-HLA-G5融合蛋白的氨基酸序列：
Amino acid sequence of B2M-HLA-G5 fusion protein:
编码B2M-HLA-G5融合蛋白的核酸序列：
Nucleic acid sequence encoding B2M-HLA-G5 fusion protein:
本发明所提供的构建方法中，将人多能干细胞的基因组中整合外源的编码B2M-HLA-G5融合蛋白的核酸的方法具体可以包括：通过慢病毒载体将编码B2M-HLA-G5融合蛋白的核酸整合入人多能干细胞的基因组，使其可以表达B2M-HLA-G5融合蛋白。In the construction method provided by the present invention, the method of integrating exogenous nucleic acid encoding B2M-HLA-G5 fusion protein into the genome of human pluripotent stem cells can specifically include: integrating the encoding B2M-HLA-G5 fusion protein through a lentiviral vector. The nucleic acid is integrated into the genome of human pluripotent stem cells, allowing them to express B2M-HLA-G5 fusion protein.
本发明所提供的人多能干细胞的构建方法可以成功地在人类多能干细胞的两个内源性B2M位点整合了HLA-G1片段序列，敲除CIITA片段序列，并且通过慢病毒载体将编码B2M-HLA-G5融合蛋白的序列整合入了人多能干细胞的基因组中。构建获得的人多能干细胞细胞系内源性HLA-A，-B，-C蛋白无法到达细胞膜表面。同时，还进行了CIITA的敲除，获得免疫兼容性非常优异、且多能性以及增殖的能力并未受到影响的多能干细胞；进一步地，该多能干细胞可作为细胞移植的目的细胞来源。The construction method of human pluripotent stem cells provided by the present invention can successfully integrate the HLA-G1 fragment sequence into two endogenous B2M sites of human pluripotent stem cells, knock out the CIITA fragment sequence, and encode the human pluripotent stem cell through a lentiviral vector. The sequence of the B2M-HLA-G5 fusion protein is integrated into the genome of human pluripotent stem cells. The endogenous HLA-A, -B, and -C proteins of the constructed human pluripotent stem cell line cannot reach the cell membrane surface. At the same time, CIITA was also knocked out to obtain pluripotent stem cells with excellent immune compatibility and unaffected pluripotency and proliferation ability; further, the pluripotent stem cells can be used as a source of target cells for cell transplantation.
在一些实施方案中，破坏多能干细胞中基因的表达在本文中通常称为基因“敲除”。通常，完成两个破坏的技术是相同的。可以实现基因敲除的手段包括但不限于ZFN、TALEN和CRISPR/Cas9技术。其中，特别有用的实施方案是使用CRISPR/Cas9技术破坏基因。在一些情况下，CRISPR/Cas9技术用于将小的缺失/***引入基因的编码区，使得不产生功能性蛋白，例如发生移码突变，其导致终止密码子的产生，使得产生截短的、非功能性蛋白质。In some embodiments, disrupting the expression of a gene in a pluripotent stem cell is generally referred to herein as a gene "knockout." Usually, the technique for accomplishing both breaches is the same. Means that can achieve gene knockout include but are not limited to ZFN, TALEN and CRISPR/Cas9 technology. Among these, a particularly useful embodiment is the use of CRISPR/Cas9 technology to disrupt genes. In some cases, CRISPR/Cas9 technology is used to introduce small deletions/insertions into the coding region of a gene such that no functional protein is produced, such as when a frameshift mutation occurs, which results in the creation of a stop codon, resulting in the production of a truncated, Non-functional proteins.
为了使得CIITA或B2M作为目的基因不表达，其它一些下调目的基因表达的技术也是可选的。作为一种可选方式，利用目的基因特异性的干扰RNA分子(如siRNA、shRNA、miRNA等)，根据本发明中提供的CIITA或B2M序列信息，可以制备获得此类干扰RNA分子。所述的干扰RNA可通过采用适当的转染试剂被输送到细胞内，或还可采用本领域已知的多种 技术被输送到细胞内。siRNAs的长度通常约为21个核苷酸(例如21-23个核苷酸)。在将小RNA或RNAi导入细胞后，该序列被传递到称为RISC(RNA诱导沉默复合物)的酶复合物，RISC识别目标并用核酸内切酶切割它。作为另一种可选方式，利用shRNA技术进行干扰作用。shRNA是一种RNA序列，它可以使一个紧密的发夹旋转，可以用来沉默基因表达通过RNA干扰。shRNA使用导入细胞的载体并利用启动子(如U6)来确保shRNA始终被表达。作为另一种可选方式，利用与目的基因特异性杂交的反义化合物来调节目的基因表达。寡聚物与其靶核酸的特异性杂交干扰了核酸的正常功能。这种通过与靶核酸特异性杂交的化合物对靶核酸功能的调节通常被称为“反义”。作为另一种可选方式，可采用同源重组的方法，特异性地靶向于目的基因，使之发生表达缺陷或缺失表达。尽管如此，基于CRISPR的方案为优选的实施方案。本领域技术人员可以理解，由本发明所提供的人多能干细胞的构建方法构建获得的人多能干细胞也可以被包含在本发明中。In order to prevent the expression of CIITA or B2M as the target gene, other techniques for down-regulating the expression of the target gene are also optional. As an alternative, using target gene-specific interfering RNA molecules (such as siRNA, shRNA, miRNA, etc.), such interfering RNA molecules can be prepared according to the CIITA or B2M sequence information provided in the present invention. The interfering RNA can be delivered into cells by using appropriate transfection reagents, or various methods known in the art can be used. Technology is delivered into cells. siRNAs are typically approximately 21 nucleotides in length (eg, 21-23 nucleotides). After small RNA, or RNAi, is introduced into a cell, the sequence is passed to an enzyme complex called RISC (RNA-induced silencing complex), which recognizes the target and cleaves it with an endonuclease. As another alternative, shRNA technology is used for interference. shRNA is an RNA sequence that twists a tight hairpin and can be used to silence gene expression through RNA interference. shRNA uses a vector introduced into cells and uses a promoter (such as U6) to ensure that the shRNA is always expressed. As another alternative, antisense compounds that specifically hybridize to the gene of interest are used to modulate the expression of the gene of interest. Specific hybridization of the oligomer to its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of the function of a target nucleic acid by a compound that specifically hybridizes to the target nucleic acid is often referred to as "antisense." As another alternative, homologous recombination can be used to specifically target the gene of interest, causing expression defects or loss of expression. Nonetheless, CRISPR-based approaches are the preferred embodiment. Those skilled in the art can understand that human pluripotent stem cells constructed by the method for constructing human pluripotent stem cells provided by the present invention can also be included in the present invention.
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，中所述的条件，或按照制造厂商所建议的条件。The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. Experimental methods without specifying specific conditions in the following examples usually follow conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experimental Guide, Third Edition, Science Press, or according to the conditions specified by the manufacturer. Suggested conditions.
除非另外说明，以下数据统计采用Graphpad软件进行，两组数据之间比较采用t检验方法检验是否具有统计学意义；3-4组数据之间的比较采用单因素方差分析(one-way ANOVA)，事后检验采用Dunnett方法。P值小于0.05被认为具有差异具有统计学意义。Unless otherwise stated, the following data statistics were performed using Graphpad software. The comparison between two groups of data was tested using the t test method to see whether there is statistical significance; the comparison between 3-4 groups of data was performed using one-way ANOVA. Post hoc testing was performed using Dunnett's method. A P value less than 0.05 was considered a difference to be statistically significant.
实施例1、B2M/CIITA双敲除hPSCs(DKO-hPSCs)的构建Example 1. Construction of B2M/CIITA double knockout hPSCs (DKO-hPSCs)
本发明的后续实施例中，所用hPSCs来自于人胚胎干细胞，为H9株系。In subsequent embodiments of the present invention, the hPSCs used are derived from human embryonic stem cells and are H9 strains.
为比较CIITA-/-B2Mm/sHLAG-hPSCs衍生细胞的免疫兼容性，构建B2M/CIITA双敲除hPSCs作为对照。利用基因编辑方法(本实施例中采用CRISPR/Cas9体系)，同时敲除B2M与CIITA基因。To compare the immune compatibility of CIITA −/− B2Mm/sHLAG-hPSCs-derived cells, B2M/CIITA double knockout hPSCs were constructed as controls. Using gene editing methods (CRISPR/Cas9 system is used in this example), the B2M and CIITA genes are knocked out simultaneously.
根据B2M与CIITA基因转录本信息，经过分析研究，本发明人优化了适合基因编辑位置的序列，基于此设计靶向该基因组位置的gRNA，gRNA序列见表1。分别将gRNA构建到U6启动子启动的质粒上，获得gRNA表达质粒gRNA-B2M与gRNA-CIITA。在构建DKO-hPSCs中先将gRNA-B2M质粒与Cas9-T2A-GFP(Addgene#44719)质粒一起转入hPSCs，培养4天后进行低密度单细胞铺板，单克隆生长后手动挑取并进行基因组DNA PCR及测序分析，选取存在非3整倍数碱基缺失或***的克隆为B2M敲除hPSCs。According to the B2M and CIITA gene transcript information, after analysis and research, the inventor optimized the sequence suitable for the gene editing position, and based on this, designed the gRNA targeting the genomic position. The gRNA sequence is shown in Table 1. The gRNA was constructed into the plasmid initiated by the U6 promoter respectively, and the gRNA expression plasmids gRNA-B2M and gRNA-CIITA were obtained. In constructing DKO-hPSCs, the gRNA-B2M plasmid and Cas9-T2A-GFP (Addgene#44719) plasmid are first transferred into hPSCs. After 4 days of culture, low-density single-cell plating is performed. After single clone growth, the genomic DNA is manually picked and analyzed. PCR and sequencing analysis were performed to select clones with non-three-fold base deletions or insertions as B2M knockout hPSCs.
在此基础上进一步构建CIITA敲除hPSCs，方法同上，但将gRNA质粒换成gRNA-CIITA质粒，并进一步挑取CIITA敲除的单克隆。通过以上方法构建经敲除的hPSCs。获得发生非3倍数碱基移码突变的克隆，作为B2M/CIITA双敲除hPSCs(DKO-hPSCs)(图2A)。On this basis, CIITA knockout hPSCs were further constructed using the same method as above, but the gRNA plasmid was replaced with gRNA-CIITA plasmid, and CIITA knockout single clones were further selected. Knockout hPSCs were constructed by the above method. Clones with non-3-fold base frameshift mutations were obtained as B2M/CIITA double knockout hPSCs (DKO-hPSCs) (Figure 2A).
表1、靶向于B2M、CIITA位点的gRNA序列
Table 1. gRNA sequences targeting B2M and CIITA sites
实施例2、CIITA-/-B2Mm/sHLAG-hPSCs的构建Example 2. Construction of CIITA -/- B2Mm/sHLAG-hPSCs
构建CIITA-/-B2Mm/sHLAG-hPSCs方法为，在专利申请CN113528448A所构建细胞基础上，利用基因编辑方法(本实施例中采用CRISPR/Cas9)敲除CIITA基因，具体为(图1)：The method of constructing CIITA -/- B2Mm/sHLAG-hPSCs is to use the gene editing method (CRISPR/Cas9 in this example) to knock out the CIITA gene based on the cells constructed in the patent application CN113528448A, specifically (Figure 1):
在野生型hPSCs中采用CRISPR/Cas9基因编辑方法在B2M基因编码框终止密码子之前***有编码柔性(G4S)4连接肽段连接的HLA-G1编码基因(重组表达B2M-HLA-G1融合蛋白，SEQ ID NO：4)，构建获得的细胞系只表达HLA-G1/B2M复合物，其他HLA I家族分子因为缺少B2M亚基，无法正常表达在细胞膜上。然后，在上述细胞基因组中导入B2M-(G4S)4-HLA-G5的编码框：将无终止密码子的B2M CDS序列(SEQ ID NO：2)、(Gly4Ser)4柔性连接肽编码序列和HLA-G5重链编码序列整合到具有嘌呤霉素抗性的慢病毒载体pLVX-CAG-Puro的BamHI、MluI中，将获得的重组慢病毒载体引入到细胞中，获得B2Mm/sHLAG hPSCs(参见CN113528448A中对应的阐述)。In wild-type hPSCs, the CRISPR/Cas9 gene editing method was used to insert the HLA-G1 coding gene (recombinant expression of B2M-HLA-G1 fusion protein) before the stop codon of the B2M gene coding frame, which encodes a flexible (G4S) 4-linked peptide segment. SEQ ID NO: 4), the constructed cell line only expresses the HLA-G1/B2M complex. Other HLA I family molecules cannot be expressed normally on the cell membrane due to the lack of B2M subunits. Then, introduce the B2M-(G4S)4-HLA-G5 coding frame into the above-mentioned cell genome: combine the B2M CDS sequence without stop codon (SEQ ID NO: 2), (Gly4Ser)4 flexible linker peptide coding sequence and HLA -The G5 heavy chain coding sequence was integrated into BamHI and MluI of the puromycin-resistant lentiviral vector pLVX-CAG-Puro, and the obtained recombinant lentiviral vector was introduced into the cells to obtain B2Mm/sHLAG hPSCs (see CN113528448A Corresponding explanation).
进一步地，利用基因编辑方法(本实施例中采用CRISPR/Cas9体系)敲除CIITA基因，通过LONZA nucleofection转染gRNA-CIITA质粒(其中gRNA序列如表1)与Cas9-T2A-GFP质粒，并挑取单克隆进行DNA PCR测序，选择发生非3倍数碱基移码突变的克隆，获得CIITA敲除的B2Mm/sHLAG hPSCs，构建免疫兼容型hPSCs，命名为CIITA-/-B2Mm/sHLAG-hPSCs(图2B)。后续的实施例所用的克隆中，CIITA的mRNA序列中ATGGAAGGTGATGAAGAGAC后缺失5个碱基，ATGGAAGGTGATGAAGAGACCAGGG后***1个碱基，导致CIITA蛋白质第90或92位后发生移码突变、引起CIITA蛋白在第124位或126位发生提前终止。Further, the CIITA gene was knocked out using a gene editing method (the CRISPR/Cas9 system was used in this example), and the gRNA-CIITA plasmid (the gRNA sequence is shown in Table 1) and Cas9-T2A-GFP plasmid were transfected through LONZA nucleofection, and the Single clones were taken for DNA PCR sequencing, and clones with non-3-fold base frameshift mutations were selected to obtain CIITA-knockout B2Mm/sHLAG hPSCs and construct immune-compatible hPSCs, named CIITA -/- B2Mm/sHLAG-hPSCs (Figure 2B). In the clones used in subsequent examples, in the CIITA mRNA sequence, 5 bases were deleted after ATGGAAGGTGATGAAGAGAC, and 1 base was inserted after ATGGAAGGTGATGAAGAGACCAGGG, resulting in a frameshift mutation after position 90 or 92 of the CIITA protein, causing the CIITA protein to change at position 124. Bit or 126 early termination occurred.
该细胞可进一步利用组织特异性细胞系分化为如心肌细胞、内皮细胞、神经细胞、NK细胞等免疫兼容型功能细胞。The cells can be further differentiated into immune-compatible functional cells such as cardiomyocytes, endothelial cells, neural cells, NK cells, etc. using tissue-specific cell lines.
实施例3、多能性标志物鉴定Example 3. Identification of pluripotency markers
为了鉴定DKO-hPSCs和CIITA-/-B2Mm/sHLAG-hPSCs自我更新能力，使用野生型hPSCs(wt-hPSCs)作为对照。采用流式细胞术及免疫荧光鉴定wt-hPSCs，DKO-hPSCs和CIITA-/-B2Mm/sHLAG-hPSCs的多能性标志物表达情况。To identify the self-renewal ability of DKO-hPSCs and CIITA −/- B2Mm/sHLAG-hPSCs, wild-type hPSCs (wt-hPSCs) were used as a control. Flow cytometry and immunofluorescence were used to identify the expression of pluripotency markers in wt-hPSCs, DKO-hPSCs and CIITA -/- B2Mm/sHLAG-hPSCs.
流式细胞术鉴定方法：Flow cytometry identification method:
培养的wt-hPSCs，DKO-hPSCs和CIITA-/-B2Mm/sHLAG-hPSCs生长至70％汇合度，Accutase消化成单细胞，离心收集细胞后固定、破膜并用hPSCs多能性细胞标志物OCT4及SSEA4抗体孵育，然后利用APC荧光基团偶联的二抗染色，采用流式细胞术分析。对照组采用同型对照抗体。检测结果见图3A-B。The cultured wt-hPSCs, DKO-hPSCs and CIITA-/-B2Mm/sHLAG-hPSCs were grown to 70% confluence, digested with Accutase into single cells, collected by centrifugation, fixed, ruptured, and used with hPSCs pluripotent cell markers OCT4 and The cells were incubated with SSEA4 antibodies, then stained with APC fluorophore-conjugated secondary antibodies, and analyzed by flow cytometry. Isotype control antibodies were used in the control group. The test results are shown in Figure 3A-B.
细胞免疫荧光鉴定方法： Cell immunofluorescence identification method:
培养的wt-hPSCs，DKO-hPSCs和CIITA-/-B2Mm/sHLAG-hPSCs克隆生长至合适大小，吸去培养基后固定、破膜并用多能性细胞标志物OCT4及SOX2抗体孵育，然后利用Alexa 488荧光基团偶联的二抗染色，采用激光共聚焦显微镜进行图像采集。检测结果见图4。The cultured wt-hPSCs, DKO-hPSCs and CIITA-/-B2Mm/sHLAG-hPSCs clones were grown to a suitable size, the culture medium was aspirated, fixed, ruptured and incubated with pluripotent cell markers OCT4 and SOX2 antibodies, and then used Alexa 488 fluorophore-coupled secondary antibody staining, and image collection using laser confocal microscopy. The test results are shown in Figure 4.
如图3所示，三种细胞中多能性标志物OCT4及SSEA4表达阳性率均在98％以上；如图4所示，多能性标志物OCT4和SOX2均有良好的核定位特征。As shown in Figure 3, the positive rates of expression of pluripotency markers OCT4 and SSEA4 in the three types of cells were above 98%; as shown in Figure 4, the pluripotency markers OCT4 and SOX2 both had good nuclear localization characteristics.
以上数据证明，所获得的DKO-hPSCs及CIITA-/-B2Mm/sHLAG-hPSCs与wt-hPSCs在多能性标志物表达方面没有差异，表明自我更新能力均良好。The above data prove that there is no difference in the expression of pluripotency markers between the obtained DKO-hPSCs and CIITA -/- B2Mm/sHLAG-hPSCs and wt-hPSCs, indicating that both have good self-renewal abilities.
实施例4、HLA分子表达鉴定Example 4. HLA molecular expression identification
为了鉴定利用以上方法构建的细胞是否表达HLA分子，本发明人首先采用流式细胞术进行鉴定。In order to identify whether the cells constructed using the above method express HLA molecules, the inventors first used flow cytometry for identification.
流式细胞术鉴定方法：Flow cytometry identification method:
wt-hPSCs，DKO-hPSCs和CIITA-/-B2Mm/sHLAG-hPSCs细胞在培养体系中加入干扰素gamma(IFN-γ)，终浓度为100ng/ml，培养48小时后，采用Accutase消化成单细胞，离心收集细胞用于检测HLA分子表达。因HLA I及HLA II家族成员众多，采用具代表性的HLA-A，-B，-C(属于HLA I家族代表)及HLA-DR(属于HLA II家族代表)表征wt-hPSCs，DKO-hPSCs和CIITA-/-B2Mm/sHLAG-hPSCs中HLA I及HLA II分子的表达情况。HLA-A/B/C抗体偶联FITC荧光基团，HLA-DR抗体偶联PE荧光基团。将单细胞收集后直接与对应抗体共孵育，并用流式细胞术检测HLA I及HLA II分子表达。HLA-G1鉴定也采用上述流式细胞术方法。Interferon gamma (IFN-γ) was added to the culture system of wt-hPSCs, DKO-hPSCs and CIITA -/- B2Mm/sHLAG-hPSCs at a final concentration of 100ng/ml. After 48 hours of culture, Accutase was used to digest them into single cells. , cells were collected by centrifugation for detection of HLA molecule expression. Since there are many members of the HLA I and HLA II families, the representative HLA-A, -B, -C (representatives of the HLA I family) and HLA-DR (representatives of the HLA II family) are used to characterize wt-hPSCs and DKO-hPSCs. and the expression of HLA I and HLA II molecules in CIITA -/- B2Mm/sHLAG-hPSCs. HLA-A/B/C antibodies are coupled to FITC fluorophore, and HLA-DR antibodies are coupled to PE fluorophore. Single cells were collected and directly incubated with corresponding antibodies, and flow cytometry was used to detect the expression of HLA I and HLA II molecules. HLA-G1 identification also uses the flow cytometry method described above.
HLA-A，-B，-C是经典HLA I家族分子成员，比较wt-hPSCs、DKO-hPSCs及CIITA-/-B2Mm/sHLAG-hPSCs中HLA-A，-B，-C的表达，发现HLA-A，-B，-C仅在wt-hPSCs中表达，其表达未在DKO-hPSCs及CIITA-/-B2Mm/sHLAG-hPSCs中检测到(图5A)；而具有免疫抑制作用的HLA-G1分子仅在CIITA-/-B2Mm/sHLAG-hPSCs中表达(图5B)。利用干扰素gamma(IFN-γ)处理hPSCs 48小时候，流式检测HLA-DR的表达，显示在wt中荧光信号值显著高于DKO-hPSCs及CIITA-/-B2Mm/sHLAG-hPSCs细胞(图5C)。HLA-A, -B, and -C are members of the classic HLA I family. Comparing the expression of HLA-A, -B, and -C in wt-hPSCs, DKO-hPSCs, and CIITA -/- B2Mm/sHLAG-hPSCs, we found that HLA -A, -B, -C are only expressed in wt-hPSCs, and their expression is not detected in DKO-hPSCs and CIITA -/- B2Mm/sHLAG-hPSCs (Figure 5A); while HLA-G1 has immunosuppressive effects The molecule was expressed only in CIITA −/− B2Mm/sHLAG-hPSCs (Fig. 5B ). hPSCs were treated with interferon gamma (IFN-γ) for 48 hours, and the expression of HLA-DR was detected by flow cytometry, which showed that the fluorescence signal value in wt was significantly higher than that in DKO-hPSCs and CIITA -/- B2Mm/sHLAG-hPSCs (Figure 5C ).
利用免疫印迹方法鉴定分泌型HLA-G5分子表达。The expression of secreted HLA-G5 molecules was identified using immunoblotting method.
免疫印迹鉴定分泌型HLA-G5表达方法：Western blotting method to identify secretory HLA-G5 expression:
wt-hPSCs，DKO-hPSCs和CIITA-/-B2Mm/sHLAG-hPSCs细胞在hPSCs培养基中培养至50％汇合度，换成DMEM/F12基础培养基培养24小时，收集培养基上清，0.25μm滤膜过滤滤去细胞碎片，利用10kd超滤管浓缩上清。进行免疫印迹鉴定之前加入loading buffer(含β-巯基乙醇)至终浓度为1x，95℃水浴10分钟使蛋白质变性，采用10％SDS-PAGE凝胶电泳对蛋白进行分离，检测抗体为5A6G7。wt-hPSCs, DKO-hPSCs and CIITA -/- B2Mm/sHLAG-hPSCs cells were cultured in hPSCs medium to 50% confluence, then replaced with DMEM/F12 basal medium and cultured for 24 hours, and the culture supernatant was collected, 0.25 μm The cell debris was removed by filtration through a membrane, and the supernatant was concentrated using a 10kd ultrafiltration tube. Before performing immunoblot identification, add loading buffer (containing β-mercaptoethanol) to a final concentration of 1x, denature the protein in a 95°C water bath for 10 minutes, and use 10% SDS-PAGE gel electrophoresis to separate the protein. The detection antibody is 5A6G7.
结果证明CIITA-/-B2Mm/sHLAG-hPSCs可以表达HLA-G5，wt-hPSCs和DKO-hPSCs不表达HLA-G5(图5D)。 The results demonstrated that CIITA −/− B2Mm/sHLAG-hPSCs could express HLA-G5, while wt-hPSCs and DKO-hPSCs did not express HLA-G5 (Fig. 5D).
实施例5、内皮细胞及心肌细胞的诱导分化和鉴定Example 5. Induced differentiation and identification of endothelial cells and cardiomyocytes
hPSCs可以通过体外诱导方法获得多种组织类型细胞，在本实施例中，本发明人将hPSCs向内皮和心肌细胞分化。hPSCs can be obtained from various tissue types through in vitro induction methods. In this example, the inventor differentiated hPSCs into endothelial and cardiomyocytes.
1、内皮细胞的分化1. Differentiation of endothelial cells
wt-hPSCs，DKO-hPSCs和CIITA-/-B2Mm/sHLAG-hPSCs细胞生长至融合度为100％时更换为分化培养基RPMI1640+0.2％BSA和6μM CHIR99021处理2天，然后更换培养基为RPMI1640+0.2％BSA+50ng/ml bFGF处理1天后，更换为RPMI1640+0.2％BSA+50ng/ml VEGF+50ng/ml BMP4处理2天。第5天采用美天旎CD144(VE-Cadherin)MicroBeads磁珠纯化内皮细胞。纯化后的细胞采用ECM培养基进一步培养。When wt-hPSCs, DKO-hPSCs and CIITA -/- B2Mm/sHLAG-hPSCs cells grow to 100% confluence, they are replaced with differentiation medium RPMI1640+0.2% BSA and 6μM CHIR99021 for 2 days, and then the medium is replaced with RPMI1640+ After 1 day of treatment with 0.2% BSA+50ng/ml bFGF, it was replaced with RPMI1640+0.2% BSA+50ng/ml VEGF+50ng/ml BMP4 for 2 days. On day 5, Miltenyi CD144 (VE-Cadherin) MicroBeads magnetic beads were used to purify endothelial cells. The purified cells were further cultured in ECM medium.
取分化的细胞(内皮细胞，第6天；心肌细胞，第14天)，Accutase消化为单细胞，采用70μm滤网过滤去掉细胞团块。内皮细胞采用APC偶联的CD144抗体直接与细胞孵育进行检测；心肌细胞经过固定、破膜，用心肌细胞标志物cTNT抗体作为一抗，APC偶联的抗小鼠IgG抗体作为二抗进行检测。Differentiated cells (endothelial cells, day 6; cardiomyocytes, day 14) were collected, digested with Accutase into single cells, and filtered with a 70 μm filter to remove cell clumps. Endothelial cells were detected by incubating the APC-conjugated CD144 antibody directly with the cells; cardiomyocytes were fixed and ruptured, and the cardiomyocyte marker cTNT antibody was used as the primary antibody, and the APC-conjugated anti-mouse IgG antibody was used as the secondary antibody for detection.
结果显示，在内皮细胞分化中，经过纯化后的内皮细胞具备良好的内皮细胞形态及极高纯度的内皮细胞标志物CD144表达比例(图6A)。因此，分化获得免疫兼容型内皮细胞。The results showed that in endothelial cell differentiation, the purified endothelial cells had good endothelial cell morphology and extremely high purity expression ratio of endothelial cell marker CD144 (Figure 6A). Thus, immune-compatible endothelial cells are obtained through differentiation.
2、心肌细胞的分化2. Differentiation of cardiomyocytes
wt-hPSCs，DKO-hPSCs和CIITA-/-B2Mm/sHLAG-hPSCs细胞生长至融合度达到100％时更换为分化培养基RPMI1640+0.2％BSA+6μM CHIR99021处理2天，然后更换RPMI1640+0.2％BSA+5μM IWR-1处理2天，之后每2天更换RPMI1640+0.2％BSA。第7天开始出现跳动心肌细胞，根据起始hPSCs的不同类型分别命名为wt-CMs，DKO-CMs和CIITA-/-B2Mm/sHLAG-hPSCs。When wt-hPSCs, DKO-hPSCs and CIITA -/- B2Mm/sHLAG-hPSCs cells grow to 100% confluence, they are replaced with differentiation medium RPMI1640+0.2%BSA+6μM CHIR99021 for 2 days, and then replaced with RPMI1640+0.2%BSA +5 μM IWR-1 for 2 days, then replaced with RPMI1640 + 0.2% BSA every 2 days. Beating cardiomyocytes began to appear on day 7, and were named wt-CMs, DKO-CMs and CIITA-/-B2Mm/sHLAG-hPSCs according to the different types of starting hPSCs.
心肌细胞的细胞免疫荧光鉴定方法同实施例3所述的hPSCs多能性标志物检测方法，将一抗替换为心肌细胞标志物cTNT抗体与间隙连接Connexin 43抗体。The cell immunofluorescence identification method of cardiomyocytes is the same as the hPSCs pluripotency marker detection method described in Example 3, except that the primary antibody is replaced with the cardiomyocyte marker cTNT antibody and the gap junction Connexin 43 antibody.
在分化的心肌细胞中，通过肌节蛋白alpha-ACTININ染色，三种hPSCs来源的心肌细胞均具备良好的肌丝结构，而且细胞之间形成良好的间隙连接(Connexin-43)(图6B)。免疫荧光鉴定肌节蛋白α-ACTININ和间隙蛋白Connexin-43在分化的心肌细胞中的表达如图6C。In differentiated cardiomyocytes, through sarcomeric protein alpha-ACTININ staining, cardiomyocytes derived from three hPSCs all have a good myofilament structure, and good gap junctions (Connexin-43) are formed between cells (Figure 6B). Immunofluorescence identification of the expression of sarcomeric protein α-ACTININ and gap protein Connexin-43 in differentiated cardiomyocytes is shown in Figure 6C.
因此，分化获得免疫兼容型心肌细胞(CIITA-/-B2Mm/sHLAG-CMs)。Therefore, immune-compatible cardiomyocytes (CIITA -/- B2Mm/sHLAG-CMs) were obtained through differentiation.
实施例6、免疫识别与杀伤作用Example 6. Immune recognition and killing effect
为探究CIITA-/-B2Mm/sHLAG-hPSCs及其衍生细胞是否具备逃逸免疫细胞识别与杀伤的作用，本发明人采用体外免疫细胞(PBMCs或NK92)与CIITA-/-B2Mm/sHLAG-hPSCs源心肌细胞共培养方式进行鉴定。T细胞进行激活标志物检测、增殖检测、对靶细胞的杀伤作用检测和分泌的IFN-γ检测。NK细胞进行靶细胞的识别与杀伤检测和分泌的IFN-γ检测。In order to explore whether CIITA -/- B2Mm/sHLAG-hPSCs and their derived cells have the ability to escape immune cell recognition and killing, the inventors used in vitro immune cells (PBMCs or NK92) and CIITA -/- B2Mm/sHLAG-hPSCs-derived myocardium. Cell co-culture method for identification. T cells were tested for activation markers, proliferation, killing effect on target cells, and secreted IFN-γ. NK cells perform target cell recognition and killing detection and secreted IFN-γ detection.
1、T细胞激活标志物检测1. T cell activation marker detection
T细胞会被不匹配的HLA分子激活，因此利用wt-CMs作为对照和 CIITA-/-B2Mm/sHLAG-hPSCs比较对T细胞的激活作用。将wt-CMs和CIITA-/-B2Mm/sHLAG-hPSCs铺于96孔板，24小时候加入终浓度100ng/ml IFN-γ作用48小时，以刺激HLA分子的表达。之后撤去IFN-γ，按照心肌细胞与人外周血单核细胞(PMBCs)数目比1∶3加入原代外周血单核细胞(PBMCs，其中富含T细胞)，共培养48小时后，收集共培养的PBMCs，孵育T细胞标志物CD3抗体与T细胞早期激活标志物CD69抗体，然后进行流式细胞检测。通过CD69阳性细胞在CD3阳性细胞中所占比例比较wt-CMs和CIITA-/-B2Mm/sHLAG-hPSCs对T细胞的激活能力。T cells are activated by mismatched HLA molecules, so wt-CMs are used as controls and Comparative activation effect of CIITA -/- B2Mm/sHLAG-hPSCs on T cells. Wt-CMs and CIITA -/- B2Mm/sHLAG-hPSCs were spread in 96-well plates, and IFN-γ at a final concentration of 100ng/ml was added for 24 hours and incubated for 48 hours to stimulate the expression of HLA molecules. Afterwards, IFN-γ was removed, and primary peripheral blood mononuclear cells (PBMCs, which are rich in T cells) were added according to the ratio of cardiomyocytes to human peripheral blood mononuclear cells (PMBCs) of 1:3. After co-culture for 48 hours, a total of Cultured PBMCs were incubated with T cell marker CD3 antibody and T cell early activation marker CD69 antibody, and then flow cytometry was performed. The T cell activation abilities of wt-CMs and CIITA -/- B2Mm/sHLAG-hPSCs were compared based on the proportion of CD69-positive cells in CD3-positive cells.
2、T细胞增殖检测2. T cell proliferation detection
前期处理同“T细胞激活标志物检测”，共培养之前将PBMCs标记上CFSE荧光标记后再与心肌细胞共培养，培养时间为7天，之后收集PBMCs，孵育T细胞标志物CD3抗体，然后流式细胞检测。CFSE在标记细胞中不会从细胞中泄露，但会随着细胞***稀释，因此增殖的T细胞中CFSE荧光信号降低，通过比较低CFSE信号的细胞占全部CD3阳性细胞的比例比较wt-CMs和CIITA-/-B2Mm/sHLAG-hPSCs对T细胞增殖作用。The preliminary treatment is the same as "T cell activation marker detection". Before co-culture, PBMCs are labeled with CFSE fluorescent label and then co-cultured with cardiomyocytes. The culture time is 7 days. Afterwards, PBMCs are collected, incubated with T cell marker CD3 antibody, and then flowed cell detection. CFSE does not leak from cells in labeled cells but is diluted as cells divide, so the CFSE fluorescence signal decreases in proliferating T cells. Compare wt-CMs and Effect of CIITA -/- B2Mm/sHLAG-hPSCs on T cell proliferation.
3、T细胞对靶细胞的杀伤作用检测3. Detection of the killing effect of T cells on target cells
前期处理同“T细胞激活标志物检测”，PBMCs与wt-CMs或CIITA-/-B2Mm/sHLAG-hPSCs共培养3-4天，然后收集共培养上清培养液，300g离心3分钟去除细胞与细胞碎片，然后利用碧云天LDH检测试剂盒检测上清中乳酸脱氢酶活性，确定细胞损伤程度。The preliminary treatment is the same as "T cell activation marker detection". PBMCs are co-cultured with wt-CMs or CIITA -/- B2Mm/sHLAG-hPSCs for 3-4 days. Then the co-culture supernatant is collected and centrifuged at 300g for 3 minutes to remove the cells and Cell debris was then used to detect the lactate dehydrogenase activity in the supernatant using the Beyotime LDH detection kit to determine the degree of cell damage.
4、PBMC分泌的IFN-γ检测4. Detection of IFN-γ secreted by PBMC
前期处理同“T细胞激活标志物检测”，PBMCs与wt-CMs或CIITA-/-B2Mm/sHLAG-hPSCs共培养3-4天，然后收集共培养上清培养液，300g离心3分钟去除细胞与细胞碎片，然后利用IFN-γ检测试剂盒检测上清中IFN-γ含量。The preliminary treatment is the same as "T cell activation marker detection". PBMCs are co-cultured with wt-CMs or CIITA -/- B2Mm/sHLAG-hPSCs for 3-4 days. Then the co-culture supernatant is collected and centrifuged at 300g for 3 minutes to remove the cells and The cells were fragmented, and then the IFN-γ content in the supernatant was detected using an IFN-γ detection kit.
5、NK细胞对靶细胞的识别与杀伤检测5. NK cell recognition and killing detection of target cells
NK细胞可识别无HLA分子表达的细胞，因此在本实施例中采用DKO-CMs作为对照。将DKO-CMs和CIITA-/-B2Mm/sHLAG-hPSCs铺于96孔板，24小时候按照CMs∶NK-92＝10∶1、3∶1、1∶1加入NK-92细胞，培养48小时候采集共培养上清培养液，300g离心3分钟去除细胞与细胞碎片，然后利用碧云天LDH检测试剂盒检测上清中乳酸脱氢酶活性，确定细胞损伤程度。NK cells can recognize cells without HLA molecule expression, so DKO-CMs were used as a control in this example. DKO-CMs and CIITA -/- B2Mm/sHLAG-hPSCs were spread on a 96-well plate, and NK-92 cells were added according to CMs:NK-92=10:1, 3:1, 1:1 for 24 hours, and collected after 48 hours of culture. Co-culture the supernatant culture medium, centrifuge at 300g for 3 minutes to remove cells and cell debris, and then use Beyotime LDH detection kit to detect lactate dehydrogenase activity in the supernatant to determine the degree of cell damage.
6、NK细胞分泌的IFN-γ检测6. Detection of IFN-γ secreted by NK cells
细胞前期处理同“NK细胞对靶细胞的识别与杀伤检测”，共培养3-4天后收集共培养上清，300g离心3分钟去除细胞与细胞碎片，然后利用IFN-γ检测试剂盒检测上清中IFN-γ的含量。The preliminary treatment of cells is the same as "NK cell identification and killing detection of target cells". After co-culture for 3-4 days, collect the co-culture supernatant, centrifuge at 300g for 3 minutes to remove cells and cell debris, and then use the IFN-γ detection kit to detect the supernatant IFN-γ content.
将人PBMCs与心肌细胞共培养，并鉴定早期T细胞激活标志物CD69表达。结果显示，相比wt心肌细胞(wt-CMs)，免疫兼容型心肌细胞(CIITA-/-B2Mm/sHLAG-CMs)对T细胞的激活显著降低；而且CIITA-/-B2Mm/sHLAG-CMs对T细胞增殖的作用也显著低于wt-CMs。同时，PBMCs对CIITA-/-B2Mm/sHLAG-CMs的杀伤作用显著低于wt-CMs，而且 CIITA-/-B2Mm/sHLAG-CMs刺激PBMCs分泌IFN-γ的能力也显著低于wt-CMs，如图7A-B。Human PBMCs were co-cultured with cardiomyocytes, and expression of the early T cell activation marker CD69 was identified. The results showed that compared with wt cardiomyocytes (wt-CMs), immune-compatible cardiomyocytes (CIITA -/- B2Mm/sHLAG-CMs) significantly reduced the activation of T cells; and CIITA -/- B2Mm/sHLAG-CMs The effect of cell proliferation was also significantly lower than that of wt-CMs. At the same time, the killing effect of PBMCs on CIITA -/- B2Mm/sHLAG-CMs was significantly lower than that of wt-CMs, and The ability of CIITA -/- B2Mm/sHLAG-CMs to stimulate PBMCs to secrete IFN-γ was also significantly lower than that of wt-CMs, as shown in Figure 7A-B.
为检验CIITA-/-B2Mm/sHLAG-CMs对NK细胞的激活作用，本发明人利用DKO-CMs作为对照，将分化的心肌细胞于NK-92细胞共培养，发现NK-92对CIITA-/-B2Mm/sHLAG-CMs细胞的杀伤作用显著低于DKO-CMs，且CIITA-/-B2Mm/sHLAG-CMs刺激NK-92分泌IFN-γ的能力也显著低于DKO-CMs，如图7C-F。In order to test the activation effect of CIITA -/- B2Mm/sHLAG-CMs on NK cells, the inventors used DKO-CMs as a control, co-cultured differentiated cardiomyocytes with NK-92 cells, and found that NK-92 had an effect on CIITA -/- The killing effect of B2Mm/sHLAG-CMs cells was significantly lower than that of DKO-CMs, and the ability of CIITA -/- B2Mm/sHLAG-CMs to stimulate NK-92 to secrete IFN-γ was also significantly lower than that of DKO-CMs, as shown in Figure 7C-F.
以上数据证明，CIITA-/-B2Mm/sHLAG-hPSCs衍生的心肌细胞具备逃逸T细胞及NK细胞的识别与杀伤作用。The above data prove that cardiomyocytes derived from CIITA -/- B2Mm/sHLAG-hPSCs are capable of evading recognition and killing by T cells and NK cells.
实施例7、心梗后移植hPSCs源心肌细胞后细胞驻留鉴定Example 7. Identification of cell residence after transplantation of hPSCs-derived cardiomyocytes after myocardial infarction
为进一步探究在体损伤环境下CIITA-/-B2Mm/sHLAG-hPSCs是否具备更好的驻留效果，本发明人利用免疫***人源化小鼠(Hu-mice)构建心脏损伤模型(本实施例中采用缺血/再灌注损伤模型)，并于建模后心肌内移植不同hPSCs来源的心肌细胞。具体步骤为：将免疫重建第10周，人免疫细胞已在小鼠外周血中占据相当比例的Hu-mice心脏行冠脉左前降支结扎后缺血60分钟后再开放复灌，构建心肌缺血/再灌注(I/R)模型。模型鼠随机分为I/R组、I/R+wt-CMs组、I/R+DKO-CMs组和I/R+CIITA-/-B2Mm/sHLAG-CMs组；在复灌时进行细胞移植，每只小鼠于心肌梗死边缘区域心肌注射5x105数量的wt-CMs，DKO-CMs和CIITA-/-B2Mm/sHLAG-CMs。In order to further explore whether CIITA -/- B2Mm/sHLAG-hPSCs have a better retention effect in an in vivo injury environment, the inventors used immune system humanized mice (Hu-mice) to construct a heart injury model (this example An ischemia/reperfusion injury model was used in the model), and cardiomyocytes derived from different hPSCs were transplanted into the myocardium after modeling. The specific steps are as follows: At the 10th week of immune reconstitution, the Hu-mice heart, where human immune cells have occupied a considerable proportion in the peripheral blood of mice, was subjected to ligation of the left anterior descending coronary artery and ischemia for 60 minutes, followed by reperfusion to construct myocardial ischemia. Blood/reperfusion (I/R) model. Model mice were randomly divided into I/R group, I/R+wt-CMs group, I/R+DKO-CMs group and I/R+CIITA -/- B2Mm/sHLAG-CMs group; cell transplantation was performed during reperfusion. , each mouse was injected with 5x10 5 amounts of wt-CMs, DKO-CMs and CIITA -/- B2Mm/sHLAG-CMs into the myocardium in the border area of myocardial infarction.
移植后第28天对小鼠心脏进行收样，通过免疫荧光及qRT-PCR鉴定移植细胞驻留。移植细胞驻留检测方法如下：Mouse hearts were collected on the 28th day after transplantation, and transplanted cell resident was identified by immunofluorescence and qRT-PCR. The method for detecting transplanted cell residence is as follows:
细胞移植后28天，收集小鼠心脏并进行OCT包埋。将小鼠心脏按照一定的间距从心尖到结扎点分为12层，并对每层细胞进行冰冻切片，然后利用免疫荧光鉴定移植细胞，同时利用Masson染色统计移植细胞区域占疤痕区域的比例。Twenty-eight days after cell transplantation, mouse hearts were collected and embedded in OCT. The mouse heart was divided into 12 layers at a certain distance from the apex to the ligation point, and the cells in each layer were frozen and sectioned. Immunofluorescence was then used to identify the transplanted cells, and Masson staining was used to count the proportion of the transplanted cell area in the scar area.
免疫荧光：冰冻切片后将组织固定、通透及封闭，然后共孵育肌节蛋白α-ACTININ抗体与人KU80抗体(C48E7，CST)。利用荧光显微镜采集图像并进行统计。Immunofluorescence: After frozen sectioning, the tissue was fixed, permeabilized and blocked, and then co-incubated with sarcomeric protein α-ACTININ antibody and human KU80 antibody (C48E7, CST). Use a fluorescence microscope to collect images and perform statistics.
qRT-PCR：收集细胞移植后28天Hu-mice心脏样本，抽提RNA后翻转为cDNA，利用人cTNT引物通过荧光定量PCR鉴定细胞驻留。qRT-PCR: Hu-mice heart samples were collected 28 days after cell transplantation. RNA was extracted and converted into cDNA. Human cTNT primers were used to identify cell residence through fluorescence quantitative PCR.
通过比较细胞驻留面积发现，在雌性与雄性小鼠中，CIITA-/-B2Mm/sHLAG-CMs的驻留面积显著高于wt-CMs及DKO-CMs。qRT-PCR结果与免疫荧光染色结果一致(图8A-D)。By comparing the cell resident area, it was found that the resident area of CIITA -/- B2Mm/sHLAG-CMs was significantly higher than that of wt-CMs and DKO-CMs in female and male mice. The qRT-PCR results were consistent with the immunofluorescence staining results (Figure 8A-D).
以上数据证明，CIITA-/-B2Mm/sHLAG-CMs在损伤模型中的长时程驻留能力显著强于wt-CMs及DKO-CMs。The above data prove that the long-term residence ability of CIITA -/- B2Mm/sHLAG-CMs in the damage model is significantly stronger than that of wt-CMs and DKO-CMs.
实施例8、移植细胞对心梗区域的修复作用Example 8. Repair effect of transplanted cells on myocardial infarction area
进一步，本发明人利用Masson染色探究移植细胞驻留对小鼠心脏损伤区域的修复作用。Furthermore, the inventors used Masson staining to explore the repair effect of transplanted cells on the injured cardiac area in mice.
Masson染色方法：冰冻切片后将组织固定，然后利用碧云天Masson染色试剂盒染色，统计再肌肉化面积及疤痕面积，计算再肌肉化比例。 Masson staining method: Fix the tissue after frozen sectioning, and then stain it with the Beyotime Masson staining kit to count the remuscularization area and scar area, and calculate the remuscularization ratio.
再肌肉化比例＝再肌肉面积/疤痕面积Remuscularization ratio = remuscle area/scar area
结果显示，小鼠心脏损伤后损伤部位细胞逐渐死亡，形成疤痕组织。比较三种细胞移植后小鼠心脏切片发现，CIITA-/-B2Mm/sHLAG-CMs移植后可在损伤部位形成心肌组织，弥补丢失心肌细胞。The results showed that after mouse heart injury, cells in the injured area gradually died and scar tissue formed. Comparing mouse heart slices after transplantation of the three types of cells, it was found that CIITA -/- B2Mm/sHLAG-CMs could form myocardial tissue at the injured site after transplantation and make up for the loss of cardiomyocytes.
统计结果显示，CIITA-/-B2Mm/sHLAG-CMs移植后损伤区域再肌肉化程度显著高于wt-CMs与DKO-CMs(图9A-B)。Statistical results showed that the degree of remuscleization of the injured area after transplantation of CIITA -/- B2Mm/sHLAG-CMs was significantly higher than that of wt-CMs and DKO-CMs (Figure 9A-B).
实施例9、利用非病毒构建体建立免疫兼容型人诱导多能干细胞及其表达Example 9. Establishment of immune-compatible human induced pluripotent stem cells and their expression using non-viral constructs
1、利用非病毒构建体建立免疫兼容型人诱导多能干细胞(iPSC-IC)1. Establish immune-compatible human induced pluripotent stem cells (iPSC-IC) using non-viral constructs
首先按照前述方法构建HLA-I与HLA-II分子缺失的人iPSCs(即：hPSCs(DKO-hPSCs))，然后采用基因定点敲入(图10A)或转座子***(图10B)，将相关质粒转导入目标人iPSCs中，经过进一步挑取单克隆并筛选表达目标基因的人iPSCs单克隆细胞系。First, human iPSCs with deleted HLA-I and HLA-II molecules (i.e., hPSCs (DKO-hPSCs)) were constructed according to the aforementioned method, and then gene site-directed knock-in (Fig. 10A) or transposon system (Fig. 10B) was used to combine the relevant The plasmid is transduced into the target human iPSCs, and monoclonal cells are further selected and screened for human iPSCs monoclonal cell lines expressing the target gene.
外源***片段结构如图10C所示，其中基因表达框中前后部分通过2A序列进行连接，这样前后部分的蛋白质在翻译时断裂为2部分，结果是一个表达框可表达两个独立的蛋白质。因2A结构前后的蛋白质表达水平存在差异，因此分别设计了HLAG1-B2M-2A-HLAG5-B2M与HLAG5-B2M-2A-HLAG1-B2M两种形式的表达框，用于筛选具备最佳表达效率的单克隆细胞系。The structure of the exogenous inserted fragment is shown in Figure 10C, in which the front and back parts of the gene expression box are connected through the 2A sequence, so that the protein in the front and back parts is broken into two parts during translation. The result is that one expression box can express two independent proteins. Because there are differences in protein expression levels before and after the 2A structure, two forms of expression cassettes, HLAG1-B2M-2A-HLAG5-B2M and HLAG5-B2M-2A-HLAG1-B2M, were designed to screen for optimal expression efficiency. Monoclonal cell lines.
2.HLAG1与HLAG5在构建的单克隆免疫兼容型人诱导多能干细胞源心肌细胞(iPSC-IC-CMs)的表达检测2. Detection of expression of HLAG1 and HLAG5 in constructed monoclonal immune-compatible human induced pluripotent stem cell-derived cardiomyocytes (iPSC-IC-CMs)
首先采用流式细胞术鉴定野生型人iPSCs(iPSC)源心肌细胞、HLA-I与HLA-II分子缺失的人iPSCs(iPSC-DKO)源心肌细胞，基因定点敲入的人iPSCs(iPSC-IC-AAVS)源心肌细胞，转座子介导的基因***的人iPSC(iPSC-IC-Pbx)源心肌细胞细胞膜上的HLAG1的表达水平。First, flow cytometry was used to identify cardiomyocytes derived from wild-type human iPSCs (iPSC), human iPSCs derived from HLA-I and HLA-II molecules (iPSC-DKO), and gene-knocked-in human iPSCs (iPSC-IC). -AAVS)-derived cardiomyocytes, and the expression level of HLAG1 on the cell membrane of transposon-mediated gene insertion human iPSC (iPSC-IC-Pbx)-derived cardiomyocytes.
如图11A(左)所示，相对于iPSC源心肌细胞及iPSC-DKO源心肌细胞，iPSC-IC-AAVS源心肌细胞及iPSC-IC-Pbx源心肌细胞显著高表达HLAG1。而Piggybac转座子***构建的、HLAG5-B2M-2A-HLAG1-B2M结构表达框的iPSC单克隆来源的心肌细胞中HLAG1的表达最高(图11A(右))。As shown in Figure 11A (left), compared with iPSC-derived cardiomyocytes and iPSC-DKO-derived cardiomyocytes, iPSC-IC-AAVS-derived cardiomyocytes and iPSC-IC-Pbx-derived cardiomyocytes significantly higher expressed HLAG1. However, the expression of HLAG1 in cardiomyocytes derived from iPSC monoclones constructed with the Piggybac transposon system and the HLAG5-B2M-2A-HLAG1-B2M structural expression cassette was the highest (Figure 11A (right)).
利用ELISA及免疫印迹检测心肌细胞培养液中HLAG5的分泌水平，发现***HLAG5-B2M-2A-HLAG1-B2M结构的表达框的单克隆iPSC源心肌细胞，其HLAG5分泌水平更高，而***HLAG1-B2M-2A-HLAG5-B2M结构的表达框的单克隆iPSC源心肌细胞，其培养上清中未检测到HLAG5的表达(图11B)。ELISA and Western blotting were used to detect the secretion level of HLAG5 in the cardiomyocyte culture medium. It was found that monoclonal iPSC-derived cardiomyocytes inserted into the expression cassette of the HLAG5-B2M-2A-HLAG1-B2M structure had higher HLAG5 secretion levels, while those inserted into the HLAG1- No expression of HLAG5 was detected in the culture supernatant of monoclonal iPSC-derived cardiomyocytes containing the expression cassette of the B2M-2A-HLAG5-B2M structure (Fig. 11B).
以上数据表明，不同基因整合方式、不同表达框结构对目的基因的表达带来显著影响，通过这两种方式的组合可以获得高表达外源基因的单克隆细胞系。The above data show that different gene integration methods and different expression box structures have significant effects on the expression of target genes. Through the combination of these two methods, monoclonal cell lines with high expression of foreign genes can be obtained.
其中，***片段序列如下：Among them, the inserted fragment sequence is as follows:
HLAG1-B2M-2A-HLAG5-B2M的DNA序列(SEQ ID NO：12)：

DNA sequence of HLAG1-B2M-2A-HLAG5-B2M (SEQ ID NO: 12):

HLAG1-B2M-2A-HLAG5-B2M的蛋白序列(SEQ ID NO：13)
Protein sequence of HLAG1-B2M-2A-HLAG5-B2M (SEQ ID NO: 13)
HLAG5-B2M-2A-HLAG1-B2M的DNA序列(SEQ ID NO：14)：

DNA sequence of HLAG5-B2M-2A-HLAG1-B2M (SEQ ID NO: 14):

HLAG5-B2M-2A-HLAG1-B2M的蛋白序列(SEQ ID NO：15)：
Protein sequence of HLAG5-B2M-2A-HLAG1-B2M (SEQ ID NO: 15):
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。同时，在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。 The above-mentioned embodiments only express several implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the patent scope of the present invention. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the scope of protection of the patent of the present invention should be determined by the appended claims. All documents mentioned in this application are herein incorporated by reference to the same extent as if each individual document was individually incorporated by reference.

Claims (14)

  1. 一种制备免疫兼容型人多能干细胞的方法，包括改造人多能干细胞，使之：A method for preparing immune-compatible human pluripotent stem cells, including modifying human pluripotent stem cells to:
    (a)不表达游离B2M蛋白，表达HLA-G1与分泌型HLA-G5；以及(a) Does not express free B2M protein, expresses HLA-G1 and secreted HLA-G5; and
    (b)不表达CIITA蛋白。(b) CIITA protein is not expressed.
  2. 如权利要求1所述的方法，其特征在于，(a)中，The method of claim 1, wherein in (a),
    改造人多能干细胞的基因组，将编码HLA-G1的多核苷酸与人多能干细胞中内源性的B2M基因融合，从而表达B2M-HLA-G1融合蛋白且不表达游离B2M蛋白；较佳地，将HLA-G1的编码基因引入到内源B2M外显子3中终止密码子前的位置，或以HLA-G1的编码基因替换位于内源B2M基因外显子3中的终止密码子；更佳地，通过基因编辑方法进行所述改造；以及Modify the genome of human pluripotent stem cells and fuse the polynucleotide encoding HLA-G1 with the endogenous B2M gene in human pluripotent stem cells, thereby expressing the B2M-HLA-G1 fusion protein and not expressing free B2M protein; preferably , introduce the HLA-G1 coding gene into the position before the stop codon in exon 3 of the endogenous B2M gene, or replace the stop codon in exon 3 of the endogenous B2M gene with the HLA-G1 coding gene; more Preferably, the transformation is carried out by gene editing methods; and
    在人多能干细胞中引入外源的编码B2M-HLA-G5融合蛋白的多核苷酸，所述B2M-HLA-G5融合蛋白包括B2M和HLA-G5。Exogenous polynucleotides encoding B2M-HLA-G5 fusion proteins are introduced into human pluripotent stem cells, and the B2M-HLA-G5 fusion proteins include B2M and HLA-G5.
  3. 如权利要求2所述的方法，其特征在于，利用重组载体表达所述B2M-HLA-G1和B2M-HLA-G5；较佳地，所述重组载体包括非病毒载体或病毒载体。The method of claim 2, characterized in that a recombinant vector is used to express the B2M-HLA-G1 and B2M-HLA-G5; preferably, the recombinant vector includes a non-viral vector or a viral vector.
  4. 如权利要求3所述的方法，其特征在于，所述非病毒载体包括：定点敲入***或转座子***；或，所述病毒载体包括：慢病毒载体。The method of claim 3, wherein the non-viral vector includes a site-directed knock-in system or a transposon system; or the viral vector includes a lentiviral vector.
  5. 如权利要求2所述的方法，其特征在于，所述B2M-HLA-G1融合蛋白中，还包括位于B2M和HLA-G1之间的柔性连接肽，优选如SEQ ID NO：3所示的柔性连接肽；和/或，所述B2M-HLA-G1融合蛋白自N端至C端依次包括：B2M和HLA-G1；较佳地，所述B2M-HLA-G1融合蛋白的氨基酸序列如SEQ ID NO：4所示；更佳地，所述B2M-HLA-G1融合蛋白的核酸序列如SEQ ID NO：9所示；和/或The method of claim 2, wherein the B2M-HLA-G1 fusion protein also includes a flexible connecting peptide between B2M and HLA-G1, preferably a flexible one as shown in SEQ ID NO: 3 Connecting peptide; and/or, the B2M-HLA-G1 fusion protein includes: B2M and HLA-G1 from the N-terminus to the C-terminus; preferably, the amino acid sequence of the B2M-HLA-G1 fusion protein is such as SEQ ID As shown in NO: 4; more preferably, the nucleic acid sequence of the B2M-HLA-G1 fusion protein is as shown in SEQ ID NO: 9; and/or
    所述B2M-HLA-G5融合蛋白中，还包括位于B2M和HLA-G5之间的柔性连接肽，优选如SEQ ID NO：3所示的柔性连接肽；和/或，所述B2M-HLA-G5融合蛋白自N端至C端依次包括：B2M和HLA-G5；较佳地，所述B2M-HLA-G5融合蛋白的氨基酸序列如SEQ ID NO：8所示；更佳地，所述B2M-HLA-G5融合蛋白的核酸序列如SEQ ID NO：10所示。The B2M-HLA-G5 fusion protein also includes a flexible connecting peptide between B2M and HLA-G5, preferably a flexible connecting peptide as shown in SEQ ID NO: 3; and/or the B2M-HLA- The G5 fusion protein includes: B2M and HLA-G5 in sequence from the N-terminus to the C-terminus; preferably, the amino acid sequence of the B2M-HLA-G5 fusion protein is shown in SEQ ID NO: 8; more preferably, the B2M -The nucleic acid sequence of the HLA-G5 fusion protein is shown in SEQ ID NO: 10.
  6. 如权利要求1所述的方法，其特征在于，(b)中，针对人多能干细胞基因组中CIITA基因的外显子3进行敲除；较佳地，通过基因编辑方法进行敲除；更佳地，以SEQ ID NO：11所示核苷酸序列的gRNA进行基因编辑。The method of claim 1, wherein in (b), exon 3 of the CIITA gene in the human pluripotent stem cell genome is deleted; preferably, the knockout is performed by a gene editing method; more preferably The gRNA with the nucleotide sequence shown in SEQ ID NO: 11 is used for gene editing.
  7. 如权利要求1～6任一所述的方法，其特征在于，其包括如下的A-D中的一个或多个：The method according to any one of claims 1 to 6, characterized in that it includes one or more of the following A-D:
    A、所述HLA-G1包括：氨基酸序列如SEQ ID NO：1所示的多肽；或，氨基酸序列与SEQ ID NO：1具有90％以上序列同一性且具有SEQ ID NO：1所示的多肽的功能的衍生多肽；A. The HLA-G1 includes: a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1; or a polypeptide with an amino acid sequence having more than 90% sequence identity with SEQ ID NO: 1 and having a polypeptide as shown in SEQ ID NO: 1 Functionally derived polypeptides;
    B、所述B2M包括：氨基酸序列如SEQ ID NO：2所示的多肽；或，氨基酸序列与SEQ ID NO：2具有90％以上序列同一性且具有SEQ ID NO：2所示的多肽的功能的衍生多肽；B. The B2M includes: a polypeptide with an amino acid sequence as shown in SEQ ID NO: 2; or an amino acid sequence with more than 90% sequence identity as SEQ ID NO: 2 and having the function of the polypeptide shown in SEQ ID NO: 2 Derived peptides;
    C、所述CIITA蛋白包括：氨基酸序列如SEQ ID NO：5所示的多肽；或，氨基酸序列与SEQ ID NO：5具有90％以上序列同一性且具有SEQ ID NO：5所示的多肽的功能的衍生多肽； C. The CIITA protein includes: a polypeptide with an amino acid sequence as shown in SEQ ID NO: 5; or a polypeptide with an amino acid sequence having more than 90% sequence identity with SEQ ID NO: 5 and a polypeptide as shown in SEQ ID NO: 5 Functional derived peptides;
    D、所述HLA-G5包括：氨基酸序列如SEQ ID NO：6所示的多肽；或，氨基酸序列与SEQ ID NO：6具有90％以上序列同一性且具有SEQ ID NO：6所示的多肽的功能的多肽片段。D. The HLA-G5 includes: a polypeptide with an amino acid sequence as shown in SEQ ID NO: 6; or a polypeptide with an amino acid sequence having more than 90% sequence identity with SEQ ID NO: 6 and having a polypeptide as shown in SEQ ID NO: 6 Functional peptide fragments.
  8. 一种免疫兼容型人多能干细胞，其(a)不表达游离B2M蛋白，表达HLA-G1与分泌型HLA-G5；以及(b)不表达CIITA蛋白；较佳地，所述人多能干细胞：基因组中内源性的B2M基因与编码HLA-G1的多核苷酸融合；包含外源的编码B2M-HLA-G5融合蛋白的多核苷酸；且，基因组中CIITA基因被敲除。An immune-compatible human pluripotent stem cell, which (a) does not express free B2M protein but expresses HLA-G1 and secreted HLA-G5; and (b) does not express CIITA protein; preferably, the human pluripotent stem cell : The endogenous B2M gene in the genome is fused to the polynucleotide encoding HLA-G1; it contains exogenous polynucleotide encoding the B2M-HLA-G5 fusion protein; and, the CIITA gene in the genome is knocked out.
  9. 如权利要求8所述的免疫兼容型人多能干细胞，其特征在于，其由如权利要求1～5任一项所述的方法构建获得。The immune-compatible human pluripotent stem cell according to claim 8, characterized in that it is constructed and obtained by the method according to any one of claims 1 to 5.
  10. 权利要求8或9所述的免疫兼容型人多能干细胞的应用，用于通过诱导分化制备适于移植的细胞；较佳地，所述适于移植的细胞为组织或器官细胞；更佳地，所述组织或器官细胞包括：心血管前体细胞、心肌细胞、内皮细胞、平滑肌细胞、神经细胞、造血干细胞、髓系细胞、淋系细胞、视网膜色素上皮细胞、胰岛B细胞、肝脏细胞、角质形成细胞、骨骼肌细胞、脂肪细胞、骨细胞、软骨细胞、间充质干细胞；更佳地，其中所述的髓系细胞包括粒细胞、单核细胞、巨噬细胞、红细胞、血小板，和/或其中所述的淋系细胞包括自然杀伤细胞、T细胞、B细胞，和/或其中所述的肝脏细胞包括肝细胞，胆管细胞，肝内皮细胞，肝星状细胞，Kupffer细胞，间皮细胞。The application of immune-compatible human pluripotent stem cells according to claim 8 or 9 is used to prepare cells suitable for transplantation by inducing differentiation; preferably, the cells suitable for transplantation are tissue or organ cells; more preferably , the tissue or organ cells include: cardiovascular precursor cells, cardiomyocytes, endothelial cells, smooth muscle cells, nerve cells, hematopoietic stem cells, myeloid cells, lymphoid cells, retinal pigment epithelial cells, pancreatic islet B cells, liver cells, Keratinocytes, skeletal muscle cells, adipocytes, osteocytes, chondrocytes, mesenchymal stem cells; more preferably, the myeloid cells include granulocytes, monocytes, macrophages, red blood cells, platelets, and /or wherein the lymphoid cells include natural killer cells, T cells, B cells, and/or where the liver cells include hepatocytes, bile duct cells, hepatic endothelial cells, hepatic stellate cells, Kupffer cells, mesothelial cells cell.
  11. 一种制备适于移植的细胞的方法，包括：(a)以权利要求1～7任一所述的方法制备获得免疫兼容型人多能干细胞；(b)将(a)的细胞进一步进行诱导分化，获得适于移植的细胞；较佳地，所述适于移植的细胞为组织或器官细胞；更佳地，所述组织或器官细胞包括：心血管前体细胞、心肌细胞、内皮细胞、平滑肌细胞、神经细胞、造血干细胞、髓系细胞、淋系细胞、视网膜色素上皮细胞、胰岛B细胞、肝脏细胞、角质形成细胞、骨骼肌细胞、脂肪细胞、骨细胞、软骨细胞、间充质干细胞；更佳地，其中所述的髓系细胞包括粒细胞、单核细胞、巨噬细胞、红细胞、血小板，和/或其中所述的淋系细胞包括自然杀伤细胞、T细胞、B细胞，和/或其中所述的肝脏细胞包括肝细胞，胆管细胞，肝内皮细胞，肝星状细胞，Kupffer细胞，间皮细胞。A method for preparing cells suitable for transplantation, comprising: (a) preparing immune-compatible human pluripotent stem cells by the method described in any one of claims 1 to 7; (b) further inducing the cells of (a) Differentiate to obtain cells suitable for transplantation; preferably, the cells suitable for transplantation are tissue or organ cells; more preferably, the tissue or organ cells include: cardiovascular precursor cells, cardiomyocytes, endothelial cells, Smooth muscle cells, nerve cells, hematopoietic stem cells, myeloid cells, lymphoid cells, retinal pigment epithelial cells, islet B cells, liver cells, keratinocytes, skeletal muscle cells, adipocytes, bone cells, chondrocytes, mesenchymal stem cells ; More preferably, the myeloid cells include granulocytes, monocytes, macrophages, red blood cells, platelets, and/or the lymphoid cells include natural killer cells, T cells, and B cells, and /Or the liver cells described therein include hepatocytes, bile duct cells, liver endothelial cells, hepatic stellate cells, Kupffer cells, and mesothelial cells.
  12. 如权利要求11所述的方法，其特征在于，所述组织或器官细胞为心肌细胞，采用先后以CHIR99021、IWR-1诱导的方法制备；或，所述组织或器官细胞为内皮细胞，采用先后以CHIR99021、bFGF、VEGF+BMP4诱导的方法制备。The method of claim 11, wherein the tissue or organ cells are cardiomyocytes, which are prepared by inducing CHIR99021 and IWR-1 successively; or, the tissue or organ cells are endothelial cells, which are prepared by inducing successively CHIR99021 and IWR-1. Prepared by induction method of CHIR99021, bFGF, VEGF+BMP4.
  13. 一种心肌细胞或内皮细胞，由权利要求1～7任一所述的方法制备获得的免疫兼容型人多能干细胞或如权利要求8或9所述的免疫兼容型人多能干细胞衍生获得。A cardiomyocyte or endothelial cell derived from an immune-compatible human pluripotent stem cell prepared by the method of any one of claims 1 to 7 or an immune-compatible human pluripotent stem cell as claimed in claim 8 or 9.
  14. 如权利要求13所述的心肌细胞在制备用于治疗心脏相关的疾病或病症的组合物或药物中的用途；较佳地，所述心脏相关的疾病或病症是心肌损伤、心肌梗死、心肌缺血、缺血再灌注损伤或其他心脏损伤；更佳地，所述心脏相关的疾病或病症是缺血/再灌注损伤。 The use of cardiomyocytes as claimed in claim 13 in the preparation of compositions or medicines for treating heart-related diseases or disorders; preferably, the heart-related diseases or disorders are myocardial injury, myocardial infarction, myocardial insufficiency. blood, ischemia-reperfusion injury or other heart injury; more preferably, the heart-related disease or disorder is ischemia/reperfusion injury.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110177869A (en) * 2017-01-13 2019-08-27 加利福尼亚大学董事会 The pluripotent cell of immunologic reconstitution
CN112342196A (en) * 2020-08-18 2021-02-09 未来智人再生医学研究院(广州)有限公司 Immune-compatible reversible universal pluripotent stem cell and application thereof
CN113528448A (en) * 2020-04-14 2021-10-22 同济大学 Construction method of human embryonic stem cells
CN114107211A (en) * 2020-12-04 2022-03-01 未来智人再生医学研究院(广州)有限公司 Pluripotent stem cell and derivative thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110177869A (en) * 2017-01-13 2019-08-27 加利福尼亚大学董事会 The pluripotent cell of immunologic reconstitution
CN113528448A (en) * 2020-04-14 2021-10-22 同济大学 Construction method of human embryonic stem cells
CN112342196A (en) * 2020-08-18 2021-02-09 未来智人再生医学研究院(广州)有限公司 Immune-compatible reversible universal pluripotent stem cell and application thereof
CN114107211A (en) * 2020-12-04 2022-03-01 未来智人再生医学研究院(广州)有限公司 Pluripotent stem cell and derivative thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
WANG XIAOKAI, LU MENG, TIAN XIAOYU, REN YANSONG, LI YIJUN, XIANG MENG, CHEN SIFENG: "Diminished expression of major histocompatibility complex facilitates the use of human induced pluripotent stem cells in monkey", STEM CELL RESEARCH & THERAPY, BIOMED CENTRAL LTD, LONDON, UK, vol. 11, no. 1, 1 December 2020 (2020-12-01), London, UK , XP093121396, ISSN: 1757-6512, DOI: 10.1186/s13287-020-01847-9 *
ZHENG, D. J. ET AL.: "Engineering of human mesenchymal stem cells resistant to multiple natural killer subtypes", INT. J. BIOL. SCI., vol. 18, no. 1, 1 January 2022 (2022-01-01), pages 426 - 440, XP093069310, DOI: 10.7150/ijbs.64640 *

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