WO2024120427A1 - Cellule universelle exprimant gsn et son procédé de préparation - Google Patents

Cellule universelle exprimant gsn et son procédé de préparation 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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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/fr

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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

L'invention concerne une cellule souche pluripotente à faible immunogénicité exprimant la gelsoline (GSN) et son procédé de préparation. La cellule souche pluripotente à faible immunogénicité peut réduire considérablement ou échapper à la reconnaissance et aux attaques du système immunitaire, les propriétés de mise à jour et de différenciation des cellules souches restant inchangées.
PCT/CN2023/136703 2022-12-07 2023-12-06 Cellule universelle exprimant gsn et son procédé de préparation WO2024120427A1 (fr)

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