WO2024002279A1 - Cellule souche pluripotente humaine immunocompatible, son procédé de préparation et son utilisation - Google Patents

Cellule souche pluripotente humaine immunocompatible, son procédé de préparation et son utilisation 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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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/fr

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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

L'invention concerne une cellule souche pluripotente humaine immunocompatible, son procédé de préparation et son utilisation. La cellule souche pluripotente humaine modifiée peut être induite et différenciée dans divers tissus/diverses cellules organiques, a une très bonne immunocompatibilité, et présente une large plage d'application.
PCT/CN2023/104109 2022-06-29 2023-06-29 Cellule souche pluripotente humaine immunocompatible, son procédé de préparation et son utilisation WO2024002279A1 (fr)

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CN113528448A (zh) * 2020-04-14 2021-10-22 同济大学 一种人胚胎干细胞的构建方法
CN114107211A (zh) * 2020-12-04 2022-03-01 未来智人再生医学研究院(广州)有限公司 一种多能干细胞及其衍生物

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CN113528448A (zh) * 2020-04-14 2021-10-22 同济大学 一种人胚胎干细胞的构建方法
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