Inducible systems for altering gene expression in hypoimmunogenic cells

Abstract

Disclosed herein are engineered and / or hypoimmunogenic cells, including engineered and / or hypoimmunogenic stem cells, engineered and / or hypoimmunogenic cells differentiated therefrom, and / or engineered and / or hypoimmunogenic CAR-T cells (either primary or differentiated from engineered and / or hypoimmunogenic stem cells), and related methods of their use and production, including engineered and / or hypoimmunogenic cells that comprise regulatable reduced expression of one or more MHC class I and / or MHC class II human leukocyte antigen molecules, and regulatable overexpression of CD47. Provided herein are cells that further exhibit reduced expression of T cell receptors.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 232,141, filed August 11, 2021, and U.S. Provisional Patent Application No. 63 / 270,454, filed October 21, 2021, the disclosures of each of which are incorporated herein in their entireties. Summary of the Invention

[0002] Off-the-shelf therapeutic cells can offer advantages over autologous cell-based strategies, including ease of production, quality control, and avoidance of harmful contamination and T cell dysfunction. However, vigorous host-versus-graft immune responses against histoincompatible cells can prevent the expansion and persistence of allogeneic cells, reducing the efficacy of this approach.

[0003] There is strong evidence in both animal models and human patients that hypoimmunogenic cell transplantation is a scientifically feasible and clinically promising approach to the treatment of numerous disorders, conditions, and diseases.

[0004] There remains a need for novel approaches, compositions, and methods for generating cell-based therapies that avoid detection by the recipient's immune system.

[0005] In some embodiments, provided herein is an engineered cell that comprises i) a modification that reduces expression of one or more MHC class I and / or MHC class II molecules, and ii) a modification that increases expression of CD47, relative to a control, wherein the engineered cell expresses CD47 at or above a threshold level.

[0006] In some embodiments, provided herein are engineered cells that contain a regulatable modification that increases expression of CD47 relative to a control.

[0007] In some embodiments, the engineered cells are selected from the group consisting of stem cells, pluripotent stem cells (PSCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), embryonic stem cells (ESCs), pancreatic islet cells, beta islet cells, immune cells, B cells, T cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophage cells, immune privileged cells, optic nerve cells, retinal pigment epithelial cells (RPE), liver cells, thyroid cells, endothelial cells, skin cells, glial progenitor cells, neuronal cells, muscle cells, cardiac cells, and blood cells.

[0008] In some embodiments, provided herein is an engineered pancreatic islet cell comprising: i) a modification that reduces expression of one or more MHC class I and / or MHC class II molecules; and ii) a modification that increases expression of CD47, relative to a control, wherein the engineered pancreatic islet cell expresses CD47 at or above a threshold level.

[0009] In some embodiments, the pancreatic islet cells are beta islet cells.

[0010] In some embodiments, provided herein is an engineered endothelial cell comprising: i) a modification that reduces expression of one or more MHC class I and / or MHC class II molecules; and ii) a modification that increases expression of CD47, relative to a control, wherein the engineered endothelial cell expresses CD47 at or above a threshold level.

[0011] In some embodiments, provided herein is an engineered cardiomyocyte comprising: i) a modification that reduces expression of one or more MHC class I and / or MHC class II molecules; and ii) a modification that increases expression of CD47 relative to a control, wherein the engineered cardiomyocyte expresses CD47 at or above a threshold level.

[0012] In some embodiments, provided herein is an engineered smooth muscle cell that comprises: i) a modification that reduces expression of one or more MHC class I and / or MHC class II molecules; and ii) a modification that increases expression of CD47, relative to a control, wherein the engineered smooth muscle cell expresses CD47 at or above a threshold level.

[0013] In some embodiments, provided herein is an engineered skeletal muscle cell that comprises: i) a modification that reduces expression of one or more MHC class I and / or MHC class II molecules; and ii) a modification that increases expression of CD47, relative to a control, wherein the engineered skeletal muscle cell expresses CD47 at or above a threshold level.

[0014] In some embodiments, provided herein is an engineered hepatocyte comprising: i) a modification that reduces expression of one or more MHC class I and / or MHC class II molecules; and ii) a modification that increases expression of CD47, relative to a control, wherein the engineered hepatocyte expresses CD47 at or above a threshold level.

[0015] In some embodiments, provided herein is an engineered glial progenitor cell that comprises: i) a modification that reduces expression of one or more MHC class I and / or MHC class II molecules; and ii) a modification that increases expression of CD47, relative to a control, wherein the engineered glial progenitor cell expresses CD47 at or above a threshold level.

[0016] In some embodiments, provided herein is an engineered dopaminergic neuron that comprises: i) a modification that reduces expression of one or more MHC class I and / or MHC class II molecules; and ii) a modification that increases expression of CD47, relative to a control, wherein the engineered dopaminergic neuron expresses CD47 at or above a threshold level.

[0017] In some embodiments, provided herein is an engineered immunoprivileged cell that comprises: i) a modification that reduces expression of one or more MHC class I and / or MHC class II molecules; and ii) a modification that increases expression of CD47, relative to a control, wherein the engineered immunoprivileged cell expresses CD47 at or above a threshold level.

[0018] In some embodiments, provided herein is an engineered retinal pigment epithelial cell comprising: i) a modification that reduces expression of one or more MHC class I and / or MHC class II molecules; and ii) a modification that increases expression of CD47, relative to a control, wherein the engineered retinal pigment epithelial cell expresses CD47 at or above a threshold level.

[0019] In some embodiments, provided herein is an engineered thyrocyte comprising: i) a modification that reduces expression of one or more MHC class I and / or MHC class II molecules; and ii) a modification that increases expression of CD47, relative to a control, wherein the engineered thyrocyte expresses CD47 at or above a threshold level.

[0020] In some embodiments, provided herein is an engineered immune cell that comprises i) a modification that reduces expression of one or more MHC class I and / or MHC class II molecules, and ii) a modification that increases expression of CD47, relative to a control, wherein the engineered immune cell expresses CD47 at or above a threshold level.

[0021] In some embodiments, the engineered immune cells comprise an exogenous polynucleotide encoding one or more chimeric antigen receptors (CARs).

[0022] In some embodiments, provided herein is an engineered T cell that comprises i) a modification that reduces expression of one or more MHC class I and / or MHC class II molecules, and ii) a modification that increases expression of CD47, relative to a control, wherein the engineered T cell expresses CD47 at or above a threshold level.

[0023] In some embodiments, the engineered T cells comprise an exogenous polynucleotide encoding one or more chimeric antigen receptors (CARs).

[0024] In some embodiments, provided herein is an engineered NK cell comprising: i) a modification that reduces expression of one or more MHC class I and / or MHC class II molecules; and ii) a modification that increases expression of CD47, relative to a control, wherein the engineered NK cell expresses CD47 at or above a threshold level.

[0025] In some embodiments, the engineered T cells comprise an exogenous polynucleotide encoding one or more chimeric antigen receptors (CARs).

[0026] In some embodiments, provided herein is an engineered macrophage cell comprising: i) a modification that reduces expression of one or more MHC class I and / or MHC class II molecules; and ii) a modification that increases expression of CD47, relative to a control, wherein the engineered macrophage cell expresses CD47 at or above a threshold level.

[0027] In some embodiments, the cells express at least about the same amount of CD47 as a control.

[0028] In some embodiments, the cell is an immunoprivileged cell.

[0029] In some embodiments, the cells express CD47 in an amount at least about 10% greater than a control.

[0030] In some embodiments, the cells express at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% greater amount of CD47 relative to a control.

[0031] In some embodiments, the cells express at least about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800% or 900% greater amount of CD47 relative to a control.

[0032] In some embodiments, the cells express at least about 1000% greater amount of CD47 relative to a control.

[0033] In some embodiments, the cells express at least about 1.1-fold the level of CD47 expressed in a control.

[0034] In some embodiments, the cells express at least about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, or about 5-fold the level of CD47 expressed in a control.

[0035] In some embodiments, the cells express at least about 4-fold, about 4.5-fold, about 5-fold, or about 5.5-fold the level of CD47 expressed in a control.

[0036] In some embodiments, the cells express at least about four-fold the level of CD47 expressed in a control.

[0037] In some embodiments, the cells express at least about 4.5-fold the level of CD47 expressed in a control.

[0038] In some embodiments, the cells express at least about 5-fold the level of CD47 expressed in a control.

[0039] In some embodiments, the cells express at least about 5.5-fold the level of CD47 expressed in a control.

[0040] In some embodiments, the cells express at least about 16-fold, about 17-fold, about 18-fold, about 19-fold, or about 20-fold the level of CD47 expressed in a control.

[0041] In some embodiments, the control is a wild-type cell, a control cell, or a baseline reference.

[0042] In some embodiments, the control cell is an unmodified or unaltered cell, optionally, the unmodified or unaltered cell is of the same cell type as the engineered cell.

[0043] In some embodiments, the control cells are starting material from a donor or a pool of starting cells from a pool of donors.

[0044] In some embodiments, the baseline reference is an isotype control or background signal level.

[0045] In some embodiments, the baseline is an isotype control, and optionally, CD47 levels are determined using an antibody-based assay.

[0046] In some embodiments, CD47 levels are determined using an antibody-based quantification method, optionally the Quantibrite™ assay.

[0047] In some embodiments, the engineered cells are beta islet cells that express at least about 200,000, 250,000, 300,000, 350,000 or 400,000 CD47 molecules per cell.

[0048] In some embodiments, the engineered cells are retinal pigment epithelial cells that express at least about a 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold or more increase in CD47 expression relative to baseline.

[0049] In some embodiments, the engineered cells are T cells that express at least about 180,000, 190,000, 200,000, 210,000, 220,000, 230,000, 240,000, 250,000, 260,000, 270,000, 280,000, 290,000, 300,000, 350,000, 400,000, 450,000, 500,000, 550,000, 600,000, 650,000 or 700,000 CD47 molecules per cell.

[0050] In some embodiments, provided herein is an engineered cell that comprises i) a modification that reduces expression of one or more MHC class I and / or MHC class II human leukocyte antigens, and ii) a modification that increases expression of one or more tolerogenic factors, relative to a control, wherein the engineered cell expresses the tolerogenic factor at or above a threshold level.

[0051] In some embodiments, the engineered cells are selected from the group consisting of stem cells, pluripotent stem cells (PSCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), embryonic stem cells (ESCs), pancreatic islet cells, beta islet cells, immune cells, B cells, T cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophage cells, immune privileged cells, optic nerve cells, retinal pigment epithelial cells (RPE), liver cells, thyroid cells, endothelial cells, skin cells, glial progenitor cells, neuronal cells, muscle cells, cardiac cells, and blood cells.

[0052] In some embodiments, the control is a wild-type cell, a control cell, or a baseline reference.

[0053] In some embodiments, the control cell is an unmodified or unaltered cell, optionally, the unmodified or unaltered cell is of the same cell type as the engineered cell.

[0054] In some embodiments, the control cells are starting material from a donor or a pool of starting cells from a pool of donors.

[0055] In some embodiments, the baseline reference is an isotype control or background signal level.

[0056] In some embodiments, the baseline is an isotype control, and optionally the amount of the tolerogenic factor is determined using an antibody-based assay.

[0057] In some embodiments, the amount of a tolerogenic factor is determined using an antibody-based quantification method, optionally a Quantibrite™ assay.

[0058] In some embodiments, the cells express at least about the same amount of the tolerogenic factor as a control.

[0059] In some embodiments, the cell is an immunoprivileged cell.

[0060] In some embodiments, the cells express an amount of the tolerogenic factor that is at least about 10% greater than a control.

[0061] In some embodiments, the cells express at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% greater amount of the tolerogenic factor relative to a control.

[0062] In some embodiments, the cells express at least about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800% or 900% greater amount of the tolerogenic factor relative to a control.

[0063] In some embodiments, the cells express an amount of the tolerogenic factor that is at least about 1000% greater relative to a control.

[0064] In some embodiments, the cells express at least about 1.1-fold the level of the tolerogenic factor expressed in a control.

[0065] In some embodiments, the cells express at least about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, or about 5-fold the level of the tolerogenic factor expressed in a control.

[0066] In some embodiments, the cells express at least about 4-fold, about 4.5-fold, about 5-fold, or about 5.5-fold the level of the tolerogenic factor expressed in a control.

[0067] In some embodiments, the cells express at least about four-fold the level of the tolerogenic factor expressed in a control.

[0068] In some embodiments, the cells express at least about 4.5-fold the level of the tolerogenic factor expressed in a control.

[0069] In some embodiments, the cells express at least about 5-fold the level of the tolerogenic factor expressed in a control.

[0070] In some embodiments, the cells express at least about 5.5-fold the level of the tolerogenic factor expressed in a control.

[0071] In some embodiments, the cells express at least about 16-fold, about 17-fold, about 18-fold, about 19-fold, or about 20-fold the level of the tolerogenic factor expressed in a control.

[0072] In some embodiments, the modification reduces expression of (a) MHC class I molecules, (b) MHC class II molecules, or (c) MHC class I molecules and MHC class II molecules.

[0073] In some embodiments, the modification reduces expression of one or more of B2M, TAP I, NLRC5, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, RFX5, RFXANK, RFXAP, NFY-A, NFY-B, and / or NFY-C relative to a control.

[0074] In some embodiments, the cells do not express MHC class I and / or MHC class II molecules.

[0075] In some embodiments, the cells do not express one or more of B2M, TAP I, NLRC5, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, RFX5, RFXANK, RFXAP, NFY-A, NFY-B and / or NFY-C relative to a control.

[0076] In some embodiments, the modification comprises knockout of one or more targets selected from the group consisting of B2M, TAP I, NLRC5, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, RFX5, RFXANK, RFXAP, NFY-A, NFY-B and / or NFY-C.

[0077] In some embodiments, the modification reduces expression of one or more targets selected from the group consisting of B2M, TAP1, NLRC5 and / or CIITA.

[0078] In some embodiments, the modification comprises knockout of one or more targets selected from the group consisting of B2M, TAP1, NLRC5 and / or CIITA.

[0079] In some embodiments, the knockout occurs in both alleles.

[0080] In some embodiments, the cells further comprise one or more modifications that reduce expression of CTLA-4, PD-1, IRF1, MIC-A, MIC-B, proteins involved in oxidative or ER stress, TRAC, TRB, CD142, ABO, CD38, PCDH11Y, NLGN4Y, and / or RHD relative to a control.

[0081] In some embodiments, the protein involved in oxidative or ER stress is selected from the group consisting of thioredoxin interacting protein (TXNIP), PKR-like ER kinase (PERK), inositol-requiring enzyme 1 alpha (IRE1α) and DJ-1 (PARK7).

[0082] In some embodiments, the modification comprises knockout of one or more targets selected from the group consisting of CTLA-4, PD-1, IRF1, MIC-A, MIC-B, proteins involved in oxidative or ER stress, TRAC, TRB, CD142, ABO, CD38, PCDH11Y, NLGN4Y, and / or RHD.

[0083] In some embodiments, the knockout occurs in both alleles.

[0084] In some embodiments, the modification reduces expression of B2M.

[0085] In some embodiments, the modification reduces expression of CIITA.

[0086] In some embodiments, the modification reduces expression of B2M and CIITA.

[0087] In some embodiments, the modification comprises knockout of B2M and / or CIITA.

[0088] In some embodiments, the B2M and / or CIITA knockout occurs in both alleles.

[0089] In some embodiments, the modification reduces expression of an NK cell ligand, optionally MIC-A and / or MIC-B.

[0090] In some embodiments, the modification comprises knockout of MIC-A and / or MIC-B.

[0091] In some embodiments, the MIC-A and / or MIC-B knockout occurs in both alleles.

[0092] In some embodiments, the cells further comprise a modification that reduces expression of one or more Y chromosome genes relative to a control.

[0093] In some embodiments, the one or more Y chromosome genes are selected from the group consisting of protocadherin 11 Y-linked and neuroligin 4 Y-linked.

[0094] In some embodiments, the modification reduces expression of TXNIP.

[0095] In some embodiments, the modification comprises a knockout of TXNIP.

[0096] In some embodiments, the TXNIP knockout occurs in both alleles.

[0097] In some embodiments, the cells further comprise a modification that reduces expression of B2M, TAP I, NLRC5, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, RFX5, RFXANK, RFXAP, NFY-A, NFY-B, NFY-C, CTLA-4, PD-1, IRF1, MIC-A, MIC-B, proteins involved in oxidative or ER stress, TRAC, TRB, CD142, ABO, CD38, PCDH11Y, NLGN4Y, and / or RHD.

[0098] In some embodiments, the cells do not express B2M, TAP I, NLRC5, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, RFX5, RFXANK, RFXAP, NFY-A, NFY-B, NFY-C, CTLA-4, PD-1, IRF1, MIC-A, MIC-B, proteins involved in oxidative or ER stress, TRAC, TRB, CD142, ABO, CD38, PCDH11Y, NLGN4Y, and / or RHD.

[0099] In some embodiments, the cells further comprise a modification that reduces expression of B2M, CIITA, NLRC5, TRAC, TRB, CD142, ABO, MIC-A / B, CD38, PCDH11Y, NLGN4Y, and / or RHD relative to a control.

[0100] In some embodiments, the cells do not express B2M, CIITA, NLRC5, TRAC, TRB, CD142, ABO, MIC-A / B, CD38, CD52, PCDH11Y, NLGN4Y, and / or RHD.

[0101] In some embodiments, the cells contain an additional modification that reduces expression of one or more tolerogenic factors.

[0102] In some embodiments, the one or more tolerogenic factors are selected from the group consisting of A20 / TNFAIP3, C1-inhibitor, CCL21, CCL22, CD16, CD16 Fc receptor, CD24, CD27, CD35, CD39, CD46, CD52, CD55, CD59, CD200, CR1, CTLA4-Ig, DUX4, FasL, H2-M3, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IDO1, IL-10, IL15-RF, IL-35, MANF, Mfge8, PD-1, PD-L1, and / or Serpinb9.

[0103] In some embodiments, the one or more tolerogenic factors are selected from the group consisting of A20 / TNFAIP3, C1-inhibitor, CCL21, CCL22, CD16, CD16 Fc receptor, CD24, CD27, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD200, CR1, CTLA4-Ig, DUX4, FasL, H2-M3, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IDO1, IL-10, IL15-RF, IL-35, MANF, Mfge8, PD-1, PD-L1, and / or Serpinb9.

[0104] In some embodiments, the one or more tolerogenic factors comprises CD47.

[0105] In some embodiments, the one or more tolerogenic factors comprises HLA-E.

[0106] In some embodiments, the one or more tolerogenic factors comprises CD24.

[0107] In some embodiments, the one or more tolerogenic factors comprises PD-L1.

[0108] In some embodiments, the one or more tolerogenic factors comprises CD46.

[0109] In some embodiments, the one or more tolerogenic factors comprises CD55.

[0110] In some embodiments, the one or more tolerogenic factors comprises CD59.

[0111] In some embodiments, the one or more tolerogenic factors comprises CR1.

[0112] In some embodiments, the one or more tolerogenic factors comprises MANF.

[0113] In some embodiments, the one or more tolerogenic factors comprises A20 / TNFAIP3.

[0114] In some embodiments, the one or more tolerogenic factors include HLA-E and CD47.

[0115] In some embodiments, the one or more tolerogenic factors include one or more of CD24, CD47, and / or PDL1.

[0116] In some embodiments, the one or more tolerogenic factors include one or more of HLA-E, CD24, CD47, and / or PDL1.

[0117] In some embodiments, the one or more tolerogenic factors include one or more of CD46, CD55, CD59, and / or CR1.

[0118] In some embodiments, the one or more tolerogenic factors include one or more of HLA-E, CD46, CD55, CD59, and / or CR1.

[0119] In some embodiments, the one or more tolerogenic factors include one or more of HLA-E, CD24, CD47, PDL1, CD46, CD55, CD59, and / or CR1.

[0120] In some embodiments, the one or more tolerogenic factors include HLA-E and PDL1.

[0121] In some embodiments, the one or more tolerogenic factors include one or more of HLA-E, PDL1, and / or A20 / TNFAIP.

[0122] In some embodiments, the one or more tolerogenic factors include one or more of HLA-E, PDL1, and / or MANF.

[0123] In some embodiments, the one or more tolerogenic factors include one or more of HLA-E, PDL1, A20 / TNFAIP, and / or MANF.

[0124] In some embodiments, the modifications include (a) reducing expression of MHC class I and / or MHC class II molecules, (b) reducing expression of MIC-A and / or MIC-B, (c) increasing expression of CD47, and optionally, increasing expression of CD24 and PD-L1, and (d) increasing expression of CD46, CD55, CD59 and CR1.

[0125] In some embodiments, the modifications (a) reduce expression of MHC class I molecules, (b) reduce expression of MIC-A and / or MIC-B, (c) reduce expression of TXNIP, and (d) increase expression of PD-L1 and HLA-E.

[0126] In some embodiments, the modification further increases expression of A20 / TNFAIP3 and MANF.

[0127] In some embodiments, the cells are derived from human cells or animal cells.

[0128] In some embodiments, the cell is a differentiated cell derived from a stem cell or its progeny.

[0129] In some embodiments, the stem cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs) and embryonic stem cells (ESCs).

[0130] In some embodiments, the cells are derived from a primary cell or its progeny.

[0131] In some embodiments, the cells avoid NK cell-mediated cytotoxicity upon administration to a recipient patient.

[0132] In some embodiments, the cells are protected from cytolysis by mature NK cells upon administration to a recipient patient.

[0133] In some embodiments, the cells avoid macrophage phagocytosis upon administration to a recipient patient.

[0134] In some embodiments, the cells do not induce an innate and / or adaptive immune response to the cells upon administration to a recipient patient.

[0135] In some embodiments, the cells do not induce an antibody-based immune response to the cells upon administration to a recipient patient.

[0136] In some embodiments, one or more of the modifications are regulatable modifications.

[0137] In some embodiments, provided herein is an engineered cell that comprises one or more regulatable modifications to alter expression of one or more targets in the engineered cell relative to a control, and optionally, the one or more regulatable modifications increase expression of CD47 relative to a control.

[0138] In some embodiments, the one or more regulatable modifications include conditional or inducible RNA-based components to i) increase, or ii) reduce or knock out, expression of one or more targets relative to a control.

[0139] In some embodiments, the conditional or inducible RNA-based component is selected from the group consisting of conditional or inducible shRNA, conditional or inducible siRNA, conditional or inducible miRNA, and conditional or inducible CRISPR interference (CRISPRi).

[0140] In some embodiments, the conditional RNA-based component is under the control of a conditional promoter selected from the group consisting of a cell cycle-specific promoter, a tissue-specific promoter, a lineage-specific promoter, and a differentiation-induced promoter.

[0141] In some embodiments, the inducible RNA-based component is under the control of an inducible promoter that is regulated by a small molecule, a ligand, a biological agent, an aptamer-mediated regulator of polyadenylation, or an aptamer-regulated riboswitch.

[0142] In some embodiments, regulatable modifications include conditional or inducible DNA-based components to i) increase, or ii) reduce or knock out, expression of one or more targets relative to a control.

[0143] In some embodiments, the conditional or inducible DNA-based component is selected from the group consisting of conditional or inducible CRISPR, conditional or inducible TALEN, conditional or inducible zinc finger nuclease, conditional or inducible homing endonuclease, conditional or inducible prime editing, conditional or inducible PASTE editing, and conditional or inducible meganuclease.

[0144] In some embodiments, the conditional DNA-based component is under the control of a conditional promoter selected from the group consisting of a cell cycle specific promoter, a tissue specific promoter, a lineage specific promoter, and a differentiation-induced promoter.

[0145] In some embodiments, the conditional DNA-based component is under the control of an inducible promoter that is regulated by a small molecule, a ligand, a biological agent, an aptamer-mediated regulator of polyadenylation, or an aptamer-regulated riboswitch.

[0146] In some embodiments, the regulatable modification comprises a conditional or inducible protein-based component to i) increase, or ii) reduce or knock out, expression of one or more targets relative to a control.

[0147] In some embodiments, the conditional or inducible protein-based component is a conditional or inducible degron component.

[0148] In some embodiments, the conditional or inducible degron component is selected from the group consisting of ligand-induced degradation (LID) using SMASH tags, a LID using Shield-1, a LID using auxin, a LID using rapamycin, a conditional or inducible peptide degron (e.g., an IKZF3-based degron), and a conditional or inducible protein degradation targeted chimera (PROTAC).

[0149] In some embodiments, the conditional protein-based component is under the control of a conditional promoter selected from the group consisting of a cell cycle specific promoter, a tissue specific promoter, a lineage specific promoter, and a differentiation-induced promoter.

[0150] In some embodiments, the protein-based component is under the control of an inducible promoter that is regulated by a small molecule, a ligand, a biological agent, an aptamer-mediated regulator of polyadenylation, or an aptamer-regulated riboswitch.

[0151] In some embodiments, the cells comprise a conditional promoter operably linked to an exogenous polynucleotide encoding one or more tolerogenic factors or CD47.

[0152] In some embodiments, the cell comprises (i) an exogenous polynucleotide comprising a conditional promoter operably linked to a transposase, and (ii) an exogenous polynucleotide comprising a transposon comprising a cargo polynucleotide encoding one or more tolerogenic factors or CD47.

[0153] In some embodiments, the conditional promoter is selected from the group consisting of a cell cycle specific promoter, a tissue specific promoter, a lineage specific promoter, and a differentiation induced promoter.

[0154] In some embodiments, the cells comprise an inducible promoter operably linked to an exogenous polynucleotide encoding one or more tolerogenic factors or CD47.

[0155] In some embodiments, the cell comprises (i) an exogenous polynucleotide comprising an inducible promoter operably linked to a transposase, and (ii) an exogenous polynucleotide comprising a transposon comprising a cargo polynucleotide encoding one or more tolerogenic factors or CD47.

[0156] In some embodiments, the inducible promoter is regulated by a small molecule, a ligand, a biological agent, an aptamer-mediated regulator of polyadenylation, or an aptamer-regulated riboswitch.

[0157] In some embodiments, the cells comprise a CD47 polypeptide having at least 80%, 85%, 90%, 95%, 98% or 100% sequence identity to the amino acid sequence of SEQ ID NO:129.

[0158] In some embodiments, the cells comprise a CD47 polypeptide having at least 80%, 85%, 90%, 95%, 98% or 100% sequence identity to the amino acid sequence of SEQ ID NO:130.

[0159] In some embodiments, the cells further comprise a regulatable modification that increases expression of one or more of A20 / TNFAIP3, C1-inhibitor, CCL21, CCL22, CD16, CD16 Fc receptor, CD24, CD27, CD35, CD39, CD46, CD52, CD55, CD59, CD200, CR1, CTLA4-Ig, DUX4, FasL, H2-M3, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IDO1, IL-10, IL15-RF, IL-35, MANF, Mfge8, PD-1, PD-L1, and / or Serpinb9 relative to a control.

[0160] In some embodiments, the cells express at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% greater amount of A20 / TNFAIP3, C1-inhibitor, CCL21, CCL22, CD16, CD16 Fc receptor, CD24, CD27, CD35, CD39, CD46, CD52, CD55, CD59, CD200, CR1, CTLA4-Ig, DUX4, FasL, H2-M3, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IDO1, IL-10, IL15-RF, IL-35, MANF, Mfge8, PD-1, PD-L1 and / or Serpinb9 relative to a control.

[0161] In some embodiments, the cells express at least about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800% or 900% greater amount of A20 / TNFAIP3, C1-inhibitor, CCL21, CCL22, CD16, CD16 Fc receptor, CD24, CD27, CD35, CD39, CD46, CD52, CD55, CD59, CD200, CR1, CTLA4-Ig, DUX4, FasL, H2-M3, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IDO1, IL-10, IL15-RF, IL-35, MANF, Mfge8, PD-1, PD-L1 and / or Serpinb9 relative to a control.

[0162] In some embodiments, the cells express at least about 1000% greater amount of A20 / TNFAIP3, C1-inhibitor, CCL21, CCL22, CD16, CD16 Fc receptor, CD24, CD27, CD35, CD39, CD46, CD52, CD55, CD59, CD200, CR1, CTLA4-Ig, DUX4, FasL, H2-M3, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IDO1, IL-10, IL15-RF, IL-35, MANF, Mfge8, PD-1, PD-L1, and / or Serpinb9 relative to a control.

[0163] In some embodiments, the control is a wild-type cell, a control cell, or a baseline reference.

[0164] In some embodiments, the control cell is an unmodified or unaltered cell, optionally, the unmodified or unaltered cell is of the same cell type as the engineered cell.

[0165] In some embodiments, the control cells are starting material from a donor or a pool of starting cells from a pool of donors.

[0166] In some embodiments, the baseline reference is an isotype control or background signal level.

[0167] In some embodiments, the one or more tolerogenic factors or CD47 are encoded by a first exogenous polynucleotide.

[0168] In some embodiments, the cells comprise a second exogenous polynucleotide encoding one or more chimeric antigen receptors (CARs).

[0169] In some embodiments, the first exogenous polynucleotide and / or the second exogenous polynucleotide is inserted within a first specific locus and / or a second specific locus of at least one allele of the cell.

[0170] In some embodiments, the first specific locus and / or the second specific locus is selected from the group consisting of a safe harbor locus, a target locus, a RHD locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus.

[0171] In some embodiments, the safe harbor locus is selected from the group consisting of the CCR5 locus, the PPP1R12C locus, the Rosa locus, the ROSA26 locus, and the CLYBL locus.

[0172] In some embodiments, the target locus is selected from the group consisting of the CXCR4 locus, the ALB locus, the SHS231 locus, the F3 (CD142) locus, the MICA locus, the MICB locus, the LRP1 (CD91) locus, the HMGB1 locus, the ABO locus, the FUT1 locus, and the KDM5D locus.

[0173] In some embodiments, the first exogenous polynucleotide and / or the second exogenous polynucleotide is introduced into the cell using a lentiviral vector.

[0174] In some embodiments, the first exogenous polynucleotide and / or the second exogenous polynucleotide are introduced into the cell using fusogen-mediated delivery or using a transposase system selected from the group consisting of a conditional or inducible transposase, a conditional or inducible PiggyBac transposon, a conditional or inducible Sleeping Beauty (SB11) transposon, a conditional or inducible Mos1 transposon, and a conditional or inducible Tol2 transposon.

[0175] In some embodiments, provided herein are pancreatic islet cells that have reduced expression of MHC class I HLA and / or reduced expression of MHC class II HLA and express at least about 1000% greater amount of CD47 relative to a control.

[0176] In some embodiments, the cells are primary beta islet cells that express at least about 16-fold, about 17-fold, about 18-fold, about 19-fold, or about 20-fold the level of CD47 expressed in a control.

[0177] In some embodiments, engineered cells are provided herein that express at least about 10% more CD47 than a control or that express at least about 1.1-fold the level of CD47 expressed in a control.

[0178] In some embodiments, provided herein are engineered cells that express at least about 10% more CD47 than a control or that express at least about 1.1-fold the level of CD47 expressed in a control.

[0179] In some embodiments, the cells express at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900% or about 1000% greater amount of CD47 relative to a control.

[0180] In some embodiments, the cells express at least about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, or about 5-fold the level of CD47 expressed in a control.

[0181] In some embodiments, the cells are primary pancreatic islet cells that express at least about 1000% or at least about 2000% greater amount of CD47 relative to a control.

[0182] In some embodiments, the control is a wild-type cell, a control cell, or a baseline reference.

[0183] In some embodiments, the control cell is an unmodified or unaltered cell, optionally, the unmodified or unaltered cell is of the same cell type as the engineered cell.

[0184] In some embodiments, the control cells are starting material from a donor or a pool of starting cells from a pool of donors.

[0185] In some embodiments, the baseline reference is an isotype control or background signal level.

[0186] In some embodiments, CD47 levels are determined using an antibody-based quantification method, optionally the Quantibrite™ assay.

[0187] In some embodiments, provided herein are engineered T cells that have reduced expression of MHC class I HLAs and / or reduced expression of MHC class II HLAs relative to a control, and express at least about 10% greater amount of CD47, express at least about 1.1-fold the level of CD47 expressed in a control, or express at least about 170,000 CD47 molecules.

[0188] In some embodiments, the cells are T cells that express at least about 300% or at least about 400% greater amount of CD47 relative to a control.

[0189] In some embodiments, the cells are T cells that express at least about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, or about 5-fold the level of CD47 expressed in a control.

[0190] In some embodiments, provided herein are engineered T cells that express at least about 170,000 CD47 molecules.

[0191] In some embodiments, the T cells express at least about 180,000 CD47 molecules, at least about 190,000 CD47 molecules, at least about 200,000 CD47 molecules, at least about 210,000 CD47 molecules, at least about 220,000 CD47 molecules, at least about 230,000 CD47 molecules, at least about 240,000 CD47 molecules, at least about 250,000 CD47 molecules, at least about 260,000 CD47 molecules, at least about 270,000 CD47 molecules, at least about 280,000 CD47 molecules, at least about 290,000 CD47 molecules, or at least about 300,000 CD47 molecules.

[0192] In some embodiments, the control is a wild-type cell, a control cell, or a baseline reference.

[0193] In some embodiments, the control cell is an unmodified or unaltered cell, optionally, the unmodified or unaltered cell is of the same cell type as the engineered cell.

[0194] In some embodiments, the control cells are starting material from a donor or a pool of starting cells from a pool of donors.

[0195] In some embodiments, the baseline reference is an isotype control or background signal level.

[0196] In some embodiments, CD47 levels are determined using an antibody-based quantification method, optionally the Quantibrite™ assay.

[0197] In some embodiments, the cells comprise one, two, three, four or five copies of an exogenous polynucleotide encoding CD47.

[0198] In some embodiments, the cells comprise a constitutive promoter operably linked to an exogenous polynucleotide encoding CD47.

[0199] In some embodiments, the exogenous polynucleotide encoding CD47 is delivered to the cell via viral-mediated integration.

[0200] In some embodiments, the viral-mediated integration is lentiviral-mediated.

[0201] In some embodiments, the exogenous polynucleotide encoding CD47 is integrated at a site in the genome of the cell via HDR.

[0202] In some embodiments, the exogenous polynucleotide encoding CD47 is integrated within a locus in the TRAC gene, a locus in the TRBC gene, or a combination thereof.

[0203] In some embodiments, the exogenous polynucleotide encoding CD47 is integrated within at least one TRAC allele, at least one TRBC allele, or a combination thereof.

[0204] In some embodiments, the exogenous polynucleotide encoding CD47 is integrated into at least two TRAC alleles, at least two TRBC alleles, or a combination thereof.

[0205] In some embodiments, the cell comprises an exogenous polynucleotide comprising a CD47 polypeptide having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO:129, at least about 85% sequence identity to the amino acid sequence of SEQ ID NO:129, at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:129, at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:129, at least about 98% sequence identity to the amino acid sequence of SEQ ID NO:129, at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:129, or the amino acid sequence of SEQ ID NO:129.

[0206] In some embodiments, the cell comprises an exogenous polynucleotide comprising a CD47 polypeptide having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO:130, at least about 85% sequence identity to the amino acid sequence of SEQ ID NO:130, at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:130, at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:130, at least about 98% sequence identity to the amino acid sequence of SEQ ID NO:130, at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:130, or the amino acid sequence of SEQ ID NO:130.

[0207] In some embodiments, the cells comprise reduced expression of one or more MHC class I and / or MHC class II molecules relative to a control.

[0208] In some embodiments, the reduced expression of one or more MHC class I and / or MHC class II molecules is caused by a constitutive modification to one or more genes encoding MHC class I and / or class II HLA.

[0209] In some embodiments, the cells comprise one or more knockouts of a target selected from the group consisting of MHC class I and MHC class II HLA.

[0210] In some embodiments, the one or more knockouts are constitutive knockouts.

[0211] In some embodiments, the cells comprise reduced expression of one or more targets selected from the group consisting of B2M and CIITA relative to a control.

[0212] In some embodiments, the reduced expression of B2M and / or CIITA is caused by a constitutive modification to the B2M gene and / or the CIITA gene.

[0213] In some embodiments, the cells comprise one or more knockouts of targets selected from the group consisting of B2M and CIITA.

[0214] In some embodiments, the cells comprise a knockout of both alleles of B2M and / or both alleles of CIITA.

[0215] In some embodiments, the one or more knockouts are constitutive knockouts.

[0216] In some embodiments, the cells further comprise an exogenous polynucleotide encoding one or more additional tolerogenic factors.

[0217] In some embodiments, the one or more additional tolerogenic factors are selected from the group consisting of HLA-C, HLA-E, HLA-F, HLA-G, PD-L1, CTLA-4-Ig, C1-inhibitor, and IL-35.

[0218] In some embodiments, the cells comprise reduced expression of B2M, CIITA, NLRC5, TRAC, TRB, CD142, ABO, MIC-A / B, CD38, CD52, PCDH11Y, NLGN4Y, and / or RHD relative to a control.

[0219] In some embodiments, the cells do not express B2M, CIITA, NLRC5, TRAC, TRB, CD142, ABO, MIC-A / B, CD38, CD52, PCDH11Y, NLGN4Y, and / or RHD.

[0220] In some embodiments, the cells are pluripotent stem cells.

[0221] In some embodiments, the pluripotent stem cells are induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs) or embryonic stem cells (ESCs).

[0222] In some embodiments, the cell is a differentiated cell derived from a pluripotent stem cell or its progeny.

[0223] In some embodiments, the differentiated cells are selected from the group consisting of pancreatic islet cells, T cells, natural killer (NK) cells, CAR-M cells, endothelial cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, hepatocytes, glial progenitor cells, dopaminergic neurons, retinal pigment epithelial cells, and thyroid cells.

[0224] In some embodiments, the cell is a primary cell or a progeny thereof.

[0225] In some embodiments, the primary cell or its progeny is a T cell or a NK cell.

[0226] In some embodiments, the T cells further comprise reduced expression of T cell receptor (TCR) alpha and / or TCR beta.

[0227] In some embodiments, the T cells do not express TCR alpha and / or TCR beta.

[0228] In some embodiments, the T cell further comprises a second exogenous polynucleotide encoding one or more chimeric antigen receptors (CARs).

[0229] In some embodiments, the cells express at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% greater CD47 expression relative to a control and express reduced expression of one or more of MHC class I and MHC class II human leukocyte antigens relative to a control.

[0230] In some embodiments, the cells express at least about two-fold, about three-fold, about four-fold, or about five-fold the level of CD47 expressed in wild-type or control cells that have no or low expression of CD47, and express reduced expression of one or more of MHC class I and MHC class II human leukocyte antigens relative to the control cells.

[0231] In some embodiments, the cells express at least about three, about four, or about five times the level of CD47 expressed in wild-type or control cells of the same cell type that have no or low expression of CD47.

[0232] In some embodiments, the control cell is a pancreatic islet cell, a T cell, a natural killer (NK) cell, a CAR-M cell, an endothelial cell, a cardiomyocyte, a smooth muscle cell, a skeletal muscle cell, a hepatocyte, a glial progenitor cell, a dopaminergic neuron, a retinal pigment epithelial cell, or a thyroid cell.

[0233] In some embodiments, the differentiated cells or their progeny, or the primary immune cells or their progeny, avoid NK cell-mediated cytotoxicity upon administration to a recipient patient, are protected from cytolysis by mature NK cells upon administration to a recipient patient, avoid macrophage phagocytosis upon administration to a recipient patient, do not induce an innate and / or adaptive immune response against the cells upon administration to a recipient patient, and / or do not induce an antibody-based immune response against the cells upon administration to a recipient patient.

[0234] In some embodiments, the cells are autologous cells.

[0235] In some embodiments, the cells are allogeneic cells.

[0236] In some embodiments, provided herein is a pharmaceutical composition comprising a population of engineered cells disclosed herein and a pharma- ceutically acceptable excipient, carrier, diluent, or excipient.

[0237] In some embodiments, the engineered cells are beta islet cells and the pharmaceutical composition further comprises one or more additional pancreatic islet cells.

[0238] In some embodiments, provided herein are methods of treating a patient having a disease or condition that would benefit from a cell-based therapy, comprising administering to the patient a clinically or therapeutically effective amount of the engineered cells disclosed herein.

[0239] In some embodiments, provided herein is a method of treating a patient having a disease or condition that would benefit from a cell-based therapy, comprising administering to the patient a population of cells that comprises the engineered cells disclosed herein.

[0240] In some embodiments, provided herein is a method of treating a patient having a disease or condition that would benefit from a cell-based therapy, comprising administering to the patient a population of cells comprising the differentiated cells disclosed herein.

[0241] In some embodiments, provided herein is a method of treating a patient having a disease or condition that would benefit from a cell-based therapy, comprising administering to the patient a pharmaceutical composition disclosed herein.

[0242] In some embodiments, the disease or condition is cancer, a genetic disorder, a chronic infectious disease, an autoimmune disorder, a neurological disorder, a cardiac disorder (pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, idiopathic cardiomyopathy, other cardiomyopathies, myocardial ischemia reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end-stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart, arterial inflammation, cardiovascular disease, myocardial infarction, myocardial ischemia, myocardial infarction, cardiac ischemia, cardiac trauma, myocardial ischemia, vascular disease, acquired heart Cardiac disease, congenital heart disease, coronary artery disease, dysfunctional conduction system, dysfunctional coronary arteries, pulmonary hypertension, cardiac arrhythmias, muscular dystrophies, abnormal muscle mass, muscle degeneration, myocarditis, infectious myocarditis, drug- or toxin-induced myopathy, hypersensitivity myocarditis, mitral valve insufficiency, autoimmune endocarditis, primary arrhythmia disorders, cardiac channelopathies, long QT syndrome, short QT syndrome, Brugada syndrome, catecholamine-induced polymorphic ventricular tachycardia, Jerber-Lange-Nielsen syndrome, myocardial infarction, heart failure, cardiomyopathy, congenital heart defects, valvular heart disease or dysfunction, endocarditis, rheumatic fever, mitral valve prolapse, infective endocarditis, hypertrophy cardiomyopathy, dilated cardiomyopathy, myocarditis, cardiac enlargement, mitral valve insufficiency), neurological disorders (Alzheimer's disease, Huntington's disease, Parkinson's disease, Pelizaeus-Merzbach disease, other neurodegenerative diseases or conditions, attention deficit hyperactivity disorder (ADHD), ischemia, multiple sclerosis, traumatic brain injury, epilepsy, catalepsy, encephalitis, meningitis, migraine, stroke, transient ischemic attack, subarachnoid hemorrhage, subdural hemorrhage, hematoma, epidural hemorrhage, spinal cord injury, cervical spondylosis, carpal tunnel syndrome, brain or spinal cord tumor, peripheral neuropathy, Guillain-Barre syndrome, neuralgia, amyotrophic lateral sclerosis (ALS) LS), tauopathy, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease, Bell's palsy, cerebral palsy, motor neuron disease, neurofibromatosis, encephalitis, meningitis, Tourette's syndrome, schizophrenia, psychosis, depression, and other neuropsychiatric disorders), vascular dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, other neurodegenerative diseases or conditions, Attention Deficit Hyperactivity Disorder (ADHD), Tourette's syndrome (TS), schizophrenia, psychosis, depression, other neuropsychiatric disorders, HIV-1 associated neurocognitive disorder, traumatic brain injury, stroke,Amyotrophic lateral sclerosis (ALS), cerebral hemorrhage, epileptic seizures, spinal cord injury, argyrophilic grain disease (AGD), amyotrophic lateral sclerosis (ALS), corticobasal degeneration (CBD), Parkinsonism chromosome 17 linkage (FTDP-17), multiple system atrophy (MSA), Parkinson's disease / diffuse Lewy body disease (PD / DLBD) or Alzheimer's disease, atherosclerosis, atherogenesis, arterial thrombosis, venous thrombosis, thrombocytopenia, vascular leakage, diffuse intravascular coagulation, diabetes, insulin resistance, cardiovascular disease, vascular disease, peripheral vascular disease, ischemic disease, myocardial infarction, congestion heart failure, peripheral vascular occlusive disease, stroke, reperfusion injury, limb ischemia, neuropathy (e.g., peripheral neuropathy or diabetic neuropathy), organ failure (e.g., liver failure and renal failure), diabetes, rheumatoid arthritis, osteoporosis, vascular injury, tissue injury, hypertension, angina and myocardial infarction due to coronary artery disease, renal vascular hypertension, renal failure due to renal artery stenosis, claudication of the lower limbs, transient ischemic attack or stroke, myocardial infarction and limb ischemia, repair of ischemic tissue, formation of blood vessels and heart valves, engineering of artificial blood vessels, repair of damaged blood vessels, and engineering tissue (e.g., prior to transplantation). Induction of blood vessel formation in tissues, repair or replacement for tissues in need of vascular cells or angiogenesis, particularly cardiac tissue, hepatic tissue, pancreatic tissue, renal tissue, muscle tissue, nervous tissue, bone tissue, which may be tissues characterized thereby as damaged by excessive cell death, tissues at risk of damage or artificially engineered tissues, coronary artery disease, cerebrovascular disease, aortic stenosis, aortic aneurysms, peripheral artery disease, atherosclerosis, varicose veins, vasculopathy, infarcted areas of the heart lacking coronary perfusion, non-healing wounds, diabetic or non-diabetic ulcers, or inducing the formation of blood vessels. , any other disease or disorder in which it is desirable to improve prosthetic implants used in vascular reconstruction surgery (e.g., blood vessels made from synthetic materials such as Dacron and Gortex); vascular injury, cardiovascular disease, vascular disease, peripheral vascular disease, ischemic disease, myocardial infarction, congestive heart failure, peripheral vascular occlusive disease, hypertension, ischemic tissue injury, reperfusion injury, limb ischemia, stroke, neuropathy (e.g., peripheral neuropathy or diabetic neuropathy), organ failure (e.g., liver failure and kidney failure), diabetes, rheumatoid arthritis, osteoporosis, cerebrovascular disease, hypertension,Vascular disorders selected from the group consisting of angina pectoris and myocardial infarction due to coronary artery disease, renal vascular hypertension, renal failure due to renal artery stenosis, claudication of the lower limbs, other vascular conditions or diseases; autoimmune thyroiditis, goiter, hyperparathyroidism, hypoparathyroidism (congenital or autoimmune), thyroiditis, Hashimoto's thyroiditis, postpartum thyroiditis, subacute thyroiditis, iatrogenic hypothyroidism, Graves' disease and thyroid eye disease, infectious hepatitis (A, B and C), autoimmune hepatitis, primary biliary cholangitis, primary sclerosing cholangitis, nonalcoholic fatty liver disease, cirrhosis, hemochromatosis, hyperoxalemia, Aciduria, Alpha 1-antitrypsin deficiency, Liver failure, Wilson's disease, Hepatic encephalopathy, Jaundice, Acute hepatic porphyria, Alagille syndrome, Biliary atresia, Budd-Chiari syndrome, Hyperbilirubinemia, Crigler-Najjar syndrome, Gilbert-Meulengracht syndrome, Dubin-Johnson syndrome, Rotor syndrome, Galactosemia, Glycogen storage disease type 1, Hepatorenal syndrome, Intrahepatic cholestasis of pregnancy, Progressive familial intrahepatic cholestasis, Reye's syndrome, Lysosomal acid lipase deficiency, Alcohol-related pancreatitis, Gallstone pancreatitis, Diabetes mellitus (types 1 and 2), Prediabetes, Gestational diabetes, Pancreatic deficiency Impaired diabetes, exocrine pancreatic insufficiency, acute pancreatitis, chronic pancreatitis, hereditary pancreatitis, hyperinsulinemia, pancreatic cysts, Zollinger-Ellison syndrome, Shwachman-Diamond syndrome, hereditary hemochromatosis, thalassemia, pancreatic iron deposition, cystic fibrosis, pancreatectomy and pancreatectomy, patients with macular degeneration or damaged RPE cells, age-related macular degeneration (AMD), early AMD, intermediate AMD, late AMD, non-neovascular age-related macular degeneration, dry macular degeneration (dry age-related macular degeneration), wet macular degeneration (wet age-related macular degeneration), adult-onset vitelliform macular degeneration (AVMD) , Best vitelliform macular degeneration, Stargardt-like macular dystrophy (STGD3), Sorsby fundus degeneration (SFD), ABCA4-related disorders, Usher type IB, autosomal recessive bestrophinosis, autosomal dominant vitreoretinochoroidopathy, juvenile macular degeneration (JMD), Leber congenital amaurosis or retinitis pigmentosa, retinal detachment, retinal breaks, severe combined immunodeficiency (SCID), Omenn syndrome, cartilage-hair hypoplasia, reticular dysplasia, Wiskott-Aldrich syndrome, ataxia-telangiectasia, DiGeorge syndrome, immune-mediated osteogenesis imperfecta, dyskeratosis congenita,Chronic mucocutaneous candidiasis, hematological malignancies, follicular lymphoma (FL), myeloid neoplasms, mature T / NK neoplasms, histiocytic neoplasms, multiple myeloma (MM), myelodysplastic syndromes (MDS), lymphoplasmacytic lymphoma (LPL), Waldenstrom's hypergammaglobulinemia, Burkitt's lymphoma (BL), primary mediastinal large B-cell lymphoma (PMBL), Hodgkin's lymphoma, mantle cell lymphoma (MCL), hairy cell leukemia (HCL), myeloproliferative / myelodysplastic syndromes (MDS), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute Myeloid leukemia (AML), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), T-cell lymphoma, B-cell lymphoma; autoimmune diseases including lupus, systemic lupus erythematosus, rheumatoid arthritis, psoriasis, psoriatic arthritis, multiple sclerosis, Crohn's disease, ulcerative colitis, Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, diabetes mellitus type 1, primary biliary cirrhosis, autoimmune hepatitis, celiac disease; B-cell acute lymphoblastic leukemia (BL), Cancers including, but not limited to, leukemia (B-ALL), diffuse large B-cell lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, lung cancer, non-small cell lung cancer, acute myeloid lymphocytic leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, neuroblastoma, squamous cell carcinoma of the lung, hepatocellular carcinoma, and bladder cancer; systemic lupus erythematosus (SLE), type 1 diabetes, autoimmune liver disease, Sjogren's syndrome, rheumatoid arthritis, systemic sclerosis (scleroderma), organ-specific autoimmune diseases (autoimmune hepatitis, primary sclerosing cholangitis), alcohol-related liver disease, and multiple sclerosis NK cell deficiency (NKD) (functional (FNKD) or classical (CNKD)), immunodeficiency-polyendocrinopathy-enteropathy-X-linked (IPEX)-like syndrome, Bloom's syndrome, Fanconi anemia, dyskeratosis congenita, Chediak-Higashi syndrome, familial hemophagocytic lymphohistiocytosis (FHL), Grischeri syndrome type 2, Hermansky-Pudlak syndrome, Papillon-Lefevre syndrome, Wiskott-Aldrich syndrome, autosomal recessive hyper-IgE syndrome, May-Hegglin anomaly, and leukocyte adhesion deficiency type I or III.

[0243] In some embodiments, the differentiated cells are selected from the group consisting of mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), pancreatic islet cells, beta islet cells, immune cells, B cells, T cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophage cells, immune privileged cells, optic nerve cells, retinal pigment epithelial cells (RPE), liver cells, thyroid cells, endothelial cells, skin cells, glial progenitor cells, neuronal cells, muscle cells, cardiac cells, and blood cells.

[0244] In some embodiments, no immunosuppressive and / or immunomodulatory agents are administered to the patient prior to administration of the population of cells.

[0245] In some embodiments, the methods further comprise administering to the patient one or more immunosuppressive agents.

[0246] In some embodiments, the patient is receiving one or more immunosuppressive agents.

[0247] In some embodiments, the one or more immunosuppressive agents is a small molecule or an antibody.

[0248] In some embodiments, the one or more immunosuppressive agents are selected from the group consisting of cyclosporine, azathioprine, mycophenolic acid, mycophenolate mofetil, corticosteroids, prednisone, methotrexate, gold salts, sulfasalazine, antimalarials, brequinar, leflunomide, mizoribine, 15-deoxyspergualin, 6-mercaptopurine, cyclophosphamide, rapamycin, tacrolimus (FK-506), OKT3, antithymocyte globulin, thymopentin (thymosin alpha), and immunosuppressant antibodies.

[0249] In some embodiments, the one or more immunosuppressants comprises cyclosporine.

[0250] In some embodiments, the one or more immunosuppressants comprises mycophenolate mofetil.

[0251] In some embodiments, the one or more immunosuppressants comprises a corticosteroid.

[0252] In some embodiments, the one or more immunosuppressants comprises cyclophosphamide.

[0253] In some embodiments, the one or more immunosuppressants comprises rapamycin.

[0254] In some embodiments, the one or more immunosuppressants comprises tacrolimus (FK-506).

[0255] In some embodiments, the one or more immunosuppressive agents comprises antithymocyte globulin.

[0256] In some embodiments, the one or more immunosuppressive agents are one or more immunomodulatory agents.

[0257] In some embodiments, the one or more immunomodulatory agents is a small molecule or an antibody.

[0258] In some embodiments, the antibody binds to one or more of the receptors or ligands selected from the group consisting of p75 of the IL-2 receptor, MHC, CD2, CD3, CD4, CD7, CD28, B7, CD40, CD45, IFN gamma, TNF alpha, IL-4, IL-5, IL-6R, IL-6, IGF, IGFR1, IL-7, IL-8, IL-10, CD11a, CD58, and the antibody binds to any of those ligands.

[0259] In some embodiments, one or more immunosuppressive agents are or have been administered to the patient prior to administration of the engineered cells.

[0260] In some embodiments, the one or more immunosuppressive agents are or have been administered to the patient at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days prior to administration of the engineered cells.

[0261] In some embodiments, the one or more immunosuppressive agents are or have been administered to the patient at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more prior to administration of the engineered cells.

[0262] In some embodiments, the one or more immunosuppressive agents are or have been administered to the patient at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after administration of the engineered cells.

[0263] In some embodiments, the one or more immunosuppressive agents are or have been administered to the patient at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more after administration of the engineered cells.

[0264] In some embodiments, the one or more immunosuppressive agents are or have been administered to the patient on the same day as the first administration of the engineered cells.

[0265] In some embodiments, one or more immunosuppressive agents are or have been administered to the patient following administration of the engineered cells.

[0266] In some embodiments, one or more immunosuppressive agents are or have been administered to the patient after the first administration and / or the second administration of the engineered cells.

[0267] In some embodiments, one or more immunosuppressive agents are or have been administered to the patient prior to the first administration and / or the second administration of the engineered cells.

[0268] In some embodiments, the one or more immunosuppressive agents are or have been administered to the patient at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days prior to the first administration and / or the second administration of the engineered cells.

[0269] In some embodiments, the one or more immunosuppressive agents are or have been administered to the patient at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more prior to the first administration and / or second administration of the engineered cells.

[0270] In some embodiments, the one or more immunosuppressive agents are or have been administered to the patient at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days after the first administration and / or the second administration of the engineered cells.

[0271] In some embodiments, the one or more immunosuppressive agents are or have been administered to the patient at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more after the first administration and / or second administration of the engineered cells.

[0272] In some embodiments, the one or more immunosuppressants are administered at a lower dosage compared to the dosage of the one or more immunosuppressants administered to reduce immune rejection of immunogenic cells that do not include engineered cellular modifications.

[0273] In some embodiments, provided herein is the use of a population of engineered cells disclosed herein to treat a disorder or condition in a recipient patient that would benefit from a cell-based therapy.

[0274] In some embodiments, provided herein are methods for producing an engineered cell as disclosed herein or a population of cells comprising an engineered cell as disclosed herein, the method comprising: (a) obtaining an isolated cell; and (b) contacting the isolated cell with one or more reagents and / or components to modify gene expression in the isolated cell, thereby producing the engineered cell or a population of cells comprising the engineered cell.

[0275] In some embodiments, the method further comprises determining the CD47 expression level of the engineered cell or population of cells.

[0276] In some embodiments, the method further comprises selecting the engineered cell or population of cells for use in the manufacture of a therapeutic product if it is determined that the engineered cell or population of cells expresses CD47 at or above a threshold level.

[0277] In some embodiments, the engineered cell or population of cells expresses at least about the same amount of CD47 as a control.

[0278] In some embodiments, the engineered cells or population of cells express at least about 10% more CD47 than a control.

[0279] In some embodiments, the engineered cells or population of cells express at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% greater amount of CD47 relative to a control.

[0280] In some embodiments, the engineered cells or population of cells express at least about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800% or 900% greater amount of CD47 relative to a control.

[0281] In some embodiments, the engineered cells or population of cells express at least about 1000% more CD47 than a control.

[0282] In some embodiments, the engineered cell or population of cells expresses at least about 1.1-fold the level of CD47 expressed in a control.

[0283] In some embodiments, the engineered cells or populations of cells express at least about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, or about 5-fold the level of CD47 expressed in a control.

[0284] In some embodiments, the engineered cells or populations of cells express at least about 4-fold, about 4.5-fold, about 5-fold, or about 5.5-fold the level of CD47 expressed in a control.

[0285] In some embodiments, the engineered cell or population of cells expresses at least about four-fold the level of CD47 expressed in a control.

[0286] In some embodiments, the engineered cell or population of cells expresses at least about 4.5-fold the level of CD47 expressed in a control.

[0287] In some embodiments, the engineered cell or population of cells expresses at least about 5-fold the level of CD47 expressed in a control.

[0288] In some embodiments, the engineered cell or population of cells expresses at least about 5.5-fold the level of CD47 expressed in a control.

[0289] In some embodiments, the engineered cells or population of cells express at least about 16-fold, about 17-fold, about 18-fold, about 19-fold, or about 20-fold the level of CD47 expressed in a control.

[0290] In some embodiments, the control is a wild-type cell or population of wild-type cells, a control cell or population of control cells, or a baseline reference.

[0291] In some embodiments, the control cell or population of control cells comprises unmodified or unaltered cells, optionally, the unmodified or unaltered cells are of the same cell type as the engineered cells.

[0292] In some embodiments, the control cell or population of control cells is starting material from a donor or a pool of starting cells from a pool of donors.

[0293] In some embodiments, the baseline reference is an isotype control or background signal level.

[0294] In some embodiments, the engineered cells are beta islet cells, and the population of cells comprises beta islet cells and additional pancreatic islet cells.

[0295] In some embodiments, the engineered cells contain a regulatable modification that alters expression of one or more targets in the engineered cells relative to a control.

[0296] In some embodiments, the regulatable modification reduces expression of one or more MHC class I and / or MHC class II molecules relative to a wild-type cell, a population of wild-type cells, a control cell, or a population of control cells.

[0297] In some embodiments, the regulatable modification increases expression of one or more tolerogenic factors relative to a wild-type cell, a population of wild-type cells, a control cell, or a population of control cells.

[0298] In some embodiments, the one or more reagents for modifying gene expression in the isolated cell comprise i) a conditional or inducible RNA-based component for altering expression of one or more targets, ii) a conditional or inducible DNA-based component for altering expression of one or more targets, or iii) a conditional or inducible protein-based component for altering expression of one or more targets.

[0299] In some embodiments, the method further comprises contacting the isolated cell with an exogenous factor or exposing the isolated cell to conditions to activate a conditional or inducible promoter, thereby causing expression of one or more targets, thereby producing an engineered cell.

[0300] In some embodiments, a method for producing an engineered cell, the engineered cell comprising: i) a regulatable modification that reduces expression of one or more MHC class I and / or MHC class II molecules; and ii) a regulatable modification that increases expression of one or more tolerogenic factors relative to a control, the method comprising: (a) obtaining an isolated cell; and (b) introducing into the cell a conditional or inducible RNA-based construct for regulatable, reduced expression of MHC class I and / or MHC class II human leukocyte molecules, a conditional or inducible DNA-based construct for regulatable, reduced expression of MHC class I and / or MHC class II human leukocyte molecules, or a conditional or inducible DNA-based construct for regulatable, reduced expression of MHC class I and / or MHC class II human leukocyte molecules. Provided herein are methods comprising: (c) introducing an inducible protein-based component into the isolated cell; (d) introducing into the isolated cell a nucleic acid comprising a conditional or inducible promoter operably linked to an exogenous polynucleotide encoding one or more tolerogenic factors for regulatable increased expression of the one or more tolerogenic factors; and (e) exposing the engineered cell to conditions or exogenous factors to activate the conditional or inducible promoter, thereby causing expression of the exogenous one or more tolerogenic factors, thereby producing an engineered cell.

[0301] In some embodiments, steps (a)-(d) are performed in any order.

[0302] In some embodiments, one or more of steps (a)-(d) are performed simultaneously.

[0303] In some embodiments, steps (b) and (c) are performed before steps (d) and (e).

[0304] In some embodiments, steps (d) and (e) are performed before steps (b) and (c).

[0305] In some embodiments, steps (c) and (e) are performed sequentially.

[0306] In some embodiments, steps (c) and (e) are performed simultaneously.

[0307] In some embodiments, provided herein are methods for identifying a population of cells, or a population of cells comprising an engineered cell as disclosed herein, that are suitable for use as a therapeutic product, the methods comprising: (a) obtaining isolated cells; (b) introducing into the cells one or more modifications that reduce expression of one or more MHC class I and / or MHC class II molecules relative to a control; (c) introducing into the cells one or more modifications that increase expression of CD47 relative to a control; (d) measuring the level of CD47 expression in the cells; and (e) selecting a population of cells that expresses at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% greater than the control, and identifying the population as suitable for use as a therapeutic product.

[0308] In some embodiments, a method for identifying a population of cells, or a population of cells comprising engineered cells as disclosed herein, that are suitable for use as a therapeutic product includes: (a) obtaining isolated cells; (b) introducing into the cells one or more modifications that reduce expression of one or more MHC class I and / or MHC class II molecules relative to a control; (c) introducing into the cells one or more modifications that increase expression of CD47 relative to a control; and (d) determining the level of CD47 expression in the cells. and (e) selecting a population of cells expressing at least about 1.1 fold, about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 3.5 fold, about 4 fold, about 4.5 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about 11 fold, about 12 fold, about 13 fold, about 14 fold, about 15 fold, about 16 fold, about 17 fold, about 18 fold, about 19 fold or about 20 fold the level of CD47 expressed in a control, identifying the population as suitable for use as a therapeutic product.

[0309] In some embodiments, step (b) is performed before step (c).

[0310] In some embodiments, step (c) is performed before step (b).

[0311] In some embodiments, steps (b) and (c) are performed simultaneously.

[0312] In some embodiments, provided herein is a method of determining whether a population of cells is suitable for use as a therapeutic product, the method comprising: (a) producing engineered cells comprising a first exogenous polynucleotide encoding CD47, optionally an engineered cell as disclosed herein; (b) measuring the expression level of CD47 in the cells; and (c) determining that the population of cells is suitable for use as a therapeutic product if the cells express an amount of CD47 that is greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% relative to a control.

[0313] In some embodiments, provided herein are methods of determining whether a population of cells is suitable for use as a therapeutic product, the methods comprising: (a) producing engineered cells comprising a first exogenous polynucleotide encoding CD47, optionally an engineered cell as disclosed herein; (b) measuring the expression level of CD47 in the cells; and (c) determining that the population of cells is suitable for use as a therapeutic product if the cells express at least about 1.1 fold, about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 3.5 fold, about 4 fold, about 4.5 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about 11 fold, about 12 fold, about 13 fold, about 14 fold, about 15 fold, about 16 fold, about 17 fold, about 18 fold, about 19 fold, or about 20 fold the level of CD47 expressed in a control.

[0314] In some embodiments, the control is a wild-type cell, a control cell, or a baseline reference.

[0315] In some embodiments, the control cell is an unmodified or unaltered cell, optionally, the unmodified or unaltered cell is of the same cell type as the engineered cell.

[0316] In some embodiments, the control cells are starting material from a donor or a pool of starting cells from a pool of donors.

[0317] In some embodiments, the baseline reference is an isotype control or background signal level.

[0318] In some embodiments, CD47 levels are determined using an antibody-based quantification method, optionally the Quantibrite™ assay.

[0319] In some embodiments, provided herein are methods for determining a threshold CD47 expression level required for immune evasion of a poorly immunogenic cell, the method comprising: (a) producing engineered cells comprising a first exogenous polynucleotide encoding CD47; (b) sorting the engineered cells based on CD47 expression level to generate a pool of cells having similar CD47 expression levels; (c) evaluating the immune response induced by the pool of cells; and (d) determining the threshold CD47 expression level required for immune evasion.

[0320] In some embodiments, CD47 levels are determined using an antibody-based quantification method, optionally the Quantibrite™ assay.

[0321] In some embodiments, step (a) of the method further comprises engineering the cells to contain reduced expression of one or more Y chromosome genes, and major histocompatibility complex (MHC) class I and / or class II human leukocyte antigens, relative to wild-type or control cells.

[0322] In some embodiments, the assessment of the immune response is performed using an in vitro or in vivo assay.

[0323] In some embodiments, assessment of the immune response is performed by measuring NK cell-mediated cytotoxicity, lysis by mature NK cells, macrophage phagocytosis, antibody-based immune responses to the cells, or by measuring the percentage of cells still present in the recipient after a period of administration to the recipient patient.

[0324] In some embodiments, provided herein is a method for identifying a population of cells, or a population of cells comprising an engineered cell as disclosed herein, that is suitable for use as a therapeutic product, the method comprising: (a) introducing into the isolated cells one or more modifications that reduce expression of one or more MHC class I and / or MHC class II molecules relative to a control; and (b) introducing into the cells one or more modifications that increase expression of CD47 relative to a control.

[0325] In some embodiments, the method further comprises measuring the expression level of CD47 in the cells in step (c).

[0326] In some embodiments, the method further comprises step (d) selecting a population of cells expressing at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% greater than a control, and identifying the population as suitable for use as a therapeutic product.

[0327] In some embodiments, the method further comprises step (d) selecting a population of cells expressing at least about 1.1 fold, about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 3.5 fold, about 4 fold, about 4.5 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about 11 fold, about 12 fold, about 13 fold, about 14 fold, about 15 fold, about 16 fold, about 17 fold, about 18 fold, about 19 fold or about 20 fold the level of CD47 expressed in the control, and identifying the population as suitable for use as a therapeutic product.

[0328] In some embodiments, step (a) is performed before step (b).

[0329] In some embodiments, step (b) is performed before step (a).

[0330] In some embodiments, steps (a) and (b) are performed simultaneously.

[0331] Detailed descriptions of hypoimmunogenic cells, methods for their production, and methods for their use can be found in U.S. Provisional Patent Application No. 63 / 065,342, filed August 13, 2020, U.S. Provisional Patent Application No. 63 / 136,152, filed December 31, 2020, U.S. Provisional Patent Application No. 63 / 175,030, filed April 14, 2021, U.S. Provisional Patent Application No. 63 / 175,003, filed April 14, 2021, and U.S. Provisional Patent Application No. 63 / 175,003, filed January 11, 2021 (Attorney Docket No. 18615-30046.00), which is incorporated herein by reference in its entirety. No. 6,399,143, the disclosures of which, including examples, sequence listings and figures, are incorporated herein by reference in their entireties. [Brief description of the drawings]

[0332] [Figure 1A] Flow cytometry data measuring CD47 levels on the cell surface of primary murine B2M- / -;CD47tg beta islet cells generated from beta islet cells isolated from B2M knockout C57BL / 6 (B6) mice and then transduced with lentivirus containing a CD47 transgene. Different MOIs were evaluated in B2M- / -;CD47tg beta islet cells. CD47 levels were compared to isotype control (left). [Figure 1B]1 shows data for NK cell-mediated killing of B2M- / -;CD47tg beta islet cells by mouse NK cells. [Figure 1C] Flow cytometry data measuring CD47 levels on the cell surface of primary murine B2M- / -;CD47tg beta islet cells generated from beta islet cells isolated from B2M knockout C57BL / 6 (B6) mice and then transduced with lentivirus containing a CD47 transgene. Different MOIs were evaluated in B2M- / -;CD47tg beta islet cells. CD47 levels were compared to isotype control (left). [Figure 1D] 1 shows data for NK cell-mediated killing of B2M- / -;CD47tg beta islet cells by mouse NK cells. [Figure 1E] Flow cytometry data measuring CD47 levels on the cell surface of primary murine B2M- / -;CD47tg beta islet cells generated from beta islet cells isolated from B2M knockout C57BL / 6 (B6) mice and then transduced with lentivirus containing a CD47 transgene. Different MOIs were evaluated in B2M- / -;CD47tg beta islet cells. CD47 levels were compared to isotype control (left). [Figure 1F] 1 shows data for NK cell-mediated killing of B2M- / -;CD47tg beta islet cells by mouse NK cells. [Figure 1G] Flow cytometry data measuring CD47 levels on the cell surface of primary murine B2M- / -;CD47tg beta islet cells generated from beta islet cells isolated from B2M knockout C57BL / 6 (B6) mice and then transduced with lentivirus containing a CD47 transgene. Different MOIs were evaluated in B2M- / -;CD47tg beta islet cells. CD47 levels were compared to isotype control (left). [Figure 1H] 1 shows data for NK cell-mediated killing of B2M- / -;CD47tg beta islet cells by mouse NK cells. [Figure 1I]Flow cytometry data measuring CD47 levels on the cell surface of primary murine B2M- / -;CD47tg beta islet cells generated from beta islet cells isolated from B2M knockout C57BL / 6 (B6) mice and then transduced with lentivirus containing a CD47 transgene. Different MOIs were evaluated in B2M- / -;CD47tg beta islet cells. CD47 levels were compared to isotype control (left). [Figure 1J] 1 shows data for NK cell-mediated killing of B2M- / -;CD47tg beta islet cells by mouse NK cells. [Figure 1K] Flow cytometry data measuring CD47 levels on the cell surface of primary murine B2M- / -;CD47tg beta islet cells generated from beta islet cells isolated from B2M knockout C57BL / 6 (B6) mice and then transduced with lentivirus containing a CD47 transgene. Different MOIs were evaluated in B2M- / -;CD47tg beta islet cells. CD47 levels were compared to isotype control (left). [Figure 1L] 1 shows data for NK cell-mediated killing of B2M- / -;CD47tg beta islet cells by mouse NK cells. [Figure 1M] Flow cytometry data measuring CD47 levels on the cell surface of primary murine B2M- / -;CD47tg beta islet cells generated from beta islet cells isolated from B2M knockout C57BL / 6 (B6) mice and then transduced with lentivirus containing a CD47 transgene. Different MOIs were evaluated in B2M- / -;CD47tg beta islet cells. CD47 levels were compared to isotype control (left). [Figure 1N] 1 shows data for NK cell-mediated killing of B2M- / -;CD47tg beta islet cells by mouse NK cells. [Figure 2A] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2B]1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2C] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2D] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2E] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2F] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2G] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2H] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2I] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2J] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2K]1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2L] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2M] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2N] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2O] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2P] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2Q] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2R] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2S] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2T]1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2U] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2V] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2W] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2X] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2Y] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2Z] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2AA] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 2AB] 1 shows data from a Xelligence assay of NK cell and macrophage mediated killing, or lack thereof, of B2M- / -;CD47tg T cells by NK cells and macrophages. [Figure 3A]1 shows data from a Xelligence assay of NK cell-mediated killing or lack thereof of B2M- / -;CD47tg T cells by NK cells. [Figure 3B] 1 shows data from a Xelligence assay of NK cell-mediated killing or lack thereof of B2M- / -;CD47tg T cells by NK cells. [Figure 3C] 1 shows data from a Xelligence assay of NK cell-mediated killing or lack thereof of B2M- / -;CD47tg T cells by NK cells. [Figure 3D] 1 shows data from a Xelligence assay of NK cell-mediated killing or lack thereof of B2M- / -;CD47tg T cells by NK cells. [Figure 3E] 1 shows data from a Xelligence assay of NK cell-mediated killing or lack thereof of B2M- / -;CD47tg T cells by NK cells. [Figure 3F] 1 shows data from a Xelligence assay of NK cell-mediated killing or lack thereof of B2M- / -;CD47tg T cells by NK cells. [Figure 3G] 1 shows data from a Xelligence assay of NK cell-mediated killing or lack thereof of B2M- / -;CD47tg T cells by NK cells. [Figure 3H] 1 shows data from a Xelligence assay of NK cell-mediated killing or lack thereof of B2M- / -;CD47tg T cells by NK cells. [Figure 3I] 1 shows data from a Xelligence assay of NK cell-mediated killing or lack thereof of B2M- / -;CD47tg T cells by NK cells. [Figure 3J] 1 shows data from a Xelligence assay of NK cell-mediated killing or lack thereof of B2M- / -;CD47tg T cells by NK cells. [Figure 3K] 1 shows data from a Xelligence assay of NK cell-mediated killing or lack thereof of B2M- / -;CD47tg T cells by NK cells. [Figure 3L] 1 shows data from a Xelligence assay of NK cell-mediated killing or lack thereof of B2M- / -;CD47tg T cells by NK cells. [Figure 4] AC show flow cytometry data measuring HLA-I, HLA-II and CD47 levels on the cell surface of unmodified primary RPE cells. [Figure 5A] Shown are cell morphology (A) and flow cytometry (B-D) data measuring HLA-I, HLA-II, and CD47 levels on the cell surface of B2M- / -;CIITA- / -;CD47tg primary RPE cells. [Figure 5B] Shown are cell morphology (A) and flow cytometry (B-D) data measuring HLA-I, HLA-II, and CD47 levels on the cell surface of B2M- / -;CIITA- / -;CD47tg primary RPE cells. [Figure 5C] Shown are cell morphology (A) and flow cytometry (B-D) data measuring HLA-I, HLA-II, and CD47 levels on the cell surface of B2M- / -;CIITA- / -;CD47tg primary RPE cells. [Figure 5D] Shown are cell morphology (A) and flow cytometry (B-D) data measuring HLA-I, HLA-II, and CD47 levels on the cell surface of B2M- / -;CIITA- / -;CD47tg primary RPE cells. [Figure 6A] Flow cytometry data measuring HLA-I, HLA-II and CD47 levels on the cell surface of unmodified (A-C), B2M- / -;CIITA- / - (D-F) and B2M- / -;CIITA- / -;CD47tg (G-I) primary RPE cells are shown. [Figure 6B] Flow cytometry data measuring HLA-I, HLA-II and CD47 levels on the cell surface of unmodified (A-C), B2M- / -;CIITA- / - (D-F) and B2M- / -;CIITA- / -;CD47tg (G-I) primary RPE cells are shown. [Figure 6C]Flow cytometry data measuring HLA-I, HLA-II and CD47 levels on the cell surface of unmodified (A-C), B2M- / -;CIITA- / - (D-F) and B2M- / -;CIITA- / -;CD47tg (G-I) primary RPE cells are shown. [Figure 6D] Flow cytometry data measuring HLA-I, HLA-II and CD47 levels on the cell surface of unmodified (A-C), B2M- / -;CIITA- / - (D-F) and B2M- / -;CIITA- / -;CD47tg (G-I) primary RPE cells are shown. [Figure 6E] Flow cytometry data measuring HLA-I, HLA-II and CD47 levels on the cell surface of unmodified (A-C), B2M- / -;CIITA- / - (D-F) and B2M- / -;CIITA- / -;CD47tg (G-I) primary RPE cells are shown. [Figure 6F] Flow cytometry data measuring HLA-I, HLA-II and CD47 levels on the cell surface of unmodified (A-C), B2M- / -;CIITA- / - (D-F) and B2M- / -;CIITA- / -;CD47tg (G-I) primary RPE cells are shown. [Figure 6G] Flow cytometry data measuring HLA-I, HLA-II and CD47 levels on the cell surface of unmodified (A-C), B2M- / -;CIITA- / - (D-F) and B2M- / -;CIITA- / -;CD47tg (G-I) primary RPE cells are shown. [Figure 6H] Flow cytometry data measuring HLA-I, HLA-II and CD47 levels on the cell surface of unmodified (A-C), B2M- / -;CIITA- / - (D-F) and B2M- / -;CIITA- / -;CD47tg (G-I) primary RPE cells are shown. [Figure 6I] Flow cytometry data measuring HLA-I, HLA-II and CD47 levels on the cell surface of unmodified (A-C), B2M- / -;CIITA- / - (D-F) and B2M- / -;CIITA- / -;CD47tg (G-I) primary RPE cells are shown. [Figure 7A]Shown are data from a Xelligence assay of NK cell- and macrophage-mediated killing, or lack thereof, of unmodified (A-C), B2M- / -;CIITA- / - (D-F) and B2M- / -;CIITA- / -;CD47tg (GI) primary RPE cells by NK cells and macrophages. [Figure 7B] Shown are data from a Xelligence assay of NK cell- and macrophage-mediated killing, or lack thereof, of unmodified (A-C), B2M- / -;CIITA- / - (D-F) and B2M- / -;CIITA- / -;CD47tg (GI) primary RPE cells by NK cells and macrophages. [Figure 7C] Shown are data from a Xelligence assay of NK cell- and macrophage-mediated killing, or lack thereof, of unmodified (A-C), B2M- / -;CIITA- / - (D-F) and B2M- / -;CIITA- / -;CD47tg (GI) primary RPE cells by NK cells and macrophages. [Figure 7D] Shown are data from a Xelligence assay of NK cell- and macrophage-mediated killing, or lack thereof, of unmodified (A-C), B2M- / -;CIITA- / - (D-F) and B2M- / -;CIITA- / -;CD47tg (GI) primary RPE cells by NK cells and macrophages. [Figure 7E] Shown are data from a Xelligence assay of NK cell- and macrophage-mediated killing, or lack thereof, of unmodified (A-C), B2M- / -;CIITA- / - (D-F) and B2M- / -;CIITA- / -;CD47tg (GI) primary RPE cells by NK cells and macrophages. [Figure 7F] Shown are data from a Xelligence assay of NK cell- and macrophage-mediated killing, or lack thereof, of unmodified (A-C), B2M- / -;CIITA- / - (D-F) and B2M- / -;CIITA- / -;CD47tg (GI) primary RPE cells by NK cells and macrophages. [Figure 7G]Shown are data from a Xelligence assay of NK cell- and macrophage-mediated killing, or lack thereof, of unmodified (A-C), B2M- / -;CIITA- / - (D-F) and B2M- / -;CIITA- / -;CD47tg (GI) primary RPE cells by NK cells and macrophages. [Figure 7H] Shown are data from a Xelligence assay of NK cell- and macrophage-mediated killing, or lack thereof, of unmodified (A-C), B2M- / -;CIITA- / - (D-F) and B2M- / -;CIITA- / -;CD47tg (GI) primary RPE cells by NK cells and macrophages. [Figure 7I] Shown are data from a Xelligence assay of NK cell- and macrophage-mediated killing, or lack thereof, of unmodified (A-C), B2M- / -;CIITA- / - (D-F) and B2M- / -;CIITA- / -;CD47tg (GI) primary RPE cells by NK cells and macrophages. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0333] Other objects, advantages and embodiments of the present disclosure will become apparent from the following detailed description.

[0334] I. Introduction Described herein are engineered or modified immune evasive cells based in part on the hypo-immune editing platform described in WO2018132783 and PCT / US21 / 65157 filed December 23, 2021, each of which is incorporated herein by reference in its entirety, and the engineered or modified immune evasive cells include, but are not limited to, human immune evasive cells. To overcome the problem of subject immune rejection of these primary and / or stem cell derived grafts, the inventors have developed and described herein hypo-immunogenic cells (e.g., hypo-immunogenic pluripotent cells, differentiated cells and primary cells derived therefrom) that represent a viable source for any transplantable cell type. Such cells are protected from adaptive and / or innate immune rejection upon administration to a recipient subject. Advantageously, the cells disclosed herein are protected from adaptive and innate immune rejection upon administration to a recipient subject, and therefore are not rejected by the subject's immune system, regardless of the recipient subject's genetic makeup. In some embodiments, the hypoimmunogenic cells regulatably lack expression of one or more MHC class I and class II antigen molecules, and / or T cell receptors. In certain embodiments, the hypoimmunogenic cells regulatably lack expression of major histocompatibility complex (MHC) I and II antigen molecules, and / or T cell receptors, and regulatably overexpress one or more tolerogenic factors. In certain embodiments, the hypoimmunogenic cells, e.g., hypoimmunogenic T cells, regulatably lack expression of one or more MHC I and II antigen molecules, and / or T cell receptors, regulatably overexpress CD47, and regulatably express a CAR. In some embodiments, the hypoimmunogenic cells regulatably lack expression of one or more MHC I and II antigen molecules, and / or T cell receptors, and / or one or more Y chromosome genes. In certain embodiments, the hypoimmunogenic cells regulatably lack expression of one or more MHC I and II antigen molecules, and / or T cell receptors, and / or one or more Y chromosome genes, and regulatably overexpress CD47. In certain embodiments, the hypoimmunogenic cells regulatably lack expression of one or more MHC I and II antigen molecules, and / or T cell receptors, and / or RHD, and regulatably overexpress CD47 protein.In certain embodiments, the hypoimmunogenic cells controllably lack expression of one or more MHC I and II antigen molecules, and / or T cell receptors, and / or ABO, and controllably overexpress CD47 protein. In certain embodiments, the hypoimmunogenic cells controllably lack expression of one or more MHC I and II antigen molecules, and / or T cell receptors, and / or MICA, and controllably overexpress CD47 protein. In certain embodiments, the hypoimmunogenic cells controllably lack expression of one or more MHC I and II antigen molecules, and / or T cell receptors, and / or MICB, and controllably overexpress CD47 protein. In certain embodiments, the hypoimmunogenic cells, e.g., hypoimmunogenic T cells, controllably lack expression of one or more MHC I and II antigen molecules, and / or T cell receptors, and / or one or more Y chromosome genes, controllably overexpress CD47, and controllably express CAR.

[0335] In some embodiments, the hypoimmunogenic cells outlined herein are not subject to natural immune cell rejection. In some cases, the hypoimmunogenic cells are not subject to NK cell-mediated lysis. In some cases, the hypoimmunogenic cells are not subject to macrophage phagocytosis. In some embodiments, the hypoimmunogenic cells are useful as a source of universally compatible cells or tissues (e.g., universal donor cells or tissues) that are transplanted into a recipient subject with little or no need for immunosuppressants. Such hypoimmunogenic cells retain cell-specific characteristics and features upon transplantation, including, for example, pluripotency and the ability to engraft and function similarly to the corresponding native cells.

[0336] The techniques disclosed herein use regulatable expression of tolerogenic factors, as well as regulatable modulation (e.g., reduction or elimination) of MHC I molecule, MHC II molecule, and / or TCR expression in human cells. In some embodiments, regulatable genome editing techniques using regulatable rare-cutting endonucleases (e.g., CRISPR / Cas, TALEN, zinc finger nucleases, meganucleases, and homing endonuclease systems) are also used to reduce or eliminate expression of genes involved in innate and / or adaptive immune responses in cells (e.g., by deleting genomic DNA of genes involved in innate and / or adaptive immune responses or by inserting genomic DNA into such genes such that gene expression is affected). In some embodiments, regulatable genome editing techniques or other gene modulation techniques are used to insert tolerance-inducing (tolerogenic) factors in human cells to enable the cells and their progeny (including any differentiated cells prepared therefrom) to evade immune recognition upon engraftment into a recipient subject. Thus, the cells described herein exhibit regulatable, coordinated expression of one or more genes and factors that affect MHC I molecule, MHC II molecule and / or TCR expression, evading the immune system of a recipient subject.

[0337] Surprisingly, it has been found that some transgenes that overexpress exogenous polynucleotides can be silenced during differentiation of, for example, iPSCs and primary cells into engineered, hypoimmunogenic differentiated cells.Thus, the present disclosure provides a system that allows for regulatable expression of exogenous polynucleotides.It has also been found that reduced expression of one or more MHC I molecules, MHC II molecules and / or TCRs is not required prior to generation of differentiated cells, for example, engineered, hypoimmunogenic differentiated cells.Thus, the present disclosure also provides a system that allows for regulatable knockout or knockdown of MHC I molecules, MHC II molecules and / or TCRs.

[0338] Genome editing techniques allow for double-stranded DNA breaks at desired loci. These controlled double-stranded breaks promote homologous recombination at specific loci. This process focuses on targeting specific sequences of nucleic acid molecules, such as chromosomes, with endonucleases that recognize and bind to the sequence and induce double-stranded breaks in the nucleic acid molecule. The double-stranded breaks are repaired by either error-prone non-homologous end joining (NHEJ) or homologous recombination (HR).

[0339] The practice of many of the embodiments will employ, unless specifically indicated to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology, and cell biology that are within the skill of those in the art, many of which are described below by way of illustration, and such techniques are explained more fully in the literature.For example, Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2001), Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989), Maniatis et al., Molecular Cloning: A Laboratory Manual (1982), Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I&II (IRL Press, Oxford, 1985), Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992), Transcription and See monographs in journals such as: A Practical Guide to Molecular Cloning (B. Hames & S. Higgins, Eds., 1984), Perbal, A Practical Guide to Molecular Cloning (1984), Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998), Current Protocols in Immunology QE Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991), Annual Review of Immunology, and Advances in Immunology.

[0340] II. Definition As described in this disclosure, the following terms are employed and are defined as set forth below.

[0341] As used herein, the term "antigen" refers to a molecule capable of eliciting an immune response. Antigens include, but are not limited to, cells, cell extracts, proteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, peptidic and non-peptidic mimetics of polysaccharides and other molecules, small molecules, lipids, glycolipids, carbohydrates, viruses and viral extracts, and multicellular organisms such as parasites and allergens. The term antigen broadly includes any type of molecule that is recognized as foreign by the host immune system.

[0342] The term "autoimmune disease" or "autoimmune disorder" or "inflammatory disease" or "inflammatory disorder" refers to any disease or disorder in which a subject mounts an innate and / or adaptive immune response against its own tissues and / or cells. Autoimmune disorders can affect almost every organ system of a subject (e.g., a human), including, but not limited to, diseases of the nervous system, gastrointestinal system, and endocrine system, as well as skin and other connective tissues, eyes, blood and blood vessels. Examples of autoimmune diseases include, but are not limited to, Hashimoto's thyroiditis, systemic lupus erythematosus, Sjogren's syndrome, Graves' disease, scleroderma, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and diabetes.

[0343] As used herein, the term "cancer" is defined as the hyperproliferation of cells whose unique characteristics (e.g., loss of normal control) result in unregulated growth, lack of differentiation, local tissue invasion, and metastasis. In the context of the methods of the present invention, cancer includes acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, anal, anal canal, or anorectal cancer, eye cancer, intrahepatic cholangiocarcinoma, joint cancer, cervical, nasal cavity, or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, Hodgkin's lymphoma. The tumor may be any cancer, including any of the following: , hypopharyngeal cancer, renal cancer, laryngeal cancer, leukemia, liquid tumors, liver cancer, lung cancer, lymphoma, malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, and / or bladder cancer. As used herein, the term "tumor" refers to an abnormal growth of cells or tissue of a malignant type, and does not include benign type tissue, unless specifically indicated otherwise.

[0344] The term "chronic infectious disease" refers to a disease caused by an infectious agent in which the infection persists. Such diseases can include hepatitis (A, B, or C), herpes viruses (such as VZV, HSV-1, HSV-6, HSV-II, CMV, and EBV), and HIV / AIDS. Non-viral examples can include chronic fungal diseases such as aspergillosis, candidiasis, coccidioidomycosis, and diseases associated with cryptococcosis and histoplasmosis. Non-limiting examples of chronic bacterial infectious agents can be Chlamydia pneumoniae, Listeria monocytogenes, and Mycobacterium tuberculosis. In some embodiments, the disorder is human immunodeficiency virus (HIV) infection. In some embodiments, the disorder is acquired immune deficiency syndrome (AIDS).

[0345] As used herein, a "clinically effective amount" refers to an amount sufficient to provide a clinical benefit in the treatment and / or management of a disease, disorder, or condition. In some embodiments, a clinically effective amount is an amount shown to produce at least one improved clinical endpoint relative to the standard of care for the disease, disorder, or condition. In some embodiments, a clinically effective amount is an amount that has been demonstrated, for example, in a clinical trial, to be sufficient to provide statistically significant and meaningful efficacy for treating a disease, disorder, or condition. In some embodiments, a clinically effective amount is also a therapeutically effective amount. In other embodiments, a clinically effective amount is not a therapeutically effective amount.

[0346] As used herein, a "conditional promoter" is active under a certain cellular state or under a certain cellular phase. As used herein, conditional promoters include, for example, cell-specific promoters, tissue-specific promoters, lineage-specific promoters, development-specific promoters, cell differentiation-specific promoters, differentiation-induced promoters, cell cycle-specific promoters, and cell phase-specific promoters. "Cell-specific promoters," "tissue-specific promoters," and "lineage-specific promoters" are promoters that cause a nucleotide sequence to be expressed in a particular cell, tissue, or lineage type, such as respiratory, prostate, pancreatic, breast, renal, intestinal, nervous, skeletal, vascular, hepatic, hematopoietic, muscle, endothelial, epithelial, or cardiac cells. A promoter that causes a nucleotide sequence to be expressed during a specific phase of development or cell differentiation is generally referred to as a "development-specific promoter", "cell differentiation-specific promoter" or "differentiation-induced promoter", and includes, for example, a promoter that is activated or inactivated when a cell transitions from one cell type to another, for example, from an undifferentiated cell to a differentiated cell, for example, from a stem cell to a multipotent progenitor cell, from a multipotent progenitor cell to a lineage-committed progenitor cell, from a lineage-committed progenitor cell to a precursor cell, or from a precursor cell to a mature cell. A promoter that causes a nucleotide sequence to be expressed during a specific phase of the cell cycle is generally referred to as a "cell cycle-specific promoter" or "cell phase-specific promoter". Many standard conditional promoters are known to those skilled in the art.

[0347] A "constitutive promoter" is typically active, i.e., promotes transcription, under most conditions. In some instances, a constitutive promoter is capable of directing the transcription of an operably linked nucleic acid sequence in the absence of a stimulus (e.g., heat shock, chemicals, etc.). In some instances, a constitutive promoter is active in most cell types at most times. Many standard conditional promoters are known to those skilled in the art. Constitutive promoters are included herein as a type of "regulatable promoter."

[0348] In some embodiments, the changes or modifications described herein (including, for example, genetic changes or modifications) result in reduced expression of a target or selected polynucleotide sequence. In some embodiments, the changes or modifications described herein result in reduced expression of a target or selected polypeptide sequence. In some embodiments, the changes or modifications described herein result in increased expression of a target or selected polynucleotide sequence. In some embodiments, the changes or modifications described herein result in increased expression of a target or selected polypeptide sequence. The terms "decrease," "reduced," "reduction," and "decrease" are all used herein generally to mean a decrease by a statistically significant amount. However, for the avoidance of doubt, "reduce," "decreased," "reduction," and "reduction" refer to a reduction of at least 10% compared to a reference level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% reduction (i.e., levels of absence compared to a reference sample), or any reduction between 10-100% compared to a reference level. In some embodiments, the cells are engineered to have reduced expression of one or more targets compared to an unaltered or unmodified wild-type cell.

[0349] In additional or alternative embodiments, the present disclosure contemplates changing the target polynucleotide sequence in any manner available to those skilled in the art, for example, using TALEN system or RNA-guided transposase.Although examples of methods using CRISPR / Cas (e.g., Cas9 and Cas12a) and TALEN are described in detail herein, it should be understood that the present disclosure is not limited to the use of these methods / systems.Other methods of targeting to reduce or eliminate expression in target cells known to those skilled in the art, such as B2M, can be utilized herein.

[0350] As used herein, a "degron element" refers to a subunit of a protein that regulates the degradation of a protein. In some cases, a degron contains a sequence of amino acids that provides a degradation signal that directs a polypeptide for cellular degradation. A degron can promote the degradation of a bound polypeptide through either the proteasome pathway or the autophagy-lysosomal pathway. In a fusion protein, a degron must be operably linked to a polypeptide of interest, but need not be contiguous with it, as long as the degron still functions to direct the degradation of the polypeptide of interest. Preferably, the degron induces rapid degradation of the polypeptide of interest. For a discussion of degrons and their function in protein degradation, see, e.g., Kanemaki et al. (2013) Pflugers Arch. 465(3):419-425; Erales et al. (2014) Biochim Biophys Acta 1843(1):216-221; Schrader et al. (2009) Nat. Chem. Biol. 5(11):815-822; Ravid et al. (2008) Nat. Rev. Mol. Cell. Biol. 9(9):679-690; Tasaki et al. (2007) Trends Biochem Sci. 32(11):520-528; Meinnel et al. (2006) Biol. Chem. 387(7):839-851; Kim et al. (2013) Autophagy 9(7):1100-1103, Varshaysky (2012) Methods Mol. Biol. 832:1-11, and Fayadat et al. (2003) Mol Biol Cell. 14(3):1268-1278, the contents of which are incorporated by reference in their entireties.

[0351] In some embodiments, the engineered and hypoimmunogenic cells described are derived from iPSCs or their progeny. As used herein, the term "derived from iPSCs or their progeny" encompasses the initial iPSC generated and any subsequent progeny thereof. As used herein, the term "progeny" encompasses, for example, first generation progeny, i.e., progeny directly derived from, obtained from, obtainable from, or derived from the initial iPSC, e.g., by conventional propagation methods. The term "progeny" also encompasses further generations, such as second, third, fourth, fifth, sixth, seventh, or higher generations, i.e., generations of cells derived from, obtained from, obtainable from, or derived from the previous generation, e.g., by conventional propagation methods. The term "progeny" also encompasses modified cells resulting from modification or alteration of the initial iPSC or its progeny.

[0352] The term "donor subject" refers to an animal, e.g., a human, from which cells can be obtained. "Non-human animal" and "non-human mammal", used interchangeably herein, include mammals, e.g., rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term "donor subject" also encompasses any vertebrate, including, but not limited to, mammals, reptiles, amphibians, and fish. However, advantageously, the donor subject is a mammal, such as a human, or other mammal, such as a domesticated mammal, e.g., a dog, cat, horse, or a production mammal, e.g., a cow, sheep, pig, etc. "Donor subject" can also refer to more than one donor, e.g., one or more human or non-human animals or non-human mammals.

[0353] The term "endogenous" refers to a referent molecule or polypeptide that is naturally present in a cell. Similarly, when used in reference to the expression of a coding nucleic acid, the term refers to the expression of a coding nucleic acid that is naturally contained in a cell and not exogenously introduced. Similarly, when used in reference to a promoter sequence, the term refers to a promoter sequence that is naturally contained in a cell and not exogenously introduced.

[0354] As used herein, the term "engineered cell" refers to a cell that has been altered in at least some way by human intervention, including, for example, genetic change or modification, such that the engineered cell differs from a wild-type cell.

[0355] As used herein, the term "exogenous" in the context of polynucleotide or polypeptide expression is intended to mean that the reference molecule or polypeptide is introduced into the cell of interest. The polypeptide can be introduced, for example, by introducing the coding nucleic acid into the genetic material of the cell, such as by integration into a chromosome or as non-chromosomal genetic material such as a plasmid or expression vector. Thus, when used in reference to the expression of a coding nucleic acid, the term refers to the introduction of the coding nucleic acid into the cell in an expressible form.

[0356] An "exogenous" molecule is a molecule, construct, factor, etc. that is not normally present in a cell, but that can be introduced into the cell by one or more genetic, biochemical, or other methods. "Normal presence in a cell" is determined for the particular developmental stage and environmental conditions of the cell. Thus, for example, a molecule that is present only during embryonic development of a neuron is an exogenous molecule to an adult neuronal cell. Exogenous molecules can include, for example, a functional version of a dysfunctional endogenous molecule, or a dysfunctional version of a normally functioning endogenous molecule.

[0357] Exogenous molecules or factors can be small molecules, such as those produced by combinatorial chemistry processes, or macromolecules, such as proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, polysaccharides, any modified derivatives of the above molecules, or any complexes containing one or more of the above molecules, among others. Nucleic acids include DNA and RNA, can be single-stranded or double-stranded, linear, branched, or circular, and can be of any length. Nucleic acids include nucleic acids capable of forming duplexes, as well as triplex-forming nucleic acids. See, for example, U.S. Patent Nos. 5,176,996 and 5,422,251. Proteins include, but are not limited to, DNA-binding proteins, transcription factors, chromatin remodeling factors, methylated DNA-binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases, and helicases.

[0358] The exogenous molecule or construct can be the same type of molecule as the endogenous molecule, for example, an exogenous protein or nucleic acid. In such cases, the exogenous molecule is introduced into the cell at a concentration higher than the concentration of the endogenous molecule in the cell. In some cases, the exogenous nucleic acid can include an infectious viral genome, a plasmid or episome introduced into the cell, or a chromosome that is not normally present in the cell. Methods for introducing exogenous molecules into cells are known to those skilled in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes containing neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran mediated transfer, and viral vector mediated transfer.

[0359] As used herein, "fusosome" includes a gene therapy vector comprising a retroviral vector pseudotyped with an engineered fusogen, where the engineered fusogen comprises a G protein modified to include a targeting moiety and an F protein that is adapted so that it no longer recognizes its cognate receptor. In some embodiments, the fusogen protein complex is from a paramyxovirus, optionally where the paramyxovirus is a Nipah virus. In some embodiments, the retroviral vector is a lentiviral vector.

[0360] For purposes of this disclosure, a "gene" includes a DNA region that encodes a gene product, as well as all DNA regions that control the production of the gene product (whether or not such control sequences are adjacent to the coding and / or transcribed sequence). Thus, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translation control sequences, such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and / or locus control regions.

[0361] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. A gene product can be the direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or other types of RNA) or a protein produced by translation of an mRNA. Gene products also include RNA that is modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins that are modified, for example, by methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation, and / or glycosylation.

[0362] As used herein, the term "genetic modification" and its grammatical equivalents may refer to one or more changes in a nucleic acid, for example, a nucleic acid in the genome of an organism. For example, genetic modification may refer to a change, addition, and / or deletion of a gene or a portion of a gene or other nucleic acid sequence. A genetically modified cell may also refer to a cell having an addition, deletion, and / or change of a gene or a portion of a gene. A genetically modified cell may also refer to a cell having an addition of a nucleic acid sequence that is not a gene or a portion of a gene. Genetic modifications include, for example, both transient knock-in or knock-down mechanisms and mechanisms that result in a permanent knock-in, knock-down, or knock-out of a target gene or a portion of a gene or nucleic acid sequence. Genetic modifications include, for example, both transient knock-in and mechanisms that result in a permanent knock-in of a nucleic acid sequence. Genetic modifications also include, for example, reduced or increased transcription, reduced or increased mRNA stability, reduced or increased translation, and reduced or increased protein stability.

[0363] As used herein, the terms "graft," "administration," "introduction," "implantation," and "transplantation," as well as grammatical variations thereof, are used interchangeably in the context of placement of cells (e.g., cells described herein) into a subject by a method or route that results in localization or at least partial localization of the introduced cells at a desired site, or by systemic introduction (e.g., into the circulation). The cells may be directly implanted at a desired site within the subject, or alternatively, may be administered by any suitable route that results in delivery to the desired site, where at least a portion of the implanted cells or components of the cells remain viable. The survival period of the cells after administration to the subject may be as short as a few hours, e.g., 24 hours to several days, or as long as several years. In some embodiments, the cells may also be administered (e.g., injected) at a location other than the desired site, e.g., intracerebrally or subcutaneously, e.g., in a capsule, to maintain the transplanted cells at the transplanted location and avoid migration of the transplanted cells.

[0364] "HLA" or "human leukocyte antigen" or "HLA molecule" or "human leukocyte antigen molecule" complex is a complex of genes that code for MHC proteins in humans. These cell surface proteins that make up the HLA complex are responsible for controlling the immune response to antigens. In humans, there are two MHCs, class I and class II molecules, "HLA-I" and "HLA-II", or "HLA-I molecule" and "HLA-II molecule". HLA-I contains three proteins, HLA-A, HLA-B, and HLA-C, that present peptides from inside the cell, and the antigens presented by the HLA-I complex attract killer T cells (also known as CD8+ T cells or cytotoxic T cells). HLA-I proteins are associated with beta-2 microglobulin (B2M). HLA-II contains five proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ, and HLA-DR, that present antigens to T lymphocytes from outside the cell. It stimulates CD4+ cells (also known as helper T cells). It should be understood that the use of either "MHC" or "HLA" is not meant to be limiting, as it depends on whether the gene is human (HLA) or mouse (MHC). Thus, these terms may be used interchangeably herein when it comes to mammalian cells.

[0365] As used herein to characterize cells, the terms "immune privilege" and "low immunogenicity" are used interchangeably and generally mean that such cells are less prone to natural or adaptive immune rejection by a subject into which such cells are transplanted, e.g., that the cells are less prone to allogeneic rejection by a subject into which such cells are transplanted. For example, relative to cells of the same cell type that do not contain modifications, such low immunogenic cells can be about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more less prone to natural or adaptive immune rejection by a subject into which such cells are transplanted. In some embodiments, genome editing techniques are used to modulate the expression of one or more MHC I and MHC II genes, thus contributing to the generation of low immunogenic cells. In some embodiments, low immunogenic cells avoid immune rejection in MHC-mismatched allogeneic recipients. In some cases, differentiated cells produced from the hypoimmunogenic stem cells outlined herein avoid immune rejection when administered (e.g., transplanted or grafted) to an MHC-mismatched allogeneic recipient. In some embodiments, the hypoimmunogenic cells are protected from T cell-mediated adaptive immune rejection and / or innate immune cell rejection. Detailed descriptions of hypoimmunogenic cells, their methods of production and their methods of use are found in WO2016183041, filed May 9, 2015; WO2018132783, filed January 14, 2018; WO2018176390, filed March 20, 2018; WO2020018615, filed July 17, 2019; WO2020018620, filed July 3, 2020; and WO2020018637, filed July 17, 2020. The disclosures found in PCT / US2020 / 44635 filed on 1, WO2021022223 filed on July 31, 2020, WO2021041316 filed on August 24, 2020, WO2021222285 filed on April 27, 2021, and WO2021222285 filed on April 27, 2021, including examples, sequence listings and figures, are hereby incorporated by reference in their entireties.

[0366] The low immunogenicity of a cell can be determined by assessing the immunogenicity of the cell, such as the ability of the cell to elicit adaptive and innate immune responses or to avoid eliciting such adaptive and innate immune responses. Such immune responses can be measured using assays recognized by those skilled in the art. In some embodiments, the innate and / or adaptive immune response assays measure the effect of the low immunogenic cells on T cell proliferation, T cell activation, T cell killing, donor-specific antibody production, NK cell proliferation, NK cell activation, and macrophage activity. In some cases, the low immunogenic cells and their derivatives undergo reduced killing by T cells and / or NK cells upon administration to a subject. In some cases, the cells and their derivatives exhibit reduced macrophage phagocytosis compared to unmodified or wild-type cells. In some embodiments, the low immunogenic cells elicit a reduced or diminished immune response in a recipient subject compared to the corresponding unmodified wild-type cells. In some embodiments, the low immunogenic cells are non-immunogenic or unable to elicit an innate and / or adaptive immune response in a recipient subject.

[0367] The term "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that have a certain percentage of nucleotides or amino acid residues that are the same when compared and aligned to their closest counterparts, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP, and BLASTN, or other algorithms available to those skilled in the art) or by visual inspection. Depending on the application, the percent "identity" may be over a region of the sequences being compared, e.g., over a functional domain, or alternatively, over the entire length of the two sequences being compared. For sequence comparison, typically one sequence serves as a reference sequence, and this sequence is compared to a test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated, and sequence algorithm program parameters are designated, if necessary. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence(s) relative to the reference sequence based on the designated program parameters.

[0368] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA (Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.)), or by visual inspection (see generally, Ausubel et al., infra).

[0369] One example of a suitable algorithm for determining percent sequence identity and sequence similarity is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0370] As used herein, "immune signaling factor" refers, in some cases, to a molecule, protein, peptide, etc. that activates an immune signaling pathway.

[0371] As used herein, "immunosuppressive factors" or "immunoregulatory factors" or "tolerogenic factors" include hypoimmune factors, complement inhibitors, and other factors that modulate or affect the ability of cells to be recognized by the immune system of a host or recipient subject upon administration, transplantation, or engraftment, optionally in combination with additional genetic modifications.

[0372] The terms "increased", "increase", or "enhance" or "activate" are all used herein to generally mean an increase of a statistically significant amount. For the avoidance of doubt, the terms "increased", "increase", or "enhance" or "activate" mean an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to a reference level, or an increase up to and including 100%, or any increase of 10-100%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase of 2-fold to more than 10-fold compared to a reference level. In some embodiments, the reference level, also referred to as the basal level, is 0.

[0373] In some embodiments, the change is an indel. As used herein, "indel" refers to a mutation resulting from an insertion, deletion, or a combination thereof. As will be understood by those skilled in the art, an indel in the coding region of a genome sequence will result in a frameshift mutation unless the length of the indel is a multiple of three. In some embodiments, the change is a point mutation. As used herein, "point mutation" refers to a substitution that replaces one of the nucleotides. The gene editing (e.g., CRISPR / Cas) system of the present disclosure can be used to induce indels or point mutations of any length in a target polynucleotide sequence.

[0374] An "inducible promoter" is active only under certain conditions, such as the presence of a given molecular factor (e.g., a drug, biomolecule, chemical, or ligand) or a given environmental condition (e.g., the presence of a particular CO 2Inducible promoters can be induced by a variety of conditions, including but not limited to, conditions such as concentration, nutrient levels, light, heat. In the absence of such conditions, inducible promoters typically do not allow significant or measurable levels of transcriptional activity. For example, inducible promoters can be induced according to temperature, pH, hormones, metabolites (e.g., lactose, mannitol, amino acids), light (e.g., wavelength-specific), osmotic potential (e.g., salt-inducible), heavy metals, or antibiotics. Many standard inducible promoters will be known to those skilled in the art. Inducible promoters are included herein as a type of "regulatable promoter".

[0375] In some cases, the inducible gene expression system can turn transcription on or off in the presence of ligands, small molecules, peptides, factors, drugs, etc. In some cases, the inducible gene expression system can activate protein degradation pathways in response to the presence of ligands, small molecules, peptides, factors, drugs, etc.

[0376] As used herein, "knockdown" refers to the reduction of expression of target mRNA or corresponding target protein. Knockdown is generally reported relative to the level present after administration or expression of a non-control molecule (e.g., non-target control shRNA, siRNA or miRNA) that does not mediate a reduction in the expression level of the RNA. In some embodiments, knockdown of target gene is achieved by conditional or inducible shRNA, conditional or inducible siRNA, conditional or inducible miRNA, or conditional or inducible CRISPR interference (CRISPRi). In some embodiments, knockdown of target gene is achieved by protein-based components, such as conditional or inducible degron methods. In some embodiments, knockdown of target gene is achieved by genetic modification, which includes the use of shRNA, siRNA or miRNA, or gene editing system (e.g., CRISPR / Cas).

[0377] Knockdown is generally assessed by measuring mRNA levels using quantitative polymerase chain reaction (qPCR) amplification, or by measuring protein levels by Western blot or enzyme-linked immunosorbent assay (ELISA). Analysis of protein levels provides an assessment of both mRNA cleavage and translation inhibition. Additional techniques for measuring knockdown include RNA solution hybridization, nuclease protection, Northern hybridization, gene expression monitoring using microarrays, antibody binding, radioimmunoassay, and fluorescence-activated cell analysis. Based on the details described herein, those skilled in the art will easily understand how to use the gene editing system (e.g., CRISPR / Cas) of the present disclosure to knock out a target polynucleotide sequence or a portion thereof.

[0378] "Knock-in" or "knock-in" as used herein refers to a genetic modification resulting from the insertion of a DNA sequence into a chromosomal locus in a host cell. This causes the initiation or increased level of expression of the knocked-in gene, part of a gene, or nucleic acid sequence insertion product, e.g., an increase in RNA transcript level and / or encoded protein level. As will be understood by those skilled in the art, this can be accomplished in several ways, including inserting or adding one or more additional copies of the gene or part thereof into the host cell, or altering the regulatory components of an endogenous gene to increase the expression of the protein to be made, or inserting a specific nucleic acid sequence whose expression is desired. This may be accomplished by modifying the promoter, adding a different promoter, adding enhancers, adding other regulatory elements, or modifying other gene expression sequences.

[0379] As used herein, "knock out" or "knock-out" includes deleting all or a part of a target polynucleotide sequence to prevent translation or function of the target polynucleotide sequence. For example, knock-out can be achieved by altering the target polynucleotide sequence by inducing an insertion or deletion ("indel") in the target polynucleotide sequence, including a functional domain (e.g., a DNA-binding domain) of the target polynucleotide sequence. Based on the details described herein, it will be easily understood by those skilled in the art how to use the gene editing system (e.g., CRISPR / Cas) of the present disclosure to knock out a target polynucleotide sequence or a part thereof.

[0380] In some embodiments, the genetic modification or alteration results in knocking out or knocking down of the target polynucleotide sequence or a portion thereof. Knocking out the target polynucleotide sequence or a portion thereof using the gene editing system (e.g., CRISPR / Cas) of the present disclosure can be useful for various applications. For example, knocking out the target polynucleotide sequence in a cell can be performed in vitro for research purposes. For ex vivo purposes, knocking out the target polynucleotide sequence in a cell can be useful for treating or preventing disorders associated with the expression of the target polynucleotide sequence (e.g., by knocking out mutant alleles in cells ex vivo and introducing these cells containing the knocked out mutant alleles into a subject), or for modifying the genotype or phenotype of a cell.

[0381] "Modulation" of gene expression refers to a change in the expression level of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression. Modulation can also be complete, i.e., gene expression is completely inactivated or activated above wild-type levels, or modulation can be partial, where gene expression is partially reduced or partially activated to a percentage of wild-type levels. As used herein, the term "modifying gene expression" refers to introducing any of the modifications disclosed herein into a cell to create an engineered cell as disclosed herein.

[0382] In additional or alternative aspects, the present disclosure contemplates altering the target polynucleotide sequence in any manner available to one of skill in the art, for example, using a nuclease system, for example, a TAL effector nuclease (TALEN) or a zinc finger nuclease (ZFN) system. Examples of methods using CRISPR / Cas (e.g., Cas9 and Cas12a) and TALEN are described in detail herein, but it should be understood that the present disclosure is not limited to the use of these methods / systems. Other methods known to one of skill in the art that aim to reduce or eliminate expression in a target cell can be used herein. The methods provided herein can be used to alter the target polynucleotide sequence in a cell. The present disclosure contemplates altering the target polynucleotide sequence in a cell for any purpose. In some embodiments, the target polynucleotide sequence in a cell is altered to produce a mutant cell. As used herein, a "mutant cell" refers to a cell whose resulting genotype is different from its original genotype. In some cases, a "mutated cell" exhibits a mutant phenotype, for example, when a normally functioning gene is altered using a gene editing system of the present disclosure (e.g., CRISPR / Cas). In other cases, a "mutated cell" exhibits a wild-type phenotype, for example, when a gene editing system of the present disclosure (e.g., CRISPR / Cas) is used to correct a mutant genotype. In some embodiments, a target polynucleotide sequence in a cell is modified to correct or repair a genetic mutation (e.g., to restore a normal phenotype to a cell). In some embodiments, a target polynucleotide sequence in a cell is modified to induce a genetic mutation (e.g., to disrupt the function of a gene or genomic element).

[0383] As used herein, the term "native cell" refers to a cell that has not otherwise been modified (e.g., engineered). In some embodiments, a native cell is a naturally occurring wild-type or control cell.

[0384] The terms "operably linked" or "operably linked" are used interchangeably with respect to the juxtaposition of two or more components (such as sequence elements) that are positioned to allow for the possibility that both components function normally and that at least one of the components may mediate a function on at least one of the other components. By way of illustration, a transcription control sequence, such as a promoter, is operably linked to a coding sequence if the transcription control sequence controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcription control factors. A transcription control sequence is generally operably linked in cis with a coding sequence, but need not be directly adjacent to it. For example, an enhancer is a transcription control sequence that is operably linked to a coding sequence even if they are not contiguous.

[0385] As used herein, a "pluripotent stem cell" has the ability to differentiate into any of three germ layers: endoderm (e.g., stomach wall, gastrointestinal tract, lungs, etc.), mesoderm (e.g., muscle, bone, blood, urogenital tissue, etc.), or ectoderm (e.g., epithelial tissue and nervous system tissue). As used herein, the term "pluripotent stem cell" also encompasses "induced pluripotent stem cells" or "iPSCs", a type of pluripotent stem cell derived from a non-pluripotent cell. In some embodiments, pluripotent stem cells are produced or generated from cells that are not pluripotent cells. In other words, pluripotent stem cells can be direct or indirect descendants of non-pluripotent cells. 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 generated by inducing the expression of certain regulatory genes or by exogenous application of certain proteins. Methods for the derivation of iPS cells are known in the art and are further described below (see, e.g., Zhou et al., Stem Cells 27(11):2667-74(2009); Huangfu et al., Nature Biotechnol. 26(7):795(2008); Woltjen et al., Nature 458(7239):766-770(2009); and Zhou et al., Cell Stem Cell 8:381-384(2009); each of which is incorporated herein by reference in its entirety.) The generation of induced pluripotent stem cells (iPSCs) is outlined below. As used herein, "hiPSCs" refers to human induced pluripotent stem cells. In some embodiments, as used herein, "pluripotent stem cells" also encompass mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs) and / or embryonic stem cells (ESCs).

[0386] As used herein, a "promoter", "promoter sequence" or "promoter region" refers to a DNA regulatory region / sequence that is capable of binding RNA polymerase and is involved in initiating transcription of downstream coding or non-coding sequences. In some instances, a promoter sequence includes a transcription initiation site and extends upstream to include the minimum number of bases or elements required to initiate transcription at a level detectable above background. In some embodiments, a promoter sequence includes a transcription initiation site and a protein binding domain that contributes to the binding of RNA polymerase. Eukaryotic promoters often, but not always, contain "TATA" boxes and "CAT" boxes.

[0387] In some embodiments, the described engineered and hypoimmunogenic cells are expanded from primary T cells or their progeny. As used herein, the term "expanded from primary T cells or their progeny" includes primary T cells isolated from a donor subject and any subsequent progeny thereof. As used herein, the term "progeny" includes, for example, first generation progeny, i.e., progeny are directly derived, obtained, obtainable, or derived from the original primary T cell, e.g., by conventional propagation methods. The term "progeny" also includes further generations, such as second, third, fourth, fifth, sixth, seventh, or higher generations, i.e., generations of cells that are derived, obtained, obtainable, or derived from the previous generation, e.g., by conventional propagation methods. The term "progeny" also includes modified cells resulting from modification or alteration of primary T cells or their progeny.

[0388] The term "recipient patient" refers to an animal, e.g., a human, to which treatment, including prophylactic treatment, with the cells described herein is provided. For treatment of those infections, conditions, or pathologies specific to a particular animal, such as a human patient, the term patient refers to that particular animal. The term "recipient patient" also encompasses any vertebrate, including, but not limited to, mammals, reptiles, amphibians, and fish. Advantageously, however, the recipient patient is a mammal, such as a human, or other mammal, such as a domestic mammal, e.g., dog, cat, horse, or a production mammal, e.g., cow, sheep, pig, etc. In some embodiments, the recipient patient has an infection, condition, disease, or disorder. In some embodiments, the recipient patient is suspected of having an infection, condition, disease, or disorder.

[0389] As used herein, "regulatable modification" refers to any modification of a cell that occurs under certain conditions, such as, but not limited to, a cell state or stage, or an external condition. In an embodiment, the regulatable modification comprises a regulatable knockout of a target gene. In an embodiment, the regulatable modification comprises a regulatable reduced expression of one or more target genes. In an embodiment, the regulatable modification comprises a regulatable increased expression of one or more endogenous or exogenous genes. In an embodiment, the regulatable modification comprises a conditional or inducible DNA-based component, a conditional or inducible RNA-based component, or a conditional or inducible protein-based component for increasing, decreasing, or knocking out the expression of a target gene.

[0390] As used herein, a "regulatable promoter" is active only under certain conditions, such as, but not limited to, a cell state or phase, or an external condition. As used herein, a regulatable promoter includes conditional promoters and inducible promoters. In some cases, an inducible regulatable gene expression system can turn transcription on or off in the presence of a ligand, small molecule, peptide, factor, drug, etc. In some cases, a regulatable gene expression system can activate a protein degradation pathway in response to the presence of a ligand, small molecule, peptide, factor, drug, etc.

[0391] As used herein, the terms "regulatory sequence," "regulatory element," and "control element" are interchangeable and refer to polynucleotide sequences that are upstream (5' non-coding sequences), within, or downstream (3' non-translated sequences) of a polynucleotide target to be expressed. Regulatory sequences affect, for example, but not limited to, the timing of transcription, the amount or level of transcription, RNA processing or stability, and / or translation of associated structural nucleotide sequences. Regulatory sequences may include activator binding sequences, enhancers, introns, polyadenylation recognition sequences, promoters, repressor binding sequences, stem-loop structures, translation initiation sequences, translation leader sequences, transcription termination sequences, translation termination sequences, primer binding sites, and the like. Since in most cases the exact boundaries of regulatory sequences have not been completely defined, it will be recognized that nucleotide sequences of different lengths may have identical regulatory or promoter activity.

[0392] As used herein, a "safe harbor locus" refers to a locus that allows expression of a transgene or exogenous gene in a manner that allows the newly inserted genetic element to function as expected and does not cause changes in the host genome in a manner that poses a risk to the host cell. Exemplary "safe harbor" loci include, but are not limited to, the CCR5 gene, the PPP1R12C gene (also known as AAVS1), the CLYBL gene, and / or the Rosa gene (e.g., ROSA26). A "target locus" as used herein refers to a locus that allows expression of a transgene or exogenous gene. Exemplary "target loci" include, but are not limited to, the CXCR4 gene, the albumin gene, the SHS231 locus, the F3 gene (also known as CD142), the MICA gene, the MICB gene, the LRP1 gene (also known as CD91), the HMGB1 gene, the ABO gene, the RHD gene, the FUT1 gene, and / or the KDM5D gene (also known as HY). The exogenous polynucleotide encoding an exogenous gene may be inserted in the CDS region for B2M, CIITA, TRAC, TRBC, CCR5, F3 (i.e., CD142), MICA, MICB, LRP1, HMGB1, ABO, RHD, FUT1, KDM5D (i.e., HY), PDGFRa, OLIG2, and / or GFAP. The exogenous polynucleotide encoding an exogenous gene may be inserted in intron 1 or 2 for PPP1R12C (i.e., AAVS1) or CCR5. The exogenous polynucleotide encoding an exogenous gene may be inserted in exon 1 or 2 or 3 for CCR5. The exogenous polynucleotide encoding an exogenous gene may be inserted in intron 2 for CLYBL. The exogenous polynucleotide encoding an exogenous gene may be inserted in a 500 bp window in Ch-4:58,976,613 (i.e., SHS231).An exogenous polynucleotide encoding an exogenous gene may be inserted at a suitable region of any of the above-mentioned safe harbor or target loci that will allow expression of the exogenous gene, suitable regions including, for example, introns, exons or coding sequence regions in the safe harbor or target locus.

[0393] As used herein, "target" may refer to a gene, a portion of a gene, a portion of a genome, or a protein that is subject to controllable reduced expression by the methods described herein.

[0394] As used herein, a "therapeutically effective amount" refers to an amount sufficient to provide a therapeutic benefit in the treatment and / or management of a disease, disorder, or condition. In some embodiments, a therapeutically effective amount is an amount sufficient to ameliorate, alleviate, stabilize, reverse, slow down, attenuate, or delay the progression of a disease, disorder, or condition, or the symptoms or side effects of a disease, disorder, or condition. In some embodiments, a therapeutically effective amount is also a clinically effective amount. In other embodiments, a therapeutically effective amount is not a clinically effective amount.

[0395] As used herein, the terms "treating" and "treatment" include administering to a subject a therapeutically or clinically effective amount of cells described herein such that the subject has a reduction in at least one symptom of the disease or an improvement in the disease, e.g., a beneficial or desired therapeutic or clinical result. For purposes of the present technology, a beneficial or desired therapeutic or clinical result includes, but is not limited to, relief of one or more symptoms, whether detectable or undetectable, attenuation of the extent of the disease, a stable (i.e., not worsening) state of the disease, a delay or slowing of disease progression, remission or alleviation of the disease state, and remission (whether partial or complete). Treating may refer to extending survival compared to the expected survival in the absence of treatment. Thus, one of skill in the art recognizes that treatment may improve the pathology, but may not be a complete cure for the disease. In some embodiments, one or more symptoms of a condition, disease, or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% by treatment of the condition, disease, or disorder.

[0396] For purposes of the present technology, beneficial or desired therapeutic or clinical results of disease treatment include, but are not limited to, alleviation of one or more symptoms, whether detectable or undetectable, attenuation of the extent of the disease, a stabilized (i.e., not worsening) disease state, a delay or slowing of disease progression, an improvement or palliation of the disease state, and remission (whether partial or complete).

[0397] A "vector" or "construct" is capable of transferring a gene sequence into a target cell. Typically, "vector construct", "expression vector" and "gene transfer vector" refer to any nucleic acid construct capable of directing the expression of a gene of interest and transferring a gene sequence into a target cell. Thus, the term includes cloning and expression vehicles as well as integration vectors. Methods for introducing vectors or constructs into cells are known to those skilled in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes containing neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran mediated transfer and / or viral vector mediated transfer.

[0398] In some embodiments, the cells are engineered to have reduced or increased expression of one or more targets compared to unaltered or unmodified wild-type cells. In some embodiments, the cells are engineered to have constitutive reduced or increased expression of one or more targets compared to unaltered or unmodified wild-type cells. In some embodiments, the cells are engineered to have controllably reduced or increased expression of one or more targets compared to unaltered or unmodified wild-type cells. In some embodiments, the cells comprise increased expression of CD47 compared to wild-type or control cells of the same cell type. By "wild-type" or "wild-type (wt)" or "control" in the context of a cell is meant any cell found in nature. Examples of wild-type or control cells include primary cells and T cells found in nature. However, as an example, in the context of engineered cells, as used herein, "wild type" or "control" can also refer to engineered cells that may contain nucleic acid modifications that result in reduced expression of one or more MHC class I and / or class II molecules and / or T cell receptors, but have not been subjected to a gene editing procedure to result in overexpression of the CD47 protein. For example, as used herein, "wild type" or "control" refers to engineered cells that contain reduced or knocked-out expression of B2M, CIITA, and / or TRAC. Also, as used herein, "wild type" or "control" refers to engineered cells that contain reduced or knocked-out expression of B2M, CIITA, TRAC, and / or TRBC. As used herein, "wild type" or "control" can also refer to engineered cells that may contain nucleic acid modifications that result in overexpression of the CD47 protein, but have not been subjected to a gene editing procedure to result in reduced expression of one or more MHC class I and / or class II molecules and / or T cell receptors.In the context of iPSCs or their progeny, "wild type" or "control" also refers to iPSCs or their progeny that may contain nucleic acid modifications that result in pluripotency, but have not been subjected to the gene editing procedures of the present disclosure to achieve reduced expression of one or more MHC class I and / or class II molecules and / or T cell receptors, and / or overexpression of CD47 protein. For example, as used herein, "wild type" or "control" refers to iPSCs or their progeny that contain reduced or knocked out expression of B2M, CIITA, and / or TRAC. Also, as used herein, "wild type" or "control" refers to iPSCs or their progeny that contain reduced or knocked out expression of B2M, CIITA, TRAC, and / or TRBC. In the context of primary T cells or their progeny, "wild type" or "control" also refers to primary T cells or their progeny that may contain nucleic acid modifications that result in reduced expression of one or more MHC class I and / or class II molecules and / or T cell receptors, but have not been subjected to the gene editing procedures to achieve overexpression of CD47 protein. For example, as used herein, "wild type" or "control" refers to a primary T cell or its progeny that includes reduced or knocked-out expression of B2M, CIITA, and / or TRAC. Also, as used herein, "wild type" or "control" refers to a primary T cell or its progeny that includes reduced or knocked-out expression of B2M, CIITA, TRAC, and / or TRBC. In the context of a primary T cell or its progeny, "wild type" or "control" also refers to a primary T cell or its progeny that may contain a nucleic acid alteration that results in overexpression of CD47 protein, but has not been subjected to a gene editing procedure to result in reduced expression of one or more MHC class I and / or class II molecules and / or T cell receptors. In some embodiments, the cells are engineered to have a controllable reduced or increased expression of one or more targets compared to cells of the same cell type that do not contain the alteration. In some embodiments, wild type or control cells are the starting material.In some embodiments, the starting material is otherwise modified or engineered to have altered expression of one or more genes to generate engineered cells. In some embodiments, the control cells are from the same starting material as the cells described herein. In some embodiments, the control cells are from a reference starting material. In some embodiments, the starting material is from a single donor. In some embodiments, the starting material is from a pool of donors.

[0399] In some embodiments, the cells are engineered to express a higher amount of the tolerogenic factor relative to a control. As used herein, the term "control" may be used in the context of a cell, a population of cells, a sample, or a measurement. In some embodiments, the cells are engineered to express a higher amount of the tolerogenic factor relative to a control cell. In some embodiments, the cells are engineered to express a higher amount of the tolerogenic factor relative to a population of control cells. In some embodiments, the cells are engineered to express a higher amount of the tolerogenic factor relative to a control sample. In some embodiments, the cells are engineered to express a higher amount of the tolerogenic factor relative to a control measurement, the control measurement including, but not limited to, a baseline reference or control signal in an assay or test. As used herein, "baseline reference" refers to any suitable reference value or signal level known to one of skill in the art in view of the present disclosure, suitable reference values ​​or signal levels include those used in the examples presented herein. In some embodiments, the baseline reference refers to a control level of expression, some levels and normal levels against which a test level of expression can be compared. In some embodiments, the baseline reference refers to a control or background level that is appropriate for the particular test or assay used. In some embodiments, the baseline reference refers to a control signal, including but not limited to an isotype control value from any suitable test or assay known in the art that can be used to assess expression levels. In some embodiments, the baseline reference refers to a background signal from any suitable test or assay known in the art that can be used to assess expression levels. In some embodiments, the cells are engineered to express a tolerogenic factor at or above a threshold level. In some embodiments, the cells are engineered to express CD47 at or above a threshold level. The threshold value can be determined using any suitable method known to one of skill in the art in light of the present specification, including, for example, those disclosed herein.In some embodiments, the baseline reference is specific to the engineered cells or a population of cells that includes the engineered cells.

[0400] It should be noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a predicate for the use of exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements, or for the use of "negative" limitations. As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and features that can be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the disclosure. Any recited method may be carried out in the order of events recited or in any other order that is logically possible. Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present disclosure, representative exemplary methods and materials are described below.

[0401] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. When a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to one tenth of the unit of the lower limit, as well as any other stated or intervening value in that stated range, is encompassed within the disclosure unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included within the smaller ranges, and may also be encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. Certain ranges are presented herein with the term "about" preceding the numerical values. The term "about" is used herein to provide literal support for the number it precedes, as well as a number that is close to or approximately the number it precedes. In determining whether a number is close to or approximate to a specifically recited number, the close or approximate unrecited number may be a number that, in the context presented, provides a substantial equivalent value to the specifically recited number. The term about is used herein to mean plus or minus ten percent (10%) of a value. For example, "about 100" refers to any number between 90 and 110.

[0402] All publications, patents, and patent applications cited herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Furthermore, each cited publication, patent, or patent application is incorporated by reference herein to disclose and describe the subject matter in connection with which the publication is cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the technology described herein is not entitled to antedate such publication by virtue of prior art. Further, dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0403] Before further describing the present technology, it should be understood that the present technology is not limited to the specific embodiments described, and thus can of course vary. It should also be understood that the terminology used herein is only for describing specific embodiments, and is not intended to be limiting, since the scope of the present disclosure is limited only by the scope of the appended claims. It should also be understood that the headings used herein are not limiting, but are only intended to provide the reader with orientation, but that the contents generally apply to the technology disclosed herein.

[0404] III. MODE FOR CARRYING OUT THE DISCLOSURE A. Low immunogenic cells In some embodiments, the disclosure provides engineered (e.g., modified and genetically modified) cells comprising a regulatable modification: i) a regulatable modification that reduces expression of one or more MHC class I and / or MHC class II human leukocyte antigen molecules relative to cells of the same cell type not comprising the modification, wherein the regulatable, reduced expression is via an RNA-based component, a DNA-based component, or a protein-based component; and / or ii) a regulatable modification that increases expression of a first exogenous polynucleotide encoding one or more tolerogenic factors relative to cells of the same cell type not comprising the modification, wherein the regulatable overexpression is via a conditional or inducible promoter. In some embodiments, the cells are capable of evading an activated NK cell-mediated and / or antibody-based immune response.

[0405] In some embodiments, the cells are induced pluripotent stem cells, any type of differentiated cells thereof, primary immune cells, and other primary cells of any tissue. In some embodiments, the differentiated cells are cardiac cells and their subpopulations, neural cells and their subpopulations, brain endothelial cells and their subpopulations, dopaminergic neurons and their subpopulations, glial progenitor cells and their subpopulations, endothelial cells and their subpopulations, thyroid cells and their subpopulations, hepatic cells and their subpopulations, pancreatic islet cells and their subpopulations, or retinal pigment epithelial cells and their subpopulations. In some embodiments, the differentiated cells are T cells and their subpopulations, NK cells and their subpopulations. In some embodiments, the primary immune cells are T cells and their subpopulations, and NK cells and their subpopulations. In some embodiments, the primary tissue cells include primary endothelial cells and their subpopulations.

[0406] In some embodiments, the cells described herein comprise regulatable reduced expression of one or more MHC class I and / or MHC class II human leukocyte antigen molecules relative to cells of the same cell type not containing the modification, and the regulatable reduced expression is due to an RNA-based component. In some embodiments, the RNA-based component is selected from the group consisting of conditional or inducible shRNA, conditional or inducible siRNA, conditional or inducible miRNA, and conditional or inducible CRISPR interference (CRISPRi). In some embodiments, the RNA-based component is under the control of a conditional promoter, and the conditional promoter is a cell cycle specific promoter, a tissue specific promoter, a lineage specific promoter, or a differentiation-induced promoter. In some embodiments, the RNA-based component is under the control of an inducible promoter, and the inducible promoter is regulated by a small molecule, a ligand, a biological agent, an aptamer-mediated regulator of polyadenylation, or an aptamer-regulated riboswitch.

[0407] In some embodiments, the cells described herein comprise regulatable reduced expression of one or more MHC class I and / or MHC class II human leukocyte antigen molecules relative to cells of the same cell type not containing the modification, and the regulatable reduced expression is due to a DNA-based component. In some embodiments, the DNA-based component is a knockout or knockdown using a method selected from the group consisting of conditional or inducible CRISPR, conditional or inducible TALEN, conditional or inducible zinc finger nuclease, conditional or inducible homing endonuclease, and conditional or inducible meganuclease. In some embodiments, the DNA-based component is under the control of a conditional promoter, and the conditional promoter is a cell cycle specific promoter, a tissue specific promoter, a lineage specific promoter, or a differentiation-induced promoter. In some embodiments, the DNA-based component is under the control of an inducible promoter, and the inducible promoter is regulated by a small molecule, a ligand, a biological agent, an aptamer-mediated regulator of polyadenylation, or an aptamer-regulated riboswitch.

[0408] In some embodiments, the cells described herein comprise regulatable reduced expression of one or more MHC class I and / or MHC class II human leukocyte antigen molecules relative to cells of the same cell type not containing the modification, and the regulatable reduced expression is due to a protein-based component. In some embodiments, the protein-based component is a conditional or inducible degron method. In some embodiments, the degron method is selected from the group consisting of ligand-induced degradation (LID) using SMASH tags, LID using Shield-1, LID using auxin, LID using rapamycin, conditional or inducible peptide degrons (e.g., IKZF3-based degrons), and conditional or inducible protein degradation targeting chimeras (PROTACs). In some embodiments, the protein-based component is under the control of a conditional promoter, and the conditional promoter is a cell cycle-specific promoter, a tissue-specific promoter, a lineage-specific promoter, or a differentiation-induced promoter. In some embodiments, the protein-based component is under the control of an inducible promoter, and the inducible promoter is regulated by a small molecule, a ligand, a biological agent, an aptamer-mediated regulator of polyadenylation, or an aptamer-regulated riboswitch.

[0409] In some embodiments, the cells described herein comprise regulatable overexpression of a first exogenous polynucleotide encoding one or more tolerogenic factors, and the regulatable overexpression is by a conditional or inducible promoter. In some embodiments, the regulatable overexpression is by a conditional promoter, and the conditional promoter is a cell cycle specific promoter, a tissue specific promoter, a lineage specific promoter, or a differentiation-inducing promoter. In some embodiments, the regulatable overexpression is by an inducible promoter, and the inducible promoter is regulated by a small molecule, a ligand or a biological agent, an aptamer-mediated regulator of polyadenylation, or an aptamer-regulated riboswitch.

[0410] In some embodiments, the present disclosure is directed to pluripotent stem cells (e.g., pluripotent stem cells and induced pluripotent stem cells (iPSCs)), differentiated cells derived from such pluripotent stem cells (e.g., but not limited to, T cells, NK cells, cardiac cells, neuronal cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, pancreatic islet cells, and retinal pigment epithelial cells), and primary cells (e.g., but not limited to, primary T cells and primary NK cells). In some embodiments, pluripotent stem cells, differentiated cells derived therefrom, such as T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, pancreatic islet cells, and retinal pigment epithelial cells, and primary cells, such as primary T cells and primary NK cells, are engineered for regulatable reduced expression or lack of expression of one or more MHC class I and / or MHC class II human leukocyte antigen molecules, and in some cases, for regulatable reduced expression or lack of expression of the T cell receptor (TCR) complex. In some embodiments, the hypoimmune T cells and primary T cells, in addition to (i) regulatable reduced expression or lack of expression of one or more MHC class I and / or MHC class II human leukocyte antigen molecules, and (ii) regulatable reduced expression or lack of expression of the T cell receptor (TCR) complex, regulatable overexpress CD47 and, optionally, regulatable overexpress a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an antigen binding domain that binds to any one selected from the group consisting of CD19, CD22, CD38, CD123, CD138, and BCMA. In some embodiments, the CAR is a CD19-specific CAR. In some embodiments, the CAR is a CD22-specific CAR. In some cases, the CAR is a CD38-specific CAR. In some embodiments, the CAR is a CD123-specific CAR. In some embodiments, the CAR is a CD138-specific CAR. In some cases, the CAR is a BCMA-specific CAR. In some embodiments, the CAR is a bispecific CAR.In some embodiments, the bispecific CAR is a CD19 / CD22 bispecific CAR. In some embodiments, the bispecific CAR is a BCMA / CD38 bispecific CAR. In some embodiments, the described cells express a CD19-specific CAR and a different CAR, such as but not limited to a CD22-specific CAR, a CD38-specific CAR, a CD123-specific CAR, a CD138-specific CAR, and a BCMA-specific CAR. In some embodiments, the described cells express a CD22-specific CAR and a different CAR, such as but not limited to a CD19-specific CAR, a CD38-specific CAR, a CD123-specific CAR, a CD138-specific CAR, and a BCMA-specific CAR. In some embodiments, the described cells express a CD38-specific CAR and a different CAR, such as but not limited to a CD22-specific CAR, a CD18-specific CAR, a CD123-specific CAR, a CD138-specific CAR, and a BCMA-specific CAR. In some embodiments, the described cells express a CD123-specific CAR and a different CAR, such as, but not limited to, a CD22-specific CAR, a CD38-specific CAR, a CD19-specific CAR, a CD138-specific CAR, and a BCMA-specific CAR. In some embodiments, the described cells express a CD138-specific CAR and a different CAR, such as, but not limited to, a CD22-specific CAR, a CD38-specific CAR, a CD123-specific CAR, a CD19-specific CAR, and a BCMA-specific CAR. In some embodiments, the described cells express a BCMA-specific CAR and a different CAR, such as, but not limited to, a CD22-specific CAR, a CD38-specific CAR, a CD123-specific CAR, a CD138-specific CAR, and a CD19-specific CAR.

[0411] In some embodiments, hypoimmune cells derived from iPSCs, such as, but not limited to, T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, pancreatic islet cells, and retinal pigment epithelial cells, regulatably overexpress CD47 and contain a regulatable genomic modification, or a regulatable knockout or knockdown of the B2M gene. In some embodiments, hypoimmune cells derived from iPSCs, such as, but not limited to, T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, pancreatic islet cells, and retinal pigment epithelial cells, regulatably overexpress CD47 and contain a regulatable genomic modification, or a regulatable knockout or knockdown of the CIITA gene. In some embodiments, the cells regulatably overexpress CD47 and contain a regulatable genomic modification, or a regulatable knockout or knockdown of the CIITA gene. - / - In some embodiments, the cell is a CIITA cell. - / - In some embodiments, the cells are regulatably CD47tg cells. In some embodiments, the cells are regulatably B2M インデル / インデル In some embodiments, the cell is a CIITA cell. インデル / インデル In some embodiments, the cells are regulatably CD47tg cells. In some embodiments, the cells are regulatably B2M ノックダウン In some embodiments, the cell is a CIITA cell. ノックダウン In some embodiments, the cells are regulatably CD47tg cells. In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / -In some embodiments, the cell is regulatably B2M - / - In some embodiments, the cells are regulatable CIITA cells. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル In some embodiments, the cell is regulatably B2M インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン In some embodiments, the cell is regulatably B2M ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - ,TRAC - / - In some embodiments, the cell is regulatably B2M - / - ,TRAC - / - In some embodiments, the cells are regulatable CIITA cells. - / - ,TRAC - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル In some embodiments, the cell is regulatably B2M インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン In some embodiments, the cell is regulatably expressing B2M ノックダウン ,TRACノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - ,TRAC - / - In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M - / - ,TRAC - / - In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. - / - ,TRAC - / - In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. - / - , C.I.T.A. - / - , T.R.B.C. - / -In some embodiments, the cell is regulatably expressing B2M - / - , T.R.B.C. - / - In some embodiments, the cells are regulatable CIITA cells. - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is regulatably expressing B2M インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is regulatably expressing B2M ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - , T.R.B.C. - / - In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M - / - , T.R.B.C. - / - In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. インデル / インデル , C.I.T.A. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M インデル / インデル , T.R.B.C. インデル / インデルIn some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. ノックダウン , C.I.T.A. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. - / - , C.I.T.A. - / - , T.R.B.C. - / - ,TRAC - / - In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデルIn some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン , CD47tg cells, which also express CAR. In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cell is regulatably expressing B2M - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cells are regulatable CIITA cells. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is regulatably expressing B2M インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン, C.I.T.A. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is regulatably B2M ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン , CD47tg cells.

[0412] In some embodiments, hypoimmune cells derived from iPSCs are produced by differentiating induced pluripotent stem cells, for example hypoimmunogenic induced pluripotent stem cells.

[0413] In some embodiments, hypoimmune cells derived from ESCs, such as, but not limited to, T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, pancreatic islet cells, and retinal pigment epithelial cells, regulatably overexpress CD47 and comprise a regulatable genomic modification, or a regulatable knockout or knockdown of the B2M gene. In some embodiments, hypoimmune cells derived from ESCs, such as, but not limited to, T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, pancreatic islet cells, and retinal pigment epithelial cells, regulatably overexpress CD47 and comprise a regulatable genomic modification, or a regulatable knockout or knockdown of the CIITA gene. In some embodiments, the cells regulatably overexpress CD47 and comprise a regulatable genomic modification, or a regulatable knockout or knockdown of the CIITA gene. - / - In some embodiments, the cell is a CIITA cell. - / - In some embodiments, the cells are regulatably CD47tg cells. In some embodiments, the cells are regulatably B2M インデル / インデル In some embodiments, the cell is a CIITA cell. インデル / インデルIn some embodiments, the cells are regulatably CD47tg cells. In some embodiments, the cells are regulatably B2M ノックダウン In some embodiments, the cell is a CIITA cell. ノックダウン In some embodiments, the cells are regulatably CD47tg cells. In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - In some embodiments, the cell is regulatably B2M - / - In some embodiments, the cells are regulatable CIITA cells. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル In some embodiments, the cell is regulatably B2M インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン In some embodiments, the cell is regulatably B2M ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - ,TRAC - / - In some embodiments, the cell is regulatably B2M - / - ,TRAC - / -In some embodiments, the cells are regulatable CIITA cells. - / - ,TRAC - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル In some embodiments, the cell is regulatably expressing B2M インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン In some embodiments, the cell is regulatably expressing B2M ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - ,TRAC - / - In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M - / - ,TRAC - / - In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. - / - ,TRAC - / - In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR.インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. - / - , C.I.T.A. - / - , T.R.B.C. - / - In some embodiments, the cell is regulatably B2M - / - , T.R.B.C. - / - In some embodiments, the cells are regulatable CIITA cells. - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is regulatably B2M インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is regulatably B2M ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン , T.R.B.C. ノックダウンIn some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - , T.R.B.C. - / - In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M - / - , T.R.B.C. - / - In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. インデル / インデル , C.I.T.A. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. ノックダウン , C.I.T.A. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. - / - , C.I.T.A. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M - / - ,TRAC - / - , T.R.B.C.- / - In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン , CD47tg cells, which also express CAR. In an embodiment, the cells are regulatably B2M - / - , C.I.T.A. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cell is regulatably B2M - / - ,TRAC- / - , T.R.B.C. - / - In some embodiments, the cells are regulatable CIITA cells. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is regulatably B2M インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is regulatably B2M ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン , CD47tg cells. In some embodiments, the iPSC-derived hypoimmune cells are produced by differentiating pluripotent stem cells, such as hypoimmunogenic embryonic stem cells.

[0414] In some embodiments, hypoimmune T cells derived from iPSCs and primary T cells regulatably overexpress one or more tolerogenic factors and a chimeric antigen receptor (CAR), and include a regulatable genomic modification, or a regulatable knockout or knockdown of the B2M gene. In some embodiments, hypoimmune T cells derived from iPSCs and primary T cells regulatably overexpress one or more tolerogenic factors and a regulatable genomic modification, or a regulatable knockout or knockdown of the CIITA gene. In some embodiments, hypoimmune T cells derived from iPSCs and primary T cells regulatably overexpress one or more tolerogenic factors and a CAR, and include a regulatable genomic modification, or a regulatable knockout or knockdown of the TRAC gene. In some embodiments, hypoimmune T cells derived from iPSCs and primary T cells regulatably overexpress one or more tolerogenic factors and a CAR, and include a regulatable genomic modification, or a regulatable knockout or knockdown of the TRB gene. In some embodiments, hypoimmune T cells derived from iPSCs and primary T cells regulatably overexpress one or more tolerogenic factors and CARs, and include one or more regulatable genomic modifications, or regulatable knockout or knockdown, selected from the group consisting of B2M, CIITA, TRAC, and TRB genes. In some embodiments, hypoimmune T cells derived from iPSCs and primary T cells regulatably overexpress one or more tolerogenic factors and CARs, and include regulatable genomic modifications, or regulatable knockout or knockdown, of B2M, CIITA, TRAC, and TRB genes. In some embodiments, hypoimmune T cells derived from iPSCs and primary T cells regulatably overexpress CD47 and chimeric antigen receptor (CAR), and include regulatable genomic modifications, or regulatable knockout or knockdown, of B2M genes. In some embodiments, hypoimmune T cells derived from iPSCs and primary T cells regulatably overexpress CD47, and include regulatable genomic modifications, or regulatable knockout or knockdown, of CIITA genes.In some embodiments, hypoimmune T cells derived from iPSCs and primary T cells regulatably overexpress CD47 and CAR and comprise a regulatable genomic modification, or a regulatable knockout or knockdown of the TRAC gene. In some embodiments, hypoimmune T cells derived from iPSCs and primary T cells regulatably overexpress CD47 and CAR and comprise a regulatable genomic modification, or a regulatable knockout or knockdown of the TRB gene. In some embodiments, hypoimmune T cells derived from iPSCs and primary T cells regulatably overexpress CD47 and CAR and comprise one or more regulatable genomic modifications, or a regulatable knockout or knockdown of the B2M, CIITA, TRAC and TRB genes. In some embodiments, hypoimmune T cells derived from iPSCs and primary T cells regulatably overexpress CD47 and CAR and comprise a regulatable genomic modification, or a regulatable knockout or knockdown of the B2M, CIITA, TRAC and TRB genes. In some embodiments, the cells regulatably overexpress CD47 and CAR and comprise a regulatable genomic modification, or a regulatable knockout or knockdown of the B2M, CIITA, TRAC and TRB genes. In some embodiments, the cells regulatably overexpress B2M. - / - In some embodiments, the cell is a CIITA cell. - / - In some embodiments, the cells are regulatably CD47tg cells. In some embodiments, the cells are regulatably B2M インデル / インデル In some embodiments, the cell is a CIITA cell. インデル / インデル In some embodiments, the cells are regulatably CD47tg cells. In some embodiments, the cells are regulatably B2M ノックダウン In some embodiments, the cell is a CIITA cell. ノックダウン In some embodiments, the cells are regulatably CD47tg cells. In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン, C.I.T.A. ノックダウン In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - In some embodiments, the cell is regulatably expressing B2M - / - In some embodiments, the cells are regulatable CIITA cells. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル In some embodiments, the cell is regulatably expressing B2M インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン In some embodiments, the cell is regulatably expressing B2M ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - ,TRAC - / - In some embodiments, the cell is regulatably expressing B2M - / - ,TRAC - / - In some embodiments, the cells are regulatable CIITA cells. - / - ,TRAC - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル In some embodiments, the cell is regulatably expressing B2M インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン, C.I.T.A. ノックダウン ,TRAC ノックダウン In some embodiments, the cell is regulatably expressing B2M ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - ,TRAC - / - In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M - / - ,TRAC - / - In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. - / - ,TRAC - / - In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. ノックダウン ,TRAC ノックダウンIn some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. - / - , C.I.T.A. - / - , T.R.B.C. - / - In some embodiments, the cell is regulatably B2M - / - , T.R.B.C. - / - In some embodiments, the cells are regulatable CIITA cells. - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is regulatably B2M インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is regulatably B2M ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - , T.R.B.C. - / - In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M - / - , T.R.B.C. - / - In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. インデル / インデル , C.I.T.A.インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. ノックダウン , C.I.T.A. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M ノックダウン , T.R.B.C. ノックダウン , CD47tg cells, which also In some embodiments, the cells express CIITA AR in a regulatable manner. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. - / - , C.I.T.A. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデルIn some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. - / - , C.I.T.A. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cell is regulatably B2M - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cells are regulatable CIITA cells. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is regulatably B2M インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデルIn some embodiments, the cells are regulatable CIITA cells. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is regulatably B2M ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン , CD47tg cells. In some embodiments, hypoimmune T cells are produced by differentiating induced pluripotent stem cells, such as hypoimmunogenic induced pluripotent stem cells.

[0415] In some embodiments, hypoimmune T cells derived from iPSCs and primary T cells are regulatable B2M - / - , C.I.T.A. - / - ,TRAC - / - In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M - / - ,TRAC - / - In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. - / - ,TRAC - / - In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. インデル / インデル ,TRAC インデル / インデルIn some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. - / - , C.I.T.A. - / - , T.R.B.C. - / - In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M - / - , T.R.B.C. - / - In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. インデル / インデル , C.I.T.A. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. ノックダウン , C.I.T.A. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M ノックダウン , T.R.B.C. ノックダウンIn some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. - / - , C.I.T.A. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウンIn some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン , CD47tg cells, which also express CAR.

[0416] In some embodiments, the described engineered or modified cells are pluripotent stem cells, induced pluripotent stem cells, NK cells differentiated from such pluripotent stem cells and induced pluripotent stem cells, T cells differentiated from such pluripotent stem cells and induced pluripotent stem cells, or primary T cells. Non-limiting examples of primary T cells include CD3+ T cells, CD4+ T cells, CD8+ T cells, naive T cells, regulatory T (Treg) cells, non-regulatory T cells, Th1 cells, Th2 cells, Th9 cells, Th17 cells, follicular helper T (Tfh) cells, cytotoxic T lymphocytes (CTLs), effector T (Teff) cells, central memory T (Tcm) cells, effector memory T (Tem) cells, effector memory T cells expressing CD45RA (TEMRA cells), tissue resident memory (Trm) cells, virtual memory T cells, natural memory T cells, memory stem cells (Tsc), γδ T cells, and any other subtype of T cells. In some embodiments, the primary T cells are selected from the group including cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, tumor infiltrating lymphocytes, and combinations thereof. Non-limiting examples of NK cells and primary NK cells include immature NK cells and mature NK cells. In some embodiments, the cells are modified or engineered relative to wild-type or control cells, including unaltered or unmodified wild-type or control cells. In some embodiments, the wild-type or control cells are the starting material. In some embodiments, the starting material has been otherwise modified or engineered to have altered expression of one or more genes to generate the engineered cells.

[0417] In some embodiments, the primary T cells are from a pool of primary T cells from one or more donor subjects different from the recipient subject (e.g., the patient to whom the cells were administered). The primary T cells can be obtained and pooled together from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, or more donor subjects. The primary T cells can be obtained and pooled together from 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 50 or more, or 100 or more donor subjects. In some embodiments, the primary T cells are harvested from one or more individuals, and in some cases, the primary T cells or pool of primary T cells are cultured in vitro. In some embodiments, the primary T cells or pool of primary T cells are engineered to exogenously express CD47 in a regulatable manner and are cultured in vitro.

[0418] In many embodiments, the primary T cells or pool of primary T cells are engineered to regulatably express a chimeric antigen receptor (CAR). The CAR can be any known to those skilled in the art. Useful CARs include those that bind to an antigen selected from the group including CD19, CD20, CD22, CD38, CD123, CD138, and BCMA. In some cases, the CAR is the same or equivalent as those used in FDA-approved CAR-T cell therapies, such as, but not limited to, tisagenlecleucel and axicabtagenecilorucel, or others under investigation in clinical trials.

[0419] In some embodiments, the primary T cells or pool of primary T cells are engineered to regulatably display reduced expression of an endogenous T cell receptor compared to unmodified primary T cells. In certain embodiments, the primary T cells or pool of primary T cells are engineered to display reduced expression of CTLA-4, PD-1, or both CTLA-4 and PD-1 compared to unmodified primary T cells. Methods for genetically modifying cells, including T cells, are detailed, for example, in WO2020 / 018620 and WO2016 / 183041, the disclosures of which are incorporated by reference in their entirety, including tables, appendices, sequence listings, and figures.

[0420] In some embodiments, the CAR-T cells comprise a CAR selected from the group comprising: (a) a first generation CAR comprising an antigen binding domain, a transmembrane domain, and a signaling domain; (b) a second generation CAR comprising an antigen binding domain, a transmembrane domain, and at least two signaling domains; (c) a third generation CAR comprising an antigen binding domain, a transmembrane domain, and at least three signaling domains; and (d) a fourth generation CAR comprising an antigen binding domain, a transmembrane domain, three or four signaling domains, and a domain that induces expression of a cytokine gene upon successful signaling of the CAR.

[0421] In some embodiments, the CAR-T cell comprises a CAR comprising an antigen binding domain, a transmembrane, and one or more signaling domains. In some embodiments, the CAR also comprises a linker. In some embodiments, the CAR comprises a CD19 antigen binding domain. In some embodiments, the CAR comprises a CD28 or CD8α transmembrane domain. In some embodiments, the CAR comprises a CD8α signal peptide. In some embodiments, the CAR comprises the Whitlow linker GSTSGSGKPGSGEGSTKG (SEQ ID NO: 15). In some embodiments, the antigen binding domain of the CAR is selected from the group including, but not limited to, (a) an antigen binding domain that targets an antigen characteristic of a neoplastic cell, (b) an antigen binding domain that targets an antigen characteristic of a T cell, (c) an antigen binding domain that targets an antigen characteristic of an autoimmune or inflammatory disorder, (d) an antigen binding domain that targets an antigen characteristic of a senescent cell, (e) an antigen binding domain that targets an antigen characteristic of an infectious disease, and (f) an antigen binding domain that binds to a cell surface antigen of a cell.

[0422] In some embodiments, the CAR further comprises one or more linkers. The format of an scFv is generally two variable domains linked by a flexible peptide sequence or "linker" in either a VH-linker-VL or VL-linker-VH orientation. Any suitable linker known to one of skill in the art in view of the present specification may be used in the CAR. Examples of suitable linkers include, but are not limited to, GS-based linker sequences, and the Whitlow linker GSTSGSGKPGSGEGSTKG (SEQ ID NO: 15). In some embodiments, the linker is a GS or gly-ser linker. An exemplary gly-ser polypeptide linker is a GS or gly-ser linker having the amino acid sequence Ser (Gly 4 Ser) n , and (Gly 4 Ser) n and / or (Gly 4 Ser 3 ) nIn some embodiments, n=2. In some embodiments, n=3, i.e., Ser(Gly 4 Ser) 3 In some embodiments, n=4, i.e., Ser(Gly 4 Ser) 4 In some embodiments, n=5. In some embodiments, n=6. In some embodiments, n=7. In some embodiments, n=8. In some embodiments, n=9. In some embodiments, n=10. Another exemplary gly-ser polypeptide linker is the amino acid sequence Ser (Gly 4 Ser) n In some embodiments, n=2. In some embodiments, n=3. In other embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker is (Gly 4 Ser) n In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker is (Gly 3 Ser) n In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In other embodiments, n=5. In yet other embodiments, n=6. Another exemplary gly-ser polypeptide linker is (Gly 4 Ser 3 ) n In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker is (Gly3 Ser) n In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In other embodiments, n=5. In yet other embodiments, n=6.

[0423] In some embodiments, the antigen binding domain is selected from the group including an antibody, an antigen binding portion or fragment thereof, an scFv, and a Fab. In some embodiments, the antigen binding domain binds to CD19, CD20, CD22, CD38, CD123, CD138, or BCMA. In some embodiments, the antigen binding domain is an anti-CD19 scFv, such as, but not limited to, FMC63.

[0424] In some embodiments, the transmembrane domain comprises one selected from the group comprising the transmembrane regions of TCR alpha, TCR beta, TCR zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD3 zeta, CD4, CD5, CD8 alpha, CD8 beta, CD9, CD16, CD28, CD45, CD22, CD33, CD34, CD37, CD40, CD40L / CD154, CD45, CD64, CD80, CD86, OX40 / CD134, 4-1BB / CD137, CD154, Fc epsilon RI gamma, VEGFR2, FAS, FGFR2B, and functional variants thereof.

[0425] In some embodiments, the signaling domain(s) of the CAR comprises a costimulatory domain(s). For example, the signaling domain can include a costimulatory domain. Or, the signaling domain can include one or more costimulatory domains. In certain embodiments, the signaling domain comprises one costimulatory domain. In other embodiments, the signaling domain comprises a costimulatory domain. In some cases, when a CAR comprises more than one costimulatory domain, the two costimulatory domains are not the same. In some embodiments, the costimulatory domain comprises two costimulatory domains that are not the same. In some embodiments, the costimulatory domain enhances cytokine production, CAR-T cell proliferation, and / or CAR-T cell persistence during T cell activation. In some embodiments, the costimulatory domain enhances cytokine production, CAR-T cell proliferation, and / or CAR-T cell persistence during T cell activation.

[0426] As described herein, a fourth generation CAR may contain an antigen binding domain, a transmembrane domain, three or four signaling domains, and a domain that induces expression of a cytokine gene upon successful signaling of the CAR. In some cases, the cytokine gene is an endogenous or exogenous cytokine gene of the hypoimmunogenic cell. In some cases, the cytokine gene encodes a proinflammatory cytokine. In some embodiments, the proinflammatory cytokine is selected from the group including IL-1, IL-2, IL-9, IL-12, IL-18, TNF, IFN-gamma, and functional fragments thereof. In some embodiments, the domain that induces expression of a cytokine gene upon successful signaling of the CAR comprises a transcription factor or a functional domain or fragment thereof.

[0427] In some embodiments, the CAR comprises a CD3 zeta (CD3ζ) domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, and (ii) a CD28 domain, or a 4-1BB domain, or a functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-dependent activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain, or a functional variant thereof, and (iii) a 4-1BB domain, or a CD134 domain, or a functional variant thereof. In certain embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-dependent activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain, or a functional variant thereof, (iii) a 4-1BB domain, or a CD134 domain, or a functional variant thereof, and (iv) a cytokine or costimulatory ligand transgene. In some embodiments, the CAR comprises (i) an anti-CD19 scFv, (ii) a CD8α hinge and transmembrane domain, or a functional variant thereof, (iii) a 4-1BB costimulatory domain, or a functional variant thereof, and (iv) a CD3ζ signaling domain, or a functional variant thereof.

[0428] Methods for introducing CAR constructs or generating CAR-T cells are well known to those skilled in the art. Detailed descriptions can be found, for example, in Vormittag et al., Curr Opin Biotechnol, 2018, 53, 162-181, and Eyquem et al., Nature, 2017, 543, 113-117.

[0429] In some embodiments, the cells derived from the primary T cells comprise reduced expression of an endogenous T cell receptor, for example, by disruption of an endogenous T cell receptor gene (e.g., T cell receptor alpha constant region (TRAC) or T cell receptor beta constant region (TRB)). In some embodiments, an exogenous nucleic acid encoding a polypeptide disclosed herein (e.g., a chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein) is inserted at the disrupted T cell receptor gene. In some embodiments, the exogenous nucleic acid encoding a polypeptide is inserted at the TRAC or TRB locus.

[0430] In some embodiments, the cells derived from the primary T cells comprise reduced expression of cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and / or programmed cell death (PD1). Methods for reducing or eliminating expression of CTLA4, PD1, and both CTLA4 and PD1 may include any method recognized by those skilled in the art, such as, but not limited to, genetic modification techniques utilizing rare-cutting endonucleases, and RNA silencing or RNA interference techniques. Non-limiting examples of rare-cutting endonucleases include any Cas protein, TALEN, zinc finger nucleases, meganucleases, and homing endonucleases. In some embodiments, an exogenous nucleic acid encoding a polypeptide disclosed herein (e.g., a chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein) is inserted at the CTLA4 and / or PD1 locus.

[0431] In some embodiments, a transgene encoding one or more tolerogenic factors with regulatable expression is inserted into a preselected locus of the cell. In some embodiments, a transgene encoding a CAR is inserted into a preselected locus of the cell. In certain embodiments, a transgene encoding one or more tolerogenic factors with regulatable expression and a transgene encoding a CAR are inserted into a preselected locus of the cell. The preselected locus can be a safe harbor locus or a target locus. Non-limiting examples of safe harbor loci include, but are not limited to, the CCR5 locus, the PPP1R12C locus (also known as AAVS1) and the CLYBL locus, the Rosa locus (e.g., the ROSA26 locus). Non-limiting examples of target loci include, but are not limited to, the CXCR4 locus, the albumin locus, the SHS231 locus, the F3 locus (also known as CD142), the MICA locus, the MICB locus, the LRP1 locus (also known as the CD91 locus), the HMGB1 locus, the ABO locus, the RHD locus, the FUT1 locus, and the KDM5D locus. A transgene encoding one or more tolerogenic factors may be inserted in intron 1 or 2 for PPP1R12C (i.e., AAVS1) or CCR5. A transgene encoding one or more tolerogenic factors may be inserted in intron 1 or 2 for PPP1R12C (i.e., AAVS1) or CCR5. A transgene encoding one or more tolerogenic factors may be inserted in exon 1 or 2 or 3 for CCR5. A transgene encoding one or more tolerogenic factors may be inserted in intron 2 for CLYBL. A transgene encoding one or more tolerogenic factors can be inserted in a 500 bp window in Ch-4:58,976,613 (ie, SHS231).A transgene encoding one or more tolerogenic factors may be inserted in a suitable region of any of the above safe harbor or target loci that allows expression of the exogenous gene, suitable regions including, for example, introns, exons or coding sequence regions in the safe harbor or target locus. In some embodiments, the preselected locus is selected from the group consisting of the B2M locus, the CIITA locus, the TRAC locus and the TRB locus. In some embodiments, the preselected locus is the B2M locus. In some embodiments, the preselected locus is the CIITA locus. In some embodiments, the preselected locus is the TRAC locus. In some embodiments, the preselected locus is the TRB locus.

[0432] In some embodiments, the transgene encoding one or more tolerogenic factors with regulatable expression and the transgene encoding the CAR are inserted into the same locus. In some embodiments, the transgene encoding one or more tolerogenic factors with regulatable expression and the transgene encoding the CAR are inserted into different loci. In many cases, the transgene encoding one or more tolerogenic factors is inserted into a safe harbor or target locus. In many cases, the transgene encoding the CAR is inserted into a safe harbor or target locus. In some cases, the transgene encoding one or more tolerogenic factors is inserted into the B2M locus. In some cases, the transgene encoding the CAR is inserted into the B2M locus. In certain cases, the transgene encoding one or more tolerogenic factors is inserted into the CIITA locus. In certain cases, the transgene encoding the CAR is inserted into the CIITA locus. In certain cases, the transgene encoding one or more tolerogenic factors is inserted into the TRAC locus. In certain cases, the transgene encoding the CAR is inserted into the TRAC locus. In many other cases, the transgene encoding one or more tolerogenic factors is inserted within the TRB locus. In many other cases, the transgene encoding the CAR is inserted within the TRB locus. In some embodiments, the transgene encoding one or more tolerogenic factors and the transgene encoding the CAR are inserted within a safe harbor or target locus (e.g., the CCR5 locus, the CXCR4 locus, the PPP1R12C locus, the albumin locus, the SHS231 locus, the CLYBL locus, the ROSA locus, the F3 (CD142) locus, the MICA locus, the MICB locus, the LRP1 (CD91) locus, the HMGB1 locus, the ABO locus, the RHD locus, the FUT1 locus, and the KDM5D locus).

[0433] In many embodiments, the transgenes encoding one or more tolerogenic factors with regulatable expression and the transgene encoding the CAR are inserted into a safe harbor or target locus. In certain embodiments, the transgenes encoding one or more tolerogenic factors with regulatable expression and the transgene encoding the CAR are controlled by a single promoter and inserted into a safe harbor or target locus. In certain embodiments, the transgenes encoding one or more tolerogenic factors with regulatable expression and the transgene encoding the CAR are controlled by their own promoters and inserted into a safe harbor or target locus. In certain embodiments, the transgenes encoding one or more tolerogenic factors and the transgene encoding the CAR are inserted into the TRAC locus. In certain embodiments, the transgenes encoding one or more tolerogenic factors and the transgene encoding the CAR are controlled by a single promoter and inserted into the TRAC locus. In certain embodiments, the transgenes encoding one or more tolerogenic factors and the transgene encoding the CAR are controlled by their own promoters and inserted into the TRAC locus. In some embodiments, the transgenes encoding one or more tolerogenic factors and the transgene encoding the CAR are inserted into the TRB locus. In some embodiments, the transgenes encoding one or more tolerogenic factors and the transgene encoding the CAR are controlled by a single promoter and inserted into the TRB locus. In some embodiments, the transgenes encoding one or more tolerogenic factors and the transgene encoding the CAR are controlled by their own promoters and inserted into the TRB locus. In other embodiments, the transgenes encoding one or more tolerogenic factors and the transgene encoding the CAR are inserted into the B2M locus. In other embodiments, the transgenes encoding one or more tolerogenic factors and the transgene encoding the CAR are controlled by a single promoter and inserted into the B2M locus.In other embodiments, the transgenes encoding one or more tolerogenic factors and the transgene encoding the CAR are controlled by their own promoters and are inserted into the B2M locus. In various embodiments, the transgenes encoding one or more tolerogenic factors and the transgene encoding the CAR are inserted into the CIITA locus. In various embodiments, the transgenes encoding one or more tolerogenic factors and the transgene encoding the CAR are controlled by a single promoter and are inserted into the CIITA locus. In various embodiments, the transgenes encoding one or more tolerogenic factors and the transgene encoding the CAR are controlled by their own promoters and are inserted into the CIITA locus.

[0434] In some cases, the promoter controlling expression of any of the described transgenes is a constitutive promoter. In some cases, the promoter controlling expression of any of the described transgenes is a conditional promoter. In other cases, the promoter for any of the described transgenes is an inducible promoter. In some embodiments, the promoter is an EF1α promoter. In some embodiments, the promoter is a CAG promoter. In some embodiments, the transgene encoding one or more tolerogenic factors is controlled by a constitutive promoter. In some embodiments, the transgene encoding one or more tolerogenic factors is controlled by a conditional promoter. In some embodiments, the transgene encoding one or more tolerogenic factors is controlled by a cell cycle specific promoter. In some embodiments, the transgene encoding one or more tolerogenic factors is controlled by a tissue specific promoter. In some embodiments, the transgene encoding one or more tolerogenic factors is controlled by a lineage specific promoter. In some embodiments, the transgene encoding one or more tolerogenic factors is controlled by a differentiation-inducing promoter. In some embodiments, the transgene encoding one or more tolerogenic factors is controlled by an inducible promoter. In some embodiments, the transgene encoding one or more tolerogenic factors is controlled by an inducible promoter regulated by a small molecule. In some embodiments, the transgene encoding one or more tolerogenic factors is controlled by an inducible promoter regulated by a ligand. In some embodiments, the transgene encoding one or more tolerogenic factors is controlled by an inducible promoter regulated by a biological agent. In some embodiments, the transgene encoding one or more tolerogenic factors is controlled by an inducible promoter regulated by an aptamer-mediated regulator of polyadenylation.In some embodiments, the transgene encoding one or more tolerogenic factors is controlled by an inducible promoter regulated by an aptamer-regulated riboswitch. In some embodiments, the CAR transgene is controlled by a constitutive promoter. In some embodiments, the CAR transgene is controlled by a conditional promoter. In some embodiments, the CAR transgene is controlled by a cell cycle specific promoter. In some embodiments, the CAR transgene is controlled by a tissue specific promoter. In some embodiments, the CAR transgene is controlled by a lineage specific promoter. In some embodiments, the CAR transgene is controlled by a differentiation-induced promoter. In some embodiments, the CAR transgene is controlled by an inducible promoter. In some embodiments, the CAR transgene is controlled by an inducible promoter that is regulated by a small molecule. In some embodiments, the CAR transgene is controlled by an inducible promoter that is regulated by a ligand. In some embodiments, the CAR transgene is controlled by an inducible promoter that is regulated by a biologic agent. In some embodiments, the CAR transgene is controlled by an inducible promoter that is regulated by an aptamer-mediated regulator of polyadenylation. In some embodiments, the CAR transgene is controlled by an inducible promoter regulated by an aptamer-regulated riboswitch. In some embodiments, the transgene encoding one or more tolerogenic factors and the transgene encoding the CAR are both controlled by conditional promoters. In some embodiments, the transgene encoding one or more tolerogenic factors and the transgene encoding the CAR are both controlled by inducible promoters. In some embodiments, the transgene encoding one or more tolerogenic factors is controlled by a constitutive promoter and the transgene encoding the CAR is controlled by an inducible promoter.In some embodiments, the transgenes encoding one or more tolerogenic factors are controlled by a constitutive promoter and the transgene encoding the CAR is controlled by a conditional promoter. In some embodiments, the transgenes encoding one or more tolerogenic factors are controlled by a conditional promoter and the transgene encoding the CAR is controlled by an inducible promoter. In some embodiments, the transgenes encoding one or more tolerogenic factors are controlled by a conditional promoter and the transgene encoding the CAR is controlled by a constitutive promoter. In some embodiments, the transgenes encoding one or more tolerogenic factors are controlled by an inducible promoter and the transgene encoding the CAR is controlled by a conditional promoter. In various embodiments, the transgenes encoding one or more tolerogenic factors are controlled by an EF1 alpha promoter and the transgene encoding the CAR is controlled by an EF1 alpha promoter. In some embodiments, the transgenes encoding one or more tolerogenic factors are controlled by a CAG promoter and the transgene encoding the CAR is controlled by a CAG promoter. In some embodiments, the transgenes encoding one or more tolerogenic factors are controlled by a CAG promoter and the transgene encoding a CAR is controlled by an EF1 alpha promoter. In some embodiments, the transgenes encoding one or more tolerogenic factors are controlled by an EF1 alpha promoter and the transgene encoding a CAR is controlled by a CAG promoter. In some embodiments, the expression of both the transgenes encoding one or more tolerogenic factors and the transgene encoding a CAR is controlled by a single EF1 alpha promoter. In some embodiments, the expression of both the transgenes encoding one or more tolerogenic factors and the transgene encoding a CAR is controlled by a single CAG promoter.

[0435] In another embodiment, the disclosure disclosed herein is directed to pluripotent stem cells (e.g., pluripotent stem cells and induced pluripotent stem cells (iPSCs)), differentiated cells derived from such pluripotent stem cells (e.g., hypoimmune T cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, pancreatic islet cells, and retinal pigment epithelial cells), and primary cells that regulatably overexpress CD47 (e.g., regulatably express CD47 protein), have regulatably reduced expression or lack of expression of one or more MHC class I and / or MHC class II human leukocyte antigen molecules, and have regulatably reduced expression or lack of expression of the T cell receptor (TCR) complex. In some embodiments, the hypoimmune T cells and primary T cells regulatably overexpress CD47 (e.g., regulatably exogenously express CD47 protein), have regulatably reduced expression or lack of expression of one or more MHC class I and / or MHC class II human leukocyte antigen molecules, and have regulatably reduced expression or lack of expression of the T cell receptor (TCR) complex.

[0436] In some embodiments, pluripotent stem cells (e.g., pluripotent stem cells and induced pluripotent stem cells (iPSCs)), differentiated cells derived from such pluripotent stem cells (e.g., hypoimmune T cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, pancreatic islet cells, and retinal pigment epithelial cells), and primary cells regulatably overexpress CD47 and comprise a regulatable genomic modification of the B2M gene. In some embodiments, pluripotent stem cells, differentiated cells derived from such pluripotent stem cells, and primary T cells regulatably overexpress CD47 and comprise a regulatable genomic modification of the CIITA gene. In some embodiments, pluripotent stem cells, and differentiated cells derived from such pluripotent stem cells, for example, but not limited to, T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, pancreatic islet cells, and retinal pigment epithelial cells, regulatably overexpress CD47 and comprise a regulatable genomic modification of the CIITA gene. - / - In some embodiments, the cell is a CIITA cell. - / - In some embodiments, the cells are regulatably CD47tg cells. In some embodiments, the cells are regulatably B2M インデル / インデル In some embodiments, the cell is a CIITA cell. インデル / インデル In some embodiments, the cells are regulatably CD47tg cells. In some embodiments, the cells are regulatably B2M ノックダウン In some embodiments, the cell is a CIITA cell. ノックダウン In some embodiments, the cells are regulatably CD47tg cells. In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン In some embodiments, the cells are regulatably B2M- / - , C.I.T.A. - / - In some embodiments, the cell is regulatably B2M - / - In some embodiments, the cells are regulatable CIITA cells. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル In some embodiments, the cell is regulatably B2M インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン In some embodiments, the cell is regulatably B2M ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - ,TRAC - / - In some embodiments, the cell is regulatably B2M - / - ,TRAC - / - In some embodiments, the cells are regulatable CIITA cells. - / - ,TRAC - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル In some embodiments, the cell is regulatably B2M インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウンIn some embodiments, the cell is regulatably B2M ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - , T.R.B.C. - / - In some embodiments, the cell is regulatably B2M - / - , T.R.B.C. - / - In some embodiments, the cells are regulatable CIITA cells. - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is regulatably B2M インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is regulatably B2M ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cell is regulatably B2M - / - ,TRAC - / - , T.R.B.C. - / -In some embodiments, the cells are regulatable CIITA cells. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is regulatably B2M インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is regulatably B2M ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン, CD47tg cells. In some embodiments, the pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells regulatably overexpress CD47 and comprise a regulatable genomic modification in the TRAC gene. In some embodiments, the pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells regulatably overexpress CD47 and comprise a regulatable genomic modification in the TRB gene. In some embodiments, the pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells regulatably overexpress CD47 and comprise one or more regulatable genomic modifications selected from the group consisting of B2M, CIITA, TRAC, and TRB genes. In some embodiments, the pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells regulatably overexpress CD47 and comprise a regulatable genomic modification in the B2M, CIITA, and TRAC genes. In some embodiments, the pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells regulatably overexpress CD47 and comprise regulatable genomic modifications in the B2M, CIITA, and TRB genes. In some embodiments, the pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells regulatably overexpress CD47 and comprise regulatable genomic modifications in the B2M, CIITA, TRAC, and TRB genes. In certain embodiments, the pluripotent stem cells, differentiated cells derived from such pluripotent stem cells, and primary T cells regulatably overexpress CD47 and comprise regulatable genomic modifications in the B2M, CIITA, TRAC, and TRB genes. - / - In some embodiments, the cell is a CIITA cell. - / - In some embodiments, the cells are regulatably CD47tg cells. In some embodiments, the cells are regulatably B2M インデル / インデル In some embodiments, the cell is a CIITA cell. インデル / インデル In some embodiments, the cells are regulatably CD47tg cells. In some embodiments, the cells are regulatably B2M ノックダウン In some embodiments, the cell is a CIITA cell. ノックダウンIn some embodiments, the cells are regulatably CD47tg cells. In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - In some embodiments, the cell is regulatably B2M - / - In some embodiments, the cells are regulatable CIITA cells. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル In some embodiments, the cell is regulatably B2M インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン In some embodiments, the cell is regulatably B2M ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン In some embodiments, the cells are regulatably B2M - / - , C IITA - / - ,TRAC - / - In some embodiments, the cell is regulatably B2M - / - ,TRAC - / - In some embodiments, the cells are regulatable CIITA cells. - / - ,TRAC- / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル In some embodiments, the cell is regulatably expressing B2M インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン In some embodiments, the cell is regulatably expressing B2M ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - ,TRAC - / - In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M - / - ,TRAC - / - In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. - / - ,TRAC - / - In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. インデル / インデル ,TRAC インデル / インデルIn some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. - / - , C.I.T.A. - / - , T.R.B.C. - / - In some embodiments, the cell is regulatably expressing B2M - / - , T.R.B.C. - / - In some embodiments, the cells are regulatable CIITA cells. - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is regulatably expressing B2M インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is regulatably expressing B2M ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatably B2M - / - , C.I.T.A.- / - , T.R.B.C. - / - In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M - / - , T.R.B.C. - / - In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. インデル / インデル , C.I.T.A. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. ノックダウン , C.I.T.A. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. - / - , C.I.T.A. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M - / - ,TRAC - / - , T.R.B.C. - / -In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. - / - , C.I.T.A. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cell is regulatably B2M - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cells are regulatable CIITA cells.- / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is regulatably expressing B2M インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is regulatably expressing B2M ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン, CD47tg cells. In some embodiments, the engineered or modified cells described are pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells), T cells differentiated from such pluripotent stem cells, or primary T cells. Non-limiting examples of primary T cells include CD3+ T cells, CD4+ T cells, CD8+ T cells, naive T cells, regulatory T (Treg) cells, non-regulatory T cells, Th1 cells, Th2 cells, Th9 cells, Th17 cells, follicular helper T (Tfh) cells, cytotoxic T lymphocytes (CTLs), effector T (Teff) cells, central memory T (Tcm) cells, effector memory T (Tem) cells, effector memory T cells expressing CD45RA (TEMRA cells), tissue resident memory (Trm) cells, virtual memory T cells, natural memory T cells, memory stem cells (Tsc), γδ T cells, and any other subtype of T cells. In some embodiments, the cells are modified or engineered compared to wild-type or control cells, including unaltered or unmodified wild-type or control cells. In some embodiments, the wild-type or control cells are the starting material. In some embodiments, the starting material is otherwise modified or engineered to have altered expression of one or more genes to generate engineered cells.

[0437] In some embodiments, a transgene encoding one or more tolerogenic factors with regulatable expression is inserted into a preselected locus of the cell. The preselected locus can be a safe harbor or a target locus. Non-limiting examples of safe harbor loci include the CCR5 locus, the PPP1R12C locus, the CLYBL locus, and the Rosa locus. Non-limiting examples of target loci include the CXCR4 locus, the albumin locus, the SHS231 locus, the F3 (CD142) locus, the MICA locus, the MICB locus, the LRP1 (CD91) locus, the HMGB1 locus, the ABO locus, the RHD locus, the FUT1 locus, and the KDM5D locus. In some embodiments, the preselected locus is the TRAC locus. In some embodiments, the transgene encoding one or more tolerogenic factors is inserted into a safe harbor or target locus (e.g., the CCR5 locus, the CXCR4 locus, the PPP1R12C locus, the albumin locus, the SHS231 locus, the CLYBL locus, the ROSA locus, the F3 (CD142) locus, the MICA locus, the MICB locus, the LRP1 (CD91) locus, the HMGB1 locus, the ABO locus, the RHD locus, the FUT1 locus, and the KDM5D locus). In certain embodiments, the transgene encoding one or more tolerogenic factors is inserted into the B2M locus. In certain embodiments, the transgene encoding one or more tolerogenic factors is inserted into the B2M locus. In certain embodiments, the transgene encoding one or more tolerogenic factors is inserted into the TRAC locus. In certain embodiments, the transgene encoding one or more tolerogenic factors is inserted into the TRB locus.

[0438] In some cases, expression of the transgene encoding one or more tolerogenic factors is controlled by a conditional promoter, while in other cases, expression of the transgene encoding one or more tolerogenic factors is controlled by an inducible promoter.

[0439] In yet another embodiment, the disclosure disclosed herein is directed to pluripotent stem cells (e.g., pluripotent stem cells and induced pluripotent stem cells (iPSCs)), T cells (e.g., hypoimmune T cells) and primary T cells derived from such pluripotent stem cells, which have regulatable reduced expression or lack of expression of one or more MHC class I and / or MHC class II human leukocyte antigen molecules, and have regulatable reduced expression or lack of expression of T cell receptor (TCR) complexes. In some embodiments, the cells have regulatable reduced expression or lack of expression of one or more MHC class I antigen molecules, MHC class II antigen molecules, and TCR complexes.

[0440] In some embodiments, pluripotent stem cells (e.g., iPSCs), differentiated cells derived from such pluripotent stem cells (e.g., T cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, pancreatic islet cells, and retinal pigment epithelial cells differentiated from such pluripotent stem cells), and primary T cells comprise a regulatable genomic modification or regulatable knockdown of the B2M gene. In some embodiments, pluripotent stem cells (e.g., iPSCs), differentiated cells derived from such pluripotent stem cells (e.g., T cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, pancreatic islet cells, and retinal pigment epithelial cells differentiated from such pluripotent stem cells), and primary T cells comprise a regulatable genomic modification or regulatable knockdown of the CIITA gene. In some embodiments, cells, including iPSCs and differentiated cells derived from such pluripotent stem cells, such as, but not limited to, T cells, NK cells, cardiac cells, neuronal cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, pancreatic islet cells, and retinal pigment epithelial cells, are expressed in a regulatable B2M - / - In some embodiments, the cell is a CIITA cell. - / -In some embodiments, the cells are regulatably CD47tg cells. In some embodiments, the cells are regulatably B2M インデル / インデル In some embodiments, the cell is a CIITA cell. インデル / インデル In some embodiments, the cells are regulatably CD47tg cells. In some embodiments, the cells are regulatably B2M ノックダウン In some embodiments, the cell is a CIITA cell. ノックダウン In some embodiments, the cells are regulatably CD47tg cells. In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - In some embodiments, the cell is regulatably expressing B2M - / - In some embodiments, the cells are regulatable CIITA cells. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル In some embodiments, the cell is regulatably expressing B2M インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン In some embodiments, the cell is regulatably expressing B2M ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウンIn some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - ,TRAC - / - In some embodiments, the cell is regulatably B2M - / - ,TRAC - / - In some embodiments, the cells are regulatable CIITA cells. - / - ,TRAC - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル In some embodiments, the cell is regulatably B2M インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン In some embodiments, the cell is regulatably B2M ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - , T.R.B.C. - / - In some embodiments, the cell is regulatably B2M - / - , T.R.B.C. - / - In some embodiments, the cells are regulatable CIITA cells. - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is regulatably B2M インデル / インデル, T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is regulatably B2M ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cell is regulatably B2M - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cells are regulatable CIITA cells. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is regulatably B2M インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is regulatably B2M ノックダウン,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン , CD47tg cells. In some embodiments, the pluripotent stem cells (e.g., ESCs or iPSCs), T cells differentiated from such pluripotent stem cells, and primary T cells comprise a regulatable genomic modification or regulatable knockdown of the TRAC gene. In some embodiments, the pluripotent stem cells (e.g., iPSCs), T cells differentiated from such pluripotent stem cells, and primary T cells comprise a regulatable genomic modification or regulatable knockdown of the TRB gene. In some embodiments, the pluripotent stem cells (e.g., iPSCs), T cells differentiated from such pluripotent stem cells, and primary T cells comprise a regulatable genomic modification or regulatable knockdown of one or more genes selected from the group consisting of B2M, CIITA, and TRAC. In some embodiments, the pluripotent stem cells (e.g., iPSCs), T cells differentiated from such pluripotent stem cells, and primary T cells comprise a regulatable genomic modification or regulatable knockdown of one or more genes selected from the group consisting of B2M, CIITA, and TRB. In some embodiments, pluripotent stem cells (e.g., iPSCs), T cells differentiated from such pluripotent stem cells, and primary T cells comprise a regulatable genomic modification or regulatable knockdown of one or more selected from the group consisting of B2M, CIITA, TRAC, and TRB genes. In certain embodiments, cells, including iPSCs, T cells differentiated from such iPSCs, and primary T cells, comprise a regulatable genomic modification or regulatable knockdown of one or more selected from the group consisting of B2M, CIITA, TRAC, and TRB genes. - / - In some embodiments, the cell is a CIITA cell. - / - In some embodiments, the cells are regulatably CD47tg cells. In some embodiments, the cells are regulatably B2M インデル / インデル In some embodiments, the cell is a CIITA cell. インデル / インデル In some embodiments, the cells are regulatably CD47tg cells. In some embodiments, the cells are regulatably B2Mノックダウン In some embodiments, the cell is a CIITA cell. ノックダウン In some embodiments, the cells are regulatably CD47tg cells. In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - In some embodiments, the cell is regulatably expressing B2M - / - In some embodiments, the cells are regulatable CIITA cells. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル In some embodiments, the cell is regulatably expressing B2M インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン In some embodiments, the cell is regulatably expressing B2M ノックダウン , CD47tg cells. In this state, cells express CIITA in a regulatable manner. ノックダウン In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - ,TRAC - / - In some embodiments, the cell is regulatably expressing B2M - / - ,TRAC - / -In some embodiments, the cells are regulatable CIITA cells. - / - ,TRAC - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル In some embodiments, the cell is regulatably B2M インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン In some embodiments, the cell is regulatably B2M ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - ,TRAC - / - In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M - / - ,TRAC - / - In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. - / - ,TRAC - / - In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR.インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. ノックダウン ,TRAC ノックダウン In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. - / - , C.I.T.A. - / - , T.R.B.C. - / - In some embodiments, the cell is regulatably B2M - / - , T.R.B.C. - / - In some embodiments, the cells are regulatable CIITA cells. - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is regulatably B2M インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is regulatably B2M ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン , T.R.B.C. ノックダウンIn some embodiments, the cells are regulatably B2M - / - , C.I.T.A. - / - , T.R.B.C. - / - In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M - / - , T.R.B.C. - / - In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. インデル / インデル , C.I.T.A. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. ノックダウン , C.I.T.A. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. - / - , C.I.T.A. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M - / - ,TRAC - / - , T.R.B.C.- / - In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is a CAR-expressing cell, which also expresses a CAR. In some embodiments, the cell is regulatably expressing B2M ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatable CIITA cells, which are CD47tg cells and which also express a CAR. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatably B2M, CD47tg cells, which also express a CAR. - / - , C.I.T.A. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cell is regulatably expressing B2M - / - ,TRAC - / - , T.R.B.C. - / -In some embodiments, the cells are regulatable CIITA cells. - / - ,TRAC - / - , T.R.B.C. - / - In some embodiments, the cells are regulatably B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cell is regulatably expressing B2M インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatable CIITA cells. インデル / インデル ,TRAC インデル / インデル , T.R.B.C. インデル / インデル In some embodiments, the cells are regulatably B2M ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cell is regulatably expressing B2M ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン In some embodiments, the cells are regulatable CIITA cells. ノックダウン ,TRAC ノックダウン , T.R.B.C. ノックダウン, CD47tg cells. In some embodiments, the modified cells described are pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from such pluripotent stem cells and induced pluripotent stem cells, or primary T cells. Non-limiting examples of primary T cells include CD3+ T cells, CD4+ T cells, CD8+ T cells, naive T cells, regulatory T (Treg) cells, non-regulatory T cells, Th1 cells, Th2 cells, Th9 cells, Th17 cells, follicular helper T (Tfh) cells, cytotoxic T lymphocytes (CTL), effector T (Teff) cells, central memory T (Tcm) cells, effector memory T (Tem) cells, effector memory T cells expressing CD45RA (TEMRA cells), tissue resident memory (Trm) cells, virtual memory T cells, natural memory T cells, memory stem cells (Tsc), γδ T cells, and any other subtype of T cells. In some embodiments, the cells are modified or engineered compared to wild-type or control cells, including unaltered or unmodified wild-type or control cells. In some embodiments, the wild-type or control cells are the starting material. In some embodiments, the starting material is otherwise modified or engineered to have altered expression of one or more genes to generate engineered cells.

[0441] The cells of the present disclosure exhibit regulatably reduced expression or regulatably lack of expression of one or more MHC class I antigen molecules, MHC class II antigen molecules, and / or TCR complexes. Reduction of MHC I and / or MHC II expression can be achieved, for example, by: (1) direct targeting of polymorphic HLA alleles (HLA-A, HLA-B, HLA-C) and MHC-II genes, (2) elimination of B2M, which prevents surface trafficking of all MHC-I molecules, (3) elimination of CIITA, which prevents surface trafficking of all MHC-II molecules, and / or (4) deletion of components of the MHC enhanceosome that are important for HLA expression, such as LRC5, RFX5, RFXANK, RFXAP, IRFl, NF-Y (including NFY-A, NFY-B, NFY-C), and CIITA.

[0442] In some embodiments, HLA expression is interfered with by targeting individual HLA (e.g., knocking out, knocking down or reducing expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ and / or HLA-DR), targeting transcriptional regulators of HLA expression (e.g., knocking out or reducing expression of NLRC5, CIITA, RFX5, RFXAP, RFXANK, NFY-A, NFY-B, NFY-C and / or IRF-1), blocking surface trafficking of MHC class I molecules (e.g., knocking out or reducing expression of B2M and / or TAP1), and / or targeting HLA-Razor (see, e.g., WO2016183041).

[0443] In some embodiments, the cells disclosed herein, including but not limited to pluripotent stem cells, induced pluripotent stem cells, differentiated cells derived from such stem cells, and primary T cells, are characterized as not regulatably expressing one or more human leukocyte antigen molecules corresponding to MHC-I and / or MHC-II (e.g., HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and / or HLA-DR), and therefore being hypoimmunogenic. For example, in certain embodiments, the disclosed pluripotent stem cells and induced pluripotent stem cells are modified such that the stem cells or differentiated cells prepared therefrom do not regulatably express, or regulatably display reduced expression of, one or more of the following MHC-I molecules, HLA-A, HLA-B, and HLA-C. In some embodiments, one or more of HLA-A, HLA-B, and HLA-C can be regulatably "knocked out" from the cells. Cells with regulatable knockout HLA-A, HLA-B and / or HLA-C genes can regulatably show reduced or eliminated expression of each knocked-out gene. In some embodiments, one or more of HLA-A, HLA-B and HLA-C can be regulatably knocked down or knocked out in cells. Cells with knocked-down HLA-A, HLA-B and / or HLA-C genes can regulatably show reduced or eliminated expression of each knocked-down gene.

[0444] In some embodiments, the guide RNA, shRNA, siRNA, or miRNA that allows simultaneous deletion of all MHC class I alleles by targeting conserved regions in HLA genes is identified as HLA Razor. In some embodiments, gRNA is part of a CRISPR system, for example, a regulatable CRISPR system, for example, a conditional or inducible CRISPR system. In alternative embodiments, gRNA is part of a TALEN system, for example, a regulatable TALEN system, for example, a conditional or inducible TALEN system. In some embodiments, shRNA, siRNA, or mRNA is part of a regulatable RNAi system, for example, a conditional or inducible RNAi system. In some embodiments, the HLA Razor that targets the identified conserved regions in HLA is described in WO2016183041. In some embodiments, multiple HLA Razors that target the identified conserved regions are utilized. It is generally understood that any guide, siRNA, shRNA, or miRNA molecule that targets conserved regions in HLA can act as HLA Razor.

[0445] The provided methods are useful for regulatable inactivation or elimination of MHC class I and / or MHC class II expression in cells, including but not limited to pluripotent stem cells, differentiated cells, and primary T cells. In some embodiments, regulatable genome editing techniques using rare-cutting endonucleases (e.g., CRISPR / Cas, TALEN, zinc finger nucleases, meganucleases, and homing endonuclease systems) are also used to reduce or eliminate expression of genes involved in innate and / or adaptive immune responses in cells (e.g., by deleting genomic DNA of genes involved in innate and / or adaptive immune responses or by inserting genomic DNA into such genes such that gene expression is affected). In certain embodiments, regulatable genome editing techniques or other gene modulation techniques are used to insert tolerance inducers in human cells to make these human cells and differentiated cells prepared therefrom hypoimmunogenic cells. Thus, hypoimmunogenic cells have reduced or eliminated expression of one or more MHC I and MHC II. In some embodiments, the cells are non-immunogenic (eg, do not induce an innate and / or adaptive immune response) in a recipient subject.

[0446] In some embodiments, the cells comprise modifications to regulatably increase expression of CD47 and one or more factors selected from the group consisting of DUX4, CD24, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, FasL, CCL21, CCL22, Mfge8, CD16, CD52, H2-M3, CD16 Fc receptor, IL15-RF, and / or Serpinb9.

[0447] In some embodiments, the cells comprise a regulatable genomic modification or regulatable knockdown of one or more target polynucleotide sequences that regulate the expression of either an MHC class I molecule, an MHC class II molecule, or an MHC class I and an MHC class II molecule. In some embodiments, a regulatable gene editing system is used to modify the one or more target polynucleotide sequences. In some embodiments, a regulatable RNAi system is used to knockdown the expression of the one or more target polynucleotide sequences. In some embodiments, the target polynucleotide sequences are one or more selected from the group comprising B2M, CIITA, and NLRC5. In some embodiments, the cells comprise a regulatable gene editing modification to the B2M gene. In some embodiments, the cells comprise a regulatable gene editing modification to the CIITA gene. In some embodiments, the cells comprise a regulatable gene editing modification to the NLRC5 gene. In some embodiments, the cells comprise a regulatable gene editing modification to the B2M and CIITA genes. In some embodiments, the cells comprise a regulatable gene editing modification to the B2M and NLRC5 genes. In some embodiments, the cells comprise a regulatable gene editing modification to the CIITA and NLRC5 genes. In many embodiments, the cell comprises regulatable gene editing modifications to the B2M, CIITA and NLRC5 genes. In some embodiments, the cell comprises a regulatable RNAi system targeting the B2M gene. In some embodiments, the cell comprises a regulatable RNAi system targeting the CIITA gene. In some embodiments, the cell comprises a regulatable RNAi system targeting the NLRC5 gene. In some embodiments, the cell comprises a regulatable RNAi system targeting the B2M and CIITA genes. In some embodiments, the cell comprises a regulatable RNAi system targeting the B2M and NLRC5 genes. In some embodiments, the cell comprises a regulatable RNAi system targeting the CIITA and NLRC5 genes. In many embodiments, the cell comprises a regulatable RNAi system targeting the B2M, CIITA and NLRC5 genes.In certain embodiments, the genome of the cell is modified to reduce or delete essential components of HLA expression. In certain embodiments, the cell comprises a regulatable RNAi system that targets key components of HLA expression. In some embodiments, the cell is modified or engineered in comparison with wild-type or control cells, including unaltered or unmodified wild-type or control cells. In some embodiments, the wild-type or control cells are the starting material. In some embodiments, the starting material is otherwise modified or engineered to have altered expression of one or more genes to generate engineered cells.

[0448] In some embodiments, the disclosure provides a cell or population thereof comprising a genome (e.g., a stem cell, an induced pluripotent stem cell, a differentiated cell, such as a cardiac cell, a neuronal cell, a brain endothelial cell, a dopaminergic neuron, a glial progenitor cell, an endothelial cell, a thyroid cell, a hepatic cell, a pancreatic islet cell or a retinal pigment epithelial cell, a hematopoietic stem cell, a primary NK cell, a CAR-NK cell, a primary T cell, or a CAR-T cell), in which genes have been regulatably edited to delete contiguous stretches of genomic DNA, thus reducing or eliminating surface expression of one or more MHC class I molecules in the cell or population thereof. In certain embodiments, the disclosure provides a cell or population thereof comprising a genome (e.g., a stem cell, an induced pluripotent stem cell, a differentiated cell, such as a cardiac cell, a neuronal cell, a brain endothelial cell, a dopaminergic neuron, a glial progenitor cell, an endothelial cell, a thyroid cell, a hepatic cell, a pancreatic islet cell or a retinal pigment epithelial cell, a hematopoietic stem cell, a primary NK cell, a CAR-NK cell, a primary T cell, or a CAR-T cell), in which genes have been regulatably edited to delete contiguous stretches of genomic DNA, thus reducing or eliminating surface expression of one or more MHC class II molecules in the cell or population thereof. In numerous embodiments, the disclosure provides a cell or population thereof (e.g., a stem cell, an induced pluripotent stem cell, a differentiated cell, such as a cardiac cell, a neuronal cell, a brain endothelial cell, a dopaminergic neuron, a glial progenitor cell, an endothelial cell, a thyroid cell, a hepatic cell, a pancreatic islet cell or a retinal pigment epithelial cell, a hematopoietic stem cell, a primary NK cell, a CAR-NK cell, a primary T cell, or a CAR-T cell) comprising a genome, in which one or more genes have been regulatably edited to delete contiguous stretches of genomic DNA, thus reducing or eliminating surface expression of one or more MHC class I and II molecules in the cell or population thereof.

[0449] In many embodiments, expression of one or more MHC I and / or MHC II molecules is controllably regulated by targeting and deleting a contiguous stretch of genomic DNA, thereby reducing or eliminating expression of a target gene selected from the group consisting of B2M, CIITA, and NLRC5. In some embodiments, described herein are gene-edited cells (e.g., modified human cells) that contain a regulatable exogenous CD47 protein and a controllably inactivated or modified CIITA gene sequence, and in some cases, an additional genetic modification that controllably inactivates or modifies the B2M gene sequence. In some embodiments, described herein are gene-edited cells that contain a regulatable exogenous CD47 protein and a controllably inactivated or modified CIITA gene sequence, and in some cases, an additional genetic modification that controllably inactivates or modifies the NLRC5 gene sequence. In some embodiments, described herein are gene-edited cells that comprise a regulatable exogenous CD47 protein and a regulatable inactivated or modified B2M gene sequence, and in some cases, additional genetic modifications that regulatably inactivate or modify the NLRC5 gene sequence. In some embodiments, described herein are gene-edited cells that comprise a regulatable exogenous CD47 protein and a regulatably inactivated or modified B2M gene sequence, and in some cases, additional genetic modifications that regulatably inactivate or modify the CIITA gene sequence and the NLRC5 gene sequence.

[0450] Provided herein are cells that exhibit one or more target polynucleotide sequence modifications, where the modifications regulatably regulate expression of any one of the following: (a) an MHC I antigen molecule, (b) an MHC II antigen molecule, (c) a TCR complex, (d) both MHC I and II antigen molecules, and (e) an MHC I and II antigen molecule and a TCR complex. In certain embodiments, the modifications include regulatably increasing expression of CD47. In some embodiments, the cells comprise an exogenous or recombinant CD47 polypeptide. In certain embodiments, the modifications include regulatably expressing a chimeric antigen receptor. In some embodiments, the cells comprise an exogenous or recombinant chimeric antigen receptor polypeptide.

[0451] In some embodiments, the cells comprise genomic modifications of one or more target polynucleotide sequences that regulatably regulate the expression of one or more MHC I antigen molecules, MHC II antigen molecules, and / or TCR complexes. In some embodiments, a gene editing system is used to regulatably modify one or more target polynucleotide sequences. In some embodiments, the polynucleotide sequences target one or more genes selected from the group consisting of B2M, CIITA, TRAC, and TRB. In certain embodiments, the genome of T cells (e.g., T cells differentiated from hypoimmunogenic iPSCs and primary T cells) is altered to regulatably reduce or delete key components of HLA and TCR expression, such as HLA-A antigens, HLA-B antigens, HLA-C antigens, HLA-DP antigens, HLA-DQ antigens, HLA-DR antigens, TCR alpha, and TCR beta.

[0452] In some embodiments, the present disclosure provides a cell or a population thereof comprising a genome, in which a gene is regulatably edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating the surface expression of one or more MHC class I molecules in the cell or a population thereof. In certain embodiments, the present disclosure provides a cell or a population thereof comprising a genome, in which a gene is regulatably edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating the surface expression of one or more MHC class II molecules in the cell or a population thereof. In certain embodiments, the present disclosure provides a cell or a population thereof comprising a genome, in which a gene is regulatably edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating the surface expression of TCR molecules in the cell or a population thereof. In numerous embodiments, the disclosure provides a cell or population thereof comprising a genome in which one or more genes have been controllably edited to delete contiguous stretches of genomic DNA, thus reducing or eliminating surface expression of one or more MHC class I and II molecules, and TCR complex molecules in the cell or population thereof.

[0453] In some embodiments, the cells and methods described herein include adjustably editing a human cell to cleave a CIITA gene sequence and adjustably editing the genome of such a cell to alter one or more additional target polynucleotide sequences, the one or more additional target polynucleotide sequences being, for example, but not limited to, B2M, TRAC, and TRB. In some embodiments, the cells and methods described herein include adjustably editing a human cell to cleave a B2M gene sequence and edit the genome of such a cell to alter one or more additional target polynucleotide sequences, the one or more additional target polynucleotide sequences being, for example, but not limited to, CIITA, TRAC, and TRB. In some embodiments, the cells and methods described herein include adjustably editing a human cell to cleave a TRAC gene sequence and edit the genome of such a cell to alter one or more additional target polynucleotide sequences, the one or more additional target polynucleotide sequences being, for example, but not limited to, B2M, CIITA, and TRB. In some embodiments, the cells and methods described herein include regulatably editing a human cell's genome to cleave a TRB gene sequence, and editing the genome of such a cell to alter one or more additional target polynucleotide sequences, such as, but not limited to, B2M, CIITA, and TRAC.

[0454] Provided herein is a hypoimmunogenic stem cell comprising: i) a regulatable reduced expression of HLA-A, HLA-B, HLA-C, CIITA, TCR alpha, and TCR beta relative to a wild-type stem cell, wherein the regulatable reduced expression is by an RNA-based component, a DNA-based component, or a protein-based component; and ii) a set of exogenous genes comprising a first regulatable gene encoding one or more tolerogenic factors and a second regulatable gene encoding a chimeric antigen receptor (CAR), wherein the first regulatable gene and / or the second regulatable gene is inserted within a specific locus of at least one allele of the cell. Also provided herein is a hypoimmunogenic primary T cell, including a primary T cell of any subtype, comprising: i) a regulatable, reduced expression of HLA-A, HLA-B, HLA-C, CIITA, TCR alpha, and TCR beta relative to a wild-type primary T cell, wherein the regulatable, reduced expression is by an RNA-based component, a DNA-based component, or a protein-based component; and ii) a set of exogenous genes comprising a first regulatable gene encoding one or more tolerogenic factors and a second regulatable gene encoding a chimeric antigen receptor (CAR), wherein the first regulatable gene and / or the second regulatable gene is inserted within a specific locus of at least one allele of the cell.Further provided herein is a hypoimmunogenic T cell differentiated from a hypoimmunogenic induced pluripotent stem cell, comprising: i) a regulatable, reduced expression of HLA-A, HLA-B, HLA-C, CIITA, TCR alpha, and TCR beta relative to a wild-type primary T cell, wherein the regulatable, reduced expression is by an RNA-based component, a DNA-based component, or a protein-based component; and ii) a set of exogenous genes comprising a first regulatable gene encoding one or more tolerogenic factors and a second regulatable gene encoding a chimeric antigen receptor (CAR), wherein the first regulatable gene and / or the second regulatable gene is inserted within a specific locus of at least one allele of the cell.

[0455] In some embodiments, the population of engineered cells described avoid NK cell-mediated cytotoxicity upon administration to a recipient patient. In some embodiments, the population of engineered cells avoid NK cell-mediated cytotoxicity by one or more subpopulations of NK cells. In some embodiments, the population of engineered cells are protected from cytolysis by NK cells, including immature and / or mature NK cells, upon administration to a recipient patient. In some embodiments, the population of engineered cells avoid macrophage phagocytosis upon administration to a recipient patient. In some embodiments, the population of engineered cells do not induce an innate and / or adaptive immune response against the cells upon administration to a recipient patient. In some embodiments, the population of engineered cells avoid NK cell-mediated cytotoxicity by one or more subpopulations of NK cells, as determined by in vitro or in vivo assays. In some embodiments, the population of engineered cells are protected from cytolysis by NK cells, including immature and / or mature NK cells, upon administration to a recipient patient, as determined by in vitro or in vivo assays. In some embodiments, the population of engineered cells avoids macrophage phagocytosis upon administration to a recipient patient as determined by in vitro or in vivo assays, hi some embodiments, the population of engineered cells do not induce an innate and / or adaptive immune response to the cells upon administration to a recipient patient as determined by in vitro or in vivo assays.

[0456] In some embodiments, the cells described herein comprise a safety switch. The term "safety switch" as used herein refers to a system for controlling expression of a gene or protein of interest that, when down-regulated or up-regulated, results in cell clearance or death, for example, via recognition by the host's immune system. The safety switch can be designed to be triggered by an exogenous molecule upon an adverse clinical event. The safety switch can be manipulated by controlling expression at the DNA, RNA, and protein levels. The safety switch includes proteins or molecules that allow for control of cellular activity in response to an adverse event. In one embodiment, the safety switch is a "kill switch" that is expressed in an inactivated state, and activation of the switch by an externally provided selective agent is lethal to the cell expressing the safety switch. In one embodiment, the safety switch gene is cis-acting relative to the gene of interest in the construct. Activation of the safety switch causes the cell to kill only itself, or itself and neighboring cells by apoptosis or necrosis. In some embodiments, the cells described herein, e.g., stem cells, induced pluripotent stem cells, hematopoietic stem cells, primary cells, or differentiated cells, including but not limited to cardiac cells, cardiac progenitor cells, neuronal cells, glial progenitor cells, endothelial cells, T cells, B cells, pancreatic islet cells, retinal pigment epithelial cells, liver cells, thyroid cells, skin cells, blood cells, plasma cells, platelets, kidney cells, epithelial cells, CAR-T cells, NK cells, and / or CAR-NK cells, comprise a safety switch.

[0457] In some embodiments, the safety switch comprises a therapeutic agent that inhibits or blocks the interaction of CD47 and SIRPα. In some aspects, the CD47-SIRPα blocking agent is an agent that neutralizes, blocks, antagonizes, or interferes with cell surface expression of CD47, SIRPα, or both. In some embodiments, the CD47-SIRPα blocking agent inhibits or blocks the interaction of CD47, SIRPα, or both. In some embodiments, the CD47-SIRPα blocking agent (e.g., a CD47-SIRPα blocking, inhibiting, reducing, antagonizing, neutralizing, or interfering agent) comprises an agent selected from the group including an antibody or fragment thereof that binds CD47, a bispecific antibody that binds CD47, an immunocytokine fusion protein that binds CD47, a CD47-containing fusion protein, an antibody or fragment thereof that binds SIRPα, a bispecific antibody that binds SIRPα, an immunocytokine fusion protein that binds SIRPα, a SIRPα-containing fusion protein, and combinations thereof.

[0458] In some embodiments, the cells described herein contain a "suicide gene" (or "suicide switch"). The suicide gene can cause the death of hypoimmunogenic cells if they are allowed to grow and divide in an undesired manner. The "suicide gene" ablation approach includes a suicide gene in a gene transfer vector that encodes a protein that results in cell killing only when activated by a specific compound. The suicide gene may encode an enzyme that selectively converts non-toxic compounds into highly toxic metabolites. In some embodiments, the cells described herein, such as stem cells, induced pluripotent stem cells, hematopoietic stem cells, primary cells, or differentiated cells, including but not limited to cardiac cells, cardiac progenitor cells, neuronal cells, glial progenitor cells, endothelial cells, T cells, B cells, pancreatic islet cells, retinal pigment epithelial cells, hepatocytes, thyroid cells, skin cells, blood cells, plasma cells, platelets, kidney cells, epithelial cells, CAR-T cells, NK cells, and / or CAR-NK cells, contain a suicide gene.

[0459] In some embodiments, the population of engineered cells described induces reduced levels of immune activation or no immune activation upon administration to a recipient subject. In some embodiments, the cells induce reduced levels of systemic TH1 activation or no systemic TH1 activation in the recipient subject. In some embodiments, the cells induce reduced levels of peripheral blood mononuclear cell (PBMC) immune activation or no PBMC immune activation in the recipient subject. In some embodiments, the cells induce reduced levels of donor-specific IgG antibodies against the cells or no donor-specific IgG antibodies upon administration to the recipient subject. In some embodiments, the cells induce reduced levels of IgM and IgG antibody production against the cells or no IgM and IgG antibody production in the recipient subject. In some embodiments, the cells induce reduced levels of cytotoxic T cell killing of the cells upon administration to the recipient subject.

[0460] B. Conditional HIP Cells and Methods for Conditional Downregulation of Target Genes The introduction of regulatable reduced expression of target genes improves the safety of cell therapies developed using hypoimmunogenic cells (HIP cells). In some embodiments, the regulatable reduced expression of target genes allows to avoid potential difficulties when differentiating cells from pluripotent stem cells. In some embodiments, the regulatable reduced expression of target genes includes regulatable reduced expression, e.g., regulatable knockout or knockdown, of B2M, CIITA, NLRC5, TRAC, TRB, CD142, ABO, MIC-A / B, CD38, CD52, PCDH11Y, NLGN4Y and / or RHD. The regulatable reduced expression of one or more of the target genes functions to control the innate and / or adaptive immune response by the recipient subject to the engrafted hypoimmunogenic cells.

[0461] Described herein is a method for reducing expression of target genes, which involves a mechanism for turning off the expression of target genes in a controlled manner.Also described is a HIP cell with regulatable reduced expression of one or more target genes.In some cases, cells can be induced to knock out or knock down the expression of one or more target genes.

[0462] In some embodiments, hypoimmunity of cells introduced into a recipient subject is achieved through overexpression of immunosuppressive molecules, including hypoimmune factors and complement inhibitors, with suppression or genetic disruption of HLA-I and HLA-II loci. These modifications hide the cells from effector cells of the recipient immune system, such as T cells, B cells, NK cells, and macrophages, which are involved in the clearance of infected, malignant, or non-self cells. Hiding the cells from the immune system allows the presence and persistence of allogeneic cells in the body. The expression level of any of the described immunosuppressive molecules can be controlled in cells at the protein, mRNA, or DNA level. Similarly, the expression level of any of the described immune signaling molecules can be controlled in cells at the protein, mRNA, or DNA level.

[0463] In some embodiments, any of the regulatable reduced expression methods described (e.g., RNA-level, DNA-level, and protein-level methods) are used to reduce the level of a target protein in a cell such that the lower level of the target protein is below a threshold level. In some embodiments, the level of the target protein in a cell is reduced by about 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, 1-fold, or 0.5-fold below a threshold level of expression. In some embodiments, the level of the target protein in a cell is reduced by about 10-fold to 5-fold, 10-fold to 3-fold, 9-fold to 1-fold, 8-fold to 1-fold, 7-fold to 0.5-fold, 6-fold to 1-fold, 5-fold to 0.5-fold, 4-fold to 0.5-fold, 3-fold to 0.5-fold, 2-fold to 0.5-fold, or 1-fold to 0.5-fold below a threshold level of expression. In some embodiments, a threshold level of expression of a target protein is established based on the expression of such factors in induced pluripotent stem cells. In some embodiments, the threshold level of target protein expression is established based on the expression level of the target protein in corresponding hypoimmune cells, e.g., MHC I and MHC II knockout cells, or MHC I / MHC II / TCR knockout cells.

[0464] 1. RNA-based components Target gene can be targeted by shRNA, siRNA or miRNA, thereby causing the degradation of the transcript that codes for the factor.shRNA, siRNA or miRNA can be exogenously provided or genetically encoded to provide control over the transcription of inhibitory RNA.shRNA, siRNA or miRNA can anneal to the transcript of target gene, causing degradation by RISC complex.

[0465] In some embodiments, methods for inducible RNA regulation to downregulate expression of target genes include, but are not limited to, conditional or inducible shRNA, conditional or inducible siRNA, conditional or inducible miRNA, conditional or inducible CRISPR interference (CRISPRi), and conditional or inducible RNA-targeted nucleases.

[0466] In some embodiments, the method includes shRNA, siRNA or miRNA targeting the RNA of the target gene. In some cases, the expression of shRNA, siRNA or miRNA is induced by small molecules or biological agents. In some cases, the expression of shRNA, siRNA or miRNA is induced by cell conditions.

[0467] In some embodiments, a method is provided for controlling the immunogenicity of mammalian cells (e.g., human cells) by obtaining isolated cells and introducing a construct containing a conditional or inducible RNA polymerase promoter, the conditional or inducible RNA polymerase promoter being operably linked to an shRNA, siRNA or miRNA sequence targeting a target gene, the shRNA, siRNA or miRNA sequence being operably linked to a constitutive promoter, the constitutive promoter being operably linked to a transactivator element, and the transactivator ...

Claims

1. An engineered cell comprising: i) a modification that reduces the expression of one or more MHC class I and / or MHC class II molecules; and ii) a modification that increases the expression of CD47, relative to a control, wherein the engineered cell expresses CD47 at or above a threshold level.

2. 2. The engineered cell of claim 1, wherein the engineered cell is selected from the group consisting of stem cells, pluripotent stem cells (PSCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), embryonic stem cells (ESCs), pancreatic islet cells, beta islet cells, immune cells, B cells, T cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophage cells, immune privileged cells, optic nerve cells, retinal pigment epithelial cells (RPE), hepatocytes, thyroid cells, endothelial cells, skin cells, glial progenitor cells, nerve cells, muscle cells, cardiac cells, and blood cells. (i) the cells express at least about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800% or 900% greater amount of CD47 than the control; (ii) the cells express at least about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, or about 5-fold the level of CD47 expressed in the control; or (iii) the cells express at least about 16-fold, about 17-fold, about 18-fold, about 19-fold, or about 20-fold the level of CD47 expressed in the control; The engineered cell of claim 1 .

4. 2. The engineered cell of claim 1, wherein the control is a wild-type cell, a control cell, or a baseline reference.

5. 5. The engineered cell of claim 4, wherein the baseline is an isotype control and the CD47 level is determined using an antibody-based assay. (i) the engineered cells are beta islet cells that express at least about 200,000, 250,000, 300,000, 350,000, or 400,000 CD47 molecules per cell; (ii) the engineered cells are retinal pigment epithelial cells that express at least about a 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold or more increase in CD47 expression relative to baseline; or (iii) the engineered cells are T cells that express at least about 180,000, 190,000, 200,000, 210,000, 220,000, 230,000, 240,000, 250,000, 260,000, 270,000, 280,000, 290,000, 300,000, 350,000, 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, or 700,000 CD47 molecules per cell; The engineered cell of claim 4.

7. The engineered cell of claim 1 , wherein the cell does not express one or more MHC class I molecules and / or one or more MHC class II molecules relative to a control.

8. 2. The engineered cell of claim 1, wherein the MHC class I molecule and MHC class II molecule are selected from B2M, TAP I, NLRC5, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, RFX5, RFXANK, RFXAP, NFY-A, NFY-B, and / or NFY-C. (i) the cell is a differentiated cell derived from a stem cell or its progeny; or (ii) the cell is derived from a primary cell or its progeny; The engineered cell of claim 1 .

10. The engineered cell of claim 1, comprising one or more tunable modifications to alter expression of CD47 in the engineered cell relative to a control.

11. The one or more adjustable modifications include: (a) a conditional or inducible RNA-based component for i) increasing, or ii) reducing or knocking out, expression of said one or more targets relative to a control; (b) a conditional or inducible DNA-based construct for i) increasing, or ii) reducing or knocking out, expression of said one or more targets relative to a control; or (c) a conditional or inducible protein-based component for i) increasing expression of said one or more targets, or ii) reducing or knocking it out, relative to a control; 11. The engineered cell of claim 10, comprising:

12. The cells (i) a conditional promoter operably linked to an exogenous polynucleotide encoding CD47, or (ii) a conditional promoter operably linked to an exogenous polynucleotide encoding CD47.

12. The engineered cell of claim 11, comprising:

13. 13. The engineered cell of claim 12, wherein the conditional promoter is selected from a cell cycle-specific promoter, a tissue-specific promoter, a lineage-specific promoter, and a differentiation-induced promoter.

14. 13. The engineered cell of claim 12, wherein the inducible promoter is regulated by a small molecule, a ligand, a biological agent, an aptamer-mediated regulator of polyadenylation, or an aptamer-regulated riboswitch.

15. A pharmaceutical composition comprising the population of engineered cells of any one of claims 1 to 14 and a pharmaceutically acceptable excipient, carrier, diluent or excipient.

16. 15. Use of the population of engineered cells of any one of claims 1 to 14 to treat a disorder or condition in a recipient patient that would benefit from cell-based therapy.

17. 1. A method for producing an engineered cell, wherein the engineered cell comprises: i) a regulatable modification that reduces expression of one or more MHC class I and / or MHC class II molecules; and ii) a regulatable modification that increases expression of one or more tolerogenic factors relative to a control, the method comprising: (a) introducing into a cell a conditional or inducible RNA-based construct for regulatable, reduced expression of MHC class I and / or MHC class II human leukocyte molecules, a conditional or inducible DNA-based construct for regulatable, reduced expression of MHC class I and / or MHC class II human leukocyte molecules, or a conditional or inducible protein-based construct for regulatable, reduced expression of MHC class I and / or MHC class II human leukocyte molecules; (b) exposing the cell to a condition or exogenous factor to activate the conditional or inducible component, thereby causing reduced expression of the MHC class I and / or MHC class C molecules; (c) introducing into the isolated cells a nucleic acid comprising a conditional or inducible promoter operably linked to an exogenous polynucleotide encoding the one or more tolerogenic factors for regulatable increased expression of the one or more tolerogenic factors; (d) inducing the engineered cell to activate the conditional or inducible promoter. and exposing the engineered cells to conditions or exogenous factors to induce expression of one or more exogenous tolerogenic factors, thereby producing the engineered cells.

18. 15. A method for identifying a population of cells or a population of engineered cells according to any one of claims 1 to 14, which is suitable for use as a therapeutic product, comprising: (a) introducing into the isolated cell one or more modifications that reduce expression of one or more MHC class I and / or MHC class II molecules relative to a control; (b) introducing into said cells one or more modifications that increase expression of CD47 relative to a control; (c) measuring the expression level of the CD47 in the cells; (d) selecting a population of cells expressing at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% greater than a control, and identifying said population as suitable for use as a therapeutic product.

19. 19. The method of claim 18, wherein the control is a wild-type cell, a control cell, or a baseline reference.

20. 20. The method of claim 19, wherein the control cell is an unmodified or unaltered cell, optionally the unmodified or unaltered cell is of the same cell type as the engineered cell.

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