WO2024120427A1 - Gsn-expressing universal cell and preparation method therefor - Google Patents

Gsn-expressing universal cell and preparation method therefor Download PDF

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WO2024120427A1
WO2024120427A1 PCT/CN2023/136703 CN2023136703W WO2024120427A1 WO 2024120427 A1 WO2024120427 A1 WO 2024120427A1 CN 2023136703 W CN2023136703 W CN 2023136703W WO 2024120427 A1 WO2024120427 A1 WO 2024120427A1
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protein
pluripotent stem
acid sequence
cells
gsn
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PCT/CN2023/136703
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French (fr)
Chinese (zh)
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李翔
朱珉喆
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士泽生物医药(上海)有限公司
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Publication of WO2024120427A1 publication Critical patent/WO2024120427A1/en

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  • the present invention belongs to the field of genetic engineering and stem cell technology, and specifically relates to a universal cell expressing GSN and a preparation method thereof.
  • Stem cells are a type of cells that have the ability to self-renew and differentiate into specific functional somatic cells. Based on the degree of differences in stem cell characteristics, stem cells are mainly divided into: totipotent stem cells, pluripotent stem cells and adult stem cells. Induced pluripotent stem cells (iPSCs) have the potential to proliferate indefinitely, self-renew and differentiate into various types of cells, and have important application prospects in the treatment of cancer, neurological, cardiovascular and other diseases.
  • iPSCs Induced pluripotent stem cells
  • key issues such as immune incompatibility and immune rejection of transplanted cells have hindered the clinical application of transplanted allogeneic functional cells for treatment.
  • MHC human major histocompatibility complex
  • HLA human leukocyte antigen
  • cells can express non-classical HLA-I class molecules such as HLA-E/G, or express immunosuppressive checkpoint proteins such as PD-L1, CTLA4-Ig, CD47, CD24, etc., which can effectively escape the killing of NK cells (WO2021041316A1).
  • the present invention provides a low immunogenic pluripotent stem cell, comprising: reduced endogenous major histocompatibility class I antigen (MHC-I) function compared to a parental pluripotent stem cell; reduced endogenous major histocompatibility class II antigen (MHC-II) function compared to a parental pluripotent stem cell; and reduced sensitivity to NK cell killing compared to a parental pluripotent stem cell, wherein the reduced sensitivity to NK cell killing is caused by increased expression of GSN protein.
  • MHC-I major histocompatibility class I antigen
  • MHC-II reduced endogenous major histocompatibility class II antigen
  • the MHC-I function is reduced by reducing the activity of an MHC-I class protein or an MHC-I transcriptional regulator.
  • the MHC-I function is reduced by reducing the activity of the B2M protein.
  • the B2M protein is a human B2M protein, which comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence that has 90% identity with the amino acid sequence shown in SEQ ID NO:1.
  • the B2M protein is a crab-eating macaque B2M protein, which comprises the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence that has 90% identity with the amino acid sequence shown in SEQ ID NO:9.
  • the MHC-II function is reduced by reducing the activity of an MHC-II class protein or an MHC-II transcriptional regulator.
  • the MHC-II function is reduced by reducing the activity of the CIITA protein.
  • the CIITA protein is a cynomolgus monkey CIITA protein, which comprises the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO:10.
  • the GSN protein is a human GSN protein, which comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence that has 90% identity with the amino acid sequence shown in SEQ ID NO:3.
  • the GSN protein is a cynomolgus monkey GSN protein, which comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO:7.
  • the low immunogenic pluripotent stem cells comprise: one or more changes that reduce the activity of endogenous B2M protein; one or more changes that reduce the activity of endogenous CIITA protein; and one or more changes that cause increased expression of GSN protein in the low immunogenic pluripotent stem cells.
  • the low immunogenic pluripotent stem cells comprise: one or more changes that inactivate both alleles of the endogenous B2M gene; one or more changes that inactivate both alleles of the endogenous CIITA gene; and one or more changes that cause increased expression of the GSN gene in the low immunogenic pluripotent stem cells.
  • the present invention also provides a method for producing the low immunogenic pluripotent stem cells of the present invention, the method comprising: reducing the endogenous major histocompatibility class I antigen (MHC-I) function in the pluripotent stem cells; reducing the endogenous major histocompatibility class II antigen (MHC-II) function in the pluripotent stem cells; and increasing the expression of a protein that reduces the sensitivity of the pluripotent stem cells to NK cell killing, wherein the protein is GSN protein.
  • MHC-I major histocompatibility class I antigen
  • MHC-II major histocompatibility class II antigen
  • the method comprises: eliminating the activity of both alleles of the B2M gene in the pluripotent stem cells; eliminating the activity of both alleles of the CIITA gene in the pluripotent stem cells; and increasing the expression of the GSN gene in the pluripotent stem cells.
  • the activity of B2M protein in the pluripotent stem cells is reduced by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • the activity of CIITA protein in the pluripotent stem cells is reduced by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • expression of the GSN protein is increased by expression of a transgene.
  • a nucleic acid sequence encoding the GSN protein is synthesized and constructed into a lentiviral vector, and then at least one copy of the GSN gene under the control of a promoter is introduced into the pluripotent stem cells via the lentiviral vector to increase the expression of the GSN protein.
  • the nucleic acid sequence encoding the GSN protein comprises the nucleic acid sequence shown in SEQ ID NO:4 or a nucleic acid sequence that has at least 80% identity with the nucleic acid sequence shown in SEQ ID NO:4.
  • the nucleic acid sequence encoding the GSN protein comprises the nucleic acid sequence shown in SEQ ID NO:8 or a nucleic acid sequence that has at least 80% identity with the nucleic acid sequence shown in SEQ ID NO:8.
  • the present invention also provides the low immunogenic pluripotent stem cells of the present invention or the cells prepared by the method of the present invention.
  • FIG. 1 shows the knockout strategy of the B2M gene in the B2M and CIITA double knockout cell lines (DKO) and the results of B2M gene knockout verification by PCR.
  • FIG. 2 shows the knockout strategy of the CIITA gene in B2M and CIITA double knockout cell lines (DKO) and the results of the CIITA gene knockout verification by PCR.
  • FIG. 3 shows the results of qPCR detection of the expression of B2M and CIITA at the RNA level in B2M/CIITA biallelic knockout clones DKO.
  • FIG. 4 shows the results of Western blot detection of B2M protein levels in B2M/CIITA biallelic knockout clones DKO.
  • Figure 5 shows the FACS detection results of WT and DKO cells stimulated with IFN- ⁇ to detect HLA class I/II molecules on the surface of H1 cells.
  • FIG. 6 shows the karyotype detection results of B2M/CIITA biallelic knockout clones DKO.
  • Figures 7A, 7B and 7C show the results of detecting the expression of stemness genes in DKO cells.
  • Figure 7A is the result of detecting the protein levels of stemness genes POU5F1 and NANOG in WT and DKO cells by immunofluorescence
  • Figure 7B is the result of detecting the expression of stemness genes POU5F1, NANOG and SOX2 in WT and DKO cells at the RNA level by RT-qPCR
  • Figure 7C is the result of detecting the expression of stemness genes SSEA-4 and Tra1-81 on the surface of WT and DKO cells by flow cytometry.
  • FIG. 8 shows the results of detecting the differentiation ability of B2M/CIITA biallelic knockout DKO cells to form teratomas and differentiate into cells of the three germ layers, endomeseoblasts and ectoblasts, in vivo by hematoxylin and eosin staining.
  • Figure 9 shows the results of detecting the immune escape function of WT and DKO cells by RTCA.
  • the top three figures are the results of detecting the killing rate of NK cells against WT and DKO cells by RTCA, that is, the detection results of the immune escape function of WT and DKO cells against NK cells; the bottom three figures are the results of detecting the killing rate of T cells against WT and DKO cells by RTCA, that is, the detection results of the immune escape function of WT and DKO cells against T cells.
  • FIG. 10 shows a schematic diagram of the structure of the lentiviral vector pGC-EF1a.
  • Figures 11A and 11B show the results of verifying the overexpression of CD47 in DKO+CD47 cells.
  • Figure 11A is the result of detecting the expression level of CD47 in DKO+CD47 cell lines by FACS;
  • Figure 11B is the result of detecting the expression level of CD47 in DKO+CD47 cell lines by qPCR.
  • FIG. 12 shows the results of detecting the immune escape function of WT and DKO+CD47 cells by RTCA.
  • FIG. 13 shows the results of detecting the overexpression level of mRNA in the constructed DKO+GSN cell line by RT-PCR.
  • FIG. 14 shows the results of immunofluorescence detection of the protein levels of stemness genes OCT4, NANOG, SOX2, TRA-1-60, and TRA-1-81 in the constructed DKO+GSN cell line.
  • FIG. 15 shows the results of detecting the expression levels of cell surface stemness genes SSEA-4, TRA-1-60, Tra1-81, and OCT4 in the constructed DKO+GSN cell line by flow cytometry.
  • FIG. 16 shows the results of immunofluorescence detection of the three-germ layer differentiation ability of the constructed DKO+GSN cell line.
  • FIG. 17 shows the results of testing the teratoma-forming ability of the DKO+GSN cell line.
  • Figures 18A-18D show the results of the escape function of the constructed DKO+GSN cell line on different immune cells detected by RTCA.
  • Figures 18A and 18B are the results of the NK cell killing experiment on DKO+GSN cells detected by RTCA, wherein Figure 18B is a multiple killing statistical graph;
  • Figures 18C and 18D are the results of the T cell+NK cell killing experiment on DKO+GSN cells detected by RTCA, wherein Figure 18D is a multiple killing statistical graph.
  • FIG. 19 shows the cell status of the constructed DKO+GSN cell line after co-culture with NK cells for 24 hours.
  • FIG. 20A and 20B show the results of Elispot detection of IFN- ⁇ spot secretion by the constructed DKO+GSN cell line after co-culture with NK cells for 24 hours, wherein FIG. 20B is a statistical histogram of IFN- ⁇ spot frequency.
  • FIG. 21 shows the results of FACS detection of the expression of CD107a, an indicator of NK cell activity, in the constructed DKO+GSN cell line.
  • FIG. 22A and FIG. 22B show the results of detecting the escape function of differentiated cells of the constructed DKO+GSN cell line against NK cells by RTCA, wherein FIG. 22B is a statistical diagram of multiple killings.
  • Figure 23 shows the FACS detection results of NHP iPSC-WT, NHP iPSC-DKO1 and NHP iPSC-DKO2 cells stimulated with IFN- ⁇ to detect HLA class I/II molecules on the cell surface.
  • Figure 24 shows the results of the RTCA test to detect the killing ability of T cells against NHP iPSC-WT, NHP iPSC-DKO1 and NHP iPSC-DKO2 cells.
  • Figure 25 shows the results of detecting the GSN expression level in the NHP iPSC-DKO+GSN cell line by qPCR.
  • Figure 26 shows the results of the RTCA test to detect the killing ability of PBMC cells on NHP iPSC-DKO+GSN cells.
  • the expressions “comprises,” “comprising,” “containing,” and “having” are open ended, meaning the inclusion of the listed elements, steps, or components but not the exclusion of other unlisted elements, steps, or components.
  • the expression “consisting of” excludes any element, step, or component not specified.
  • the expression “consisting essentially of” means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions “consisting essentially of” and “consisting of” are encompassed within the meaning of the expression “comprising.”
  • labels such as 1), 2), ..., i), ii), ..., a), b), ... are merely examples of distinction and do not imply that the method steps described are performed in such an order.
  • pluripotent cell refers to a cell that is capable of self-renewal and proliferation while remaining in an undifferentiated state and that can be induced to differentiate into a specialized cell type under appropriate conditions.
  • pluripotent stem cell has 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 (e.g., epidermal tissue and nervous system tissue).
  • endoderm e.g., gastric junction, gastrointestinal tract, lung, etc.
  • mesoderm e.g., muscle, bone, blood, urogenital tissue, etc.
  • ectoderm e.g., epidermal tissue and nervous system tissue.
  • the term “pluripotent stem cell” also includes "induced pluripotent stem cells” or "iPSCs,” a pluripotent stem cell derived from a non-pluripotent cell.
  • iPSCs a pluripotent stem cell derived from a non-pluripotent cell.
  • Exemplary human pluripotent stem cell lines include the
  • pluripotent 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 (as described in Cowan CA, et al. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med. 2004 Mar 25; 350(13): 1353-6.).
  • totipotency refers to the ability of a cell to form a complete organism.
  • totipotency refers to the ability of a cell to form a complete organism.
  • the pluripotent stem cells described herein do not have totipotency and will not form a complete organism.
  • universal cells refers to cells that are modified using gene editing technology to eliminate immune rejection and achieve universalization.
  • the cell can be from, for example, a human or non-human mammal.
  • 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 cell is from an adult or a non-human mammal. In some embodiments, the cell is from a newborn human, an adult, or a non-human mammal.
  • immune rejection or “immune incompatibility” refers to the fact that foreign cells, tissues or organs will be attacked by the recipient's own immune cells after being transplanted into the recipient, thereby failing to ensure their normal physiological functions.
  • the human major histocompatibility complex i.e., human leukocyte antigen (HLA)
  • HLA human leukocyte antigen
  • the term "subject” or “patient” refers to any animal, such as a domesticated animal, a zoo animal, or a human.
  • a “subject” or “patient” can be a mammal, such as a dog, a cat, a bird, livestock, or a human.
  • Specific examples of “subjects” and “patients” include, but are not limited to, individuals (particularly humans) with diseases or conditions associated with the liver, heart, lungs, kidneys, pancreas, brain, nervous tissue, blood, bones, bone marrow, etc.
  • hypoimmunogenic pluripotent stem cells herein refer to pluripotent stem cells, which retain the characteristics of their pluripotent stem cells, and produce a reduced immune rejection reaction when transferred to an allogeneic host.
  • low immunogenic pluripotent stem cells do not produce an immune response. Therefore, " low immunogenicity " refers to an immune response that is significantly reduced or eliminated compared to the immune response of the parent (“ WT ”) stem cell before immune modification. For example, relative to wild-type cells that have not been immune modified, such low immunogenic cells may be about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more than 99% less likely to be immune rejected.
  • MHC major histocompatibility complex
  • MHC-I refers to the major histocompatibility complex class I proteins or genes. Within the human MHC-I region, there are the HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, CD1a, CD1b, and CD1c subregions. MHC class I proteins are present on the surface of almost all cells, including most tumor cells. MHC-I proteins are loaded with antigens, which are usually derived from endogenous proteins or pathogens present within the cell, and then presented to cytotoxic T lymphocytes (CTLs, also known as CD8+ T cells). T cell receptors are able to recognize and bind peptides complexed with MHC-I class molecules. Each cytotoxic T lymphocyte expresses a unique T cell receptor that is able to bind to a specific MHC/peptide complex. MHC class I molecules primarily mediate the presentation of endogenous antigens.
  • CTLs cytotoxic T lymphocytes
  • MHC-II refers to major histocompatibility complex class II proteins or genes.
  • MHC II includes 5 proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ and HLA-DR.
  • MHC class II proteins are mainly expressed on antigen presenting cells such as B cells, monocytes and macrophages, and dendritic cells.
  • MHC class II molecules mainly mediate the presentation of exogenous antigens. They present exogenous antigen polypeptide molecules to Th cells (helper T cells), that is, stimulate CD4+T cells.
  • MHC/peptide complex relates to a non-covalent complex of a binding domain of an MHC class I or MHC class II molecule and an MHC class I or MHC class II bound peptide.
  • Kernetout herein refers to the process of making a specific gene inactive in the host cell in which it is located, which results in the non-production of the target protein or an inactive form. As will be appreciated by those skilled in the art and described further below, this can be achieved in a variety of different ways, including removing the nucleic acid sequence from the gene, or interrupting the sequence with other sequences, changing the reading frame, or changing the regulatory elements of the nucleic acid. For example, all or part of the coding region of the target gene can be removed or replaced with a "nonsense" sequence, all or part of the regulatory sequence (e.g., a promoter) can be removed or replaced, the translation initiation sequence can be removed or replaced, etc.
  • a "nonsense" sequence all or part of the regulatory sequence (e.g., a promoter) can be removed or replaced, the translation initiation sequence can be removed or replaced, etc.
  • the terms “reduce” and “reduce” are generally used to represent a statistically significant amount of reduction.
  • “reduce”, “reduce” includes reducing at least 10% compared to a reference level, such as reducing at least about 20% or at least about 30% compared to a reference level, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% reduction (i.e., a level that does not exist compared to a reference sample), or any reduction between 10-100%.
  • Kerne-in or “overexpression” herein refers to the process of adding genetic functions to a host cell. This results in an increase in the level of the encoded protein. As will be appreciated by those skilled in the art, this can be achieved 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 the expression of the protein. This can be achieved by modifying the promoter, adding a different promoter, adding an enhancer, or modifying other gene expression sequences.
  • the term “increase” is generally used to indicate an increase by a statistically significant amount; to avoid any doubt, the term “increase” refers to an increase of at least 10% compared to a reference level, such as an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% increase or any increase between 10-100%, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold or at least about 10-fold compared to a reference level, or any increase between 2-fold and 10-fold or greater than 10-fold.
  • Beta-2 microglobulin or " ⁇ 2M” or “B2M” protein is a component of MHC-I class molecules. B2M protein is expressed in all nucleated cells (except red blood cells) and can non-covalently bind to the ⁇ chain of MHC-I molecules, attach to the cell membrane, and can also be released into various tissue fluids.
  • CD47 protein or “Integrin-associated protein (IAP)” is an important self-signal that can inhibit the phagocytosis of macrophages and cause immune escape by binding to the N-terminus of the ligand signal regulatory protein ⁇ (SIRP ⁇ ) on immune cells.
  • SIRP ⁇ ligand signal regulatory protein ⁇
  • MHC-II transactivator protein (CIITA) protein is a key molecule that regulates the expression of MHC-II.
  • the body mainly regulates the expression level of MHC II genes by controlling the expression of CIITA.
  • GSN protein refers to gelsolin, which is an extracellular protein. GSN protein has been reported to reduce the binding of DNGR-1 to F-actin and the cross-presentation of dead cell-associated antigens by type 1 conventional dendritic cells (cDC1).
  • the term “syngeneic” refers to the genetic similarity or identity of the host organism and the cell transplant wherein there is immunological compatibility; eg, no immune response is generated.
  • allogeneic refers to the genetic differences of the host organism and the cells transplanted into which an immune response is generated.
  • B2M-/- refers to a diploid cell having an inactivated B2M gene in both chromosomes.
  • CIITA-/- refers to a diploid cell having an inactivated CIITA gene in both chromosomes.
  • polypeptide refers to a polymer comprising two or more amino acids covalently linked by peptide bonds.
  • a “protein” may comprise one or more polypeptides, wherein the polypeptides interact with each other covalently or non-covalently. Unless otherwise indicated, “polypeptide” and “protein” may be used interchangeably.
  • wild type refers to cells found in nature.
  • pluripotent stem cells as used herein, it also refers to pluripotent stem cells that have not undergone a gene editing procedure to achieve low immunogenicity, e.g., the parental pluripotent stem cells (WT) described herein.
  • WT parental pluripotent stem cells
  • % identity refers to the percentage of identical nucleotides or amino acids in an optimal alignment between the sequences to be compared.
  • the differences between the two sequences can be distributed over local regions (segments) or over the entire length of the sequences to be compared.
  • the identity between the two sequences is usually determined after optimal alignment of a segment or "comparison window".
  • Optimal alignment can be performed manually or with the aid of algorithms known in the art, including but not limited to the local homology algorithm described by Smith and Waterman, 1981, Ads App. Math. 2, 482 and Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, the similarity search method described by Pearson and Lipman, 1988, Proc. Natl Acad.
  • the percent identity of two sequences can be determined using the BLASTN or BLASTP algorithms publicly available on the website of the National Center for Biotechnology Information (NCBI).
  • the % identity is obtained by determining the number of identical positions corresponding to the sequences being compared, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100.
  • at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of a region exhibits a degree of identity.
  • the degree of homogeneity is given for the entire length of the reference sequence.
  • Comparisons for determining sequence homogeneity can be performed using tools known in the art, preferably using optimal sequence alignments, e.g., using Align, using standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.
  • optimal sequence alignments e.g., using Align
  • standard settings preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.
  • nucleotide includes deoxyribonucleotides and ribonucleotides and their derivatives.
  • ribonucleotide is a constituent substance of ribonucleic acid (RNA), consisting of one molecule of base, one molecule of pentose, and one molecule of phosphoric acid, which refers to a nucleotide with a hydroxyl group at the 2' position of the ⁇ -D-ribofuranosyl group.
  • Deoxyribonucleotide is a constituent substance of deoxyribonucleic acid (DNA), also consisting of one molecule of base, one molecule of pentose, and one molecule of phosphoric acid, which refers to a nucleotide in which the hydroxyl group at the 2' position of the ⁇ -D-ribofuranosyl group is replaced by hydrogen, and is the main chemical component of chromosomes.
  • DNA deoxyribonucleic acid
  • Nucleotide is usually referred to by a single letter representing the base: "A (a)” refers to deoxyadenosine or adenylic acid containing adenine, “C (c)” refers to deoxycytidine or cytidine containing cytosine, “G (g)” refers to deoxyguanosine or guanylate containing guanine, “U (u)” refers to uridine containing uracil, and “T (t)” refers to deoxythymidylate containing thymine.
  • polynucleotide and “nucleic acid” are used interchangeably to refer to a polymer of deoxyribonucleotides (deoxyribonucleic acid, DNA) or a polymer of ribonucleotides (ribonucleic acid, RNA).
  • Polynucleotide sequence and “nucleotide sequence” are used interchangeably to refer to the order of nucleotides in a polynucleotide.
  • DNA coding strand sense strand
  • RNA it encodes can be considered to have the same nucleotide sequence, and the deoxythymidylic acid in the DNA coding strand sequence corresponds to the uridine acid in the RNA sequence it encodes.
  • the term "expression” includes transcription and/or translation of a nucleotide sequence. Thus, expression may involve the production of transcripts and/or polypeptides.
  • transcription refers to the process of transcribing the genetic code in a DNA sequence into RNA (transcript).
  • in vitro transcription refers to the in vitro synthesis of RNA, particularly mRNA, in a cell-free system (e.g., in an appropriate cell extract) (see, e.g., Pardi N., Muramatsu H., Weissman D., Karikó K. (2013). In: Rabinovich P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation.
  • a vector that can be used to produce a transcript is also referred to as a "transcription vector,” which contains regulatory sequences required for transcription.
  • transcription encompasses "in vitro transcription.”
  • encoding refers to the inherent properties of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA can be used as a template to synthesize polymers and macromolecules in other biological processes, as long as there is a clear nucleotide sequence or a clear amino acid sequence. Therefore, a gene encodes a protein when the gene's mRNA produces a protein in a cell or other biological system through transcription and translation.
  • the present invention provides a low immunogenic pluripotent stem cell, comprising:
  • MHC-I major histocompatibility class I antigen
  • MHC-II Reduced endogenous major histocompatibility class II antigen
  • parental pluripotent stem cells refer to parental (also referred to herein as “WT”) pluripotent stem cells before immune modification that have not undergone a gene editing procedure to achieve low immunogenicity.
  • the reduced sensitivity to NK cell killing is caused by increased expression of the GSN protein.
  • reduction of function can be achieved in a variety of ways, including removal of nucleic acid sequences from a gene, interruption of a sequence with another sequence, or alteration of a regulatory component of a nucleic acid.
  • all or part of the coding region of a target gene can be removed or replaced with a "nonsense" sequence, frameshift mutations can be performed, all or part of a regulatory sequence such as a promoter can be removed or replaced, a translation initiation sequence can be deleted or replaced, etc.
  • the reduction in MHC I (HLA I when the cells are derived from human cells) function in pluripotent stem cells can be measured using techniques known in the art and as described below; for example, using FACS techniques with labeled antibodies that bind to the HLA complex; for example, using commercially available HLA-A, HLA-B, HLA-C antibodies that bind to human major histocompatibility HLA class I.
  • MHC II when the cells are derived from human cells
  • the reduction in MHC II (HLA II when the cells are derived from human cells) function in pluripotent stem cells can be measured using techniques known in the art and as described below; for example, using FACS techniques with labeled antibodies that bind to the HLA complex; for example, using commercially available HLA-DQ, HLA-DR, HLA-DP antibodies that bind to human major histocompatibility HLA class II.
  • the MHC-I function is reduced by reducing the activity of MHC-I class proteins.
  • the MHC-I class protein comprises a human leukocyte antigen-A (HLA-A) protein, a human leukocyte antigen-B (HLA-B) protein, or a human leukocyte antigen-C (HLA-C) protein.
  • HLA-A human leukocyte antigen-A
  • HLA-B human leukocyte antigen-B
  • HLA-C human leukocyte antigen-C
  • the MHC-I function is reduced by reducing the activity of an MHC-I transcriptional regulator.
  • the MHC-I transcriptional regulator may be selected from one or more of: ⁇ 2 microglobulin (B2M), transporter associated with antigen processing 1 (TAP1), transporter associated with antigen processing 2 (TAP2), transporter associated with antigen processing (TAP)-associated glycoprotein (Tapasin), or NOD-like receptor family caspase recruitment domain 5 (NLRC5).
  • the MHC-I function is reduced by reducing the activity of the HLA-A protein.
  • the MHC-I function is reduced by knocking out the gene encoding the HLA-A protein.
  • the MHC-I function is reduced by reducing the activity of the HLA-B protein.
  • the MHC-I function is reduced by knocking out the gene encoding the HLA-B protein.
  • the MHC-I function is reduced by reducing the activity of the HLA-C protein.
  • the MHC-I function is reduced by knocking out the gene encoding the HLA-C protein.
  • the MHC-I function is reduced by reducing the activity of TAP1 protein.
  • the MHC-I function is reduced by knocking out the gene encoding the TAP1 protein.
  • the MHC-I function is reduced by reducing the activity of the TAP2 protein.
  • the MHC-I function is reduced by knocking out the gene encoding the TAP2 protein.
  • the MHC-I function is reduced by reducing the activity of the Tapasin protein.
  • the MHC-I function is reduced by knocking out the gene encoding the Tapasin protein.
  • the MHC-I function is reduced by reducing the activity of the NLRC5 protein.
  • the MHC-I function is reduced by knocking out the gene encoding the NLRC5 protein.
  • the MHC-I function is reduced by reducing the activity of the B2M protein.
  • the B2M protein is a human B2M protein, which comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:1.
  • the B2M protein is a crab-eating macaque B2M protein, which comprises the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:9.
  • the MHC-I function is reduced by knocking out the gene encoding the B2M protein.
  • the MHC-II function is reduced by reducing the activity of MHC class II proteins.
  • the MHC-II class protein comprises a human leukocyte antigen-DR (HLA-DR) protein, a human leukocyte antigen-DQ (HLA-DQ) protein, or a human leukocyte antigen-DP (HLA-DP) protein.
  • HLA-DR human leukocyte antigen-DR
  • HLA-DQ human leukocyte antigen-DQ
  • HLA-DP human leukocyte antigen-DP
  • the MHC-II function is reduced by reducing the activity of an MHC-II transcriptional regulator.
  • the MHC-II transcriptional regulator may be selected from: one or more of MHC-II transactivator protein (CIITA), regulatory factor X-associated anchor protein (RFXANK), regulatory factor X5 (RFX5), and regulatory factor X-associated protein (RFXAP).
  • CIITA MHC-II transactivator protein
  • RFXANK regulatory factor X-associated anchor protein
  • RFX5 regulatory factor X5
  • RFXAP regulatory factor X-associated protein
  • the MHC-II function is reduced by reducing the activity of the HLA-DR protein.
  • the MHC-II function is reduced by knocking out the gene encoding the HLA-DR protein.
  • the MHC-II function is reduced by reducing the activity of the HLA-DQ protein.
  • the MHC-II function is reduced by knocking out the gene encoding the HLA-DQ protein.
  • the MHC-II function is reduced by reducing the activity of the HLA-DP protein.
  • the MHC-II function is reduced by knocking out the gene encoding the HLA-DP protein.
  • the MHC-II function is reduced by reducing the activity of the RFXANK protein.
  • the MHC-II function is reduced by knocking out the gene encoding the RFXANK protein.
  • the MHC-II function is reduced by reducing the activity of the RFX5 protein.
  • the MHC-II function is reduced by knocking out the gene encoding the RFX5 protein.
  • the MHC-II function is reduced by reducing the activity of the RFXAP protein.
  • the MHC-II function is reduced by knocking out the gene encoding the RFXAP protein.
  • the MHC-II function is reduced by reducing the activity of the CIITA protein.
  • the CIITA protein is a human CIITA protein, which comprises the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:2.
  • the CIITA protein is a cynomolgus monkey CIITA protein, which comprises the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:10.
  • the MHC-II function is reduced by knocking out the gene encoding the CIITA protein.
  • the gene is knocked out using CRISPR technology.
  • CRISPR technology is used to introduce small deletions/insertions into the coding region of a gene so that no functional protein is produced, usually as a result of a frameshift mutation, which results in the generation of a stop codon, resulting in a truncated, non-functional protein.
  • MHC-I HLA-I when the cells are derived from human cells
  • MHC-II HLA-II when the cells are derived from human cells
  • pluripotent stem cells can be measured using techniques known in the art, such as using protein Western blotting, FACS technology, RT-PCR, qPCR technology, etc.
  • the reduced sensitivity to NK cell killing is caused by increased expression of GSN protein in pluripotent stem cells. This is accomplished in several ways, as will be appreciated by those skilled in the art, and can use "knock-in" or transgenic techniques. In some cases, increased GSN expression is caused by one or more GSN transgenes.
  • one or more copies of the GSN gene are added to pluripotent stem cells under the control of an inducible or constitutive promoter.
  • a lentiviral construct is used as described herein or known in the art.
  • the GSN gene can be integrated into the genome of a host cell under the control of a suitable promoter.
  • the increased GSN protein expression is caused by a GSN transgene.
  • the GSN protein is a human GSN protein, which comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:3.
  • the GSN protein is a cynomolgus monkey GSN protein, which comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:7.
  • sufficient GSN protein expression can be determined using known techniques, such as those described in the Examples, for example using Western blot, ELISA assay or FACS assay.
  • "sufficient" in this context means an increase in GSN protein expression on the surface of pluripotent stem cells, which silences NK cell killing.
  • the present invention further provides a low immunogenic pluripotent stem cell comprising:
  • One or more alterations result in increased expression of GSN protein in the low immunogenic pluripotent stem cells.
  • the low immunogenic pluripotent stem cells comprise:
  • One or more alterations result in increased GSN gene expression in the low immunogenic pluripotent stem cells.
  • modifying a genome refers to modifying a nucleic acid sequence in a cell or under a cell-free condition to produce a transformed pluripotent cell and a pluripotent stem cell.
  • Exemplary "changes” or “genetic changes” include, but are not limited to, homologous recombination, knock-in, ZFN (zinc finger nuclease), TALEN (transcription activator-like effector nuclease), CRISPR (clustered regularly spaced short palindromic repeats)/Cas9 and other site-specific nuclease technologies. These technologies enable double-stranded DNA breaks to be performed at the desired gene locus. These controlled double-strand breaks promote homologous recombination at specific gene locus points.
  • alteration or “genetic alteration” techniques also include the introduction of gene expression modifying molecules, including but not limited to siRNA, shRNA, microRNA, antisense RNA, antisense oligonucleotides ASO (antisense oligonucleotides) or anti-miRNA oligonucleotides AMO (Anti-miRNA oligonucleotides).
  • gene expression modifying molecules including but not limited to siRNA, shRNA, microRNA, antisense RNA, antisense oligonucleotides ASO (antisense oligonucleotides) or anti-miRNA oligonucleotides AMO (Anti-miRNA oligonucleotides).
  • pluripotent cells and pluripotent cells of the present invention stem cells to render them less immunogenic.
  • CRISPR technology is used to reduce the expression of active B2M and/or CIITA proteins in modified cells
  • viral technology e.g., lentivirus
  • GSN gene is used to knock in the GSN gene.
  • these genes can be manipulated in different orders using different techniques.
  • the one or more changes contained in the low immunogenic pluripotent stem cells of the present invention can reduce endogenous major histocompatibility class I antigen (MHC-I) function. In some embodiments, the one or more changes contained in the low immunogenic pluripotent stem cells of the present invention can reduce endogenous major histocompatibility class II antigen (MHC-II) function. In some embodiments, the one or more changes contained in the low immunogenic pluripotent stem cells of the present invention can reduce sensitivity to NK cell killing.
  • MHC-I major histocompatibility class I antigen
  • MHC-II major histocompatibility class II antigen
  • the one or more changes contained in the low immunogenic pluripotent stem cells of the present invention can reduce the activity of endogenous B2M protein. In some embodiments, the one or more changes contained in the low immunogenic pluripotent stem cells of the present invention can reduce the activity of endogenous CIITA protein. In some embodiments, the one or more changes contained in the low immunogenic pluripotent stem cells of the present invention can increase the expression of GSN protein.
  • the low immunogenic pluripotent stem cells of the present invention comprise one or more changes that inhibit the expression of endogenous B2M protein. In some embodiments, the low immunogenic pluripotent stem cells of the present invention comprise one or more changes that inhibit the expression of endogenous CIITA protein.
  • the low immunogenic pluripotent stem cells of the present invention comprise one or more changes that can interfere with the expression of endogenous B2M protein. In some embodiments, the low immunogenic pluripotent stem cells of the present invention comprise one or more changes that can interfere with the expression of endogenous CIITA protein.
  • the low immunogenic pluripotent stem cells of the present invention comprise one or more changes that can reduce the expression of endogenous B2M protein. In some embodiments, the low immunogenic pluripotent stem cells of the present invention comprise one or more changes that can reduce the expression of endogenous CIITA protein.
  • the low immunogenic pluripotent stem cells of the present invention comprise one or more changes that can knock out endogenous B2M protein. In some embodiments, the low immunogenic pluripotent stem cells of the present invention comprise one or more changes that can knock out endogenous CIITA protein.
  • the pluripotent stem cells are altered using Clustered Regularly Interspaced Short Palindromic Repeats/Cas (“CRISPR”) technology known in the art to reduce the activity of endogenous B2M protein.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats/Cas
  • the pluripotent stem cells are altered using Clustered Regularly Interspaced Short Palindromic Repeats/Cas ("CRISPR”) technology known in the art to reduce the activity of endogenous CIITA protein.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats/Cas
  • the pluripotent stem cells are altered to inactivate both alleles of the endogenous B2M gene using Clustered Regularly Interspaced Short Palindromic Repeats/Cas (“CRISPR”) technology known in the art.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats/Cas
  • the assay is a Western blot of cell lysates probed with antibodies against the B2M protein or the CIITA protein.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • the use of viral techniques known in the art can be used to cause increased expression of the GSN gene in the low immunogenic pluripotent stem cells.
  • the viral techniques include, but are not limited to, the use of retroviral vectors, lentiviral vectors, adenoviral vectors, and Sendai viral vectors.
  • the nucleic acid sequence encoding the GSN protein is introduced into a selected site of the cell; the selected site of the cell is a safe harbor gene site such as AAVS1, CCR5, etc.
  • a "safe harbor gene site” refers to a site that can be used for safe gene knock-in and can ensure normal and stable expression of the introduced gene.
  • a lentiviral vector is used to induce increased expression of the GSN gene in the low immunogenic pluripotent stem cells.
  • the low immunogenic pluripotent stem cells are human pluripotent stem cells.
  • the B2M protein is a human B2M protein, which comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 1.
  • the CIITA protein is a human CIITA protein, which comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 2.
  • the GSN protein is a human GSN protein, which comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:3.
  • the low immunogenic pluripotent stem cells are cynomolgus monkey pluripotent stem cells.
  • the B2M protein is a cynomolgus monkey B2M protein, which comprises the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:9.
  • the CIITA protein is a cynomolgus monkey CIITA protein, which comprises the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:10.
  • the GSN protein is a cynomolgus monkey GSN protein, which comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:7.
  • the low immunogenicity pluripotent stem cells are low immunogenicity induced pluripotent stem cells (iPSCs).
  • the low immunogenic pluripotent stem cells comprise:
  • MHC-I major histocompatibility class I antigen
  • MHC-II Reduced endogenous major histocompatibility class II antigen
  • the T cell response elicited by the low immunogenic pluripotent stem cells is lower than the T cell response elicited by the parental pluripotent stem cells, and the parental pluripotent stem cells do not contain the changes that reduce the activity of B2M and CIITA proteins and the changes that cause increased GSN protein expression.
  • the T cell response is measured by measuring the killing of T cells to the low immunogenic pluripotent stem cells or parental pluripotent stem cells by real-time label-free dynamic cell analysis (RTCA).
  • RTCA real-time label-free dynamic cell analysis
  • the natural killer (NK) cell response elicited by the low immunogenic pluripotent stem cells is low
  • the DKO cell comprises the changes that reduce the activity of the B2M and CIITA proteins but does not comprise the changes that cause increased GSN protein expression.
  • the NK cell response is measured by determining the IFN- ⁇ level of NK cells incubated in vitro with the low immunogenic pluripotent stem cells or DKO cells.
  • the NK cell response is measured by measuring the killing of NK cells to the low immunogenic pluripotent stem cells or DKO cells by real-time label-free dynamic cell analysis (RTCA).
  • RTCA real-time label-free dynamic cell analysis
  • the present invention also provides a method for producing the low immunogenic pluripotent stem cells of the present invention, the method comprising: reducing the endogenous major histocompatibility class I antigen (MHC-I) function in the pluripotent stem cells; reducing the endogenous major histocompatibility class II antigen (MHC-II) function in the pluripotent stem cells; and increasing the expression of a protein that reduces the sensitivity of the pluripotent stem cells to NK cell killing, wherein the protein is GSN protein.
  • MHC-I major histocompatibility class I antigen
  • MHC-II major histocompatibility class II antigen
  • the low immunogenicity pluripotent stem cells are low immunogenicity induced pluripotent stem cells (iPSCs).
  • the method comprises: eliminating the activity of both alleles of the B2M gene in the pluripotent stem cells; eliminating the activity of both alleles of the CIITA gene in the pluripotent stem cells; and increasing the expression of the GSN gene in the pluripotent stem cells.
  • the activity of the B2M protein in the pluripotent stem cells can be reduced by the technique of "alteration” or “genetic alteration” as described above.
  • the activity of the CIITA protein in the pluripotent stem cells can be reduced by the technique of "alteration” or “genetic alteration” as described above.
  • the techniques for example, introduce gene expression modifying molecules, clustered regularly interspaced short palindromic repeats (CRISPR) technology, transcription activator-like effector nuclease (TALEN) technology, zinc finger nuclease (ZFN) technology or homologous recombination technology.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • TALEN transcription activator-like effector nuclease
  • ZFN zinc finger nuclease
  • the gene expression modifying molecules comprise siRNA, shRNA, microRNA, antisense RNA, antisense oligonucleotides ASO (antisense oligonucleotides) or anti-miRNA oligonucleotides AMO (Anti-miRNA oligonucleotides).
  • the CRISPR/Cas system includes a Cas protein or a nucleic acid sequence encoding a Cas protein and at least one to two ribonucleic acids (e.g., gRNA), which can guide the Cas protein to a target motif of a target polynucleotide sequence and hybridize with the target motif.
  • a Cas protein or a nucleic acid sequence encoding a Cas protein and at least one to two ribonucleic acids (e.g., gRNA), which can guide the Cas protein to a target motif of a target polynucleotide sequence and hybridize with the target motif.
  • gRNA ribonucleic acids
  • the CRISPR/Cas system includes a Cas protein or a nucleic acid sequence encoding a Cas protein and a single ribonucleic acid or at least one ribonucleic acid (e.g., gRNA) pair, which can guide the Cas protein to a target motif of a target polynucleotide sequence and hybridize with the target motif.
  • a Cas protein or a nucleic acid sequence encoding a Cas protein and a single ribonucleic acid or at least one ribonucleic acid (e.g., gRNA) pair, which can guide the Cas protein to a target motif of a target polynucleotide sequence and hybridize with the target motif.
  • the Cas protein comprises one or more amino acid substitutions or modifications. In some embodiments, one or more amino acid substitutions comprise conservative amino acid substitutions. In some cases, substitutions and/or modifications can prevent or reduce proteolytic degradation and/or extend the half-life of polypeptides in cells.
  • the Cas protein may comprise peptide bond replacements (e.g., urea, thiourea, carbamate, sulfonylurea, etc.). In some embodiments, the Cas protein may comprise naturally occurring amino acids. In some embodiments, the Cas protein may comprise optional amino acids (e.g., D-amino acids, ⁇ -amino acids, homocysteine, phosphoserine, etc.). In some embodiments, the Cas protein may comprise modifications to include portions (e.g., pegylation, glycosylation, lipidation, acetylation, capping, etc.).
  • the Cas protein comprises a core Cas protein.
  • Exemplary Cas core proteins include but are not limited to In Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8 and Cas9.
  • the Cas protein comprises a Cas protein of an Escherichia coli (E.coli) subtype (also referred to as CASS2).
  • Exemplary Cas proteins of E. coli subtypes include, but are not limited to, Cse1, Cse2, Cse3, Cse4 and Cas5e.
  • the Cas protein comprises a Cas protein of a Ypest subtype (also referred to as CASS3).
  • Exemplary Cas proteins of Ypest subtypes include, but are not limited to, Csy1, Csy2, Csy3 and Csy4.
  • the Cas protein comprises a Cas protein of an Nmeni subtype (also referred to as CASS4).
  • Exemplary Cas proteins of Nmeni subtypes include, but are not limited to, Csn1 and Csn2.
  • the Cas protein comprises a Cas protein of a Dvulg subtype (also referred to as CASS1).
  • Exemplary Cas proteins of Dvulg subtypes include, but are not limited to, Csd1, Csd2 and Cas5d.
  • the Cas protein comprises a Cas protein of a Tneap subtype (also referred to as CASS7).
  • Exemplary Cas proteins of the Tneap subtype include, but are not limited to, Cst1, Cst2, and Cas5t.
  • the Cas protein comprises a Cas protein of the Hmari subtype.
  • Exemplary Cas proteins of the Hmari subtype include, but are not limited to, Csh1, Csh2, and Cas5h.
  • the Cas protein comprises a Cas protein of the Apern subtype (also known as CASS5).
  • Exemplary Cas proteins of the Apern subtype include, but are not limited to, Csa1, Csa2, Csa3, Csa4, Csa5, and Cas5a.
  • the Cas protein comprises a Cas protein of the Mtube subtype (also known as CASS6).
  • Exemplary Cas proteins of the Mtube subtype include, but are not limited to, Csm1, Csm2, Csm3, Csm4, and Csm5.
  • the Cas protein comprises a RAMP-type Cas protein.
  • Exemplary RAMP-type Cas proteins include, but are not limited to, Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6.
  • the Cas protein is a Streptococcus pyogenes Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Streptococcus aureus Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Streptococcus thermophilus Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Neisseria meningitides Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Treponema denticola Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Cas9 protein or a functional portion thereof from any bacterial species.
  • the Cas9 protein is a member of a type II CRISPR system, which typically includes a trans-encoded small RNA (tracrRNA), an endogenous ribonuclease 3 (rnc), and a Cas protein.
  • the Cas9 protein (also known as a CRISPR-associated endonuclease Cas9/Csn1) is a polypeptide comprising 1368 amino acids.
  • the activity of B2M protein in the pluripotent stem cells is reduced by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • the activity of both alleles of the B2M gene in the pluripotent stem cells is eliminated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • the activity of CIITA protein in the pluripotent stem cells is reduced by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • the activity of both alleles of the CIITA gene in the pluripotent stem cells is eliminated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • the expression of GSN protein is increased by modification of the endogenous locus.
  • the endogenous locus is modified by the technique of "alteration” or “genetic alteration” as described above.
  • the technique for example, gene knock-in, clustered regularly interspaced short palindromic repeats (CRISPR) technology, transcription activator-like effector nuclease (TALEN) technology, zinc finger nuclease (ZFN) technology or homologous recombination technology.
  • the expression of GSN protein is increased by expression of a transgene.
  • the transgenic expression technology used to increase the expression of GSN protein includes but is not limited to viral technology, Piggybac transposon technology, and Sleeping Beauty transposon technology.
  • the nucleic acid sequence encoding the target protein can be operably connected to one or more regulatory nucleotide sequences in the expression construct.
  • the regulatory nucleotide sequence is generally suitable for host cells and subjects to be treated.
  • suitable expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.
  • one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader sequences or signal sequences, ribosome binding sites, transcription start and stop sequences, translation start and stop sequences, and enhancers or activator sequences.
  • the expression construct used herein can use constitutive or inducible promoters known in the art.
  • the promoter can be a naturally occurring promoter, or a hybrid promoter combining elements of more than one promoter.
  • the expression construct can be present in the cell on an episome (e.g., a plasmid), or the expression construct can be inserted into a chromosome.
  • the expression vector includes a selectable marker gene to allow selection of transformed host cells.
  • Some embodiments include an expression vector comprising a nucleotide sequence encoding the target protein operably connected to at least one regulatory sequence.
  • the regulatory sequences used herein include promoters, enhancers, and other expression control elements.
  • the expression vector is designed to select the host cell to be transformed, the desired protein to be expressed, the copy number of the vector, the ability to control the copy number, or the expression of any other protein encoded by the vector, such as an antibiotic marker.
  • the promoter is the EF1a promoter.
  • Viral techniques can be used to induce increased expression of the GSN gene in the low immunogenic pluripotent stem cells.
  • the viral techniques include, but are not limited to, the use of retroviral vectors, lentiviral vectors, adenoviral vectors, and Sendai virus vectors.
  • a nucleic acid sequence encoding the GSN protein is synthesized and constructed into a lentiviral vector, and then at least one copy of the GSN gene under the control of a promoter is introduced into the pluripotent stem cells via the lentiviral vector to increase the expression of the GSN protein.
  • a nucleic acid sequence encoding a GSN protein is introduced into a selected site of the pluripotent stem cell genome.
  • the selected site is a safe harbor gene site such as AAVS1, CCR5, etc.
  • a "safe harbor gene site” refers to a site that can be used for safe gene knock-in and can ensure normal and stable expression of the transferred gene.
  • the GSN protein is a human GSN protein.
  • the nucleic acid sequence encoding the GSN protein comprises the nucleic acid sequence shown in SEQ ID NO:4 or a nucleic acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in SEQ ID NO:4.
  • the GSN protein is a cynomolgus monkey GSN protein.
  • the nucleic acid sequence encoding the GSN protein comprises the nucleic acid sequence shown in SEQ ID NO:8 or a nucleic acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in SEQ ID NO:8.
  • the present invention further provides use of the low-immunogenic pluripotent stem cells of the present invention or the low-immunogenic pluripotent stem cells prepared by the method of the present invention in preparing a drug for preventing or treating a disease requiring cell transplantation.
  • the low immunogenicity pluripotent stem cells of the present invention or the low immunogenicity pluripotent stem cells prepared by the method of the present invention can be Differentiate into different cells with induction, it can be used for different prevention or treatment purposes, to prevent or treat different diseases.
  • differentiation method depends on the required cell type using known technology.
  • cell suspension differentiation can be used, then made into gel matrix form, such as matrigel, gelatin or fibrin/thrombin form, to promote cell survival.
  • gel matrix form such as matrigel, gelatin or fibrin/thrombin form
  • cell differentiation can be into cardiomyocyte, nerve cell, glial cell, endothelial cell, T cell, NK cell, NKT cell, macrophage, hematopoietic progenitor cell, mesenchymal cell, islet cell, chondrocyte, retinal pigment epithelial cell, nephrocyte, hepatocyte, thyroid cell, skin cell, blood cell or epithelial cell under
  • the disease is cancer
  • the cancer comprises solid tumors and blood tumors.
  • the solid tumor comprises small cell lung cancer, breast cancer, testicular cancer, neuroblastoma, ovarian cancer or melanoma.
  • the blood tumor comprises acute leukemia, chronic leukemia, lymphoma, myelodysplastic syndrome or multiple myeloma.
  • the disease comprises aplastic anemia.
  • the disease comprises a congenital immunodeficiency disease.
  • the disease is an autoimmune disease comprising systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, or type I diabetes.
  • the disease is a neurodegenerative disease comprising Parkinson's disease, Alzheimer's disease, spinal cord injury, retinal degeneration, stroke, Huntington's disease, or amyotrophic lateral sclerosis.
  • the disease is a cardiovascular disease comprising atherosclerosis, hypertension, rheumatic heart disease, cardiomyopathy, arrhythmia, congenital heart disease, valvular heart disease, carditis, myocardial infarction, heart failure, aortic aneurysm, or peripheral arterial disease.
  • the disease is a metabolism-related disease comprising type II diabetes, scurvy, hypoglycemia, hyperlipidemia, or osteoporosis.
  • the pluripotent stem cells of the present invention and the low-immunogenic pluripotent stem cells prepared by the method of the present invention can exhibit excellent effects, such as but not limited to: (1) having good self-renewal and differentiation abilities; (2) being able to escape T cell killing; (3) being able to escape NK cell killing; and/or (4) the differentiated cells can also escape NK cell killing; thereby showing excellent application potential.
  • human pluripotent stem cell lines H1 (Wicell, WA01) or H9 (Wicell, WA09) were used to construct target cell lines.
  • the cell culture and gene knockout reagents used are shown in Table 1.
  • CRISPR/CAS9 is used to knock out ⁇ -2-microglobulin (B2M) in the endoplasmic reticulum, preventing the cell surface MHC-I from forming functional molecules, thereby escaping the killing of allogeneic CD8 + T cells; escaping the killing of CD4+ T cells is achieved by knocking out CIITA, a positive regulator of MHC-II gene transcription, thereby reducing the expression of MHC-II class molecules.
  • B2M ⁇ -2-microglobulin
  • B2M-gRNA1 and B2M-gRNA2 (EasyEdit sgRNA, GenScript) were used to directly knock out the B2M exon segment at both ends, and then two pairs of PCR primers, B2M-F1/R1 and B2M-F2/R2, were used to verify the knockout of the genomic sequence.
  • CIITA-gRNA1 and CIITA-gRNA2 (EasyEdit sgRNA, GenScript) were used to directly knock out the CIITA exon segment at both ends, and then the genome sequence knockout was verified using two pairs of PCR primers, CIITA-F1/R1 and CIITA-F2/R2, respectively.
  • Human pluripotent stem cells were cultured in mTeSR1 medium supplemented with Y-27632 on a Matrigel-coated 6-well plate to 80% density. After digestion with TrypLE, the cells were neutralized with DMEM/F12 and counted. 2 ⁇ 10 6 cells were taken out and plated on EP After centrifugation, discard the supernatant.
  • Neon transfection system (ThermoFisher) 100 ⁇ L electroporation system, 15 ⁇ g TrueCut TM Cas9 Protein + 3 ⁇ g gRNA (B2M-gRNA1 + B2M-gRNA2 + CIITA-gRNA1 + CIITA-gRNA2) was added to form a ribonucleoprotein complex (RNP) system, mixed and placed at room temperature for 20 minutes.
  • RNP ribonucleoprotein complex
  • qPCR was used to detect the expression of B2M and CIITA at the RNA level in the B2M/CIITA biallelic knockout clone DKO.
  • the primers used are shown below.
  • reaction system is as follows:
  • Amplification 95°C 10s, 60°C 30s, 40 cycles.
  • IFN- ⁇ (PeproTech, Cat#300-02) was used to stimulate WT and DKO to detect HLA class I/II molecules on the surface of H1 cells.
  • the specific detection method is as follows:
  • the cells were plated, and the medium containing IFN- ⁇ was added to the cells when the medium was changed on the next day. After 48 hours of action, the cells were digested and the expression of HLA-I/II was detected using a flow cytometer (Agilent Technologies, NovoCyte).
  • the obtained B2M/CIITA biallelic knockout positive clones (DKO) were subjected to karyotype detection, and the specific detection method is as follows:
  • the chromosome specimen fixed on the slide is treated with trypsin and then stained with Giemsa stain. Based on the chromosome length, centromere position, long-short arm ratio, satellite presence and other characteristics, the chromosome number and morphological structure of the metaphase chromosome are analyzed to determine whether its karyotype is consistent with the normal karyotype.
  • the results of karyotype detection are shown in Figure 6 .
  • the DKO karyotype is normal, with no significant changes compared with the normal karyotype.
  • This example further tests whether the stemness and immune function of pluripotent stem cells change after knocking out the B2M/CIITA biallelic genes.
  • the protein levels of stemness genes POU5F1 and NANOG in WT and DKO cells were detected by immunofluorescence, the expression of stemness genes POU5F1, NANOG and SOX2 in WT and DKO cells at the RNA level was detected by RT-qPCR, and the expression of stemness genes SSEA-4 and Tra1-81 on the surface of WT and DKO cells was detected by flow cytometry.
  • the specific detection methods are as follows:
  • Immunofluorescence detection WT or DKO cells were plated in 12-well plates. After the cells grew to a density of 60-80%, the medium was aspirated and fixed with 4% paraformaldehyde. After the cells were permeabilized, the primary antibodies of POU5F1 and NANOG were used for overnight incubation at 4°C. After the primary antibodies were washed off, the secondary antibodies with fluorescent labels were incubated at room temperature, and then photographed using a fluorescence microscope (Nikon Ts2R-FL).
  • reaction system is as follows:
  • Amplification 95°C 10s, 60°C 30s, 40 cycles.
  • MSC Mesenchymal stem cells
  • FIG. 7A The results of immunofluorescence detection are shown in Figure 7A. Both WT and DKO cells express stemness genes POU5F1 and NANOG at the protein level, and there is no significant difference in the expression of stemness genes POU5F1 and NANOG in DKO cells compared with WT cells.
  • the results of RT-qPCR detection are shown in Figure 7B. WT and DKO cells express stemness genes POU5F1, NANOG and SOX2 at the RNA level, and there is no significant difference in the expression of stemness genes POU5F1, NANOG and SOX2 in DKO cells compared with WT cells.
  • the results of flow cytometry detection are shown in Figure 7C. Both WT and DKO cells highly express stemness genes SSEA-4 (WT 100% and DKO 99.98%) and Tra1-81 (WT 96.75% and DKO 99.13%).
  • the specific detection method is as follows: 100 ⁇ L of a suspension containing 5 ⁇ 10 5 DKO cells was subcutaneously injected into immunodeficient mice (SCID Beige, Vital River), and the teratomas were removed when the volume was greater than 1.5 cm 3 , and paraffin sections and hematoxylin and eosin staining were performed.
  • the staining results are shown in Figure 8 , and the DKO cells with B2M/CIITA biallelic knockout can form teratomas in vivo and differentiate into cells of the inner, middle and outer germ layers, and the DKO cells have normal differentiation ability of the three germ layers.
  • the xCELLigence RTCA Instrument was used to perform T cell and NK cell cytotoxicity experiments to detect changes in the immune function of DKO cells.
  • the reagents used in the cytotoxicity experiments are shown in Table 3.
  • T cells XC11228, purchased from SAILYBIO
  • NK cells XC11013, purchased from SAILYBIO
  • T cells will be subjected to CD3, CD4 and CD8 flow cytometry before use
  • NK cells will be subjected to CD16 and CD56 flow cytometry before use to ensure the function of the T cells and NK cells used.
  • RTCA detection data were analyzed using xCELLigence software to calculate the killing rate and escape function.
  • RTCA results are shown in Figure 9.
  • WT cells escape NK cell killing due to the expression of HLA-I, but are killed by T cells.
  • DKO cells can escape T cell killing and are more sensitive to NK cell killing.
  • the nucleic acid sequence encoding CD47 protein (SEQ ID NO: 6) was constructed in a lentiviral vector (pGC-EF1a) initiated by EF1a and carrying a puromycin selection marker.
  • pGC-EF1a lentiviral vector
  • the structure of the pGC-EF1a vector is shown in Figure 10.
  • the specific operation method is as follows:
  • the lentiviral vector was digested with BamHI/NheI, and the nucleic acid sequence encoding CD47 protein (SEQ ID NO: 6) was ligated to After successful connection, Sanger sequencing was used to verify the correctness of the inserted sequence and virus packaging was performed.
  • the lentiviral vector was transfected into the DKO cells constructed in Example 1, and the medium was changed after 24 hours, and after 48 hours, the medium was changed to a medium containing puromycin for screening.
  • the constructed stable cell line DKO+CD47 was subjected to flow cytometry (CD47 antibody was purchased from FACS: Biolegend, catalog number: 323108) and qPCR detection.
  • the qPCR primers were CD47-F: AGAAGGTGAAACGATCATCGAGC (SEQ ID NO: 36); CD47-R: CTCATCCATACCACCGGATCT (SEQ ID NO: 37).
  • the test results are shown in Figures 11A and 11B.
  • the expression level of CD47 in the constructed DKO+CD47 cell line was significantly higher than that in the WT cell.
  • the validated DKO+CD47 cell line was then subjected to cell expansion and subsequent functional testing.
  • NK cells can effectively kill DKO cells, while WT and DKO+CD47 overexpressing cells can escape NK killing.
  • the nucleic acid sequence encoding the GSN protein (the amino acid sequence of the GSN protein is shown in SEQ ID NO:3) was directly synthesized.
  • the nucleic acid sequence is shown in SEQ ID NO:4.
  • a lentiviral vector (pGC-EF1a) initiated by EF1a and carrying a puromycin selection marker was constructed.
  • the structure of the pGC-EF1a vector is shown in Figure 10.
  • the vector was digested with BamHI/NheI, and the nucleic acid sequence of the synthesized GSN was connected to the lentiviral vector. After successful connection, Sanger sequencing was used to verify the correctness of the inserted sequence and perform viral packaging.
  • the lentiviral vector was transfected into the DKO cells constructed in Example 1, and the medium was changed after 24 hours, and the medium with puromycin was changed after 48 hours for screening.
  • the expression of stemness genes in the DKO+GSN cell line was detected by immunofluorescence and flow cytometry. For specific detection methods, see Example 2.1.
  • the flow cytometry results are shown in Figure 15.
  • the stemness genes SSEA-4, TRA-1-60, Tra1-81 and OCT4 were highly expressed on the surface of DKO+GSN cells, and the proportions of each stemness gene were 98.89%, 98.73%, 95.87% and 98.92%, respectively.
  • DKO+GSN cells were used to detect the ability of three germ layers to differentiate.
  • the dissociated DKO+GSN single cells were resuspended in three germ layer culture media supplemented with Y27632, and an appropriate amount of cells were attached to a well plate with a cell crawler coated with matrix gel. After 24 hours, the preheated differentiation medium was replaced, and the medium was changed every day until the seventh day to obtain mesoderm, endoderm, and ectoderm cells.
  • the expression of three germ layer marker proteins was detected by immunofluorescence to detect the three germ layer differentiation ability of the DKO+GSN cell line.
  • DKO+GSN cells express ectoderm marker proteins: PAX6 and GAD1; mesoderm marker proteins: Brachyury and NCAM; endoderm marker proteins: SOX17 and FOXA2 at the protein level.
  • This example detects the differentiation ability of DKO+GSN cells.
  • the specific detection method is as follows: 100 ⁇ L of a suspension containing 5 ⁇ 10 5 DKO+GSN cells is subcutaneously injected into immunodeficient mice (SCID Beige), and the teratomas are removed when the volume is greater than 1.5 cm 3 , and paraffin sections are made and stained with hematoxylin and eosin.
  • the escape function of DKO+GSN cells on different immune cells was detected by RTCA. See Example 2.3 for specific detection methods.
  • the PBNK cells used were obtained by adding IL-2 to PBMC (peripheral blood mononuclear cells, from SAILYBIO) during in vitro culture to increase the proportion of NK cells.
  • the test results are shown in Figures 18A-18D.
  • the DKO cells constructed in Example 1 were completely killed by NK cells, while WT cells and DKO+GSN cells successfully escaped ( Figures 18A and 18B).
  • Elispot was used to detect the secretion of IFN- ⁇ spots by NK cells to determine the immune escape function of DKO+GSN cells.
  • the specific operation method is as follows:
  • NK cells collected after 24 hours were plated in a 96-well plate coated with IFN- ⁇ antibody and incubated in a 37°C incubator for 24 hours; affinity antibody and streptavidin were added and incubated for color detection of IFN- ⁇ secretion spots.
  • WT cells, DKO cells, and DKO+GSN cells were plated in 6-well plates at a specific density. After 24 hours, the culture medium was discarded and a specific number of NK cells were added for co-culture. After 24 hours, NK cells were collected and their cell surface activation indicator CD107a (Biolegend, 328620) was detected by FACS.
  • the FACS detection results are shown in Figure 21. Compared with DKO cells, DKO+GSN cells can reduce the activation of NK cells.
  • the NK cell escape function of differentiated cells of DKO+GSN cells was detected by RTCA.
  • the specific detection method is shown in Example 2.3.
  • Monkey iPSC-DKO cells i.e., non-human primate (NHP) iPSC-DKO cells
  • NEP non-human primate
  • Monkey iPSC-DKO cells were generated according to the method described in Example 1. Specifically, adult cynomolgus monkey cells (collected from 5-10 year old male cynomolgus monkeys) were collected and monkey iPSC cells were prepared by CTS TM CytoTune TM -iPS 2.1 Sendai virus reprogramming kit (Cat. No.: A34546).
  • NHP-B2M-gRNA1 and NHP-B2M-gRNA2 were used to directly knock out the B2M exon segment at both ends, and then a pair of PCR primers NHP-B2M-F/R were used to verify the genome sequence knockout (gRNA sequence and identification primer sequence are shown in Table 5).
  • NHP-CIITA-gRNA1 and NHP-CIITA-gRNA2 were used to directly knock out the CIITA exon segment at both ends, and the genome sequence knockout was verified by genomic PCR using a pair of PCR primers NHP-CIITA-F/R (gRNA sequence and identification primer sequence are shown in Table 5).
  • Two monoclonal NHP iPSC-DKO ie, NHP iPSC-DKO1 and NHP iPSC-DKO2 cells below) were selected for subsequent experiments.
  • IFN- ⁇ (PeproTech, Cat#300-02) was used to stimulate NHP iPSC-WT, NHP iPSC-DKO1 and NHP iPSC-DKO2 cells to detect HLA class I/II molecules on the cell surface.
  • the specific detection method is as follows:
  • the cells were plated and the medium containing IFN- ⁇ was added to the cells when the medium was changed the next day. After 48 hours, the cells were digested and the expression of HLA-I/II was detected using a flow cytometer (Agilent Technologies, NovoCyte).
  • T cell killing assay was performed on the xCELLigence platform (ACEA BioSciences) to detect changes in the immune function of NHP iPSC-DKO cells. The specific detection methods and results are described as follows.
  • NHP iPSC-WT and NHP iPSC-DKO cells were resuspended in 100 ⁇ l of cell culture medium and plated on 96-well E-plates (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index (reflecting the number of cells) reached 1, T cells (isolated from the blood of collected cynomolgus monkeys) were added at an E:T ratio (effector: target ratio) of 2:1. The data were normalized and analyzed using RTCA software (ACEA). As shown in Figure 24, NHP iPSC-DKO cells clearly escaped T cell killing, while NHP iPSC-WT cells were killed by T cells.
  • Example 4.1 To prepare NHP iPSC-DKO+GSN cells. Specifically, directly synthesize the nucleic acid sequence encoding the GSN protein (the amino acid sequence of the GSN protein is shown in SEQ ID NO:7). And connect the synthesized GSN nucleic acid sequence to the lentiviral vector. After successful connection, verify the correctness of the inserted sequence and perform viral packaging. Transfect the lentiviral vector into the NHP iPSC-DKO cells constructed in Example 7. And use qPCR to detect the overexpression level of cynomolgus monkey GSN mRNA, WT cells are negative controls, and the primers used are shown in Table 6.
  • PBMC cell killing assay was performed on the XCelligence platform (ACEA BioSciences) to detect the immune escape function of universal cells NHP iPSC-DKO+GSN.
  • the specific detection method and results are described as follows.
  • NHP iPSC-WT, NHP iPSC-DKO and NHP iPSC-DKO+GSN cells were resuspended in 100 ⁇ l of cell-specific culture medium and plated on a 96-well E-plate (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index value reached 1, PBMC (isolated from the blood of collected crab-eating macaques) was added at an E:T ratio of 1:1. The data were standardized and analyzed using RTCA software (ACEA). The results are shown in Figure 26. NHP iPSC-DKO+GSN cells can obviously escape the killing of PBMC cells.

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Abstract

Provided are a gelsolin (GSN)-expressing low-immunogenicity pluripotent stem cell and a preparation method therefor. The low-immunogenicity pluripotent stem cell can significantly reduce or escape recognition and attacks by the immune system with the updating and differentiation properties of stem cells unchanged.

Description

一种表达GSN的通用型细胞及其制备方法A universal cell expressing GSN and preparation method thereof 技术领域Technical Field
本发明属于基因工程与干细胞技术领域，具体涉及一种表达GSN的通用型细胞及其制备方法。The present invention belongs to the field of genetic engineering and stem cell technology, and specifically relates to a universal cell expressing GSN and a preparation method thereof.
背景技术Background technique
干细胞是一类具备自我更新能力以及向特定功能体细胞分化能力的细胞，依据干细胞特性的程度差异，主要将干细胞分为：全能干细胞、多能干细胞和成体干细胞。诱导性多能干细胞(iPSC)具有无限增殖、自我更新和分化到各种类型细胞的潜力，在治疗癌症、神经相关、心血管等疾病具有重要应用前景。但是免疫不相容和移植细胞遭受的免疫排斥等关键问题阻碍了移植异体功能细胞进行治疗的临床应用。Stem cells are a type of cells that have the ability to self-renew and differentiate into specific functional somatic cells. Based on the degree of differences in stem cell characteristics, stem cells are mainly divided into: totipotent stem cells, pluripotent stem cells and adult stem cells. Induced pluripotent stem cells (iPSCs) have the potential to proliferate indefinitely, self-renew and differentiate into various types of cells, and have important application prospects in the treatment of cancer, neurological, cardiovascular and other diseases. However, key issues such as immune incompatibility and immune rejection of transplanted cells have hindered the clinical application of transplanted allogeneic functional cells for treatment.
人主要组织相容性复合体(MHC)，即人白细胞抗原(HLA)，是导致免疫不相容的主要原因。MHC由一系列基因组成，可分为I类、II类和III类。MHC-I基因在几乎所有的组织细胞类型中表达，表达“非己”MHC-I类分子的移植细胞将刺激CD8+T细胞的活化而被消除。CD4+辅助T细胞识别“非己”细胞的MHC-II基因，从而进行免疫排斥，而III类分子不参与免疫活动。The human major histocompatibility complex (MHC), also known as human leukocyte antigen (HLA), is the main cause of immune incompatibility. MHC is composed of a series of genes and can be divided into class I, class II and class III. MHC-I genes are expressed in almost all tissue cell types. Transplanted cells expressing "non-self" MHC-I class molecules will stimulate the activation of CD8+T cells and be eliminated. CD4+ helper T cells recognize the MHC-II genes of "non-self" cells, thereby performing immune rejection, while class III molecules do not participate in immune activities.
近年已有报道通过敲除B2M、CIITA等基因，可以实现MHC-I和MHC-II细胞表面或本身基因的缺失表达，进而使细胞具备免疫耐受或逃逸T细胞/B细胞特异性免疫应答，产生免疫兼容的通用型多能干细胞。In recent years, it has been reported that by knocking out genes such as B2M and CIITA, the expression of MHC-I and MHC-II cell surface or genes themselves can be deleted, thereby making the cells immune tolerant or escaping T cell/B cell-specific immune responses, and producing immune-compatible universal pluripotent stem cells.
目前已报道在破坏MHC-I和MHC-II类基因表达的基础上，可以使细胞表达HLA-E/G等非经典HLA-I类分子，或表达PD-L1、CTLA4-Ig、CD47、CD24等免疫抑制检查点蛋白，可有效逃逸NK细胞的杀伤(WO2021041316A1)。It has been reported that on the basis of destroying the expression of MHC-I and MHC-II class genes, cells can express non-classical HLA-I class molecules such as HLA-E/G, or express immunosuppressive checkpoint proteins such as PD-L1, CTLA4-Ig, CD47, CD24, etc., which can effectively escape the killing of NK cells (WO2021041316A1).
发明内容Summary of the invention
在一方面，本发明提供一种低免疫原性多能干细胞，其包含：与亲本多能干细胞相比降低的内源主要组织相容性I类抗原(MHC-I)功能；与亲本多能干细胞相比降低的内源主要组织相容性II类抗原(MHC-II)功能；和与亲本多能干细胞相比降低的对NK细胞杀伤的敏感性，其中所述降低的对NK细胞杀伤的敏感性是由增加的GSN蛋白的表达引起的。In one aspect, the present invention provides a low immunogenic pluripotent stem cell, comprising: reduced endogenous major histocompatibility class I antigen (MHC-I) function compared to a parental pluripotent stem cell; reduced endogenous major histocompatibility class II antigen (MHC-II) function compared to a parental pluripotent stem cell; and reduced sensitivity to NK cell killing compared to a parental pluripotent stem cell, wherein the reduced sensitivity to NK cell killing is caused by increased expression of GSN protein.
在一些实施方案中，所述MHC-I功能通过降低MHC-I类蛋白或MHC-I转录调节因子的活性而降低。In some embodiments, the MHC-I function is reduced by reducing the activity of an MHC-I class protein or an MHC-I transcriptional regulator.
在一实施方案中，所述MHC-I功能通过降低B2M蛋白的活性而降低。In one embodiment, the MHC-I function is reduced by reducing the activity of the B2M protein.
在一实施方案中，所述B2M蛋白为人B2M蛋白，其包含SEQ ID NO:1所示的氨基酸序列或与SEQ ID NO:1所示的氨基酸序列具有90％相同性的氨基酸序列。In one embodiment, the B2M protein is a human B2M protein, which comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence that has 90% identity with the amino acid sequence shown in SEQ ID NO:1.
在一实施方案中，所述B2M蛋白为食蟹猴B2M蛋白，其包含SEQ ID NO:9所示的氨基酸序列或与SEQ ID NO:9所示的氨基酸序列具有90％相同性的氨基酸序列。In one embodiment, the B2M protein is a crab-eating macaque B2M protein, which comprises the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence that has 90% identity with the amino acid sequence shown in SEQ ID NO:9.
在一些实施方案中，所述MHC-II功能通过降低MHC-II类蛋白或MHC-II转录调节因子的活性而降低。 In some embodiments, the MHC-II function is reduced by reducing the activity of an MHC-II class protein or an MHC-II transcriptional regulator.
在一实施方案中，所述MHC-II功能通过降低CIITA蛋白的活性而降低。In one embodiment, the MHC-II function is reduced by reducing the activity of the CIITA protein.
在一实施方案中，所述CIITA蛋白为人CIITA蛋白，其包含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％相同性的氨基酸序列。In one embodiment, the CIITA protein is a human CIITA protein, which comprises the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence that is 90% identical to the amino acid sequence shown in SEQ ID NO:2.
在一实施方案中，所述CIITA蛋白为食蟹猴CIITA蛋白，其包含SEQ ID NO:10所示的氨基酸序列或与SEQ ID NO:10所示的氨基酸序列具有至少90％相同性的氨基酸序列。In one embodiment, the CIITA protein is a cynomolgus monkey CIITA protein, which comprises the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO:10.
在一些实施方案中，所述GSN蛋白为人GSN蛋白，其包含SEQ ID NO:3所示的氨基酸序列或与SEQ ID NO:3所示的氨基酸序列具有90％相同性的氨基酸序列。In some embodiments, the GSN protein is a human GSN protein, which comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence that has 90% identity with the amino acid sequence shown in SEQ ID NO:3.
在一些实施方案中，所述GSN蛋白为食蟹猴GSN蛋白，其包含SEQ ID NO:7所示的氨基酸序列或与SEQ ID NO:7所示的氨基酸序列具有至少90％相同性的氨基酸序列。In some embodiments, the GSN protein is a cynomolgus monkey GSN protein, which comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO:7.
在一实施方案中，所述低免疫原性多能干细胞包含：使内源B2M蛋白活性降低的一个或多个改变；使内源CIITA蛋白活性降低的一个或多个改变；和在所述低免疫原性多能干细胞中引起增加的GSN蛋白表达的一个或多个改变。In one embodiment, the low immunogenic pluripotent stem cells comprise: one or more changes that reduce the activity of endogenous B2M protein; one or more changes that reduce the activity of endogenous CIITA protein; and one or more changes that cause increased expression of GSN protein in the low immunogenic pluripotent stem cells.
在一实施方案中，所述低免疫原性多能干细胞包含：使内源B2M基因的两个等位基因失活的一个或多个改变；使内源CIITA基因的两个等位基因失活的一个或多个改变；和在所述低免疫原性多能干细胞中引起增加的GSN基因表达的一个或多个改变。In one embodiment, the low immunogenic pluripotent stem cells comprise: one or more changes that inactivate both alleles of the endogenous B2M gene; one or more changes that inactivate both alleles of the endogenous CIITA gene; and one or more changes that cause increased expression of the GSN gene in the low immunogenic pluripotent stem cells.
在另一方面，本发明还提供一种产生本发明的低免疫原性多能干细胞的方法，所述方法包括：降低所述多能干细胞中内源主要组织相容性I类抗原(MHC-I)功能；降低所述多能干细胞中内源主要组织相容性II类抗原(MHC-II)功能；和增加降低所述多能干细胞对NK细胞杀伤的敏感性的蛋白的表达，其中所述蛋白为GSN蛋白。On the other hand, the present invention also provides a method for producing the low immunogenic pluripotent stem cells of the present invention, the method comprising: reducing the endogenous major histocompatibility class I antigen (MHC-I) function in the pluripotent stem cells; reducing the endogenous major histocompatibility class II antigen (MHC-II) function in the pluripotent stem cells; and increasing the expression of a protein that reduces the sensitivity of the pluripotent stem cells to NK cell killing, wherein the protein is GSN protein.
在一实施方案中，所述方法包括：降低所述多能干细胞中B2M蛋白的活性；降低所述多能干细胞中CIITA蛋白的活性；和增加所述多能干细胞中GSN蛋白的表达。In one embodiment, the method comprises: reducing the activity of B2M protein in the pluripotent stem cells; reducing the activity of CIITA protein in the pluripotent stem cells; and increasing the expression of GSN protein in the pluripotent stem cells.
在一实施方案中，所述方法包括：消除所述多能干细胞中B2M基因的两个等位基因的活性；消除所述多能干细胞中CIITA基因的两个等位基因的活性；和增加所述多能干细胞中GSN基因的表达。In one embodiment, the method comprises: eliminating the activity of both alleles of the B2M gene in the pluripotent stem cells; eliminating the activity of both alleles of the CIITA gene in the pluripotent stem cells; and increasing the expression of the GSN gene in the pluripotent stem cells.
在一实施方案中，通过成簇规律间隔短回文重复序列(CRISPR)/Cas9基因编辑技术降低所述多能干细胞中B2M蛋白的活性。In one embodiment, the activity of B2M protein in the pluripotent stem cells is reduced by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology.
在一实施方案中，通过成簇规律间隔短回文重复序列(CRISPR)/Cas9基因编辑技术降低所述多能干细胞中CIITA蛋白的活性。In one embodiment, the activity of CIITA protein in the pluripotent stem cells is reduced by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology.
在一实施方案中，通过转基因的表达增加GSN蛋白的表达。In one embodiment, expression of the GSN protein is increased by expression of a transgene.
在一优选实施方案中，通过合成编码GSN蛋白的核酸序列，以构建至慢病毒载体中，再通过慢病毒载体，将至少一个拷贝的在启动子控制下的GSN基因引入所述多能干细胞中增加GSN蛋白的表达。In a preferred embodiment, a nucleic acid sequence encoding the GSN protein is synthesized and constructed into a lentiviral vector, and then at least one copy of the GSN gene under the control of a promoter is introduced into the pluripotent stem cells via the lentiviral vector to increase the expression of the GSN protein.
在一实施方案中，所述编码GSN蛋白的核酸序列包含SEQ ID NO:4所示的核酸序列或与SEQ ID NO:4所示的核酸序列具有至少80％相同性的核酸序列。In one embodiment, the nucleic acid sequence encoding the GSN protein comprises the nucleic acid sequence shown in SEQ ID NO:4 or a nucleic acid sequence that has at least 80% identity with the nucleic acid sequence shown in SEQ ID NO:4.
在一实施方案中，所述编码GSN蛋白的核酸序列包含SEQ ID NO:8所示的核酸序列或与SEQ ID NO:8所示的核酸序列具有至少80％相同性的核酸序列。In one embodiment, the nucleic acid sequence encoding the GSN protein comprises the nucleic acid sequence shown in SEQ ID NO:8 or a nucleic acid sequence that has at least 80% identity with the nucleic acid sequence shown in SEQ ID NO:8.
在另一方面，本发明还提供本发明的低免疫原性多能干细胞或本发明的方法制备的 低免疫原性多能干细胞在制备用于预防或治疗需要细胞移植的疾病的药物中的用途。In another aspect, the present invention also provides the low immunogenic pluripotent stem cells of the present invention or the cells prepared by the method of the present invention. Use of low-immunogenic pluripotent stem cells in the preparation of a medicament for preventing or treating a disease requiring cell transplantation.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1示出B2M及CIITA双敲除细胞系(DKO)的B2M基因的敲除策略和通过PCR进行B2M基因敲除验证的结果。FIG. 1 shows the knockout strategy of the B2M gene in the B2M and CIITA double knockout cell lines (DKO) and the results of B2M gene knockout verification by PCR.
图2示出B2M及CIITA双敲除细胞系(DKO)的CIITA基因的敲除策略和通过PCR进行CIITA基因敲除验证的结果。FIG. 2 shows the knockout strategy of the CIITA gene in B2M and CIITA double knockout cell lines (DKO) and the results of the CIITA gene knockout verification by PCR.
图3示出qPCR检测B2M/CIITA双等位基因敲除的克隆DKO的B2M和CIITA在RNA水平的表达的结果。FIG. 3 shows the results of qPCR detection of the expression of B2M and CIITA at the RNA level in B2M/CIITA biallelic knockout clones DKO.
图4示出蛋白印迹检测B2M/CIITA双等位基因敲除的克隆DKO的B2M蛋白水平的结果。FIG. 4 shows the results of Western blot detection of B2M protein levels in B2M/CIITA biallelic knockout clones DKO.
图5示出使用IFN-γ刺激WT及DKO细胞以检测H1细胞表面的HLA I/II型分子的FACS检测结果。Figure 5 shows the FACS detection results of WT and DKO cells stimulated with IFN-γ to detect HLA class I/II molecules on the surface of H1 cells.
图6示出B2M/CIITA双等位基因敲除的克隆DKO的核型检测结果。FIG. 6 shows the karyotype detection results of B2M/CIITA biallelic knockout clones DKO.
图7A、图7B和图7C示出DKO细胞中干性基因的表达的检测结果。图7A为通过免疫荧光检测WT和DKO细胞中干性基因POU5F1和NANOG的蛋白水平的结果；图7B为通过RT-qPCR检测WT和DKO细胞中干性基因POU5F1、NANOG和SOX2在RNA水平上的表达的结果；图7C为通过流式细胞术检测WT和DKO细胞表面干性基因SSEA-4和Tra1-81的表达的结果。Figures 7A, 7B and 7C show the results of detecting the expression of stemness genes in DKO cells. Figure 7A is the result of detecting the protein levels of stemness genes POU5F1 and NANOG in WT and DKO cells by immunofluorescence; Figure 7B is the result of detecting the expression of stemness genes POU5F1, NANOG and SOX2 in WT and DKO cells at the RNA level by RT-qPCR; Figure 7C is the result of detecting the expression of stemness genes SSEA-4 and Tra1-81 on the surface of WT and DKO cells by flow cytometry.
图8示出通过苏木精伊红染色检测B2M/CIITA双等位基因敲除的DKO细胞在体内形成畸胎瘤并分化出内中外三胚层的细胞的分化能力的结果。FIG. 8 shows the results of detecting the differentiation ability of B2M/CIITA biallelic knockout DKO cells to form teratomas and differentiate into cells of the three germ layers, endomeseoblasts and ectoblasts, in vivo by hematoxylin and eosin staining.
图9示出通过RTCA检测WT和DKO细胞的免疫逃逸功能的结果。其中上方三张图为通过RTCA检测NK细胞对WT和DKO细胞的杀伤率的结果，即WT和DKO细胞对NK细胞的免疫逃逸功能的检测结果；下方三张图为通过RTCA检测T细胞对WT和DKO细胞的杀伤率的结果，即WT和DKO细胞对T细胞的免疫逃逸功能的检测结果。Figure 9 shows the results of detecting the immune escape function of WT and DKO cells by RTCA. The top three figures are the results of detecting the killing rate of NK cells against WT and DKO cells by RTCA, that is, the detection results of the immune escape function of WT and DKO cells against NK cells; the bottom three figures are the results of detecting the killing rate of T cells against WT and DKO cells by RTCA, that is, the detection results of the immune escape function of WT and DKO cells against T cells.
图10示出慢病毒载体pGC-EF1a载体的结构示意图。FIG. 10 shows a schematic diagram of the structure of the lentiviral vector pGC-EF1a.
图11A和图11B示出验证DKO+CD47细胞中CD47过表达的结果。图11A为通过FACS检测DKO+CD47细胞系中CD47表达水平的结果；图11B为通过qPCR检测DKO+CD47细胞系中CD47表达水平的结果。Figures 11A and 11B show the results of verifying the overexpression of CD47 in DKO+CD47 cells. Figure 11A is the result of detecting the expression level of CD47 in DKO+CD47 cell lines by FACS; Figure 11B is the result of detecting the expression level of CD47 in DKO+CD47 cell lines by qPCR.
图12示出通过RTCA检测WT和DKO+CD47细胞的免疫逃逸功能的结果。FIG. 12 shows the results of detecting the immune escape function of WT and DKO+CD47 cells by RTCA.
图13示出通过RT-PCR检测构建的DKO+GSN细胞系中mRNA的过表达水平的结果。FIG. 13 shows the results of detecting the overexpression level of mRNA in the constructed DKO+GSN cell line by RT-PCR.
图14示出通过免疫荧光检测构建的DKO+GSN细胞系中干性基因OCT4、NANOG、SOX2、TRA-1-60、TRA-1-81的蛋白水平的结果。FIG. 14 shows the results of immunofluorescence detection of the protein levels of stemness genes OCT4, NANOG, SOX2, TRA-1-60, and TRA-1-81 in the constructed DKO+GSN cell line.
图15示出通过流式细胞术检测构建的DKO+GSN细胞系中细胞表面干性基因SSEA-4、TRA-1-60、Tra1-81和OCT4的表达水平的结果。FIG. 15 shows the results of detecting the expression levels of cell surface stemness genes SSEA-4, TRA-1-60, Tra1-81, and OCT4 in the constructed DKO+GSN cell line by flow cytometry.
图16示出通过免疫荧光检测构建的DKO+GSN细胞系的三胚层分化能力的结果。FIG. 16 shows the results of immunofluorescence detection of the three-germ layer differentiation ability of the constructed DKO+GSN cell line.
图17示出DKO+GSN细胞系的畸胎瘤形成能力的检测结果。 FIG. 17 shows the results of testing the teratoma-forming ability of the DKO+GSN cell line.
图18A-图18D示出通过RTCA检测构建的DKO+GSN细胞系对不同免疫细胞的逃逸功能的结果。图18A和图18B为通过RTCA检测的NK细胞对DKO+GSN细胞杀伤性实验的结果，其中图18B为多次杀伤统计图；图18C和图18D为通过RTCA检测的T细胞+NK细胞对DKO+GSN细胞杀伤性实验的结果，其中图18D为多次杀伤统计图。Figures 18A-18D show the results of the escape function of the constructed DKO+GSN cell line on different immune cells detected by RTCA. Figures 18A and 18B are the results of the NK cell killing experiment on DKO+GSN cells detected by RTCA, wherein Figure 18B is a multiple killing statistical graph; Figures 18C and 18D are the results of the T cell+NK cell killing experiment on DKO+GSN cells detected by RTCA, wherein Figure 18D is a multiple killing statistical graph.
图19示出构建的DKO+GSN细胞系在和NK细胞共培养24小时后的细胞状况。FIG. 19 shows the cell status of the constructed DKO+GSN cell line after co-culture with NK cells for 24 hours.
图20A和图20B示出通过Elispot检测构建的DKO+GSN细胞系在和NK细胞共培养24小时后IFN-γ斑点分泌的结果，其中图20B为IFN-γ斑点频率的统计直方图。20A and 20B show the results of Elispot detection of IFN-γ spot secretion by the constructed DKO+GSN cell line after co-culture with NK cells for 24 hours, wherein FIG. 20B is a statistical histogram of IFN-γ spot frequency.
图21示出通过FACS检测构建的DKO+GSN细胞系中的NK细胞活性指标CD107a的表达的结果。FIG. 21 shows the results of FACS detection of the expression of CD107a, an indicator of NK cell activity, in the constructed DKO+GSN cell line.
图22A和图22B示出通过RTCA检测构建的DKO+GSN细胞系的分化细胞对NK细胞的逃逸功能的结果，其中图22B为多次杀伤统计图。FIG. 22A and FIG. 22B show the results of detecting the escape function of differentiated cells of the constructed DKO+GSN cell line against NK cells by RTCA, wherein FIG. 22B is a statistical diagram of multiple killings.
图23示出使用IFN-γ刺激NHP iPSC-WT及NHP iPSC-DKO1和NHP iPSC-DKO2细胞以检测细胞表面的HLA I/II型分子的FACS检测结果。Figure 23 shows the FACS detection results of NHP iPSC-WT, NHP iPSC-DKO1 and NHP iPSC-DKO2 cells stimulated with IFN-γ to detect HLA class I/II molecules on the cell surface.
图24示出通过RTCA检测T细胞对NHP iPSC-WT及NHP iPSC-DKO1和NHP iPSC-DKO2细胞杀伤性实验的结果。Figure 24 shows the results of the RTCA test to detect the killing ability of T cells against NHP iPSC-WT, NHP iPSC-DKO1 and NHP iPSC-DKO2 cells.
图25示出通过qPCR检测NHP iPSC-DKO+GSN细胞系中GSN表达水平的结果。Figure 25 shows the results of detecting the GSN expression level in the NHP iPSC-DKO+GSN cell line by qPCR.
图26示出通过RTCA检测PBMC细胞对NHP iPSC-DKO+GSN细胞杀伤性实验的结果。Figure 26 shows the results of the RTCA test to detect the killing ability of PBMC cells on NHP iPSC-DKO+GSN cells.
具体实施方案Specific implementation plan
一般定义和术语General Definitions and Terminology
本文引用的所有专利、专利申请、科学出版物、制造商的说明书和指南等，无论上文或下文，均整体援引加入本文。本文中的任何内容均不应理解为承认本公开无权先于这样的公开。All patents, patent applications, scientific publications, manufacturer's instructions and guidelines, etc., cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publication.
除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白和核酸化学、分子生物学、细胞和组织培养、微生物学相关术语均为相应领域内广泛使用的术语(参见，例如，Molecular Cloning:A Laboratory Manual,2nd Edition,J.Sambrook et al.eds.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor 1989)。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。Unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, and microbiology related terms used herein are widely used terms in the corresponding fields (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). At the same time, in order to better understand the present invention, the definitions and explanations of the related terms are provided below.
如本文所用，表述“包括”、“包含”、“含有”和“具有”是开放式的，表示包括所列举的元素、步骤或组分但不排除其他未列举的元素、步骤或组分。表述“由……组成”不包括未指定的任何元素、步骤或组分。表述“基本上由……组成”是指范围限于指定的元素、步骤或组分，加上不显著影响要求保护的主题的基本和新颖性质的任选存在的元素、步骤或组分。应当理解，表述“基本上由……组成”和“由……组成”涵盖在表述“包含”的含义之内。As used herein, the expressions "comprises," "comprising," "containing," and "having" are open ended, meaning the inclusion of the listed elements, steps, or components but not the exclusion of other unlisted elements, steps, or components. The expression "consisting of" excludes any element, step, or component not specified. The expression "consisting essentially of" means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions "consisting essentially of" and "consisting of" are encompassed within the meaning of the expression "comprising."
如本文所用，除非上下文另外指明，单数形式的表述“一个”、“一种”或“这个”包括复数指代。术语“一个或多个”或者“至少一个”涵盖1、2、3、4、5、6、7、8、9个或更多个。As used herein, the singular forms "a", "an" or "the" include plural references unless the context indicates otherwise. The terms "one or more" or "at least one" encompass 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.
本文中值的范围的列举仅为了用作单独提到落在所述范围内的每个不同值的速记 方法。除非本文另有说明，否则每个单独的值如其在本文中单独列举地加入本说明书。除非明确指出相反，在本文示出的数值或范围均由“约”修饰，表示所列举或声称的数值或范围±20％、±10％、±5％或±3％。Recitation of ranges of values herein is merely intended to serve as a shorthand for referring individually to each separate value falling within the range. Method. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. Unless expressly indicated to the contrary, the values or ranges set forth herein are modified by "about", meaning ±20%, ±10%, ±5%, or ±3% of the recited or claimed value or range.
除非另外指明，本文所述的方法步骤中，诸如1)、2)…，i)、ii)..，a)、b)、…的标识仅作为区分的示例，并不表示所述的方法步骤以这样的顺序进行。Unless otherwise specified, in the method steps described herein, labels such as 1), 2), ..., i), ii), ..., a), b), ... are merely examples of distinction and do not imply that the method steps described are performed in such an order.
术语“多能细胞”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。The term "pluripotent cell" refers to a cell that is capable of self-renewal and proliferation while remaining in an undifferentiated state and that can be induced to differentiate into a specialized cell type under appropriate conditions.
如本文所用，术语“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系统组织)。如本文所用的术语“多能干细胞”还包括“诱导性多能干细胞”或“iPSC”，一种衍生自非多能细胞的多能干细胞。示例性人多能干细胞系包括H1人多能干细胞系和H9人多能干细胞系。另外的示例性多能干细胞系包括可通过National Institutes of Health HumanEmbryonic Stem Cell Registry和Howard Hughes Medical Institute HUES集合获得的那些(如Cowan CA,et al.Derivation of embryonic stem-cell lines from human blastocysts.N Engl J Med.2004 Mar 25；350(13):1353-6.所述)。As used herein, the term "pluripotent stem cell" has 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 (e.g., epidermal tissue and nervous system tissue). As used herein, the term "pluripotent stem cell" also includes "induced pluripotent stem cells" or "iPSCs," a pluripotent stem cell derived from a non-pluripotent cell. Exemplary human pluripotent stem cell lines include the H1 human pluripotent stem cell line and the H9 human pluripotent stem cell line. Additional exemplary pluripotent 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 (as described in Cowan CA, et al. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med. 2004 Mar 25; 350(13): 1353-6.).
如本文所用，术语“全能”是指细胞形成完整生物体的能力。例如，在哺乳动物中，只有受精卵和第一卵裂期卵裂球是全能的。在一实施方案中，本文所述的多能干细胞不具有全能性，也不会形成完整生物体。As used herein, the term "totipotency" refers to the ability of a cell to form a complete organism. For example, in mammals, only the fertilized egg and the first cleavage stage blastomere are totipotent. In one embodiment, the pluripotent stem cells described herein do not have totipotency and will not form a complete organism.
如本文所用，术语“通用型细胞”是指利用基因编辑技术改造异体细胞以消除免疫排斥，实现通用化的细胞。As used herein, the term "universal cells" refers to cells that are modified using gene editing technology to eliminate immune rejection and achieve universalization.
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，细胞来自新生儿人、成年人或非人哺乳动物。The cell can be from, for example, a human or non-human mammal. 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 cell is from an adult or a non-human mammal. In some embodiments, the cell is from a newborn human, an adult, or a non-human mammal.
如本文所用，术语“免疫排斥”或“免疫不相容”是指异体的细胞、组织或器官在移植给受体之后会受到受体本身的免疫细胞的攻击，从而无法保证其正常的生理功能。人主要组织相容性复合体(MHC)，即人白细胞抗原(HLA)，是导致“免疫排斥”或“免疫不相容”的主要原因。As used herein, the term "immune rejection" or "immune incompatibility" refers to the fact that foreign cells, tissues or organs will be attacked by the recipient's own immune cells after being transplanted into the recipient, thereby failing to ensure their normal physiological functions. The human major histocompatibility complex (MHC), i.e., human leukocyte antigen (HLA), is the main cause of "immune rejection" or "immune incompatibility".
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、牲畜或人。“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)。As used herein, the term "subject" or "patient" refers to any animal, such as a domesticated animal, a zoo animal, or a human. A "subject" or "patient" can be a mammal, such as a dog, a cat, a bird, livestock, or a human. Specific examples of "subjects" and "patients" include, but are not limited to, individuals (particularly humans) with diseases or conditions associated with the liver, heart, lungs, kidneys, pancreas, brain, nervous tissue, blood, bones, bone marrow, etc.
本文的“低免疫原性多能干细胞”是指多能干细胞，其保留其多能干细胞的特征，并且当转移到同种异体宿主中时产生降低的免疫排斥反应。在优选的实施方案中，低免疫原性多能干细胞不产生免疫应答。因此，“低免疫原性”是指与免疫改造之前的亲本(“WT”)干细胞的免疫应答相比，显著降低或消除的免疫应答。例如，相对于未进行免疫改造的野生型细胞，这种低免疫原性细胞可能约2.5％、5％、10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、97.5％、99％或大于99％更不易被产生免疫排斥。" Hypoimmunogenic pluripotent stem cells " herein refer to pluripotent stem cells, which retain the characteristics of their pluripotent stem cells, and produce a reduced immune rejection reaction when transferred to an allogeneic host. In a preferred embodiment, low immunogenic pluripotent stem cells do not produce an immune response. Therefore, " low immunogenicity " refers to an immune response that is significantly reduced or eliminated compared to the immune response of the parent (" WT ") stem cell before immune modification. For example, relative to wild-type cells that have not been immune modified, such low immunogenic cells may be about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more than 99% less likely to be immune rejected.
术语“主要组织相容性复合体(MHC)”涉及发生在所有脊椎动物中的基因复合体。 MHC蛋白或分子在正常免疫反应中在淋巴细胞和抗原呈递细胞之间的信号传导中发挥的功能。人MHC，也称为HLA即人白细胞抗原，位于第6号染色体上，包括MHC-I和MHC-II。The term "major histocompatibility complex (MHC)" refers to a gene complex that occurs in all vertebrates. The function of MHC proteins or molecules in signaling between lymphocytes and antigen-presenting cells in normal immune responses. Human MHC, also known as HLA or human leukocyte antigen, is located on chromosome 6 and includes MHC-I and MHC-II.
术语“MHC-I”或“MHC I类”涉及主要组织相容性复合物I类蛋白或基因。在人MHC-I区域内，有HLA-A、HLA-B、HLA-C、HLA-E、HLA-F、CD1a、CD1b和CD1c亚区。MHC I类蛋白存在于几乎所有细胞表面，包括大多数肿瘤细胞。MHC-I蛋白负载有抗原，这些抗原通常来源于内源性蛋白或细胞内存在的病原体，然后呈递给细胞毒性T淋巴细胞(CTL，也称为CD8+T细胞)。T细胞受体能够识别和结合与MHC-I类分子复合的肽。每个细胞毒性T淋巴细胞表达一个独特的T细胞受体，能够结合特异性MHC/肽复合物。MHC I类分子主要介导内源性抗原的呈递过程。The term "MHC-I" or "MHC class I" refers to the major histocompatibility complex class I proteins or genes. Within the human MHC-I region, there are the HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, CD1a, CD1b, and CD1c subregions. MHC class I proteins are present on the surface of almost all cells, including most tumor cells. MHC-I proteins are loaded with antigens, which are usually derived from endogenous proteins or pathogens present within the cell, and then presented to cytotoxic T lymphocytes (CTLs, also known as CD8+ T cells). T cell receptors are able to recognize and bind peptides complexed with MHC-I class molecules. Each cytotoxic T lymphocyte expresses a unique T cell receptor that is able to bind to a specific MHC/peptide complex. MHC class I molecules primarily mediate the presentation of endogenous antigens.
术语“MHC-II”或“MHC II类”涉及主要组织相容性复合物II类蛋白或基因。MHC II包括5种蛋白，HLA-DP、HLA-DM、HLA-DOB、HLA-DQ和HLA-DR。MHC II类蛋白主要表达在B细胞、单核巨噬细胞、树突状细胞等抗原呈递细胞上。MHC II类分子主要介导外源性抗原的呈递过程，它们呈递外源性抗原多肽分子至Th细胞(辅助性T细胞)，即刺激CD4+T细胞。The term "MHC-II" or "MHC class II" refers to major histocompatibility complex class II proteins or genes. MHC II includes 5 proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ and HLA-DR. MHC class II proteins are mainly expressed on antigen presenting cells such as B cells, monocytes and macrophages, and dendritic cells. MHC class II molecules mainly mediate the presentation of exogenous antigens. They present exogenous antigen polypeptide molecules to Th cells (helper T cells), that is, stimulate CD4+T cells.
术语“MHC/肽复合物”涉及MHC I类或MHC II类分子的结合域和MHC I类或MHC II类结合肽的非共价复合物。The term "MHC/peptide complex" relates to a non-covalent complex of a binding domain of an MHC class I or MHC class II molecule and an MHC class I or MHC class II bound peptide.
本文的“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，包括从基因中去除核酸序列，或用其他序列中断序列，改变阅读框，或改变核酸的调节成分。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。"Knockout" herein refers to the process of making a specific gene inactive in the host cell in which it is located, which results in the non-production of the target protein or an inactive form. As will be appreciated by those skilled in the art and described further below, this can be achieved in a variety of different ways, including removing the nucleic acid sequence from the gene, or interrupting the sequence with other sequences, changing the reading frame, or changing the regulatory elements of the nucleic acid. For example, all or part of the coding region of the target gene can be removed or replaced with a "nonsense" sequence, all or part of the regulatory sequence (e.g., a promoter) can be removed or replaced, the translation initiation sequence can be removed or replaced, etc.
在本文中，术语“减少”和“降低”通常都用于表示减少统计学显著的量。然而，为了避免疑义，“减少”、“降低”包括与参考水平相比减少至少10％，例如与参考水平相比减少至少约20％或至少约30％，或至少约40％，或至少约50％，或至少约60％，或至少约70％，或至少约80％，或至少约90％，或高达并且包括100％减少(即与参考样品相比不存在的水平)，或在10-100％之间的任何减少。In this article, the terms "reduce" and "reduce" are generally used to represent a statistically significant amount of reduction. However, for the avoidance of doubt, "reduce", "reduce" includes reducing at least 10% compared to a reference level, such as reducing at least about 20% or at least about 30% compared to a reference level, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% reduction (i.e., a level that does not exist compared to a reference sample), or any reduction between 10-100%.
本文的“敲入”或“过表达”是指向宿主细胞添加遗传功能的过程。这导致编码的蛋白水平增加。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。"Knock-in" or "overexpression" herein refers to the process of adding genetic functions to a host cell. This results in an increase in the level of the encoded protein. As will be appreciated by those skilled in the art, this can be achieved 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 the expression of the protein. This can be achieved by modifying the promoter, adding a different promoter, adding an enhancer, or modifying other gene expression sequences.
在本文中，术语“增加”通常都用于表示增加统计学显著的量；为了避免任何疑义，术语“增加”是指与参考水平相比增加至少10％，例如与参考水平相比增加至少约20％，或至少约30％，或至少约40％，或至少约50％，或至少约60％，或至少约70％，或至少约80％，或至少约90％，或高达并且包括100％增加或在10-100％之间的任何增加，或与参考水平相比至少约2倍，或者至少约3倍，或者至少约4倍，或者至少约5倍或至少约10倍增加，或在2倍至10倍或大于10倍之间的任何增加。 As used herein, the term "increase" is generally used to indicate an increase by a statistically significant amount; to avoid any doubt, the term "increase" refers to an increase of at least 10% compared to a reference level, such as an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% increase or any increase between 10-100%, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold or at least about 10-fold compared to a reference level, or any increase between 2-fold and 10-fold or greater than 10-fold.
“β-2微球蛋白”或“β2M”或“B2M”蛋白是MHC-I类分子的组成部分。B2M蛋白在所有有核细胞(除红细胞外)中都有表达可以和MHC-I分子的α链非共价结合，附着细胞膜上，也可以释放到多种组织液内。"Beta-2 microglobulin" or "β2M" or "B2M" protein is a component of MHC-I class molecules. B2M protein is expressed in all nucleated cells (except red blood cells) and can non-covalently bind to the α chain of MHC-I molecules, attach to the cell membrane, and can also be released into various tissue fluids.
“CD47蛋白”或“整合素相关蛋白(Integrin-associated protein,IAP)”是一种重要的自我信号，它能够通过与免疫细胞上的配体信号调节蛋白α(SIRPα)的N末端结合，进而抑制巨噬细胞的吞噬作用，引起免疫逃逸。"CD47 protein" or "Integrin-associated protein (IAP)" is an important self-signal that can inhibit the phagocytosis of macrophages and cause immune escape by binding to the N-terminus of the ligand signal regulatory protein α (SIRPα) on immune cells.
“MHC-II反式激活蛋白(CIITA)蛋白”是调控MHC-II表达的关键分子，机体主要通过控制CIITA的表达来调节MHC II基因的表达水平。"MHC-II transactivator protein (CIITA) protein" is a key molecule that regulates the expression of MHC-II. The body mainly regulates the expression level of MHC II genes by controlling the expression of CIITA.
“GSN蛋白”是指凝溶胶蛋白，其为一种细胞外蛋白。据报道，GSN蛋白可以减少DNGR-1与F-肌动蛋白的结合以及1型常规树突状细胞(cDC1)对死细胞相关抗原的交叉呈递。"GSN protein" refers to gelsolin, which is an extracellular protein. GSN protein has been reported to reduce the binding of DNGR-1 to F-actin and the cross-presentation of dead cell-associated antigens by type 1 conventional dendritic cells (cDC1).
如本文所用，术语“同基因”是指宿主生物和细胞移植物的遗传相似性或同一性，其中具有免疫相容性；例如不产生免疫应答。As used herein, the term "syngeneic" refers to the genetic similarity or identity of the host organism and the cell transplant wherein there is immunological compatibility; eg, no immune response is generated.
如本文所用，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。As used herein, the term "allogeneic" refers to the genetic differences of the host organism and the cells transplanted into which an immune response is generated.
如本文所用，术语“B2M-/-”是指二倍体细胞在两条染色体中都具有失活的B2M基因。As used herein, the term "B2M-/-" refers to a diploid cell having an inactivated B2M gene in both chromosomes.
如本文所用，术语“CIITA-/-”是指二倍体细胞在两条染色体中都具有失活的CIITA基因。As used herein, the term "CIITA-/-" refers to a diploid cell having an inactivated CIITA gene in both chromosomes.
如本文所用，术语“多肽”指包含通过肽键共价连接的两个以上氨基酸的聚合物。“蛋白”可以包含一条或多条多肽，其中多肽之间通过共价或非共价方式相互作用。除非另有说明，“多肽”和“蛋白”可以互换使用。As used herein, the term "polypeptide" refers to a polymer comprising two or more amino acids covalently linked by peptide bonds. A "protein" may comprise one or more polypeptides, wherein the polypeptides interact with each other covalently or non-covalently. Unless otherwise indicated, "polypeptide" and "protein" may be used interchangeably.
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，它还指不经历基因编辑程序以实现低免疫原性的多能干细胞，例如，本文所述的亲本多能干细胞(WT)。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 refers to pluripotent stem cells that have not undergone a gene editing procedure to achieve low immunogenicity, e.g., the parental pluripotent stem cells (WT) described herein.
如本文所用，关于序列的术语“％相同性”是指在待比较的序列之间的最佳比对中相同的核苷酸或氨基酸的百分比。两个序列之间的差异可以分布在待比较序列的局部区域(区段)或整个长度上。通常在对区段或“比较窗口”最佳比对之后，确定两个序列之间的相同性。最佳比对可以手动进行，或者借助于本领域已知算法，包括但不限于Smith and Waterman,1981,Ads App.Math.2,482和Neddleman and Wunsch,1970,J.Mol.Biol.48,443描述的局部同源性算法，Pearson and Lipman,1988,Proc.Natl Acad.Sci.USA 88,2444描述的相似性搜索方法，或使用计算机程序，例如Wisconsin Genetics Software Package,Genetics Computer Group,575 Science Drive,Madison,Wis.中的GAP、BESTFIT、FASTA、BLAST P、BLAST N和TFASTA进行。例如，可以利用美国国家生物技术信息中心(NCBI)网站公共可用的BLASTN或BLASTP算法确定两个序列的百分比相同性。As used herein, the term "% identity" with respect to a sequence refers to the percentage of identical nucleotides or amino acids in an optimal alignment between the sequences to be compared. The differences between the two sequences can be distributed over local regions (segments) or over the entire length of the sequences to be compared. The identity between the two sequences is usually determined after optimal alignment of a segment or "comparison window". Optimal alignment can be performed manually or with the aid of algorithms known in the art, including but not limited to the local homology algorithm described by Smith and Waterman, 1981, Ads App. Math. 2, 482 and Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, the similarity search method described by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or using computer programs such as GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. For example, the percent identity of two sequences can be determined using the BLASTN or BLASTP algorithms publicly available on the website of the National Center for Biotechnology Information (NCBI).
通过确定待比较的序列对应的相同位置的数目，用这个数目除以比较的位置数目(例如，参考序列中的位置数目)，并将这个结果乘以100，获得％相同性。在一些实施方案中，至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％或约100％的区域给出相同性程度。 在一些实施方案中，对参考序列的整个长度给出相同性程度。可以用本领域已知的工具进行确定序列相同性的比对，优选利用最佳序列比对，例如，利用Align，利用标准设置，优选EMBOSS::needle、Matrix:Blosum62、Gap Open 10.0、Gap Extend 0.5。The % identity is obtained by determining the number of identical positions corresponding to the sequences being compared, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100. In some embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of a region exhibits a degree of identity. In some embodiments, the degree of homogeneity is given for the entire length of the reference sequence. Comparisons for determining sequence homogeneity can be performed using tools known in the art, preferably using optimal sequence alignments, e.g., using Align, using standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.
在本文中，“核苷酸”包括脱氧核糖核苷酸和核糖核苷酸及其衍生物。如本文所用，“核糖核苷酸”是核糖核酸(RNA)的构成物质，由一分子碱基，一分子五碳糖，一分子磷酸组成，其是指在β-D-呋喃核糖(β-D-ribofuranosyl)基团的2’位置具有羟基的核苷酸。而“脱氧核糖核苷酸”是脱氧核糖核酸(DNA)的构成物质，也由一分子碱基，一分子五碳糖，一分子磷酸构成，其是指在β-D-呋喃核糖(β-D-ribofuranosyl)基团的2’位置的羟基被氢取代的核苷酸，是染色体的主要化学成分。“核苷酸”通常由代表其中碱基的单字母来指代：“A(a)”指含有腺嘌呤的脱氧腺苷酸或腺苷酸，“C(c)”指含有胞嘧啶的脱氧胞苷酸或胞苷酸，“G(g)”指含有鸟嘌呤的脱氧鸟苷酸或鸟苷酸，“U(u)”指含有尿嘧啶的尿苷酸，“T(t)”指含有胸腺嘧啶的脱氧胸苷酸。In this article, "nucleotide" includes deoxyribonucleotides and ribonucleotides and their derivatives. As used herein, "ribonucleotide" is a constituent substance of ribonucleic acid (RNA), consisting of one molecule of base, one molecule of pentose, and one molecule of phosphoric acid, which refers to a nucleotide with a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. "Deoxyribonucleotide" is a constituent substance of deoxyribonucleic acid (DNA), also consisting of one molecule of base, one molecule of pentose, and one molecule of phosphoric acid, which refers to a nucleotide in which the hydroxyl group at the 2' position of the β-D-ribofuranosyl group is replaced by hydrogen, and is the main chemical component of chromosomes. "Nucleotide" is usually referred to by a single letter representing the base: "A (a)" refers to deoxyadenosine or adenylic acid containing adenine, "C (c)" refers to deoxycytidine or cytidine containing cytosine, "G (g)" refers to deoxyguanosine or guanylate containing guanine, "U (u)" refers to uridine containing uracil, and "T (t)" refers to deoxythymidylate containing thymine.
如本文所用，术语“多核苷酸”和“核酸”可以互换使用，用于指脱氧核糖核苷酸的聚合物(脱氧核糖核酸，DNA)或核糖核苷酸的聚合物(核糖核酸，RNA)。“多核苷酸序列”、“核酸序列”和“核苷酸序列”可以互换使用，用来表示多核苷酸中核苷酸的排序。本领域人员应当理解，DNA编码链(有义链)与其编码的RNA可以看作具有相同的核苷酸序列，DNA编码链序列中的脱氧胸苷酸对应其编码的RNA序列中的尿苷酸。As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to a polymer of deoxyribonucleotides (deoxyribonucleic acid, DNA) or a polymer of ribonucleotides (ribonucleic acid, RNA). "Polynucleotide sequence", "nucleic acid sequence" and "nucleotide sequence" are used interchangeably to refer to the order of nucleotides in a polynucleotide. It should be understood by those skilled in the art that a DNA coding strand (sense strand) and the RNA it encodes can be considered to have the same nucleotide sequence, and the deoxythymidylic acid in the DNA coding strand sequence corresponds to the uridine acid in the RNA sequence it encodes.
如本文所用，术语“表达”包括核苷酸序列的转录和/或翻译。因此，表达可以涉及转录物和/或多肽的产生。术语“转录”涉及将DNA序列中的遗传密码转录为RNA(转录物)的过程。术语“体外转录”指在不含细胞的系统中(例如在适当的细胞提取物中)体外合成RNA，特别是mRNA(参见，例如Pardi N.,Muramatsu H.,Weissman D.,KarikóK.(2013).In:Rabinovich P.(eds)Synthetic Messenger RNA and Cell Metabolism Modulation.Methods in Molecular Biology(Methods and Protocols),vol 969.Humana Press,Totowa,NJ.)。可以用于产生转录物的载体又称为“转录载体”，其中包含转录所需的调控序列。术语“转录”涵盖“体外转录”。As used herein, the term "expression" includes transcription and/or translation of a nucleotide sequence. Thus, expression may involve the production of transcripts and/or polypeptides. The term "transcription" refers to the process of transcribing the genetic code in a DNA sequence into RNA (transcript). The term "in vitro transcription" refers to the in vitro synthesis of RNA, particularly mRNA, in a cell-free system (e.g., in an appropriate cell extract) (see, e.g., Pardi N., Muramatsu H., Weissman D., Karikó K. (2013). In: Rabinovich P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation. Methods in Molecular Biology (Methods and Protocols), vol 969. Humana Press, Totowa, NJ.). A vector that can be used to produce a transcript is also referred to as a "transcription vector," which contains regulatory sequences required for transcription. The term "transcription" encompasses "in vitro transcription."
如本文所用，“编码”是指多核苷酸中特定核苷酸序列的固有特性，比如基因，cDNA或者mRNA都能作为模板去合成其他生物过程中的多聚物和大分子，只要已经有明确的核苷酸序列或者有明确的氨基酸序列。因此一个基因编码蛋白是指基因的mRNA通过转录和翻译在细胞中或其它生物系统中产生蛋白。As used herein, "encoding" refers to the inherent properties of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA can be used as a template to synthesize polymers and macromolecules in other biological processes, as long as there is a clear nucleotide sequence or a clear amino acid sequence. Therefore, a gene encodes a protein when the gene's mRNA produces a protein in a cell or other biological system through transcription and translation.
除非另有说明，否则本文描述的所有方法可以以任何合适的顺序进行。Unless otherwise stated, all methods described herein can be performed in any suitable order.
多能干细胞Pluripotent stem cells
在一方面，本发明提供一种低免疫原性多能干细胞，所述低免疫原性多能干细胞包含：In one aspect, the present invention provides a low immunogenic pluripotent stem cell, comprising:
与亲本多能干细胞相比降低的内源主要组织相容性I类抗原(MHC-I)功能；reduced endogenous major histocompatibility class I antigen (MHC-I) function compared to parental pluripotent stem cells;
与亲本多能干细胞相比降低的内源主要组织相容性II类抗原(MHC-II)功能；和Reduced endogenous major histocompatibility class II antigen (MHC-II) function compared to the parental pluripotent stem cell; and
与亲本多能干细胞相比降低的对NK细胞杀伤的敏感性。Reduced sensitivity to NK cell killing compared to parental pluripotent stem cells.
在本文中，亲本多能干细胞指免疫改造之前的，未经历基因编辑程序以实现低免疫原性的亲本(在本文中又称为“WT”)多能干细胞。 Herein, parental pluripotent stem cells refer to parental (also referred to herein as “WT”) pluripotent stem cells before immune modification that have not undergone a gene editing procedure to achieve low immunogenicity.
在一特别优选的实施方案中，降低的对NK细胞杀伤的敏感性是由增加的GSN蛋白的表达引起的。In a particularly preferred embodiment, the reduced sensitivity to NK cell killing is caused by increased expression of the GSN protein.
如本领域技术人员所理解的，功能的降低可以通过多种方式实现，包括从基因中去除核酸序列、用其他序列中断序列、或改变核酸的调节组分。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，可以进行移码突变，可以去除或替换调节序列例如启动子的全部或部分，可以删除或替换翻译启动序列等。As will be appreciated by those skilled in the art, reduction of function can be achieved in a variety of ways, including removal of nucleic acid sequences from a gene, interruption of a sequence with another sequence, or alteration of a regulatory component of a nucleic acid. For example, all or part of the coding region of a target gene can be removed or replaced with a "nonsense" sequence, frameshift mutations can be performed, all or part of a regulatory sequence such as a promoter can be removed or replaced, a translation initiation sequence can be deleted or replaced, etc.
如本领域技术人员所理解的，可以使用本领域已知的和如下所述的技术测量多能干细胞中MHC I(当细胞来源于人细胞时HLA I)功能的降低；例如，使用结合HLA复合物的标记抗体的FACS技术；例如，使用市售的HLA-A、HLA-B、HLA-C抗体，其结合人主要组织相容性HLA I类。可以使用本领域已知的和如下所述的技术测量多能干细胞中MHC II(当细胞来源于人细胞时HLA II)功能的降低；例如，使用结合HLA复合物的标记抗体的FACS技术；例如，使用市售的HLA-DQ、HLA-DR、HLA-DP抗体，其结合人主要组织相容性HLA II类。As will be appreciated by those skilled in the art, the reduction in MHC I (HLA I when the cells are derived from human cells) function in pluripotent stem cells can be measured using techniques known in the art and as described below; for example, using FACS techniques with labeled antibodies that bind to the HLA complex; for example, using commercially available HLA-A, HLA-B, HLA-C antibodies that bind to human major histocompatibility HLA class I. The reduction in MHC II (HLA II when the cells are derived from human cells) function in pluripotent stem cells can be measured using techniques known in the art and as described below; for example, using FACS techniques with labeled antibodies that bind to the HLA complex; for example, using commercially available HLA-DQ, HLA-DR, HLA-DP antibodies that bind to human major histocompatibility HLA class II.
在一些实施方案中，所述MHC-I功能通过降低MHC-I类蛋白的活性而降低。In some embodiments, the MHC-I function is reduced by reducing the activity of MHC-I class proteins.
在一实施方案中，所述MHC-I类蛋白包含人类白细胞抗原-A(HLA-A)蛋白、人类白细胞抗原-B(HLA-B)蛋白或人类白细胞抗原-C(HLA-C)蛋白。In one embodiment, the MHC-I class protein comprises a human leukocyte antigen-A (HLA-A) protein, a human leukocyte antigen-B (HLA-B) protein, or a human leukocyte antigen-C (HLA-C) protein.
在一些实施方案中，所述MHC-I功能通过降低MHC-I转录调节因子的活性而降低。在一些优选的实施方案中，所述MHC-I的转录调节因子可以选自：β2微球蛋白(B2M)、抗原加工相关转运体1(TAP1)、抗原加工相关转运体2(TAP2)、抗原加工相关转运体(TAP)相关糖蛋白(Tapasin)或NOD样受体家族半胱天冬酶募集结构域5(NLRC5)中的一个或多个。In some embodiments, the MHC-I function is reduced by reducing the activity of an MHC-I transcriptional regulator. In some preferred embodiments, the MHC-I transcriptional regulator may be selected from one or more of: β2 microglobulin (B2M), transporter associated with antigen processing 1 (TAP1), transporter associated with antigen processing 2 (TAP2), transporter associated with antigen processing (TAP)-associated glycoprotein (Tapasin), or NOD-like receptor family caspase recruitment domain 5 (NLRC5).
在一实施方案中，所述MHC-I功能通过降低HLA-A蛋白的活性而降低。In one embodiment, the MHC-I function is reduced by reducing the activity of the HLA-A protein.
在一实施方案中，所述MHC-I功能通过敲除编码所述HLA-A蛋白的基因而降低。In one embodiment, the MHC-I function is reduced by knocking out the gene encoding the HLA-A protein.
在一实施方案中，所述MHC-I功能通过降低HLA-B蛋白的活性而降低。In one embodiment, the MHC-I function is reduced by reducing the activity of the HLA-B protein.
在一实施方案中，所述MHC-I功能通过敲除编码所述HLA-B蛋白的基因而降低。In one embodiment, the MHC-I function is reduced by knocking out the gene encoding the HLA-B protein.
在一实施方案中，所述MHC-I功能通过降低HLA-C蛋白的活性而降低。In one embodiment, the MHC-I function is reduced by reducing the activity of the HLA-C protein.
在一实施方案中，所述MHC-I功能通过敲除编码所述HLA-C蛋白的基因而降低。In one embodiment, the MHC-I function is reduced by knocking out the gene encoding the HLA-C protein.
在一实施方案中，所述MHC-I功能通过降低TAP1蛋白的活性而降低。In one embodiment, the MHC-I function is reduced by reducing the activity of TAP1 protein.
在一实施方案中，所述MHC-I功能通过敲除编码所述TAP1蛋白的基因而降低。In one embodiment, the MHC-I function is reduced by knocking out the gene encoding the TAP1 protein.
在一实施方案中，所述MHC-I功能通过降低TAP2蛋白的活性而降低。In one embodiment, the MHC-I function is reduced by reducing the activity of the TAP2 protein.
在一实施方案中，所述MHC-I功能通过敲除编码所述TAP2蛋白的基因而降低。In one embodiment, the MHC-I function is reduced by knocking out the gene encoding the TAP2 protein.
在一实施方案中，所述MHC-I功能通过降低Tapasin蛋白的活性而降低。In one embodiment, the MHC-I function is reduced by reducing the activity of the Tapasin protein.
在一实施方案中，所述MHC-I功能通过敲除编码所述Tapasin蛋白的基因而降低。In one embodiment, the MHC-I function is reduced by knocking out the gene encoding the Tapasin protein.
在一实施方案中，所述MHC-I功能通过降低NLRC5蛋白的活性而降低。In one embodiment, the MHC-I function is reduced by reducing the activity of the NLRC5 protein.
在一实施方案中，所述MHC-I功能通过敲除编码所述NLRC5蛋白的基因而降低。In one embodiment, the MHC-I function is reduced by knocking out the gene encoding the NLRC5 protein.
在一优选实施方案中，所述MHC-I功能通过降低B2M蛋白的活性而降低。In a preferred embodiment, the MHC-I function is reduced by reducing the activity of the B2M protein.
在一实施方案中，所述B2M蛋白为人B2M蛋白，其包含SEQ ID NO:1所示的氨基酸序列或与SEQ ID NO:1所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同性的氨基酸序列。 In one embodiment, the B2M protein is a human B2M protein, which comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:1.
在一实施方案中，所述B2M蛋白为食蟹猴B2M蛋白，其包含SEQ ID NO:9所示的氨基酸序列或与SEQ ID NO:9所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同性的氨基酸序列。In one embodiment, the B2M protein is a crab-eating macaque B2M protein, which comprises the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:9.
在一实施方案中，所述MHC-I功能通过敲除编码所述B2M蛋白的基因而降低。In one embodiment, the MHC-I function is reduced by knocking out the gene encoding the B2M protein.
在一些实施方案中，所述MHC-II功能通过降低MHC-II类蛋白的活性而降低。In some embodiments, the MHC-II function is reduced by reducing the activity of MHC class II proteins.
在一实施方案中，所述MHC-II类蛋白包含人类白细胞抗原-DR(HLA-DR)蛋白、人类白细胞抗原-DQ(HLA-DQ)蛋白或人类白细胞抗原-DP(HLA-DP)蛋白。In one embodiment, the MHC-II class protein comprises a human leukocyte antigen-DR (HLA-DR) protein, a human leukocyte antigen-DQ (HLA-DQ) protein, or a human leukocyte antigen-DP (HLA-DP) protein.
在一些实施方案中，所述MHC-II功能通过降低MHC-II转录调节因子的活性而降低。在一些优选的实施方案中，所述MHC-II的转录调节因子可以选自：MHC-II反式激活蛋白(CIITA)、调节因子X相关锚蛋白(RFXANK)、调节因子X5(RFX5)、调节因子X相关蛋白(RFXAP)中的一个或多个。In some embodiments, the MHC-II function is reduced by reducing the activity of an MHC-II transcriptional regulator. In some preferred embodiments, the MHC-II transcriptional regulator may be selected from: one or more of MHC-II transactivator protein (CIITA), regulatory factor X-associated anchor protein (RFXANK), regulatory factor X5 (RFX5), and regulatory factor X-associated protein (RFXAP).
在一实施方案中，所述MHC-II功能通过降低HLA-DR蛋白的活性而降低。In one embodiment, the MHC-II function is reduced by reducing the activity of the HLA-DR protein.
在一实施方案中，所述MHC-II功能通过敲除编码所述HLA-DR蛋白的基因而降低。In one embodiment, the MHC-II function is reduced by knocking out the gene encoding the HLA-DR protein.
在一实施方案中，所述MHC-II功能通过降低HLA-DQ蛋白的活性而降低。In one embodiment, the MHC-II function is reduced by reducing the activity of the HLA-DQ protein.
在一实施方案中，所述MHC-II功能通过敲除编码所述HLA-DQ蛋白的基因而降低。In one embodiment, the MHC-II function is reduced by knocking out the gene encoding the HLA-DQ protein.
在一实施方案中，所述MHC-II功能通过降低HLA-DP蛋白的活性而降低。In one embodiment, the MHC-II function is reduced by reducing the activity of the HLA-DP protein.
在一实施方案中，所述MHC-II功能通过敲除编码所述HLA-DP蛋白的基因而降低。In one embodiment, the MHC-II function is reduced by knocking out the gene encoding the HLA-DP protein.
在一实施方案中，所述MHC-II功能通过降低RFXANK蛋白的活性而降低。In one embodiment, the MHC-II function is reduced by reducing the activity of the RFXANK protein.
在一实施方案中，所述MHC-II功能通过敲除编码所述RFXANK蛋白的基因而降低。In one embodiment, the MHC-II function is reduced by knocking out the gene encoding the RFXANK protein.
在一实施方案中，所述MHC-II功能通过降低RFX5蛋白的活性而降低。In one embodiment, the MHC-II function is reduced by reducing the activity of the RFX5 protein.
在一实施方案中，所述MHC-II功能通过敲除编码所述RFX5蛋白的基因而降低。In one embodiment, the MHC-II function is reduced by knocking out the gene encoding the RFX5 protein.
在一实施方案中，所述MHC-II功能通过降低RFXAP蛋白的活性而降低。In one embodiment, the MHC-II function is reduced by reducing the activity of the RFXAP protein.
在一实施方案中，所述MHC-II功能通过敲除编码所述RFXAP蛋白的基因而降低。In one embodiment, the MHC-II function is reduced by knocking out the gene encoding the RFXAP protein.
在一优选实施方案中，所述MHC-II功能通过降低CIITA蛋白的活性而降低。In a preferred embodiment, the MHC-II function is reduced by reducing the activity of the CIITA protein.
在一实施方案中，所述CIITA蛋白为人CIITA蛋白，其包含SEQ ID NO:2所示的氨基酸序列或与SEQ ID NO:2所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同性的氨基酸序列。In one embodiment, the CIITA protein is a human CIITA protein, which comprises the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:2.
在一实施方案中，所述CIITA蛋白为食蟹猴CIITA蛋白，其包含SEQ ID NO:10所示的氨基酸序列或与SEQ ID NO:10所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同性的氨基酸序列。In one embodiment, the CIITA protein is a cynomolgus monkey CIITA protein, which comprises the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:10.
在一实施方案中，所述MHC-II功能通过敲除编码所述CIITA蛋白的基因而降低。In one embodiment, the MHC-II function is reduced by knocking out the gene encoding the CIITA protein.
在一优选实施方案中，使用CRISPR技术敲除基因。在一些情况下，CRISPR技术用于将小的缺失/插入引入基因的编码区，使得不产生功能性蛋白，通常是移码突变的结果，其导致终止密码子的产生，使得产生截短的、非功能性蛋白。In a preferred embodiment, the gene is knocked out using CRISPR technology. In some cases, CRISPR technology is used to introduce small deletions/insertions into the coding region of a gene so that no functional protein is produced, usually as a result of a frameshift mutation, which results in the generation of a stop codon, resulting in a truncated, non-functional protein.
可以使用本领域已知的技术测量多能干细胞中MHC-I(当细胞来源于人细胞时为HLA-I)功能和MHC-II(当细胞来源于人细胞时为HLA-II)功能的成功降低，例如使用蛋 白抗体的蛋白印迹、FACS技术、RT-PCR、qPCR技术等。The successful reduction of MHC-I (HLA-I when the cells are derived from human cells) function and MHC-II (HLA-II when the cells are derived from human cells) function in pluripotent stem cells can be measured using techniques known in the art, such as using protein Western blotting, FACS technology, RT-PCR, qPCR technology, etc.
在一些实施方案中，降低的对NK细胞杀伤的敏感性是由多能干细胞中的GSN蛋白表达增加引起的。这通过几种方式完成，如本领域技术人员所理解的，可以使用“敲入”或转基因技术。在一些情况下，GSN表达增加是由一种或多种GSN转基因引起的。In some embodiments, the reduced sensitivity to NK cell killing is caused by increased expression of GSN protein in pluripotent stem cells. This is accomplished in several ways, as will be appreciated by those skilled in the art, and can use "knock-in" or transgenic techniques. In some cases, increased GSN expression is caused by one or more GSN transgenes.
因此，在一些实施方案中，在诱导型或组成型启动子的控制下将GSN基因的一个或多个拷贝添加至多能干细胞。在一些实施方案中，如本文所述或本领域已知的使用慢病毒构建体。如本领域已知的，GSN基因可以在合适的启动子的控制下整合到宿主细胞的基因组中。Thus, in some embodiments, one or more copies of the GSN gene are added to pluripotent stem cells under the control of an inducible or constitutive promoter. In some embodiments, a lentiviral construct is used as described herein or known in the art. As known in the art, the GSN gene can be integrated into the genome of a host cell under the control of a suitable promoter.
在一实施方案中，所述增加的GSN蛋白表达是由GSN转基因引起的。In one embodiment, the increased GSN protein expression is caused by a GSN transgene.
在一实施方案中，所述GSN蛋白为人GSN蛋白，其包含SEQ ID NO:3所示的氨基酸序列或与SEQ ID NO:3所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同性的氨基酸序列。In one embodiment, the GSN protein is a human GSN protein, which comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:3.
在一实施方案中，所述GSN蛋白为食蟹猴GSN蛋白，其包含SEQ ID NO:7所示的氨基酸序列或与SEQ ID NO:7所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同性的氨基酸序列。In one embodiment, the GSN protein is a cynomolgus monkey GSN protein, which comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:7.
可以使用已知技术测定足够的GSN蛋白表达的存在，例如实施例中描述的那些，例如使用蛋白印迹、ELISA测定或FACS测定。通常，在此上下文中的“足够”意指多能干细胞表面上GSN蛋白表达的增加，其沉默NK细胞的杀伤。The presence of sufficient GSN protein expression can be determined using known techniques, such as those described in the Examples, for example using Western blot, ELISA assay or FACS assay. Generally, "sufficient" in this context means an increase in GSN protein expression on the surface of pluripotent stem cells, which silences NK cell killing.
在又一方面，本发明还提供一种低免疫原性多能干细胞，其包含：In yet another aspect, the present invention further provides a low immunogenic pluripotent stem cell comprising:
使内源B2M蛋白活性降低的一个或多个改变；One or more changes that reduce the activity of endogenous B2M protein;
使内源CIITA蛋白活性降低的一个或多个改变；和One or more changes that reduce the activity of endogenous CIITA protein; and
在所述低免疫原性多能干细胞中引起增加的GSN蛋白表达的一个或多个改变。One or more alterations result in increased expression of GSN protein in the low immunogenic pluripotent stem cells.
在一实施方案中，所述低免疫原性多能干细胞包含：In one embodiment, the low immunogenic pluripotent stem cells comprise:
使内源B2M基因的两个等位基因失活的一个或多个改变；one or more alterations that inactivate both alleles of the endogenous B2M gene;
使内源CIITA基因的两个等位基因失活的一个或多个改变；和One or more alterations that inactivate both alleles of the endogenous CIITA gene; and
在所述低免疫原性多能干细胞中引起增加的GSN基因表达的一个或多个改变。One or more alterations result in increased GSN gene expression in the low immunogenic pluripotent stem cells.
如本文所用，术语“改变”或“遗传改变”是指引起细胞，例如本文所述的多能干细胞的变化，其可以例如通过修饰基因组或引入新的基因片段实现。在本文中，修饰基因组是指修饰细胞内或无细胞条件下的核酸序列以产生改造的多能细胞和多能干细胞。示例性的“改变”或“遗传改变”的技术包括但不限于同源重组、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。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。示例性的“改变”或“遗传改变”的技术还包括引入基因表达修饰分子，所述基因表达修饰分子包括但不限于siRNA、shRNA、microRNA、反义RNA、反义寡核苷酸ASO(antisense oligonucleotides)或抗miRNA寡核苷酸AMO(Anti-miRNA oligonucleotides)。As used herein, the term "change" or "genetic change" refers to causing a cell, such as a change in a pluripotent stem cell as described herein, which can be achieved, for example, by modifying a genome or introducing a new gene fragment. In this article, modifying a genome refers to modifying a nucleic acid sequence in a cell or under a cell-free condition to produce a transformed pluripotent cell and a pluripotent stem cell. Exemplary "changes" or "genetic changes" include, but are not limited to, homologous recombination, knock-in, ZFN (zinc finger nuclease), TALEN (transcription activator-like effector nuclease), CRISPR (clustered regularly spaced short palindromic repeats)/Cas9 and other site-specific nuclease technologies. These technologies enable double-stranded DNA breaks to be performed at the desired gene locus. These controlled double-strand breaks promote homologous recombination at specific gene locus points. The process focuses on targeting specific sequences of nucleic acid molecules with endonucleases, such as chromosomes, and the endonucleases recognize and bind sequences and induce double-strand breaks in nucleic acid molecules. Double-strand breaks are repaired by fallible non-homologous end joining (NHEJ) or by homologous recombination (HR). Exemplary "alteration" or "genetic alteration" techniques also include the introduction of gene expression modifying molecules, including but not limited to siRNA, shRNA, microRNA, antisense RNA, antisense oligonucleotides ASO (antisense oligonucleotides) or anti-miRNA oligonucleotides AMO (Anti-miRNA oligonucleotides).
本领域技术人员应当理解，可以使用许多不同的技术来改造本发明的多能细胞和多 能干细胞，以使其变得低免疫原性。It will be appreciated by those skilled in the art that many different techniques can be used to engineer the pluripotent cells and pluripotent cells of the present invention. stem cells to render them less immunogenic.
通常，这些技术可以单独使用或组合使用。例如，使用CRISPR技术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)敲入GSN基因。此外，本领域技术人员应当理解，这些基因可以使用不同的技术以不同的顺序操作。Generally, these techniques can be used alone or in combination. For example, CRISPR technology is used to reduce the expression of active B2M and/or CIITA proteins in modified cells, and viral technology (e.g., lentivirus) is used to knock in the GSN gene. In addition, it should be understood by those skilled in the art that these genes can be manipulated in different orders using different techniques.
在一些实施方案中，本发明的低免疫原性多能干细胞所包含的一个或多个改变能降低内源主要组织相容性I类抗原(MHC-I)功能。在一些实施方案中，本发明的低免疫原性多能干细胞所包含的一个或多个改变能降低内源主要组织相容性II类抗原(MHC-II)功能。在一些实施方案中，本发明的低免疫原性多能干细胞所包含的一个或多个改变能降低对NK细胞杀伤的敏感性。In some embodiments, the one or more changes contained in the low immunogenic pluripotent stem cells of the present invention can reduce endogenous major histocompatibility class I antigen (MHC-I) function. In some embodiments, the one or more changes contained in the low immunogenic pluripotent stem cells of the present invention can reduce endogenous major histocompatibility class II antigen (MHC-II) function. In some embodiments, the one or more changes contained in the low immunogenic pluripotent stem cells of the present invention can reduce sensitivity to NK cell killing.
在一些实施方案中，本发明的低免疫原性多能干细胞所包含的一个或多个改变能使内源B2M蛋白活性降低。在一些实施方案中，本发明的低免疫原性多能干细胞所包含的一个或多个改变能使内源CIITA蛋白活性降低。在一些实施方案中，本发明的低免疫原性多能干细胞所包含的一个或多个改变能增加GSN蛋白表达。In some embodiments, the one or more changes contained in the low immunogenic pluripotent stem cells of the present invention can reduce the activity of endogenous B2M protein. In some embodiments, the one or more changes contained in the low immunogenic pluripotent stem cells of the present invention can reduce the activity of endogenous CIITA protein. In some embodiments, the one or more changes contained in the low immunogenic pluripotent stem cells of the present invention can increase the expression of GSN protein.
在一些实施方案中，本发明的低免疫原性多能干细胞所包含的一个或多个改变能使内源B2M基因的两个等位基因失活。在一些实施方案中，本发明的低免疫原性多能干细胞所包含的一个或多个改变能使内源CIITA基因的两个等位基因失活。在一些实施方案中，本发明的低免疫原性多能干细胞所包含的一个或多个改变能增加GSN基因表达。In some embodiments, the one or more changes contained in the low immunogenic pluripotent stem cells of the present invention can inactivate both alleles of the endogenous B2M gene. In some embodiments, the one or more changes contained in the low immunogenic pluripotent stem cells of the present invention can inactivate both alleles of the endogenous CIITA gene. In some embodiments, the one or more changes contained in the low immunogenic pluripotent stem cells of the present invention can increase the expression of the GSN gene.
在一些实施方案中，本发明的低免疫原性多能干细胞所包含的一个或多个改变能使内源B2M蛋白表达受抑制。在一些实施方案中，本发明的低免疫原性多能干细胞所包含的一个或多个改变能使内源CIITA蛋白表达受抑制。In some embodiments, the low immunogenic pluripotent stem cells of the present invention comprise one or more changes that inhibit the expression of endogenous B2M protein. In some embodiments, the low immunogenic pluripotent stem cells of the present invention comprise one or more changes that inhibit the expression of endogenous CIITA protein.
在一些实施方案中，本发明的低免疫原性多能干细胞所包含的一个或多个改变能使内源B2M蛋白表达受干扰。在一些实施方案中，本发明的低免疫原性多能干细胞所包含的一个或多个改变能使内源CIITA蛋白表达受干扰。In some embodiments, the low immunogenic pluripotent stem cells of the present invention comprise one or more changes that can interfere with the expression of endogenous B2M protein. In some embodiments, the low immunogenic pluripotent stem cells of the present invention comprise one or more changes that can interfere with the expression of endogenous CIITA protein.
在一些实施方案中，本发明的低免疫原性多能干细胞所包含的一个或多个改变能使内源B2M蛋白表达降低。在一些实施方案中，本发明的低免疫原性多能干细胞所包含的一个或多个改变能使内源CIITA蛋白表达降低。In some embodiments, the low immunogenic pluripotent stem cells of the present invention comprise one or more changes that can reduce the expression of endogenous B2M protein. In some embodiments, the low immunogenic pluripotent stem cells of the present invention comprise one or more changes that can reduce the expression of endogenous CIITA protein.
在一些实施方案中，本发明的低免疫原性多能干细胞所包含的一个或多个改变能敲除内源B2M蛋白。在一些实施方案中，本发明的低免疫原性多能干细胞所包含的一个或多个改变能敲除内源CIITA蛋白。In some embodiments, the low immunogenic pluripotent stem cells of the present invention comprise one or more changes that can knock out endogenous B2M protein. In some embodiments, the low immunogenic pluripotent stem cells of the present invention comprise one or more changes that can knock out endogenous CIITA protein.
在一实施方案中，使用本领域已知的成簇规律间隔短回文重复序列/Cas(“CRISPR”)技术改变所述多能干细胞，使内源B2M蛋白活性降低。In one embodiment, the pluripotent stem cells are altered using Clustered Regularly Interspaced Short Palindromic Repeats/Cas ("CRISPR") technology known in the art to reduce the activity of endogenous B2M protein.
在一实施方案中，使用本领域已知的成簇规律间隔短回文重复序列/Cas(“CRISPR”)技术改变所述多能干细胞，使内源CIITA蛋白活性降低。In one embodiment, the pluripotent stem cells are altered using Clustered Regularly Interspaced Short Palindromic Repeats/Cas ("CRISPR") technology known in the art to reduce the activity of endogenous CIITA protein.
在一实施方案中，使用本领域已知的成簇规律间隔短回文重复序列/Cas(“CRISPR”)技术改变所述多能干细胞，使内源B2M基因的两个等位基因失活。In one embodiment, the pluripotent stem cells are altered to inactivate both alleles of the endogenous B2M gene using Clustered Regularly Interspaced Short Palindromic Repeats/Cas ("CRISPR") technology known in the art.
在一实施方案中，使用本领域已知的成簇规律间隔短回文重复序列/Cas(“CRISPR”)技术改变所述多能干细胞，使内源CIITA基因的两个等位基因失活。In one embodiment, the pluripotent stem cells are altered using Clustered Regularly Interspaced Short Palindromic Repeats/Cas ("CRISPR") technology known in the art to inactivate both alleles of the endogenous CIITA gene.
测试基因是否已经失活的测定法是已知的并在本文中描述。在一个实施方案中，该测定是用针对B2M蛋白或CIITA蛋白的抗体探测细胞裂解物的蛋白印迹。在另一个实 施方案中，逆转录酶聚合酶链反应(RT-PCR)证实存在失活改变。Assays for testing whether a gene has been inactivated are known and described herein. In one embodiment, the assay is a Western blot of cell lysates probed with antibodies against the B2M protein or the CIITA protein. In another embodiment, In this embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) confirms the presence of the inactivating change.
在一实施方案中，使用本领域已知的病毒技术可用于在所述低免疫原性多能干细胞中引起增加的GSN基因的表达。所述病毒技术包括但不限于逆转录病毒载体、慢病毒载体、腺病毒载体和仙台病毒载体的使用。在一些实施方案中，将编码GSN蛋白的核酸序列导入所述细胞的选定位点；所述细胞的选定位点是AAVS1、CCR5等安全港基因位点。如本文所用，“安全港基因位点”是指能用于基因安全敲入并能保证转入基因的正常稳定表达的位点。In one embodiment, the use of viral techniques known in the art can be used to cause increased expression of the GSN gene in the low immunogenic pluripotent stem cells. The viral techniques include, but are not limited to, the use of retroviral vectors, lentiviral vectors, adenoviral vectors, and Sendai viral vectors. In some embodiments, the nucleic acid sequence encoding the GSN protein is introduced into a selected site of the cell; the selected site of the cell is a safe harbor gene site such as AAVS1, CCR5, etc. As used herein, a "safe harbor gene site" refers to a site that can be used for safe gene knock-in and can ensure normal and stable expression of the introduced gene.
在一优选实施方案中，使用慢病毒载体在所述低免疫原性多能干细胞中引起增加的GSN基因的表达。In a preferred embodiment, a lentiviral vector is used to induce increased expression of the GSN gene in the low immunogenic pluripotent stem cells.
在一实施方案中，所述低免疫原性多能干细胞为人多能干细胞。在一实施方案中，所述B2M蛋白为人B2M蛋白，其包含SEQ ID NO:1所示的氨基酸序列或与SEQ ID NO:1所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同性的氨基酸序列。在一实施方案中，所述CIITA蛋白为人CIITA蛋白，其包含SEQ ID NO:2所示的氨基酸序列或与SEQ ID NO:2所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同性的氨基酸序列。在一实施方案中，所述GSN蛋白为人GSN蛋白，其包含SEQ ID NO:3所示的氨基酸序列或与SEQ ID NO:3所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同性的氨基酸序列。In one embodiment, the low immunogenic pluripotent stem cells are human pluripotent stem cells. In one embodiment, the B2M protein is a human B2M protein, which comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 1. In one embodiment, the CIITA protein is a human CIITA protein, which comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 2. In one embodiment, the GSN protein is a human GSN protein, which comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:3.
在一实施方案中，所述低免疫原性多能干细胞为食蟹猴多能干细胞。在一实施方案中，所述B2M蛋白为食蟹猴B2M蛋白，其包含SEQ ID NO:9所示的氨基酸序列或与SEQ ID NO:9所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同性的氨基酸序列。在一实施方案中，所述CIITA蛋白为食蟹猴CIITA蛋白，其包含SEQ ID NO:10所示的氨基酸序列或与SEQ ID NO:10所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同性的氨基酸序列。在一实施方案中，所述GSN蛋白为食蟹猴GSN蛋白，其包含SEQ ID NO:7所示的氨基酸序列或与SEQ ID NO:7所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同性的氨基酸序列。In one embodiment, the low immunogenic pluripotent stem cells are cynomolgus monkey pluripotent stem cells. In one embodiment, the B2M protein is a cynomolgus monkey B2M protein, which comprises the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:9. In one embodiment, the CIITA protein is a cynomolgus monkey CIITA protein, which comprises the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:10. In one embodiment, the GSN protein is a cynomolgus monkey GSN protein, which comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:7.
在一些实施方案中，所述低免疫原性多能干细胞为低免疫原性的诱导性多能干细胞(iPSC)。In some embodiments, the low immunogenicity pluripotent stem cells are low immunogenicity induced pluripotent stem cells (iPSCs).
在一实施方案中，所述低免疫原性多能干细胞包含：In one embodiment, the low immunogenic pluripotent stem cells comprise:
与亲本多能干细胞相比降低的内源主要组织相容性I类抗原(MHC-I)功能；reduced endogenous major histocompatibility class I antigen (MHC-I) function compared to parental pluripotent stem cells;
与亲本多能干细胞相比降低的内源主要组织相容性II类抗原(MHC-II)功能；和Reduced endogenous major histocompatibility class II antigen (MHC-II) function compared to the parental pluripotent stem cell; and
与亲本多能干细胞相比降低的对NK细胞杀伤的敏感性。Reduced sensitivity to NK cell killing compared to parental pluripotent stem cells.
在一实施方案中，所述低免疫原性多能干细胞引发的T细胞应答，其低于由亲本多能干细胞引发的T细胞应答，所述亲本多能干细胞不包含所述使B2M和CIITA蛋白活性降低的改变以及所述引起增加的GSN蛋白表达的改变。在一实施方案中，所述T细胞应答是通过实时无标记动态细胞分析技术(RTCA)测定T细胞对所述低免疫原性多能干细胞或亲本多能干细胞的杀伤性来测量的。In one embodiment, the T cell response elicited by the low immunogenic pluripotent stem cells is lower than the T cell response elicited by the parental pluripotent stem cells, and the parental pluripotent stem cells do not contain the changes that reduce the activity of B2M and CIITA proteins and the changes that cause increased GSN protein expression. In one embodiment, the T cell response is measured by measuring the killing of T cells to the low immunogenic pluripotent stem cells or parental pluripotent stem cells by real-time label-free dynamic cell analysis (RTCA).
在一实施方案中，所述低免疫原性多能干细胞引发的天然杀伤(NK)细胞应答，其低 于由B2M/CIITA双等位基因敲除的克隆DKO细胞引发的NK细胞应答，所述DKO细胞包含所述使B2M和CIITA蛋白活性降低的改变但不包含所述引起增加的GSN蛋白表达的改变。在一实施方案中，所述NK细胞应答是通过测定与所述低免疫原性多能干细胞或DKO细胞体外孵育的NK细胞的IFN-γ水平来测量的。在一实施方案中，所述NK细胞应答是通过实时无标记动态细胞分析技术(RTCA)测定NK细胞对所述低免疫原性多能干细胞或DKO细胞的杀伤性来测量的。In one embodiment, the natural killer (NK) cell response elicited by the low immunogenic pluripotent stem cells is low In an NK cell response initiated by a cloned DKO cell with a B2M/CIITA biallelic knockout, the DKO cell comprises the changes that reduce the activity of the B2M and CIITA proteins but does not comprise the changes that cause increased GSN protein expression. In one embodiment, the NK cell response is measured by determining the IFN-γ level of NK cells incubated in vitro with the low immunogenic pluripotent stem cells or DKO cells. In one embodiment, the NK cell response is measured by measuring the killing of NK cells to the low immunogenic pluripotent stem cells or DKO cells by real-time label-free dynamic cell analysis (RTCA).
产生本发明的低免疫原性多能干细胞的方法Method for producing low immunogenic pluripotent stem cells of the present invention
本发明还提供一种产生本发明的低免疫原性多能干细胞的方法，所述方法包括：降低所述多能干细胞中内源主要组织相容性I类抗原(MHC-I)功能；降低所述多能干细胞中内源主要组织相容性II类抗原(MHC-II)功能；和增加降低所述多能干细胞对NK细胞杀伤的敏感性的蛋白的表达，其中所述蛋白为GSN蛋白。The present invention also provides a method for producing the low immunogenic pluripotent stem cells of the present invention, the method comprising: reducing the endogenous major histocompatibility class I antigen (MHC-I) function in the pluripotent stem cells; reducing the endogenous major histocompatibility class II antigen (MHC-II) function in the pluripotent stem cells; and increasing the expression of a protein that reduces the sensitivity of the pluripotent stem cells to NK cell killing, wherein the protein is GSN protein.
在一些实施方案中，所述低免疫原性多能干细胞为低免疫原性的诱导性多能干细胞(iPSC)。In some embodiments, the low immunogenicity pluripotent stem cells are low immunogenicity induced pluripotent stem cells (iPSCs).
在一些实施方案中，所述方法包括：降低所述多能干细胞中B2M蛋白的活性；降低所述多能干细胞中CIITA蛋白的活性；和增加所述多能干细胞中GSN蛋白的表达。In some embodiments, the method comprises: reducing the activity of B2M protein in the pluripotent stem cells; reducing the activity of CIITA protein in the pluripotent stem cells; and increasing the expression of GSN protein in the pluripotent stem cells.
在一实施方案中，所述方法包括：消除所述多能干细胞中B2M基因的两个等位基因的活性；消除所述多能干细胞中CIITA基因的两个等位基因的活性；和增加所述多能干细胞中GSN基因的表达。In one embodiment, the method comprises: eliminating the activity of both alleles of the B2M gene in the pluripotent stem cells; eliminating the activity of both alleles of the CIITA gene in the pluripotent stem cells; and increasing the expression of the GSN gene in the pluripotent stem cells.
在一些实施方案中，可以通过如上所述的“改变”或“遗传改变”的技术降低所述多能干细胞中B2M蛋白的活性。在一些实施方案中，可以通过如上所述的“改变”或“遗传改变”的技术降低所述多能干细胞中CIITA蛋白的活性。所述技术例如，引入基因表达修饰分子、成簇规律间隔短回文重复序列(CRISPR)技术、转录激活因子样效应核酸酶(TALEN)技术、锌指核酸酶(ZFN)技术或同源重组技术。在一优选实施方案中，所述基因表达修饰分子包含siRNA、shRNA、microRNA、反义RNA、反义寡核苷酸ASO(antisense oligonucleotides)或抗miRNA寡核苷酸AMO(Anti-miRNA oligonucleotides)。In some embodiments, the activity of the B2M protein in the pluripotent stem cells can be reduced by the technique of "alteration" or "genetic alteration" as described above. In some embodiments, the activity of the CIITA protein in the pluripotent stem cells can be reduced by the technique of "alteration" or "genetic alteration" as described above. The techniques, for example, introduce gene expression modifying molecules, clustered regularly interspaced short palindromic repeats (CRISPR) technology, transcription activator-like effector nuclease (TALEN) technology, zinc finger nuclease (ZFN) technology or homologous recombination technology. In a preferred embodiment, the gene expression modifying molecules comprise siRNA, shRNA, microRNA, antisense RNA, antisense oligonucleotides ASO (antisense oligonucleotides) or anti-miRNA oligonucleotides AMO (Anti-miRNA oligonucleotides).
在一些实施方式中，CRISPR/Cas系统包括Cas蛋白或编码Cas蛋白的核酸序列和至少一种至两种核糖核酸(例如gRNA)，所述核糖核酸能够将Cas蛋白引导至靶多核苷酸序列的靶基序并与所述靶基序杂交。在一些实施方式中，CRISPR/Cas系统包括Cas蛋白或编码Cas蛋白的核酸序列和单个核糖核酸或至少一个核糖核酸(例如gRNA)对，所述核糖核酸能够将Cas蛋白引导至靶多核苷酸序列的靶基序并与所述靶基序杂交。In some embodiments, the CRISPR/Cas system includes a Cas protein or a nucleic acid sequence encoding a Cas protein and at least one to two ribonucleic acids (e.g., gRNA), which can guide the Cas protein to a target motif of a target polynucleotide sequence and hybridize with the target motif. In some embodiments, the CRISPR/Cas system includes a Cas protein or a nucleic acid sequence encoding a Cas protein and a single ribonucleic acid or at least one ribonucleic acid (e.g., gRNA) pair, which can guide the Cas protein to a target motif of a target polynucleotide sequence and hybridize with the target motif.
在一些实施方式中，Cas蛋白包含一个或多个氨基酸取代或修饰。在一些实施方式中，一个或多个氨基酸取代包含保守氨基酸取代。在一些情况下，取代和/或修饰可以防止或降低蛋白水解降解和/或延长细胞中多肽的半衰期。在一些实施方式中，Cas蛋白可以包含肽键置换(例如脲、硫脲、氨基甲酸酯、磺酰脲等)。在一些实施方式中，Cas蛋白可以包含天然存在的氨基酸。在一些实施方式中，Cas蛋白可以包含可选的氨基酸(例如D-氨基酸、β-氨基酸、高半胱氨酸、磷酸丝氨酸等)。在一些实施方式中，Cas蛋白可以包含修饰以包括部分(例如聚乙二醇化、糖基化、脂化、乙酰化、封端等)。In some embodiments, the Cas protein comprises one or more amino acid substitutions or modifications. In some embodiments, one or more amino acid substitutions comprise conservative amino acid substitutions. In some cases, substitutions and/or modifications can prevent or reduce proteolytic degradation and/or extend the half-life of polypeptides in cells. In some embodiments, the Cas protein may comprise peptide bond replacements (e.g., urea, thiourea, carbamate, sulfonylurea, etc.). In some embodiments, the Cas protein may comprise naturally occurring amino acids. In some embodiments, the Cas protein may comprise optional amino acids (e.g., D-amino acids, β-amino acids, homocysteine, phosphoserine, etc.). In some embodiments, the Cas protein may comprise modifications to include portions (e.g., pegylation, glycosylation, lipidation, acetylation, capping, etc.).
在一些实施方式中，Cas蛋白包含核心Cas蛋白。示例性Cas核心蛋白包括但不限 于Cas1、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8和Cas9。在一些实施方式中，Cas蛋白包含大肠杆菌(E.coli)亚型的Cas蛋白(也称为CASS2)。大肠杆菌亚型的示例性Cas蛋白包括但不限于Cse1、Cse2、Cse3、Cse4和Cas5e。在一些实施方式中，Cas蛋白包含Ypest亚型的Cas蛋白(也称为CASS3)。Ypest亚型的示例性Cas蛋白包括但不限于Csy1、Csy2、Csy3和Csy4。在一些实施方式中，Cas蛋白包含Nmeni亚型的Cas蛋白(也称为CASS4)。Nmeni亚型的示例性Cas蛋白包括但不限于Csn1和Csn2。在一些实施方式中，Cas蛋白包含Dvulg亚型的Cas蛋白(也称为CASS1)。Dvulg亚型的示例性Cas蛋白包括但不限于Csd1、Csd2和Cas5d。在一些实施方式中，Cas蛋白包含Tneap亚型的Cas蛋白(也称为CASS7)。Tneap亚型的示例性Cas蛋白包括但不限于Cst1、Cst2、Cas5t。在一些实施方式中，Cas蛋白包含Hmari亚型的Cas蛋白。Hmari亚型的示例性Cas蛋白包括但不限于Csh1、Csh2和Cas5h。在一些实施方式中，Cas蛋白包含Apern亚型的Cas蛋白(也称为CASS5)。Apern亚型的示例性Cas蛋白包括但不限于Csa1、Csa2、Csa3、Csa4、Csa5和Cas5a。在一些实施方式中，Cas蛋白包含Mtube亚型的Cas蛋白(也称为CASS6)。Mtube亚型的示例性Cas蛋白包括但不限于Csm1、Csm2、Csm3、Csm4和Csm5。在一些实施方式中，Cas蛋白包含RAMP型Cas蛋白。示例性RAMP型Cas蛋白包括但不限于Cmr1、Cmr2、Cmr3、Cmr4、Cmr5和Cmr6。In some embodiments, the Cas protein comprises a core Cas protein. Exemplary Cas core proteins include but are not limited to In Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8 and Cas9. In some embodiments, the Cas protein comprises a Cas protein of an Escherichia coli (E.coli) subtype (also referred to as CASS2). Exemplary Cas proteins of E. coli subtypes include, but are not limited to, Cse1, Cse2, Cse3, Cse4 and Cas5e. In some embodiments, the Cas protein comprises a Cas protein of a Ypest subtype (also referred to as CASS3). Exemplary Cas proteins of Ypest subtypes include, but are not limited to, Csy1, Csy2, Csy3 and Csy4. In some embodiments, the Cas protein comprises a Cas protein of an Nmeni subtype (also referred to as CASS4). Exemplary Cas proteins of Nmeni subtypes include, but are not limited to, Csn1 and Csn2. In some embodiments, the Cas protein comprises a Cas protein of a Dvulg subtype (also referred to as CASS1). Exemplary Cas proteins of Dvulg subtypes include, but are not limited to, Csd1, Csd2 and Cas5d. In some embodiments, the Cas protein comprises a Cas protein of a Tneap subtype (also referred to as CASS7). Exemplary Cas proteins of the Tneap subtype include, but are not limited to, Cst1, Cst2, and Cas5t. In some embodiments, the Cas protein comprises a Cas protein of the Hmari subtype. Exemplary Cas proteins of the Hmari subtype include, but are not limited to, Csh1, Csh2, and Cas5h. In some embodiments, the Cas protein comprises a Cas protein of the Apern subtype (also known as CASS5). Exemplary Cas proteins of the Apern subtype include, but are not limited to, Csa1, Csa2, Csa3, Csa4, Csa5, and Cas5a. In some embodiments, the Cas protein comprises a Cas protein of the Mtube subtype (also known as CASS6). Exemplary Cas proteins of the Mtube subtype include, but are not limited to, Csm1, Csm2, Csm3, Csm4, and Csm5. In some embodiments, the Cas protein comprises a RAMP-type Cas protein. Exemplary RAMP-type Cas proteins include, but are not limited to, Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6.
在一些实施方式中，Cas蛋白是化脓链球菌(Streptococcus pyogenes)Cas9蛋白或其功能部分。在一些实施方式中，Cas蛋白是金黄色葡萄球菌(Streptococcus aureus)Cas9蛋白或其功能部分。在一些实施方式中，Cas蛋白是嗜热链球菌(Streptococcusthermophilus)Cas9蛋白或其功能部分。在一些实施方式中，Cas蛋白是脑膜炎奈瑟氏球菌(Neisseria meningitides)Cas9蛋白或其功能部分。在一些实施方案中，Cas蛋白是齿垢密螺旋体(Treponema denticola)Cas9蛋白或其功能部分。在一些实施方案中，Cas蛋白是来自任何细菌种的Cas9蛋白或其功能部分。Cas9蛋白是II型CRISPR系统的成员，所述II型CRISPR系统通常包括反式编码的小RNA(tracrRNA)、内源核糖核酸酶3(rnc)和Cas蛋白。Cas9蛋白(也称为CRISPR相关核酸内切酶Cas9/Csn1)是包含1368个氨基酸的多肽。In some embodiments, the Cas protein is a Streptococcus pyogenes Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Streptococcus aureus Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Streptococcus thermophilus Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Neisseria meningitides Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Treponema denticola Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Cas9 protein or a functional portion thereof from any bacterial species. The Cas9 protein is a member of a type II CRISPR system, which typically includes a trans-encoded small RNA (tracrRNA), an endogenous ribonuclease 3 (rnc), and a Cas protein. The Cas9 protein (also known as a CRISPR-associated endonuclease Cas9/Csn1) is a polypeptide comprising 1368 amino acids.
在一实施方案中，通过成簇规律间隔短回文重复序列(CRISPR)/Cas9基因编辑技术降低所述多能干细胞中B2M蛋白的活性。In one embodiment, the activity of B2M protein in the pluripotent stem cells is reduced by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology.
在一实施方案中，通过成簇规律间隔短回文重复序列(CRISPR)/Cas9基因编辑技术消除所述多能干细胞中B2M基因的两个等位基因的活性。In one embodiment, the activity of both alleles of the B2M gene in the pluripotent stem cells is eliminated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology.
在一实施方案中，通过成簇规律间隔短回文重复序列(CRISPR)/Cas9基因编辑技术降低所述多能干细胞中CIITA蛋白的活性。In one embodiment, the activity of CIITA protein in the pluripotent stem cells is reduced by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology.
在一实施方案中，通过成簇规律间隔短回文重复序列(CRISPR)/Cas9基因编辑技术消除所述多能干细胞中CIITA基因的两个等位基因的活性。In one embodiment, the activity of both alleles of the CIITA gene in the pluripotent stem cells is eliminated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology.
在一实施方案中，通过对内源基因座的修饰增加GSN蛋白的表达。在一些实施方案中，通过如上所述的“改变”或“遗传改变”的技术对内源基因座进行修饰。所述技术例如，基因敲入、成簇规律间隔短回文重复序列(CRISPR)技术、转录激活因子样效应核酸酶(TALEN)技术、锌指核酸酶(ZFN)技术或同源重组技术。In one embodiment, the expression of GSN protein is increased by modification of the endogenous locus. In some embodiments, the endogenous locus is modified by the technique of "alteration" or "genetic alteration" as described above. The technique, for example, gene knock-in, clustered regularly interspaced short palindromic repeats (CRISPR) technology, transcription activator-like effector nuclease (TALEN) technology, zinc finger nuclease (ZFN) technology or homologous recombination technology.
在一实施方案中，通过转基因的表达增加GSN蛋白的表达。可以使用本领域已知 的转基因表达技术增加GSN蛋白的表达，包括但不限于病毒技术、Piggybac转座子技术、Sleeping Beauty转座子技术。In one embodiment, the expression of GSN protein is increased by expression of a transgene. The transgenic expression technology used to increase the expression of GSN protein includes but is not limited to viral technology, Piggybac transposon technology, and Sleeping Beauty transposon technology.
在本文中，可以使用公知的重组技术来产生如本文所述的表达构建体。在某些实施方案中，编码目的蛋白的核酸序列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。本文所用的表达构建体可以使用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插入染色体中。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目的蛋白的核苷酸序列。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、期望表达的目的蛋白、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如抗生素标志物的表达。在一些实施方案中，所述启动子为EF1a启动子。In this article, known recombinant techniques can be used to produce expression constructs as described herein. In certain embodiments, the nucleic acid sequence encoding the target protein can be operably connected to one or more regulatory nucleotide sequences in the expression construct. The regulatory nucleotide sequence is generally suitable for host cells and subjects to be treated. Various types of suitable expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. Generally, one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader sequences or signal sequences, ribosome binding sites, transcription start and stop sequences, translation start and stop sequences, and enhancers or activator sequences. The expression construct used herein can use constitutive or inducible promoters known in the art. The promoter can be a naturally occurring promoter, or a hybrid promoter combining elements of more than one promoter. The expression construct can be present in the cell on an episome (e.g., a plasmid), or the expression construct can be inserted into a chromosome. In a specific embodiment, the expression vector includes a selectable marker gene to allow selection of transformed host cells. Some embodiments include an expression vector comprising a nucleotide sequence encoding the target protein operably connected to at least one regulatory sequence. The regulatory sequences used herein include promoters, enhancers, and other expression control elements. In certain embodiments, the expression vector is designed to select the host cell to be transformed, the desired protein to be expressed, the copy number of the vector, the ability to control the copy number, or the expression of any other protein encoded by the vector, such as an antibiotic marker. In some embodiments, the promoter is the EF1a promoter.
病毒技术可用于在所述低免疫原性多能干细胞中引起增加的GSN基因的表达。所述病毒技术包括但不限于逆转录病毒载体、慢病毒载体、腺病毒载体和仙台病毒载体的使用。Viral techniques can be used to induce increased expression of the GSN gene in the low immunogenic pluripotent stem cells. The viral techniques include, but are not limited to, the use of retroviral vectors, lentiviral vectors, adenoviral vectors, and Sendai virus vectors.
在一优选实施方案中，通过合成编码GSN蛋白的核酸序列，以构建至慢病毒载体中，再通过慢病毒载体，将至少一个拷贝的在启动子控制下的GSN基因引入所述多能干细胞中增加GSN蛋白的表达。In a preferred embodiment, a nucleic acid sequence encoding the GSN protein is synthesized and constructed into a lentiviral vector, and then at least one copy of the GSN gene under the control of a promoter is introduced into the pluripotent stem cells via the lentiviral vector to increase the expression of the GSN protein.
在一实施方案中，将编码GSN蛋白的核酸序列导入所述多能干细胞基因组的选定位点。在一优选实施方案中，所述选定位点是AAVS1、CCR5等安全港基因位点。如本文所用，“安全港基因位点”是指能用于基因安全敲入并能保证转入基因的正常稳定表达的位点。In one embodiment, a nucleic acid sequence encoding a GSN protein is introduced into a selected site of the pluripotent stem cell genome. In a preferred embodiment, the selected site is a safe harbor gene site such as AAVS1, CCR5, etc. As used herein, a "safe harbor gene site" refers to a site that can be used for safe gene knock-in and can ensure normal and stable expression of the transferred gene.
在一实施方案中，所述GSN蛋白为人GSN蛋白。In one embodiment, the GSN protein is a human GSN protein.
在一实施方案中，所述编码GSN蛋白的核酸序列包含SEQ ID NO:4所示的核酸序列或与SEQ ID NO:4所示的核酸序列具有至少70％、80％、85％、90％、95％、96％、97％、98％或99％相同性的核酸序列。In one embodiment, the nucleic acid sequence encoding the GSN protein comprises the nucleic acid sequence shown in SEQ ID NO:4 or a nucleic acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in SEQ ID NO:4.
在一实施方案中，所述GSN蛋白为食蟹猴GSN蛋白。In one embodiment, the GSN protein is a cynomolgus monkey GSN protein.
在一实施方案中，所述编码GSN蛋白的核酸序列包含SEQ ID NO:8所示的核酸序列或与SEQ ID NO:8所示的核酸序列具有至少70％、80％、85％、90％、95％、96％、97％、98％或99％相同性的核酸序列。In one embodiment, the nucleic acid sequence encoding the GSN protein comprises the nucleic acid sequence shown in SEQ ID NO:8 or a nucleic acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in SEQ ID NO:8.
预防或治疗Prevention or treatment
本发明进一步提供了本发明的低免疫原性多能干细胞或本发明的方法制备的低免疫原性多能干细胞在制备用于预防或治疗需要细胞移植的疾病的药物中的用途。The present invention further provides use of the low-immunogenic pluripotent stem cells of the present invention or the low-immunogenic pluripotent stem cells prepared by the method of the present invention in preparing a drug for preventing or treating a disease requiring cell transplantation.
本发明的低免疫原性多能干细胞或本发明的方法制备的低免疫原性多能干细胞可 以诱导分化成不同的细胞，其可以用于不同的预防或治疗目的，以预防或治疗不同的疾病。如本领域技术人员应当理解，分化方法取决于使用已知技术的所需细胞类型。例如，可以将细胞悬浮分化，然后制成凝胶基质形式，例如基质胶、明胶或纤维蛋白/凝血酶形式，以促进细胞存活。通常可以通过评估细胞特异性标志物的存在来如本领域已知的那样确定分化。例如，可以在一定分化条件下将细胞分化为心肌细胞、神经细胞、胶质细胞、内皮细胞、T细胞、NK细胞、NKT细胞、巨噬细胞、造血祖细胞、间充质细胞、胰岛细胞、软骨细胞、视网膜色素上皮细胞、肾细胞、肝细胞、甲状腺细胞、皮肤细胞、血细胞或上皮细胞。The low immunogenicity pluripotent stem cells of the present invention or the low immunogenicity pluripotent stem cells prepared by the method of the present invention can be Differentiate into different cells with induction, it can be used for different prevention or treatment purposes, to prevent or treat different diseases.As will be appreciated by those skilled in the art, differentiation method depends on the required cell type using known technology.For example, cell suspension differentiation can be used, then made into gel matrix form, such as matrigel, gelatin or fibrin/thrombin form, to promote cell survival.Usually can determine differentiation as known in the art by the presence of assessment cell specific markers.For example, cell differentiation can be into cardiomyocyte, nerve cell, glial cell, endothelial cell, T cell, NK cell, NKT cell, macrophage, hematopoietic progenitor cell, mesenchymal cell, islet cell, chondrocyte, retinal pigment epithelial cell, nephrocyte, hepatocyte, thyroid cell, skin cell, blood cell or epithelial cell under certain differentiation conditions.
在一些实施方案中，所述疾病为癌症，所述癌症包含实体肿瘤和血液肿瘤。在一些实施方案中，所述实体肿瘤包含小细胞肺癌、乳腺癌、睾丸癌、神经母细胞瘤、卵巢癌或黑色素瘤。在一些实施方案中，所述血液肿瘤包含急性白血病、慢性白血病、淋巴瘤、骨髓增生异常综合征或多发性骨髓瘤。In some embodiments, the disease is cancer, and the cancer comprises solid tumors and blood tumors. In some embodiments, the solid tumor comprises small cell lung cancer, breast cancer, testicular cancer, neuroblastoma, ovarian cancer or melanoma. In some embodiments, the blood tumor comprises acute leukemia, chronic leukemia, lymphoma, myelodysplastic syndrome or multiple myeloma.
在一实施方案中，所述疾病包含再生障碍性贫血。In one embodiment, the disease comprises aplastic anemia.
在一实施方案中，所述疾病包含先天免疫缺陷性疾病。In one embodiment, the disease comprises a congenital immunodeficiency disease.
在一些实施方案中，所述疾病为自身免疫性疾病，其包含系统性红斑狼疮、类风湿性关节炎、强直性脊柱炎或I型糖尿病。In some embodiments, the disease is an autoimmune disease comprising systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, or type I diabetes.
在一些实施方案中，所述疾病为神经退行性疾病，其包含帕金森病、阿尔茨海默病、脊髓损伤、视网膜变性疾病、脑卒中、亨廷顿病或肌萎缩性侧索硬化症。In some embodiments, the disease is a neurodegenerative disease comprising Parkinson's disease, Alzheimer's disease, spinal cord injury, retinal degeneration, stroke, Huntington's disease, or amyotrophic lateral sclerosis.
在一些实施方案中，所述疾病为心血管疾病，其包含动脉粥样硬化、高血压、风湿性心脏病、心肌病、心律失常、先天性心脏病、瓣膜性心脏病、心脏炎、心肌梗塞、心力衰竭、主动脉瘤或外周动脉疾病。In some embodiments, the disease is a cardiovascular disease comprising atherosclerosis, hypertension, rheumatic heart disease, cardiomyopathy, arrhythmia, congenital heart disease, valvular heart disease, carditis, myocardial infarction, heart failure, aortic aneurysm, or peripheral arterial disease.
在一些实施方案中，所述疾病为代谢相关疾病，其包含II型糖尿病、坏血症、低血糖症、高血脂或骨质疏松。In some embodiments, the disease is a metabolism-related disease comprising type II diabetes, scurvy, hypoglycemia, hyperlipidemia, or osteoporosis.
有益效果Beneficial Effects
本发明的多能干细胞以及本发明的方法制备的低免疫原性多能干细胞可以呈现出优异的效果，例如但不限于：(1)具有良好的自我更新和分化能力；(2)能逃逸T细胞杀伤；(3)能逃逸NK细胞杀伤；和/或(4)所分化的细胞也能逃逸NK细胞的杀伤；从而表现出优异的应用潜力。The pluripotent stem cells of the present invention and the low-immunogenic pluripotent stem cells prepared by the method of the present invention can exhibit excellent effects, such as but not limited to: (1) having good self-renewal and differentiation abilities; (2) being able to escape T cell killing; (3) being able to escape NK cell killing; and/or (4) the differentiated cells can also escape NK cell killing; thereby showing excellent application potential.
实施例Example
通过参考以下实施例进一步描述本发明。应当理解，这些实施例仅作为示例，而不对本发明构成限制。以下材料和仪器均是可商购的或根据本领域公知的方法制备。以下实验均按照制造商的说明书或根据本领域公知的方法和步骤进行。The present invention is further described by reference to the following examples. It should be understood that these examples are intended to be illustrative only and are not intended to limit the present invention. The following materials and instruments are commercially available or prepared according to methods known in the art. The following experiments were performed according to the manufacturer's instructions or according to methods and procedures known in the art.
实施例1.构建B2M及CIITA双敲除细胞系(DKO)Example 1. Construction of B2M and CIITA double knockout cell line (DKO)
1.1构建B2M及CIITA双敲除细胞系(DKO)1.1 Construction of B2M and CIITA double knockout cell line (DKO)
以下实施例选用人多能干细胞系H1(Wicell，WA01)或H9(Wicell，WA09)进行目标细胞系的构建，所用的细胞培养和基因敲除试剂如表1所示。 In the following examples, human pluripotent stem cell lines H1 (Wicell, WA01) or H9 (Wicell, WA09) were used to construct target cell lines. The cell culture and gene knockout reagents used are shown in Table 1.
表1.细胞培养和基因敲除试剂
Table 1. Cell culture and gene knockout reagents
使用CRISPR/CAS9敲除内质网中β-2-微球蛋白(B2M)，使细胞表面MHC-I不能形成功能性分子，从而逃逸同种异体CD8+T细胞的杀伤；逃逸CD4+T细胞的杀伤则是通过敲除MHC-II基因转录的正调节因子CIITA，而降低MHC-II类分子表达。CRISPR/CAS9 is used to knock out β-2-microglobulin (B2M) in the endoplasmic reticulum, preventing the cell surface MHC-I from forming functional molecules, thereby escaping the killing of allogeneic CD8 + T cells; escaping the killing of CD4+ T cells is achieved by knocking out CIITA, a positive regulator of MHC-II gene transcription, thereby reducing the expression of MHC-II class molecules.
B2M的CRISPR/CAS9基因敲除策略以及所用gRNA序列和鉴定引物如图1和表2所示，使用B2M-gRNA1和B2M-gRNA2(EasyEdit sgRNA，金斯瑞)对B2M外显子区段进行两端直接敲除，然后分别使用B2M-F1/R1和B2M-F2/R2两对PCR引物进行基因组序列的敲除验证。The CRISPR/CAS9 gene knockout strategy of B2M, the gRNA sequences used, and the identification primers are shown in Figure 1 and Table 2. B2M-gRNA1 and B2M-gRNA2 (EasyEdit sgRNA, GenScript) were used to directly knock out the B2M exon segment at both ends, and then two pairs of PCR primers, B2M-F1/R1 and B2M-F2/R2, were used to verify the knockout of the genomic sequence.
另外，CIITA的CRISPR/CAS9基因敲除策略以及所用gRNA序列和鉴定引物如图2和表2所示，使用CIITA-gRNA1和CIITA-gRNA2(EasyEdit sgRNA，金斯瑞)对CIITA外显子区段进行两端直接敲除，然后分别使用CIITA-F1/R1和CIITA-F2/R2两对PCR引物进行基因组序列敲除验证。In addition, the CRISPR/CAS9 gene knockout strategy of CIITA, the gRNA sequences used, and the identification primers are shown in Figure 2 and Table 2. CIITA-gRNA1 and CIITA-gRNA2 (EasyEdit sgRNA, GenScript) were used to directly knock out the CIITA exon segment at both ends, and then the genome sequence knockout was verified using two pairs of PCR primers, CIITA-F1/R1 and CIITA-F2/R2, respectively.
表2.gRNA序列和鉴定引物
Table 2. gRNA sequences and identification primers
具体操作如下：The specific operations are as follows:
1)在Matrigel包被的6孔板上正常使用添加有Y-27632的mTeSR1培养基培养人多能干细胞至80％密度。使用TrypLE消化后加入DMEM/F12中和，计数。吸取2×106细胞于EP 管中，离心后弃上清。1) Human pluripotent stem cells were cultured in mTeSR1 medium supplemented with Y-27632 on a Matrigel-coated 6-well plate to 80% density. After digestion with TrypLE, the cells were neutralized with DMEM/F12 and counted. 2×10 6 cells were taken out and plated on EP After centrifugation, discard the supernatant.
2)根据Neon转染系统(ThermoFisher)100μL电转体系，加入15μg TrueCutTM Cas9Protein+3μg gRNA(B2M-gRNA1+B2M-gRNA2+CIITA-gRNA1+CIITA-gRNA2)组成核糖核蛋白复合物(RNP)体系，混匀后在室温下放置20min。2) According to the Neon transfection system (ThermoFisher) 100 μL electroporation system, 15 μg TrueCut TM Cas9 Protein + 3 μg gRNA (B2M-gRNA1 + B2M-gRNA2 + CIITA-gRNA1 + CIITA-gRNA2) was added to form a ribonucleoprotein complex (RNP) system, mixed and placed at room temperature for 20 minutes.
3)用100μL RNP电转体系重悬细胞，并用Neon转染系统进行电转，电转参数为1200V，30ms，1pause。迅速添加提前预热的培养基至电转后细胞，再均匀接种于1孔包被了Matrigel的6孔板中。3) Resuspend the cells with 100 μL RNP electroporation system and electroporate using Neon transfection system with electroporation parameters of 1200 V, 30 ms, 1 pause. Quickly add preheated culture medium to the electroporated cells and then evenly inoculate in one well of a 6-well plate coated with Matrigel.
4)每天更换新鲜mTeSR1培养基。待单细胞生长起来，挑取单个克隆于48孔板内，待克隆扩增后，收取基因组样品进行PCR检测基因编辑情况，PCR结果如图1和2所示。PCR阳性克隆送公司做Sanger测序进一步验证。4) Replace the mTeSR1 medium with fresh medium every day. When the single cell grows up, pick a single clone in a 48-well plate. After the clone is amplified, collect the genome sample for PCR detection of gene editing. The PCR results are shown in Figures 1 and 2. The PCR positive clones are sent to the company for Sanger sequencing for further verification.
5)扩增培养及冻存鉴定阳性的B2M/CIITA双等位基因敲除的克隆DKO。5) Expand, culture and freeze the positive B2M/CIITA biallelic knockout clones.
1.2检测DKO的B2M和CIITA的RNA水平的表达情况1.2 Detection of RNA expression of B2M and CIITA in DKO
使用FastPure Cell/Tissue Total RNA Isolation Kit V2(诺唯赞，RC112-01)提取细胞总RNA，然后根据制造商说明使用HiScript III RT SuperMix for qPCR(诺唯赞，R323-01)将RNA反转为cDNA。Total cellular RNA was extracted using FastPure Cell/Tissue Total RNA Isolation Kit V2 (Novagen, RC112-01), and then RNA was converted to cDNA using HiScript III RT SuperMix for qPCR (Novagen, R323-01) according to the manufacturer's instructions.
使用qPCR检测B2M/CIITA双等位基因敲除的克隆DKO的B2M和CIITA在RNA水平的表达情况，所用引物如下所示。qPCR was used to detect the expression of B2M and CIITA at the RNA level in the B2M/CIITA biallelic knockout clone DKO. The primers used are shown below.
B2M-F:AAGATGAGTATGCCTGCCGT(SEQ ID NO:23)B2M-F:AAGATGAGTATGCCTGCCGT (SEQ ID NO:23)
B2M-R:ATGCGGCATCTTCAAACCTC(SEQ ID NO:24)B2M-R:ATGCGGCATCTTCAAACCTC (SEQ ID NO:24)
CIITA-F:CCTGGAGCTTCTTAACAGCGA(SEQ ID NO:25)CIITA-F:CCTGGAGCTTCTTAACAGCGA (SEQ ID NO:25)
CIITA-R:TGTGTCGGGTTCTGAGTAGAG(SEQ ID NO:26)CIITA-R:TGTGTCGGGTTCTGAGTAGAG (SEQ ID NO:26)
使用Roche 480II仪器，反应体系如下：Using the Roche 480II instrument, the reaction system is as follows:
Pre-incubation，95℃，30s。Pre-incubation, 95℃, 30s.
Amplification，95℃ 10s，60℃ 30s，40个循环数。Amplification, 95℃ 10s, 60℃ 30s, 40 cycles.
Melting curve和Cooling为默认程序。Melting curve and Cooling are the default programs.
qPCR结果如图3所示，相对于未经处理的野生型人多能干细胞，B2M/CIITA双等位基因敲除的克隆DKO的B2M和CIITA在RNA水平的表达近乎没有，确定成功敲除B2M和CIITA。The qPCR results are shown in Figure 3. Compared with untreated wild-type human pluripotent stem cells, the expression of B2M and CIITA at the RNA level in the B2M/CIITA biallelic knockout clone DKO was almost absent, confirming the successful knockout of B2M and CIITA.
1.3检测DKO的B2M的蛋白水平的表达情况1.3 Detection of protein expression of B2M in DKO
使用蛋白印迹(Western-Blot)检测B2M/CIITA双等位基因敲除的克隆DKO的B2M蛋白水平的表达情况(B2M抗体的货号为ab75853；内参抗体GAPDH的货号为ab181602，都购自Abcam)。Western blot was used to detect the expression level of B2M protein in the B2M/CIITA biallelic knockout clone DKO (the B2M antibody is ab75853; the internal reference antibody GAPDH is ab181602, both purchased from Abcam).
蛋白印迹结果如图4所示，相对于野生型人多能干细胞，DKO中的B2M蛋白显著下降，确定成功敲除B2M蛋白。The results of western blot are shown in Figure 4. Compared with wild-type human pluripotent stem cells, the B2M protein in DKO was significantly decreased, confirming that the B2M protein was successfully knocked out.
1.4检测DKO的HLA-I/II类分子1.4 Detection of HLA-I/II molecules in DKO
使用IFN-γ(PeproTech,Cat#300-02)刺激WT及DKO以检测H1细胞表面的HLA I/II型分子，其具体检测方法如下：IFN-γ (PeproTech, Cat#300-02) was used to stimulate WT and DKO to detect HLA class I/II molecules on the surface of H1 cells. The specific detection method is as follows:
细胞铺板，第二天换液时将含有IFN-γ的培养基加入到细胞中，作用48h后将细胞消化后使用流式细胞仪(Agilent Technologies，NovoCyte)检测HLA-I/II的表达情况。 The cells were plated, and the medium containing IFN-γ was added to the cells when the medium was changed on the next day. After 48 hours of action, the cells were digested and the expression of HLA-I/II was detected using a flow cytometer (Agilent Technologies, NovoCyte).
流式检测结果如图5所示，其中左侧HLA-ABC检测HLA-I类分子；右侧HLA-DR，DQ，DP检测HLA-II类分子；T细胞为检测的阳性对照。观察到B2M/CIITA双等位基因敲除的克隆DKO不能响应IFN-γ的刺激而表达HLA-I/II类分子，证明DKO HLA-I和HLA-II功能降低。The flow cytometry results are shown in Figure 5, where HLA-ABC on the left detects HLA-I molecules; HLA-DR, DQ, and DP on the right detect HLA-II molecules; T cells are the positive control for the detection. It was observed that the clone DKO with B2M/CIITA biallelic knockout could not express HLA-I/II molecules in response to IFN-γ stimulation, proving that the functions of DKO HLA-I and HLA-II were reduced.
1.5 DKO的核型检测1.5 Karyotype detection of DKO
对获得的B2M/CIITA双等位基因敲除的阳性克隆(DKO)进行核型检测，其具体检测方法如下：The obtained B2M/CIITA biallelic knockout positive clones (DKO) were subjected to karyotype detection, and the specific detection method is as follows:
用胰蛋白酶处理固定于载玻片上的染色体标本，再用Giemsa染液染色。根据染色体的长度、着丝点位置、长短臂比例、随体的有无等特征，将分裂中期染色体进行染色体数目和形态结构的分析，以确定其核型是否与正常核型一致。The chromosome specimen fixed on the slide is treated with trypsin and then stained with Giemsa stain. Based on the chromosome length, centromere position, long-short arm ratio, satellite presence and other characteristics, the chromosome number and morphological structure of the metaphase chromosome are analyzed to determine whether its karyotype is consistent with the normal karyotype.
核型检测结果如图6所示，DKO核型正常，与正常核型相比无明显变化。The results of karyotype detection are shown in Figure 6 . The DKO karyotype is normal, with no significant changes compared with the normal karyotype.
实施例2.DKO细胞系的干性和免疫功能Example 2. Stemness and immune function of DKO cell lines
本实施例进一步检测在敲除B2M/CIITA双等位基因后，多能干细胞的干性和免疫功能是否发生变化。This example further tests whether the stemness and immune function of pluripotent stem cells change after knocking out the B2M/CIITA biallelic genes.
2.1 DKO细胞中干性基因的表达2.1 Expression of stemness genes in DKO cells
通过免疫荧光检测WT和DKO细胞中干性基因POU5F1和NANOG的蛋白水平，通过RT-qPCR检测WT和DKO细胞中干性基因POU5F1、NANOG和SOX2在RNA水平上的表达情况，以及通过流式细胞术检测WT和DKO细胞表面干性基因SSEA-4和Tra1-81的表达情况，其具体检测方法如下：The protein levels of stemness genes POU5F1 and NANOG in WT and DKO cells were detected by immunofluorescence, the expression of stemness genes POU5F1, NANOG and SOX2 in WT and DKO cells at the RNA level was detected by RT-qPCR, and the expression of stemness genes SSEA-4 and Tra1-81 on the surface of WT and DKO cells was detected by flow cytometry. The specific detection methods are as follows:
免疫荧光检测：将WT或DKO细胞铺板在12孔板中，待细胞长到60-80％的密度后吸弃培养基，加入4％多聚甲醛进行固定。细胞破膜后使用POU5F1和NANOG的一抗在4℃过夜孵育，洗去一抗后室温孵育带有荧光标记的二抗，随后使用荧光显微镜(Nikon Ts2R-FL)进行拍照。Immunofluorescence detection: WT or DKO cells were plated in 12-well plates. After the cells grew to a density of 60-80%, the medium was aspirated and fixed with 4% paraformaldehyde. After the cells were permeabilized, the primary antibodies of POU5F1 and NANOG were used for overnight incubation at 4°C. After the primary antibodies were washed off, the secondary antibodies with fluorescent labels were incubated at room temperature, and then photographed using a fluorescence microscope (Nikon Ts2R-FL).
RT-qPCR检测：RT-qPCR detection:
使用Roche 480II仪器，反应体系如下：Using the Roche 480II instrument, the reaction system is as follows:
Pre-incubation，95℃，30s。Pre-incubation, 95℃, 30s.
Amplification，95℃ 10s，60℃ 30s，40个循环数。Amplification, 95℃ 10s, 60℃ 30s, 40 cycles.
Melting curve和Cooling为默认程序。Melting curve and Cooling are the default programs.
其中间充质干细胞(MSC)为干性基因表达的阴性对照。Mesenchymal stem cells (MSC) were used as a negative control for stemness gene expression.
流式细胞术：Flow Cytometry:
收下细胞后在EP管中4℃避光孵育抗体30min，然后根据抗体信息选择合适的荧光采集通道，上机检测荧光。所有抗体均来自BD Biosciences。After collecting the cells, incubate the antibodies in an EP tube at 4°C in the dark for 30 minutes, then select the appropriate fluorescence acquisition channel based on the antibody information and detect the fluorescence on the machine. All antibodies are from BD Biosciences.
免疫荧光检测结果如图7A所示，WT和DKO细胞在蛋白水平都表达干性基因POU5F1和NANOG，并且DKO细胞中的干性基因POU5F1和NANOG的表达与WT细胞相比无明显差异。RT-qPCR的检测结果如图7B所示，WT和DKO细胞在RNA水平表达干性基因POU5F1、NANOG和SOX2，并且DKO细胞中的干性基因POU5F1、NANOG和SOX2的表达与WT细胞相比无明显差异。流式细胞术检测结果如图7C所示，WT和DKO细胞表面均高表达干性基因SSEA-4(WT 100％与DKO 99.98％)和Tra1-81(WT 96.75％与DKO  99.13％)。The results of immunofluorescence detection are shown in Figure 7A. Both WT and DKO cells express stemness genes POU5F1 and NANOG at the protein level, and there is no significant difference in the expression of stemness genes POU5F1 and NANOG in DKO cells compared with WT cells. The results of RT-qPCR detection are shown in Figure 7B. WT and DKO cells express stemness genes POU5F1, NANOG and SOX2 at the RNA level, and there is no significant difference in the expression of stemness genes POU5F1, NANOG and SOX2 in DKO cells compared with WT cells. The results of flow cytometry detection are shown in Figure 7C. Both WT and DKO cells highly express stemness genes SSEA-4 (WT 100% and DKO 99.98%) and Tra1-81 (WT 96.75% and DKO 99.13%).
2.2 DKO细胞的分化能力2.2 Differentiation capacity of DKO cells
本实施例检测DKO细胞的分化能力。具体检测方法如下：在免疫缺陷小鼠(SCID Beige，维通利华)皮下注射100μL含5x105DKO细胞的悬液，待畸胎瘤体积大于1.5cm3后取出，并进行石蜡切片和苏木精伊红染色。In this example, the differentiation ability of DKO cells was detected. The specific detection method is as follows: 100 μL of a suspension containing 5×10 5 DKO cells was subcutaneously injected into immunodeficient mice (SCID Beige, Vital River), and the teratomas were removed when the volume was greater than 1.5 cm 3 , and paraffin sections and hematoxylin and eosin staining were performed.
染色结果如图8所示，B2M/CIITA双等位基因敲除的DKO细胞可以在体内形成畸胎瘤并分化出内中外三胚层的细胞，DKO细胞具有正常的三胚层分化能力。The staining results are shown in Figure 8 , and the DKO cells with B2M/CIITA biallelic knockout can form teratomas in vivo and differentiate into cells of the inner, middle and outer germ layers, and the DKO cells have normal differentiation ability of the three germ layers.
2.3 DKO细胞的免疫功能2.3 Immune function of DKO cells
通过使用xCELLigence RTCA Instrument进行T细胞及NK细胞的杀伤性实验以检测DKO细胞免疫功能的变化。杀伤性实验所用的试剂如表3所示。The xCELLigence RTCA Instrument was used to perform T cell and NK cell cytotoxicity experiments to detect changes in the immune function of DKO cells. The reagents used in the cytotoxicity experiments are shown in Table 3.
使用含有人IL-2的Essential 8培养基重悬相同量的WT和DKO细胞系，并接种于用基质胶包被的96孔E-plates，加入活化的T细胞(XC11228，购自SAILYBIO)或NK细胞(XC11013，购自SAILYBIO)进行杀伤检测。T细胞在使用前将进行CD3、CD4和CD8流式检测，NK细胞在使用前将进行CD16和CD56流式检测，确保使用的T细胞及NK细胞的功能。RTCA检测数据使用xCELLigence软件进行分析，计算杀伤率和逃逸功能。The same amount of WT and DKO cell lines were resuspended in Essential 8 medium containing human IL-2 and inoculated into 96-well E-plates coated with matrix gel, and activated T cells (XC11228, purchased from SAILYBIO) or NK cells (XC11013, purchased from SAILYBIO) were added for killing detection. T cells will be subjected to CD3, CD4 and CD8 flow cytometry before use, and NK cells will be subjected to CD16 and CD56 flow cytometry before use to ensure the function of the T cells and NK cells used. RTCA detection data were analyzed using xCELLigence software to calculate the killing rate and escape function.
表3.杀伤性实验所用试剂
Table 3. Reagents used in the killing experiment
RTCA结果如图9所示，WT细胞因为有HLA-I的表达而逃逸NK细胞的杀伤，但会被T细胞杀伤。DKO细胞能逃逸T细胞杀伤，同时对NK细胞的杀伤更为敏感。The RTCA results are shown in Figure 9. WT cells escape NK cell killing due to the expression of HLA-I, but are killed by T cells. DKO cells can escape T cell killing and are more sensitive to NK cell killing.
实施例3.DKO+CD47细胞系的构建及免疫功能验证Example 3. Construction of DKO+CD47 cell line and verification of immune function
3.1 DKO+CD47细胞系的构建3.1 Construction of DKO+CD47 cell line
本实施例使用慢病毒载体在实施例1获得的DKO细胞中过表达CD47(NM_198793)，所述CD47的氨基酸序列如SEQ ID NO:5所示。In this example, a lentiviral vector is used to overexpress CD47 (NM_198793) in the DKO cells obtained in Example 1. The amino acid sequence of CD47 is shown in SEQ ID NO:5.
将编码CD47蛋白的核酸序列(SEQ ID NO:6)构建在由EF1a启动，且带嘌呤霉素筛选标记的慢病毒载体中(pGC-EF1a)，pGC-EF1a载体的结构如图10所示。具体操作方法如下：The nucleic acid sequence encoding CD47 protein (SEQ ID NO: 6) was constructed in a lentiviral vector (pGC-EF1a) initiated by EF1a and carrying a puromycin selection marker. The structure of the pGC-EF1a vector is shown in Figure 10. The specific operation method is as follows:
慢病毒载体用BamHI/NheI酶切，并将编码CD47蛋白的核酸序列(SEQ ID NO:6)连接 至慢病毒载体，连接成功后采用Sanger测序验证插入序列正确性并进行病毒包装。转染慢病毒载体至实施例1中构建的DKO细胞，24h后换液，48h后换成带嘌呤霉素的培养基进行筛选。The lentiviral vector was digested with BamHI/NheI, and the nucleic acid sequence encoding CD47 protein (SEQ ID NO: 6) was ligated to After successful connection, Sanger sequencing was used to verify the correctness of the inserted sequence and virus packaging was performed. The lentiviral vector was transfected into the DKO cells constructed in Example 1, and the medium was changed after 24 hours, and after 48 hours, the medium was changed to a medium containing puromycin for screening.
对构建的稳转株细胞DKO+CD47的进行流式细胞检测(CD47抗体购自FACS:Biolegend，货号：323108)和qPCR检测。qPCR引物为CD47-F：AGAAGGTGAAACGATCATCGAGC(SEQ ID NO:36)；CD47-R：CTCATCCATACCACCGGATCT(SEQ ID NO:37)。The constructed stable cell line DKO+CD47 was subjected to flow cytometry (CD47 antibody was purchased from FACS: Biolegend, catalog number: 323108) and qPCR detection. The qPCR primers were CD47-F: AGAAGGTGAAACGATCATCGAGC (SEQ ID NO: 36); CD47-R: CTCATCCATACCACCGGATCT (SEQ ID NO: 37).
检测结果如图11A和11B所示，构建的DKO+CD47细胞系中，CD47的表达水平显著高于WT细胞。并对验证后的DKO+CD47细胞系进行细胞扩增和后续功能性检测。The test results are shown in Figures 11A and 11B. The expression level of CD47 in the constructed DKO+CD47 cell line was significantly higher than that in the WT cell. The validated DKO+CD47 cell line was then subjected to cell expansion and subsequent functional testing.
3.2 DKO+CD47的免疫功能的验证3.2 Verification of the immune function of DKO+CD47
使用RTCA检测过表达的DKO+CD47细胞株是否可以在逃逸T细胞杀伤的同时成功逃逸NK细胞的杀伤，具体检测方法参见实施例2.3。RTCA was used to detect whether the overexpressed DKO+CD47 cell line could successfully escape the killing of NK cells while escaping the killing of T cells. For the specific detection method, see Example 2.3.
RTCA检测如图12所示，NK细胞可有效杀伤DKO细胞，而WT和DKO+CD47过表达细胞可逃逸NK杀伤。RTCA assay As shown in Figure 12 , NK cells can effectively kill DKO cells, while WT and DKO+CD47 overexpressing cells can escape NK killing.
实施例4.DKO+GSN细胞系的构建及干性和分化能力检测Example 4. Construction of DKO+GSN cell line and detection of stemness and differentiation ability
4.1 DKO+GSN细胞系的构建及过表达检测4.1 Construction of DKO+GSN cell line and overexpression detection
直接合成编码GSN蛋白(GSN蛋白的氨基酸序列如SEQ ID NO:3所示)的核酸序列。核酸序列如SEQ ID NO:4所示。The nucleic acid sequence encoding the GSN protein (the amino acid sequence of the GSN protein is shown in SEQ ID NO:3) was directly synthesized. The nucleic acid sequence is shown in SEQ ID NO:4.
如实施例3.1所述，构建由EF1a启动，且带嘌呤霉素筛选标记的慢病毒载体(pGC-EF1a)中，pGC-EF1a载体的结构如图10所示。将载体用BamHI/NheI酶切，并将上述合成的GSN的核酸序列连接至慢病毒载体中。连接成功后采用Sanger测序验证插入序列正确性并进行病毒包装。转染慢病毒载体至实施例1中构建的DKO细胞，24h后换液，48h后换成带嘌呤霉素的培养基进行筛选。As described in Example 3.1, a lentiviral vector (pGC-EF1a) initiated by EF1a and carrying a puromycin selection marker was constructed. The structure of the pGC-EF1a vector is shown in Figure 10. The vector was digested with BamHI/NheI, and the nucleic acid sequence of the synthesized GSN was connected to the lentiviral vector. After successful connection, Sanger sequencing was used to verify the correctness of the inserted sequence and perform viral packaging. The lentiviral vector was transfected into the DKO cells constructed in Example 1, and the medium was changed after 24 hours, and the medium with puromycin was changed after 48 hours for screening.
对构建的DKO+GSN细胞系使用qPCR检测mRNA的过表达水平，RT-PCR检测mRNA的过表达水平，WT细胞为阴性对照，所用引物如表4所示。The constructed DKO+GSN cell line was tested for mRNA overexpression level using qPCR and RT-PCR, and WT cells were used as negative control. The primers used are shown in Table 4.
表4.GSN的qPCR检测引物
Table 4. qPCR detection primers for GSN
检测结果如图13所示，构建的DKO+GSN细胞系中有GSN的高表达。The detection results are shown in FIG13 , and GSN is highly expressed in the constructed DKO+GSN cell line.
4.2 DKO+GSN细胞系中干性基因的表达4.2 Expression of stemness genes in DKO+GSN cell lines
通过免疫荧光和流式细胞术检测DKO+GSN细胞系中干性基因的表达，具体检测方法参见实施例2.1。The expression of stemness genes in the DKO+GSN cell line was detected by immunofluorescence and flow cytometry. For specific detection methods, see Example 2.1.
免疫荧光检测结果如图14所示，构建的DKO+GSN细胞在蛋白水平表达干性基因OCT4、NANOG、SOX2、TRA-1-60、TRA-1-81。The results of immunofluorescence detection are shown in Figure 14. The constructed DKO+GSN cells expressed stemness genes OCT4, NANOG, SOX2, TRA-1-60, and TRA-1-81 at the protein level.
流式细胞术结果如图15所示，DKO+GSN细胞表面高表达干性基因SSEA-4、TRA-1-60、Tra1-81和OCT4，各干性基因占比分别为98.89％、98.73％、95.87％和98.92％。 The flow cytometry results are shown in Figure 15. The stemness genes SSEA-4, TRA-1-60, Tra1-81 and OCT4 were highly expressed on the surface of DKO+GSN cells, and the proportions of each stemness gene were 98.89%, 98.73%, 95.87% and 98.92%, respectively.
4.3 DKO+GSN细胞系的三胚层分化能力4.3 Tri-germ layer differentiation ability of DKO+GSN cell line
使用DKO+GSN细胞进行三胚层分化能力检测。为了生成中、内、外胚层细胞，需要分别使用添加有Y27632的三个胚层培养基对解离的DKO+GSN单细胞进行重悬，并将适量细胞附着于基质胶包被的带有细胞爬片的孔板中。24h后，更换为预热的分化培养基，每天换液至第七天可得到中、内、外三个胚层细胞。通过免疫荧光检测三胚层标志蛋白的表达情况以检测DKO+GSN细胞系的三胚层分化能力。DKO+GSN cells were used to detect the ability of three germ layers to differentiate. In order to generate mesoderm, endoderm, and ectoderm cells, the dissociated DKO+GSN single cells were resuspended in three germ layer culture media supplemented with Y27632, and an appropriate amount of cells were attached to a well plate with a cell crawler coated with matrix gel. After 24 hours, the preheated differentiation medium was replaced, and the medium was changed every day until the seventh day to obtain mesoderm, endoderm, and ectoderm cells. The expression of three germ layer marker proteins was detected by immunofluorescence to detect the three germ layer differentiation ability of the DKO+GSN cell line.
免疫荧光检测结果如图16所示，DKO+GSN细胞在蛋白水平表达外胚层标志蛋白：PAX6和GAD1；中胚层标志蛋白：Brachyury和NCAM；内胚层标志蛋白：SOX17和FOXA2。The results of immunofluorescence detection are shown in Figure 16. DKO+GSN cells express ectoderm marker proteins: PAX6 and GAD1; mesoderm marker proteins: Brachyury and NCAM; endoderm marker proteins: SOX17 and FOXA2 at the protein level.
4.4 DKO+GSN细胞系的畸胎瘤形成能力4.4 Teratoma-forming ability of DKO+GSN cell line
本实施例检测DKO+GSN细胞的分化能力。具体检测方法如下：向免疫缺陷小鼠(SCID Beige)皮下注射100μL含5x105DKO+GSN细胞的悬液，待畸胎瘤体积大于1.5cm3后取出，并进行石蜡切片和苏木精伊红染色。This example detects the differentiation ability of DKO+GSN cells. The specific detection method is as follows: 100 μL of a suspension containing 5×10 5 DKO+GSN cells is subcutaneously injected into immunodeficient mice (SCID Beige), and the teratomas are removed when the volume is greater than 1.5 cm 3 , and paraffin sections are made and stained with hematoxylin and eosin.
图17示出畸胎瘤形成能力检测结果，可以看到DKO+GSN细胞在体内形成畸胎瘤并分化出内中外三胚层的细胞。FIG. 17 shows the results of the teratoma formation ability test, and it can be seen that DKO+GSN cells form teratomas in vivo and differentiate into cells of the three germ layers: endogenous, mesodermal and ectogenetic.
实施例5 DKO+GSN细胞系的免疫逃逸功能Example 5 Immune escape function of DKO+GSN cell line
为验证DKO+GSN细胞对不同免疫细胞的逃逸功能，使用NK细胞以及T细胞+NK细胞混合进行实验。To verify the escape function of DKO+GSN cells against different immune cells, experiments were conducted using NK cells and a mixture of T cells + NK cells.
5.1使用RTCA检测DKO+GSN细胞的免疫逃逸功能5.1 Detection of immune escape function of DKO+GSN cells using RTCA
通过RTCA检测DKO+GSN细胞对不同免疫细胞的逃逸功能。具体检测方法参见实施例2.3。所用的PBNK细胞由PBMC(外周血单个核细胞，来自SAILYBIO)在体外培养过程中添加IL-2增加NK细胞占比后获得。检测结果如图18A-图18D所示，在RTCA检测的NK细胞杀伤性实验中，实施例1构建的DKO细胞被NK细胞完全杀伤，而WT细胞以及DKO+GSN细胞成功逃逸(图18A和图18B)。在RTCA检测的T细胞+NK细胞的混合细胞(PBNK)杀伤性实验中只有DKO+GSN细胞成功逃逸，WT细胞和DKO细胞均被不同程度杀伤，其中DKO细胞被完全杀伤(图18C和图18D)。其中图18B和18D为多次杀伤统计图。The escape function of DKO+GSN cells on different immune cells was detected by RTCA. See Example 2.3 for specific detection methods. The PBNK cells used were obtained by adding IL-2 to PBMC (peripheral blood mononuclear cells, from SAILYBIO) during in vitro culture to increase the proportion of NK cells. The test results are shown in Figures 18A-18D. In the NK cell killing experiment detected by RTCA, the DKO cells constructed in Example 1 were completely killed by NK cells, while WT cells and DKO+GSN cells successfully escaped (Figures 18A and 18B). In the mixed cell (PBNK) killing experiment of T cells + NK cells detected by RTCA, only DKO+GSN cells successfully escaped, and WT cells and DKO cells were killed to varying degrees, among which DKO cells were completely killed (Figures 18C and 18D). Figures 18B and 18D are multiple killing statistics.
5.2使用Elispot检测NK细胞IFN-γ斑点分泌5.2 Detection of NK cell IFN-γ spot secretion using Elispot
通过Elispot检测NK细胞IFN-γ斑点分泌以测定DKO+GSN细胞的免疫逃逸功能。具体操作方法如下：Elispot was used to detect the secretion of IFN-γ spots by NK cells to determine the immune escape function of DKO+GSN cells. The specific operation method is as follows:
将WT细胞、DKO细胞和DKO+GSN细胞按照特定密度铺于6孔板，24h后弃除培养基并加入特定数量的NK细胞进行培养。24h后收集NK细胞进行后续IFN-γ分泌检测，同时观察去除NK细胞后剩余的WT细胞、DKO细胞和DKO+GSN细胞，结果如图19所示，相较DKO细胞，WT细胞以及DKO+GSN细胞被NK细胞杀伤的细胞更少，说明DKO+GSN细胞可逃逸NK细胞杀伤的能力显著高于DKO细胞。WT cells, DKO cells and DKO+GSN cells were plated in 6-well plates at a specific density. After 24 hours, the culture medium was discarded and a specific number of NK cells were added for culture. After 24 hours, NK cells were collected for subsequent IFN-γ secretion detection, and the remaining WT cells, DKO cells and DKO+GSN cells after the removal of NK cells were observed. The results are shown in Figure 19. Compared with DKO cells, fewer WT cells and DKO+GSN cells were killed by NK cells, indicating that the ability of DKO+GSN cells to escape NK cell killing is significantly higher than that of DKO cells.
将24h后收集的NK细胞在包被有IFN-γ抗体的96孔板中铺板，并放置于37℃培养箱孵育24h；加入亲和抗体和链霉亲和素孵后进行IFN-γ分泌斑点的显色检测。The NK cells collected after 24 hours were plated in a 96-well plate coated with IFN-γ antibody and incubated in a 37°C incubator for 24 hours; affinity antibody and streptavidin were added and incubated for color detection of IFN-γ secretion spots.
Elispot检测结果如图20A和图20B所示，WT细胞以及DKO+GSN细胞与NK细胞共培养后刺激NK细胞分泌的IFN-γ所形成的斑点数相似，都显著低于DKO细胞，说明过表达GSN可抵消B2M/CIITA敲除带来的NK细胞活化。 The results of Elispot detection are shown in Figures 20A and 20B. The number of spots formed by IFN-γ secreted by NK cells stimulated by co-culture of WT cells and DKO+GSN cells with NK cells is similar, and is significantly lower than that of DKO cells, indicating that overexpression of GSN can offset the NK cell activation caused by B2M/CIITA knockout.
5.3使用FACS检测NK细胞活性指标5.3 Detection of NK cell activity indicators using FACS
将WT细胞、DKO细胞、DKO+GSN细胞按照特定密度铺于6孔板，24h后弃除培养基并加入特定数量的NK细胞进行共培养。24h后收集NK细胞，利用FACS检测其细胞表面激活指标CD107a(Biolegend，328620)。WT cells, DKO cells, and DKO+GSN cells were plated in 6-well plates at a specific density. After 24 hours, the culture medium was discarded and a specific number of NK cells were added for co-culture. After 24 hours, NK cells were collected and their cell surface activation indicator CD107a (Biolegend, 328620) was detected by FACS.
FACS检测结果如图21所示，DKO+GSN细胞与DKO细胞相比，均可降低NK细胞的活化。The FACS detection results are shown in Figure 21. Compared with DKO cells, DKO+GSN cells can reduce the activation of NK cells.
实施例6.DKO+GSN细胞的分化细胞的免疫逃逸验证Example 6. Verification of immune escape of differentiated cells of DKO+GSN cells
将细胞铺在基质胶上，当汇和度达到40％时更换为分化培养基并每天换液。长满后传代到0.1％明胶包被的培养皿上，每3天进行一次传代操作，期间仍需每天换液，分化周期为10天。The cells were plated on matrigel and replaced with differentiation medium when the confluence reached 40% and the medium was changed every day. After confluence, the cells were subcultured on 0.1% gelatin-coated culture dishes and subcultured every 3 days. The medium was changed every day during the differentiation period, and the differentiation cycle was 10 days.
通过RTCA检测DKO+GSN细胞的分化细胞对NK细胞的逃逸功能。具体检测方法参见实施例2.3。The NK cell escape function of differentiated cells of DKO+GSN cells was detected by RTCA. The specific detection method is shown in Example 2.3.
结果如图22A和图22B所示，其中图22B为多次杀伤统计图。DKO细胞分化后依旧被NK细胞完全杀伤，而WT细胞的分化细胞和DKO+GSN细胞的分化细胞成功逃逸NK细胞的杀伤。The results are shown in Figures 22A and 22B, where Figure 22B is a multiple killing statistical graph. After differentiation, DKO cells were still completely killed by NK cells, while differentiated cells of WT cells and differentiated cells of DKO+GSN cells successfully escaped the killing of NK cells.
实施例7.NHP iPSC-DKO细胞的构建及验证Example 7. Construction and verification of NHP iPSC-DKO cells
7.1 NHP iPSC-DKO细胞的构建7.1 Construction of NHP iPSC-DKO cells
根据实施例1所描述的方法产生猴iPSC-DKO细胞(即，非人灵长类(NHP)iPSC-DKO细胞)。具体地，采集食蟹猴成体细胞(采集自5-10岁雄性食蟹猴)，通过CTSTM CytoTuneTM-iPS 2.1仙台病毒重编程试剂盒(货号:A34546)制备得到猴iPSC细胞。使用NHP-B2M-gRNA1和NHP-B2M-gRNA2对B2M外显子区段进行两端直接敲除，然后使用NHP-B2M-F/R一对PCR引物进行基因组序列敲除验证(gRNA序列和鉴定引物序列如表5所示)。并且，使用NHP-CIITA-gRNA1和NHP-CIITA-gRNA2对CIITA外显子区段进行两端直接敲除，通过NHP-CIITA-F/R一对PCR引物进行基因组PCR验证基因组序列敲除(gRNA序列和鉴定引物序列如表5所示)。选取两个单克隆NHP iPSC-DKO(即，下文的NHP iPSC-DKO1和NHP iPSC-DKO2细胞)进行后续实验。Monkey iPSC-DKO cells (i.e., non-human primate (NHP) iPSC-DKO cells) were generated according to the method described in Example 1. Specifically, adult cynomolgus monkey cells (collected from 5-10 year old male cynomolgus monkeys) were collected and monkey iPSC cells were prepared by CTS ™ CytoTune ™ -iPS 2.1 Sendai virus reprogramming kit (Cat. No.: A34546). NHP-B2M-gRNA1 and NHP-B2M-gRNA2 were used to directly knock out the B2M exon segment at both ends, and then a pair of PCR primers NHP-B2M-F/R were used to verify the genome sequence knockout (gRNA sequence and identification primer sequence are shown in Table 5). In addition, NHP-CIITA-gRNA1 and NHP-CIITA-gRNA2 were used to directly knock out the CIITA exon segment at both ends, and the genome sequence knockout was verified by genomic PCR using a pair of PCR primers NHP-CIITA-F/R (gRNA sequence and identification primer sequence are shown in Table 5). Two monoclonal NHP iPSC-DKO (ie, NHP iPSC-DKO1 and NHP iPSC-DKO2 cells below) were selected for subsequent experiments.
表5.gRNA序列和鉴定引物序列

Table 5. gRNA sequences and identification primer sequences

7.2检测NHP iPSC-DKO的HLA-I/II分子表达7.2 Detection of HLA-I/II molecule expression in NHP iPSC-DKO
使用IFN-γ(PeproTech,Cat#300-02)刺激NHP iPSC-WT及NHP iPSC-DKO1和NHP iPSC-DKO2细胞以检测细胞表面的HLA I/II型分子，其具体检测方法如下：IFN-γ (PeproTech, Cat#300-02) was used to stimulate NHP iPSC-WT, NHP iPSC-DKO1 and NHP iPSC-DKO2 cells to detect HLA class I/II molecules on the cell surface. The specific detection method is as follows:
细胞铺板，第二天换液时将含有IFN-γ的培养基加入到细胞中。待48h后将细胞消化，使用流式细胞仪(Agilent Technologies，NovoCyte)检测HLA-I/II的表达情况。The cells were plated and the medium containing IFN-γ was added to the cells when the medium was changed the next day. After 48 hours, the cells were digested and the expression of HLA-I/II was detected using a flow cytometer (Agilent Technologies, NovoCyte).
流式检测结果如图23所示，B2M/CIITA双等位基因敲除的NHP iPSC-DKO1和NHP iPSC-DKO2细胞不表达HLA-I/II，并且不能响应IFN-γ的刺激而上调HLA-I/II。The flow cytometry results are shown in Figure 23. B2M/CIITA biallelic knockout NHP iPSC-DKO1 and NHP iPSC-DKO2 cells do not express HLA-I/II and cannot upregulate HLA-I/II in response to IFN-γ stimulation.
7.3检测NHP iPSC-DKO的免疫逃逸功能7.3 Detection of immune escape function of NHP iPSC-DKO
在xCELLigence平台(ACEA BioSciences)上进行T细胞杀伤测定以检测NHP iPSC-DKO细胞免疫功能的变化。具体检测方法和结果如下所述。T cell killing assay was performed on the xCELLigence platform (ACEA BioSciences) to detect changes in the immune function of NHP iPSC-DKO cells. The specific detection methods and results are described as follows.
将NHP iPSC-WT和NHP iPSC-DKO细胞重悬于100μl细胞培养基中，并铺于包被有基质胶(Matrigel，Sigma‐Aldrich)的96孔E‐板(ACEA BioSciences)上。在细胞指数(体现细胞数量)达到1之后，以2:1的E:T比(效应物:靶标比)添加T细胞(分离自采集的食蟹猴的血液)。用RTCA软件(ACEA)对数据进行标准化和分析。结果如图24所示，NHP iPSC-DKO细胞明显逃逸T细胞的杀伤，而NHP iPSC-WT细胞会被T细胞杀伤。NHP iPSC-WT and NHP iPSC-DKO cells were resuspended in 100 μl of cell culture medium and plated on 96-well E-plates (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index (reflecting the number of cells) reached 1, T cells (isolated from the blood of collected cynomolgus monkeys) were added at an E:T ratio (effector: target ratio) of 2:1. The data were normalized and analyzed using RTCA software (ACEA). As shown in Figure 24, NHP iPSC-DKO cells clearly escaped T cell killing, while NHP iPSC-WT cells were killed by T cells.
实施例8.NHP iPSC--DKO+GSN细胞系的构建及验证Example 8. Construction and verification of NHP iPSC--DKO+GSN cell line
8.1 NHP iPSC-DKO+GSN细胞系的构建及过表达检测8.1 Construction of NHP iPSC-DKO+GSN cell line and overexpression detection
参见实施例4.1的方法制备NHP iPSC-DKO+GSN细胞。具体地，直接合成编码GSN蛋白(GSN蛋白的氨基酸序列如SEQ ID NO:7所示)的核酸序列。并将合成的GSN的核酸序列连接至慢病毒载体中。连接成功后验证插入序列正确性并进行病毒包装。转染慢病毒载体至实施例7中构建的NHP iPSC-DKO细胞中。并使用qPCR检测食蟹猴GSN mRNA的过表达水平，WT细胞为阴性对照，所用引物如表6所示。Refer to the method in Example 4.1 to prepare NHP iPSC-DKO+GSN cells. Specifically, directly synthesize the nucleic acid sequence encoding the GSN protein (the amino acid sequence of the GSN protein is shown in SEQ ID NO:7). And connect the synthesized GSN nucleic acid sequence to the lentiviral vector. After successful connection, verify the correctness of the inserted sequence and perform viral packaging. Transfect the lentiviral vector into the NHP iPSC-DKO cells constructed in Example 7. And use qPCR to detect the overexpression level of cynomolgus monkey GSN mRNA, WT cells are negative controls, and the primers used are shown in Table 6.
表6.食蟹猴GSN的qPCR检测引物
Table 6. qPCR primers for cynomolgus monkey GSN
检测结果如图25所示，构建的NHP iPSC-DKO+GSN细胞系中有GSN的高表达。The detection results are shown in Figure 25, and GSN is highly expressed in the constructed NHP iPSC-DKO+GSN cell line.
8.2检测NHP iPSC-DKO+GSN细胞系的免疫逃逸功能8.2 Detection of immune escape function of NHP iPSC-DKO+GSN cell line
在XCelligence平台(ACEA BioSciences)上进行PBMC细胞杀伤测定以检测通用型细胞NHP iPSC-DKO+GSN的免疫逃逸功能。具体检测方法和结果如下所述。PBMC cell killing assay was performed on the XCelligence platform (ACEA BioSciences) to detect the immune escape function of universal cells NHP iPSC-DKO+GSN. The specific detection method and results are described as follows.
将NHP iPSC-WT、NHP iPSC-DKO和NHP iPSC-DKO+GSN细胞重悬于100μl细胞特异性培养基中，并铺于包被有基质胶(Matrigel，Sigma‐Aldrich)的96孔E‐板(ACEA BioSciences)上。在细胞指标值达到1之后，以1:1的E:T比添加PBMC(分离自采集的食蟹猴的血液)。用RTCA软件(ACEA)对数据进行标准化和分析。结果如图26所示，NHP iPSC-DKO+GSN细胞可明显逃逸PBMC细胞的杀伤。可能因为食蟹猴与人的PBMC的免疫亚群的组成比例不同，杀伤能力及识别存在差异性，并且本实施例使用未 经过活化的PBMC进行杀伤测定，PBMC在此效靶比下未有效杀伤WT。NHP iPSC-WT, NHP iPSC-DKO and NHP iPSC-DKO+GSN cells were resuspended in 100 μl of cell-specific culture medium and plated on a 96-well E-plate (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index value reached 1, PBMC (isolated from the blood of collected crab-eating macaques) was added at an E:T ratio of 1:1. The data were standardized and analyzed using RTCA software (ACEA). The results are shown in Figure 26. NHP iPSC-DKO+GSN cells can obviously escape the killing of PBMC cells. It may be because the composition ratio of the immune subpopulations of PBMCs in crab-eating macaques and humans is different, there are differences in killing ability and recognition, and this example uses untreated cells. Killing assay was performed on activated PBMCs, and PBMCs did not effectively kill WT at this effector-target ratio.
虽然本发明已以较佳的实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明精神和范围内，都可以做各种的改动与修饰，因此，本发明的保护范围应该以权利要求书所界定的为准。Although the present invention has been disclosed as above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.
序列表




Sequence Listing




Claims (23)

  1. 一种低免疫原性多能干细胞，其包含：A low-immunogenic pluripotent stem cell comprising:
    与亲本多能干细胞相比降低的内源主要组织相容性I类抗原(MHC-I)功能；reduced endogenous major histocompatibility class I antigen (MHC-I) function compared to parental pluripotent stem cells;
    与亲本多能干细胞相比降低的内源主要组织相容性II类抗原(MHC-II)功能；和Reduced endogenous major histocompatibility class II antigen (MHC-II) function compared to the parental pluripotent stem cell; and
    与亲本多能干细胞相比降低的对NK细胞杀伤的敏感性，其中所述降低的对NK细胞杀伤的敏感性是由增加的GSN蛋白的表达引起的。Reduced sensitivity to NK cell killing compared to a parental pluripotent stem cell, wherein the reduced sensitivity to NK cell killing is caused by increased expression of GSN protein.
  2. 权利要求1的低免疫原性多能干细胞，其中所述MHC-I功能通过降低MHC-I类蛋白或MHC-I转录调节因子的活性而降低。The low immunogenic pluripotent stem cell of claim 1, wherein the MHC-I function is reduced by reducing the activity of MHC-I class proteins or MHC-I transcriptional regulators.
  3. 权利要求2的低免疫原性多能干细胞，其中所述MHC-I类蛋白包含HLA-A蛋白、HLA-B蛋白或HLA-C蛋白。The low immunogenic pluripotent stem cell of claim 2, wherein the MHC-I class protein comprises an HLA-A protein, an HLA-B protein or an HLA-C protein.
  4. 权利要求2的低免疫原性多能干细胞，其中所述MHC-I转录调节因子包含B2M蛋白、TAP1蛋白、TAP2蛋白、TAP相关糖蛋白或NLRC5蛋白。The low immunogenic pluripotent stem cell of claim 2, wherein the MHC-I transcriptional regulator comprises a B2M protein, a TAP1 protein, a TAP2 protein, a TAP-associated glycoprotein or a NLRC5 protein.
  5. 权利要求2的低免疫原性多能干细胞，其中所述MHC-I功能通过降低B2M蛋白的活性而降低。The low immunogenic pluripotent stem cell of claim 2, wherein the MHC-I function is reduced by reducing the activity of the B2M protein.
  6. 权利要求5的低免疫原性多能干细胞，其中所述B2M蛋白为人B2M蛋白，其包含SEQ ID NO:1所示的氨基酸序列或与SEQ ID NO:1所示的氨基酸序列具有至少90％相同性的氨基酸序列；或者The low immunogenic pluripotent stem cell of claim 5, wherein the B2M protein is a human B2M protein, which comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1; or
    所述B2M蛋白为食蟹猴B2M蛋白，其包含SEQ ID NO:9所示的氨基酸序列或与SEQ ID NO:9所示的氨基酸序列具有至少90％相同性的氨基酸序列。The B2M protein is a crab-eating macaque B2M protein, which comprises the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO:9.
  7. 权利要求1-6中任一项的低免疫原性多能干细胞，其中所述MHC-II功能通过降低MHC-II类蛋白或MHC-II转录调节因子的活性而降低。The low immunogenic pluripotent stem cell according to any one of claims 1 to 6, wherein the MHC-II function is reduced by reducing the activity of MHC-II class proteins or MHC-II transcriptional regulators.
  8. 权利要求7的低免疫原性多能干细胞，其中所述MHC-II类蛋白包含HLA-DR蛋白、HLA-DQ蛋白或HLA-DP蛋白。The low immunogenic pluripotent stem cell of claim 7, wherein the MHC-II class protein comprises an HLA-DR protein, an HLA-DQ protein or an HLA-DP protein.
  9. 权利要求7的低免疫原性多能干细胞，其中所述MHC-II转录调节因子包含CIITA蛋白、RFXANK蛋白、RFX5蛋白或RFXAP蛋白。The low immunogenic pluripotent stem cell of claim 7, wherein the MHC-II transcriptional regulator comprises CIITA protein, RFXANK protein, RFX5 protein or RFXAP protein.
  10. 权利要求7的低免疫原性多能干细胞，其中所述MHC-II功能通过降低CIITA蛋白的活性而降低。The low immunogenic pluripotent stem cell of claim 7, wherein the MHC-II function is reduced by reducing the activity of the CIITA protein.
  11. 权利要求10的低免疫原性多能干细胞，其中所述CIITA蛋白为人CIITA蛋白， 其包含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至少90％相同性的氨基酸序列；或者The low immunogenic pluripotent stem cell according to claim 10, wherein the CIITA protein is a human CIITA protein, It comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 2; or
    所述CIITA蛋白为食蟹猴CIITA蛋白，其包含SEQ ID NO:10所示的氨基酸序列或与SEQ ID NO:10所示的氨基酸序列具有至少90％相同性的氨基酸序列。The CIITA protein is the crab-eating monkey CIITA protein, which comprises the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO:10.
  12. 权利要求1-11中任一项的低免疫原性多能干细胞，其中所述GSN蛋白为人GSN蛋白，其包含SEQ ID NO:3所示的氨基酸序列或与SEQ ID NO:3所示的氨基酸序列具有至少90％相同性的氨基酸序列；或者The low immunogenic pluripotent stem cell of any one of claims 1 to 11, wherein the GSN protein is a human GSN protein comprising the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 3; or
    所述GSN蛋白为食蟹猴GSN蛋白，其包含SEQ ID NO:7所示的氨基酸序列或与SEQ ID NO:7所示的氨基酸序列具有至少90％相同性的氨基酸序列。The GSN protein is a crab-eating macaque GSN protein, which comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO:7.
  13. 权利要求1的低免疫原性多能干细胞，其包含：The low immunogenic pluripotent stem cell of claim 1, comprising:
    使内源B2M蛋白活性降低的一个或多个改变；One or more changes that reduce the activity of endogenous B2M protein;
    使内源CIITA蛋白活性降低的一个或多个改变；和One or more changes that reduce the activity of endogenous CIITA protein; and
    在所述低免疫原性多能干细胞中引起增加的GSN蛋白表达的一个或多个改变。One or more alterations result in increased expression of GSN protein in the low immunogenic pluripotent stem cells.
  14. 权利要求13的低免疫原性多能干细胞，其包含：The low immunogenic pluripotent stem cell of claim 13, comprising:
    使内源B2M基因的两个等位基因失活的一个或多个改变；one or more alterations that inactivate both alleles of the endogenous B2M gene;
    使内源CIITA基因的两个等位基因失活的一个或多个改变；和One or more alterations that inactivate both alleles of the endogenous CIITA gene; and
    在所述低免疫原性多能干细胞中引起增加的GSN基因表达的一个或多个改变。One or more alterations result in increased GSN gene expression in the low immunogenic pluripotent stem cells.
  15. 一种产生权利要求1-14中任一项的低免疫原性多能干细胞的方法，所述方法包括：A method for producing the low immunogenic pluripotent stem cell according to any one of claims 1 to 14, the method comprising:
    降低所述多能干细胞中内源主要组织相容性I类抗原(MHC-I)功能；reducing endogenous major histocompatibility class I antigen (MHC-I) function in the pluripotent stem cells;
    降低所述多能干细胞中内源主要组织相容性II类抗原(MHC-II)功能；和reducing endogenous major histocompatibility class II antigen (MHC-II) function in the pluripotent stem cells; and
    增加降低所述多能干细胞对NK细胞杀伤的敏感性的蛋白的表达，其中所述蛋白为GSN蛋白。The expression of a protein that reduces the sensitivity of the pluripotent stem cells to NK cell killing is increased, wherein the protein is GSN protein.
  16. 权利要求15的方法，所述方法包括：The method of claim 15, comprising:
    降低所述多能干细胞中B2M蛋白的活性；reducing the activity of B2M protein in the pluripotent stem cells;
    降低所述多能干细胞中CIITA蛋白的活性；和Reducing the activity of CIITA protein in the pluripotent stem cells; and
    增加所述多能干细胞中GSN蛋白的表达。The expression of GSN protein in the pluripotent stem cells is increased.
  17. 权利要求16的方法，所述方法包括：The method of claim 16, comprising:
    消除所述多能干细胞中B2M基因的两个等位基因的活性；Eliminating the activity of two alleles of the B2M gene in the pluripotent stem cells;
    消除所述多能干细胞中CIITA基因的两个等位基因的活性；和Eliminating the activity of both alleles of the CIITA gene in the pluripotent stem cells; and
    增加所述多能干细胞中GSN基因的表达。The expression of the GSN gene in the pluripotent stem cells is increased.
  18. 权利要求16或17的方法，其中通过选自以下的技术降低所述多能干细胞中 B2M蛋白的活性和/或降低所述多能干细胞中CIITA蛋白的活性：The method of claim 16 or 17, wherein the pluripotent stem cells are reduced by a technique selected from the group consisting of The activity of B2M protein and/or the activity of CIITA protein in the pluripotent stem cells is reduced:
    引入基因表达修饰分子、成簇规律间隔短回文重复序列(CRISPR)技术、转录激活因子样效应核酸酶(TALEN)技术、锌指核酸酶(ZFN)技术或同源重组技术；优选地，所述基因表达修饰分子包含siRNA、shRNA、microRNA、反义RNA、反义寡核苷酸ASO或抗miRNA寡核苷酸AMO。Introduce gene expression modifying molecules, clustered regularly interspaced short palindromic repeats (CRISPR) technology, transcription activator-like effector nuclease (TALEN) technology, zinc finger nuclease (ZFN) technology or homologous recombination technology; preferably, the gene expression modifying molecules comprise siRNA, shRNA, microRNA, antisense RNA, antisense oligonucleotide ASO or anti-miRNA oligonucleotide AMO.
  19. 权利要求18的方法，其中The method of claim 18, wherein
    通过成簇规律间隔短回文重复序列(CRISPR)/Cas9基因编辑技术降低所述多能干细胞中B2M蛋白的活性；和/或Reducing the activity of B2M protein in the pluripotent stem cells by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology; and/or
    通过成簇规律间隔短回文重复序列(CRISPR)/Cas9基因编辑技术降低所述多能干细胞中CIITA蛋白的活性。The activity of CIITA protein in the pluripotent stem cells is reduced by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology.
  20. 权利要求15-19中任一项的方法，其中通过对内源基因座的修饰增加GSN蛋白的表达。The method of any one of claims 15 to 19, wherein the expression of the GSN protein is increased by modification of an endogenous locus.
  21. 权利要求15-19中任一项的方法，其中通过转基因的表达增加GSN蛋白的表达；优选地，通过合成编码GSN蛋白的核酸序列，以构建至慢病毒载体中，再通过慢病毒载体，将至少一个拷贝的在启动子控制下的GSN基因引入所述多能干细胞中增加GSN蛋白的表达。The method of any one of claims 15 to 19, wherein the expression of the GSN protein is increased by expression of a transgene; preferably, by synthesizing a nucleic acid sequence encoding the GSN protein to construct it into a lentiviral vector, and then introducing at least one copy of the GSN gene under the control of a promoter into the pluripotent stem cells through the lentiviral vector to increase the expression of the GSN protein.
  22. 权利要求21的方法，其中所述编码GSN蛋白的核酸序列包含SEQ ID NO:4所示的核酸序列或与SEQ ID NO:4所示的核酸序列具有至少80％相同性的核酸序列；或者The method of claim 21, wherein the nucleic acid sequence encoding the GSN protein comprises the nucleic acid sequence shown in SEQ ID NO:4 or a nucleic acid sequence having at least 80% identity with the nucleic acid sequence shown in SEQ ID NO:4; or
    所述编码GSN蛋白的核酸序列包含SEQ ID NO:8所示的核酸序列或与SEQ ID NO:8所示的核酸序列具有至少80％相同性的核酸序列。The nucleic acid sequence encoding the GSN protein comprises the nucleic acid sequence shown in SEQ ID NO:8 or a nucleic acid sequence that has at least 80% identity with the nucleic acid sequence shown in SEQ ID NO:8.
  23. 权利要求1-14中任一项的低免疫原性多能干细胞或权利要求15-22中任一项的方法制备的低免疫原性多能干细胞在制备用于预防或治疗需要细胞移植的疾病的药物中的用途；优选地，所述疾病包含急性白血病、慢性白血病、淋巴瘤、骨髓增生异常综合征、多发性骨髓瘤、小细胞肺癌、乳腺癌、睾丸癌、神经母细胞瘤、卵巢癌、黑色素瘤、再生障碍性贫血、先天性免疫性缺陷、系统性红斑狼疮、类风湿性关节炎、强直性脊柱炎、I型糖尿病、帕金森病、阿尔茨海默病、脊髓损伤、视网膜变性疾病、脑卒中、亨廷顿病、肌萎缩性侧索硬化症、动脉粥样硬化、高血压、风湿性心脏病、心肌病、心律失常、先天性心脏病、瓣膜性心脏病、心脏炎、心肌梗塞、心力衰竭、主动脉瘤、外周动脉疾病、II型糖尿病、坏血症、低血糖症、高血脂或骨质疏松。 Use of the low immunogenic pluripotent stem cells according to any one of claims 1 to 14 or the low immunogenic pluripotent stem cells prepared by the method of any one of claims 15 to 22 in the preparation of a medicament for preventing or treating a disease requiring cell transplantation; preferably, the disease comprises acute leukemia, chronic leukemia, lymphoma, myelodysplastic syndrome, multiple myeloma, small cell lung cancer, breast cancer, testicular cancer, neuroblastoma, ovarian cancer, melanoma, aplastic anemia, congenital immunodeficiency, systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, type I diabetes, Parkinson's disease, Alzheimer's disease, spinal cord injury, retinal degeneration, stroke, Huntington's disease, amyotrophic lateral sclerosis, atherosclerosis, hypertension, rheumatic heart disease, cardiomyopathy, arrhythmia, congenital heart disease, valvular heart disease, carditis, myocardial infarction, heart failure, aortic aneurysm, peripheral arterial disease, type II diabetes, scurvy, hypoglycemia, hyperlipidemia or osteoporosis.
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