Modified cell

Abstract

Provided are hypoimmunogenic cells and methods of manufacturing such.

Description

Modified cell1 BACKGROUND

[0001] A number of different strategies have been employed to generate donor stem cell products that can be administered to multiple patients. These include direct knock-out of the high HLA class I and II molecules and / or transcription factors control expression of HLA genes, and the introduction of immunoregulatory tolerogenic factors. However, the introduction of extensive genome modification introduces additional risks to the final cell therapy product, e.g. off-target integration and structural variants associated with genetic modification [1], Furthermore, genetic modifications made to stem cells may be unstable and not retained in progeny differentiated cells.

[0002] The usefulness of stem cells comes from their ability to be differentiated into a range of different specialised cell types that can then be used in tissue regeneration or stem cell-derived immune cell therapies. A stem cell may be fully differentiated in vitro before administration to the patient, or partly differentiated into specialised progenitor cells that are administered to the patient and then undergo further differentiation in vivo. To be safe for administration to a patient, the hypoimmunogenic properties of a stem cell must be retained by the differentiated cells at all stages of the differentiation pathway. During differentiation, cells will undergo radical changes in their gene expression profile, including changes in expression of the genes modified for the purposes of induced immuno-tolerance. Additionally, gene silencing represents a major obstacle to efficient expression of introduced genes in pluripotent stem cells and thereof derived tissues / cell types.

[0003] The present disclosure solves one or more of the above-mentioned problems.2 SUMMARY

[0004] The present disclosure relates to a modified cell that has been modified to have increased or introduced expression of an HLA-E gene and an HLA-G gene, and deleted for decreased expression of p2M, as compared to an unmodified cell. The disclosure is supported by data, provided herein for the first time, surprisingly demonstrating that modifying a cell to overexpress of HLA-E and HLA-G in combination (including in the absence of CD4 overexpression) permits the cell to avoid NK cell mediated allo-immunity.3 BRIEF DESRIPTION OF THE DRAWINGSFigure 1 : Effect of TAP1 , TAP2 and B2M knock-out on HLA A / B / C expression

[0005] KO: Knock-out. WT: Wild-type.Figure 2: Alloreactive cytotoxic T lymphocytes (allo-CTL) dependent cytolysis of modified HeLa cells (Luciferase based killing assay)

[0006] Luciferase based killing assay (xCelligence). KO: Knock-out; MFI: mean fluorescence intensity; WT: Wild Type. Allo-CTLs derived from two donors were used.Figure 3: Allo-CTL dependent cytolysis of modified HeLa cells (xCelligence real-time based killing assay)

[0007] KO: Knock-out; WT: Wild Type.Figure 4: HLA class I and HLA class II expression in a naturally non-HLA class Il-positive cell type (HeLa cells) in the presence and absence of IFN-y stimulation.

[0008] KO: Knock-out.Figure 5: Reduced HLA class II expression in a naturally HLA class Il-positive antigen presenting cells demonstrated additional protection from HLA class Il-specific alloreactive CTL killing.

[0009] Figure 5A: HLA class II expression following gene knock-out in an HLA class l-ablated B cell line with B2M gene KO (RajiB2M / _cell, clone A5). Figure 5B: Reduced HLA class II expression by RFXANK, RFXAP and CIITA led to increased resistance to alloreactive CTL enriched for HLA class Il- specific killing. CTLs enriched in RajiB2M / _cell, clone A5 from two donors were used. KO: Knock-out. C2TA: CIITA. Cell type = Raji cells.Figure 6: HLA-E and HLA-G overexpression

[0010] Generation of p2M -fused HLA-E and HLA-G expressing B2M-KO iPSC lines through lentiviral transduction. HLA-E and HLA-G surface expression was confirmed by anti-PE-HLA-E and anti-APC- HLA-G antibody staining and flow analysis.Figure 7: HLA-E and HLA-G synergy

[0011] Figure 7A: NK killing of engineered stem cells by 4 different donors. NK mediated cytolysis was measured by the XCELLigence assay. E:T ratio at 1 :2 was used for each NK donor. Graphs represent the data at 20 hours after adding the effector NK cells to different iPSC cells. Figure 7B: Box plot showing distribution of NK killing of engineered stem cell by all donors from data in 7A.Figure 8: P2M and HLA class I expression in a modified stem cell line

[0012] Modified cells: Clone 1 E11. Control cells: SA121.Figure 9: HLA class II, HLA-E and CD47 expression in a modified stem cell line after differentiation into endothelial cells

[0013] Modified cells: Clone 1 E11. Control cells: SA121. HLA class II expression was induced after interferon-gamma (IFNg) treatment of unmodified, wildtype ESC-derived, CD144+ endothelial cells. RFXAP KO USC-derived, CD144+ endothelial cells demonstrated no HLA class II induction upon IFNy treatment. Lower panels: HLA-E and CD47 expression in a USC line.Figure 10: Resistance to alloreactive CTL mediated cytolysis by modified stem cells

[0014] CD8+ T cell killing assay. E:T = 4.1 . WT: Wild Type.Figure 11 : Resistance to alloreactive NK cell killing by modified stem cells

[0015] NK cell killing assay E:T = 1 :2. WT: Wild Type.Figure 12: Pluripotency of modified stem cells

[0016] Universal stem cell clone (1 E11) modified with p2M KO, RFXAP KO, HLA-E overexpression and CD47 overexpression were successfully differentiated into endoderm, mesoderm and ectoderm cells. ESC: embryonic stem cell.Figure 13: Differentiation of modified stem cells into beating cardiomyocytes

[0017] Day 15 differentiation, representative clone. Universal stem cell clone (1 E11) modified with B2M KO, RFXAP KO, HLA-E overexpression and CD47 overexpression. WT: Wild Type; ESC: embryonic stem cell.Figure 14: Stable CD47 and HLA-E overexpression following differentiation of modified stem cells into cardiomyocytes

[0018] Universal stem cell line (1 E11) modified with p2M KO, RFXAP KO, HLA-E overexpression and CD47 overexpression. SA121 : unmodified parental control cells. WT: Wild Type; ESC: embryonic stem cell.Figure 15: Differentiation of modified stem cells into hepatocytes

[0019] Day 22 differentiation. Universal stem cell line (1 E11) modified with p2M KO, RFXAP KO, HLA- E overexpression and CD47 overexpression. WT: Wild Type.Figure 16: Stable CD47 and HLA-E overexpression following differentiation of modified stem cells into hepatocytes

[0020] Day 22 differentiation. Universal stem cell line (1 E11) modified with p2M KO, RFXAP KO, HLA- E overexpression and CD47 overexpression. WT: Wild Type; heap: hepatocyte.4 DETAILED DESCRIPTION4.1 General

[0021] The disclosure relates to a modified cell, wherein relative to an unmodified cell of the same cell type, the modified cell comprises a) introduced or increased expression of a human leukocyte antigen (HLA)-E gene and an HLA-G gene; and b) deleted or decreased expression of a beta-2 microglobulin (p2M) gene.

[0022] The disclosure also relates to a modified cell comprising an exogenous polynucleotide encoding an HLA-E gene and an exogenous polynucleotide encoding an HLA-G gene.

[0023] Relative to an unmodified cell of the same cell type, the modified cell may comprise deleted or decreased expression of: a Class II Major Histocompatibility Complex Transactivator (CIITA) gene, a Regulatory factor X-associated ankyrin-containing protein (RFXANK) gene, and / or a Regulatory Factor X Associated Protein (RFXAP) gene. The CIITA gene, RFXANK gene and / or RFXAP gene may be human.

[0024] Relative to an unmodified cell of the same cell type, the modified cell may comprise a) introduced or increased expression of a human leukocyte antigen (HLA)-E polypeptide and an HLA-G polypeptide; and b) deleted or decreased expression of a beta-2 microglobulin (p2M) polypeptide.

[0025] The HLA-E polypeptide may comprise an amino acid sequence which is at least 80% identical to SEQ ID NO: 10 or a functional fragment thereof. The HLA-E polypeptide may comprise an amino acid sequence which is at least 90% identical to SEQ ID NO: 10 or a functional fragment thereof. The HLA-E polypeptide may comprise an amino acid sequence which is at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 10 or a functional fragment thereof. The HLA-E polypeptide may comprise an amino acid sequence which is at least 100% identical to SEQ ID NO: 10 or a functional fragment thereof. The HLA-E polypeptide may comprise an amino acid sequence which is at least 80% identical to SEQ ID NO: 10 or a functional fragment thereof. The HLA-E polypeptide may comprise an amino acid sequence which is at least 90% identical to SEQ ID NO: 10 or a functional fragment thereof. The HLA-E polypeptide may comprise an amino acid sequence which is at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 10 or a functional fragment thereof. The HLA-E polypeptide may comprise an amino acid sequence which is at least 100% identical to SEQ ID NO: 10 or a functional fragment thereof.

[0026] The HLA-G polypeptide may comprise an amino acid sequence which is at least 80% identical to SEQ ID NO: 11 or a functional fragment thereof. The HLA-G polypeptide may comprise an amino acid sequence which is at least 90% identical to SEQ ID NO: 11 or a functional fragment thereof. The HLA-G polypeptide may comprise an amino acid sequence which is at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 11 or a functional fragment thereof. The HLA-G polypeptide may comprise an amino acid sequence which is at least 100% identical to SEQ ID NO: 11 or a functional fragment thereof. The HLA-G polypeptide may comprise an amino acid sequence which is at least 80% identical to SEQ ID NO: 11 or a functional fragment thereof. The HLA-E polypeptide may comprise an amino acid sequence which is at least 90% identical to SEQ ID NO: 1 1 or a functional fragment thereof. The HLA-G polypeptide may comprise an amino acid sequence which is at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 11 or a functional fragment thereof. The HLA-G polypeptide may comprise an amino acid sequence which is at least 100% identical to SEQ ID NO: 11 or a functional fragment thereof.

[0027] Relative to an unmodified cell of the same cell type, the modified cell may comprise introduced or increased expression of a CD47 gene. Relative to an unmodified cell of the same cell type, the modified cell comprises introduced or increased expression of a CD47 polypeptide. The CD47 polypeptide may comprise an amino acid sequence which is at least 80% identical to SEQ ID NO: 12 or a functional fragment thereof. The CD47 gene may be human. The CD47 polypeptide may comprise an amino acid sequence which is at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 12 or a functional fragment thereof. The CD47 polypeptide may comprise an amino acid sequence which is at least 100% identical to SEQ ID NO: 12 or a functional fragment thereof. The CD47 polypeptide may comprise an amino acid sequence which is at least 80% identical to SEQ ID NO: 12 or a functional fragment thereof. The CD47 polypeptide may comprise an amino acid sequence which is at least 90% identical to SEQ ID NO: 12 or a functional fragment thereof. The CD47 polypeptide may comprise an amino acid sequence which is at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99% identical toSEQ ID NO: 12 or a functional fragment thereof. The CD47 polypeptide may comprise an amino acid sequence which is at least 100% identical to SEQ ID NO: 12 or a functional fragment thereof.

[0028] Relative to an unmodified cell of the same cell type, the modified cell may not comprise introduced or increased expression of a CD47 gene. The CD47 gene may be human.

[0029] Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of a CD24 gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of a CD74 gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of a CIITA gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of an HLA-A gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of an HLA-B gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of an HLA-C gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of an MIC-1 gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of an MIC-2 gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of an NLRC5 gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of a RFX5 gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of a RFXAP gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of a TAP1 gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of a TAP2 gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of a Tapasin gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of a TXNIP. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of a RFXANK gene.

[0030] Relative to an unmodified cell of the same cell type, the modified cell may not comprise increased or introduced expression of a CD47 gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise increased or introduced expression of a C1 -inhibitor gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise increased or introduced expression of a CD24 gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise increased or introduced expression of a CD46 gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise increased or introduced expression of a CD55 gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise increased or introduced expression of a CD59 gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise increased or introduced expression of a CR1 gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise increased or introducedexpression of a CTLA-4-lg gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise increased or introduced expression of a MANF gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise increased or introduced expression of a TNFAIP3 gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise increased or introduced expression of a PD-L1 gene.

[0031] Expression of the HLA-E gene and HLA-G gene in the modified cell may be driven by an elongation factor 1 alpha (EF1 a) promoter. Expression of the HLA-E gene and HLA-G gene in the modified cell may be driven by the same elongation factor 1 alpha (EF1 a) promoter.

[0032] The modified cell may be a mammalian cell. The modified cell may be a human cell. The modified cell may be an undifferentiated cell. The modified cell may be a cell capable of self-renewal. The modified cell may be a stem cell. The modified cell may be a pluripotent stem cell. The modified cell may be an embryonic stem cell (ESC). After differentiation into a different or mature cell type the cell may retain the modification or modifications. After differentiation into a different cell type the modified cell may continue to overexpress or have increased expression of an HLA-E gene and an HLA-G gene.

[0033] The modified cell may be an induced pluripotent stem cell (iPSC). The modified cell may be a multipotent stem cell. The modified cell may be a haematopoietic stem cell (HSC). The modified cell may be a differentiated cell. The modified cell may be an endothelial cell. The modified cell may be a cardiomyocyte. The modified cell may be a hepatocyte. The modified cell may be an isolated cell. The modified cell may be an engineered cell. The modified cell may be a cell that has been modified or engineered in vitro. The modified cell may be a cell that has been modified or engineered ex vivo. The modified cell may not occur in nature.

[0034] Compared to an unmodified cell of the same cell type, the modified cell may comprise reduced HLA class II protein expression following stimulation with IFN-y, optionally wherein HLA class II protein expression is determined by flow cytometry. Compared to an unmodified cell of the same cell type, the modified cell may comprise reduced HLA-DR, HLA-DQ and / or HLA-DR expression following stimulation with IFN-y, optionally as determined by flow cytometry.

[0035] Compared to an unmodified cell of the same cell type, the modified cell may comprise reduced HLA class I expression, optionally as determined by flow cytometry. The modified cell may not express HLA class I protein, optionally as determined by flow cytometry.

[0036] Compared to an unmodified cell of the same cell type, the modified cell may comprise increased expression of an HLA-E protein and an HLA-G protein, optionally as determined by flow cytometry. Compared to an unmodified cell of the same cell type, the modified cell may comprise reduced HLA-A, B and C protein expression, optionally as determined by FACS or flow cytometry.

[0037] The modified cell may not express HLA-A, B or C protein, optionally as determined by FACS.

[0038] Compared to an unmodified cell of the same cell type, the modified cell may be resistant to a llo- cytotoxic lymphocyte mediated killing, optionally as determined by real-time based killing assay. Compared to an unmodified cell of the same cell type, the cell may be resistant to allo-NK cell cytolysis, optionally as determined by real-time based killing assay. The cell may be a universal donor cell. The cell may be administered to a subject without the need of immunosuppressive therapy. The cell may be a hypoimmunogenic cell. The cell may be suitable for use in allogenic cell therapy. The modified cell may not activate NK cell mediated immunity, optionally as determined by real-time based killing assay. The modified cell may not activate The NK cell immunity. The cell may not activate T cell immunity. The cell may not activate macrophage mediated immunity.

[0039] The modified cell may not comprise a cytomegalovirus (CMV) promoter. Introduced or increased expression of the immune-tolerising genes in the cell may not be driven by a CMV promoter.

[0040] The cell may not be a mesenchymal stem cell. The cell may not be an immune cell. The cell may not be a terminally differentiated cell. The cell may not be a T cell. The cell may not be an NK cell. The cell may not be a NKT cell. The cell may not be a B cell.

[0041] The cell may express a chimeric antigen receptor or T cell receptor. The cell may not express a chimeric antigen receptor or T-cell receptor (TCR).

[0042] Compared to a unmodified cell of the same type the modified cell may not have been modified to be resistant to replicative senescence (RRS). Compared to a unmodified cell of the same type the modified cell may not have been modified to delete or reduce expression of cyclin dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-51- thioadenosine phosphorylase (MTAP). Compared to a unmodified cell of the same type the modified cell may not have been modified to delete or reduce expression of T-cell receptor a constant (TRAC). Compared to a unmodified cell of the same type the modified cell may not have been modified to increase expression of B-cell lymphoma-extra large (Bcl-xL) or B-cell lymphoma 2 (Bcl-2). Compared to a unmodified cell of the same type the modified cell may not have been modified to deleted or reduce expression of cluster of differentiation 38 (CD38) and / or phosphatase and tensin homolog (PTEN).

[0043] Introduced or increased gene expression may be determined by techniques known in the art such as flow cytometry, western blot, qPCR or immunofluorescence. Deleted or decreased gene expression. Deleted or decreased gene expression may be determined by flow cytometry, western blot, qPCR or immunofluorescence. Introduced or increased expression of the HLA-E gene and the HLA-G gene may be determined by flow cytometry. p2M gene expression may be deleted or decreased by methods comprising gene editing the cell. Gene editing techniques are known in the art, for example CRISPR-Cas9.

[0044] The disclosure also relates to a population of cells comprising one or more of the modified cells of the disclosure.

[0045] The disclosure also relates to pharmaceutical compositions comprise in the modified cell of the population of cells of the disclosure. The pharmaceutical composition may be suitable for administrationto a subject. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier. The pharmaceutical composition may comprise a pharmaceutical excipient and the modified cell or population of cells.

[0046] The disclosure also relates to the use of a modified cell in the manufacture of a medicament for the treatment of prevention of disease in a subject.

[0047] The disclosure also relates to a method for generating a modified cell, comprising a) increasing the expression of HLA-G in the cell, b) increasing the expression of HLA-E in the cell, and c) eliminating or decreasing the expression of one or both alleles of the B2M gene in a cell, wherein the cell is an undifferentiated cell. The method may also comprise eliminating the expression of both alleles of the CIITA, RFXANK and / or RFXAP genes in the cell. The cell used in the method may be an embryonic stem cell. The cell may be an induced pluripotent stem cell.

[0048] The disclosure also relates to a lentivector. The lentivector may be an adenovirus vector. The factor may comprise an HLA-E gene. The vector may comprise an HLA-G gene. The vector may encode an HLA-E and an HLA-G gene. Expression of the HLA-E and HLA-G genes may be under the control of the same promoter, optionally an EF1-alpha promoter. The lentivector may comprise a gRNA having a nucleotide sequence corresponding to the sequence of any of SEQ ID NO: 1-8.

[0049] The disclosure also relates to a method of treating or preventing a disease in a subject in need thereof, the method comprises administering to the subject the modified cell or a differentiated cell, progeny, daughter cell, or population of cells derived from the modified cell. The modified cell may be autologous with respect to the subject, i.e. a cell, or a cell derived from a cell, isolated from the subject. The modified cell may be allogenic with respect to the subject, i.e. a cell, or a cell derived from a cell, isolated from a different subject.

[0050] The method of treatment may include isolating a cell from a first subject, modifying the cell and then administering the modified cell to a second subject.

[0051] The disclosure also relates to a method of treating or prevent a disease in a subject, the method comprising: a) producing in vitro a modified cell according to a method of the disclosure, and b) and administering modified cell to the subject.

[0052] The disclosure also relates to pharmaceutical composition for use in the methods of treatment.

[0053] The disclosure also relates to a population of engineered or modified cells comprising an engineered cell of the disclosure. The population of engineered or modified cells may comprise between 104and 1010engineered immune cells provided herein. In various embodiments, the population of engineered immune cells comprises 104, 105, 106, 107, 108, 109, or 1010engineered immune cells of the disclosure.

[0054] The population of engineered cells or a population of cells comprising engineered cells as disclosed herein comprises at least 10% engineered cells, at least 20% engineered cells, at least 30%engineered cells, at least 40% engineered cells, at least 50% engineered cells, at least 75% engineered cells, at least 90% engineered cells or 100% engineered T cells.

[0055] The population of modified cells or a population of cells comprising modified cells as disclosed herein comprises at least 10% modified cells, at least 20% modified cells, at least 30% modified cells, at least 40% modified cells, at least 50% modified cells, at least 75% modified cells, at least 90% modified cells or 100% modified cells.

[0056] The disclosure relates to a cell wherein, relative to a cell of the same cell type that does not comprise the modifications and which has been subjected to otherwise identical conditions, the modified cell comprises modifications that: introduce or increase expression of an HLA-E gene and an HLA-G gene; and knockout or decrease expression of a p2M gene.

[0057] The cell may also comprise modifications, wherein relative to a cell of the same cell type that does not comprise the modifications and which has been subjected to otherwise identical conditions, the cell comprises modifications that knockout or decrease expression of a CIITA gene.

[0058] The cell may also comprise modifications, wherein relative to a cell of the same cell type that does not comprise the modifications and which has been subjected to otherwise identical conditions, the cell comprises modifications that knockout or decrease expression of a RFXANK gene.

[0059] The cell may also comprise modifications, wherein relative to a cell of the same cell type that does not comprise the modifications and which has been subjected to otherwise identical conditions, the cell comprises modifications that knockout or decrease expression of a CIITA gene.

[0060] The cell may also comprise modifications that, relative to a cell of the same cell type that does not comprise the modifications and which has been subjected to otherwise identical conditions, introduced or increase expression of a Cluster of Differentiation 47 (CD47). The CD47 gene may be human.

[0061] The modified cell may be derived from a cell or cell comprising stem cells. The modified cell may be derived from an embryonic stem cell. The modified cell may be derived from an induced pluripotent stem cell. The modified cell may be derived from a mesoderm cells. The modified cell may be derived from an intermediate mesoderm cell.

[0062] The cell population may be is derived from a cell or cells that have been expanded ex vivo. The cell population may be derived from cell or cells isolated from a subject. The cell population may be artificial. The cell population may not exist in nature. The cell population may comprise a cell or cells that have been genetically modified or gene edited. The cell population may be isolated from the body and may be comprised in a pharmaceutical composition.

[0063] The cell population may be derived from cells comprising stem cells. The cell population may be derived from cells comprising embryonic stem cells (ES cells). The ES cells may be from a mammal source, including rodents such as a mouse (mES), and primates such as a human (hES). The ES cell may be an ES cell line selected from SEES-1 , -2, -3, -4, -5, -6, and -7 reported in H. Akutsu, RegenTher. 1 :18-29 (2015). Other known ES cells may also be used. Further genetic modifications may be made to such known ES cell lines. The cell population may be derived from cells comprising induced pluripotent stem cells (iPSCs). Production of iPSCs is reported by Yamanaka in e.g., WO 2007 / 069666. Other groups including Rudolf Jaenisch of MIT, James Thomson of Wisconsin University, Konrad Hochedlinger of Harvard have also reported production of iPSCs. For a review, see Nature Reviews Drug Discovery, vol. 16, pp. 115-130 (2017). iPSCs may be induced from any somatic cell. iPSCs may be from a mammal source, including rodents such as a mouse, and primates such as a human. Somatic cells may include fibroblasts, epithelial cells, endothelial cells, nerve cells, pancreatic cells, blood cells, bone marrow cells, muscle cells, hepatic cells, adipocytes, periodontal cells, osteoblasts, and the like. iPSCs may be produced by first introducing one or more reprogramming genes into a somatic cell. Combinations of reprogramming genes include (i) Oct gene, Klf gene, Sox gene and Myc gene: (ii) Oct gene, Sox gene, NANOG gene, and LIN28 gene; (iii) Oct gene, Klf gene, Sox gene, Myc gene, hTERT gene, and SV40 large T gene; and (iv) Oct gene, Klf gene, and Sox gene. The cell population may be derived from cells comprising mesoderm cells. The cell population may be derived from cells comprising intermediate mesoderm cells. Mesoderm cells and intermediate mesoderm cells can be derived from ES cells or iPSCs. Mesoderm cells and intermediate mesoderm cells can be obtained from natural sources during embryonic development for non-human sources. Differentiation into mesoderm cells or intermediate mesoderm cells can be confirmed by examining mesoderm specific markers. Gene markers for mesoderm cells include FKL-1 , COL2A1 , FLT1 , HBZ, MYF5, MYOD1 , RUNX2, PECAM1 and the like. Gene markers for mesoderm cells also include GATA4, GATA6, and T(Brachyury). The cells may be derived from cells comprising intermediate mesoderm cells.5 DEFINITIONS5.1 Cell potency

[0064] Pluripotent cells are cells that can self-renew and proliferate while remaining in an undifferentiated state and that can, under the proper conditions, be induced to differentiate into specialized cell types. The term "pluripotent cells," as used herein, encompass embryonic stem cells and other types of stem cells, including fetal, amnionic, or somatic stem cells. Exemplary human stem cell lines include the H9 human embryonic stem cell line. Additional exemplary stem cell lines include those made available through the National Institutes of Health Human Embryonic Stem Cell Registry and the Howard Hughes Medical Institute HUES collection [2],

[0065] Pluripotent stem cells have the potential to differentiate into any of the three germ layers: endoderm (e.g. the stomach linking, gastrointestinal tract, lungs, etc), mesoderm (e.g. muscle, bone, blood, urogenital tissue, etc) or ectoderm (e.g. epidermal tissues and nervous system tissues). The term "pluripotent stem cells," as used herein, also encompasses "induced pluripotent stem cells", or "iPSCs", a type of pluripotent stem cell derived from a non-pluripotent cell. Examples of parent cells include somatic cells that have been reprogrammed to induce a pluripotent, undifferentiated phenotype by various means. Such "iPS" or "iPSC" cells can be created by inducing the expression of certainregulatory genes or by the exogenous application of certain proteins. Methods for the induction of iPS cells are known in the art [3-6],

[0066] The ability to give rise to progeny that can undergo differentiation, under the appropriate conditions, into cell types that collectively demonstrate characteristics associated with cell lineages from all of the three germinal layers (endoderm, mesoderm, and ectoderm) is a pluripotent stem cell characteristic. Expression or non- expression of certain combinations of molecular markers are also pluripotent stem cell characteristics. For example, human pluripotent stem cells express at least several, and in some embodiments, all of the markers from the following non-limiting list: SSEA-3, SSEA- 4, TRA-1-60, TRA-1-81 , TRA-2-49 / 6E, ALP, Sox2, E-cadherin, UTF-1 , Oct4, Rexl, and Nanog.

[0067] Multipotent cells can give rise to a limited number of other particular cell types. For example, induced multipotent cells are capable of forming endodermal cells. Additionally, multipotent blood stem cells can differentiate itself into several types of blood cells, including lymphocytes, monocytes, neutrophils, etc. Oligopotent cells are adult stem cells that can differentiate into only a few different cell types. For example, lymphoid or myeloid stem cells are capable of forming cells of either the lymphoid or myeloid lineages, respectively. Unipotent cells form a single cell type. For example, spermatogonia! stem cells are only capable of forming sperm cells. Totipotent cells have the ability form entire organism. For example, in mammals, only the zygote and the first cleavage stage blastomeres are totipotent. Differentiated cells include, but are not limited to, multipotent cells, oligopotent cells, unipotent cells, progenitor cells, and terminally differentiated cells. Differentiated cells include, but are not limited to, multipotent cells, oligopotent cells, unipotent cells, progenitor cells, and terminally differentiated cells. In particular

[0068] Non-pluripotent cells are not pluripotent cells. Examples of such cells include differentiated cells as well as progenitor cells. Examples of differentiated cells include, but are not limited to, cells from a tissue selected from bone marrow, skin, skeletal muscle, fat tissue and peripheral blood. Exemplary cell types include, but are not limited to, fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T-cells. The starting cells employed for generating the induced multipotent cells, the endodermal progenitor cells, and the hepatocytes can be non-pluripotent cells.5.2 Gene editing

[0069] Cells of the disclosure may be modified, engineered or manipulated using clustered regularly interspaced short palindromic repeats) / Cas ("CRISPR") technologies as is known in the art. There are a large number of techniques based on CRISPR

[0012] , CRISPRtechniques and kits are sold commercially e.g. Alt-R™ S.p. Cas9 Nuclease V3 purchased from IDT.

[0070] TALEN are restriction enzymes combined with a nuclease that can be engineered to bind to and cut practically any desired DNA sequence.

[0071] Zn finger nucleases are artificial restriction enzymes generated by fusing a zinc finger DNA- binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired a DNA sequence and this enables zinc-finger nucleases to target unique sequences withincomplex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms, similar to CRISPR and TALENs.5.3 GenesTable 1: Summary of genes5.3.1 Human leukocyte antigen (HLA)

[0072] The MHC gene family is divided into three groups: class I, class II and class III. In humans, MHC is referred to as human leukocyte antigen (HLA). HLA class II molecules (HLA-II) are transmembrane protein found only on professional antigen- presenting cells (APCs) including macrophages, dendritic cells and B cells. In addition, solid organ may sometimes express HLA class II genes that participate in immune rejection. The HLA class I (HLA-I) protein is expressed on all nucleated cells and consists of an HLA class I heavy chain (or a chain) and p-2 microglobulin (B2M, 2M). HLA class I protein presents peptides on the cell surface to CD8+ cytotoxic T cells. Six HLA class I a chains have been identified todate, including three classical (HLA-A, HLA-B and HLA-C) and three non- classical (HLA-E, HLA-F and HLA-G) a chains. The specificity for peptide binding on the HLA class I molecule peptide binding cleft is determined by the a chain. Recognition by CD8+ T cells of the peptides presented by the HLA class I molecule mediates cellular immunity.

[0073] HLA class II molecules and class I molecules are both heterodimers. Class I molecules consist of an alpha chain (or heavy chain) and p-2 microglobuin (B2M), whereas the class II molecules consist of two homologous subunits: the alpha subunit and beta subunit.

[0074] HLA class II (HLA-II) molecules or proteins present on the cell surface peptide antigens from extracellular proteins including proteins of an extracellular pathogen, while HLA class I proteins present peptides from intracellular proteins or pathogens. Loaded HLA class II proteins on the cell surface interact with CD4+ helper T cells. The interaction leads to recruitment of phagocytes, local inflammation, and / or humoral responses through the activation of B cells. Several HLA class II gene loci have been identified to date, including HLA-DM (HLA-DMA and HLA-DMB that encode HLA-DM a chain and HLA- DM p chain, respectively), HLA-DO (HLA-DOA and HLA-DOB that encode HLA-DO a chain and HLA- DO p chain, respectively), HLA-DP (HLA-DPA and HLA-DPB that encode HLA- DP a chain and HLA- DP p chain, respectively), HLA-DQ (HLA-DQA and HLA-DQB that encode HLA-DQ a chain and HLA- DQ p chain, respectively), and HLA-DR (HLA-DRA and HLA-DRB that encode HLA-DR a chain and H LA-DR p chain, respectively).

[0075] The HLA class I and / or class II proteins from an allogeneic source constitutes a foreign antigen in the context of transplantation. The recognition of non-self HLA class I and / or class II proteins is a major hurdle in using pluripotent cells for transplantation or replacement therapies.

[0076] HLA class II deficient cells, HLA class I deficient cells, or HLA class l / class II deficient cells can be used as universal donor cells.Table 2: Protein sequences5.4 Modification methods

[0077] Methods of gene modification and editing are well known in the art, see for example [9],

[0078] Deleting or decreasing the expression of immune-activating genes in a cell can be achieved in a number of ways, e.g. by gene knockout. Gene knockout is a process that renders a particular gene inactive in the cell in which it resides, resulting either in no protein of interest being produced or an inactive form. Gene knockout can be achieved in a number of different ways, including removing nucleic acid sequences from a gene, or interrupting the sequence with other sequences, altering the reading frame, or altering the regulatory components of the nucleic acid. For example, all or part of a coding region of the gene of interest can be removed or replaced with "nonsense" sequences, all or part of a regulatory sequence such as a promoter can be removed or replaced, translation initiation sequences can be removed or replaced, etc. Gene editing technologies for reducing gene expression of target genes include TALEN, zinc fingers, Cas-CLOVER, and a CRISPR / Cas system, and / or the use of any known gene knockdown methods e.g. those that employ any of various RNA-based techniques (e.g. shRNA, antisense RNA, miRNA, siRNA [10,11],

[0079] Introducing or increasing expression of immune-tolerising genes can be achieved in a number of ways, e.g. by "gene knock in". Gene knock-in means a process that adds a genetic function to a host cell. This causes increased levels of the encoded protein, e.g. by adding one or more additional copies of the gene to the host cell or altering a regulatory component of the endogenous gene increasing expression of the protein is made. This may be accomplished by modifying the promoter, adding a different promoter, adding an enhancer, or modifying other gene expression sequences.

[0080] By "knock-out" in the context of a gene means that the host cell harbouring the knock-out does not produce a functional protein product of the gene. As outlined herein, a knock-out can result in a variety of ways, from removing all or part of the coding sequence, introducing frameshift mutations such that a functional protein is not produced (either truncated or nonsense sequence), removing or altering a regulatory component (e.g. a promoter) such that the gene is not transcribed, preventing translation through binding to mRNA, etc. Generally, the knock-out is affected at the genomic DNA level, such that the cells' offspring also carry the knock-out permanently.

[0081] By "knock in" in the context of a gene means that the host cell harbouring the knock in has more functional protein active in the cell. As outlined herein, a knock in can be done in a variety of ways, usually by the introduction of at least one copy of a transgene (tg) encoding the protein into the cell, although this can also be done by replacing regulatory components as well, for example by adding a constitutive promoterto the endogenous gene. In general, knock in technologies result in the integration of the extra copy of the transgene into the host cell.5.5 Modified cell

[0082] A cell may be modified by methods including gene modification (GM), gene editing (e.g. CRISPR-Cas, TALEN, zinc fingers, Cas-CLOVER), introduction of gene expression cassettes, lentiviral transduction, gene silencing technologies and RNA interference, RNA-based techniques (e.g. shR A, antisense RNA, miRNA, siRNA). Modified cells are artificial and do not occur in nature. The cell may be modified by non-essentially biological processes.5.6 Promoters

[0083] The human elongation factor 1 alpha (EF1a) promoter is described in Kim et al. [7], The CMV promoter is described in Barrow et al. [8], The CMV promoter is a powerful promoter and is non- selective for tissue type [9],5.7 Stem cells

[0084] For the avoidance of doubt, the human embryonic cells used in the disclosure can be derived from parthenogenetically activated human oocytes.

[0085] The stem cells may be pluripotent. The stem cells may not be totipotent.5.8 Unmodified cell

[0086] An unmodified cell may be a wild-type cell. A wild-type (WT) cell is a cell found in nature, particularly a cell that has not undergone engineering or modification. Promoters6 EXAMPLE 1 : METHODS6.1 Cell lines

[0087] Human ESC line 121 (SA121) was obtained from Takara Bio Europe AB. iPSC lines were a clone of the in-house derived iPSC clone B and a B2M-KO clone derived from clone B.6.2 HLA-E and HLA-G overexpressionB2M-fused HLA-E-T2A-Puro and B2M-fused HLA-G-T2A-Blast were cloned into a lentiviral construct. 50X concentrated lentivirus were generated by standard ultracentrifugation protocol. HLA-E and HLA- G expressing B2M-KO iPSC lines were then generated through lentiviral transduction. On day 0, 0.2x106 B2M-KO iPSCs were seeded in Matrigel coated 6-well plate in StemFlex medium (Gibco) supplemented with 10 pM of Y-27632 (StemCell Technologies 72302). On day 1 , 20 pL of 50X concentrated lentivirus were mixed with 2 pL polybrene (8 mg / ml) and then added to each well of iPSCs in 2 ml of StemFlex medium (final concentration of polybrene is 8 pg / ml). On day 2, medium was changed with fresh StemFlex medium. From day 5, antibiotic selection was performed with 0.5 pg / ml Puromycin and / or 5 pg / ml Blasticidin in StemFlex medium. Cells were split as needed during the 7-day selection period before checking expression and fluorescence cell sorting (FSCS).6.3 Fluorescence cell sorting (FACS)6.3.1 HLA-A,B,C expression (HeLa)

[0088] The wild type or gene knocked-out HeLa cells were pre-treated with or without 25 ng / mL interferon-gamma (IFNg) for 48-hour before being detached from culture plates using the TrypLE™ Express Enzyme (Gibco), spined down at 350 g for 5 minutes, and rinsed using FACS buffer (DPBS containing 1 % FBS). Cells were resuspended in FACS buffer at a density of <1x106cells per 100 pL. For each 100 pL of cell suspension, 5 pL Alexa Flour 647-conjugated mouse-anti-human HLA-A,B,C antibody (BioLegend 311414) and PE-conjugated mouse-anti-human HLA-DR antibody (BioLegend 307606) were added and well-mixed with cells. The cells were then incubated at 4 °C for 15 minutes, spined down at 350 g for 5 minutes and rinsed using 1 mL of FACS buffer. The stained cells were finally resuspended in FACS buffer at a density of <1x106cells per 100 pL for flow cytometry on the BD LSRFortessa™ cell analyzer to measure the HLA class I and HLA class II expression. The Flowjo software (v10.7) were used to analyse the data.6.3.2 HLA-E and HLA-G overexpression (iPSC & SA121)

[0089] The modified stem cells were detached from culture plates using the enzyme-free cell dissociation buffer (Gibco), spined down at 300 g for 5 minutes, and rinsed using FACS buffer (DPBS containing 1 % FBS) supplemented with 10 pM of Y-27632 (StemCell Technologies 72302). Cells were resuspended in FACS buffer supplemented with 10 pM of Y-27632 at a density of <1x106cells per 100 pL. For each 100 pL of cell suspension, 5 pL of each corresponding antibody (listed below, Table 2) were added and well-mixed with cells. The cells were incubated at 4 °C for 30 minutes, spined down at 300 g for 5 minutes and rinsed using 1 mL of FACS buffer. The stained cells were finally resuspended in FACS buffer supplemented with 10 pM of Y-27632 at a density of <1x106cells per 100 pL for Fluorescent cell sorting on the BD FACSAria™ Fusion Cell Sorter. The collected cells were seeded on Matrigel coated tissue culture-treated plates for expanding and further experiments. The Flowjo software (v10.7) were used to analyse the flow cytometry data.Table 3: Antibodies6.4 Flow cytometric analysis for trilineage, cardiomyocyte, endothelial cell and hepatocyte differentiation

[0090] For trilineage differentiation, the differentiated cells were washed once with DPBS and dissociated with Accutase for 3 min at 37 °C. Dissociated cells were transferred to U-bottom 96-well plates, washed twice in DPBS and stained using True-Nuclear Transcription Buffer Set (424401 , BioLegend) following the manufacturer’s instruction for 96-well U-bottom Plate. After permeabilization, the cells were incubated overnight with antibodies against lineage specific markers. Fluorophore- conjugated antibodies against SOX17, FOXA2 (for endoderm), CD56, Brachyury (for mesoderm), and PAX6 (for ectoderm) were used. The cells were washed 3 times and resuspended in DPBS for flow cytometric analysis. Cardiomyocytes were dissociated using STEMdiff Cardiomyocyte Dissociation Kit (05025, STEMCELL Technologies) following manufacturer’s instructions. Resuspended cardiomyocytes were stained using Human Cardiomyocyte Immunocytochemistry Kit (A25973, Thermo Fisher Scientific). Briefly, the cardiomyocytes were fixed in Fixative Solution for 15 min at room temperature, spun down, aspirated and then resuspended in Permeabilization Solution. The cells were permeabilized for 15 min at room temperature, spun down, aspirated, and resuspended and incubated in Blocking Solution for 30 min at room temperature. Primary antibodies were added directly to the Blocking Solution, mixed gently and incubated overnight at 4 °C. The cells were washed 3 times with Wash Buffer, resuspended in Blocking Buffer containing Secondary Antibodies (Alexa Fluor 488 donkey anti-mouse, A25972, or Alexa Fluor 647 donkey anti-mouse, A32787, and Alexa Fluor 594 donkey antirabbit, A25970). The cells were incubated at room temperature for 30 min in the dark. The cells were washed 3 times before analysis using flow cytometer. Dissociated cardiomyocytes were also stained with FITC anti-human CD47 Antibody (323106, BioLegend) and APC anti-human HLA-E Antibody (342605, BioLegend) for 10 min at room temperature in Cell Staining Buffer (420201 , BioLegend), washed once in Cell Staining buffer and resuspended in DPBS for flow cytometric analysis. After 22 days of hepatocyte differentiation, cells were dissociated with TrypLE Express Enzyme (Gibco) for 5- 10 min at 37 °C. Dissociated cells were stained with PE anti-human ASGPR1 antibody (563655, BD Pharmingen) using FACS buffer (DPBS containing 1 % FBS) for 15 minutes at room temperature. After washing with FACS buffer, the stained cells were analyzed on the BD LSRFortessa cell analyzer.6.5 xCelligence real-time based killing assay (NK cell & CTLs)

[0091] NK donor cells were isolated by the GB blood core from in-house RSCP donor material and grown at least 2 days in Advanced RPMI+10% FBS+1x GlutaMax supplemented with 200U / mL IL2. Stem cells were plated on 96-well Matrigel-coated plates at 35K / well in stem cell media (StemFlex) and left to recover and adhere overnight after plating. The next day, NK cells were added at a 1 :1 or 1 :2 E:Tratio and XCELLigence assay was run for 24-48 hours. The results were analysed by RTCA software Pro.6.6 Trilineage, cardiomyocyte and endothelial differentiation protocol

[0092] The human pluripotent stem cells (hPSCs, ESCs or iPSCs) were maintained in StemFlex Medium (A3349401 , Gibco). The cells were passaged in the medium supplemented with RevitaCell Supplement (100X) (A2644501 , Gibco) or 10 pM Y-27632 and onto the culture vessels coated with either hESC-qualified Matrigel (354277, Corning) or truncated recombinant human vitronectin (rhVTN- N, A14701 SA, Gibco). For trilineage differentiation, STEMdiff Definitive Endoderm Kit (05110, STEMCELL Technologies), STEMdiff Trilineage Etoderm Medium (05231 , STEMCELL Technologies) Mesoderm Induction Medium (05221 , STEMCELL Technologies) and STEMdiff trilineage differentiation kit were used for endoderm, mesoderm, and ectoderm differentiation, respectively. hPSCs were differentiated into cardiomyocyte using STEMdiff Cardiomyocyte Differentiation Kit (now STEMdiff Ventricular Cardiomyocyte Differentiation Kit, 05010, STEMCELL Technologies) and maintained by STEMdiff Cardiomyocyte Maintenance Kit (05020, STEMCELL Technologies). Briefly, dissociated cells were seeded onto culture vessels coated with hESC-qualified Matrigel. The medium was replaced following the manufacturer’s instructions and derived cells were analyzed after day 15. Endothelial differentiation and expansion was performed using STEMdiff Endothelial Differentiation Kit (08005, STEMCELL Technologies) and STEMdiff Mesoderm Induction Medium (05220, STEMCELL Technologies) following the manufacturer’s instruction with modifications. Briefly, hPSCs were seeded onto culture vessels coated with rhVTN-N. Mesoderm and endothelial induction, passaging and expansion were performed following the manufacturer’s instructions. MHC class II receptors’ expression were analysed after day 14 differentiation with 48 h 50 nM IFN-y treatment.6.7 Hepatocyte differentiation protocol

[0093] Human PSCs were seeded onto 12-well plate coated with CellAdhere™ Laminin-521 (200- 0117, STEMCELL Technologies) at a density of one million viable cells per well. Next day (day 0), RPMI medium supplemented with 3 uM CHIR99021 , 30 ng / ml activin A, and 0.2 % B27 (12587010, Thermo Fisher Scientific) was added to the cells to induce endoderm. CHIR99021 was removed from this medium on day 1 of differentiation. On day 5, hepatic progenitor formation was initiated with RPMI medium supplemented with 10 ng / ml FGF4, 10 ng / ml HGF, and 0.2 % B27 supplement for 4 days. At day 9, the hepatocyte maturation was induced by Williams’ E medium supplemented with 10 ng / ml HGF, 10 ng / ml oncostatin M, 10-7M dexamethasone, 15 mM Hepes, 1X ITS (41400045, Thermo Fisher Scientific) until day 22.6.8 Alloreactive cytotoxic T lymphocyte (allo-CTL) mediated killing assay

[0094] For evaluate the T cell resistance led by the ablation of the HLA class I Mitomycin C treated wild-type HeLa cells, wild-type stem cell-derived endothelial cells were used as feeder cells to develop the allo-CTLs. To further evaluate the additional resistance against the HLA-II resitricted T cells, A B2M knocked-out B cell line (Raji-A5) was used as feeder cells to develop the HLA-II restricted allo-CTL. For the stimulation of the allo-CTLs, the feeder cells were prepared as cell suspension with aconcentration of 4x105cells / mL and treated with Mitomycin C (50 pg / mL) for 10 minutes at 37 °C. Then, the cells were rinsed with RPMI-1640 basal medium and spined down for 5 times to completely remove the Mitomycin C. Primary human Naive CD8+ T cells were then co-cultured with the Mitomycin C treated feeder cells at a ratio of 2.5:1 for 8-10 days. Media exchange was performed every three days by removing half volume of the culture media and adding the same volume of fresh media (RPMI-1640 media supplanted with 10% FBS, 1 % penicillin / streptomycin, Ix GlutaMax) containing 20 unit / mL IL-2. After 10 days, the T cells were collected and co-cultured with freshly prepared feeder cells at a ratio of 2.5:1 for an additional 8-10 days to further enrich the allo-CTLs. After 2-round of stimulation, the allo- CTLs were used for allo-CTL killing assay or cryopreserved at -80 °C.

[0095] The xCELLigence Real-Time Cell Analysis (RTCA) system (Agilent) and the Celltiter-glo® 2.0 assay (Promega, G9241) was used to evaluate the cytolysis for the killing assay using adherent cells (e.g. the edited HeLa, iPS or ES cells) as target cells. Briefly, the cells were seeded on the RTCA E- Plate 96 (RTCA-based assay) or normal 96-well plate (Celltiter-glo) at a density of 15,000 cells / well (HeLa cells) or 35,000 cells / well (iPS or ES cells). After one day, the allo-CTL effector cells were added into each well at various effector-to-target (E-to-T) ratios. For RTCA-based assay, the real-time system monitored the cell index and calculate the % cytolysis automatically; for Celltiter-glo assay, the % cytolysis were calculated by the below equation:

[0096] The Celltiter-glo® 2.0 assay (Promega, G9241) was used to evaluate the cytolysis for the killing assay using edited Raji cells as target cells. Briefly, the HLA-II resitricted allo-CTLs (stimulated by HLA class I ablated Raji-A5 cells) were co-cultured with the HLA class l&ll ablated cells (in which the CIITA, RFXAP or RFXANK gene was further knocked out based on the Raji-A5 line) for 24 hours. TheCelltiter- glo® 2.0 assay was then performed following the manufacturer’s instruction. The % cytolysis were calculated by the abovementioned equation.6.9 Gene knock-out

[0097] CRISPR gene editing was performed according to standard protocols. The Alt-R™ S.p. Cas9 Nuclease V3 purchased from IDT was used for all CRISPR experiments. The target sequences for gene knockout are shown in Table 2.Table 4: Target Sequences for gene knockout

[0098] HeLa cells were detached from culture plates using the TrypLE™ Express Enzyme (Gibco), spined down at 350 g for 5 minutes, and rinsed using DPBS. Cells were resuspended in DMEM media (Gibco) containing 10% FBS at a density of 400,000 cells / mL. The Alt-R™ CRISPR sgRNAs targeting the TAP1 , TAP2 or B2M (target sequence see table 2) and Alt-R™ S.p. Cas9 Nuclease V3 were purchased from the Integrated DNA Technologies (IDT). To perform experiment in a well of 24-well plate, the ribonucleoprotein (RNP) complex containing the relevant sgRNA were incubated in 25 pL of opti-MEM media at room temperature for 5 minutes following by mixed 25 pL of opti-MEM media containing 3 uL of the Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen 13778075). The mixed solution was incubated at room temperature for 10-15 minutes before being added into 500 uL of the HeLa cell suspension. The well-mixed cell suspension containing a final concentration of 10 nM Cas9 and 10 nM of sgRNA were seeded on a well of 24-well plate and cultured for 24-48 hours before media exchange. After 5 days of regular culture and passaging, the engineered cells can be assessed for the HLA class I expression.

[0099] iPSCs or ES cells (SA121) were seeded on a Vitronectin (VTN-N, Gibco) coated 24-well plate at a density of 50,000 cells per well. Transfection of CRISPR components were conducted after 48 hours, when the cells reach ~70% confluency. To perform the experiment, the ribonucleoprotein (RNP) complex containing the relevant sgRNA were incubated in 25 pL of opti-MEM media at room temperature for 5 minutes following by mixed 25 pL of opti-MEM media containing 3 uL of the Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen 13778075) or Lipofectamine™ Stem Transfection Reagent (Invitrogen STEM00001). The mixed solution was incubated at room temperature for 10-15 minutes before being added into the wells of stem cells containing 500 uL of fresh StemFlex media (Gibco A3349401) containing 10 ug / mL Rock inhibitor (Y-27632, StemCell technologies Y- 27632). The cells were cultured in the well-mixed media containing a final concentration of 10 nM Cas9 and 10 nM of sgRNA for 24 hours before media exchange with 500 uL of fresh StemFlex media.

[0100] Raji cells were collected from normal culture and resuspended in Lonza SG 4D-Nucleofector™ X Solution (Lonza V4XC-3024) at a density of 500,000-1 ,000,000 cells per 80 uL. For transfection of CRISPR components, the ribonucleoprotein (RNP) complex containing approximately 25 pM of the relevant sgRNA and 20 pM of Cas9 were incubated at room temperature for 10 minutes following by being mixed well with the cell suspension. The cell solution was then transferred to the Lonza Single Nucleocuvette™ and the electroporation was conducted using the Lonza 4D-Nucleofector following thepre-defined protocol. After electroporation, the cells were transferred to 24-well plate containing 1 mL / well of the RPMI-1640 media containing 10% FBS for further recovering and regular culture.7 EXAMPLE 2: Reducing cytotoxic T cell mediated immunity7.1 H LA-class I mediated allo-immunity

[0101] To determine the minimal modifications required to produce a hypo-immunogenic cell, HeLa cells were modified by CRISPR gene editing to reduce expression of TAP1 , TAP2 and p2M. HLA-A (gRNA target sequences are provided in Table 2)., HLA-B and HLA-C expression was measured in the HeLa cells. Individual deletion I knock-out of all of the genes reduced HLA-A / B / C expression in the modified HeLa cells compared to wild-type HeLa cells (Figure 1). Deletion I knock-out of p2M reduced HLA-A / B / C expression in the modified HeLa cells compared to wild-type / non-modified HeLa cells by the greatest amount (Figure 1). Knock-out of p2M alone effectively abolished HLA-A / B / C expression. These data demonstrated that deletion of p2M gene expression was sufficient to abolish HLA-A / B / C expression without the need for further modification to the cell.

[0102] The ablation of HLA A / B / C expression by p2M knock-out translated into decreased susceptibility by the modified HeLa cells to alloreactive CD8 T cells (CTL) mediated cell killing. Knock out of p2M protected the modified HeLa cells from CTL medicated cytolysis (Figure 2 & Figure 3). Knock-out of p2M alone was therefore sufficient to tolerise the modified cells to CTL-mediated immunity. Surprisingly, TAP2 or TAP1 knock-out did not protect the cells from HLA class l-specific CTL-mediated immunity.7.2 HLA-class II mediated allo-immunity

[0103] HeLa cells, which are naturally non-HLA class II positive, were modified to reduce expression of RFXAP- and RFXANK and CIITA. RFXAP- and RFXANK-KO but not CIITA-KO resulted in decrease of HLA class I expression (HLA A / B / C). CIITA-, RFXAP- or RFXANK-KO all resulted in reduction of HLA class II expression in the presence of IFN-y stimulation (Figure 4).

[0104] The CIITA-, RFXAP- or RFXANK-KO also resulted in similar level of decrease of the HLA-DR expression in a naturally HLA class Il-positive cell type, Raji-A5 cells (Figure 5A). The CIITA-, RFXAP- or RFXANK-KO reduced the HLA-II restricted allo-CTL mediated cell killing at a similar level (Figure 5B). The Knock-out of any of CIITA, RFXAP or RFXANK alone therefore protected the cells from HLA class Il-specific killing.8 EXAMPLE 3: Reducing NK cell mediated immunity

[0105] iPSCs were modified to knock-out expression of p2M (using CRISPR-Cas9 gene editing) and to overexpress HLA-E, HLA-G or CD47 via lentiviral transduction. HLA-E and HLA-G overexpression in the context of p2M knock-out was demonstrated (Figure 6).

[0106] CD47 is often used to induce immunotolerance against NK cell mediated allo-immunity. Surprisingly, overexpression of an HLA-E gene and an HLA-G gene in combination protected againstNK cell mediated cytolysis by a greater extent than overexpression of a CD47 gene and an HLA-E gene in combination (Figure 7). Furthermore, the immune-tolerising effects of HLA-E and HLA-G overexpression were surprisingly non-redundant and synergistic, such that overexpression of only the combination of HLA-E and HLA-G abolished NK cell mediated allo-immunity without the need to additional overexpress a CD74 gene (Figure 7B).9 EXAMPLE 4: Pluripotent hypoimmunogenic stem cells

[0107] HLA class II is typically expressed only in antigen presenting cells (APCs) but not other somatic cells or stem cells. HLA class II can be induced upon inflammation (e.g. following stimulation with IFN- y exposure). Minimal HLA class II expression was observed in embryonic stem cells upon IFN-y treatment. As a positive control, modified and unmodified ESC were therefore differentiated into endothelial cells and treated with IFN-y to assess the effect of the modification on induction of HLA class II expression following stimulation with IFN-y. The embryonic stem cell line (SA121) was modified by knock-out of the p2M and RFXAP and to overexpress HLA-E and CD47 (clone 1 E11) (gRNA target sequences are provided in Table 2). As previously demonstrated in HeLa cells, p2M knockout abolished HLA class I (HLA-A / B / C) expression in the modified cells (Figure 8).

[0108] In contrast to the control cells, the RFXAP KO cells differentiated into endothelial (CD144+) cells did not show express HLA class II following IFN-y stimulation. Particularly, RFXAP knock-out reduced HLA class II (HLA-DP, DQ and DR) expression in response to IFN-y stimulation (Figure 9). Additionally, HLA-E and CD47 were successfully overexpressed using the EF1 a promoter, and overexpression was retained following differentiation (Figure 9).

[0109] Stem cells (ESC) that had been modified by p2M knockout, RFXAP knockout and HLA-E and CD47 overexpression were resistant to primed alloreactive T cell killing (Figure 10) and NK cell killing (Figure 11).

[0110] The modified cells also retained their pluripotency and were capable of differentiation into endoderm, mesoderm and ecotoderm with control wild-type or unmodified cells (Figure 12).

[0111] The modified cells were further capable of differentiation into cardiomyocytes, as measured by NKX2.5 and TNNT2 expression (Figure 13). Following differentiation into hepatocytes, the cells retained overexpression of the immune-tolerising genes CD47 and HLA-E (Figure 14).

[0112] The modified cells were also capable of differentiation into hepatocytes, as measured by ASGPR1 expression, a mature hepatocyte marker (Figure 15). Following differentiation into hepatocytes, the cells retained overexpression of the immune-tolerising genes CD47 and HLA-E (Figure 16).REFERENCES

[0113] All publications mentioned in the specification are herein incorporated by reference in their entirety.[1] J. L. Dashnau et al., Cytotherapy 25, 1 (2023).[2] C. A. Cowan et al., N Engl J Med 350, 1353 (2004).[3] W. Zhou and C. R. Freed, Stem Cells 27, 2667 (2009).[4] D. Huangfu et al., Nat Biotechnol 26, 795 (2008).[5] K. Woltjen et al., Nature 458, 766 (2009).[6] H. Zhou et al., Cell Stem Cell 4, 381 (2009).[7] D. W. Kim et al. , Gene 91 , 217 (1990).[8] K. M. Barrow, F. M. Perez-Campo, and C. M. Ward, Methods Mol Biol 329, 283 (2006).[9] M. I. Phillips and Y. L. Tang, Adv Drug Deliv Rev 60, 160 (2008).

[0010] J. K. W. Lam eta!., Mol Ther Nucleic Acids 4, e252 (2015).

[0011] K. Sridharan and N. J. Gogtay, Br J Clin Pharmacol 82, 659 (2016).

[0012] J. A. Doudna and E. Charpentier, Science 346, 1258096 (2014).

Claims

CLAIMS1 . A modified cell, wherein relative to an unmodified cell of the same cell type, the modified cell comprises: a) introduced or increased expression of a human leukocyte antigen (HLA)-E gene and an HLA-G gene; and b) deleted or decreased expression of a beta-2 microglobulin (p2M) gene.

2. A modified cell comprising an exogenous polynucleotide encoding an HLA-E gene and an exogenous polynucleotide encoding an HLA-G gene.

3. The modified cell of claim 1 or 2, wherein expression of the HLA-E gene and HLA-G gene is driven by an elongation factor 1 alpha (EF1 a) promoter.

4. The modified cell of any of claims 1 to 3, wherein expression of the HLA-E gene and HLA-G gene is driven by the same elongation factor 1 alpha (EF1 a) promoter.

5. A cell, wherein relative to an unmodified cell of the same cell type, the modified cell comprises: a) introduced or increased expression of a human leukocyte antigen (HLA)-E polypeptide and an HLA-G polypeptide; and b) deleted or decreased expression of a beta-2 microglobulin (p2M) polypeptide.

6. The modified cell of claim 5, wherein the HLA-E polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO 10 or a functional fragment thereof.

7. The modified cell of claim 5 or 6, wherein the HLA-G polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO: 11 or a functional fragment thereof.

8. The modified cell of any preceding claim, wherein relative to an unmodified cell of the same cell type, the modified cell comprises deleted or decreased expression of: a) a Class II Major Histocompatibility Complex Transactivator (CIITA) gene, b) a Regulatory factor X-associated ankyrin-containing protein (RFXANK) gene, and / or c) a Regulatory Factor X Associated Protein (RFXAP) gene.

9. The modified cell of claim 8, wherein the CIITA gene, RFXANK gene and / or RFXAP gene is human.

10. The modified cell of any preceding claim, wherein relative to an unmodified cell of the same cell type, the modified cell comprises introduced or increased expression of a CD47 gene.11 . The modified cell of any preceding claim, wherein relative to an unmodified cell of the same cell type, the modified cell comprises introduced or increased expression of a CD47 polypeptide.

12. The cell of claim 11 , wherein the CD47 polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO: 12 or a functional fragment thereof.

13. The modified cell of claims 1 to 9, wherein relative to an unmodified cell of the same cell type, the modified cell does not comprise introduced or increased expression of a CD47 gene.

14. The modified cell of any of claims 10 to 13, wherein the CD47 gene is human.

15. The modified cell of any preceding claim, wherein relative to an unmodified cell of the same cell type, the modified cell does not comprise decreased or deleted expression of: a) a CD24 gene, b) a CD74 gene, c) a CIITA gene, d) an HLA-A gene, e) an HLA-B gene, f) an HLA-C gene, g) an MIC-1 gene, h) an MIC-2 gene, i) an NLRC5 gene, j) a RFX5 gene, k) a RFXAP gene, l) a TAP1 gene, m) a TAP2 gene, n) a Tapasin gene, o) a TXNIP, and / or p) a RFXANK gene.

16. The modified cell of any preceding claim, wherein relative to an unmodified cell of the same cell type, the modified cell does not comprise increased or introduced expression of: a) a Ci-inhibitor gene, b) a CD24 gene, c) a CD46 gene, d) a CD55 gene,e) a CD59 gene, f) a CR1 gene, g) a CTLA-4-lg gene, h) a MANF gene, i) a TNFAIP3 gene, and / or j) a PD-L1 gene.

17. The modified cell of any preceding claim, wherein the modified cell is a mammalian cell.

18. The modified cell of any preceding claim, wherein the modified cell is a human cell.

19. The modified cell of any preceding claim, wherein the modified cell is an undifferentiated cell.

20. The modified cell of any preceding claim, wherein the modified cell is capable of self-renewal.21 . The modified cell of any preceding claim, wherein the modified cell is a stem cell.

22. The modified cell of any preceding claim, wherein the modified cell is a pluripotent stem cell.

23. The modified cell of any preceding claims, wherein the modified cell is an embryonic stem cell (ESC).

24. The modified cell of any preceding claim, wherein after differentiation into a different cell type the modified cell retains the modification or modifications.

25. The modified cell of claim 21 , wherein the cell is an induced pluripotent stem cell (iPSC).

26. The modified cell of claim 21 , wherein the cell is a multipotent stem cell.

27. The modified cell of claim 21 , wherein the cell is a haematopoietic stem cell (HSC).

28. The modified cell of any of claims 1 to 19, wherein the cell is a differentiated cell.

29. The modified cell of claim 28, wherein the cell is an endothelial cell.

30. The modified cell of claim 28, wherein the cell is a cardiomyocyte.

31. The modified cell of claim 28, wherein the cell is a hepatocyte.

32. The modified cell of claim 28, wherein the modified cell is a B cell.

33. The modified cell of any preceding claim, wherein the modified cell is an isolated cell.

34. The modified cell of any preceding claim, wherein the modified cell is an engineered cell.

35. The modified cell of any preceding claim, wherein the modified cell has been modified or engineered in vitro.

36. The modified cell of any preceding claim, wherein the modified cell has been modified or engineered ex vivo.

37. The modified cell of any preceding claim, wherein the modified cell does not occur in nature.

38. The modified cell of any preceding claim, wherein relative to an unmodified cell of the same cell type, the modified cell comprises reduced HLA class II protein expression following stimulation with IFN-y, optionally wherein HLA class II protein expression is determined by flow cytometry.

39. The modified cell of any preceding claim, wherein relative to an unmodified cell of the same cell type, the modified cell comprises reduced HLA-DR, HLA-DQ and / or HLA-DR expression following stimulation with IFN-y, optionally as determined by flow cytometry.

40. The modified cell of any preceding claim, wherein relative to an unmodified cell of the same cell type, the modified cell comprises reduced HLA class I expression, optionally as determined by flow cytometry.41 . The modified cell of any preceding claim, wherein the modified cell does not express HLA class I protein, optionally as determined by flow cytometry.

42. The modified cell of any preceding claim, wherein relative to an unmodified cell of the same cell type, the modified cell comprises increased expression of an HLA-E protein and an HLA-G protein, optionally as determined by flow cytometry.

43. The modified cell of any preceding claim, wherein relative to an unmodified cell of the same cell type, the modified cell comprises reduced HLA-A, B and C protein expression, optionally as determined by FACS.

44. The modified cell of any preceding claim, wherein the modified cell does not express HLA-A, B or C protein, optionally as determined by FACS.

45. The modified cell of any preceding claim, wherein relative to an unmodified cell of the same cell type, the modified cell is resistant to allo-cytotoxic lymphocyte mediated killing, optionally as determined by real-time based killing assay.

46. The modified cell of any preceding claim, wherein relative to an unmodified cell of the same cell type, the cell is resistant to allo-NK cell cytolysis, optionally as determined by real-time based killing assay.

47. The modified cell of any preceding claim, wherein the modified cell is hypoimmunogenic.

48. The modified cell of any preceding claim, wherein the modified cell does not activate NK cell mediated immunity, optionally as determined by real-time based killing assay.

49. The modified cell of any preceding claim, wherein the modified cell does not activate NK cell, T cell and / or macrophages mediated immunity.

50. The modified cell of any preceding claim, wherein the modified cell does not comprise a cytomegalovirus (CMV) promoter.51 . The modified cell of any preceding claim, wherein the modified cell is not a mesenchymal stem cell.

52. The modified cell of any preceding claim, wherein the modified cell is not an immune cell.

53. The modified cell of any preceding claim, wherein the modified cell is not a terminally differentiated cell.

54. The modified cell of any preceding claim, wherein the modified cell is not a T cell, NK cell, NKT cell or B cell.

55. The modified cell of any preceding claim, wherein the modified cell does not express a chimeric antigen receptor (CAR) or T-cell receptor (TCR).

56. The modified cell of any preceding claim, wherein relative to an unmodified cell of the same cell type, the modified cell has not been modified to be resistant to replicative senescence (RRS).

57. The modified cell of any preceding claim, wherein relative to a unmodified cell of the same cell type, the cell has not been modified to delete or reduce expression of cyclin dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-51- thioadenosine phosphorylase (MTAP).

58. The modified cell of any preceding claim, wherein relative to an unmodified cell of the same cell type, the modified cell has not been modified to delete or reduce expression of T-cell receptor a constant (TRAC).

59. The modified cell of any preceding claim, wherein relative to an unmodified cell of the same cell type, the modified cell has not been modified to increase expression of B-cell lymphoma-extra large (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

60. The modified cell of any preceding claim, wherein relative to a unmodified cell of the same cell type, the modified cell has not been modified to deleted or reduce expression of cluster of differentiation 38 (CD38) and / or phosphatase and tensin homolog (PTEN).

61. The modified cell of any preceding claim, wherein introduced or increased gene expression is determined by flow cytometry, western blot, qPCR or immunofluorescence.

62. The modified cell of any preceding claim wherein deleted or decreased gene expression is determined by flow cytometry, western blot, qPCR or immunofluorescence.

63. The modified cell of any preceding claim, wherein introduced or increased expression of the HLA-E gene and the HLA-G gene is determined by flow cytometry.

64. The modified cell of any preceding claim, wherein p2M gene expression is deleted or decreased by methods comprising gene editing the cell.

65. The modified cell of any preceding claim, wherein the gene editing comprises CRISPR-Cas9.

66. A population of cells comprising the modified cell of any of claims 1 to 65.

67. A pharmaceutical composition comprising the modified cell of any of claims 1 to 65 or the cell population of claim 66.

68. A pharmaceutical composition of claim 67, comprising a pharmaceutical excipient.

69. A method of making a pharmaceutical composition comprising combining the modified cell of an of claims 1 to 65 with a pharmaceutical excipient.

70. Use of a modified cell of any of claims 1 to 65 in the manufacture of a medicament for the treatment of prevention of disease in a subject.71 . A method for generating a modified cell, comprising a) introducing or increasing the expression of HLA-G in the cell, b) introducing or increasing the expression of HLA-E in the cell, and c) eliminating or decreasing the expression of one or both alleles of the p2M gene in a cell, wherein the cell is an undifferentiated cell.

72. The method of claim 71 , comprising eliminating the expression of both alleles of the CIITA, RFXANK and / or RFXAP genes in the cell.

73. The method of claim 71 or 72, wherein the cell is a stem cell, optionally an embryonic stem cell.

74. A modified cell, wherein the modified cell is manufactured according to the method of any of claims 71 to 73.

75. A lentivector for use in any of method of any of the claims 71 to 73.

76. The lentivector of claim 75, wherein the lentivector is an adenovirus vector.

77. A lentivector of claim 75 or 76 comprising an HLA-E gene and an HLA-G gene.

78. The lentivector of claim 77, wherein expression of the HLA-E gene and the HLA-G gene is controlled by a single EF1 a promoter.

79. The lentivector of any of claims 75 to 77, wherein the lentivector comprises a gRNA having the nucleotide sequence corresponding to any of SEQ ID NO: 1-8.

80. A method of treating or preventing a disease in a subject in need thereof, the method comprises administering to the subject the modified cell of any of claims 1-65, or a differentiated cell,progeny, daughter cell, or population of cells derived from the modified cell of any of claims 1- 65.81 . The method of claim 80, wherein the modified cell is derived from a cell that is not isolated from the subject.

82. The method of claim 80, wherein the modified cell is derived a cell isolated from the subject.

83. A method of treating or prevent a disease in a subject, the method comprising: a) producing in vitro a modified cell according to the method of any of claims 71 to 74, and b) administering modified cell to the subject.

84. A pharmaceutical composition for use in the method of claim 83, wherein the pharmaceutical composition comprises the modified cell of any of claims 1 to 65 and a pharmaceutical excipient.

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