Engineered cells expressing modified HLA-e polypeptide

Abstract

Provided are hypoimmunogenic cells and methods of manufacturing such.

Description

ENGINEERED CELLS EXPRESSING MODIFIED HLA-E POLYPEPTIDE1 REFERENCE TO THE SEQUENCE LISTING

[0001] This application incorporates by reference a Sequence Listing submitted with this application as text file entitled USC-101_seq listing, created on February 6, 2024, and having a size of 42.2 kilobytes.2 BACKGROUND

[0002] There are considerable challenges associated with generating allogenic cell therapies. After administration into immune-competent host, allogeneic cell therapies are recognized by recipient immune system, triggering an immune response that limits their survival and jeopardizes product efficacy. Different strategies have been employed to generate cell therapy products that can be administered to multiple patients without being rejected by host immune system. 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 cells may be unstable and not retained in progeny differentiated cells.

[0003] The present disclosure solves one or more of the above-mentioned problems.3 SUMMARY

[0004] The present disclosure relates to a modified HLA-E heavy chain polypeptide, a modified human leukocyte antigen (HLA)-E protein comprising the modified HLA-E heavy chain polypeptide and cells engineered to express the modified HLA-E protein. The disclosure is supported by data, provided herein for the first time, surprisingly demonstrating that cells engineered to express the modified HLA-E protein have a greater ability to avoid NK cell mediated allo-immunity than a cell of the type modified to overexpress wild-type HLA-E.4 BRIEF DESRIPTION OF THE DRAWINGSFigure 1 : Effect of TAP1, TAP2 and B2M knock-out on HLA A / B / C expression

[0005] Cell surface expression of classical HLA class I molecules following gene editing of HLA class I associated genes (TAP1 , TAP2, p2M). knock-out of p2M alone effectively abolished HLA-A / B / C expression. 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] T cell mediated killing as measured by Luciferase based killing assay (xCelligence). Knock out of p2M protected the modified HeLa cells from cytotoxic T cell (CTL) medicated cytolysis. KO: Knockout; MFI: mean fluorescence intensity; WT: Wild Type. Allo-CTLs derived from two donors were used.Figure 3: 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.

[0007] RFXAP- and RFXANK-KO but not CIITA-KO resulted in decrease of classical HLA class I expression (HLA A / B / C), as assessed by flow cytometry. CHTA-, RFXAP- or RFXANK-KO all resulted in reduction of HLA class II expression (HLA-DR) in the presence of IFN-y stimulation. KO: Knock-out.Figure 4: 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.

[0008] Figure 4A: HLA class II expression following gene knock-out in an HLA class l-ablated B cell line with p2M gene KO (RajiB2M Acell, clone A5). Figure 4B: 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 Acell, clone A5 from two donors were used. KO: Knock-out. C2TA: CIITA. Cell type = Raji cells.Figure 5: HLA-E and HLA-G overexpression

[0009] 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 6: HLA-E and HLA-G synergy

[0010] 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. Box plot shows distribution of NK killing of engineered stem cell by all donors from data in 7A.Figure 7: Engineering and expression of truncated HLA-E

[0011] Structure of HLA-E and truncated cytoplasmic domains. ECD: Extracellular domain. TM: transmembrane domain.Figure 8: Expression of truncated HLA-E in iPSC

[0012] The modified HLA-E molecules were expressed in IPSC. Expression was assessed by flow cytometry using PE-HLA-E antibody at 1 :20.Figure 9: Truncated HLA-E improved NK evasion

[0013] HLA-E-C2 was more potent than full-length HLA-E at evasion of NK-mediated immunity, as measured by NK-induced cytolysis (XCELLigence assay) of modified iPSC. Data from 5 NK donors. Graphs represent the data at 20 hours after adding the effector NK cells to the iPSC, ET: 1 :2. CD47 expression did not improve NK evasion more than the HLA-E / HLA-G combination.Figure 10: Effect of an alternative modified HLA-E on NK evasion

[0014] The modified HLA-E-C1 did not improve NK-resistance compared to wild-type HLA-E. 2x NK donors, NK-induced cytolysis (XCELLigence assay), E:T=1 :2.Figure 11 : (32M and HLA class I expression in a modified stem cell line

[0015] Flow cytometry assessing p2M and HLA class I expression. Modified cells: Clone 1 E11 . Control cells: SA121 (embryonic stem cell line).Figure 12: HLA class II, HLA-E and CD47 expression in a modified stem cell line after differentiation into endothelial cells

[0016] Modified cells: Clone 1 E11. Control cells: SA121. HLA class II expression was induced after interferon-gamma (IFNy) 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 13: Resistance to alloreactive CTL mediated cytolysis by modified stem cells

[0017] CD8+ T cell killing assay. 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 E:T = 4.1.E:T = 4.1. WT: Wild Type.Figure 14: Resistance to alloreactive NK cell killing by modified stem cells

[0018] Stem cells (ESC) that had been modified by p2M knockout, RFXAP knockout and HLA-E and CD47 overexpression were resistant to primed alloreactive NK cell killing. NK cell killing assay E:T = 1 :2. WT: Wild Type.Figure 15: Pluripotency of modified stem cells

[0019] 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 16: Differentiation of modified stem cells into cardiomyocytes

[0020] The modified cells were capable of differentiation into cardiomyocytes, as measured by NKX2.5 and TNNT2 expression. 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 17: Stable CD47 and HLA-E overexpression following differentiation of modified stem cells into cardiomyocytes

[0021] 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 18: Differentiation of modified stem cells into hepatocytes

[0022] The modified cells were capable of differentiation into hepatocytes, as measured by ASGPR1 expression, a mature hepatocyte marker. 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 19: Stable CD47 and HLA-E overexpression following differentiation of modified stem cells into hepatocytes

[0023] Following differentiation into hepatocytes, the cells retained overexpression of the immune- tolerising genes CD47 and HLA-E. 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.5 DETAILED DESCRIPTION

[0024] The disclosure relates to a modified human leukocyte antigen (HLA)-E heavy chain polypeptide comprising a truncated cytoplasmic domain relative to a wild-type HLA-E heavy chain polypeptide. The wild-type HLA-E heavy chain molecule may be human. The wild-type HLA-E heavy chain molecule may have an amino acid sequence identical to SEQ ID NO: 10. The wild-type HLA-E heavy chain molecule may have three regions: a cytoplasmic region, a transmembrane region and a highly conserved Ig-like domain which binds CD8. The modified HLA-E heavy chain polypeptide may comprise a cytoplasmic region, a transmembrane region and a highly conserved Ig-like domain. The truncated cytoplasmic domain may be truncated by at least 21 amino acid residues compared to the HLA-E heavy chain wildtype cytoplasmic domain, optionally wherein the truncated cytoplasmic domain is truncated by 21 amino acid residues compared to the HLA-E wild-type cytoplasmic domain. The HLA-E heavy chain wild-type cytoplasmic domain may consist of an amino acid sequence identical to SEQ ID NO: 15, or comprising 1 , 2, or 3 amino acid substitutions compared to said sequence. The truncated cytoplasmic domain may comprise an amino acid sequence consisting of less than 8 amino acid residues. The truncated cytoplasmic domain may comprise an amino acid sequence consisting of less than 7 amino acid residues. The truncated cytoplasmic domain may comprise or consists of about 6 amino acids residues. The amino acid sequence of the truncated cytoplasmic domain may consist of an amino acid sequence which is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17 or a functional fragment thereof, optionally wherein the amino acid sequence of the truncated cytoplasmic domain consists of an amino acid sequence which is 100% identical to SEQ ID NO: 17, or comprising 1 , 2, or 3 amino acid substitutions compared to said sequence. The amino acid sequence of the truncated cytoplasmic domain may consist of the amino acid sequence RAASSD, or comprising 1 , 2, or 3 amino acid substitutions compared to said sequence. The HLA-E polypeptide may comprise the amino acid sequence which is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 14 or a functional fragment thereof, optionally 100% identical to SEQ ID NO: 14, or comprising 1 , 2, or 3 amino acid substitutions compared to said sequence. The modified HLA-E heavy chain polypeptide may be isolated from the body. The modified HLA-E heavy chain polypeptide may be human. The wildtype HLA-E heavy chain polypeptide may be human.

[0025] The disclosure also relates to a modified HLA-E protein comprising the modified HLA-E heavy chain polypeptide of the disclosure. An HLA-E protein may include the HLA-E heavy chain and a light chain e.g., p2M, and be expressed on the cell surface with a signal peptide (also referred to as a single chain binding peptide). The modified HLA-E protein may comprise comprising a beta-2 microglobulin(p2M) polypeptide. In other words, the modified HLA-E protein may comprise a modified heavy chain and a p2M polypeptide. The p2M polypeptide may have an amino acid sequence at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18, optionally wherein the p2M polypeptide has an amino acid sequence 100% identical to SEQ ID NO: 18, or comprising 1 , 2, or 3 amino acid substitutions compared to said sequence. The modified HLA-E protein may also comprise a signal peptide (single chain binding peptide). The signal peptide may be bound by the peptide binding groove of the extracellular domain of the HLA-E protein. The modified HLA-E protein may be expressed on the surface of a cell with a bound signal peptide. The signal peptide may have an amino acid sequence at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 28, optionally wherein 100% identical to SED ID NO: 28, or comprising 1 , 2 or 3 amino acid substitutions compared to said sequence.

[0026] The disclosure also relates to a vector encoding a modified HLA-E heavy chain polypeptide of the disclosure. The disclosure also relates to a vector encoding a modified HLA-E protein of the disclosure. The vector may be a lentivector or other suitable vector for use in gene therapy. The vector may comprise a polynucleotide sequence at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23, optionally 100% identical to SEQ ID NO: 23, or comprising 1 , 2, or 3 nucleotide substitutions compared to said sequence.

[0027] The disclosure also relates to a modified cell, wherein the modified cell expresses an HLA-E protein of the disclosure. The HLA-E protein may be expressed on the surface of the cell, optionally as measured by flow cytometry.

[0028] The disclosure also relates to a modified cell comprising a vector of the disclosure.

[0029] The cell may be modified to avoid HLA class I mediated allo-immunity. The modified cell may comprise deleted or decreased expression of an endogenous p2M gene relative to an unmodified cell of the same cell type. The modified cell may comprise deleted or decreased expression of both alleles of the endogenous p2M gene relative to an unmodified cell of the same cell type. The modified cell may comprise introduced or increased expression of an HLA-G gene relative to an unmodified cell of the same cell type. The modified cell may comprise an exogenous polynucleotide encoding the HLA- E protein of the disclosure. The modified cell may comprise an exogenous polynucleotide encoding an HLA-G gene. The HLA-G polypeptide may comprise an amino acid sequence which is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11 or a functional fragment thereof, optionally wherein the HLA-G polypeptide comprises an amino acid sequence 100% identical to SEQ ID NO: 11 , or comprising 1 , 2, or 3 amino acid substitutions compared to said sequence.

[0030] Other methods for avoiding HLA class I mediated allo-immunity exist in the art, for example deletion or reduced expression of NLRC5 and / or CD38.

[0031] The cell may be modified to avoid HLA class II mediated allo-CTL killing. For example, relative to an unmodified cell of the same cell type, the modified cell may comprise deleted or decreased iexpression of a Class II Major Histocompatibility Complex Transactivator (CIITA) gene, e.g. reduced by at least 70, 80, 81 , 82, 83, 84, 85, 86, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99%, optionally by 100%, e.g. as measured by flow cytometry. Relative to an unmodified cell of the same cell type, the modified cell may comprise deleted or decreased expression of a Regulatory factor X-associated ankyrin-containing protein (RFXANK) gene, e.g. reduced by at least 70, 80, 81 , 82, 83, 84, 85, 86, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99%, optionally by 100%, e.g. as measured by flow cytometry. Relative to an unmodified cell of the same cell type, the modified cell may comprise deleted or decreased expression of a Regulatory Factor X Associated Protein (RFXAP) gene, e.g. reduced by at least 70, 80, 81 , 82, 83, 84, 85, 86, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99%, optionally by 100%, e.g. as measured by flow cytometry. Relative to an unmodified cell of the same cell type, the modified cell may comprise deleted or decreased expression a CIITA gene, and a RFXANK gene or RFXAP gene, e.g. reduced by at least 70, 80, 81 , 82, 83, 84, 85, 86, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99%, optionally by 100%, e.g. as measured by flow cytometry. Relative to an unmodified cell of the same cell type, the modified cell may comprise deleted or decreased expression of a CIITA gene, and a RFXANK gene or RFXAP gene, e.g. reduced by at least 70, 80, 81 , 82, 83, 84, 85, 86, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99%, optionally by 100%, e.g. as measured by flow cytometry. Relative to an unmodified cell of the same cell type, the modified cell may comprise deleted or decreased expression of a CIITA gene, a RFXANK gene and a RFXAP gene, e.g. reduced by at least 70, 80, 81 , 82, 83, 84, 85, 86, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99%, optionally by 100%, e.g. as measured by flow cytometry. Relative to an unmodified cell of the same cell type, the modified cell may comprise introduced or increased expression of a CD47 gene, e.g., reduced by at least 70, 80, 81 , 82, 83, 84, 85, 86, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99%, optionally by 100%, e.g. as measured by flow cytometry. Relative to an unmodified cell of the same cell type, the modified cell may comprise introduced or increased expression of a CD47 polypeptide, e.g., reduced by at least 70, 80, 81 , 82, 83, 84, 85, 86, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99%, optionally by 100%, e.g. as measured by flow cytometry.

[0032] The CD47 polypeptide may comprise an amino acid sequence which is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12 or a functional fragment thereof, or comprising 1 , 2, or 3 amino acid substitutions compared to said sequence.

[0033] 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.

[0034] Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of a CD24 gene, a CD74 gene, a CIITA gene, an HLA-A gene, an HLA-B gene, an HLA-C gene, an MIC-1 gene, an MIC-2 gene, an NLRC5 gene, a RFX5 gene, a RFXAP gene, a TAP1 gene, a TAP2 gene, a Tapasin gene, a TXNIP gene, and / or a RFXANK gene, e.g. not reduced by at least 70, 80, 81 , 82, 83, 84, 85, 86, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99%, optionally by 100%, e.g. as measured by flow cytometry. 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, a CD24 gene, a CD46 gene, a CD55 gene, a CD59 gene, a CR1 gene, a CTLA-4-lg gene, a MANF gene, a TNFAIP3 gene, a PD-L1 gene, GGTA1 gene, CMAG gene, SLA-1 alpha chain gene, and / or B4galNT2 gene, .

[0035] 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 endogenous 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 gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of a RFXANK 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 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 CD46 gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of a CD55 gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of a CD59 gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of a CR1 gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of a CTLA-4-lg gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of a MANF gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of a TNFAIP3 gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of a PD- L1 gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprisedecreased or deleted expression of a GGTA1 gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of a CMAG gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of an SLA-1 alpha chain gene. Relative to an unmodified cell of the same cell type, the modified cell may not comprise decreased or deleted expression of a B4galNT2 gene.

[0036] Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type HLA-B gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type HLA-A gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type HLA-C gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type TAP gene.

[0037] Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type CD24 gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wildtype CD74 gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type a CIITA gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type HLA-A gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type HLA-B gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type HLA-C gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type MIC-1 gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type MIC-2 gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type NLRC5 gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type RFX5 gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type RFXAP gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type TAP1 gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type TAP2 gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wildtype Tapasin gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type TXNIP gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type RFXANK gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type C1 -inhibitor gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type CD24 gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type CD46 gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type CD55 gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type CD59 gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type CR1 gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type CTLA-4-lg gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type MANF gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type a TNFAIP3 gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type PD-L1 gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-typeIGGTA1 gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type CMAG gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type SLA-1 alpha chain gene. Relative to an unmodified cell of the same cell type, the modified cell may comprise a wild-type B4galNT2 gene.

[0038] The modified cell may be a mammalian cell. The modified cell may be a human cell. The modified cell may not be a pig cell. The modified cell may not be porcine cell. The modified cell may be an undifferentiated cell. The modified cell may be 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 cell type the modified cell may retain the modification or modifications. After differentiation into a different cell type the modified may still express the modified HLA-E protein on the cell surface. After terminal differentiation, the modified may still express the modified HLA-E protein on the cell surface. 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 isolated cell. The modified cell may be an engineered cell.

[0039] The modified cell may have been modified or engineered in vitro. The modified cell may have been modified or engineered ex vivo. The modified cell may not occur in nature.

[0040] Relative 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 as described in the Examples. HLA class II protein expression may be reduced by at least 70, 80, 81 , 82, 83, 84, 85, 86, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99%, optionally by 100%, e.g. as measured by flow cytometry. Relative 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 as described in the Examples. Relative 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 endogenous HLA class I protein, optionally as determined by flow cytometry. The modified cell may not express endogenous HLA-A, B and C protein, optionally as determined by flow cytometry. The modified cell may not express endogenous classical HLA class I protein. Relative to an unmodified cell of the same cell type, the modified cell may comprise increased expression of an HLA- G protein, optionally as determined by flow cytometry. Relative 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. HLA-A, B and C protein expression may be reduced by at least 70, 80, 81 , 82, 83, 84, 85, 86, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99%, optionally by 100%, e.g. as measured by flow cytometry.

[0041] The modified cell does may not express HLA-A, B or C protein, optionally as determined by FACS. Relative to an unmodified cell of the same cell type, the modified cell may be resistant to allo- cytotoxic lymphocyte mediated killing, optionally as determined by real-time based killing assay.I

[0042] Relative 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 modified cell may be hypoimmunogenic. The modified cell may be suitable for administration to a non-HLA matched donor without the need for immunosuppressive 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 NK cell mediated immunity in vitro. The modified cell may not activate NK cell mediated immunity following administration to a non-HLA matched donor. The modified cell may not activate NK cell, T cell and / or macrophages mediated immunity. The modified cell may not activate NK cell, T cell and / or macrophages mediated immunity following administration to a non-HLA matched donor. Relative to an unmodified cell of the same cell type, the modified cell may not have been modified to be resistant to replicative senescence (RRS). Relative to an unmodified cell of the same cell 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-5'-thioadenosine phosphorylase (MTAP). Relative to an unmodified cell of the same cell type, the modified cell may not have been modified to delete or reduce expression of T-cell receptor a constant (TRAC). Relative to an unmodified cell of the same cell 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). Relative to an unmodified cell of the same cell type, the modified cell may not have been modified to deleted or reduce expression of cluster of differentiation 38 (CD38). Relative to an unmodified cell of the same cell type, the modified cell may not have been modified to have 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 flow cytometry, western blot, qPCR or immunofluorescence. Deleted or decreased gene expression may be determined by flow cytometry, western blot, qPCR or immunofluorescence. p2M endogenous gene expression may be deleted or decreased by methods comprising gene editing the cell. Gene editing may comprise CRISPR-Cas9 or TALEN. The disclosure also relates to a population of cells comprising a modified cell of the disclosure.

[0044] The disclosure also relates to pharmaceutical compositions comprising a modified cell of the invention. The disclosure also relates to pharmaceutical compositions comprises the population of cells of the disclosure. The pharmaceutical composition may comprise a pharmaceutical excipient. The disclosure also relates to a method of making a pharmaceutical composition comprising combining a modified cell of the disclosure with a pharmaceutical excipient. 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. The disclosure also relates to a method for manufacturing a modified cell, comprising introducing or increasing the expression of the modified HLA-E heavy chain polypeptide of the disclosure or the modified HLA-E protein of the disclosure, into the cell. The method may comprise eliminating or decreasing the expression of one or both alleles of the endogenous p2M gene in a cell. The method may comprise eliminating the expression of both alleles of the CIITA, RFXANK and / or RFXAP genes in the cell, optionally by gene editing, optionally by CRISPR-cas9 gene editing or TALEN.The cell may be a modified a stem cell, optionally an embryonic stem cell or induced pluripotent stem cell. The method may comprise differentiating the cell into a progenitor cell, terminally differentiated cell or somatic cell. The cell may be a progenitor cell, terminally differentiated cell or somatic cell.

[0045] The disclosure also relates to a modified cell, wherein the modified cell is manufactured according to the method of the disclosure.

[0046] The disclosure also relates to a vector for use in any of method of the disclosure, optionally wherein the vector is a lentivector. The lentivector may be an adenovirus vector.

[0047] The lentivector may comprise a gRNA having the nucleotide sequence corresponding to any of SEQ ID NO: 1 to 8. 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 a modified cell of the disclosure, or a differentiated cell, progeny, daughter cell, or population of cells derived from a modified cell of the disclosure. The modified cell may be derived from a cell that is not isolated from the subject.

[0048] The disclosure also relates to a method of treating or prevent a disease in a subject, the method comprising: producing in vitro a modified cell according to the method of the disclosure, and administering modified cell to the subject.

[0049] Expression of the HLA-E protein in the modified cell may be driven by an elongation factor 1 alpha (EF1 a) promoter. Expression of the HLA-E protein in the modified cell may be driven by the same elongation factor 1 alpha (EF1 a) promoter. 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.

[0050] The cell may a mesenchymal stem cell. The cell may an immune cell. The cell be a terminally differentiated cell. The cell may be a T cell. The cell may be an NK cell. The cell be a NKT cell. The cell may be a B cell. 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.

[0051] The cell may express a chimeric antigen receptor (CAR) or T cell receptor. The cell may not express a chimeric antigen receptor or T-cell receptor (TCR).

[0052] 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-5'- 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 toa 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).

[0053] 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 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.

[0054] The disclosure also relates to a population of cells comprising one or more of the modified cells of the disclosure. 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 administration to 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. 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.

[0055] 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.

[0056] 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. 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. The disclosure also relates to pharmaceutical composition for use in the methods of treatment.

[0057] 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 T O4, T O5, T O6, T O7, T O8, T O9, or 1010engineered immune cells of the disclosure. 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. 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.

[0058] 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 a modified HLA-E protein and knockout or decrease expression of a p2M gene. 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. 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. 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. 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.

[0059] The modified cell may be derived from a cell or cell comprising a stem cell. 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.

[0060] The cell population may be 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.

[0061] 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, Regen Ther. 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, MY0D1 , 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.5.1 Cell potency

[0062] 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 foetal, 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].

[0063] 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 certain regulatory genes or by the exogenous application of certain proteins. Methods for the induction of IPS cells are known in the art [3-6].

[0064] 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.

[0065] 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

[0066] 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

[0067] 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 [7]. CRISPR techniques and kits are sold commercially e.g. Alt-R™ S.p. Cas9 Nuclease V3 purchased from IDT.

[0068] Cells of the disclosure may be modified, engineered or manipulated using TALEN technologies as is known in the art. TALEN are restriction enzymes combined with a nuclease that can be engineered to bind to and cut practically any desired DNA sequence. 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 within complex 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 Table 1 : Summary of genes5.4 Human leukocyte antigen (HLA)

[0069] 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). 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 nonself HLA class I and / or class II proteins is a major hurdle in using pluripotent cells for transplantation or replacement therapies. 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.5.4.1 HLA class I

[0070] HLA class I molecules consist of two non-covalently linked polypeptide chains, an HLA- encoded a chain or heavy chain (44 to 47 kD), and a non-HLA encoded subunit called p2microglobulin (p2M) (12 kD). The heavy chain has three extracellular domains (a1-3), a transmembrane region and a C-terminal cytoplasmic tail. The a1 and a2 domains of the a chain are the recognition region, forming a peptide antigen biding groove between them. The highly conserved a3 immunoglobulin-like domain is the region where CD8 binds. The p2M chain associates primarily with the a3 domain and is necessary for HLA stability.

[0071] The HLA class I protein is expressed on all nucleated cells. Classical HLA class I protein presents peptides on the cell surface to CD8+ cytotoxic T cells. Six HLA class I heavy (a) chains have been identified to date, including three classical (HLA-A, HLA-B and HLA-C) and three non- classical (HLA-E, HLA-F and HLA-G) heavy chains. The specificity for peptide binding on the HLA class I molecule peptide binding cleft is determined by the heavy chain. Recognition by CD8+ T cells of the peptides presented by the HLA class I molecule mediates cellular immunity.

[0072] HLA-G plays an immunomodulatory role by binding the inhibitory receptors, HLA-G inhibits the cytotoxic activity of T CD8 + and NK cells through direct interaction with leukocyte receptors, such as LILRB1 (LIR1 / ILT2), LILRB2 (ILT4), and KIR2DL4 (CD158d). The HLA-G primary transcript encodes seven different heavy isoforms through alternative splicing. 4 are membrane-associated, and 3 are secreted. HLA-G1 and -G5 are the only isoforms of HLA-G that can bind p2M [8].

[0073] The HLA-E molecules bind nonameric self-peptides derived from the leader sequences of the various HLA class I molecules. As a ligand for the CD94 / NKG2 receptors on NK cells and for the TCR on NKT cells, HLA-E molecules are involved in both innate and adaptive immunity. The HLA-E CD94 / NKG2 A / C system modulates either inhibition or activation of the NK cell-mediated cytotoxicity and cytokine production. The HLA-E molecules may bind nonameric self-peptides derived from the leader sequences of the various HLA class I molecules. Example peptide ligands of HLA-E are known in the art, see for example Nadine & Purcell, Encyclopedia of Immunology, Vol. 2, 2016, pages 215- 219 [9],

[0074] A large proportion of peptides bound to HLA-E are derived from the signal peptides of other MHC class I molecules, including HLA-A, HLA-B, HLA-C, and HLA-G. These are generally amino acid residues 3-11 of the signal sequence. These peptides are cleaved from the class I molecule during translocation into the ER and are loaded in a TAP- and tapasin-dependent fashion into the cleft of HLA- E. Loading of HLA-E with signal sequence peptides directs trafficking of HLA-E molecules and the peptides are the required cofactors for the binding of HLA-E to the NK receptors CD94-NKG2A (inhibitory), CD94-NKG2B, and CD94-NKG2C (activating). Downregulation of classical HLA class I interferes with the signal sequence peptide supply and subsequent expression of HLA-E on the cell surface. This eliminates the inhibitory signal for the CD94 / NKG2A receptor, promoting clearance of affected host cells by cytolytic effectors such as NK cells. Although HLA-E selectively presents peptides from the leader sequences of HLA class I molecules, HLA-E also binds other noncanonical peptides. When expression of HLA-E is lost, the CD94 / NKG2A receptor no longer sends inhibitory signals. Thus, the CD94 / NKG2A receptor provides a means for NK cells to monitor the overall expression of MHC class I on a tissue and prevent the escape of a tumor or virally infected cell through down-regulation of MHC class I molecules.5.4.2 HLA class II

[0075] 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.

[0076] Class II HLA molecules are composed of two non-covalently associated polypeptide chains, a 32 to 34 kD a chain, and a 29 to 32 kD p chain. The genes encoding both chains of class II molecules are polymorphic and present in the HLA locus. Both chains have three regions including an extracellular region containing TCR recognition (a1 and pi ) and CD4 binding (a2 and 2) domains, a transmembrane region containing hydrophobic amino acids by which the molecule is anchored in the cell membrane, and a short cytoplasmic region. The peptide binding groove forms between a1 and pi domains.

[0077] 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 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 HLA-DR p chain, respectively).5.5 Sequences

[0078] The polypeptide or the protein of the disclosure also relates to the sequences presented in Table 2, and sequences comprising 1 , 2, or 3 amino acid substitutions compared to said sequences. The polypeptide or the protein of the disclosure may comprise any of SEQ ID NO: 10, 13, 14, 15, 16, 17, 18, 28 and / or 29.Table 2: Amino acid sequences1 / 1 / 7": wild-type

[0079] The polypeptide or the protein of the disclosure may comprise any of the sequences presented in Table 3, and sequences comprising 1 , 2, or 3 nucleotide substitutions compared to said sequences.Table 3: Polynucleotide sequences5.6 Modification methods

[0080] Methods of gene modification and editing are well known in the art, see for example

[0010] . 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 regulatorysequence 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 [11 ,12]. 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.

[0081] 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.

[0082] 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 promoter to the endogenous gene. In general, knock in technologies result in the integration of the extra copy of the transgene into the host cell.5.7 Modified cell

[0083] 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. shRNA, antisense RNA, miRNA, siRNA). Modified cells are artificial and do not occur in nature. The cell may be modified by non-essentially biological processes.5.8 NK cell activation and non-classical HLA molecules

[0084] Human NK cells comprise the first line of defence of the innate immune system and are also involved in adaptive immunity. NK cells discriminate self and non-self using a variety of cell-surface receptors which interact with the ligands on target cells. The balance of inhibitory and activating signals determines NK cell activation or inhibition.

[0085] A strategy for avoiding T cell allo-immunity is deletion of HLA class I expression. However, HLA class I deletion can sensitise the modified cells to NK mediated immunity. Deletion of P2M, for example,prevents the surface expression of nonpolymorphic nonclassical HLA class lb molecules HLA-E and HLA-G, which are required to maintain NK cell tolerance.5.9 Promoters

[0086] The human elongation factor 1 alpha (EF1a) promoter is described in Kim et al.

[0013] . The CMV promoter is described in Barrow et al.

[0014] . The CMV promoter is a powerful promoter and is non- selective for tissue type

[0010] .5.10 Stem cells

[0087] For the avoidance of doubt, the human embryonic cells used in the disclosure can be derived from parthenogenetically activated human oocytes. The stem cells may be pluripotent. The stem cells may not be totipotent.5.11 Unmodified cell

[0088] 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.6 EXAMPLE 1 : METHODS6.1 Cell lines

[0089] 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 (or HLA-E-C1 / C2-T2A-Puro) and B2M-fused HLA-G-T2A-Blast were cloned into a lentiviral construct. The HLA-E and HLA-G proteins were expressed with a single chain peptide. 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-activated cell sorting (FACS).6.3 Fluorescence-activated cell sorting (FACS)6.3.1 HLA-A,B,C expression (HeLa)

[0090] The wild type or gene knocked-out HeLa cells were pre-treated with or without 25 ng / mL interferon-gamma (IFNy) for 48-hour before being detached from culture plates using the TrypLE™ Express Enzyme (Gibco), spun down at 350 g for 5 minutes, and rinsed using FACS buffer (DPBScontaining 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, spun 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 & SA 121)

[0091] The modified stem cells were detached from culture plates using the enzyme-free cell dissociation buffer (Gibco), spun 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 <1 x106cells per 100 pL. For each 100 pL of cell suspension, 5 pL of each corresponding antibody (listed below, Table 4) were added and well-mixed with cells. The cells were incubated at 4 °C for 30 minutes, spun 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 4: Antibodies6.4 Flow cytometric analysis for trilineage, cardiomyocyte, endothelial cell and hepatocyte differentiation

[0092] 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)

[0093] NK donor cells were isolated by the GB blood core from in-house RSCP donor material and grown at least 1 day 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:T ratio 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

[0094] 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

[0095] 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 pM 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

[0096] 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 restricted 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 development of the allo-CTLs, the feeder cells were prepared as cell suspension with a concentration 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 spun down for 5 times to completely remove the Mitomycin C. Primary human Naive CD8+ T cells were then co-cultured with the Mitomycin C treatedfeeder 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, 1x 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 development, the allo- CTLs were used for allo-CTL killing assay or cryopreserved at -80 °C.

[0097] The xCELLigence Real-Time Cell Analysis (RTCA) system (Agilent) and the Celltiter-glo® 2.0 assay (Promega, G9241) were 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:% cytolysis = ((bioluminscence no effector - bioluminscence effector) / bioluminscence no effector) X 100

[0098] 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. Brief ly, the HLA-II restricted 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. The Celltiter- 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

[0099] 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 5.Table 5: Target Sequences for gene knockout

[0100] HeLa cells were detached from culture plates using the TrypLE™ Express Enzyme (Gibco), spun 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.

[0101] 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.

[0102] 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 the pre-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 HLA-class I mediated allo-immunity

[0103] 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 or p2M(gRNA target sequences are provided in Table 5). HLA-A, HLA-B and HLA-C expression was measured in the HeLa cells following editing. Individual deletion / knock-out of the genes reduced HLA-A / B / C expression in the modified HeLa cells compared to wild-type HeLa cells, as measured by flow cytometry (Figure 1). Deletion / knock-out of the endogenous p2M gene 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). Surprisingly, 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.

[0104] The ablation of HLA A / B / C expression on the cell surface 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 mediated cytolysis (Figure 2). 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

[0105] HeLa cells were modified to reduce expression of RFXAP- and RFXANK and CIITA. HLA-II expression was assessed in the presence of IFN-y stimulation. RFXAP- and RFXANK-KO but not CIITA-KO resulted in decreased expression of classical HLA class I molecules (HLA A / B / C). CIITA-, RFXAP- or RFXANK-KO all resulted in reduction of HLA class II (HLA-DR) expression in the presence of IFN-y stimulation (Figure 3). The CIITA-, RFXAP- or RFXANK-KO also resulted in similar decrease of HLA-DR expression in a naturally HLA class Il-positive cell type, Raji-A5 cells (Figure 4A). The CIITA-, RFXAP- or RFXANK-KO reduced the HLA-II restricted allo-CTL mediated cell killing at a similar level (Figure 4B). 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

[0106] 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 5).

[0107] CD47 is often used to induce immunotolerance against NK cell mediated allo-immunity. Surprisingly, overexpression of an exogenous HLA-E protein and an exogenous HLA-G protein in combination protected against NK cell mediated cytolysis by a greater extent than overexpression of a CD47 gene and an HLA-E gene in combination (Figure 6). Furthermore, the immune-tolerizing effectsof 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 CD47 gene in this assay. However, this strategy for Immuno-tolerising required the use of two sperate overexpression vectors, encoding exogenous HLA- E protein and HLA-G protein respectively.9 EXAMPLE: 4: Truncated HLA-E for improved NK inhibitory potency

[0108] To further improve the NK-evasion of the modified cells, the inventors engineered new configurations of HLA-E, “truncated HLA-E”: HLA-E-C1 and HLA-E-C2. The sequences of HLA-E and the truncated HLA-Es are shown in in Table 2 and Table 3. HLA-E-C1 was truncated more than HLA- E-C2 and comprised 3 amino acid residue substitutions. The structures of the truncated HLA-Es are shown in Figure 7. The truncated HLA-Es were successfully expressed in iPSCs (Figure 8).

[0109] Surprisingly, truncated HLA-E-C2 was shown to be more potent than full-length HLA-E at evading NK-mediated immunity (Figure 9). The ability of the truncated HLA-E-C2 to inhibit NK immunity was compared to the HLA-G / HLA-E combination. HLA-E-C2 was as effective at inhibiting NK-induced cytolysis as the HLA-E / HLA-G combination. The use of HLA-E-C2 without overexpression of HLA-G has the advantage of reducing the amount of cell modifications needed. The addition of CD47 to the HLA-E / HLA-G combination did not provide any additional benefit to NK immunity evasion compared to the HLA-G / HLA-E combo (Figure 9).

[0110] The alternative modified HLA molecule was also tested for its ability to improve NK resistance, “HLA-E-C1 ”, in which the remaining sequence of the truncated HLA-E cytoplasmic chain was also modified. However, while HLA-E-C2 showed benefit for improving NK resistance when compared with unmodified HLA-E, HLA-E-C1 surprisingly did not (Figure 10). These data demonstrate that the sequence of the truncated cytoplasmic domain is relevant to HLA-E function.10 EXAMPLE 5: Pluripotent hypoimmunogenic stem cells

[0111] 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). Minimal HLA class II expression was observed in embryonic stem cells upon IFN-y treatment. As a positive control, modified and unmodified ESC were differentiated into endothelial cells and treated with IFN-y to assess the effect of the modification on induction of HLA class II expression. 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 5). As previously demonstrated in HeLa cells, p2M knockout abolished HLA class I (HLA-A / B / C) expression in the modified cells (Figure 11 ).

[0112] In contrast to the control cells, the RFXAP KO cells differentiated into endothelial (CD144+) cells did not show HLA class II expression 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 12).JOAdditionally, HLA-E and CD47 were successfully overexpressed using the EF1a promoter, and overexpression was retained following differentiation (Figure 12).

[0113] 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 13) and NK cell killing (Figure 14).

[0114] The modified cells also retained their pluripotency and were capable of differentiation into endoderm, mesoderm and ectoderm as control wild-type or unmodified cells (Figure 15).

[0115] The modified cells were further capable of differentiation into cardiomyocytes, as measured by NKX2.5 and TNNT2 expression (Figure 16). Following differentiation into hepatocytes, the cells retained overexpression of the immune-tolerising genes CD47 and HLA-E (Figure 17).

[0116] The modified cells were also capable of differentiation into hepatocytes, as measured by ASGPR1 expression, a mature hepatocyte marker (Figure 18). Following differentiation into hepatocytes, the cells retained overexpression of the immune-tolerising genes CD47 and HLA-E (Figure 19).11 EXAMPLE 5: EmbodimentsThe following clauses define embodiments of the disclosure.Clause 1 ) A modified human leukocyte antigen (HLA)-E heavy chain polypeptide comprising a truncated cytoplasmic domain relative to a wild-type HLA-E heavy chain polypeptide.Clause 2) The modified HLA-E heavy chain polypeptide of clause 1 , wherein the wild-type HLA- E heavy chain molecule has an amino acid sequence identical to SEQ ID NO: 10, or comprising 1 , 2, or 3 amino acid substitutions compared to said sequence.Clause 3) The modified HLA-E heavy chain polypeptide of clauses 1 or 2, wherein the truncated cytoplasmic domain is truncated by at least 21 amino acid residues compared to the HLA-E heavy chain wild-type cytoplasmic domain, optionally wherein the truncated cytoplasmic domain is truncated by 21 amino acid residues compared to the HLA-E wild-type cytoplasmic domain.Clause 4) The modified HLA-E heavy chain polypeptide of any preceding clause, wherein the HLA-E heavy chain wild-type cytoplasmic domain consists of an amino acid sequence identical to SEQ ID NO: 15, or comprising 1 , 2, or 3 amino acid substitutions compared to said sequence.Clause 5) The modified HLA-E heavy chain polypeptide of any preceding clause, wherein the truncated cytoplasmic domain comprises an amino acid sequence consisting of less than 8 amino acid residues.Clause 6) The modified HLA-E heavy chain polypeptide of any preceding clause, wherein the truncated cytoplasmic domain comprises or consists of about 6 amino acids residues.Clause 7) The modified HLA-E heavy chain polypeptide of any preceding clause, wherein the amino acid sequence of the truncated cytoplasmic domain consists of an amino acid sequence whichis at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17 or a functional fragment thereof, optionally wherein the amino acid sequence of the truncated cytoplasmic domain consists of an amino acid sequence which is 100% identical to SEQ ID NO: 17, or comprising 1 , 2, or 3 amino acid substitutions compared to said sequence.Clause 8) The modified HLA-E heavy chain polypeptide of any preceding clause, wherein the amino acid sequence of the truncated cytoplasmic domain consists of the amino acid sequence RAASSD, or comprising 1 , 2, or 3 amino acid substitutions compared to said sequence.Clause 9) The modified HLA-E heavy chain polypeptide of any preceding clause, wherein the HLA-E polypeptide comprises the amino acid sequence which is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14 or a functional fragment thereof, optionally 100% identical to SEQ ID NO: 14, or comprising 1 , 2, or 3 amino acid substitutions compared to said sequence.Clause 10) The modified HLA-E heavy chain polypeptide of any preceding clause isolated from the body.Clause 11 ) The modified HLA-E heavy chain polypeptide of any preceding clause, wherein the modified HLA-E heavy chain polypeptide is human, and the wild-type HLA-E heavy chain polypeptide is human.Clause 12) A modified HLA-E protein, comprising the modified HLA-E heavy chain polypeptide of any clauses 1 to 11 .Clause 13) The modified HLA-E protein of clause 12, comprising a beta-2 microglobulin (p2M) polypeptide.Clause 14) The modified HLA-E protein of clause 13, wherein the p2M polypeptide has an amino acid sequence at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18, optionally wherein the p2M polypeptide has an amino acid sequence 100% identical to SEQ ID NO: 18, or comprising 1 , 2, or 3 amino acid substitutions compared to said sequence.Clause 15) The modified HLA-E protein of any of clauses 12 to 14, comprising a binding peptide with amino acid sequence at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 28, optionally wherein the binding peptide has an amino acid sequence 100% identical to SEQ ID NO: 28, or comprising 1 , 2 or 3 amino acid substitutions compared to said sequence.Clause 16) A vector encoding the modified HLA-E heavy chain polypeptide of any of clauses 1 to 11 or the modified HLA-E protein of any of clauses 12 to 15.Clause 17) A vector comprising a polynucleotide sequence at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23,optionally 100% identical to SEQ ID NO: 23, or comprising 1 , 2, or 3 amino acid substitutions compared to said sequence.Clause 18) A modified cell, wherein the modified cell expresses the modified HLA-E protein of any of clauses 12 to 15.Clause 19) The modified cell of clause 18, wherein the modified HLA-E protein is expressed on the surface of the cell, optionally as measured by flow cytometry.Clause 20) A modified cell comprising the vector of clause 16 or 17.Clause 21 ) The modified cell of any of clauses 18 to 20, comprising deleted or decreased expression of an endogenous p2M gene relative to an unmodified cell of the same cell type, and / or comprising deleted or decreased expression of endogenous HLA class I protein on the cell surface, optionally comprising deleted or decreased expression of HLA-A, HLA-B and HLA-C proteins on the cell surface, optionally as measured by flow cytometry.Clause 22) The modified cell of any of clauses 18 to 21 , comprising introduced or increased expression of an exogenous HLA-G protein relative to an unmodified cell of the same cell type, optionally wherein the exogenous HLA-G protein is expressed on the cell surface, optionally as measured by flow cytometry, or wherein the modified cell does not comprise introduced or increased expression of an exogenous HLA-G protein relative to an unmodified cell of the same cell type.Clause 23) The modified cell of any of clauses 18 to 22, comprising an exogenous polynucleotide encoding the HLA-E protein of any of clauses 12 to 15.Clause 24) The modified cell of any of clauses 18 to 23, comprising an exogenous polynucleotide encoding an HLA-G protein.Clause 25) The modified cell of clause 24, wherein the HLA-G protein comprises an amino acid sequence which is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11 or a functional fragment thereof, optionally wherein the HLA-G polypeptide comprises an amino acid sequence 100% identical to SEQ ID NO: 11 , or comprising 1 , 2, or 3 amino acid substitutions compared to said sequence, optionally wherein the HLA- G protein comprises a single chain binding peptide having an amino acid sequence which is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29, optionally 100% identical to SEQ ID NO: 29 or comprising 1 , 2, or 3 amino acid substitutions compared to said sequence.Clause 26) The modified cell of any of clauses 18 to 25 , wherein relative to an unmodified cell of the same cell type, the modified cell comprises deleted or decreased expression of a Class II Major Histocompatibility Complex Transactivator (CIITA) gene.Clause 27) The modified cell of any of clauses 18 to 26, wherein relative to an unmodified cell of the same cell type, the modified cell comprises deleted or decreased expression a Regulatory factor X- associated ankyrin-containing protein (RFXANK) gene.Clause 28) The modified cell of any of clauses 18 to 27, wherein relative to an unmodified cell of the same cell type, the modified cell comprises deleted or decreased expression a Regulatory Factor X Associated Protein (RFXAP) gene.Clause 29) The modified cell of any of clauses 18 to 28, wherein relative to an unmodified cell of the same cell type, the modified cell comprises deleted or decreased expression a CIITA gene, and a RFXANK gene or RFXAP gene.Clause 30) The modified cell of any of clauses 18 to 29, wherein relative to an unmodified cell of the same cell type, the modified cell comprises deleted or decreased expression a CIITA gene, a RFXANK gene and a RFXAP gene.Clause 31 ) The modified cell of any of clauses 26 to 30, wherein relative to an unmodified cell of the same cell type, the modified cell comprises introduced or increased expression of a CD47 gene.Clause 32) The modified cell of any of clauses 26 to 31 , wherein relative to an unmodified cell of the same cell type, the modified cell comprises introduced or increased expression of a CD47 polypeptide.Clause 33) The modified cell of clause 32, wherein the CD47 polypeptide comprises an amino acid sequence which is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12 or a functional fragment thereof, optionally 100% identical to SEQ ID NO: 12, or comprising 1 , 2, or 3 amino acid substitutions compared to said sequence.Clause 34) The modified cell of any of clauses 26 to 30 , 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.Clause 35) The modified cell of any of clauses 31 to 34, wherein the CD47 gene is human.Clause 36) The modified cell of any of clauses 26 to 35, wherein relative to an unmodified cell of the same cell type, the modified cell does not comprise decreased or deleted expression of a CD24 gene, a CD74 gene, a CIITA gene, an HLA-A gene, an HLA-B gene, an HLA-C gene, an MIC-1 gene, an MIC-2 gene, an NLRC5 gene, a RFX5 gene, a RFXAP gene, a TAP1 gene, a TAP2 gene, a Tapasin gene, a TXNIP, and / or a RFXANK gene.Clause 37) The modified cell of any of clauses 26 to 36, wherein relative to an unmodified cell of the same cell type, the modified cell does not comprise increased or introduced expression of a C1 - inhibitor gene, a CD24 gene, a CD46 gene, a CD55 gene, a CD59 gene, a CR1 gene, a CTLA-4-lg gene, a MANF gene, a TNFAIP3 gene, a PD-L1 gene, GGTA1 gene, CMAG gene, SLA-1 alpha chain gene, and / or B4galNT2 gene.Clause 38) The modified cell of any of clauses 26 to 37, wherein relative to an unmodified cell of the same cell type, the modified cell does not comprise increased or introduced expression of a GGTA1 gene, a CMAH gene, a B4galNT2 gene and a SLA-1 alpha chain gene.Clause 39) The modified cell of any of clauses 26 to 38, wherein the modified cell is a mammalian cell.Clause 40) The modified cell of any of clauses 26 to 39, wherein the modified cell is a human cell.Clause 41 ) The modified cell of any of clauses 26 to 39, wherein the modified cell is not a porcine cell.Clause 42) The modified cell of any of clauses 26 to 41 , wherein the modified cell is an undifferentiated cell.Clause 43) The modified cell of any of clauses 26 to 42, wherein the modified cell is capable of self-renewal.Clause 44) The modified cell of any of clauses 26 to 43, wherein the modified cell is a stem cell.Clause 45) The modified cell of any of clauses 26 to 44, wherein the modified cell is a pluripotent stem cell.Clause 46) The modified cell of any of clauses 26 to 45, wherein the modified cell is an embryonic stem cell (ESC).Clause 47) The modified cell of any of clauses 26 to 46, wherein after differentiation into a different cell type the modified cell retains the modification or modifications.Clause 48) The modified cell of any of clauses 26 to 47, wherein the cell is an induced pluripotent stem cell (IPSC).Clause 49) The modified cell of clause 48, wherein the cell is a multipotent stem cell.Clause 50) The modified cell of clause 46, wherein the cell is a haematopoietic stem cell (HSC).Clause 51 ) The modified cell of any of clauses 26 to 41 , wherein the cell is a differentiated cell.Clause 52) The modified cell of any of clauses 26 to 51 , wherein the modified cell is an isolated cell.Clause 53) The modified cell of any of clauses 26 to 52, wherein the modified cell is an engineered cell.Clause 54) The modified cell of any of clauses 26 to 53, wherein the modified cell has been modified or engineered in vitro.Clause 55) The modified cell of any of clauses 26 to 53, wherein the modified cell has been modified or engineered ex vivo.Clause 56) The modified cell of any of clauses 26 to 55, wherein the modified cell does not occur in nature.Clause 57) The modified cell of any of clauses 26 to 56, wherein relative to an unmodified cell of the same cell type, the modified cell comprises reduced HLA class II protein expression followingstimulation with IFN-y, optionally wherein HLA class II protein expression is determined by flow cytometry.Clause 58) The modified cell of any of clauses 26 to 57, 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.Clause 59) The modified cell of any of clauses 26 to 58, 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.Clause 60) The modified cell of any of clauses 26 to 59, wherein the modified cell does not express HLA class I protein, optionally as determined by flow cytometry.Clause 61 ) The modified cell of any of clauses 26 to 60, wherein relative to an unmodified cell of the same cell type, the modified cell comprises increased expression of an HLA-G protein, optionally as determined by flow cytometry.Clause 62) The modified cell of any of clauses 26 to 61 , 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.Clause 63) The modified cell of any of clauses 26 to 62, wherein the modified cell does not express HLA-A, B or C protein, optionally as determined by FACS.Clause 64) The modified cell of any of clauses 26 to 63, 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.Clause 65) The modified cell of any of clauses 26 to 64, 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.Clause 66) The modified cell of any of clauses 26 to 65, wherein the modified cell is hypoimmunogenic.Clause 67) The modified cell of any of clauses 26 to 66, wherein the modified cell does not activate NK cell mediated immunity, optionally as determined by real-time based killing assay.Clause 68) The modified cell of any of clauses 26 to 67, wherein the modified cell does not activate NK cell, T cell and / or macrophages mediated immunity.Clause 69) The modified cell of any of clauses 26 to 68, 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).Clause 70) The modified cell of any of clauses 26 to 69, wherein relative to a unmodified cell of the same cell type, the cell has been modified to delete or reduce expression of cyclin dependent kinaseinhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'- thioadenosine phosphorylase (MTAP) relative to a wild-type cell of the same type.Clause 71 ) The modified cell of any of clauses 26 to 70, wherein relative to an unmodified cell of the same cell type, the modified cell has been modified to delete or reduce expression of T-cell receptor a constant (TRAC).Clause 72) The modified cell of any of clauses 26 to 71 , 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).Clause 73) The modified cell of any of clauses 26 to 72, 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).Clause 74) The modified cell of any of clauses 26 to 73, 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).Clause 75) The modified cell of any of clauses 26 to 74, wherein introduced or increased gene expression is determined by flow cytometry, western blot, qPCR or immunofluorescence.Clause 76) The modified cell of any of clauses 26 to 75, wherein deleted or decreased gene expression is determined by flow cytometry, western blot, qPCR or immunofluorescence.Clause 77) The modified cell of any of clauses 26 to 76, wherein introduced or increased expression of the HLA-E gene and the HLA-G gene is determined by flow cytometry.Clause 78) The modified cell of any of clauses 26 to 77, wherein p2M gene expression is deleted or decreased by methods comprising gene editing the cell.Clause 79) The modified cell of any of clauses 26 to 78, wherein the gene editing comprises CRISPR-Cas9.Clause 80) The modified cell of any of clauses 26 to 79, wherein the gene editing comprises TALEN.Clause 81 ) A population of cells comprising the modified cell of any of clauses 26 to 80.Clause 82) A pharmaceutical composition comprising the modified cell of any of clauses 26 to 80 or the cell population of clauses 81 .Clause 83) A pharmaceutical composition of clauses 82, comprising a pharmaceutical excipient.Clause 84) A method of making a pharmaceutical composition comprising combining the modified cell of an of any of clauses 26 to 80 with a pharmaceutical excipient.Clause 85) Use of a modified cell of any of any of clauses 26 to 60 in the manufacture of a medicament for the treatment of prevention of disease in a subject.Clause 86) A method for manufacturing a modified cell, comprising introducing or the expression of the modified HLA-E heavy chain polypeptide of any of clauses 1 to 11 or the modified HLA-E protein of any of clauses 11 to 15, into the cell.Clause 87) The method of clause 86 comprising eliminating or decreasing the expression of one or both alleles of the p2M gene in a cell.Clause 88) The method of clause 86 or 87, comprising eliminating the expression of both alleles of the CIITA, RFXANK and / or RFXAP genes in the cell, optionally by gene editing, optionally by CRISPR-cas9 gene editing.Clause 89) The method of any of clauses 86 to 88, wherein the modified cell is a stem cell, optionally an embryonic stem cell or induced pluripotent stem cell.Clause 90) The method of clause 89 comprising differentiating the cell into a progenitor cell, terminally differentiated cell or somatic cell.Clause 91 ) The method of any of clauses 86 to 88, wherein the modified cell is a progenitor cell, terminally differentiated cell or somatic cell.Clause 92) A modified cell, wherein the modified cell is manufactured according to the method of any of clauses 86 to 91 .Clause 93) A vector for use in any of method of any of clauses 86 to 92, optionally wherein the vector is a lentivector.Clause 94) The lentivector of clause 93, wherein the lentivector is an adenovirus vector.Clause 95) The lentivector of clauses 93 or 94, wherein the lentivector comprises a gRNA having the nucleotide sequence corresponding to any of SEQ ID NO: 1 to 8.Clause 96) 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 clauses 15 to 42, or a differentiated cell, progeny, daughter cell, or population of cells derived from the modified cell of any of clauses 15 to 42.Clause 97) The method of clause 96, wherein the modified cell is derived from a cell that is not isolated from the subject.Clause 98) A method of treating or prevent a disease in a subject, the method comprising: producing in vitro a modified cell according to the method of any of clauses 86 to 92, and administering modified cell to the subject.Clause 99) A pharmaceutical composition for use in the method of clause 98, wherein the pharmaceutical composition comprises the modified cell of any of clauses 15 to 42 and a pharmaceutical excipient.REFERENCES

[0117] All publications mentioned in the specification are herein incorporated by reference in their entirety.

[0118] [1] J. L. Dashnau et al., Cytotherapy 25, 1 (2023).

[0119] [2] C. A. Cowan et al., N Engl J Med 350, 1353 (2004).

[0120] [3] W. Zhou and C. R. Freed, Stem Cells 27, 2667 (2009).

[0121] [4] D. Huangfu et al., Nat Biotechnol 26, 795 (2008).

[0122] [5] K. Woltjen et al., Nature 458, 766 (2009).

[0123] [6] H. Zhou et al., Cell Stem Cell 4, 381 (2009).

[0124] [7] J. A. Doudna and E. Charpentier, Science 346, 1258096 (2014).

[0125] [8] D. Krijgsman et al., Int J Mol Sci 21 , 4528 (2020).

[0126] [9] N. L. Dudek and A. W. Purcell, in Encyclopedia of Immunobiology, edited by M. J. H. Ratcliffe (Academic Press, Oxford, 2016), pp. 215-219.

[0127]

[0010] M. I. Phillips and Y. L. Tang, Adv Drug Deliv Rev 60, 160 (2008).

[0128]

[0011] J. K. W. Lam et al., Mol Ther Nucleic Acids 4, e252 (2015).

[0129]

[0012] K. Sridharan and N. J. Gogtay, Br J Clin Pharmacol 82, 659 (2016).

[0130]

[0013] D. W. Kim et al., Gene 91 , 217 (1990).

[0131]

[0014] K. M. Barrow, F. M. Perez-Campo, and C. M. Ward, Methods Mol Biol 329, 283 (2006).

Claims

CLAIMS1. A modified human leukocyte antigen (HLA)-E heavy chain polypeptide comprising a truncated cytoplasmic domain relative to a wild-type HLA-E heavy chain polypeptide.

2. The modified HLA-E heavy chain polypeptide of claim 1 , wherein the wild-type HLA-E heavy chain molecule has an amino acid sequence identical to SEQ ID NO: 10.

3. The modified HLA-E heavy chain polypeptide of claim 1 or 2, wherein the truncated cytoplasmic domain is truncated by at least 21 amino acid residues compared to the HLA-E heavy chain wild-type cytoplasmic domain, optionally wherein the truncated cytoplasmic domain is truncated by 21 amino acid residues compared to the HLA-E wild-type cytoplasmic domain.

4. The modified HLA-E heavy chain polypeptide of claim 3, wherein the HLA-E heavy chain wildtype cytoplasmic domain consists of an amino acid sequence identical to SEQ ID NO: 15.

5. The modified HLA-E heavy chain polypeptide of any preceding claim, wherein the truncated cytoplasmic domain comprises an amino acid sequence consisting of 8 or less amino acid residues.

6. The modified HLA-E heavy chain polypeptide of any preceding claim, wherein the truncated cytoplasmic domain comprises or consists of about 6 amino acids residues.

7. The modified HLA-E heavy chain polypeptide of any preceding claim, wherein the amino acid sequence of the truncated cytoplasmic domain consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 17 or a functional fragment thereof, optionally wherein the amino acid sequence of the truncated cytoplasmic domain consists of an amino acid sequence which is 100% identical to SEQ ID NO: 17.

8. The modified HLA-E heavy chain polypeptide of any preceding claim, wherein the amino acid sequence of the truncated cytoplasmic domain consists of the amino acid sequence RKKSSGGK.

9. The modified HLA-E heavy chain polypeptide of any preceding claim, wherein the HLA-E heavy chain polypeptide comprises an amino acid sequence which is at least 70% identical to SEQ ID NO: 14 or a functional fragment thereof, optionally 100% identical to SEQ ID NO: 14.

10. The modified HLA-E heavy chain polypeptide of any preceding claim isolated from the body.11 . A modified HLA-E protein, comprising the modified HLA-E heavy chain molecule of any claims 1 to 10.

12. The modified HLA-E protein of claim 11 , comprising beta-2 microglobulin (p2M).

13. The modified HLA-E protein of claim 11 or 12, comprising a single chain peptide having the an amino acid sequence at least 70% identical to SEQ ID NO: 28, optionally 100% identical to SEQ ID NO: 28.

14. A vector encoding the modified HLA-E heavy chain polypeptide of any of claims 1 -10 or the modified HLA-E protein of any of claims 11 to 13.

15. A modified cell, wherein the modified cell expresses the modified HLA-E protein of any of claims 11 to 13.

16. The modified cell of claim 15, wherein the modified HLA-E protein is expressed on the surface of the cell.

17. The modified cell of claim 15 or 16, comprising deleted or decreased expression of endogenous HLA class I protein on the cell surface, optionally comprising deleted or decreased expression of HLA-A, HLA-B and HLA-C proteins on the cell surface, optionally as measured by flow cytometry.

18. The modified cell of any of claims 15 to 17, comprising deleted or decreased expression of an endogenous p2M gene relative to an unmodified cell of the same cell type.

19. The modified cell of any of claims 15 to 18, comprising introduced or increased expression of an exogenous HLA-G protein relative to an unmodified cell of the same cell type, optionally wherein the exogenous HLA-G protein is expressed on the cell surface, optionally as measured by flow cytometry.

20. The modified cell of any of claims 15 to 19, comprising an exogenous polynucleotide encoding the modified HLA-E heavy chain polypeptide of any of claims 1 to 10, or the modified HLA-E protein of any of claims 1 1 to 13.

21. The modified cell of any of claims 15 to 20, comprising an exogenous polynucleotide encoding an HLA-G protein.

22. The modified cell of claim 21 , wherein the HLA-G protein comprises a heavy chain comprising an amino acid sequence which is at least 70% identical to SEQ ID NO: 11 or a functional fragment thereof.

23. The modified cell of any of claims 15 to 22 , 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.

24. The modified cell of any of claims 15 to 23 , wherein the modified cell is a mammalian cell.

25. The modified cell of any of claims 15 to 24, wherein the modified cell is a human cell.

26. The modified cell of any of claims 15 to 25 , wherein the modified cell is an undifferentiated cell.

27. The modified cell of any of claims 15 to 26 , wherein the modified cell is a stem cell.

28. The modified cell of any of claims 15 to 25, wherein the cell is a differentiated cell.

29. The modified cell of claim 28, wherein the cell is a T cell.

30. The modified cell of any of claims 15 to 29 , wherein relative to an unmodified cell of the same cell type, the modified cell comprises reduced endogenous HLA class II protein expression on the cell surface following stimulation with IFN-y, optionally wherein HLA class II protein expression is determined by flow cytometry.

31. The modified cell of any of claims 15 to 30 , wherein relative to an unmodified cell of the same cell type, the modified cell comprises reduced HLA-DR, HLA-DQ and / or HLA-DP expression following stimulation with IFN-y, optionally as determined by flow cytometry.

32. The modified cell of any of claims 15 to 31 , wherein relative to an unmodified cell of the same cell type, the modified cell comprises reduced expression of endogenous HLA class I protein, optionally as determined by flow cytometry.

33. The modified cell of any of claims 15 to 32, wherein the modified cell does not express endogenous HLA class I protein on the cell surface, optionally as determined by flow cytometry.

34. The modified cell of any of claims 15 to 33, wherein relative to an unmodified cell of the same cell type, the modified cell comprises increased expression of HLA-G protein on the cell surface, optionally as determined by flow cytometry.

35. The modified cell of any of claims 15 to 34, wherein relative to an unmodified cell of the same cell type, the modified cell comprises reduced HLA-A, B and C polypeptide expression on the cell surface, optionally as determined by flow cytometry.

36. The modified cell of any of claims 15 to 35, wherein the modified cell does not express HLA-A, B or C polypeptide expression on the cell surface, optionally as determined by flow cytometry.

37. The modified cell of any of claims 15 to 36, 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.

38. The modified cell of any of claims 15 to 37, 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.

39. The modified cell of any of claims 15 to 38, wherein the modified cell is hypoimmunogenic.

40. The modified cell of any of claims 15 to 39, wherein the modified cell does not activate NK cell mediated immunity, optionally as determined by real-time based killing assay.41 . The modified cell of any of claims 15 to 40, wherein the modified cell does not activate NK cell, T cell and / or macrophages mediated immunity.

42. The modified cell of any of claims 15 to 41 , wherein introduced or increased gene or protein expression is determined by flow cytometry, western blot, qPCR or immunofluorescence.

43. The modified cell of any of claims 15 to 42, wherein gene or protein expression, or the lack thereof, is determined by flow cytometry, western blot, qPCR or immunofluorescence.

44. A population of cells comprising the modified cell of any of claims 15 to 43.

45. A pharmaceutical composition comprising the modified cell of any of claims 15 to 43 or the cell population of claim 44.

46. A pharmaceutical composition of claim 45, comprising a pharmaceutical excipient.

47. A method of making a pharmaceutical composition comprising combining the modified cell of any of claims 15 to 43 with a pharmaceutical excipient.

48. Use of a modified cell of any of claims 15 to 43 in the manufacture of a medicament for the treatment or prevention of disease in a subject.

49. A method for manufacturing a modified cell, comprising introducing the expression of the modified HLA-E heavy chain polypeptide of any of claims 1 to 10, or the modified HLA-E protein of any of claims 11 to 13, into the cell.

50. The method of claim 49, comprising eliminating or decreasing the expression of one or both alleles of the endogenous p2M gene in the cell.

51. The method of claim 50, comprising eliminating the expression of both alleles of the CIITA, RFXANK and / or RFXAP genes in the cell.

52. The method of claim 50 or 51 , wherein the cell is a stem cell, optionally an embryonic stem cell.

53. A modified cell, wherein the modified cell is manufactured according to the method of any of claims 49 to 52.

54. A vector for use in a method according to any of claims 49 to 52.

55. A method of treating or preventing a disease in a subject in need thereof, the method comprising administering to the subject the modified cell of any of claims 15 to 43, or a differentiated cell, progeny, daughter cell, or population of cells derived from the modified cell of any of claims 15 to 43.

56. The method of claim 55, wherein the modified cell is derived from a cell that is not isolated from the subject.

57. A method of treating or preventing a disease in a subject, the method comprising: a) producing in vitro a modified cell according to the method of any of claims 49 to 52, andb) administering the modified cell to the subject.

58. A pharmaceutical composition for use in the method of any one of claims 55 to 57, wherein the pharmaceutical composition comprises the modified cell of any of claims 15 to 43 and a pharmaceutical excipient.

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