Altered expression of Y-chromosome-linked antigens in poorly immunogenic cells.

Abstract

Disclosed herein are engineered cells and / or hypoimmunogenic cells, including engineered cells and / or hypoimmunogenic stem cells, engineered cells and / or hypoimmunogenic cells differentiated therefrom, and engineered cells and / or hypoimmunogenic CAR-T cells (primary, or differentiated from engineered and / or hypoimmunogenic stem cells), comprising reduced expression of one or more Y chromosome genes and reduced expression of MHC I and / or MHC II human leukocyte antigen molecules, and overexpression of CD47, and related methods of use and production thereof. 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 Application No. 63 / 221,887, filed July 14, 2021, and U.S. Provisional Application No. 63 / 255,914, filed October 14, 2021, the disclosures of each of which are incorporated herein by reference in their entireties. [Background technology]

[0002] Off-the-shelf CAR-T cells and other therapeutic cells may offer advantages over autologous cell-based strategies, including ease of manufacturing, quality control, and avoidance of harmful contamination and T cell dysfunction. However, robust host-versus-graft immune responses against histo-incompatible T cells hinder the proliferation and persistence of allogeneic CAR-T cells, reducing the effectiveness 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. Summary of the Invention

[0005] In some embodiments, provided herein are engineered cells that comprise reduced expression of one or more Y chromosome genes and major histocompatibility complex (MHC) class I and / or class II human leukocyte antigen molecules relative to an unchanged or unmodified wild-type or control cell, and that comprise a first exogenous polynucleotide encoding CD47, wherein the engineered cells are expanded from primary T cells or their progeny, or are derived from induced pluripotent stem cells (iPSCs) or their progeny.

[0006] In some embodiments, provided herein are hypoimmunogenic T cells that comprise reduced expression of one or more Y chromosome genes and MHC class I and / or MHC class II human leukocyte antigen molecules relative to an unchanged or unmodified wild-type or control cell, and that comprise a first exogenous polynucleotide encoding CD47, wherein the hypoimmunogenic T cells are expanded from primary T cells or their progeny or derived from iPSCs or their progeny.

[0007] In some embodiments, provided herein are resting T cells that comprise reduced expression of one or more Y chromosome genes and MHC class I and / or MHC class II human leukocyte antigen molecules relative to an unchanged or unmodified wild-type or control cell, and that comprise a first exogenous polynucleotide encoding CD47, wherein the resting T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

[0008] In some embodiments, provided herein is a pancreatic islet cell comprising reduced expression of one or more Y chromosome genes and MHC class I and / or MHC class II human leukocyte antigen molecules relative to an unchanged or unmodified wild-type or control cell, and comprising a first exogenous polynucleotide encoding CD47, wherein the pancreatic islet cell is derived from an iPSC or a progeny thereof.

[0009] In some embodiments, provided herein are cardiomyocytes comprising reduced expression of one or more Y chromosome genes and MHC class I and / or MHC class II human leukocyte antigen molecules relative to an unchanged or unmodified wild-type or control cell, and comprising a first exogenous polynucleotide encoding CD47, wherein the cardiomyocytes are derived from iPSCs or progeny thereof.

[0010] In some embodiments, provided herein are glial progenitor cells that comprise reduced expression of one or more Y chromosome genes and MHC class I and / or MHC class II human leukocyte antigen molecules relative to an unchanged or unmodified wild-type or control cell, and that comprise a first exogenous polynucleotide encoding CD47, and wherein the cardiomyocytes are derived from iPSCs or progeny thereof.

[0011] In some embodiments, provided herein are NK cells comprising reduced expression of one or more Y chromosome genes and MHC class I and / or MHC class II human leukocyte antigen molecules relative to an unchanged or unmodified wild-type or control cell, and comprising a first exogenous polynucleotide encoding CD47, wherein the cardiomyocytes are derived from iPSCs or progeny thereof.

[0012] In some embodiments, the Y chromosome gene is a Y chromosome-linked antigen or a Y chromosome-associated minor histocompatibility antigen.

[0013] In some embodiments, the one or more Y chromosome-linked antigens are protocadherin-11 Y-linked and / or neuroligin-4 Y-linked.

[0014] In some embodiments, the cells have reduced expression of protocadherin-11 Y-linked.

[0015] In some embodiments, the cells have reduced expression of neuroligin-4 Y-linked.

[0016] In some embodiments, the cells have reduced expression of protocadherin-11 Y-chain and reduced expression of neuroligin-4 Y-chain.

[0017] In some embodiments, the cells are genetically engineered to have reduced expression of protocadherin-11 Y-linked and / or neuroligin-4 Y-linked.

[0018] In some embodiments, the cells do not express protocadherin-11 Y-linked.

[0019] In some embodiments, the cells do not express neuroligin-4 Y-linked.

[0020] In some embodiments, the cells do not express protocadherin-11 Y-chain and do not express neuroligin-4 Y-chain.

[0021] In some embodiments, the cells are genetically engineered not to express protocadherin-11 Y-chain and / or neuroligin-4 Y-chain.

[0022] In some embodiments, reduced protocadherin-11 Y-linked and / or neuroligin-4 Y-linked expression is caused by knockout of the PCDH11Y and / or NLGN4Y genes, respectively.

[0023] In some embodiments, the cells are derived from human or animal cells.

[0024] In some embodiments, the human or animal cells are from a donor subject that does not have a Y chromosome.

[0025] In some embodiments, the human or animal cells are from a donor subject that has a Y chromosome, and the cells are genetically engineered to have reduced expression of protocadherin-11 Y-linked and / or neuroligin-4 Y-linked.

[0026] In some embodiments, the cells are genetically engineered not to express protocadherin-11 Y-linked.

[0027] In some embodiments, the cells are genetically engineered not to express neuroligin-4 Y-linked.

[0028] In some embodiments, the cells are genetically engineered to not express protocadherin-11 Y-chain and not express neuroligin-4 Y-chain.

[0029] In some embodiments, the cells are expanded or derived from a pool of cells isolated from one or more donor subjects different from the patient, where the one or more donor subjects optionally include one or more subjects with a Y chromosome, one or more subjects without a Y chromosome, or a mixture of subjects with and without a Y chromosome.

[0030] In some embodiments, the cells are genetically engineered to have reduced expression of protocadherin-11 Y-linked and / or neuroligin-4 Y-linked using CRISPR / Cas gene editing.

[0031] In some embodiments, CRISPR / Cas gene editing is performed using one or more guide RNAs comprising any of the sequences in Tables 2-5.

[0032] In some embodiments, CRISPR / Cas gene editing is performed using a Cas effector protein selected from the group consisting of Cas9, Cas12a, and Cas12b.

[0033] In some embodiments, CRISPR / Cas gene editing is performed using a Cas effector protein selected from the group consisting of: (a) optionally selected from the group consisting of Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, and GSU0054; (b) optionally selected from the group consisting of Cas9, Csn2, and Cas4; (c) optionally selected from the group consisting of Cas10, Csm 2, Cmr5, Cas10, Csx11, and Csx10; (d) optionally Csf1; (e) optionally selected from the group consisting of Cas12a, Cas12b, Cas12c, C2c4, C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d); and (f) optionally selected from the group consisting of Cas13, Cas13a, C2c2, Cas13b, Cas13c, and Cas13d.

[0034] In some embodiments, CRISPR / Cas gene editing is performed ex vivo from a donor subject.

[0035] In some embodiments, CRISPR / Cas gene editing is performed using a lentiviral vector.

[0036] In some embodiments, the cells comprise reduced expression of beta-2-microglobulin (B2M) and / or MHC class II transactivator (CIITA) compared to unaltered or unmodified wild-type or control cells.

[0037] In some embodiments, the cells do not express B2M and / or CIITA.

[0038] In some embodiments, the cells comprise reduced expression of RHD.

[0039] In some embodiments, the cells do not express RHD.

[0040] In some embodiments, the cell is a differentiated cell derived from an induced pluripotent stem cell or its progeny.

[0041] In some embodiments, the differentiated cells are selected from the group consisting of T cells, NK cells, endothelial cells, pancreatic islet cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, hepatocytes, glial progenitor cells, dopaminergic neurons, retinal pigment epithelial cells, and thyroid cells.

[0042] In some embodiments, the cell is a primary immune cell or its progeny.

[0043] In some embodiments, the primary immune cell or its progeny is a T cell or an NK cell.

[0044] In some embodiments, the cells comprise reduced expression of TCR-alpha and / or TCR-beta.

[0045] In some embodiments, the cells do not express TCR-alpha and / or TCR-beta.

[0046] In some embodiments, the cells further comprise a second exogenous polynucleotide encoding one or more chimeric antigen receptors (CARs), wherein the one or more CARs comprise an extracellular ligand-binding domain with specificity for CD19, CD20, CD22, or BCMA, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

[0047] In some embodiments, one or more CARs comprise a CD8α hinge domain, a CD28 hinge domain, or an IgG4 hinge domain.

[0048] In some embodiments, the one or more CARs comprise a CD8α hinge domain having the amino acid sequence of SEQ ID NO:9.

[0049] In some embodiments, one or more CARs comprise a CD28 hinge domain having the amino acid sequence of SEQ ID NO: 10 or 113.

[0050] In some embodiments, one or more CARs comprise an IgG4 hinge domain having the amino acid sequence of SEQ ID NO: 11 or 12.

[0051] In some embodiments, one or more CARs comprise a CD8α transmembrane domain or a CD28 transmembrane domain.

[0052] In some embodiments, the one or more CARs comprise a CD8α transmembrane domain having the amino acid sequence of SEQ ID NO: 14.

[0053] In some embodiments, the one or more CARs comprise a CD28 transmembrane domain having the amino acid sequence of SEQ ID NO: 15 or 114.

[0054] In some embodiments, one or more CARs comprise a 4-1BB costimulatory domain, a CD28 costimulatory domain, or a CD3ζ signaling domain.

[0055] In some embodiments, one or more CARs comprise a 4-1BB costimulatory domain having the amino acid sequence of SEQ ID NO:16.

[0056] In some embodiments, one or more CARs comprise a CD28 costimulatory domain having the amino acid sequence of SEQ ID NO: 17.

[0057] In some embodiments, the one or more CARs comprise a CD3ζ signaling domain having the amino acid sequence of SEQ ID NO: 18 or 115.

[0058] In some embodiments, one or more CARs comprise an extracellular ligand-binding domain comprising an scFv sequence of any one of SEQ ID NOs: 19, 37, 45, 54, 63, 72, 81, or 118, or and CAR has an scFv sequence comprising heavy and light chain sequences of any one of SEQ ID NOs: 20, 25, 38, 42, 46, 50, 64, 68, 73, 77, 119, or 123.

[0059] In some embodiments, one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, 117, or 128.

[0060] In some embodiments, the one or more CARs comprise the amino acid sequence set forth in SEQ ID NO: 117, or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 117, and have the following components: a CD8α signal peptide, an FMC63 scFv (VL-Whitlow linker-VH), a CD8α hinge domain, a CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain.

[0061] In some embodiments, one or more CARs comprise the amino acid sequence set forth in SEQ ID NO: 45 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 45.

[0062] In some embodiments, one or more of 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.

[0063] In some embodiments, the first specific locus and / or the second specific locus is selected from the group consisting of a safe harbor or target locus, a RHD locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus.

[0064] In some embodiments, the safe harbor or target locus is selected from the group consisting of the CCR5 locus, the CXCR4 locus, the PPP1R12C locus, the ALB 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 FUT1 locus, and the KDM5D locus.

[0065] In some embodiments, the first exogenous polynucleotide and / or the second exogenous polynucleotide is introduced into the cell using a gene therapy vector or a transposase system selected from the group consisting of a transposase, a PiggyBac transposon, a Sleeping Beauty (SB11) transposon, a Mos1 transposon, and a Tol2 transposon.

[0066] In some embodiments, the gene therapy vector is a retrovirus or a fusosome.

[0067] In some embodiments, the retrovirus is a lentiviral vector.

[0068] In some embodiments, the first exogenous polynucleotide and / or the second exogenous polynucleotide is introduced into the cell using CRISPR / Cas gene editing.

[0069] In some embodiments, CRISPR / Cas gene editing is performed using a Cas effector protein selected from the group consisting of Cas9, Cas12a, and Cas12b.

[0070] In some embodiments, CRISPR / Cas gene editing is performed using a Cas effector protein selected from the group consisting of: (a) optionally selected from the group consisting of Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, and GSU0054; (b) optionally selected from the group consisting of Cas9, Csn2, and Cas4; (c) optionally selected from the group consisting of Cas10, Csm 2, Cmr5, Cas10, Csx11, and Csx10; (d) optionally Csf1; (e) optionally selected from the group consisting of Cas12a, Cas12b, Cas12c, C2c4, C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d); and (f) optionally selected from the group consisting of Cas13, Cas13a, C2c2, Cas13b, Cas13c, and Cas13d.

[0071] In some embodiments, CRISPR / Cas gene editing is performed ex vivo from a donor subject.

[0072] In some embodiments, CRISPR / Cas gene editing is performed using a lentiviral vector.

[0073] In some embodiments, the cells or progeny thereof avoid NK cell-mediated cytotoxicity upon administration to a patient.

[0074] In some embodiments, the cells or progeny thereof are protected from cytolysis by mature NK cells upon administration to a patient.

[0075] In some embodiments, the cells or progeny thereof avoid phagocytosis by macrophages upon administration to a patient.

[0076] In some embodiments, the cells or their progeny do not induce an immune response against the cells when administered to a patient.

[0077] In some embodiments, the cells or their progeny do not induce an antibody-based immune response against the cells upon administration to a patient.

[0078] In some embodiments, wild-type or control cells are the starting material.

[0079] In some embodiments, provided herein are pharmaceutical compositions comprising a population of engineered cells, hypoimmunogenic T cells, resting T cells, pancreatic islet cells, cardiomyocytes, glial progenitor cells, or NK cells described herein, and a pharmaceutically acceptable additive, carrier, diluent, or excipient.

[0080] In some embodiments, the composition comprises one or more populations of cells selected from the group consisting of a population of hypoimmunogenic T cells, a population of resting T cells, a population of hypoimmunogenic CD19 CAR T cells, and a population of hypoimmunogenic CD22 CAR T cells, and a pharmaceutically acceptable additive, carrier, diluent, or excipient.

[0081] In some embodiments, provided herein are methods of treating a patient having a disease or condition that would benefit from a cell-based therapy, the method comprising administering to the patient a population of engineered cells, hypoimmunogenic T cells, resting T cells, pancreatic islet cells, cardiomyocytes, glial progenitor cells, or NK cells as described herein.

[0082] In some embodiments, the patient does not have a Y chromosome.

[0083] In some embodiments, the patient is not sensitized to a Y chromosome gene.

[0084] In some embodiments, the patient is sensitized to a Y chromosome gene.

[0085] In some embodiments, the patient has previously received cell therapy derived from a donor subject that has a Y chromosome or otherwise expressed one or more of the Y chromosome genes.

[0086] In some embodiments, the patient is a female patient who has previously been pregnant with a male infant.

[0087] In some embodiments, provided herein are methods of treating cancer in a patient in need thereof, the methods comprising administering to the patient a population of primary immune cells disclosed herein.

[0088] In some embodiments, the primary immune cells are selected from the group consisting of T cells and NK cells.

[0089] In some embodiments, the patient does not have a Y chromosome.

[0090] In some embodiments, the patient is not sensitized to a Y chromosome gene.

[0091] In some embodiments, the patient is sensitized to a Y chromosome gene.

[0092] In some embodiments, the patient has previously received cell therapy derived from a donor subject that has a Y chromosome or otherwise expressed one or more of the Y chromosome genes.

[0093] In some embodiments, the patient is a female patient who has previously been pregnant with a male infant.

[0094] In some embodiments, provided herein are methods for determining an appropriate cell-based therapy to administer to a patient having a disease or condition that would benefit from a cell-based therapy, the method comprising: (a) determining whether a biological sample from the patient contains antibodies to one or more Y chromosome genes by: (i) obtaining or obtaining the biological sample from the patient; (ii) performing or having performed an assay to determine whether antibodies to protocadherin-11 Y chain are present in the biological sample; and (iii) performing or having performed an assay to determine whether antibodies to neuroligin-4 Y chain are present in the biological sample; and (b) administering a population of engineered cells, hypoimmunogenic T cells, resting T cells, pancreatic islet cells, cardiomyocytes, glial progenitor cells, or NK cells as described herein; wherein (i) if antibodies to protocadherin-11 Y chain are present in the biological sample, the population of cells comprises reduced expression of protocadherin-11 Y chain, and (ii) if antibodies to neuroligin-4 Y chain are present in the biological sample, the population of cells comprises reduced expression of neuroligin-4 Y chain. (iii) if antibodies to the protocadherin-11 Y chain and antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain and reduced expression of the neuroligin-4 Y chain; and (iv) if neither antibodies to the protocadherin-11 Y chain nor antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain or reduced expression of the neuroligin-4 Y chain.

[0095] In some embodiments, provided herein are methods of identifying a patient having a disease or condition that would benefit from a cell-based therapy comprising reduced expression of one or more Y chromosome genes, the method comprising: (a) determining whether a biological sample from the patient contains antibodies to one or more Y chromosome genes by: (i) obtaining or having obtained the biological sample from the patient; (ii) performing or having performed an assay to determine whether antibodies to protocadherin-11 Y chain are present in the biological sample; and (iii) performing or having performed an assay to determine whether antibodies to neuroligin-4 Y chain are present in the biological sample; and (b) administering to the patient a population of engineered cells, hypoimmunogenic T cells, resting T cells, pancreatic islet cells, cardiomyocytes, glial progenitor cells, or NK cells as described herein; wherein (i) if antibodies to protocadherin-11 Y chain are present in the biological sample, the population of cells comprises reduced expression of protocadherin-11 Y chain and (ii) neuroligin-4 Y chain. If antibodies to the Y chain are present in the biological sample, the population of cells contains reduced expression of the neuroligin-4 Y chain; (iii) if antibodies to the protocadherin-11 Y chain and antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain and reduced expression of the neuroligin-4 Y chain; and (iv) if neither antibodies to the protocadherin-11 Y chain nor antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain or reduced expression of the neuroligin-4 Y chain.

[0096] In some embodiments, provided herein are methods for identifying a patient having a disease or condition that would benefit from a cell-based therapy comprising reduced expression of protocadherin-11 Y chain and / or reduced expression of neuroligin-4 Y chain, the method comprising: (a) determining whether a biological sample from the patient contains antibodies to protocadherin-11 Y chain and / or antibodies to neuroligin-4 Y chain by (i) obtaining or obtaining the biological sample from the patient, (ii) performing or having performed an assay to determine whether antibodies to protocadherin-11 Y chain are present in the biological sample, and (iii) performing or having performed an assay to determine whether antibodies to neuroligin-4 Y chain are present in the biological sample; and (b) administering to the patient a population of engineered cells, hypoimmunogenic T cells, resting T cells, pancreatic islet cells, cardiomyocytes, glial progenitor cells, or NK cells as described herein; If antibodies to the Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain; (ii) if antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the neuroligin-4 Y chain; (iii) if antibodies to the protocadherin-11 Y chain and antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain and reduced expression of the neuroligin-4 Y chain; and (iv) if neither antibodies to the protocadherin-11 Y chain nor antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain or reduced expression of the neuroligin-4 Y chain.

[0097] In some embodiments, provided herein are methods for determining whether a cell-based therapy that does not comprise reduced expression of protocadherin-11 Y chain and / or reduced expression of neuroligin-4 Y chain is susceptible to NK-mediated cytotoxicity upon administration to a patient, the method comprising: (a) determining whether a biological sample from the patient contains antibodies to protocadherin-11 Y chain and / or antibodies to neuroligin-4 Y chain by (i) obtaining or obtaining the biological sample from the patient, (ii) performing or having performed an assay to determine whether antibodies to protocadherin-11 Y chain are present in the biological sample, and (iii) performing or having performed an assay to determine whether antibodies to neuroligin-4 Y chain are present in the biological sample; and (b) administering to the patient a population of engineered cells, hypoimmunogenic T cells, resting T cells, pancreatic islet cells, cardiomyocytes, glial progenitor cells, or NK cells as described herein, wherein (i) the engineered cells contain antibodies to protocadherin-11 Y chain and / or neuroligin-4 Y chain. If antibodies to the Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain; (ii) if antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the neuroligin-4 Y chain; (iii) if antibodies to the protocadherin-11 Y chain and antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain and reduced expression of the neuroligin-4 Y chain; and (iv) if neither antibodies to the protocadherin-11 Y chain nor antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain or reduced expression of the neuroligin-4 Y chain.

[0098] In some embodiments, provided herein are methods for determining whether a cell-based therapy that does not comprise reduced expression of protocadherin-11 Y chain and / or reduced expression of neuroligin-4 Y chain is susceptible to lysis by mature NK cells upon administration to a patient, the method comprising: (a) determining whether a biological sample from the patient contains antibodies to protocadherin-11 Y chain and / or antibodies to neuroligin-4 Y chain by (i) obtaining or obtaining the biological sample from the patient, (ii) performing or having performed an assay to determine whether antibodies to protocadherin-11 Y chain are present in the biological sample, and (iii) performing or having performed an assay to determine whether antibodies to neuroligin-4 Y chain are present in the biological sample; and (b) administering to the patient a population of engineered cells, hypoimmunogenic T cells, resting T cells, pancreatic islet cells, cardiomyocytes, glial progenitor cells, or NK cells as described herein; If antibodies to the Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain; (ii) if antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the neuroligin-4 Y chain; (iii) if antibodies to the protocadherin-11 Y chain and antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain and reduced expression of the neuroligin-4 Y chain; and (iv) if neither antibodies to the protocadherin-11 Y chain nor antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain or reduced expression of the neuroligin-4 Y chain.

[0099] In some embodiments, provided herein are methods for determining whether a cell-based therapy that does not comprise reduced expression of protocadherin-11 Y chain and / or reduced expression of neuroligin-4 Y chain is susceptible to phagocytosis by macrophages upon administration to a patient, the method comprising: (a) determining whether a biological sample from the patient contains antibodies to protocadherin-11 Y chain and / or antibodies to neuroligin-4 Y chain by (i) obtaining or obtaining the biological sample from the patient, (ii) performing or having performed an assay to determine whether antibodies to protocadherin-11 Y chain are present in the biological sample, and (iii) performing or having performed an assay to determine whether antibodies to neuroligin-4 Y chain are present in the biological sample; and (b) administering to the patient a population of engineered cells, hypoimmunogenic T cells, resting T cells, pancreatic islet cells, cardiomyocytes, glial progenitor cells, or NK cells as described herein; If antibodies to the Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain; (ii) if antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the neuroligin-4 Y chain; (iii) if antibodies to the protocadherin-11 Y chain and antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain and reduced expression of the neuroligin-4 Y chain; and (iv) if neither antibodies to the protocadherin-11 Y chain nor antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain or reduced expression of the neuroligin-4 Y chain.

[0100] In some embodiments, provided herein are methods for determining whether a cell-based therapy that does not comprise reduced expression of protocadherin-11 Y chain and / or reduced expression of neuroligin-4 Y chain is susceptible to an induced immune response upon administration to a patient, the method comprising: (a) determining whether a biological sample from the patient contains antibodies to protocadherin-11 Y chain and / or antibodies to neuroligin-4 Y chain by (i) obtaining or obtaining the biological sample from the patient, (ii) performing or having performed an assay to determine whether antibodies to protocadherin-11 Y chain are present in the biological sample, and (iii) performing or having performed an assay to determine whether antibodies to neuroligin-4 Y chain are present in the biological sample; and (b) administering to the patient a population of engineered cells, hypoimmunogenic T cells, resting T cells, pancreatic islet cells, cardiomyocytes, glial progenitor cells, or NK cells as described herein, wherein (i) the engineered cells contain antibodies to protocadherin-11 Y chain and / or antibodies to neuroligin-4 Y chain. If antibodies to the Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain; (ii) if antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the neuroligin-4 Y chain; (iii) if antibodies to the protocadherin-11 Y chain and antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain and reduced expression of the neuroligin-4 Y chain; and (iv) if neither antibodies to the protocadherin-11 Y chain nor antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain or reduced expression of the neuroligin-4 Y chain.

[0101] In some embodiments, provided herein are methods for determining whether a cell-based therapy that does not comprise reduced expression of protocadherin-11 Y chain and / or reduced expression of neuroligin-4 Y chain is susceptible to an inducible antibody-based immune response upon administration to a patient, the method comprising: (a) determining whether a biological sample from the patient contains antibodies to protocadherin-11 Y chain and / or antibodies to neuroligin-4 Y chain by (i) obtaining or obtaining the biological sample from the patient, (ii) performing or having performed an assay to determine whether antibodies to protocadherin-11 Y chain are present in the biological sample, and (iii) performing or having performed an assay to determine whether antibodies to neuroligin-4 Y chain are present in the biological sample; and (b) administering to the patient a population of engineered cells, hypoimmunogenic T cells, resting T cells, pancreatic islet cells, cardiomyocytes, glial progenitor cells, or NK cells as described herein; If antibodies to the Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain; (ii) if antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the neuroligin-4 Y chain; (iii) if antibodies to the protocadherin-11 Y chain and antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain and reduced expression of the neuroligin-4 Y chain; and (iv) if neither antibodies to the protocadherin-11 Y chain nor antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain or reduced expression of the neuroligin-4 Y chain.

[0102] In some embodiments, provided herein are methods of treating a patient having a disease or condition that would benefit from a cell-based therapy, the method comprising: (a) determining whether a biological sample from the patient contains antibodies to one or more Y chromosome genes by: (i) obtaining or obtaining the biological sample from the patient; (ii) performing or having performed an assay to determine whether antibodies to protocadherin-11 Y chain are present in the biological sample; and (iii) performing or having performed an assay to determine whether antibodies to neuroligin-4 Y chain are present in the biological sample; and (b) administering to the patient a population of engineered cells, hypoimmunogenic T cells, resting T cells, pancreatic islet cells, cardiomyocytes, glial progenitor cells, or NK cells described herein; wherein (i) if antibodies to protocadherin-11 Y chain are present in the biological sample, the population of cells comprises reduced expression of protocadherin-11 Y chain, and (ii) if antibodies to neuroligin-4 Y chain are present in the biological sample, the population of cells comprises reduced expression of neuroligin-4 Y chain. (iii) if antibodies to the protocadherin-11 Y chain and antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain and reduced expression of the neuroligin-4 Y chain; and (iv) if neither antibodies to the protocadherin-11 Y chain nor antibodies to the neuroligin-4 Y chain are present in the biological sample, the population of cells contains reduced expression of the protocadherin-11 Y chain or reduced expression of the neuroligin-4 Y chain.

[0103] In some embodiments, the Y chromosome gene is a Y chromosome-linked antigen or a Y chromosome-associated minor histocompatibility antigen.

[0104] In some embodiments, the one or more Y chromosome-linked antigens are protocadherin-11 Y-linked and / or neuroligin-4 Y-linked.

[0105] In some embodiments, the cells have reduced expression of protocadherin-11 Y-linked.

[0106] In some embodiments, the cells have reduced expression of neuroligin-4 Y-linked.

[0107] In some embodiments, the cells have reduced expression of protocadherin-11 Y-chain and reduced expression of neuroligin-4 Y-chain.

[0108] In some embodiments, the cells are genetically engineered to have reduced expression of protocadherin-11 Y-linked and / or neuroligin-4 Y-linked.

[0109] In some embodiments, the cells do not express protocadherin-11 Y-linked.

[0110] In some embodiments, the cells do not express neuroligin-4 Y-linked.

[0111] In some embodiments, the cells do not express protocadherin-11 Y-chain and do not express neuroligin-4 Y-chain.

[0112] In some embodiments, the cells are genetically engineered not to express protocadherin-11 Y-chain and / or neuroligin-4 Y-chain.

[0113] In some embodiments, reduced protocadherin-11 Y-linked and / or neuroligin-4 Y-linked expression is caused by knockout of the PCDH11Y and / or NLGN4Y genes, respectively.

[0114] In some embodiments, the cells are derived from human or animal cells.

[0115] In some embodiments, the human or animal cells are from a donor subject that does not have a Y chromosome.

[0116] In some embodiments, the human or animal cells are from a donor subject that has a Y chromosome, and the cells are genetically engineered to have reduced expression of protocadherin-11 Y-linked and / or neuroligin-4 Y-linked.

[0117] In some embodiments, the cells are genetically engineered not to express protocadherin-11 Y-linked.

[0118] In some embodiments, the cells are genetically engineered not to express neuroligin-4 Y-linked.

[0119] In some embodiments, the cells are genetically engineered to not express protocadherin-11 Y-chain and not express neuroligin-4 Y-chain.

[0120] In some embodiments, the cells are expanded or derived from a pool of cells isolated from one or more donor subjects different from the patient, where the one or more donor subjects optionally include one or more subjects with a Y chromosome, one or more subjects without a Y chromosome, or a mixture of subjects with and without a Y chromosome.

[0121] In some embodiments, the cells are genetically engineered to have reduced expression of protocadherin-11 Y-linked and / or neuroligin-4 Y-linked using CRISPR / Cas gene editing.

[0122] In some embodiments, CRISPR / Cas gene editing is performed using one or more guide RNAs comprising any of the sequences in Tables 2-5.

[0123] In some embodiments, CRISPR / Cas gene editing is performed using a Cas effector protein selected from the group consisting of Cas9, Cas12a, and Cas12b.

[0124] In some embodiments, CRISPR / Cas gene editing is performed using a Cas effector protein selected from the group consisting of: (a) optionally selected from the group consisting of Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, and GSU0054; (b) optionally selected from the group consisting of Cas9, Csn2, and Cas4; (c) optionally selected from the group consisting of Cas10, Csm 2, Cmr5, Cas10, Csx11, and Csx10; (d) optionally Csf1; (e) optionally selected from the group consisting of Cas12a, Cas12b, Cas12c, C2c4, C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d); and (f) optionally selected from the group consisting of Cas13, Cas13a, C2c2, Cas13b, Cas13c, and Cas13d.

[0125] In some embodiments, CRISPR / Cas gene editing is performed ex vivo from a donor subject.

[0126] In some embodiments, CRISPR / Cas gene editing is performed using a lentiviral vector.

[0127] In some embodiments, the cells comprise reduced expression of B2M and / or CIITA compared to unaltered or unmodified wild-type or control cells.

[0128] In some embodiments, the cells do not express B2M and / or CIITA.

[0129] In some embodiments, the cells comprise reduced expression of RHD.

[0130] In some embodiments, the cells do not express RHD.

[0131] In some embodiments, the cell is a differentiated cell derived from an induced pluripotent stem cell or its progeny.

[0132] In some embodiments, the differentiated cells are selected from the group consisting of T cells, NK cells, endothelial cells, pancreatic islet cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, hepatocytes, glial progenitor cells, dopaminergic neurons, retinal pigment epithelial cells, and thyroid cells.

[0133] In some embodiments, the cell is a primary immune cell or its progeny.

[0134] In some embodiments, the primary immune cell or its progeny is a T cell or an NK cell.

[0135] In some embodiments, the cells comprise reduced expression of TCR-alpha and / or TCR-beta.

[0136] In some embodiments, the cells do not express TCR-alpha and / or TCR-beta.

[0137] In some embodiments, the cells further comprise a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain with specificity for CD19, CD20, CD22, or BCMA, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

[0138] In some embodiments, one or more CARs comprise a CD8α hinge domain, a CD28 hinge domain, or an IgG4 hinge domain.

[0139] In some embodiments, the one or more CARs comprise a CD8α hinge domain having the amino acid sequence of SEQ ID NO:9.

[0140] In some embodiments, one or more CARs comprise a CD28 hinge domain having the amino acid sequence of SEQ ID NO: 10 or 113.

[0141] In some embodiments, one or more CARs comprise an IgG4 hinge domain having the amino acid sequence of SEQ ID NO: 11 or 12.

[0142] In some embodiments, one or more CARs comprise a CD8α transmembrane domain or a CD28 transmembrane domain.

[0143] In some embodiments, the one or more CARs comprise a CD8α transmembrane domain having the amino acid sequence of SEQ ID NO: 14.

[0144] In some embodiments, the one or more CARs comprise a CD28 transmembrane domain having the amino acid sequence of SEQ ID NO: 15 or 114.

[0145] In some embodiments, one or more CARs comprise a 4-1BB costimulatory domain, a CD28 costimulatory domain, or a CD3ζ signaling domain.

[0146] In some embodiments, one or more CARs comprise a 4-1BB costimulatory domain having the amino acid sequence of SEQ ID NO:16.

[0147] In some embodiments, one or more CARs comprise a CD28 costimulatory domain having the amino acid sequence of SEQ ID NO: 17.

[0148] In some embodiments, the one or more CARs comprise a CD3ζ signaling domain having the amino acid sequence of SEQ ID NO: 18 or 115.

[0149] In some embodiments, one or more CARs comprise an extracellular ligand-binding domain comprising an scFv sequence of any one of SEQ ID NOs: 19, 37, 45, 54, 63, 72, 81, or 118, or the CAR has an scFv sequence comprising the heavy and light chain sequences of any one of SEQ ID NOs: 20, 25, 38, 42, 46, 50, 64, 68, 73, 77, 119, or 123.

[0150] In some embodiments, one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, 117, or 128.

[0151] In some embodiments, the one or more CARs comprise the amino acid sequence set forth in SEQ ID NO: 117, or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 117, and have the following components: a CD8α signal peptide, an FMC63 scFv (VL-Whitlow linker-VH), a CD8α hinge domain, a CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain.

[0152] In some embodiments, one or more CARs comprise the amino acid sequence set forth in SEQ ID NO: 45 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 45.

[0153] In some embodiments, one or more of 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.

[0154] In some embodiments, the first specific locus and / or the second specific locus is selected from the group consisting of a safe harbor or target locus, a RHD locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus.

[0155] In some embodiments, the safe harbor or target locus is selected from the group consisting of the CCR5 locus, the CXCR4 locus, the PPP1R12C locus, the ALB 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 FUT1 locus, and the KDM5D locus.

[0156] In some embodiments, the first exogenous polynucleotide and / or the second exogenous polynucleotide is introduced into the cell using a gene therapy vector or a transposase system selected from the group consisting of a transposase, a PiggyBac transposon, a Sleeping Beauty (SB11) transposon, a Mos1 transposon, and a Tol2 transposon.

[0157] In some embodiments, the gene therapy vector is a retrovirus or a fusosome.

[0158] In some embodiments, the retrovirus is a lentiviral vector.

[0159] In some embodiments, the first exogenous polynucleotide and / or the second exogenous polynucleotide is introduced into the cell using CRISPR / Cas gene editing.

[0160] In some embodiments, CRISPR / Cas gene editing is performed using a Cas effector protein selected from the group consisting of Cas9, Cas12a, and Cas12b.

[0161] In some embodiments, CRISPR / Cas gene editing is performed using a Cas effector protein selected from the group consisting of: (a) optionally selected from the group consisting of Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, and GSU0054; (b) optionally selected from the group consisting of Cas9, Csn2, and Cas4; (c) optionally selected from the group consisting of Cas10, Csm 2, Cmr5, Cas10, Csx11, and Csx10; (d) optionally Csf1; (e) optionally selected from the group consisting of Cas12a, Cas12b, Cas12c, C2c4, C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d); and (f) optionally selected from the group consisting of Cas13, Cas13a, C2c2, Cas13b, Cas13c, and Cas13d.

[0162] In some embodiments, CRISPR / Cas gene editing is performed ex vivo from a donor subject.

[0163] In some embodiments, CRISPR / Cas gene editing is performed using a lentiviral vector.

[0164] In some embodiments, the cells or progeny thereof avoid NK cell-mediated cytotoxicity upon administration to a patient.

[0165] In some embodiments, the cells or progeny thereof are protected from cytolysis by mature NK cells upon administration to a patient.

[0166] In some embodiments, the cells or progeny thereof avoid phagocytosis by macrophages upon administration to a patient.

[0167] In some embodiments, the cells or their progeny do not induce an immune response against the cells when administered to a patient.

[0168] In some embodiments, the cells or their progeny do not induce an antibody-based immune response against the cells upon administration to a patient.

[0169] In some embodiments, wild-type or control cells are the starting material.

[0170] In some embodiments, provided herein is a use of a population of engineered T cells to treat a disorder or condition in a patient, wherein the engineered T cells comprise reduced expression of one or more Y chromosome genes and MHC class I and / or MHC class II human leukocyte antigen molecules compared to unchanged or unmodified wild-type or control cells, and comprise a first exogenous polynucleotide encoding CD47, wherein the engineered T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

[0171] In some embodiments, provided herein is a use of a population of engineered differentiated cells for treating a disorder or condition in a patient, wherein the engineered differentiated cells comprise reduced expression of one or more Y chromosome genes and MHC class I and / or MHC class II human leukocyte antigen molecules compared to unchanged or unmodified wild-type or control cells, and comprise a first exogenous polynucleotide encoding CD47, and wherein the engineered differentiated cells are derived from iPSCs or their progeny.

[0172] In some embodiments, the Y chromosome gene is a Y chromosome-linked antigen or a Y chromosome-associated minor histocompatibility antigen.

[0173] In some embodiments, the one or more Y chromosome-linked antigens are protocadherin-11 Y-linked and / or neuroligin-4 Y-linked.

[0174] In some embodiments, the cells have reduced expression of protocadherin-11 Y-linked.

[0175] In some embodiments, the cells have reduced expression of neuroligin-4 Y-linked.

[0176] In some embodiments, the cells have reduced expression of protocadherin-11 Y-chain and reduced expression of neuroligin-4 Y-chain.

[0177] In some embodiments, the cells are genetically engineered to have reduced expression of protocadherin-11 Y-linked and / or neuroligin-4 Y-linked.

[0178] In some embodiments, the cells do not express protocadherin-11 Y-linked.

[0179] In some embodiments, the cells do not express neuroligin-4 Y-linked.

[0180] In some embodiments, the cells do not express protocadherin-11 Y-chain and do not express neuroligin-4 Y-chain.

[0181] In some embodiments, the cells are genetically engineered not to express protocadherin-11 Y-chain and / or neuroligin-4 Y-chain.

[0182] In some embodiments, reduced protocadherin-11 Y-linked and / or neuroligin-4 Y-linked expression is caused by knockout of the PCDH11Y and / or NLGN4Y genes, respectively.

[0183] In some embodiments, the cells are derived from human or animal cells.

[0184] In some embodiments, the human or animal cells are from a donor subject that does not have a Y chromosome.

[0185] In some embodiments, the human or animal cells are from a donor subject that has a Y chromosome, and the cells are genetically engineered to have reduced expression of protocadherin-11 Y-linked and / or neuroligin-4 Y-linked.

[0186] In some embodiments, the cells are genetically engineered not to express protocadherin-11 Y-linked.

[0187] In some embodiments, the cells are genetically engineered not to express neuroligin-4 Y-linked.

[0188] In some embodiments, the cells are genetically engineered to not express protocadherin-11 Y-chain and not express neuroligin-4 Y-chain.

[0189] In some embodiments, the cells are expanded or derived from a pool of cells isolated from one or more donor subjects different from the patient, where the one or more donor subjects optionally include one or more subjects with a Y chromosome, one or more subjects without a Y chromosome, or a mixture of subjects with and without a Y chromosome.

[0190] In some embodiments, the cells are genetically engineered to have reduced expression of protocadherin-11 Y-linked and / or neuroligin-4 Y-linked using CRISPR / Cas gene editing.

[0191] In some embodiments, CRISPR / Cas gene editing is performed using one or more guide RNAs comprising any of the sequences in Tables 2-5.

[0192] In some embodiments, CRISPR / Cas gene editing is performed using a Cas effector protein selected from the group consisting of Cas9, Cas12a, and Cas12b.

[0193] In some embodiments, CRISPR / Cas gene editing is performed using a Cas effector protein selected from the group consisting of: (a) optionally selected from the group consisting of Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, and GSU0054; (b) optionally selected from the group consisting of Cas9, Csn2, and Cas4; (c) optionally selected from the group consisting of Cas10, Csm 2, Cmr5, Cas10, Csx11, and Csx10; (d) optionally Csf1; (e) optionally selected from the group consisting of Cas12a, Cas12b, Cas12c, C2c4, C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d); and (f) optionally selected from the group consisting of Cas13, Cas13a, C2c2, Cas13b, Cas13c, and Cas13d.

[0194] In some embodiments, CRISPR / Cas gene editing is performed ex vivo from a donor subject.

[0195] In some embodiments, CRISPR / Cas gene editing is performed using a lentiviral vector.

[0196] In some embodiments, the cells comprise reduced expression of B2M and / or CIITA compared to unaltered or unmodified wild-type or control cells.

[0197] In some embodiments, the cells do not express B2M and / or CIITA.

[0198] In some embodiments, the cells comprise reduced expression of RHD.

[0199] In some embodiments, the cells do not express RHD.

[0200] In some embodiments, the cell is a differentiated cell derived from an induced pluripotent stem cell or its progeny.

[0201] In some embodiments, the differentiated cells are selected from the group consisting of T cells, NK cells, endothelial cells, pancreatic islet cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, hepatocytes, glial progenitor cells, dopaminergic neurons, retinal pigment epithelial cells, and thyroid cells.

[0202] In some embodiments, the cell is a primary immune cell or its progeny.

[0203] In some embodiments, the primary immune cell or its progeny is a T cell or an NK cell.

[0204] In some embodiments, the cells comprise reduced expression of TCR-alpha and / or TCR-beta.

[0205] In some embodiments, the cells do not express TCR-alpha and / or TCR-beta.

[0206] In some embodiments, the cells further comprise a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand-binding domain with specificity for CD19, CD20, CD22, or BCMA, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

[0207] In some embodiments, one or more CARs comprise a CD8α hinge domain, a CD28 hinge domain, or an IgG4 hinge domain.

[0208] In some embodiments, the one or more CARs comprise a CD8α hinge domain having the amino acid sequence of SEQ ID NO:9.

[0209] In some embodiments, one or more CARs comprise a CD28 hinge domain having the amino acid sequence of SEQ ID NO: 10 or 113.

[0210] In some embodiments, one or more CARs comprise an IgG4 hinge domain having the amino acid sequence of SEQ ID NO: 11 or 12.

[0211] In some embodiments, one or more CARs comprise a CD8α transmembrane domain or a CD28 transmembrane domain.

[0212] In some embodiments, the one or more CARs comprise a CD8α transmembrane domain having the amino acid sequence of SEQ ID NO: 14.

[0213] In some embodiments, the one or more CARs comprise a CD28 transmembrane domain having the amino acid sequence of SEQ ID NO: 15 or 114.

[0214] In some embodiments, one or more CARs comprise a 4-1BB costimulatory domain, a CD28 costimulatory domain, or a CD3ζ signaling domain.

[0215] In some embodiments, one or more CARs comprise a 4-1BB costimulatory domain having the amino acid sequence of SEQ ID NO:16.

[0216] In some embodiments, one or more CARs comprise a CD28 costimulatory domain having the amino acid sequence of SEQ ID NO: 17.

[0217] In some embodiments, the one or more CARs comprise a CD3ζ signaling domain having the amino acid sequence of SEQ ID NO: 18 or 115.

[0218] In some embodiments, one or more CARs comprise an extracellular ligand-binding domain comprising an scFv sequence of any one of SEQ ID NOs: 19, 37, 45, 54, 63, 72, 81, or 118, or the CAR has an scFv sequence comprising the heavy and light chain sequences of any one of SEQ ID NOs: 20, 25, 38, 42, 46, 50, 64, 68, 73, 77, 119, or 123.

[0219] In some embodiments, one or more CARs have the sequence of any one of SEQ ID NOs: 32, 34, 36, 117, or 128.

[0220] In some embodiments, the one or more CARs comprise the amino acid sequence set forth in SEQ ID NO: 117, or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 117, and have the following components: a CD8α signal peptide, an FMC63 scFv (VL-Whitlow linker-VH), a CD8α hinge domain, a CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain.

[0221] In some embodiments, one or more CARs comprise the amino acid sequence set forth in SEQ ID NO: 45 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 45.

[0222] In some embodiments, one or more of 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.

[0223] In some embodiments, the first specific locus and / or the second specific locus is selected from the group consisting of a safe harbor or target locus, a RHD locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus.

[0224] In some embodiments, the safe harbor or target locus is selected from the group consisting of the CCR5 locus, the CXCR4 locus, the PPP1R12C locus, the ALB 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 FUT1 locus, and the KDM5D locus.

[0225] In some embodiments, the first exogenous polynucleotide and / or the second exogenous polynucleotide is introduced into the engineered T cell using a gene therapy vector or a transposase system selected from the group consisting of a transposase, a PiggyBac transposon, a Sleeping Beauty (SB11) transposon, a Mos1 transposon, and a Tol2 transposon.

[0226] In some embodiments, the gene therapy vector is a retrovirus or a fusosome.

[0227] In some embodiments, the retrovirus is a lentiviral vector.

[0228] In some embodiments, the first exogenous polynucleotide and / or the second exogenous polynucleotide is introduced into the cell using CRISPR / Cas gene editing.

[0229] In some embodiments, CRISPR / Cas gene editing is performed using a Cas effector protein selected from the group consisting of Cas9, Cas12a, and Cas12b.

[0230] In some embodiments, CRISPR / Cas gene editing is performed using a Cas effector protein selected from the group consisting of: (a) optionally selected from the group consisting of Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, and GSU0054; (b) optionally selected from the group consisting of Cas9, Csn2, and Cas4; (c) optionally selected from the group consisting of Cas10, Csm 2, Cmr5, Cas10, Csx11, and Csx10; (d) optionally Csf1; (e) optionally selected from the group consisting of Cas12a, Cas12b, Cas12c, C2c4, C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d); and (f) optionally selected from the group consisting of Cas13, Cas13a, C2c2, Cas13b, Cas13c, and Cas13d.

[0231] In some embodiments, CRISPR / Cas gene editing is performed ex vivo from a donor subject.

[0232] In some embodiments, CRISPR / Cas gene editing is performed using a lentiviral vector.

[0233] In some embodiments, the cells or progeny thereof avoid NK cell-mediated cytotoxicity upon administration to a patient.

[0234] In some embodiments, the cells or progeny thereof are protected from cytolysis by mature NK cells upon administration to a patient.

[0235] In some embodiments, the cells or progeny thereof avoid phagocytosis by macrophages upon administration to a patient.

[0236] In some embodiments, the cells or their progeny do not induce an immune response against the cells when administered to a patient.

[0237] In some embodiments, the cells or their progeny do not induce an antibody-based immune response against the cells upon administration to a patient.

[0238] In some embodiments, wild-type or control cells are the starting material.

[0239] In some embodiments, provided herein are methods for producing an engineered cell that comprises reduced expression of one or more Y chromosome genes and MHC class I and / or MHC class II human leukocyte antigen molecules relative to an unchanged or unmodified wild-type or control cell, and that comprises a first exogenous polynucleotide encoding CD47, the method comprising: (a) obtaining an isolated cell; (b) genetically modifying the cell to reduce expression of one or more Y chromosome genes in the cell; (c) genetically modifying the cell to reduce expression of MHC class I human leukocyte antigen molecules and / or MHC class II human leukocyte antigen molecules in the cell; and (d) introducing a polynucleotide encoding CD47 into the isolated cell, thereby producing the engineered cell.

[0240] In some embodiments, provided herein are methods for producing an engineered cell that comprises reduced expression of one or more Y chromosome genes and MHC class I and / or MHC class II human leukocyte antigen molecules relative to an unchanged or unmodified wild-type or control cell, and that comprises a first exogenous polynucleotide encoding CD47, the method comprising: (a) obtaining an isolated cell; and (b) contacting the cell with a composition comprising a lentiviral vector comprising: (i) a CD4 binding agent or a CD8 binding agent, (ii) a polynucleotide encoding CRISPR / Cas gene editing components that target one or more Y chromosome loci, (iii) a polynucleotide encoding CRISPR / Cas gene editing components that target MHC class I and / or MHC class II human leukocyte antigen loci, and (iv) the first exogenous polynucleotide encoding CD47, thereby producing the engineered cell.

[0241] Detailed descriptions of the hypoimmunogenic cells, methods for their production, and methods for their 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 31, 2020, and WO20200186391 filed July 17, 2019. and WO2021022223 filed July 31, 2020, WO2021041316 filed August 24, 2020, 2021, WO2021222285 filed April 27, 2020, and WO2021222285 filed April 27, 2021, the disclosures of which, including the Examples, sequence listings, and figures, are incorporated herein by reference in their entireties. [Brief explanation of the drawings]

[0242] [Figure 1A]Illustrated is flow cytometry data measuring the levels of protocadherin-Y and neuroligin-Y on the cell surface of iPSCs derived from male donors compared to isotype controls. [Figure 1B] Illustrated is flow cytometry data measuring the levels of protocadherin-Y and neuroligin-Y on the cell surface of iPSCs derived from a female donor compared to isotype controls. [Figure 2A] Illustrated is flow cytometry data measuring the levels of protocadherin-Y and neuroligin-Y on the cell surface of CD3+ T cells from three male donors with blood type O analyzed after thawing, compared to isotype controls. [Figure 2B] Illustrated is flow cytometry data measuring the levels of protocadherin-Y and neuroligin-Y on the cell surface of CD3+ T cells from two male donors with blood type A analyzed after thawing, compared to isotype controls. [Figure 2C] Illustrated is flow cytometry data measuring the levels of protocadherin-Y and neuroligin-Y on the cell surface of CD3+ T cells from two female donors analyzed after thawing, compared to isotype controls. [Figure 3A] Figure 1 shows CDC against HIP T cells from a male donor with blood group O using sera from different volunteers. [Figure 3B] Figure 1 shows CDC against HIP T cells from a male donor with blood group O using sera from different volunteers. [Figure 3C] Figure 1 shows CDC against HIP T cells from a male donor with blood group O using sera from different volunteers. [Figure 4A] ADCC (NK cells) against HIP T cells from a male donor with blood group O using sera from different volunteers. [Figure 4B] ADCC (NK cells) against HIP T cells from a male donor with blood group O using sera from different volunteers. [Figure 4C] ADCC (NK cells) against HIP T cells from a male donor with blood group O using sera from different volunteers. [Figure 5A] CDC and ADCC (NK cells) against HIP T cells from a male donor with blood group O using serum from different volunteers and flow analysis. [Figure 5B] CDC and ADCC (NK cells) against HIP T cells from a male donor with blood group O using serum from different volunteers and flow analysis. [Figure 5C] CDC and ADCC (NK cells) against HIP T cells from a male donor with blood group O using serum from different volunteers and flow analysis.

[0243] Other objects, advantages and embodiments of the present disclosure will become apparent from the following detailed description. DETAILED DESCRIPTION OF THE INVENTION

[0244] I. Introduction Described herein are engineered or modified immune evasive cells, including but not limited to human immune evasive cells, based in part on the hypoimmune editing platform described in WO2018132783 and PCT / US21 / 65157, filed December 23, 2021, each of which is incorporated herein by reference in its entirety. To overcome the problem of rejection of these primary and / or stem cell-derived grafts by a subject's immune system, the inventors have developed hypoimmunogenic cells (e.g., hypoimmunogenic pluripotent cells, differentiated cells derived therefrom, and primary cells) that represent a viable source for any transplantable cell type, which are described herein. 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 recipient subject's immune system, regardless of the subject's genetic makeup. In some embodiments, the engineered and / or hypoimmunogenic cells do not express one or more Y chromosome genes and / or do not express MHC I and / or II antigen molecules and / or T cell receptors. In certain embodiments, the engineered and / or hypoimmunogenic cells do not express one or more Y chromosome genes and do not express MHC I antigens. In certain embodiments, the engineered and / or hypoimmunogenic cells do not express one or more Y chromosome genes, do not express MHC I and / or II antigen molecules and / or T cell receptors, and overexpress CD47 protein. In certain embodiments, the engineered and / or hypoimmunogenic cells do not express one or more Y chromosome genes, do not express MHC I and / or II antigen molecules and / or T cell receptors, and overexpress CD47 protein. In certain embodiments, the engineered and / or hypoimmunogenic cells, such as hypoimmunogenic T cells, do not express one or more Y chromosome genes, do not express MHC I and / or II antigen molecules and / or T cell receptors, overexpress CD47 protein, and express an exogenous CAR.In certain embodiments, the engineered and / or hypoimmunogenic cells, such as hypoimmunogenic T cells, do not express one or more Y chromosome genes, do not express MHC I and II antigen molecules and / or T cell receptors, overexpress CD47 protein, and express an exogenous CAR.

[0245] In some embodiments, the hypoimmunogenic cells outlined herein are not subject to rejection by innate immune cells. In some cases, hypoimmunogenic cells are not susceptible to NK cell-mediated lysis. In some cases, hypoimmunogenic cells are not susceptible to phagocytosis by macrophages. In some embodiments, hypoimmunogenic cells do not induce an immune response. In some embodiments, hypoimmunogenic cells are useful as a source of universally compatible cells or tissues (e.g., universal donor cells or tissues) that can be transplanted into a recipient subject with little to no need for immunosuppressive agents. Such hypoimmunogenic cells retain cell-specific properties and characteristics upon transplantation, including, for example, pluripotency, and the ability to engraft and function similarly to the corresponding native cells.

[0246] The technology disclosed herein utilizes expression of tolerogenic factors in human cells, as well as modulation (e.g., reduction or elimination) of expression of one or more Y chromosome genes, and optionally MHC I molecules, MHC II molecules, and / or TCRs. In some embodiments, genome editing techniques utilizing 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 the immune response in cells (e.g., by deleting genomic DNA of genes involved in the immune response or by inserting genomic DNA into such genes such that gene expression is affected). In some embodiments, genome editing techniques or other gene regulation techniques are used to insert tolerance-inducing (tolerogenic) factors in human cells, allowing the cells and their progeny (including any differentiated cells prepared therefrom) to avoid immune recognition upon transplantation into a recipient subject. Thus, the cells described herein exhibit regulated expression of one or more genes and factors that affect expression of one or more Y chromosome genes, MHC I molecules, MHC II molecules, and / or TCRs, thereby evading the immune system of a recipient subject.

[0247] Genome editing technology allows for double-stranded DNA breaks at desired gene loci. These controlled double-stranded breaks promote homologous recombination at specific gene 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).

[0248] Surprisingly, iPSCs and T cells from male donors were found to express the Y chromosome antigens protocadherin-11 Y-linked and neuroligin-4 Y-linked. These surprising findings suggest that sources of hypoimmunogenic cells, such as hypoimmunogenic donor T cells, resting T cells, pancreatic islet cells, or cardiac cells, should lack the Y chromosome or be genetically modified to reduce expression of Y chromosome antigens to avoid detection and elimination by the recipient's adaptive immune system.

[0249] The practice of many embodiments will employ, unless otherwise indicated to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology, and cell biology, which are within the skill of those in the art, many of which are described below by way of illustration and description, 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 Translation(B.Hames & See research articles in publications such as 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.

[0250] II. Definition As used herein, the following terms will be used and are defined as set forth below.

[0251] The term "antigen" as used herein 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, as well as allergens. The term antigen broadly includes any type of molecule that is recognized as foreign by the host's immune system.

[0252] The terms "autoimmune disease" or "autoimmune disorder" or "inflammatory disease" or "inflammatory disorder" refer to any disease or disorder in which a subject mounts an immune response against its own tissues and / or cells. Autoimmune disorders can affect almost every organ system in 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.

[0253] The term "cancer," as used herein, is defined as the hyperproliferation of cells whose intrinsic traits (e.g., loss of normal control) result in uncontrolled growth, lack of differentiation, local tissue invasion, and metastasis. In the context of the methods of the present invention, cancer includes acute lymphocytic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, anal, anal canal, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, oral cancer, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, Hodgkin's lymphoma, and the like. The tumor may be any cancer, including any of the following: lymphoma, hypopharyngeal cancer, kidney 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, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, and bladder cancer. As used herein, the term "tumor," unless expressly indicated otherwise, refers to an abnormal growth of cells or tissue of a malignant type, and does not include benign type tissue.

[0254] The term "cell" refers to any human or animal cell. In some embodiments, the cell is a human or animal cell from a donor subject that has a Y chromosome. In some embodiments, the cell is a human or animal cell from a donor subject that does not have a Y chromosome.

[0255] The term "chronic infectious disease" refers to a disease caused by an infectious agent in which the infection persists. Such diseases may include hepatitis (A, B, or C), herpes viruses (e.g., VZV, HSV-1, HSV-6, HSV-II, CMV, and EBV), and HIV / AIDS. Non-viral examples may include chronic fungal diseases such as aspergillosis, candidiasis, coccidioidomycosis, and diseases associated with cryptococcosis and histoplasmosis. Non-limiting examples of chronic bacterial infectious agents may 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).

[0256] 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 result in 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 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.

[0257] 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.

[0258] In additional or alternative embodiments, the present disclosure contemplates altering the target polynucleotide sequence in any manner available to those skilled in the art, for example, by using a 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 known to those skilled in the art, such as targeting B2M, can be used herein to reduce or eliminate expression in target cells.

[0259] The terms "reduce," "reduced," "reduction," and "decrease" are all used herein to generally mean a statistically significant decrease. However, for the avoidance of doubt, "reduce," "reduced," "reduction," and "decrease" refer to a decrease of at least 10% compared to a reference level, e.g., a decrease 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 a decrease of up to and including 100% (i.e., absent levels compared to a reference sample), or any decrease between 10 and 100%. In some embodiments, the cells are engineered to have reduced expression of one or more targets compared to unaltered or unmodified wild-type or control cells.

[0260] In some embodiments, the engineered and / or hypoimmunogenic cells described are derived from iPSCs or their progeny. As used herein, the term "derived from an iPSC or its 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, available from, or derivable 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 even higher generations, i.e., generations of cells derived from, obtained from, available from, or derivable from previous generations, e.g., by conventional propagation methods. The term "progeny" also encompasses modified cells resulting from modifications or alterations of the initial iPSC or its progeny.

[0261] 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 such as 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. Advantageously, however, the donor subject is a mammal, such as a human, or other mammal, such as a domestic mammal, e.g., a dog, cat, or horse, or a production mammal, e.g., a cow, sheep, or pig. "Donor subject" can also refer to more than one donor, e.g., one or more human or non-human animals or non-human mammals.

[0262] The term "endogenous" refers to a referent molecule or polypeptide that is naturally present in a cell. Similarly, when used with respect to expression of an encoding nucleic acid, the term refers to expression of an encoding nucleic acid that is naturally contained within a cell and not exogenously introduced. Similarly, when used with respect to a promoter sequence, the term refers to a promoter sequence that is naturally contained within a cell and not exogenously introduced.

[0263] 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 alteration or modification, such that the engineered cell differs from a wild-type cell.

[0264] As used herein, the term "exogenous" in the context of expressing a polynucleotide or polypeptide is intended to mean that the referred molecule or referred polypeptide is introduced into the cell of interest. A polypeptide can be introduced, for example, by introducing the encoding 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, the term, when used in reference to expression of an encoding nucleic acid, refers to the introduction of the encoding nucleic acid into the cell in an expressible form.

[0265] For purposes of this disclosure, a "gene" includes a DNA region that encodes a gene product, as well as all DNA regions that regulate the production of that gene product (whether or not such regulatory sequences flank the coding and / or transcribed sequence). Thus, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational control sequences, such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

[0266] "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 any other type of RNA) or a protein produced by translation of mRNA. Gene products also include RNAs that are 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.

[0267] As used herein, the term "genetic modification" and its grammatical equivalents may refer to one or more changes in a nucleic acid, e.g., a nucleic acid in the genome of an organism. For example, a 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 of a target gene or portion of a gene or nucleic acid sequence, and mechanisms that result in permanent knock-in, knock-down, or knock-out. Genetic modifications include, for example, both transient knock-in and permanent knock-in mechanisms 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.

[0268] As used herein, the terms "grafting," "administering," "introducing," "implanting," and "transplanting," as well as grammatical variations thereof, are used interchangeably in the context of placing cells (e.g., cells described herein) into a subject by a method or route that results in localization or at least partial localization at a desired site or systemic introduction (e.g., into the circulation) of the introduced cells. Cells can be directly implanted at a desired site within a subject, or alternatively, can be administered by any suitable route that results in delivery to a desired location, where at least a portion of the implanted cells or components of the cells remain viable. The survival period of cells after administration to a subject can be as short as a few hours, e.g., 24 hours, to as long as several days or years. In some embodiments, cells can also be administered (e.g., injected) to a site other than the desired site, such as intracerebrally or subcutaneously, e.g., in a capsule to maintain the implanted cells at the site of implantation and avoid migration of the implanted cells.

[0269] The "HLA" or "human leukocyte antigen" or "HLA molecule" or "human leukocyte antigen molecule" complex is a gene complex that encodes MHC proteins in humans. These cell surface proteins that make up the HLA complex are responsible for regulating the immune response to antigens. In humans, there are two types of MHC, "HLA-I" and "HLA-II," or "HLA-I molecules" and "HLA-II molecules," consisting of class I and class II molecules. HLA-I contains three proteins, HLA-A, HLA-B, and HLA-C, that present peptides from the inside of cells. Antigens presented by the HLA-I complex attract killer T cells (also known as CD8+ T cells or cytotoxic T cells). HLA-I proteins associate 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 the outside of cells. This stimulates CD4+ cells (also known as helper T cells). It should be understood that the use of either "MHC" or "HLA" is not intended 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 referring to mammalian cells.

[0270] The term "low immunogenicity," as used herein to characterize cells, generally means that the cells are less susceptible to innate or adaptive immune rejection by a subject receiving the cells, e.g., the cells are less susceptible to allogeneic rejection by a subject receiving the cells. For example, compared to cells of the same cell type without modification, such low immunogenic cells may be about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or more less susceptible to innate or adaptive immune rejection by a subject receiving the cells. In some embodiments, genome editing techniques are used to modulate the expression of MHC I and MHC II genes, thereby contributing to the generation of low immunogenic cells. In some embodiments, the low immunogenic cells avoid immune rejection in an MHC-mismatched allogeneic recipient. 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 and / or innate immune rejection. Detailed descriptions of the hypoimmunogenic cells, methods for their production, and methods for their 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 31, 2020, and WO20200186391 filed July 17, 2019. and WO2021022223 filed July 31, 2020, WO2021041316 filed August 24, 2020, 2021, WO2021222285 filed April 27, 2020, and WO2021222285 filed April 27, 2021, the disclosures of which, including the Examples, sequence listings, and figures, are incorporated herein by reference in their entireties.

[0271] The low immunogenicity of cells can be determined by assessing the immunogenicity of the cells, such as their ability to induce or avoid inducing adaptive and innate immune responses. Such immune responses can be measured using assays recognized by those skilled in the art. In some embodiments, immune response assays measure the effect of hypoimmunogenic 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, hypoimmunogenic 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, hypoimmunogenic cells induce a reduced or attenuated immune response in a recipient subject compared to corresponding unmodified wild-type cells. In some embodiments, hypoimmunogenic cells are non-immunogenic or fail to induce an immune response in a recipient subject.

[0272] 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 specified percentage of nucleotides or amino acid residues that are the same when compared and aligned for maximum correspondence, as determined 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" can be over a region of the sequences being compared, such as 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 to which test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. 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.

[0273] 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 similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0274] 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.

[0275] As used herein, "immune signaling factor" refers to a molecule, protein, peptide, etc. that activates an immune signaling pathway, as the case may be.

[0276] As used herein, "immunosuppressive factors" or "immunomodulatory 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.

[0277] The terms "increased," "increase," or "enhance" or "activate" are all used herein to generally mean an increase by 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, e.g., 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 of up to and including 100%, or any increase between 10 and 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 between 2-fold and more than 10-fold compared to a reference level. In some embodiments, the reference level, also referred to as the basal level, is 0.

[0278] 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.

[0279] As used herein, "knockdown" refers to the reduction of the expression of target mRNA or corresponding target protein. Knockdown is generally reported relative to the level present after the administration or expression of a non-control molecule (e.g., a non-targeting control shRNA, siRNA, or miRNA) that does not mediate the reduction of the RNA expression level. In some embodiments, the knockdown of target genes is achieved using conditional or inducible shRNA, conditional or inducible siRNA, conditional or inducible miRNA, or conditional or inducible CRISPR interference (CRISPRi). In some embodiments, the knockdown of target genes is achieved using protein-based methods, such as conditional or inducible degron methods. In some embodiments, the knockdown of target genes is achieved by genetic modification, including shRNA, siRNA, miRNA, or by using a gene editing system (e.g., CRISPR / Cas).

[0280] 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). Protein level analysis 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.

[0281] As used herein, "knock-in" or "knock-in" refers to a genetic modification resulting from the insertion of a DNA sequence into a chromosomal locus in a host cell. This results in the initiation or increased expression level of the knocked-in gene, portion of a gene, or nucleic acid sequence insertion product, e.g., increased RNA transcript levels and / or encoded protein levels. 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 a gene or portion thereof to the host cell, or altering the regulatory components of an endogenous gene to increase expression of the protein being produced, or inserting a specific nucleic acid sequence whose expression is desired. This may be accomplished by modifying the promoter, adding a different promoter, adding an enhancer, adding other regulatory elements, or modifying other gene expression sequences.

[0282] As used herein, "knock out" or "knock-out" includes deleting all or a portion of a target polynucleotide sequence to prevent translation or function of the target polynucleotide sequence. For example, knockout can be achieved by altering the target polynucleotide sequence by inducing an insertion or deletion ("indel") within the target polynucleotide sequence, including within a functional domain (e.g., a DNA-binding domain) of the target polynucleotide sequence. Based on the details described herein, one skilled in the art will readily 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.

[0283] In some embodiments, the genetic modification or alteration results in the knockout or knockdown of a target polynucleotide sequence or a portion thereof. The knockout of a 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, the knockout of a target polynucleotide sequence in a cell can be performed in vitro for research purposes. For ex vivo purposes, the knockout of a target polynucleotide sequence in a cell can be useful for treating or preventing disorders associated with the expression of a target polynucleotide sequence, or for changing the genotype or phenotype of a cell (e.g., by knocking out a mutant allele in a cell ex vivo and introducing the cells containing the knocked-out mutant allele into a subject).

[0284] "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 to wild-type levels or above), or it can be partial (gene expression is partially reduced or partially activated to a fraction of wild-type levels).

[0285] In additional or alternative aspects, the present disclosure contemplates altering a target polynucleotide sequence using any method available to one of skill in the art, for example, using a nuclease system, such as a TAL effector nuclease (TALEN) or zinc finger nuclease (ZFN) system. While exemplary methods utilizing CRISPR / Cas (e.g., Cas9 and Cas12a) and TALENs are detailed herein, it should be understood that the present disclosure is not limited to the use of these methods / systems. Other targeting methods known to those of skill in the art can be utilized herein to reduce or eliminate expression in target cells. The methods provided herein can be used to alter a target polynucleotide sequence in a cell. The present disclosure contemplates altering a target polynucleotide sequence in a cell for any purpose. In some embodiments, a 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 differs 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 (e.g., a CRISPR / Cas) system of the present disclosure. In other cases, a "mutated cell" exhibits a wild-type phenotype, for example, when a mutant genotype is corrected using a gene editing system (e.g., a CRISPR / Cas) system of the present disclosure. In some embodiments, a target polynucleotide sequence in a cell is altered to correct or repair a genetic mutation (e.g., to restore a normal phenotype to the cell). In some embodiments, a target polynucleotide sequence in a cell is altered to induce a genetic mutation (e.g., to disrupt the function of a gene or genomic element).

[0286] "Neuroligin-4 Y-linked," "Neuroligin-4-Y," and "NLGN4Y," and variations thereof, refer to the Y chromosome-linked antigen encoded by the NLGN4Y gene.

[0287] The terms "operably linked" or "operably linked" are used interchangeably in reference to the juxtaposition of two or more components (such as sequence elements) that 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 transcriptional regulatory sequence, such as a promoter, is operably linked to a coding sequence if it controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. A transcriptional regulatory sequence is generally operably linked to a coding sequence in cis, but need not be directly adjacent to it. For example, an enhancer is a transcriptional regulatory sequence that is operably linked to a coding sequence, even if they are not contiguous.

[0288] The term "patient" or "recipient patient" refers to an animal, e.g., a human, to which treatment (including prophylactic treatment) with the cells described herein is provided. In the case of treatment of an infection, condition, or disease state that is specific to a particular animal, such as a human patient, the term patient refers to that particular animal. The term "patient" also encompasses any vertebrate, including, but not limited to, mammals, reptiles, amphibians, and fish. Advantageously, however, the patient is a mammal, such as a human, or other mammal, such as a domestic mammal, e.g., a dog, cat, horse, or a production mammal, e.g., a cow, sheep, pig, etc.

[0289] As used herein, "pluripotent stem cells" have the potential to differentiate into any of three germ layers: endoderm (e.g., stomach wall, digestive tract, lungs, etc.), mesoderm (e.g., muscle, bone, blood, urogenital tissue, etc.), or ectoderm (e.g., epithelial tissue and nervous system tissue). The term "pluripotent stem cells" as used herein also encompasses "induced pluripotent stem cells" or "iPSCs," a type of pluripotent stem cell derived from a non-pluripotent cell. In some embodiments, pluripotent stem cells are produced or generated from a cell that is not a pluripotent cell. In other words, pluripotent stem cells can be the direct or indirect progeny of a non-pluripotent cell. Examples of parent cells include somatic cells that have been reprogrammed to induce a pluripotent, undifferentiated phenotype by various means. Such "iPS" or "iPSC" cells can be generated by inducing the expression of certain regulatory genes or by the 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, "pluripotent stem cells" as used herein also encompass mesenchymal stem cells (MSCs) and / or embryonic stem cells (ESCs).

[0290] As used herein, "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 a downstream coding or non-coding sequence. In some instances, a promoter sequence includes a transcription initiation site and extends upstream to include the minimum number of bases or elements necessary 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 responsible for binding RNA polymerase. Eukaryotic promoters often, but not always, contain "TATA" boxes and "CAT" boxes.

[0291] In some embodiments, the described engineered and / or 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 the initial 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 directly derived from, obtained from, obtainable from, or derivable from the initial primary T cells, e.g., by conventional propagation methods. The term "progeny" also includes further generations, such as second, third, fourth, fifth, sixth, seventh, or even higher generations, i.e., generations of cells derived from, obtained from, obtainable from, or derivable from previous generations, e.g., by conventional propagation methods. The term "progeny" also includes modified cells resulting from modifications or alterations of the initial primary T cells or their progeny.

[0292] "Protocadherin-11 Y-linked," "protocadherin-11-Y," and "PCDH11Y," and variations thereof, refer to the Y chromosome-linked antigen encoded by the PCDH11Y gene.

[0293] As used herein, the terms "regulatory sequence," "regulatory element," and "control element" are used interchangeably and refer to polynucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' untranslated sequences) of a polynucleotide target to be expressed. Regulatory sequences affect, for example, but are 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 can 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. In most cases, the exact boundaries of regulatory sequences have not been completely defined, and it is recognized that nucleotide sequences of different lengths can have identical regulatory or promoter activity.

[0294] As used herein, a "safe harbor locus" refers to a genetic locus that allows for expression of a transgene or exogenous gene in a manner that allows the newly inserted genetic element to function as expected and that is likely not to alter 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 (also known as AAVS1) gene, the CLYBL gene, and / or the Rosa gene (e.g., ROSA26).

[0295] As used herein, a "target locus" 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, albumin gene, SHS231 locus, F3 gene (also known as CD142), MICA gene, MICB gene, LRP1 gene (also known as CD91), HMGB1 gene, ABO gene, RHD gene, FUT1 gene, and / or KDM5D gene (also known as HY). An exogenous polynucleotide encoding an exogenous gene may be inserted into the CDS region of 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. An exogenous polynucleotide encoding an exogenous gene may be inserted into intron 1 or 2 of PPP1R12C (i.e., AAVS1) or CCR5. An exogenous polynucleotide encoding an exogenous gene may be inserted into exon 1, 2, or 3 of CCR5. An exogenous polynucleotide encoding an exogenous gene may be inserted into intron 2 of CLYBL. An exogenous polynucleotide encoding an exogenous gene may be inserted into a 500 bp window in Ch-4:58,976,613 (i.e., SHS231). An exogenous polynucleotide encoding an exogenous gene may be inserted into any suitable region of the aforementioned safe harbor or target locus that allows expression of the exogenous gene, including, for example, an intron, exon, or coding sequence region within the safe harbor or target locus.

[0296] The term "sensitized" as used in reference to a patient refers to a patient who has antibodies reactive to foreign cells. In some embodiments, the present disclosure contemplates the treatment of sensitized subjects. For example, subjects contemplated for the present treatment methods are sensitized to or against one or more alloantigens, including Y-chromosome-linked antigens. In some embodiments, the patient is sensitized from a previous pregnancy or a previous allogeneic transplant (e.g., including, but not limited to, allogeneic cell transplant, allogeneic blood transfusion, allogeneic tissue transplant, and allogeneic organ transplant). In some embodiments, the patient exhibits memory B cells and / or memory T cells reactive to one or more alloantigens.

[0297] In some embodiments, the present disclosure contemplates the treatment of unsensitized subjects. For example, subjects contemplated for the present treatment methods are not sensitized to or against one or more alloantigens, including Y-linked antigens. In some embodiments, the patient is not sensitized from a previous pregnancy or a previous allogeneic transplant (e.g., including, but not limited to, allogeneic cell transplant, allogeneic blood transfusion, allogeneic tissue transplant, and allogeneic organ transplant). In some embodiments, the patient does not exhibit memory B cells and / or memory T cells reactive to one or more alloantigens.

[0298] As used herein, "target" may refer to a gene, a portion of a gene, a portion of a genome, or a protein whose expression is modulatable and reduced by the methods described herein.

[0299] 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, 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.

[0300] As used herein, the terms "treating" and "treatment" include administering a therapeutically effective amount or a clinically effective amount of a cell described herein to a subject so 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, 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). Treating can also refer to extending survival compared to the expected survival in the absence of treatment. Thus, those skilled in the art recognize that treatment may improve a condition 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% upon treatment of the condition, disease, or disorder.

[0301] 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, delay or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or complete).

[0302] A "vector" or "construct" is capable of transferring a gene sequence into a target cell. Typically, the terms "vector construct," "expression vector," and "gene transfer vector" refer to any nucleic acid construct that can direct the expression of a gene of interest and transfer the gene sequence into a target cell. Thus, the term includes cloning and expression vehicles as well as integrating vectors. Methods for introducing a vector or construct into a cell 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.

[0303] 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 or control cells. "Wild-type" or "wild-type (wt)" or "control" in the context of cells refers to any cell found in nature. Examples of wild-type or control cells include primary cells and T cells found in nature. However, by way of example, as used herein, in the context of engineered T cells and / or hypoimmunogenic T cells, "wild-type" or "control" can also refer to engineered T cells and / or hypoimmunogenic T cells that may contain nucleic acid alterations that result in reduced expression of one or more MHC class I molecules and / or class II molecules and / or T cell receptors, and / or overexpression of CD47 protein, but that have not undergone the gene editing procedures of the present disclosure to achieve reduced expression of one or more Y chromosome genes. For example, as used herein, "wild-type" or "control" refers to engineered cells in which expression of B2M, CIITA, and / or TRAC has been reduced or knocked out. Also as used herein, "wild-type" or "control" refers to engineered cells in which expression of B2M, CIITA, TRAC, and / or TRBC has been reduced or knocked out. As used herein, "wild-type" or "control" also refers to engineered cells that may contain nucleic acid changes that result in overexpression of CD47 protein, but have not undergone a gene editing procedure to achieve reduced expression of one or more MHC class I molecules 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 changes that result in pluripotency, but have not undergone a gene editing procedure of the present disclosure to achieve reduced expression of one or more MHC class I molecules and / or class II molecules and / or T cell receptors and / or one or more Y chromosome genes, and / or overexpression of CD47 protein.For example, as used herein, "wild-type" or "control" refers to an iPSC or its progeny in which expression of B2M, CIITA, and / or TRAC has been reduced or knocked out. Also as used herein, "wild-type" or "control" refers to an iPSC or its progeny in which expression of B2M, CIITA, TRAC, and / or TRBC has been reduced or knocked out. 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 nucleic acid alterations that result in reduced expression of one or more MHC class I molecules and / or class II molecules and / or T cell receptors, but that has not undergone a gene editing procedure of the present disclosure to achieve reduced expression of one or more Y chromosome genes. For example, as used herein, "wild-type" or "control" refers to a primary T cell or its progeny in which expression of B2M, CIITA, and / or TRAC has been reduced or knocked out, but that has not undergone a gene editing procedure to achieve reduced expression of one or more Y chromosome genes. Also as used herein, "wild-type" or "control" refers to a primary T cell, or its progeny, in which expression of B2M, CIITA, TRAC, and / or TRBC has been reduced or knocked out, but which has not undergone a gene editing procedure to achieve the reduced expression of one or more Y chromosome genes. 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, which may contain a nucleic acid alteration that results in overexpression of CD47 protein, but which has not undergone a gene editing procedure to achieve the reduced expression of one or more Y chromosome genes. In some embodiments, the wild-type or control cell is the starting material. In some embodiments, the starting material is otherwise modified or engineered to alter the expression of one or more genes. In some embodiments, "wild-type" or "control" refers to a cell that has a Y chromosome.

[0304] As used herein, "Y chromosome-linked antigen," "Y chromosome antigen," "histocompatibility Y chromosome-linked antigen," or "male-specific antigen," and variations thereof, refer to peptides encoded by genes on the Y chromosome that are capable of eliciting an immune response. In particular, peptides can elicit an immune response when presented in the context of MHC molecules and / or when antibodies against the peptide are present. Y chromosome-linked antigens include antigens that are antigenic portions of or are the entire protein encoded by genes on the Y chromosome. Examples of Y chromosome-linked antigens include, but are not limited to, protocadherin-11 Y-linked (PCDH11Y), neuroligin-4 Y-linked (NLGN4Y), HY antigen, and the like.

[0305] It should be noted that the claims may be drafted to exclude any optional element. Accordingly, 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 a "negative" limitation. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features that may be readily separated from or combined with features of any of the other embodiments without departing from the scope or spirit of the present 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 can also be used in the practice or testing of the present disclosure, representative exemplary methods and materials are now described.

[0306] 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 technology 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 the tenth of the unit of the lower limit unless the context clearly dictates otherwise, as well as any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in 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 value. The term "about" is used herein to provide literal support for the number it precedes, as well as for a number that is close to or approximately the number it precedes. When determining whether a number is near or approximately a specifically recited number, the near or approximately unrecited number may be a number that, in the context provided, 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.

[0307] 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, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0308] Before further describing the present technology, it should be understood that the present technology is not limited to the particular embodiments described, and thus may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present disclosure is limited only by the appended claims. It should also be understood that the headings used herein are not limiting, but are intended only to provide orientation to the reader, but that their content generally applies to the technology disclosed herein.

[0309] III. MODE FOR CARRYING OUT THE INVENTION A. Low immunogenic cells In some embodiments, the present disclosure provides engineered (e.g., modified and genetically engineered) cells that comprise reduced expression of one or more Y chromosome genes and MHC class I and / or MHC class II human leukocyte antigen molecules relative to an unaltered or unmodified wild-type or control cell, and that comprise a first exogenous polynucleotide encoding CD47, wherein the engineered cells are propagated from primary T cells or their progeny, induced pluripotent stem cells (iPSCs) or their progeny. In some embodiments, the cells are also capable of avoiding activation of NK cell-mediated and / or antibody-based immune responses.

[0310] 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 subpopulations thereof, neural cells and subpopulations thereof, brain endothelial cells and subpopulations thereof, dopaminergic neurons and subpopulations thereof, glial progenitor cells and subpopulations thereof, endothelial cells and subpopulations thereof, thyroid cells and subpopulations thereof, hepatocytes and subpopulations thereof, pancreatic islet cells and subpopulations thereof, or retinal pigment epithelial cells and subpopulations thereof. In some embodiments, the differentiated cells are T cells and subpopulations thereof, NK cells and subpopulations thereof, and endothelial cells and subpopulations thereof. In some embodiments, the primary immune cells are T cells and subpopulations thereof and NK cells and subpopulations thereof. In some embodiments, the primary tissue cells include primary endothelial cells and subpopulations thereof.

[0311] In some embodiments, the cells described herein comprise reduced expression of one or more Y chromosome genes and MHC class I and / or MHC class II human leukocyte antigen molecules compared to unaltered or unmodified wild-type or control cells. In some embodiments, the cells described herein comprise reduced expression of protocadherin-11 Y chain and / or neuroligin-4 Y chain and MHC class I and / or MHC class II human leukocyte antigen molecules compared to unaltered or unmodified wild-type or control cells. In some embodiments, the cells described herein comprise reduced expression of protocadherin-11 Y chain and MHC class I and / or MHC class II human leukocyte antigen molecules compared to unaltered or unmodified wild-type or control cells. In some embodiments, the cells described herein comprise reduced expression of neuroligin-4 Y chain and MHC class I and / or MHC class II human leukocyte antigen molecules compared to unaltered or unmodified wild-type or control cells. In some embodiments, the cells described herein comprise reduced expression of protocadherin-11 Y-chain and neuroligin-4 Y-chain and MHC class I and / or MHC class II human leukocyte antigen molecules compared to an unaltered or unmodified wild-type or control cell. In some embodiments, the cells described herein comprise a first exogenous polynucleotide encoding CD47. In some embodiments, the cells described herein comprise a second exogenous polynucleotide encoding a CAR.

[0312] 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 (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), and primary cells (such as, but not limited to, primary T cells and primary NK cells). In some embodiments, pluripotent stem cells, differentiated cells derived therefrom, e.g., 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 reduced or absent expression of one or more Y chromosome genes and MHC class I and / or MHC class II human leukocyte antigen molecules, and in some cases, for reduced or absent expression of the T cell receptor (TCR) complex. In some embodiments, hypoimmune (HIP) T cells and primary T cells overexpress CD47 and a chimeric antigen receptor (CAR) and have reduced or absent expression of the T cell receptor (TCR) complex, in addition to reduced or absent expression of one or more Y chromosome genes and MHC class I and / or MHC class II human leukocyte antigen molecules. In some embodiments, the CAR comprises an antigen-binding domain that binds to any one selected from the group consisting of CD19, CD20, CD22, CD38, CD123, CD138, and BCMA. In some embodiments, the CAR is a CD19-specific CAR. In some embodiments, the CAR is a CD20-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 / CD20 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 CD20-specific CAR, 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 CD20-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, a CD19-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 CD20-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 CD20-specific CAR, 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 CD20-specific CAR, 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 CD20-specific CAR, 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 as well as a different CAR, such as, but not limited to, a CD20-specific CAR, a CD22-specific CAR, a CD38-specific CAR, a CD123-specific CAR, a CD138-specific CAR, and a CD19-specific CAR.

[0313] In some embodiments, iPSC-derived hypoimmune cells, 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, overexpress CD47 and comprise a genomic modification or knockout or knockdown of the PCDH11Y gene. In some embodiments, iPSC-derived hypoimmune cells, 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, overexpress CD47 and comprise a genomic modification or knockout or knockdown of the NLGN4Y gene. In some embodiments, iPSC-derived hypoimmune cells, including but not limited to T cells, NK cells, cardiac cells, neurons, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, pancreatic islet cells, and retinal pigment epithelial cells, overexpress CD47 and comprise a genomic modification or knockout or knockdown of the B2M gene. In some embodiments, iPSC-derived hypoimmune cells, including but not limited to T cells, NK cells, cardiac cells, neurons, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, pancreatic islet cells, and retinal pigment epithelial cells, overexpress CD47 and comprise a genomic modification or knockout or knockdown of the CIITA gene. In some embodiments, the cells are derived from iPSCs. - / - ,NLGN4Y - / - , B2M - / - , C.I.T.A. - / - In some embodiments, the cells are PCDH11Y, CD47tg cells. - / - ,NLGN4Y - / - , B2M- / - In some embodiments, the cells are PCDH11Y, CD47tg cells. インデル / インデル ,NLGN4Y インデル / インデル , 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 PCDH11Y, CD47tg cells. インデル / インデル ,NLGN4Y インデル / インデル , B2M インデル / インデル In some embodiments, the cells are PCDH11Y, CD47tg cells. ノックダウン ,NLGN4Y ノックダウン , B2M ノックダウン , C.I.T.A. ノックダウン In some embodiments, the cells are PCDH11Y, CD47tg cells. ノックダウン ,NLGN4Y ノックダウン , B2M ノックダウン , CD47tg cells. In some embodiments, iPSC-derived hypoimmune cells are produced by differentiating induced pluripotent stem cells, such as hypoimmunogenic induced pluripotent stem 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 is otherwise modified or engineered to alter the expression of one or more genes to generate the engineered cells.

[0314] 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, overexpress CD47 and comprise a genomic modification or knockout or knockdown of the PCDH11Y 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, overexpress CD47 and comprise a genomic modification or knockout or knockdown of the NLGN4Y 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, overexpress CD47 and comprise a genomic modification or 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, overexpress CD47 and comprise a genomic modification or knockout or knockdown of the CIITA gene. In some embodiments, the cells are derived from PCDH11Y - / - ,NLGN4Y - / - , B2M - / - , C.I.T.A. - / - In some embodiments, the cells are PCDH11Y, CD47tg cells. - / - ,NLGN4Y - / - , B2M - / - In some embodiments, the cells are PCDH11Y, CD47tg cells. インデル / インデル ,NLGN4Y インデル / インデル , 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 PCDH11Y, CD47tg cells. インデル / インデル ,NLGN4Yインデル / インデル , B2M インデル / インデル In some embodiments, the cells are PCDH11Y, CD47tg cells. ノックダウン ,NLGN4Y ノックダウン , B2M ノックダウン , C.I.T.A. ノックダウン In some embodiments, the cells are PCDH11Y, CD47tg cells. ノックダウン ,NLGN4Y ノックダウン , B2M ノックダウン , CD47tg cells. In some embodiments, iPSC-derived hypoimmune cells are produced by differentiating induced pluripotent stem cells, such as hypoimmunogenic embryonic stem 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 is otherwise modified or engineered to alter the expression of one or more genes to generate the engineered cells.

[0315] In some embodiments, hypoimmune (HIP) T cells and primary T cells derived from iPSCs overexpress CD47 and a chimeric antigen receptor (CAR) and comprise a genomic modification or knockout or knockdown of the PCDH11Y gene. In some embodiments, hypoimmune (HIP) T cells and primary T cells derived from iPSCs overexpress CD47 and a chimeric antigen receptor (CAR) and comprise a genomic modification or knockout or knockdown of the NLGN4Y gene. In some embodiments, hypoimmune (HIP) T cells and primary T cells derived from iPSCs overexpress CD47 and a chimeric antigen receptor (CAR) and comprise a genomic modification or knockout or knockdown of the B2M gene. In some embodiments, hypoimmune (HIP) T cells and primary T cells derived from iPSCs overexpress CD47 and comprise a genomic modification or knockout or knockdown of the CIITA gene. In some embodiments, hypoimmune (HIP) T cells and primary T cells derived from iPSCs overexpress CD47 and CAR and comprise a genomic modification or knockout or knockdown of the TRAC gene. In some embodiments, hypoimmune (HIP) T cells and primary T cells derived from iPSCs overexpress CD47 and CAR and comprise a genomic modification or knockout or knockdown of the TRB gene. In some embodiments, hypoimmune (HIP) T cells and primary T cells derived from iPSCs overexpress CD47 and CAR and comprise a genomic modification or knockout or knockdown of one or more genes selected from the group consisting of PCDH11Y, NLGN4Y, B2M, CIITA, TRAC, and TRB genes. In some embodiments, hypoimmune (HIP) T cells and primary T cells derived from iPSCs overexpress CD47 and CAR and comprise a genomic modification or knockout or knockdown of the PCDH11Y, NLGN4Y, B2M, CIITA, TRAC, and TRB genes. In some embodiments, the cells also express a CAR, B2M - / - , C.I.T.A. - / - ,TRAC - / - In some embodiments, the cells are B2M, CD47tg cells that also express a CAR. - / -,TRAC - / - , CD47tg cells. In some embodiments, hypoimmune (HIP) T cells are produced by differentiating induced pluripotent stem cells, such as hypoimmunogenic induced pluripotent stem 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 is otherwise modified or engineered to alter the expression of one or more genes to generate the engineered cells.

[0316] In some embodiments, hypoimmune (HIP) T cells and primary T cells derived from iPSCs also express a CAR, PCDH11Y - / - ,NLGN4Y - / - , B2M - / - , C.I.T.A. - / - , TRB - / - In some embodiments, iPSC-derived hypoimmune (HIP) T cells and primary T cells also express a CAR, PCDH11Y - / - ,NLGN4Y - / - , B2M - / - , TRB - / - In some embodiments, the cells are PCDH11Y, CD47tg cells that also express a CAR. - / - ,NLGN4Y - / - ,B2M - / - , C.I.T.A. - / - ,TRAC - / - , TRB - / - In some embodiments, the cells are PCDH11Y, CD47tg cells that also express a CAR. - / - ,NLGN4Y - / - ,B2M - / - ,TRAC - / - , TRB - / - In certain embodiments, the cells are PCDH11Y, CD47tg cells that also express a CAR. インデル / インデル ,NLGN4Y インデル / インデル , 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 certain embodiments, the cells are PCDH11Y, CD47tg cells that also express a CAR. インデル / インデル ,NLGN4Y インデル / インデル , B2M インデル / インデル ,TRAC インデル / インデル In certain embodiments, the cells are PCDH11Y, CD47tg cells that also express a CAR. インデル / インデル ,NLGN4Y インデル / インデル , B2M インデル / インデル , C.I.T.A. インデル / インデル , TRB インデル / インデル In certain embodiments, the cells are PCDH11Y, CD47tg cells that also express a CAR. インデル / インデル ,NLGN4Y インデル / インデル , B2M インデル / インデル , TRB インデル / インデル In certain embodiments, the cells are PCDH11Y, CD47tg cells that also express a CAR. インデル / インデル ,NLGN4Y インデル / インデル , 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 , TRB インデル / インデル In certain embodiments, the cells are PCDH11Y, CD47tg cells that also express a CAR. インデル / インデル ,NLGN4Y インデル / インデル , B2M インデル / インデル ,TRAC インデル / インデル , TRB インデル / インデル In some embodiments, the cells are PCDH11Y, CD47tg cells that also express a CAR. ノックダウン ,NLGN4Y ノックダウン , 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 certain embodiments, the cells are PCDH11Y, CD47tg cells that also express a CAR. ノックダウン ,NLGN4Y ノックダウン , B2M ノックダウン , C.I.T.A. ノックダウン , TRB ノックダウン In certain embodiments, the cells are PCDH11Y, CD47tg cells that also express a CAR. ノックダウン,NLGN4Y ノックダウン , B2M ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン , TRB ノックダウン In some embodiments, the cells are PCDH11Y, CD47tg cells that also express a CAR. ノックダウン ,NLGN4Y ノックダウン , B2M ノックダウン ,TRAC ノックダウン In certain embodiments, the cells are PCDH11Y, CD47tg cells that also express a CAR. ノックダウン ,NLGN4Y ノックダウン , B2M ノックダウン , TRB ノックダウン In certain embodiments, the cells are PCDH11Y, CD47tg cells that also express a CAR. ノックダウン ,NLGN4Y ノックダウン , B2M ノックダウン ,TRAC ノックダウン , TRB ノックダウン , CD47tg 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 is otherwise modified or engineered to alter the expression of one or more genes to generate the engineered cells.

[0317] In some embodiments, the engineered or modified cells described 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, putative memory T cells, innate 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 unchanged 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 alter the expression of one or more genes to generate the engineered cells.

[0318] 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 receiving the cells). 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 and cultured in vitro.

[0319] In certain embodiments, primary T cells or pools of primary T cells are engineered to 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 consisting of CD19, CD20, CD22, CD38, CD123, CD138, and BCMA. In some cases, the CAR is the same as or equivalent to those used in FDA-approved CAR-T cell therapies, such as, but not limited to, tisagenlecleucel and axicabtageneciloreucel, or others under investigation in clinical trials.

[0320] In some embodiments, primary T cells or pools of primary T cells are engineered to exhibit reduced expression of endogenous T cell receptors relative to unmodified primary T cells. In certain embodiments, primary T cells or pools of primary T cells are engineered to exhibit reduced expression of CTLA-4, PD-1, or both CTLA-4 and PD-1 relative 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 entireties, including tables, appendices, sequence listings, and figures.

[0321] 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.

[0322] In some embodiments, the CAR-T cells comprise 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 CD20 antigen-binding domain. In some embodiments, the CAR comprises a CD22-binding domain. In some embodiments, the CAR comprises a BCMA-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: 99). 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 disease / disorder and / or an inflammatory disease / 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.

[0323] In some embodiments, the CAR further comprises one or more linkers. The scFv format generally comprises 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 in the art in light of this specification can be used in a CAR. Examples of suitable linkers include, but are not limited to, GS-based linker sequences and the Whitlow linker GSTSGSGKPGSGEGSTKG (SEQ ID NO: 99). In some embodiments, the linker is a GS or gly-ser linker. An exemplary gly-ser polypeptide linker has the amino acid sequence Ser (Gly4Ser) n , and (Gly4Ser) nand / or (Gly4Ser3) n In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3, i.e., Ser(Gly4Ser)3. In some embodiments, n=4, i.e., Ser(Gly4Ser)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 comprises the amino acid sequence Ser(Gly4Ser) n In some embodiments, n=1. 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 comprises (Gly4Ser)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 comprises (Gly3Ser)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 comprises (Gly3Ser)n. 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 (Gly4Ser3) 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 (Gly3Ser) nIn 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.

[0324] 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. In some embodiments, the antigen-binding domain is an anti-CD20 scFv. In some embodiments, the antigen-binding domain is an anti-CD22 scFv. In some embodiments, the antigen-binding domain is an anti-BCMA scFv.

[0325] In some embodiments, the transmembrane domain comprises one selected from the group comprising the transmembrane region of TCRα, TCRβ, TCRζ, CD3ε, CD3γ, CD3δ, CD3ζ, CD4, CD5, CD8α, CD8β, CD9, CD16, CD28, CD45, CD22, CD33, CD34, CD37, CD40, CD40L / CD154, CD45, CD64, CD80, CD86, OX40 / CD134, 4-1BB / CD137, CD154, FcεRIγ, VEGFR2, FAS, FGFR2B, and functional variants thereof.

[0326] In some embodiments, the signaling domain(s) of the CAR comprise costimulatory domain(s). For example, the signaling domain(s) can contain a costimulatory domain(s). Alternatively, the signaling domain(s) can contain one or more costimulatory domains. In certain embodiments, the signaling domain(s) comprise a costimulatory domain(s). In other embodiments, the signaling domain(s) comprise costimulatory domain(s). In some cases, when a CAR comprises two or more costimulatory domains, the two costimulatory domains are not the same. In some embodiments, the costimulatory domains comprise two costimulatory domains that are not the same. In some embodiments, the costimulatory domains enhance cytokine production during T cell activation, CAR-T cell proliferation, and / or CAR-T cell persistence. In some embodiments, the costimulatory domains enhance cytokine production during T cell activation, CAR-T cell proliferation, and / or CAR-T cell persistence.

[0327] 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 CAR signaling. In some cases, the cytokine gene is an endogenous or exogenous cytokine gene of the engineered and / or 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 CAR signaling comprises a transcription factor or a functional domain or fragment thereof.

[0328] 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 other embodiments, the CAR comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-based 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-based 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.

[0329] Methods for introducing CAR constructs or producing 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.

[0330] In some embodiments, cells derived from primary T cells comprise reduced expression of an endogenous T cell receptor, e.g., by disruption of an endogenous T cell receptor gene (e.g., the T cell receptor alpha constant region (TRAC) or the T cell receptor beta constant region (TRB)). In some embodiments, an exogenous nucleic acid encoding a polypeptide as 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.

[0331] In some embodiments, cells derived from primary T cells contain 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, or both CTLA4 and PD1 can include any of those 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 nuclease, meganuclease, and / or homing endonucleases. In some embodiments, an exogenous nucleic acid encoding a polypeptide as disclosed herein (e.g., a chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein) is inserted at the CTLA4 and / or PD1 locus.

[0332] In some embodiments, the CD47 transgene 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, the CD47 transgene and the transgene encoding a CAR are inserted into preselected loci of the cell. The preselected locus can be a safe harbor or target locus. Non-limiting examples of safe harbor or target loci include, but are not limited to, the CCR5 locus, the PPP1R12C (also known as AAVS1) locus, the CLYBL locus, and 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. The CD47 transgene may be inserted into intron 1 or 2 of PPP1R12C (i.e., AAVS1) or CCR5. The CD47 transgene may be inserted into exon 1, 2, or 3 of CCR5. The CD47 transgene may be inserted into intron 2 of CLYBL. The CD47 transgene may be inserted into a 500 bp window in Ch-4:58,976,613 (i.e., SHS231). The CD47 transgene may be inserted into any suitable region of the aforementioned safe harbor or target locus that allows expression of the exogenous gene, including, for example, an intron, exon, or coding sequence region within 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.

[0333] In some embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into the same locus. In some embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into different loci. In many cases, the CD47 transgene 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 CD47 transgene is inserted into the B2M locus. In some cases, the transgene encoding the CAR is inserted into the B2M locus. In certain cases, the CD47 transgene is inserted into the CIITA locus. In certain cases, the transgene encoding the CAR is inserted into the CIITA locus. In certain cases, the CD47 transgene 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 CD47 transgene is inserted into the TRB locus. In many other cases, the transgene encoding the CAR is inserted into the TRB locus. In some embodiments, the CD47 transgene 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).

[0334] In certain embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into a safe harbor or target locus. In certain embodiments, the CD47 transgene 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 CD47 transgene 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 CD47 transgene and the transgene encoding the CAR are inserted into the TRAC locus. In certain embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by a single promoter and inserted into the TRAC locus. In certain embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by their own promoters and inserted into the TRAC locus. In some embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into the TRB locus. In some embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by a single promoter and inserted into the TRB locus. In some embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by their own promoters and inserted into the TRB locus. In other embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into the B2M locus. In other embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by a single promoter and inserted into the B2M locus. In other embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by their own promoters and inserted into the B2M locus. In various embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into the CIITA locus. In various embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by a single promoter and inserted into the CIITA locus.In various embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by their own promoters and inserted within the CIITA locus.

[0335] In some cases, the promoter controlling expression of any of the described transgenes is a constitutive 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 CD47 transgene and the transgene encoding a CAR are both controlled by a constitutive promoter. In some embodiments, the CD47 transgene and the transgene encoding a CAR are both controlled by an inducible promoter. In some embodiments, the CD47 transgene is controlled by a constitutive promoter and the transgene encoding a CAR is controlled by an inducible promoter. In some embodiments, the CD47 transgene is controlled by an inducible promoter and the transgene encoding a CAR is controlled by a constitutive promoter. In various embodiments, the CD47 transgene is controlled by an EF1α promoter and the transgene encoding a CAR is controlled by an EF1α promoter. In some embodiments, the CD47 transgene is controlled by a CAG promoter and the transgene encoding a CAR is controlled by a CAG promoter. In some embodiments, the CD47 transgene is controlled by a CAG promoter and the transgene encoding a CAR is controlled by an EF1 alpha promoter. In some embodiments, the CD47 transgene is controlled by an EF1 alpha promoter and the transgene encoding a CAR is controlled by a CAG promoter. In some embodiments, expression of both the CD47 transgene and the transgene encoding a CAR is controlled by a single EF1 alpha promoter. In some embodiments, expression of both the CD47 transgene and the transgene encoding a CAR is controlled by a single CAG promoter.

[0336] 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 (HIP) 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 T cells that overexpress CD47 (e.g., exogenously express CD47 protein), have reduced or absent expression of MHC class I and / or MHC class II human leukocyte antigen molecules, and have reduced or absent expression of T cell receptor (TCR) complexes. In some embodiments, hypoimmune (HIP) T cells and primary T cells overexpress CD47 (e.g., exogenously express CD47 protein), have reduced or absent expression of one or more MHC class I and / or MHC class II human leukocyte antigen molecules, and have reduced or absent expression of the T cell receptor (TCR) complex.

[0337] 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 (HIP) 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 T cells overexpress CD47 and comprise a genomic modification of the B2M gene. In some embodiments, pluripotent stem cells, differentiated cells derived from such pluripotent stem cells, and primary T cells overexpress CD47 and comprise a genomic modification of the CIITA gene. In some embodiments, pluripotent stem cells, differentiated cells derived from such pluripotent stem cells, including 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, are expressed as PCDH11Y. - / - ,NLGN4Y - / -, B2M - / - , C.I.T.A. - / - In some embodiments, the cells are PCDH11Y, CD47tg cells. - / - ,NLGN4Y - / - , B2M - / - In some embodiments, the cells are PCDH11Y, CD47tg cells. インデル / インデル ,NLGN4Y インデル / インデル , 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 PCDH11Y, CD47tg cells. インデル / インデル ,NLGN4Y インデル / インデル , B2M インデル / インデル In some embodiments, the cells are PCDH11Y, CD47tg cells. ノックダウン ,NLGN4Y ノックダウン , B2M ノックダウン , C.I.T.A. ノックダウン In some embodiments, the cells are PCDH11Y, CD47tg cells. ノックダウン ,NLGN4Y ノックダウン , B2M 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 overexpress CD47 and comprise a genomic modification in the PCDH11Y gene. In some embodiments, the pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells overexpress CD47 and comprise a genomic modification in the NLGN4Y gene. In some embodiments, the pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells overexpress CD47 and comprise a 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 overexpress CD47 and comprise a 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 overexpress CD47 and comprise one or more genomic modifications selected from the group consisting of PCDH11Y, NLGN4Y, 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 overexpress CD47 and comprise genomic modifications in PCDH11Y, NLGN4Y, B2M, CIITA, and TRAC genes. In some embodiments, the pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells overexpress CD47 and comprise genomic modifications in PCDH11Y, NLGN4Y, B2M, CIITA, and TRB genes. In some embodiments, the pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells overexpress CD47 and comprise genomic modifications in the PCDH11Y, NLGN4Y, 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 overexpress CD47 and comprise genomic modifications in the PCDH11Y, NLGN4Y, B2M, CIITA, TRAC, and TRB genes. - / - ,NLGN4Y - / - , B2M - / - , C.I.T.A. - / - ,TRAC - / - , CD47tg cells. In certain embodiments, the cells are PCDH11Y - / - ,NLGN4Y - / - , B2M - / - ,TRAC - / -, CD47tg cells. In certain embodiments, the cells are PCDH11Y - / - ,NLGN4Y - / - , B2M - / - , C.I.T.A. - / - , TRB - / - , CD47tg cells. In certain embodiments, the cells are PCDH11Y - / - ,NLGN4Y - / - , B2M - / - , TRB - / - , CD47tg cells. In certain embodiments, the cells are PCDH11Y - / - ,NLGN4Y - / - , B2M - / - , C.I.T.A. - / - ,TRAC - / - , TRB - / - , CD47tg cells. In certain embodiments, the cells are PCDH11Y - / - ,NLGN4Y - / - , B2M - / - ,TRAC - / - , TRB - / - In some embodiments, the cells are PCDH11Y, CD47tg cells. インデル / インデル ,NLGN4Y インデル / インデル , 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 cells are PCDH11Y, CD47tg cells. インデル / インデル ,NLGN4Y インデル / インデル , B2M インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are PCDH11Y, CD47tg cells. インデル / インデル ,NLGN4Y インデル / インデル , B2M インデル / インデル , C.I.T.A. インデル / インデル , TRB インデル / インデル In some embodiments, the cells are PCDH11Y, CD47tg cells. インデル / インデル ,NLGN4Y インデル / インデル , B2M インデル / インデル , TRB インデル / インデル In some embodiments, the cells are PCDH11Y, CD47tg cells. インデル / インデル,NLGN4Y インデル / インデル , 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 , TRB インデル / インデル In some embodiments, the cells are PCDH11Y, CD47tg cells. インデル / インデル ,NLGN4Y インデル / インデル , B2M インデル / インデル ,TRAC インデル / インデル , TRB インデル / インデル In some embodiments, the cells are PCDH11Y, CD47tg cells. ノックダウン ,NLGN4Y ノックダウン , 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 cells are PCDH11Y, CD47tg cells. ノックダウン ,NLGN4Y ノックダウン , B2M ノックダウン , C.I.T.A. ノックダウン , TRB ノックダウン In some embodiments, the cells are PCDH11Y, CD47tg cells. ノックダウン ,NLGN4Y ノックダウン , B2M ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン , TRB ノックダウン In some embodiments, the cells are PCDH11Y, CD47tg cells. ノックダウン ,NLGN4Y ノックダウン , B2M ノックダウン ,TRAC ノックダウン In some embodiments, the cells are PCDH11Y, CD47tg cells. ノックダウン ,NLGN4Y ノックダウン , B2M ノックダウン , TRB ノックダウン In some embodiments, the cells are PCDH11Y, CD47tg cells. ノックダウン ,NLGN4Y ノックダウン , B2M ノックダウン ,TRAC ノックダウン , TRB 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, putative memory T cells, innate 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 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 is otherwise modified or engineered to alter the expression of one or more genes to generate the engineered cells.

[0338] In some embodiments, the CD47 transgene 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 or target loci include the CCR5 locus, CXCR4 locus, PPP1R12C locus, albumin locus, SHS231 locus, CLYBL locus, Rosa locus, F3 (CD142) locus, MICA locus, MICB locus, LRP1 (CD91) locus, HMGB1 locus, ABO locus, RHD locus, FUT1 locus, and KDM5D locus. In some embodiments, the preselected locus is the TRAC locus. In some embodiments, the CD47 transgene 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 CD47 transgene is inserted into the B2M locus. In certain embodiments, the CD47 transgene is inserted into the B2M locus. In certain embodiments, the CD47 transgene is inserted into the TRAC locus. In certain embodiments, the CD47 transgene is inserted into the TRB locus.

[0339] In some cases, expression of the CD47 transgene is controlled by a constitutive promoter. In other cases, expression of the CD47 transgene is controlled by an inducible promoter. In some embodiments, the promoter is an EF1 alpha (EF1α) promoter. In some embodiments, the promoter is a CAG promoter.

[0340] 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 derived from such pluripotent stem cells (e.g., hypoimmune (HIP) T cells), and primary T cells that have reduced or absent expression of one or more Y chromosome genes and MHC class I and / or MHC class II human leukocyte antigen molecules, and have reduced or absent expression of T cell receptor (TCR) complexes. In some embodiments, the cells have reduced or absent expression of one or more Y chromosome genes and MHC class I antigen molecules, MHC class II antigen molecules, and TCR complexes.

[0341] In some embodiments, pluripotent stem cells (e.g., iPSCs), differentiated cells derived therefrom (e.g., 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 differentiated therefrom), and primary T cells comprise a genomic modification or knockdown of the PCDH11Y gene. In some embodiments, pluripotent stem cells (e.g., iPSCs), differentiated cells derived therefrom (e.g., 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 differentiated therefrom), and primary T cells comprise a genomic modification or knockdown of the NLGN4Y gene. In some embodiments, pluripotent stem cells (e.g., iPSCs), differentiated cells derived therefrom (e.g., 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 differentiated therefrom), and primary T cells comprise a genomic modification or knockdown of the B2M gene. In some embodiments, pluripotent stem cells (e.g., iPSCs), differentiated cells derived therefrom (e.g., 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 differentiated therefrom), and primary T cells comprise a genomic modification or 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, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, pancreatic islet cells, and retinal pigment epithelial cells, are expressed using PCDH11Y. - / - ,NLGN4Y - / - , B2M - / - , C.I.T.A. - / - In some embodiments, the cell is a PCDH11Y - / - ,NLGN4Y - / - , B2M - / - In some embodiments, the cell is a PCDH11Yインデル / インデル ,NLGN4Y インデル / インデル , 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 a PCDH11Y インデル / インデル ,NLGN4Y インデル / インデル , B2M インデル / インデル In some embodiments, the cell is a PCDH11Y ノックダウン ,NLGN4Y ノックダウン , B2M ノックダウン , C.I.T.A. ノックダウン In some embodiments, the cell is a PCDH11Y ノックダウン ,NLGN4Y ノックダウン , B2M ノックダウンIn some embodiments, the pluripotent stem cells (e.g., ESCs or iPSCs), T cells differentiated therefrom, and primary T cells comprise a genomic modification or knockdown of the PCDH11Y gene. In some embodiments, the pluripotent stem cells (e.g., ESCs or iPSCs), T cells differentiated therefrom, and primary T cells comprise a genomic modification or knockdown of the NLGN4Y gene. In some embodiments, the pluripotent stem cells (e.g., ESCs or iPSCs), T cells differentiated therefrom, and primary T cells comprise a genomic modification or knockdown of the TRAC gene. In some embodiments, the pluripotent stem cells (e.g., iPSCs), T cells differentiated therefrom, and primary T cells comprise a genomic modification or knockdown of the TRB gene. In some embodiments, the pluripotent stem cells (e.g., iPSCs), T cells differentiated therefrom, and primary T cells comprise a genomic modification or knockdown of one or more genes selected from the group consisting of B2M, CIITA, and TRAC genes. In some embodiments, the pluripotent stem cells (e.g., iPSCs), T cells differentiated therefrom, and primary T cells comprise one or more genomic modifications or knockdowns selected from the group consisting of PCDH11Y, NLGN4Y, B2M, CIITA, and TRB genes. In some embodiments, the pluripotent stem cells (e.g., iPSCs), T cells differentiated therefrom, and primary T cells comprise one or more genomic modifications or knockdowns selected from the group consisting of PCDH11Y, NLGN4Y, B2M, CIITA, TRAC, and TRB genes. In certain embodiments, cells, including iPSCs, T cells differentiated therefrom, and primary T cells, comprise one or more genomic modifications or knockdowns selected from the group consisting of PCDH11Y, NLGN4Y, B2M, CIITA, TRAC, and TRB genes. - / - ,NLGN4Y - / - , B2M - / - , C.I.T.A. - / - ,TRAC - / - In certain embodiments, the cells, including iPSCs, T cells differentiated therefrom, and primary T cells, are PCDH11Y - / - ,NLGN4Y - / - , B2M - / - ,TRAC - / -In certain embodiments, the cells, including iPSCs, T cells differentiated therefrom, and primary T cells, are PCDH11Y - / - ,NLGN4Y - / - , B2M - / - , C.I.T.A. - / - , TRB - / - In certain embodiments, the cells, including iPSCs, T cells differentiated therefrom, and primary T cells, are PCDH11Y - / - ,NLGN4Y - / - , B2M - / - , TRB - / - In certain embodiments, the cells, including iPSCs, T cells differentiated therefrom, and primary T cells, are PCDH11Y - / - ,NLGN4Y - / - , B2M - / - , C.I.T.A. - / - ,TRAC - / - , TRB - / - In certain embodiments, the cells, including iPSCs, T cells differentiated therefrom, and primary T cells, are PCDH11Y - / - ,NLGN4Y - / - , B2M - / - ,TRAC - / - , TRB - / - In some embodiments, the cells, including iPSCs, T cells differentiated therefrom, and primary T cells, are PCDH11Y インデル / インデル ,NLGN4Y インデル / インデル , 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 cells, including iPSCs, T cells differentiated therefrom, and primary T cells, are PCDH11Y インデル / インデル ,NLGN4Y インデル / インデル , B2M インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells, including iPSCs, T cells differentiated therefrom, and primary T cells, are PCDH11Y インデル / インデル ,NLGN4Y インデル / インデル , B2M インデル / インデル , C.I.T.A. インデル / インデル , TRB インデル / インデルIn some embodiments, the cells, including iPSCs, T cells differentiated therefrom, and primary T cells, are PCDH11Y インデル / インデル ,NLGN4Y インデル / インデル , B2M インデル / インデル , TRB インデル / インデル In some embodiments, the cells, including iPSCs, T cells differentiated therefrom, and primary T cells, are PCDH11Y インデル / インデル ,NLGN4Y インデル / インデル , 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 , TRB インデル / インデル In some embodiments, the cells, including iPSCs, T cells differentiated therefrom, and primary T cells, are PCDH11Y インデル / インデル ,NLGN4Y インデル / インデル , B2M インデル / インデル ,TRAC インデル / インデル , TRB インデル / インデル In some embodiments, the cells, including ESCs, iPSCs, T cells differentiated therefrom, and primary T cells, are PCDH11Y ノックダウン ,NLGN4Y ノックダウン , 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 cells, including iPSCs, T cells differentiated therefrom, and primary T cells, are PCDH11Y ノックダウン ,NLGN4Y ノックダウン , B2M ノックダウン , C.I.T.A. ノックダウン , TRB ノックダウン In some embodiments, the cells, including ESCs, iPSCs, T cells differentiated therefrom, and primary T cells, are PCDH11Y ノックダウン ,NLGN4Y ノックダウン , B2M ノックダウン , C.I.T.A. ノックダウン ,TRAC ノックダウン , TRB ノックダウン In some embodiments, the cells, including ESCs, iPSCs, T cells differentiated therefrom, and primary T cells, are PCDH11Y ノックダウン ,NLGN4Y ノックダウン , B2M ノックダウン,TRAC ノックダウン In some embodiments, the cells, including iPSCs, T cells differentiated therefrom, and primary T cells, are PCDH11Y ノックダウン ,NLGN4Y ノックダウン , B2M ノックダウン , TRB ノックダウン In some embodiments, the cells, including ESCs, iPSCs, T cells differentiated therefrom, and primary T cells, are PCDH11Y ノックダウン ,NLGN4Y ノックダウン , B2M ノックダウン ,TRAC ノックダウン , TRB ノックダウン 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 (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, putative memory T cells, innate memory T cells, memory stem cells (Tsc), γδ T cells, and any other subtype of T cells.

[0342] 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 is otherwise modified or engineered to have reduced expression or lack expression of one or more Y chromosome genes, including, but not limited to, PCDH11Y and / or NLGN4Y. Reducing expression of PCDH11Y and / or NLGN4Y can be accomplished, for example, by directly targeting the PCDH11Y and NLGN4Y genes and / or by targeting components essential for their transcription, translation, or protein stability.

[0343] The cells of the present disclosure exhibit reduced or absent expression of MHC class I antigen molecules, MHC class II antigen molecules, and / or TCR complexes. Reduction of expression of one or more MHC class I and / or MHC class II HLA molecules can be achieved, for example, by one or more of the following: (1) directly targeting polymorphic HLA alleles (HLA-A, HLA-B, HLA-C) and MHC-II genes, (2) abolishing surface trafficking of all MHC-I molecules by deleting B2M, (3) abolishing surface trafficking of all MHC-II molecules by deleting CIITA, and / or (4) deleting components of the MHC enhanceosome, such as LRC5, RFX5, RFXANK, RFXAP, IRF1, NF-Y (including NFY-A, NFY-B, NFY-C), and CIITA, which are essential for HLA expression.

[0344] In some embodiments, HLA expression is disrupted by targeting individual HLAs (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, knocking down, 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, knocking down, or reducing expression of B2M and / or TAP1), and / or targeting with HLA-Razor (see, e.g., WO2016183041).

[0345] 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, do not express one or more human leukocyte antigen molecules corresponding to MHC-I and / or MHC-II molecules (e.g., HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and / or HLA-DR), and are therefore characterized as being hypoimmunogenic. For example, in certain embodiments, the disclosed pluripotent stem cells and induced pluripotent stem cells have been modified such that the stem cells or differentiated stem cells prepared therefrom do not express or exhibit 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 "knocked out" from the cells. Cells in which the HLA-A, HLA-B, and / or HLA-C genes have been knocked out can exhibit reduced expression or elimination of the respective gene that has been knocked out.

[0346] In some embodiments, guide RNA, shRNA, siRNA, or miRNA that targets conserved regions in HLA genes and thereby allows simultaneous deletion of all MHC class I alleles is identified as HLA Razor. In some embodiments, gRNA is part of a CRISPR system. In alternative embodiments, gRNA is part of a TALEN system. In some embodiments, HLA Razor that targets identified conserved regions in HLA is described in WO2016183041. In some embodiments, multiple HLA Razors that target 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.

[0347] The provided methods are useful for inactivating or eliminating expression of MHC class I molecules and / or MHC class II molecules in cells, including, but not limited to, pluripotent stem cells, differentiated cells, and primary T cells. In some embodiments, genome editing techniques utilizing 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 immune responses in cells (e.g., by deleting genomic DNA of genes involved in immune responses or by inserting genomic DNA into such genes such that gene expression is affected). In certain embodiments, genome editing techniques or other gene regulation techniques are used to insert tolerance-inducing factors in human cells to render those cells and differentiated cells prepared therefrom hypoimmunogenic. Thus, the engineered and / or hypoimmunogenic cells have reduced or eliminated expression of MHC I molecules and / or MHC II molecules. In some embodiments, the cells are non-immunogenic (eg, do not induce an innate and / or adaptive immune response) in a recipient subject.

[0348] In some embodiments, the cells comprise a modification that increases expression of CD47 and one or more factors selected from the group consisting of DUX4, CD24, CD27, 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, IL-39, FasL, CCL21, CCL22, Mfge8, and Serpinb9.

[0349] In some embodiments, the cells comprise genomic modifications of one or more target polynucleotide sequences that regulate the expression of either an MHC class I molecule, an MHC class II molecule, or both an MHC class I and an MHC class II molecule. In some embodiments, a gene editing system is used to modify the one or more target polynucleotide sequences. In some embodiments, an RNAi system is used to knock down 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 consisting of B2M, CIITA, and NLRC5. In some embodiments, the cells comprise a gene editing modification to the B2M gene. In some embodiments, the cells comprise a gene editing modification to the CIITA gene. In some embodiments, the cells comprise a gene editing modification to the NLRC5 gene. In some embodiments, the cells comprise gene editing modifications to the B2M and CIITA genes. In some embodiments, the cells comprise gene editing modifications to the B2M and NLRC5 genes. In some embodiments, the cells comprise gene editing modifications to the CIITA and NLRC5 genes. In certain embodiments, the cells comprise gene editing modifications to the B2M, CIITA, and NLRC5 genes. In certain embodiments, the genome of the cells has been altered to reduce or delete essential components of HLA expression. 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 is otherwise modified or engineered to alter the expression of one or more genes to generate the engineered cell.

[0350] In some embodiments, the present disclosure provides a cell (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 hepatocyte, 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) or a population thereof comprising a genome that has been genetically edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of an MHC class I molecule in the cell or population thereof. In certain embodiments, the present disclosure provides a cell (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 hepatocyte, 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) or a population thereof comprising a genome that has been genetically edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of an MHC class II molecule in the cell or population thereof. In numerous embodiments, the disclosure provides a cell (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 hepatocyte, 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) or a population thereof comprising a genome in which one or more genes have been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I and II molecules in the cell or population thereof.

[0351] In certain embodiments, expression of one or more MHC I and / or MHC II molecules (including one or more MHC class I and / or MHC class II HLA molecules) is regulated by targeted deletion of 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) comprising an exogenous CD47 protein and an inactivated or modified CIITA gene sequence, and in some cases, an additional genetic modification that inactivates or modifies the B2M gene sequence. In some embodiments, described herein are gene-edited cells comprising an exogenous CD47 protein and an inactivated or modified CIITA gene sequence, and in some cases, an additional genetic modification that inactivates or modifies the NLRC5 gene sequence. In some embodiments, described herein are gene-edited cells comprising an exogenous CD47 protein and an inactivated or modified B2M gene sequence, and in some cases, additional genetic modifications that inactivate or modify the NLRC5 gene sequence. In some embodiments, described herein are gene-edited cells comprising an exogenous CD47 protein and an inactivated or modified B2M gene sequence, and in some cases, additional genetic modifications that inactivate or modify the CIITA gene sequence and the NLRC5 gene sequence.

[0352] Provided herein are cells that exhibit modifications of one or more target polynucleotide sequences that regulate the 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 an MHC I antigen molecule and an MHC II antigen molecule, and (e) an MHC I and II antigen molecule and a TCR complex. In certain embodiments, the modification comprises increasing expression of CD47. In some embodiments, the cell comprises an exogenous or recombinant CD47 polypeptide. In certain embodiments, the modification comprises expression of a chimeric antigen receptor. In some embodiments, the cell comprises an exogenous or recombinant chimeric antigen receptor polypeptide.

[0353] In some embodiments, the cells comprise genomic modifications of one or more target polynucleotide sequences that 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 modify the 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) has been altered to reduce or delete essential 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.

[0354] In some embodiments, the present disclosure provides a cell or population thereof comprising a genome in which a gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of an MHC class I molecule in the cell or population thereof. In some embodiments, the present disclosure provides a cell or population thereof comprising a genome in which a gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of an MHC class II molecule in the cell or population thereof. In some embodiments, the present disclosure provides a cell or population thereof comprising a genome in which a gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of a TCR molecule in the cell or population thereof. In many embodiments, the present disclosure provides a cell or population thereof comprising a genome in which one or more genes have been edited to delete a contiguous stretch of genomic DNA, thereby 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.

[0355] In some embodiments, the cells and methods described herein involve genome editing a human cell to cut a CIITA 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, PCDH11Y, NLGN4Y, B2M, TRAC, and TRB. In some embodiments, the cells and methods described herein involve genome editing a human cell to cut a B2M 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, PCDH11Y, NLGN4Y, CIITA, TRAC, and TRB. In some embodiments, the cells and methods described herein involve genome editing a human cell to cut a TRAC 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, PCDH11Y, NLGN4Y, B2M, CIITA, and TRB. In some embodiments, the cells and methods described herein involve genome editing of human cells to ablate a TRB gene sequence, as well as editing the genome of such cells to alter one or more additional target polynucleotide sequences, such as, but not limited to, PCDH11Y, NLGN4Y, B2M, CIITA, and TRAC.

[0356] Provided herein are hypoimmunogenic stem cells comprising reduced expression of PCDH11Y and / or NLGN4Y and HLA-A, HLA-B, HLA-C, CIITA, TCR-alpha, and TCR-beta compared to wild-type stem cells, wherein the hypoimmunogenic stem cells further comprise a set of exogenous polynucleotides comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a chimeric antigen receptor (CAR), wherein the first exogenous polynucleotide and / or the second exogenous polynucleotide is inserted within a specific locus of at least one allele of the cell. Also provided herein are hypoimmunogenic primary T cells, including any subtype of primary T cells, comprising reduced expression of PCDH11Y and / or NLGN4Y and HLA-A, HLA-B, HLA-C, CIITA, TCR-alpha, and TCR-beta relative to wild-type primary T cells, wherein the hypoimmunogenic stem cells further comprise a set of exogenous polynucleotides, including a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a chimeric antigen receptor (CAR), wherein the first exogenous polynucleotide and / or the second exogenous polynucleotide are inserted within a specific locus of at least one allele of the cell. Further provided herein are hypoimmunogenic T cells differentiated from hypoimmunogenic induced pluripotent stem cells, which comprise reduced expression of PCDH11Y and / or NLGN4Y and HLA-A, HLA-B, HLA-C, CIITA, TCR-alpha, and TCR-beta relative to wild-type primary T cells, wherein the hypoimmunogenic stem cells further comprise a set of exogenous polynucleotides, including a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a chimeric antigen receptor (CAR), wherein the first exogenous polynucleotide and / or the second exogenous polynucleotide is inserted within a specific locus of at least one allele of the cell.

[0357] In some embodiments, the described populations of engineered cells avoid NK cell-mediated cytotoxicity upon administration to a patient. In some embodiments, the populations of engineered cells avoid NK cell-mediated cytotoxicity by one or more subpopulations of NK cells. In some embodiments, the populations of engineered cells are protected from cytolysis by NK cells, including immature and / or mature NK cells, upon administration to a patient. In some embodiments, the populations of engineered cells avoid phagocytosis by macrophages upon administration to a patient. In some embodiments, the populations of engineered cells do not induce an innate and / or adaptive immune response against the cells upon administration to a patient.

[0358] In some embodiments, the cells described herein comprise a safety switch. As used herein, the term "safety switch" refers to a system for controlling the expression of a gene or protein of interest that, when down- or up-regulated, triggers elimination or death of the cell, for example, through recognition by the host's immune system. Safety switches can be designed to be activated by exogenous molecules in the event of an adverse clinical event. Safety switches can be manipulated by regulating expression at the DNA, RNA, and protein levels. Safety switches include proteins or molecules that allow for the control of cellular activity in response to adverse events. In one embodiment, the safety switch is a "kill switch" that is expressed in an inactivated state; 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 itself alone, 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 T cells, CAR-T cells, NK cells, and / or CAR-NK cells, comprise a safety switch.

[0359] 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.

[0360] In some embodiments, the cells described herein comprise a "suicide gene" (or "suicide switch") that can cause the death of hypoimmunogenic cells if they are allowed to grow and divide in an undesired manner. The "suicide gene" ablation approach comprises 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, for example, stem cells, induced pluripotent stem cells, hematopoietic stem cells, primary cells, or differentiated cells, including but not limited to T cells, CAR-T cells, NK cells, and / or CAR-NK cells, comprise a suicide gene.

[0361] In some embodiments, the populations of engineered cells described elicit reduced levels of immune activation or no immune activation upon administration to a recipient subject. In some embodiments, the cells elicit reduced levels of systemic TH1 activation or no systemic TH1 activation in the recipient subject. In some embodiments, the cells elicit reduced levels of peripheral blood mononuclear cell (PBMC) immune activation or no PBMC immune activation in the recipient subject. In some embodiments, the cells elicit reduced levels of donor-specific IgG antibodies or no donor-specific IgG antibodies against the cells upon administration to a recipient subject. In some embodiments, the cells elicit reduced levels of IgM and IgG antibody production or no IgM and IgG antibody production against the cells in the recipient subject. In some embodiments, the cells elicit reduced levels of cytotoxic T cell killing of the cells upon administration to a recipient subject.

[0362] B.CIITA In some embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) expression of MHC II genes by targeting and modulating (e.g., reducing or eliminating) expression of class II transactivator (CIITA). In some embodiments, the modulation occurs using a gene editing system (e.g., a CRISPR / Cas system).

[0363] CIITA is a member of the LR, or nucleotide-binding domain (NBD) leucine-rich repeat (LRR) family of proteins, which regulates MHC II transcription by associating with the MHC enhanceosome.

[0364] In some embodiments, the target polynucleotide sequence of the present disclosure is a variant of CIITA. In some embodiments, the target polynucleotide sequence is a homolog of CIITA. In some embodiments, the target polynucleotide sequence is an ortholog of CIITA.

[0365] In some embodiments, the reduced expression or elimination of CIITA reduces or eliminates the expression of one or more of the following MHC class II molecules: HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR.

[0366] In some embodiments, the cells described herein comprise a genetic modification at a locus encoding a CIITA protein. In other words, the cells comprise a genetic modification at the CIITA locus. In some cases, the nucleotide sequence encoding the CIITA protein is set forth in RefSeq. No. NM_000246.4 and NCBI GenBank No. U18259. In some cases, the CIITA locus is set forth in NCBI Gene ID No. 4261. In certain instances, the amino acid sequence of CIITA is set forth as NCBI GenBank No. AAA88861.1. Additional descriptions of the CIITA protein and locus can be found in Uniprot No. P33076, HGNC Reference No. 7067, and OMIM Reference No. 600005.

[0367] In some embodiments, the engineered and / or hypoimmunogenic cells outlined herein comprise a genetic modification targeting the CIITA gene. In some embodiments, the genetic modification targeting the CIITA gene using a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein and at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene. In some embodiments, the at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene is selected from the group consisting of SEQ ID NOs: 5184-36352 in Table 12 of WO2016183041, which is incorporated herein by reference. In some embodiments, the cells have a reduced ability to induce innate and / or adaptive immune responses in a recipient subject. In some embodiments, an exogenous nucleic acid encoding a polypeptide as disclosed herein (e.g., a chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein) is inserted into the CIITA gene.

[0368] In some embodiments, the engineered and / or hypoimmunogenic cells outlined herein comprise a knockout of CIITA expression, such that the cells express CIITA - / - In some embodiments, the engineered and / or hypoimmunogenic cells outlined herein introduce an indel within the CIITA locus, such that the cells express CIITA インデル / インデル In some embodiments, the engineered and / or hypoimmunogenic cells outlined herein comprise knockdown of CIITA expression, such that the cells express CIITA ノックダウン is.

[0369] Assays for testing whether the CIITA gene is inactivated are known and are described herein. In some embodiments, the resulting genetic modification of the CIITA gene can be assayed by PCR, and the reduction of HLA-II expression can be assayed by FACS analysis. In another embodiment, CIITA protein expression is detected using Western blot of cell lysate probed with an antibody against CIITA protein. In another embodiment, the presence of the inactivating genetic modification is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0370] C.B2M In some embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) expression of MHC-I genes by targeting and modulating (e.g., reducing or eliminating) the expression of the accessory chain B2M. In some embodiments, the modulation occurs using a gene editing system (e.g., a CRISPR / Cas system).

[0371] Modulating (e.g., reducing or deleting) the expression of B2M blocks surface trafficking of MHC-I molecules, rendering the cells less immunogenic, and in some embodiments, the cells have a reduced ability to induce an innate and / or adaptive immune response in a recipient subject.

[0372] In some embodiments, the target polynucleotide sequence of the present disclosure is a variant of B2M. In some embodiments, the target polynucleotide sequence is a homolog of B2M. In some embodiments, the target polynucleotide sequence is an ortholog of B2M.

[0373] In some embodiments, reducing or eliminating the expression of B2M reduces or eliminates the expression of one or more of the following MHC I molecules: HLA-A, HLA-B, and HLA-C.

[0374] In some embodiments, the cells described herein comprise a genetic modification at a locus encoding a B2M protein. In other words, the cells comprise a genetic modification at the B2M locus. In some cases, the nucleotide sequence encoding the B2M protein is set forth in RefSeq. No. NM_004048.4 and GenBank No. AB021288.1. In some cases, the B2M locus is set forth in NCBI Gene ID No. 567. In one particular instance, the amino acid sequence of B2M is set forth as NCBI GenBank No. BAA35182.1. Additional description of the B2M protein and locus can be found in Uniprot No. P61769, HGNC Reference No. 914, and OMIM Reference No. 109700.

[0375] In some embodiments, the engineered and / or hypoimmunogenic cells outlined herein comprise a genetic modification targeting the B2M gene. In some embodiments, the genetic modification targeting the B2M gene using a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein and at least one guide ribonucleic acid sequence for specifically targeting the B2M gene. In some embodiments, the at least one guide ribonucleic acid sequence for specifically targeting the B2M gene is selected from the group consisting of SEQ ID NOs: 81240-85644 in Table 15 of WO2016183041, which is incorporated herein by reference. In some embodiments, an exogenous nucleic acid encoding a polypeptide as disclosed herein (e.g., a chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein) is inserted into the B2M gene.

[0376] Assays for testing whether the B2M gene is inactivated are known and described herein. In some embodiments, the resulting genetic modification of the B2M gene can be assayed by PCR, and the reduction in HLA-I expression can be assayed by FACS analysis. In another embodiment, B2M protein expression is detected using Western blot of cell lysates probed with an antibody against the B2M protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of the inactivating genetic modification.

[0377] In some embodiments, the engineered and / or hypoimmunogenic cells outlined herein comprise a knockout of B2M expression, such that the cells express B2M - / - In some embodiments, the engineered and / or hypoimmunogenic cells outlined herein introduce an indel within the B2M locus, such that the cells express B2M インデル / インデル In some embodiments, the engineered and / or hypoimmunogenic cells outlined herein comprise knockdown of B2M expression, such that the cells express B2M ノックダウン is.

[0378] D.NLRC5 In certain embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) the expression of MHC-I genes by targeting and modulating (e.g., reducing or eliminating) the expression of the NLR family, CARD domain-containing 5 / NOD27 / CLR16.1 (NLRC5). In some embodiments, the modulation occurs using a gene editing system (e.g., a CRISPR / Cas system).

[0379] NLRC5 is a regulator of MHC-I-mediated immune responses. Like CIITA, NLRC5 is highly inducible by IFN-γ and can translocate to the nucleus. NLRC5 activates the promoter of MHC-I genes, inducing the transcription of not only MHC-I but also related genes involved in MHC-I antigen presentation.

[0380] In some embodiments, the target polynucleotide sequence is a variant of NLRC5. In some embodiments, the target polynucleotide sequence is a homolog of NLRC5. In some embodiments, the target polynucleotide sequence is an ortholog of NLRC5.

[0381] In some embodiments, reducing or eliminating the expression of NLRC5 reduces or eliminates the expression of one or more of the following MHC I molecules: HLA-A, HLA-B, and HLA-C.

[0382] In some embodiments, the cells described herein comprise a genetic modification targeting the NLRC5 gene. In some embodiments, the genetic modification targeting the NLRC5 gene using a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding the Cas protein and at least one guide ribonucleic acid sequence for specifically targeting the NLRC5 gene. In some embodiments, the at least one guide ribonucleic acid sequence for specifically targeting the NLRC5 gene is selected from the group consisting of SEQ ID NOs: 36353 to 81239 in Appendix 3 or Table 14 of WO2016183041, the disclosure of which is incorporated by reference in its entirety.

[0383] Assays for testing whether the NLRC5 gene is inactivated are known and described herein. In some embodiments, the resulting genetic modification of the NLRC5 gene can be assayed by PCR, and the reduction in HLA-I expression can be assayed by FACS analysis. In another embodiment, NLRC5 protein expression is detected using Western blot of cell lysates probed with an antibody against the NLRC5 protein. In another embodiment, the presence of the inactivating genetic modification is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0384] In some embodiments, the engineered and / or hypoimmunogenic cells outlined herein comprise a knockout of NLRC5 expression, such that the cells express NLRC5 - / - In some embodiments, the engineered and / or hypoimmunogenic cells outlined herein introduce an indel into the NLRC5 locus, such that the cells express NLRC5 インデル / インデル In some embodiments, the engineered and / or hypoimmunogenic cells outlined herein comprise knockdown of NLRC5 expression, such that the cells express NLRC5. ノックダウン is.

[0385] E.TRAC In certain embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) expression of TCR genes, including the TRAC gene, by targeting and modulating (e.g., reducing or eliminating) the expression of the constant region of the T cell receptor alpha chain. In some embodiments, modulation occurs using a gene editing system (e.g., a CRISPR / Cas system).

[0386] By modulating (e.g., reducing or deleting) the expression of TRAC, surface trafficking of TCR molecules is blocked. In some embodiments, the cells also have a reduced ability to induce an innate and / or adaptive immune response in a recipient subject.

[0387] In some embodiments, the target polynucleotide sequence of the present disclosure is a variant of TRAC. In some embodiments, the target polynucleotide sequence is a homolog of TRAC. In some embodiments, the target polynucleotide sequence is an ortholog of TRAC.

[0388] In some embodiments, reducing or eliminating the expression of TRAC reduces or eliminates the surface expression of TCR.

[0389] In some embodiments, cells, such as, but not limited to, pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, comprise a genetic modification at a locus encoding a TRAC protein. In other words, the cells comprise a genetic modification at the TRAC locus. In some cases, the nucleotide sequence encoding the TRAC protein is set forth in Genbank No. X02592.1. In some cases, the TRAC locus is set forth in RefSeq. No. NG_001332.3 and NCBI Gene ID No. 28755. In certain instances, the amino acid sequence of TRAC is set forth as Uniprot No. P01848. Additional description of the TRAC protein and locus can be found in Uniprot No. P01848, HGNC Reference No. 12029, and OMIM Reference No. 186880.

[0390] In some embodiments, the engineered and / or hypoimmunogenic cells outlined herein comprise a genetic modification that targets the TRAC gene. In some embodiments, the genetic modification that targets the TRAC gene using a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the TRAC gene. In some embodiments, the at least one guide ribonucleic acid sequence for specifically targeting the TRAC gene is selected from the group consisting of SEQ ID NOs: 532-609 and 9102-9797 of US20160348073, which is incorporated herein by reference.

[0391] Assays for testing whether the TRAC gene is inactivated are known and described herein. In some embodiments, the resulting genetic modification of the TRAC gene can be assayed by PCR, and the reduction in TCR expression can be assayed by FACS analysis. In another embodiment, TRAC protein expression is detected using Western blot of cell lysates probed with an antibody against the TRAC protein. In another embodiment, the presence of the inactivating genetic modification is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0392] In some embodiments, the engineered and / or hypoimmunogenic cells outlined herein comprise a knockout of TRAC expression, such that the cells express TRAC - / - In some embodiments, the engineered and / or hypoimmunogenic cells outlined herein introduce an indel into the TRAC locus, such that the cells express TRAC インデル / インデル In some embodiments, the engineered and / or hypoimmunogenic cells outlined herein comprise a knockdown of TRAC expression, such that the cells express TRAC. ノックダウン is.

[0393] F.TRB In certain embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) expression of TCR genes, including genes encoding the T cell antigen receptor, beta chain (e.g., TRB, TRBC, or TCRB genes), by targeting and modulating (e.g., reducing or eliminating) the expression of the constant region of the T cell receptor beta chain. In some embodiments, modulation occurs using a gene editing system (e.g., a CRISPR / Cas system).

[0394] By modulating (e.g., reducing or deleting) the expression of TRB, surface trafficking of TCR molecules is blocked. In some embodiments, the cells also have a reduced ability to induce an innate and / or adaptive immune response in a recipient subject.

[0395] In some embodiments, the target polynucleotide sequence of the present disclosure is a variant of TRB. In some embodiments, the target polynucleotide sequence is a homolog of TRB. In some embodiments, the target polynucleotide sequence is an ortholog of TRB.

[0396] In some embodiments, reducing or eliminating expression of TRB reduces or eliminates surface expression of TCR.

[0397] In some embodiments, cells, such as, but not limited to, pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, comprise a genetic modification at a locus encoding a TRB protein. In other words, the cells comprise a genetic modification at the TRB locus. In some cases, the nucleotide sequence encoding the TRB protein is set forth in UniProt No. P0DSE2. In some cases, the TRB locus is set forth in RefSeq. No. NG_001333.2 and NCBI Gene ID No. 6957. In one particular instance, the amino acid sequence of TRB is set forth as Uniprot No. P01848. Additional description of TRB proteins and loci can be found in GenBank No. L36092.2, Uniprot No. P0DSE2, and HGNC Reference No. 12155.

[0398] In some embodiments, the engineered and / or hypoimmunogenic cells described herein comprise a genetic modification targeting the TRB gene. In some embodiments, the genetic modification targeting the TRB gene with a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein and at least one guide ribonucleic acid sequence for specifically targeting the TRB gene. In some embodiments, the at least one guide ribonucleic acid sequence for specifically targeting the TRB gene is selected from the group consisting of SEQ ID NOs: 610-765 and 9798-10532 of US20160348073, which is incorporated herein by reference.

[0399] Assays for testing whether the TRB gene is inactivated are known and are described herein. In some embodiments, the resulting genetic modification of the TRB gene can be assayed by PCR, and the reduction in TCR expression can be assayed by FACS analysis. In another embodiment, TRB protein expression is detected using Western blot of cell lysates probed with an antibody against the TRB protein. In another embodiment, the presence of the inactivating genetic modification is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0400] In some embodiments, the engineered and / or hypoimmunogenic cells outlined herein comprise a knockout of TRB expression, such that the cells express TRB - / - In some embodiments, the engineered and / or hypoimmunogenic cells outlined herein introduce an indel into the TRB locus, such that the cells express TRB インデル / インデル In some embodiments, the engineered and / or hypoimmunogenic cells outlined herein comprise a knockdown of TRB expression, such that the cells express TRB ノックダウン is.

[0401] G.CD142 In certain embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) the expression of CD142 (also known as tissue factor, factor III, and F3). In some embodiments, the modulation occurs using a gene editing system (e.g., a CRISPR / Cas system).

[0402] In some embodiments, the target polynucleotide sequence is CD142 or a variant of CD142. In some embodiments, the target polynucleotide sequence is a homolog of CD142. In some embodiments, the target polynucleotide sequence is an ortholog of CD142.

[0403] In some embodiments, the cells described herein comprise a genetic modification targeting the CD142 gene. In some embodiments, the genetic modification targeting the CD142 gene with a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding the Cas protein, and at least one guide ribonucleic acid (gRNA) sequence for specifically targeting the CD142 gene. Useful methods for identifying gRNA sequences targeting CD142 are described below.

[0404] Assays for testing whether the CD142 gene is inactivated are known and described herein. In some embodiments, the resulting genetic modification of the CD142 gene can be assayed by PCR, and the reduction in CD142 expression can be assayed by FACS analysis. In another embodiment, CD142 protein expression is detected using Western blot of cell lysate probed with an antibody against CD142 protein. In another embodiment, the presence of the inactivating genetic modification is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0405] Useful genome, polynucleotide, and polypeptide information for human CD142 is provided, for example, under GeneCard identifier GC01M094530, HGNC number 3541, NCBI gene ID 2152, NCBI RefSeq numbers NM_001178096.1, NM_001993.4, NP_001171567.1, and NP_001984.1, UniProt number P13726, etc.

[0406] H.RHD In some embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) expression of the RhD antigen by targeting and modulating (e.g., reducing or eliminating) expression of the RHD gene. In some embodiments, the modulation occurs using a gene editing system (e.g., a CRISPR / Cas system). In some embodiments, the cells have a reduced ability to induce an innate and / or adaptive immune response in a recipient subject.

[0407] In some embodiments, the target polynucleotide sequence of the present disclosure is a variant of the RHD gene. In some embodiments, the target polynucleotide sequence is a homolog of the RHD gene. In some embodiments, the target polynucleotide sequence is an ortholog of the RHD gene.

[0408] In some embodiments, the cells described herein comprise a genetic modification at a locus encoding a RhD antigen protein. In other words, the cells comprise a genetic modification at the RHD locus. In some cases, the nucleotide sequence encoding the RhD antigen protein is set forth in RefSeq. Nos. NM_001127691.2, NM_001282868.1, NM_001282869.1, NM_001282871.1, or NM_016124.4, or GenBank No. L08429. In some cases, the RHD locus is set forth in NCBI Gene ID No. 6007. In certain instances, the amino acid sequence of the RhD antigen protein is set forth as NCBI GenBank No. AAA02679.1. Additional descriptions of RhD proteins and loci can be found in Uniprot No. Q02161, HGNC Reference No. 10009, and OMIM Reference No. 111680.

[0409] In some embodiments, the cells described herein comprise a genetic modification that targets the RHD gene. In some embodiments, the genetic modification that targets the RHD gene is generated by gene editing the RHD gene using a gene editing tool, such as but not limited to, CRISPR / Cas, TALE-nuclease, zinc finger nuclease, other virus-based gene editing system, or RNA interference. In some embodiments, the gene editing targets the coding sequence of the RHD gene. In some cases, the cells do not produce functional RHD gene products. In the absence of RHD gene products, the cells completely lack Rh blood group antigens.

[0410] In some embodiments, the genetic modification targeting the RHD gene with a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding the Cas protein and at least one guide ribonucleic acid (gRNA) sequence for specifically targeting the RHD gene. Useful methods for identifying gRNA sequences that target the RHD are described below.

[0411] Assays for testing whether the RHD gene is inactivated are known and described herein. In some embodiments, the resulting genetic modification of the RHD gene can be assayed by PCR, and the reduction in RHD expression can be assayed by FACS analysis. In another embodiment, RHD protein expression is detected using Western blot of cell lysates probed with an antibody against the RHD protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of the inactivating genetic modification.

[0412] I.CTLA-4 In some embodiments, the target polynucleotide sequence is CTLA-4 or a variant of CTLA-4. In some embodiments, the target polynucleotide sequence is a homolog of CTLA-4. In some embodiments, the target polynucleotide sequence is an ortholog of CTLA-4.

[0413] In some embodiments, the cells outlined herein comprise a genetic modification targeting the CTLA-4 gene. In certain embodiments, primary T cells comprise a genetic modification targeting the CTLA-4 gene. The genetic modification can reduce expression of CTLA-4 polynucleotides and CTLA-4 polypeptides in T cells, including primary T cells and CAR-T cells. In some embodiments, the genetic modification targeting the CTLA-4 gene using a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding the Cas protein and at least one guide ribonucleic acid (gRNA) sequence for specifically targeting the CTLA-4 gene. Useful methods for identifying gRNA sequences targeting CTLA-4 are described below.

[0414] Assays for testing whether the CTLA-4 gene is inactivated are known and are described herein. In some embodiments, the resulting genetic modification of the CTLA-4 gene can be assayed by PCR, and the reduction in CTLA-4 expression can be assayed by FACS analysis. In another embodiment, CTLA-4 protein expression is detected using Western blot of cell lysate probed with an antibody against CTLA-4 protein. In another embodiment, the presence of the inactivating genetic modification is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0415] Useful genome, polynucleotide, and polypeptide information for human CTLA-4 is provided, for example, under GeneCard identifier GC02P203867, HGNC number 2505, NCBI gene ID 1493, NCBI RefSeq numbers NM_005214.4, NM_001037631.2, NP_001032720.1 and NP_005205.2, UniProt number P16410, etc.

[0416] J.PD-1 In some embodiments, the target polynucleotide sequence is PD-1 or a variant of PD-1. In some embodiments, the target polynucleotide sequence is a homolog of PD-1. In some embodiments, the target polynucleotide sequence is an ortholog of PD-1.

[0417] In some embodiments, the cells outlined herein comprise a gene encoding the programmed cell death protein 1 (PD-1) protein or a genetic modification targeting the PDCD1 gene. In certain embodiments, primary T cells comprise a genetic modification targeting the PDCD1 gene. The genetic modification may reduce expression of PD-1 polynucleotides and PD-1 polypeptides in T cells, including primary T cells and CAR-T cells. In some embodiments, the genetic modification targeting the PDCD1 gene with a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein and at least one guide ribonucleic acid (gRNA) sequence for specifically targeting the PDCD1 gene. Useful methods for identifying gRNA sequences targeting PD-1 are described below.

[0418] Assays for testing whether the PDCD1 gene is inactivated are known and described herein. In some embodiments, the resulting genetic alteration of the PDCD1 gene can be assayed by PCR, and the reduction in PD-1 expression can be assayed by FACS analysis. In another embodiment, PD-1 protein expression is detected using Western blot of cell lysates probed with an antibody against the PD-1 protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of the inactivating genetic alteration.

[0419] Useful genomic, polynucleotide, and polypeptide information for human PD-1, including the PDCD1 gene, is provided, for example, under GeneCard identifier GC02M241849, HGNC number 8760, NCBI gene ID 5133, Uniprot number Q15116, and NCBI RefSeq numbers NM_005018.2 and NP_005009.2.

[0420] K.CD47 In some embodiments, the present disclosure provides a cell or population thereof modified to express the tolerogenic factor (e.g., immunomodulatory polypeptide) CD47. In some embodiments, the disclosure provides a method for altering the genome of a cell to express CD47. In some embodiments, the stem cell expresses exogenous CD47. In some cases, the cell expresses an expression vector comprising a nucleotide sequence encoding a human CD47 polypeptide. In some embodiments, the cell is genetically modified using homology-directed repair to comprise an integrated exogenous polynucleotide encoding CD47. In some cases, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide such that the nucleotide sequence is inserted into at least one allele of a safe harbor or target locus. In some cases, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide such that the nucleotide sequence is inserted into at least one allele of the AAVS1 locus. In some cases, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide such that the nucleotide sequence is inserted into at least one allele of a safe harbor or target locus. In some cases, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide, such that the nucleotide sequence is inserted into at least one allele of the CCR5 locus. In some cases, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide, wherein the nucleotide sequence is inserted into at least one allele of the AAVS1 locus. In some cases, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide, wherein the nucleotide sequence is inserted into at least one allele of the CCR5 locus.In some cases, the cells express a nucleotide sequence encoding a human CD47 polypeptide, wherein the nucleotide sequence is inserted within at least one allele of a safe harbor or target locus, such as, but not limited to, the CCR5 locus, CXCR4 locus, PPP1R12C locus, albumin locus, SHS231 locus, CLYBL locus, ROSA locus, F3 (CD142) locus, MICA locus, MICB locus, LRP1 (CD91) locus, HMGB1 locus, ABO locus, RHD locus, FUT1 locus, and KDM5D locus. In some cases, the cells express a nucleotide sequence encoding a human CD47 polypeptide, wherein the nucleotide sequence is inserted within at least one allele of the TRAC locus.

[0421] CD47 is a leukocyte surface antigen that plays a role in cell adhesion and regulation of integrins. It is expressed on the surface of cells and signals circulating macrophages to stop them from phagocytosing the cells.

[0422] In some embodiments, the cells outlined herein comprise a nucleotide sequence encoding a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in NCBI Ref.Sequence Nos. NP_001768.1 and NP_942088.1. In some embodiments, the cells outlined herein comprise a nucleotide sequence encoding a CD47 polypeptide having the amino acid sequence set forth in NCBI Ref.Sequence Nos. NP_001768.1 and NP_942088.1. In some embodiments, the cells comprise a nucleotide sequence for CD47 having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the sequence set forth in NCBI Ref. Nos. NM_001777.3 and NM_198793.2. In some embodiments, the cells comprise a nucleotide sequence for CD47 set forth in NCBI Ref. Sequence Nos. NM_001777.3 and NM_198793.2. In some embodiments, the nucleotide sequence encoding the CD47 polynucleotide is a codon-optimized sequence. In some embodiments, the nucleotide sequence encoding the CD47 polynucleotide is a human codon-optimized sequence.

[0423] In some embodiments, the cells comprise a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to the amino acid sequence set forth in NCBI Ref.Sequence Nos. NP_001768.1 and NP_942088.1. In some embodiments, the cells outlined herein comprise a CD47 polypeptide having an amino acid sequence set forth in NCBI Ref.Sequence Nos. NP_001768.1 and NP_942088.1.

[0424] Exemplary amino acid sequences of human CD47 with and without the signal sequence are provided in Table 1. [Table 1]

[0425] In some embodiments, the cells comprise a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the cells comprise a CD47 polypeptide having the amino acid sequence of SEQ ID NO: 97. In some embodiments, the cells comprise a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the cells comprise a CD47 polypeptide having the amino acid sequence of SEQ ID NO: 98.

[0426] In some embodiments, the cell comprises a nucleotide sequence encoding a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the cell comprises a nucleotide sequence encoding a CD47 polypeptide having the amino acid sequence of SEQ ID NO: 97. In some embodiments, the cell comprises a nucleotide sequence encoding a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the cell comprises a nucleotide sequence encoding a CD47 polypeptide having the amino acid sequence of SEQ ID NO: 98. In some embodiments, the nucleotide sequence is codon-optimized for expression in a particular cell.

[0427] In some embodiments, a suitable gene editing system (e.g., a CRISPR / Cas system, or any of the gene editing systems described herein) is used to facilitate insertion of a polynucleotide encoding CD47 into a genomic locus of a hypoimmunogenic cell. In some cases, the polynucleotide encoding CD47 is inserted into a safe harbor or target locus, such as, but not limited to, the AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (CD142), MICA, MICB, LRP1 (CD91), HMGB1, ABO, RHD, FUT1, or KDM5D locus. In some embodiments, the polynucleotide encoding CD47 is inserted into the B2M locus, CIITA locus, TRAC locus, or TRB locus. In some embodiments, the polynucleotide encoding CD47 is inserted into any one of the loci set forth in Table 21 provided herein. In certain embodiments, the polynucleotide encoding CD47 is operably linked to a promoter.

[0428] In another embodiment, CD47 protein expression is detected using Western blots of cell lysates probed with an antibody against CD47 protein, hi another embodiment, the presence of exogenous CD47 mRNA is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0429] L.CD24 In some embodiments, the present disclosure provides cells or populations thereof that have been modified to express the tolerogenic factor (e.g., immunomodulatory polypeptide) CD24. In some embodiments, the present disclosure provides methods for altering the genome of a cell to express CD24. In some embodiments, the stem cell expresses exogenous CD24. In some cases, the cell expresses an expression vector comprising a nucleotide sequence encoding a human CD24 polypeptide.

[0430] CD24, also known as the heat-stable antigen or small cell lung cancer cluster 4 antigen, is a glycosylated glycosylphosphatidylinositol-anchored surface protein (Pirruccello et al., J Immunol, 1986, 136, 3779-3784; Chen et al., Glycobiology, 2017, 57, 800-806). It binds to Siglec-10 on innate immune cells. Recently, it has been shown that CD24 functions as an innate immune checkpoint via Siglec-10 (Barkal et al., Nature, 2019, 572, 392-396).

[0431] In some embodiments, the cells outlined herein comprise a nucleotide sequence encoding a CD24 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in NCBI reference numbers NP_001278666.1, NP_001278667.1, NP_001278668.1, and NP_037362.1. In some embodiments, the cells outlined herein comprise a nucleotide sequence encoding a CD24 polypeptide having the amino acid sequence set forth in NCBI reference numbers NP_001278666.1, NP_001278667.1, NP_001278668.1, and NP_037362.1.

[0432] In some embodiments, the cell comprises a nucleotide sequence having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to a sequence set forth in NCBI reference numbers NM_00129737.1, NM_00129738.1, NM_001291739.1, and NM_013230.3. In some embodiments, the cell comprises a nucleotide sequence set forth in NCBI reference numbers NM_00129737.1, NM_00129738.1, NM_001291739.1, and NM_013230.3.

[0433] In some embodiments, a suitable gene editing system (e.g., a CRISPR / Cas system, or any of the gene editing systems described herein) is used to facilitate insertion of a polynucleotide encoding CD24 into a genomic locus of a hypoimmunogenic cell. In some cases, the polynucleotide encoding CD24 is inserted into a safe harbor or target locus, such as, but not limited to, the AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (CD142), MICA, MICB, LRP1 (CD91), HMGB1, ABO, RHD, FUT1, or KDM5D locus. In some embodiments, the polynucleotide encoding CD24 is inserted into the B2M locus, CIITA locus, TRAC locus, or TRB locus. In some embodiments, the polynucleotide encoding CD24 is inserted into any one of the loci set forth in Table 20 provided herein. In certain embodiments, the polynucleotide encoding CD24 is operably linked to a promoter.

[0434] In another embodiment, CD24 protein expression is detected using Western blots of cell lysates probed with an antibody against CD24 protein, hi another embodiment, the presence of exogenous CD24 mRNA is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0435] In some embodiments, a suitable gene editing system (e.g., a CRISPR / Cas system, or any of the gene editing systems described herein) is used to facilitate insertion of a polynucleotide encoding CD24 into a genomic locus of a hypoimmunogenic cell. In some cases, the polynucleotide encoding CD24 is inserted into a safe harbor or target locus, such as, but not limited to, the AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (also known as CD142), MICA, MICB, LRP1 (also known as CD91), HMGB1, ABO, RHD, FUT1, or KDM5D locus. In some embodiments, the polynucleotide encoding CD24 is inserted into the B2M locus, CIITA locus, TRAC locus, or TRB locus. In some embodiments, the polynucleotide encoding CD24 is inserted into any one of the loci set forth in Table 20 provided herein. In certain embodiments, the polynucleotide encoding CD24 is operably linked to a promoter.

[0436] M.DUX4 In some embodiments, the present disclosure provides a cell (e.g., a stem cell, an induced pluripotent stem cell, a differentiated cell, a hematopoietic stem cell, a primary T cell, or a CAR-T cell) or population thereof comprising a genome modified to increase expression of a tolerogenic or immunosuppressive factor such as DUX4. In some embodiments, the present disclosure provides a method for altering the genome of a cell to result in increased expression of DUX4. In some embodiments, the present disclosure provides a cell or population thereof comprising an exogenously expressed DUX4 protein. In some embodiments, the increased expression of DUX4 suppresses, reduces, or eliminates expression of one or more of the following MHC I molecules: HLA-A, HLA-B, and HLA-C.

[0437] DUX4 is a transcription factor active in embryonic tissues and induced pluripotent stem cells but silent in normal, healthy somatic tissues (Feng et al., 2015, ELife4; De Iaco et al., 2017, Nat Genet., 49, 941-945; Hendrickson et al., 2017, Nat Genet., 49, 925-934; Snider et al., 2010, PLoS Genet., e1001181; Whiddon et al., 2017, Nat Genet.). DUX4 expression acts to block IFN-gamma-mediated induction of MHC class I gene expression (e.g., B2M, HLA-A, HLA-B, and HLA-C). DUX4 expression has been implicated in the suppression of antigen presentation by MHC class I (Chew et al., Developmental Cell, 2019, 50, 1-14). DUX4 functions as a transcription factor in the cleavage-stage gene expression (transcription) program. Its target genes include, but are not limited to, coding genes, non-coding genes, and repetitive elements.

[0438] At least two isoforms of DUX4 exist, the longest of which contains a transcriptional activation domain at the C-terminus of DUX4. These isoforms are generated by alternative splicing. See, e.g., Geng et al., 2012, Dev Cell, 22, 38-51; Snider et al., 2010, PLoS Genet., e1001181. The active isoform of DUX4 contains a DNA-binding domain at its N-terminus and an activation domain at its C-terminus. See, e.g., Choi et al., 2016, Nucleic Acid Res, 44, 5161-5173.

[0439] It has been shown that reducing the number of CpG motifs in DUX4 reduces silencing of the DUX4 transgene (Jagannathan et al., Human Molecular Genetics, 2016, 25(20):4419-4431). The nucleic acid sequence provided in Jagannathan et al. (see above) represents a codon-altered sequence of DUX4 containing one or more base substitutions that reduce the total number of CpG sites while preserving the DUX4 protein sequence. This nucleic acid sequence is commercially available from Addgene (Catalog No. 99281).

[0440] In many embodiments, at least one or more polynucleotides may be utilized to facilitate exogenous expression of DUX4 by a cell, e.g., a stem cell, an induced pluripotent stem cell, a differentiated cell, a hematopoietic stem cell, a primary T cell, or a CAR-T cell.

[0441] In some embodiments, a suitable gene editing system (e.g., a CRISPR / Cas system, or any of the gene editing systems described herein) is used to facilitate insertion of a polynucleotide encoding DUX4 into a genomic locus of a hypoimmunogenic cell. In some cases, the polynucleotide encoding DUX4 is inserted into a safe harbor or target locus, such as, but not limited to, the AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (CD142), MICA, MICB, LRP1 (CD91), HMGB1, ABO, RHD, FUT1, or KDM5D locus. In some embodiments, the polynucleotide encoding DUX4 is inserted into the B2M locus, CIITA locus, TRAC locus, or TRB locus. In some embodiments, the polynucleotide encoding DUX4 is inserted into any one of the loci set forth in Table 20 provided herein. In certain embodiments, the polynucleotide encoding DUX4 is operably linked to a promoter.

[0442] In some embodiments, the polynucleotide sequence encoding DUX4 comprises a polynucleotide sequence comprising a nucleotide sequence in which the codons of DUX4 are altered, comprising one or more base substitutions that reduce the total number of CpG sites while preserving the DUX4 protein sequence. In some embodiments, the polynucleotide sequence encoding DUX4 comprising one or more base substitutions that reduce the total number of CpG sites has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 1 of PCT / US2020 / 44635, filed July 31, 2020. In some embodiments, the polynucleotide sequence encoding DUX4 is SEQ ID NO: 1 of PCT / US2020 / 44635.

[0443] In some embodiments, the polynucleotide sequence encoding DUX4 is a nucleotide sequence encoding a polypeptide sequence having at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, as provided in PCT / US2020 / 44635. In some embodiments, the polynucleotide sequence encoding DUX4 is a nucleotide sequence encoding a polypeptide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29. The amino acid sequences set forth as SEQ ID NOs:2-29 are set forth in Figures 1A-1G of PCT / US2020 / 44635.

[0444] In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to, or the amino acid sequence set forth in GenBank Accession No. ACN62209.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to, or the amino acid sequence set forth in GenBank Accession No. ACN62209.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to, or the amino acid sequence set forth in NCBI RefSeq No. NP_001280727.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to, or the amino acid sequence set forth in GenBank Accession No. ACP30489.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to, or the amino acid sequence set forth in UniProt No. P0CJ85.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to, or the amino acid sequence set forth in GenBank Accession No. AUA60622.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to, or the amino acid sequence set forth in GenBank Accession No. AUA60622.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to, or the amino acid sequence set forth in GenBank Accession No. ADK24683.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to, or the amino acid sequence set forth in GenBank Accession No. ACN62210.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to, or the amino acid sequence set forth in GenBank Accession No. ADK24706.1.In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in GenBank Accession No. ADK24685.1, or the amino acid sequence set forth in GenBank Accession No. ADK24685.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in GenBank Accession No. ACP30488.1, or the amino acid sequence set forth in GenBank Accession No. ACP30488.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in GenBank Accession No. ADK24687.1, or the amino acid sequence set forth in GenBank Accession No. ADK24687.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to, or the amino acid sequence set forth in GenBank Accession No. ACP30487.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to, or the amino acid sequence set forth in GenBank Accession No. ADK24717.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to, or the amino acid sequence set forth in GenBank Accession No. ADK24717.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to, or the amino acid sequence set forth in GenBank Accession No. ADK24690.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in GenBank Accession No. ADK24689.1, or the amino acid sequence set forth in GenBank Accession No. ADK24689.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in GenBank Accession No. ADK24692.1, or the amino acid sequence set forth in GenBank Accession No. ADK24692.1.In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in GenBank Accession No. ADK24693.1, or the amino acid sequence set forth in GenBank Accession No. ADK24693.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in GenBank Accession No. ADK24712.1, or the amino acid sequence set forth in GenBank Accession No. ADK24712.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in GenBank Accession No. ADK24691.1, or the amino acid sequence set forth in GenBank Accession No. ADK24691.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in UniProt No. P0CJ87.1, or the amino acid sequence set forth in UniProt No. P0CJ87.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in GenBank Accession No. ADK24714.1, or the amino acid sequence set forth in GenBank Accession No. ADK24714.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in GenBank Accession No. ADK24684.1, or the amino acid sequence set forth in GenBank Accession No. ADK24684.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in GenBank Accession No. ADK24695.1, or the amino acid sequence set forth in GenBank Accession No. ADK24695.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in GenBank Accession No. ADK24699.1, or the amino acid sequence set forth in GenBank Accession No. ADK24699.1.In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in NCBI RefSeq No. NP_001768.1, or the amino acid sequence set forth in NCBI RefSeq No. NP_001768. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in NCBI RefSeq No. NP_942088.1, or the amino acid sequence set forth in NCBI RefSeq No. NP_942088.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 28 as provided in PCT / US2020 / 44635, or the amino acid sequence of SEQ ID NO: 28 as provided in PCT / US2020 / 44635. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to, or the amino acid sequence of, SEQ ID NO: 29 provided in PCT / US2020 / 44635.

[0445] In other embodiments, expression of the tolerogenic factor is facilitated using an expression vector. In some embodiments, the expression vector comprises a polynucleotide sequence encoding DUX4, which is a codon-altered sequence comprising one or more base substitutions to reduce the total number of CpG sites while preserving the DUX4 protein sequence. In some cases, the codon-altered sequence of DUX4 comprises SEQ ID NO: 1 of PCT / US2020 / 44635. In some cases, the codon-altered sequence of DUX4 is SEQ ID NO: 1 of PCT / US2020 / 44635. In other embodiments, the expression vector comprises a polynucleotide sequence encoding DUX4 comprising SEQ ID NO: 1 of PCT / US2020 / 44635. In some embodiments, the expression vector comprises a polynucleotide sequence encoding a DUX4 polypeptide sequence having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29 of PCT / US2020 / 44635. In some embodiments, the expression vector comprises a polynucleotide sequence encoding a DUX4 polypeptide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29 of PCT / US2020 / 44635.

[0446] Increased DUX4 expression can be assayed using known techniques such as Western blot, ELISA assay, FACS assay, immunoassay, and the like.

[0447] N. Additional Tolerogenic Factors In certain embodiments, one or more tolerogenic factors can be inserted or reinserted into the genome-edited cells to create immune-privileged universal donor cells, such as universal donor stem cells, universal donor T cells, or universal donor cells. In certain embodiments, the engineered and / or hypoimmunogenic cells disclosed herein are further modified to express one or more tolerogenic factors. Exemplary tolerogenic factors include, but are not limited to, one or more of CD47, 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 Serpinb9. In some embodiments, the tolerogenic factor is selected from the group consisting of CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FasL, Serpinb9, CCL21, CCL22, and Mfge8. In some embodiments, the tolerogenic factor is selected from the group consisting of DUX4, HLA-C, HLA-E, HLA-F, HLA-G, PD-L1, CTLA-4-Ig, C1-inhibitor, and IL-35. In some embodiments, the tolerogenic factor is selected from the group consisting of HLA-C, HLA-E, HLA-F, HLA-G, PD-L1, CTLA-4-Ig, C1-inhibitor, and IL-35. In some embodiments, the tolerogenic factor is selected from the group comprising CD47, 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 Serpinb9.

[0448] Useful genomic, polynucleotide, and polypeptide information for human CD27 (also known as CD27L receptor, tumor necrosis factor receptor superfamily member 7, TNFSF7, T cell activation antigen S152, Tp55, and T14) is provided, for example, under GeneCard identifier GC12P008144, HGNC number 11922, NCBI gene ID 939, Uniprot number P26842, and NCBI RefSeq numbers NM_001242.4 and NP_001233.1.

[0449] Useful genome, polynucleotide, and polypeptide information for human CD46 is available, for example, under GeneCard identifier GC01P207752, HGNC number 6953, NCBI gene ID 4179, Uniprot number P15529, and NCBI RefSeq numbers NM_002389.4, NM_153826.3, NM_172350.2, NM_172351.2, NM_172352.2. Available in NP_758860.1, NM_172353.2, NM_172359.2, NM_172361.2, NP_002380.3, NP_722548.1, NP_758860.1, NP_758861.1, NP_758862.1, NP_758863.1, NP_758869.1, and NP_758871.1.

[0450] Useful genomic, polynucleotide, and polypeptide information for human CD55 (also known as complement decay-accelerating factor) is provided, for example, under GeneCard identifier GC01P207321, HGNC number 2665, NCBI Gene ID 1604, Uniprot number P08174, and NCBI RefSeq numbers NM_000574.4, NM_001114752.2, NM_001300903.1, NM_001300904.1, NP_000565.1, NP_001108224.1, NP_001287832.1, and NP_001287833.1.

[0451] Useful genome, polynucleotide, and polypeptide information for human CD59 is available, for example, under GeneCard identifier GC11M033704, HGNC number 1689, NCBI gene ID 966, Uniprot number P13987, and NCBI The sequences are provided in RefSeq numbers NP_000602.1, NM_000611.5, NP_001120695.1, NM_001127223.1, NP_001120697.1, NM_001127225.1, NP_001120698.1, NM_001127226.1, NP_001120699.1, NM_001127227.1, NP_976074.1, NM_203329.2, NP_976075.1, NM_203330.2, NP_976076.1, and NM_203331.2.

[0452] Useful genomic, polynucleotide, and polypeptide information for human CD200 is provided, for example, under GeneCard identifier GC03P112332, HGNC number 7203, NCBI gene ID 4345, Uniprot number P41217, and NCBI RefSeq numbers NP_001004196.2, NM_001004196.3, NP_001305757.1, NM_001318828.1, NP_005935.4, NM_005944.6, XP_005247539.1, and XM_005247482.2.

[0453] Useful genomic, polynucleotide, and polypeptide information for human HLA-C is provided, for example, under GeneCard identifier GC06M031272, HGNC number 4933, NCBI gene ID 3107, Uniprot number P10321, and NCBI RefSeq numbers NP_002108.4 and NM_002117.5.

[0454] Useful genomic, polynucleotide, and polypeptide information for human HLA-E is provided, for example, under GeneCard identifier GC06P047281, HGNC number 4962, NCBI gene ID 3133, Uniprot number P13747, and NCBI RefSeq numbers NP_005507.3 and NM_005516.5.

[0455] Useful genomic, polynucleotide, and polypeptide information for human HLA-G is provided, for example, under GeneCard identifier GC06P047256, HGNC number 4964, NCBI gene ID 3135, Uniprot number P17693, and NCBI RefSeq numbers NP_002118.1 and NM_002127.5.

[0456] Useful genomic, polynucleotide, and polypeptide information for human PD-L1 or CD274 is provided, for example, under GeneCard identifier GC09P005450, HGNC number 17635, NCBI Gene ID 29126, Uniprot number Q9NZQ7, and NCBI RefSeq numbers NP_001254635.1, NM_001267706.1, NP_054862.1, and NM_014143.3.

[0457] Useful genome, polynucleotide, and polypeptide information for human IDO1 is provided, for example, under GeneCard identifier GC08P039891, HGNC number 6059, NCBI gene ID 3620, Uniprot number P14902, and NCBI RefSeq numbers NP_002155.1 and NM_002164.5.

[0458] Useful genomic, polynucleotide, and polypeptide information for human IL-10 is provided, for example, under GeneCard identifier GC01M206767, HGNC number 5962, NCBI gene ID 3586, Uniprot number P22301, and NCBI RefSeq numbers NP_000563.1 and NM_000572.2.

[0459] Useful genomic, polynucleotide, and polypeptide information for human Fas Ligand (also known as FasL, FASLG, CD178, TNFSF6, etc.) is provided, for example, under GeneCard identifier GC01P172628, HGNC number 11936, NCBI Gene ID 356, Uniprot number P48023, and NCBI RefSeq numbers NP_000630.1, NM_000639.2, NP_001289675.1, and NM_001302746.1.

[0460] Useful genome, polynucleotide, and polypeptide information for human CCL21 is provided, for example, under GeneCard identifier GC09M034709, HGNC number 10620, NCBI gene ID 6366, Uniprot number O00585, and NCBI RefSeq numbers NP_002980.1 and NM_002989.3.

[0461] Useful genome, polynucleotide, and polypeptide information for human CCL22 is provided, for example, under GeneCard identifier GC16P057359, HGNC number 10621, NCBI gene ID 6367, Uniprot number O00626, and NCBI RefSeq numbers NP_002981.2, NM_002990.4, XP_016879020.1, and XM_017023531.1.

[0462] Useful genome, polynucleotide, and polypeptide information for human Mfge8 is provided, for example, under GeneCard identifier GC15M088898, HGNC number 7036, NCBI Gene ID 4240, Uniprot number Q08431, and NCBI RefSeq numbers NP_001108086.1, NM_001114614.2, NP_001297248.1, NM_001310319.1, NP_001297249.1, NM_001310320.1, NP_001297250.1, NM_001310321.1, NP_005919.2, and NM_005928.3.

[0463] Useful genome, polynucleotide, and polypeptide information for human SerpinB9 is provided, for example, under GeneCard identifier GC06M002887, HGNC number 8955, NCBI gene ID 5272, Uniprot number P50453, and NCBI RefSeq numbers NP_004146.1, NM_004155.5, XP_005249241.1, and XM_005249184.4.

[0464] Methods for regulating gene and factor (protein) expression include genome editing techniques, RNA or protein expression techniques, etc. For all of these techniques, well-known recombinant techniques are used to generate recombinant nucleic acids as outlined herein.

[0465] In some embodiments, the cells (e.g., stem cells, induced pluripotent stem cells, differentiated cells, hematopoietic stem cells, primary T cells, or CAR-T cells) have genetic modifications that inactivate the B2M and CIITA genes, and are further comprised of CD47 and DUX4, CD47 and CD24, CD47 and CD27, CD47 and CD46, CD47 and CD55, CD47 and CD59, CD47 and CD200, CD47 and HLA-C, CD47 and HLA-E, CD47 and HLA-E heavy chain, The cells express multiple exogenous polypeptides selected from the group including CD47 and HLA-G, CD47 and PD-L1, CD47 and IDO1, CD47 and CTLA4-Ig, CD47 and C1-inhibitor, CD47 and IL-10, CD47 and IL-35, CD47 and IL-39, CD47 and FasL, CD47 and CCL21, CD47 and CCL22, CD47 and Mfge8, and CD47 and Serpinb9, and any combination thereof. In some cases, the cells also have a genetic modification that inactivates the CD142 gene.

[0466] In some cases, a gene editing system, such as a CRISPR / Cas system, is used to facilitate insertion of a tolerogenic factor, such as a tolerogenic factor, into a safe harbor or target locus, such as the AAVS1 locus, to actively inhibit immune rejection. In some cases, the tolerogenic factor is inserted into the safe harbor or target locus using an expression vector. In some embodiments, the safe harbor or target locus is the AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (also known as CD142), MICA, MICB, LRP1 (also known as CD91), HMGB1, ABO, RHD, FUT1, or KDM5D locus.

[0467] In some embodiments, expression of a target gene (e.g., CD47, DUX4, or another tolerogenic factor gene) is increased by expression of a fusion protein or protein complex containing (1) a site-specific binding domain specific for the endogenous target gene (e.g., CD47, DUX4, or another tolerogenic factor gene) and (2) a transcriptional activator.

[0468] In some embodiments, the regulatory element is comprised of a site-specific DNA-binding nucleic acid molecule, such as a guide RNA (gRNA). In some embodiments, the method is achieved by a site-specific DNA-binding targeting protein, such as a zinc finger protein (ZFP), also known as a zinc finger nuclease (ZFN), or a fusion protein containing a ZFP.

[0469] In some embodiments, the regulatory factor comprises a site-specific binding domain, e.g., using a DNA-binding protein or DNA-binding nucleic acid, that specifically binds or hybridizes to a gene at a target region. In some embodiments, the provided polynucleotide or polypeptide is linked to or complexed with a site-specific nuclease, such as a modified nuclease. For example, in some embodiments, administration is achieved using a modified nuclease, e.g., a meganuclease or an RNA-guided nuclease, e.g., a fusion comprising a DNA-targeting protein of a clustered regularly interspersed short palindromic nucleic acid (CRISPR)-Cas system, e.g., a CRISPR-Cas9 system. In some embodiments, the nuclease is modified to lack nuclease activity. In some embodiments, the modified nuclease is a catalytically dead dCas9.

[0470] In some embodiments, the site-specific binding domain can be derived from a nuclease, for example, the recognition sequences of homing endonucleases and meganucleases such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII, and I-TevIII. See also U.S. Pat. No. 5,420,032, U.S. Pat. No. 6,833,252, Belfort et al., (1997) Nucleic Acids Res. 25:3379-3388, Dujon et al., (1989) Gene 82:115-118, Perler et al., (1994) Nucleic Acids Res. 22, 1125-1127, Jasin (1996) Trends Genet. 12:224-228, Gimble et al., (1996) J. Mol. Biol. 263:163-180, Argast et al., (1998) J. Mol. Biol. 280:345-353, and the New England Biolabs catalog. In addition, the DNA binding specificity of homing endonucleases and meganucleases can be engineered to bind to non-natural target sites (see, e.g., Chevalier et al., (2002) Molec. Cell 10:895-905; Epinat et al., (2003) Nucleic Acids Res. 31:2952-2962; Ashworth et al., (2006) Nature 441:656-659; Paques et al., (2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No. 2007 / 0117128).

[0471] The binding domains of zinc finger, TALE, and CRISPR systems can be "engineered" to bind to a predetermined nucleotide sequence, for example, by manipulating the recognition helix region of a naturally occurring zinc finger or TALE protein (changing one or more amino acids). Engineered DNA-binding proteins (zinc finger or TALE) are proteins that do not occur in nature. Rational criteria for design include the application of substitution rules and computerized algorithms for processing information in databases that store information on existing ZFP and / or TALE designs and binding data. See, for example, U.S. Patent Nos. 6,140,081, 6,453,242, and 6,534,261; WO98 / 53058, WO98 / 53059, WO98 / 53060, WO02 / 016536, and WO03 / 016496; and U.S. Publication No. 20110301073.

[0472] In some embodiments, the site-specific binding domain comprises one or more zinc finger proteins (ZFPs) or domains thereof that bind to DNA in a sequence-specific manner. A ZFP or domain thereof is a protein or domain within a larger protein that binds to DNA in a sequence-specific manner via one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized by the coordination of a zinc ion.

[0473] Among ZFPs are artificial ZFP domains, typically 9-18 nucleotides in length, that target specific DNA sequences generated by the assembly of individual fingers. ZFPs include those with two, three, four, five, or six fingers, each with a single finger domain approximately 30 amino acids in length and containing an alpha helix containing two invariant histidine residues coordinated to zinc along with two cysteines in a single beta turn. In general, the sequence specificity of a ZFP can be altered by making amino acid substitutions at four helical positions (-1, 2, 3, and 6) on the recognition helix of the zinc finger. Thus, in some embodiments, the ZFP or ZFP-containing molecule is non-naturally occurring and, for example, engineered to bind to a target site of choice. For example, Beerli et al. (2002) Nature Biotechnol.20:135-141, Pabo et al. (2001) Ann.Rev.Biochem.70:313-340, Isalan et al. (2001) Nature Biotechnol.19:656-660, Segal et al. al. (2001) Curr. Opin. Biotechnol. 12:632-637, Choo et al. al.(2000)Curr.Opin.Struct.Biol.10:411-416, U.S. Patent No. 6,453,242, U.S. Patent No. 6,534,261, U.S. Pat. No. 6,599,692, No. 6,503,717, No. 6,689,558, No. 7,030,215, No. 6,794,136, No. 7,067, 317, 7,262,054, 7,070,934, 7,361,635, 7,253,273, and U.S. Patent Publication Nos. 2005 / 0064474, 2007 / 0218528, and 2005 / 0267061, all of which are incorporated herein by reference in their entireties.

[0474] Many gene-specific engineered zinc fingers are commercially available. For example, Sangamo Biosciences (Richmond, CA, USA) has partnered with Sigma-Aldrich (St. Louis, MO, USA) to develop a platform for zinc finger construction (CompoZr) that allows researchers to completely bypass the construction and validation of zinc fingers and provides specifically targeted zinc fingers for thousands of proteins (Gaj et al., Trends in Biotechnology, 2013, 31(7), 397-405). In some embodiments, commercially available zinc fingers are used or custom-designed.

[0475] In some embodiments, the site-specific binding domain comprises the DNA binding domain of a naturally occurring or engineered (non-naturally occurring) transcription activator-like protein (TAL), such as in a transcription activator-like protein effector (TALE) protein. See, e.g., U.S. Patent Publication No. 20110301073, which is incorporated herein by reference in its entirety.

[0476] In some embodiments, the site-specific binding domain is derived from a CRISPR / Cas system. Generally, a "CRISPR system" refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr mate sequences (including "direct repeats" and partial direct repeats processed by tracrRNA in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" or "targeting sequences" in the context of endogenous CRISPR systems), and / or other sequences and transcripts from the CRISPR locus.

[0477] Generally, the guide sequence comprises a targeting domain, which comprises a polynucleotide sequence that has sufficient complementarity with the target polynucleotide sequence to hybridize with the target sequence and direct the sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is about 50% or more, about 60% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97.5% or more, about 99% or more, or more, when optimally aligned using a suitable alignment algorithm. In some examples, the targeting domain of the gRNA is complementary to the target sequence on the target nucleic acid, for example, at least 80, 85, 90, 95, 98, or 99% complementary, for example, fully complementary.

[0478] In some embodiments, the target site is upstream of the transcription start site of the target gene. In some embodiments, the target site is adjacent to the transcription start site of the gene. In some embodiments, the target site is adjacent to an RNA polymerase pause site downstream of the transcription start site of the gene.

[0479] In some embodiments, the targeting domain is configured to target the promoter region of the target gene to promote transcription initiation, one or more transcriptional enhancers or activators, and / or RNA polymerase binding. One or more gRNAs can be used to target the promoter region of the gene. In some embodiments, one or more regions of the gene can be targeted. In certain aspects, the target site is within 600 base pairs on either side of the transcription start site (TSS) of the gene.

[0480] It is within the skill level of those skilled in the art to design or identify a gRNA sequence that targets a gene or that includes the sequence, including exon sequences and regulatory region sequences including promoters and activators. A genome-wide gRNA database for CRISPR genome editing is publicly available, and includes exemplary single guide RNA (sgRNA) target sequences within the constitutive exons of genes in the human genome or mouse genome (see, for example, genescript.com / gRNA-database.html; also see Sanjana et al. (2014) Nat. Methods, 11:783-4, www.e-crisp.org / E-CRISP / , crispr.mit.edu / ). In some embodiments, the gRNA sequence is or includes a sequence that minimizes off-target binding to non-target genes.

[0481] In some embodiments, the regulatory element further comprises a functional domain, for example, a transcriptional activator.

[0482] In some embodiments, the transcriptional activator is or contains one or more regulatory elements, such as one or more transcriptional regulatory elements, of a target gene, whereby a site-specific domain, as provided above, is recognized to drive expression of such a gene. In some embodiments, the transcriptional activator drives expression of a target gene. In some cases, the transcriptional activator can be or contain all or a portion of a heterologous transactivation domain. For example, in some embodiments, the transcriptional activator is selected from a herpes simplex-derived transactivation domain, a Dnmt3a methyltransferase domain, p65, VP16, and VP64.

[0483] In some embodiments, the regulatory factor is a zinc finger transcription factor (ZF-TF). In some embodiments, the regulatory factor is VP64-p65-Rta (VPR).

[0484] In certain embodiments, the regulator further comprises a transcriptional regulatory domain. Common domains include, for example, transcription factor domains (activators, repressors, coactivators, corepressors), silencers, oncogenes (e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos family members, etc.); DNA repair enzymes and their associated factors and modifiers; DNA remodeling enzymes and their associated factors and modifiers; chromatin-associated proteins and their modifiers (e.g., kinases, acetylases, and deacetylases); and DNA-modifying enzymes (e.g., methyltransferases such as members of the DNMT family (e.g., DNMT1, DNMT3A, DNMT3B, DNMT3L, etc., topoisomerases, helicases, ligases, kinases, phosphatases, polymerases, endonucleases) and their associated factors and modifiers. See, for example, U.S. Publication No. 2013 / 0253040, which is incorporated herein by reference in its entirety.

[0485] Suitable domains for achieving activation include the HSV VP16 activation domain (see, e.g., Hagmann et al., J. Virol. 71, 5952-5962 (197)), nuclear hormone receptors (see, e.g., Torchia et al., Curr. Opin. Cell. Biol. 10:373-383 (1998)), the p65 subunit of nuclear factor kappa B (Bitko & Bank, J. Virol. 72:5610-5618 (1998) and Doyle & Hunt, Neuroreport 8:2937-2942 (1997)), Liu et al., Cancer Gene Ther. 5:3-28 (1998)), or artificial chimeric functional domains such as VP64 (Beerli et al., (1998) Proc. Natl. Acad. Sci. USA 95:14623-33), and degrons (Molinari et al., (1999) EMBO J.18,6439-6447). Additional exemplary activation domains include Oct1, Oct-2A, Spl, AP-2, and CTF1 (Seipel et al., EMBO J. 11, 4961-4968 (1992)), as well as p300, CBP, PCAF, SRC1 PvALF, AtHD2A, and ERF-2. See, e.g., Robyr et al. (2000) Mol. Endocrinol. 14:329-347; Collingwood et al. (1999) J. Mol. Endocrinol 23:255-275; Leo et al. (2000) Gene 245:1-11; Manteuffel-Cymborowska (1999) Acta Biochem. Pol. 46:77-89; McKenna et al. (1999) J. Steroid. See Biochem. Mol. Biol. 69:3-12, Malik et al. (2000) Trends Biochem. Sci. 25:277-283, and Lemon et al. (1999) Curr. Opin. Genet. Dev. 9:499-504.Additional exemplary activation domains include, but are not limited to, OsGAI, HALF-1, Cl, AP1, ARF-5, -6, -1, and -8, CPRF1, CPRF4, MYC-RP / GP, and TRAB1. For example, Ogawa et al, (2000) Gene 245:21-29, Okanami et al, (1996) Genes Cells 1:87-99, Goff et al, (1991) Genes Dev.5:298-309, Cho et al, (1999) Plant Mol Biol 40:419-429, Ulmason et al. al,(1999)Proc.Natl.Acad.Sci.USA 96:5844-5849, Sprenger-Haussels et al,(2000)Plant J.22:1-8, Gong et al,(1999)Plant Mol.Biol.41:33-44, and Hobo et al.,(1999)Proc.Natl.Acad.Sci.USA See 96:15,348-15,353.

[0486] Exemplary repression domains that can be used to generate gene repressors include, but are not limited to, KRAB A / B, KOX, TGF-beta inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, members of the DNMT family (e.g., DNMT1, DNMT3A, DNMT3B, DNMT3L, etc.), Rb, and MeCP2. See, e.g., Bird et al. (1999) Cell 99:451-454, Tyler et al. (1999) Cell 99:443-446, Knoepfler et al. (1999) Cell 99:447-450, and Robertson et al. (2000) Nature Genet. 25:338-342. Additional exemplary repression domains include, but are not limited to, ROM2 and AtHD2A. See, for example, Chem et al., (1996) Plant Cell 8:305-321, and Wu et al., (2000) Plant J. 22:19-27.

[0487] In some cases, the domain is involved in epigenetic regulation of chromosomes. In some embodiments, the domain is a nuclear-localized histone acetyltransferase (HAT) (e.g., type A), such as the MYST family members MOZ, Ybf2 / Sas3, MOF, and Tip60, the GNAT family members Gcn5 or pCAF, the p300 family members CBP, p300, or Rtt109 (Bemdsen and Denu (2008) Curr Opin Struct Biol 18(6):682-689). In other cases, the domain is a histone deacetylase (HDAC), such as class I (HDAC-1, 2, 3, and 8), class II (HDAC IIA (HDAC-4, 5, 7, and 9), HDAC IIB (HDAC 6 and 10)), class IV (HDAC-1 1), or class III (also known as sirtuins (SIRTs); SIRT1-7) (see Mottamal et al., (2015) Molecules 20(3):3898-3941). Another domain used in some embodiments is a histone phosphorylase or kinase, examples of which include MSK1, MSK2, ATR, ATM, DNA-PK, Bubl, VprBP, IKK-a, PKCpi, Dik / Zip, JAK2, PKC5, WSTF, and CK2. In some embodiments, a methylation domain is used, which may be selected from the group including Ezh2, PRMT1 / 6, PRMT5 / 7, PRMT 2 / 6, CARM1, set7 / 9, MLL, ALL-1, Suv 39h, G9a, SETDB1, Ezh2, Set2, Dot1, PRMT1 / 6, PRMT5 / 7, PR-Set7, and Suv4-20h. Domains involved in sumoylation and biotinylation (Lys9, 13, 4, 18, and 12) may also be used in some embodiments (for review, see Kousarides (2007) Cell 128:693-705).

[0488] Fusion molecules are constructed by cloning and biochemical conjugation methods well known to those skilled in the art. Fusion molecules contain a DNA-binding domain and a functional domain (e.g., a transcriptional activation or repression domain). Optionally, the fusion molecule also contains a nuclear localization signal (e.g., a signal from SV40 middle T antigen) and an epitope tag (e.g., FLAG and hemagglutinin). Fusion proteins (and the nucleic acids encoding them) are designed to preserve the translational reading frame between the fusion components.

[0489] Fusions between a polypeptide component of a functional domain (or functional fragment thereof) on the one hand and a non-protein DNA-binding domain (e.g., an antibiotic, an intercalator, a minor groove binder, a nucleic acid) on the other hand are constructed by biochemical conjugation methods known to those skilled in the art. See, e.g., Pierce Chemical Company (Rockford, IL) Catalogue. Methods and compositions for creating fusions between minor groove binders and polypeptides are described. Mapp et al. (2000) Proc. Natl. Acad. Sci. USA 97:3930-3935. Similarly, CRISPR / Cas TFs and nucleases comprising a component that is an sgRNA nucleic acid in association with a functional domain that is a polypeptide component are also known to those skilled in the art and are described in detail herein.

[0490] In some embodiments, the present disclosure provides cells (e.g., primary T cells and hypoimmunogenic stem cells and their derivatives) or populations thereof comprising a genome in which the genome of the cell has been modified to express CD47. In some embodiments, the present disclosure provides methods for altering the genome of a cell to express CD47. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate insertion of CD47 into a cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 200784-231885 in Table 29 of WO2016183041, which is incorporated herein by reference.

[0491] In some embodiments, the present disclosure provides cells (e.g., primary T cells and hypoimmunogenic stem cells and their derivatives) or populations thereof comprising a genome in which the genome of the cell has been modified to express HLA-C. In some embodiments, the present disclosure provides methods for altering the genome of a cell to express HLA-C. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate insertion of HLA-C into a cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 3278-5183 in Table 10 of WO2016183041, which is incorporated herein by reference.

[0492] In some embodiments, the present disclosure provides cells (e.g., primary T cells and hypoimmunogenic stem cells and their derivatives) or populations thereof comprising a genome in which the genome of the cell has been modified to express HLA-E. In some embodiments, the present disclosure provides methods for altering the genome of a cell to express HLA-E. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate insertion of HLA-E into a cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 189859-193183 in Table 19 of WO2016183041, which is incorporated herein by reference.

[0493] In some embodiments, the present disclosure provides cells (e.g., primary T cells and hypoimmunogenic stem cells and their derivatives) or populations thereof comprising a genome in which the genome of the cell has been modified to express HLA-F. In some embodiments, the present disclosure provides methods for altering the genome of a cell to express HLA-F. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate insertion of HLA-F into a cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 688808-399754 in Table 45 of WO2016183041, which is incorporated herein by reference.

[0494] In some embodiments, the present disclosure provides cells (e.g., primary T cells and hypoimmunogenic stem cells and their derivatives) or populations thereof comprising a genome in which the genome of the cell has been modified to express HLA-G. In some embodiments, the present disclosure provides methods for altering the genome of a cell to express HLA-G. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate insertion of HLA-G into a stem cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 188372-189858 in Table 18 of WO2016183041, which is incorporated herein by reference.

[0495] In some embodiments, the present disclosure provides cells (e.g., primary T cells and hypoimmunogenic stem cells and their derivatives) or populations thereof comprising a genome in which the genome of the cell has been modified to express PD-L1. In some embodiments, the present disclosure provides methods for altering the genome of a cell to express PD-L1. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate insertion of PD-L1 into a stem cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 193184-200783 in Table 21 of WO2016183041, which is incorporated herein by reference.

[0496] In some embodiments, the present disclosure provides cells (e.g., primary T cells and hypoimmunogenic stem cells and their derivatives) or populations thereof comprising a genome in which the genome of the cell has been modified to express CTLA4-Ig. In some embodiments, the present disclosure provides methods for altering the genome of a cell to express CTLA4-Ig. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate insertion of CTLA4-Ig into a stem cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from any one disclosed in WO2016183041, including the sequence listing.

[0497] In some embodiments, the present disclosure provides cells (e.g., primary T cells and hypoimmunogenic stem cells and their derivatives) or populations thereof, comprising a genome in which the genome of the cell has been modified to express a CI-inhibitor. In some embodiments, the present disclosure provides methods for altering the genome of a cell to express a CI-inhibitor. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate the insertion of a CI-inhibitor into a stem cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from any one of those disclosed in WO2016183041, including the sequence listing.

[0498] In some embodiments, the present disclosure provides cells (e.g., primary T cells and hypoimmunogenic stem cells and derivatives thereof) or populations thereof comprising a genome in which the genome of the cell has been modified to express IL-35. In some embodiments, the present disclosure provides methods for altering the genome of a cell to express IL-35. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate insertion of IL-35 into a stem cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from any one disclosed in WO2016183041, including the sequence listing.

[0499] In some embodiments, the tolerogenic factor is expressed in cells using an expression vector. For example, an expression vector for expressing CD47 in cells comprises a polynucleotide sequence encoding CD47. The expression vector may be an inducible expression vector. The expression vector may be a viral vector, such as, but not limited to, a lentiviral vector. In some embodiments, the tolerogenic factor is introduced into cells using fusogen-mediated delivery or 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.

[0500] In some embodiments, a suitable gene editing system (e.g., a CRISPR / Cas system or any of the gene editing systems described herein) is used to facilitate insertion of a polynucleotide encoding a tolerogenic factor into a genomic locus of a hypoimmunogenic cell. In some cases, the polynucleotide encoding the tolerogenic factor is inserted into a safe or target harbor locus, such as, but not limited to, the AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (CD142), MICA, MICB, LRP1 (CD91), HMGB1, ABO, RHD, FUT1, or KDM5D locus. In some embodiments, the polynucleotide encoding the tolerogenic factor is inserted into the B2M locus, CIITA locus, TRAC locus, or TRB locus. In some embodiments, the polynucleotide encoding the tolerogenic factor is inserted into any one of the loci set forth in Tables 2-5 provided herein. In certain embodiments, the polynucleotide encoding the tolerogenic factor is operably linked to a promoter.

[0501] O. Protocadherin-11 Y-linked In certain embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) expression of one or more Y chromosome genes by targeting and modulating (e.g., reducing or eliminating) the expression of the Y chromosome genes. In some embodiments, the modulation occurs using a gene editing system (e.g., a CRISPR / Cas system). In some embodiments, the cells have a reduced ability to induce an innate and / or adaptive immune response in a recipient subject.

[0502] In certain embodiments, the technology disclosed herein regulates (e.g., reduces or eliminates) the expression of a protocadherin-11 Y-linked antigen by targeting and modulating (e.g., reducing or eliminating) the expression of a protocadherin-11 Y-linked gene, such as PCDH11Y. In some embodiments, the modulation occurs using a CRISPR / Cas system. In some embodiments, the cells have a reduced ability to induce an immune response in a recipient subject.

[0503] In some embodiments, the target polynucleotide sequence of the present disclosure is a variant of the PCDH11Y gene. In some embodiments, the target polynucleotide sequence is a homolog of the PCDH11Y gene. In some embodiments, the target polynucleotide sequence is an ortholog of the PCDH11Y gene.

[0504] In some embodiments, the cells described herein comprise a genetic modification at a locus encoding the protocadherin-11 Y-linked antigen protein. In other words, the cells comprise a genetic modification at the PCDH11Y locus. In some cases, the nucleotide sequence encoding the protocadherin-11 Y-linked antigen protein is set forth in RefSeq. No. NM_001278619.1, NM_032971.2, NM_032972.2, NM_032973.2, or XM_017030082.1, or GenBank No. AJ276803, AF277053, AF332216, AF332217, AJ564958, AJ564959, AJ564960, AJ564961, AJ564962, AJ564963, AJ564966, or AJ56496. In some cases, the PCDH11Y locus is set forth in NCBI Gene ID No. 83259. In certain instances, the amino acid sequence of the protocadherin-11 Y-linked antigen is set forth as NCBI GenBank No. CAC13122.1, AAL55729.1, AAK13468.1, AAK13469.1, CAD92429.1, CAD92430.1, CAD92431.1, CAD92432.1, CAD92433.1, CAD92434.1, CAD92437.1, or CAD92440.1. Additional descriptions of the protocadherin-11 Y-linked antigen protein and gene locus can be found in Uniprot No. Q9BZA8, HGNC Reference No. 15813, and OMIM Reference No. 400022.

[0505] In some embodiments, the engineered and / or hypoimmunogenic cells described herein comprise a genetic modification targeting the PCDH11Y gene. In some embodiments, the genetic modification targeting the PCDH11Y gene is generated by gene editing the PCDH11Y gene using a gene editing tool, such as, but not limited to, CRISPR / Cas, TALE-nuclease, zinc finger nuclease, other virus-based gene editing systems, or RNA interference. In some embodiments, the gene editing targets the coding sequence of the PCDH11Y gene. In some cases, the cells do not produce a functional PCDH11Y gene product. In the absence of the PCDH11Y gene product, the cells completely lack the protocadherin-11 Y-linked antigen.

[0506] In some embodiments, a Cas9 or Cas12a editing system is used to target a sequence of the PCDH11Y gene to introduce an insertion or deletion into the gene to disrupt its function, or in some cases, to inactivate it. In some embodiments, a single guide RNA is used. In some embodiments, a dual guide RNA is used. In some embodiments, any one of the gRNA target sequences in Table 2A or Table 2B is used. In some cases, more than one gRNA target sequence in Table 2A and / or Table 2B is used for gene editing. In some embodiments, the Cas9 editing system comprises a Cas9 protein or fragment thereof, a tracrRNA, and a crRNA. In some embodiments, the Cas12a editing system comprises a Cas12a protein or fragment thereof and a crRNA.

[0507] In some embodiments, a frameshift insertion-deletion is introduced into any coding sequence of the gene. In some embodiments, modifications are made within the UTRs, introns, or exons of the gene to disrupt function of the PCDH11Y gene. In some embodiments, CRISPR / Cas editing is utilized that includes any one or more of the gRNA target sequences in Table 2A and / or Table 2B.

[0508] In some embodiments, a modification is introduced into the PCDH11Y gene to inactivate the gene. In some embodiments, a coding exon, such as exon 1 or exon 2 or exon 3 of the PCDH11Y gene, is targeted. In some cases, a deletion is created using a Cas editing system and a guide RNA target sequence that targets a sequence 5' of the PCDH11Y gene and a guide RNA target sequence for an exon, such as, but not limited to, exon 1 or 2. In some embodiments, the cells described herein comprise a homozygous modification of the PCDH11Y gene, thereby inactivating the gene. [Table 2-1] [Table 2-2] [Table 2-3] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11]

[0509] In some embodiments, the gRNA target sequence is directed against exon 1 or exon 2 of the PCDH11Y gene. In some embodiments, the gRNA target sequence is a gRNA from Table 2A and / or Table 2B that introduces a frameshift mutation to inactivate exon 1 or exon 2.

[0510] In some embodiments, expression of the PCDH11Y gene is partially or completely inactivated by an insertion or deletion within exon 1 or exon 2 of the PCDH11Y gene.

[0511] Assays for testing whether the PCDH11Y gene is inactivated are known and described herein. In one embodiment, the resulting genetic modification of the PCDH11Y gene can be assayed by PCR, and the reduction in protocadherin-11 Y-linked antigen protein expression can be assayed by FACS analysis. In another embodiment, protocadherin-11 Y-linked antigen protein expression is detected using Western blot of cell lysates probed with an antibody against the protocadherin-11 Y-linked antigen protein. In another embodiment, the presence of the inactivating genetic modification is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0512] P. neuroligin-4 Y-linked In certain embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) expression of a neuroligin-4 Y-linked antigen by targeting and modulating (e.g., reducing or eliminating) expression of a neuroligin-4 Y-linked gene, e.g., NLGN4Y. In some embodiments, the modulation occurs using a gene editing system (e.g., a CRISPR / Cas system). In some embodiments, the cells have a reduced ability to induce an innate and / or adaptive immune response in a recipient subject.

[0513] In some embodiments, the target polynucleotide sequence of the present disclosure is a variant of the NLGN4Y gene. In some embodiments, the target polynucleotide sequence is a homolog of the NLGN4Y gene. In some embodiments, the target polynucleotide sequence is an ortholog of the NLGN4Y gene.

[0514] In some embodiments, the cells described herein comprise a genetic modification at a locus encoding a neuroligin-4 Y-linked antigen protein. In other words, the cells comprise a genetic modification at the NLGN4Y locus. In some cases, the nucleotide sequence encoding the neuroligin-4 Y-linked antigen protein is set forth in RefSeq. No. N NM_001164238.1, NM_001206850.1, NM_014893.4, XM_017030034.1, XM_017030035.1, XM_017030036.1, XM_017030037.1, XM_017030038.1, XM_017030040.1, or XM_017030041.1, or as set forth in Genbank numbers AF376804, AB023168, BX537428, AC010726, AC010879, AC010979, AC011903, BC032567, BC113525, or BC113551. In some instances, the NLGN4Y locus is set forth in NCBI Gene ID number 22829. In certain instances, the amino acid sequence of the neuroligin-4 Y-linked antigen is set forth as NCBI GenBank number AAM46113.1, BAA76795.2, CAD97670.1, AAH32567.1, AAI13526.1, or AAI13552.1. Additional descriptions of the neuroligin-4 Y-linked antigen protein and locus can be found in Uniprot number Q8NFZ3, HGNC reference number 15529, and OMIM reference number 400028.

[0515] In some embodiments, the engineered and / or hypoimmunogenic cells described herein comprise a genetic modification targeting the NLGN4Y gene. In some embodiments, the genetic modification targeting the NLGN4Y gene is generated by gene editing the NLGN4Y gene using a gene editing tool, such as, but not limited to, CRISPR / Cas, TALE-nuclease, zinc finger nuclease, other viral-based gene editing systems, or RNA interference. In some embodiments, the gene editing targets the coding sequence of the NLGN4Y gene. In some cases, the cells do not produce a functional NLGN4Y gene product. In the absence of the NLGN4Y gene product, the cells completely lack the neuroligin-4 Y-linked antigen.

[0516] In some embodiments, a Cas9 or Cas12a editing system is used to target a sequence of the NLGN4Y gene to introduce an insertion or deletion into the gene to disrupt its function, or in some cases, to inactivate it. In some embodiments, a single guide RNA is used. In some embodiments, a dual guide RNA is used. In some embodiments, any one of the gRNA target sequences in Table 3, Table 4, and / or Table 5 is used. In some cases, more than one gRNA target sequence in Table 3, Table 4, and / or Table 5 is used for gene editing. In some embodiments, the Cas9 editing system comprises a Cas9 protein or fragment thereof, a tracrRNA, and a crRNA. In some embodiments, the Cas12a editing system comprises a Cas12a protein or fragment thereof and a crRNA.

[0517] In some embodiments, frameshift insertion-deletions are introduced into any coding sequence of the gene. In some embodiments, modifications are made within the UTRs, introns, or exons of the gene to disrupt function of the NLGN4Y gene. In some embodiments, CRISPR / Cas editing is utilized that includes any one or more of the gRNA target sequences in Table 3, Table 4, and / or Table 5.

[0518] In some embodiments, a modification is introduced into the NLGN4Y gene to inactivate the gene. In some embodiments, a coding exon, such as exon 3 or exon 4 or exon 5 of the NLGN4Y gene, is targeted. In some cases, a deletion is created using a Cas editing system and a guide RNA target sequence that targets a sequence 5' of the NLGN4Y gene and a guide RNA target sequence to an exon, such as, but not limited to, exon 3 or exon 4 or exon 5. In some embodiments, the cells described herein comprise a homozygous modification of the NLGN4Y gene, thereby inactivating the gene. [Table 4] [Table 5-1] [Table 5-2] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7]

[0519] In some embodiments, the gRNA target sequence is directed to exon 1 or exon 2 of the NLGN4Y gene. In some embodiments, the gRNA target sequence is a gRNA from Table 3, Table 4, and / or Table 5 that introduces a frameshift mutation to inactivate exon 3, exon 4, or exon 5.

[0520] In some embodiments, expression of the NLGN4Y gene is partially or completely inactivated by an insertion or deletion within exon 3, exon 4, or exon 5 of the NLGN4Y gene.

[0521] Assays for testing whether the NLGN4Y gene is inactivated are known and are described herein. In one embodiment, the resulting genetic modification of the NLGN4Y gene can be assayed for by FACS analysis to determine the reduction in neuroligin-4 Y-linked antigen protein expression. In another embodiment, neuroligin-4 Y-linked antigen protein expression is detected using Western blot of cell lysates probed with an antibody against neuroligin-4 Y-linked antigen protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm t...

Claims

1. An engineered cell comprising one or more genetic modifications that reduce the expression of one or more Y chromosome genes and major histocompatibility complex (MHC) class I and / or class II human leukocyte antigen molecules compared to unaltered or unmodified wild-type or control cells, and comprising a first exogenous polynucleotide encoding CD47.

2. The engineered cell according to claim 1, wherein the engineered cell is selected from the group consisting of T cells, hypoimmunogenic T cells, inactivated T cells, pancreatic islet cells, cardiomyocytes, glial progenitor cells, smooth muscle cells, skeletal muscle cells, hepatocytes, dopaminergic neurons, retinal pigment epithelial cells, thyroid cells, and NK cells.

3. The engineered cell according to claim 1, wherein the engineered cell is derived from primary cells or their progeny, or from pluripotent stem cells or their progeny.

4. The engineered cell according to claim 3, wherein the engineered cell is an embryonic stem cell or an induced pluripotent stem cell (iPSC).

5. The engineered cell according to claim 1, wherein the one or more Y chromosome genes are protocadherin-11 Y-linked, neurexin-4 Y-linked, or a combination thereof.

6. The engineered cell according to claim 1, wherein the cell is derived from human cells or animal cells.

7. The engineered cell according to claim 1, wherein the human cells or animal cells are from a donor subject without a Y chromosome.

8. The engineered cell according to claim 1, wherein the cell comprises reduced expression of beta-2-microglobulin (B2M) and / or MHC class II transactivator (CIITA) compared to unaltered or unmodified wild-type or control cells.

9. The engineered cell according to claim 8, wherein the cell does not express B2M and / or CIITA.

10. The cell is (i) one or more genetic modifications that reduce the expression of RHD, TCR-alpha and / or TCR-beta, (ii) a second exogenous polynucleotide encoding one or more chimeric antigen receptors (CARs), and / or (iii) one or more genetic modifications that knockout the PCDH11Y gene and / or the NLGN4Y gene The engineered cell according to claim 1, comprising.

11. The one or more CARs are (i) an extracellular ligand-binding domain, a hinge domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain having specificity for CD19, CD20, CD22, or BCMA, (ii) a CD8α hinge domain, a CD28 hinge domain, or an IgG4 hinge domain, (iii) a CD8α hinge domain having the amino acid sequence of SEQ ID NO: 9, (iv) a CD28 hinge domain having the amino acid sequence of SEQ ID NO: 10 or 113, (v) an IgG4 hinge domain having the amino acid sequence of SEQ ID NO: 11 or 12, (vi) a CD8α transmembrane domain or a CD28 transmembrane domain, (vii) a CD8α transmembrane domain having the amino acid sequence of SEQ ID NO: 14, (viii) a CD28 transmembrane domain having the amino acid sequence of SEQ ID NO: 15 or 114, (ix) a 4-1BB co-stimulatory domain, a CD28 co-stimulatory domain, or a CD3ζ signaling domain, (x) a 4-1BB co-stimulatory domain having the amino acid sequence of SEQ ID NO: 16, (xi) a CD28 co-stimulatory domain having the amino acid sequence of SEQ ID NO: 17, (xii) a CD3ζ signaling domain having the amino acid sequence of SEQ ID NO: 18 or 115, (xiii) an extracellular ligand-binding domain comprising any one of the scFv sequences of SEQ ID NO: 19, 37, 45, 54, 63, 72, 81, or 118, or the CAR has an scFv sequence comprising any one of the heavy and light chain sequences of SEQ ID NO: 20, 25, 38, 42, 46, 50, 64, 68, 73, 77, 119, or 123, (xiv) any one of the sequences of SEQ ID NO: 32, 34, 36, 117, or 128, (xv) the following components, namely a CD8α signal peptide, FMC63 scFv (VL-Whitlow linker-VH), a CD8α hinge domain, a CD8α transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain, the amino acid sequence defined in SEQ ID NO: 117, or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence defined in SEQ ID NO: 117, and / or (xvi) the amino acid sequence defined in SEQ ID NO: 45, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence defined in SEQ ID NO: 45 The engineered cell according to claim 10, comprising

12. The engineered cell according to claim 10, wherein one or more of the first exogenous polynucleotide and / or the second exogenous polynucleotide are inserted into the first specific locus and / or the second specific locus of at least one allele of the cell.

13. The first specific locus and / or the second specific locus is a safe harbor or a target locus, RHD locus, B2M locus, CIITA locus, TRAC locus, and TRB locus, the engineered cell according to claim 12.

14. The engineered cell according to claim 13, wherein the safe harbor or the target locus is selected from the group consisting of CCR5 locus, CXCR4 locus, PPP1R12C locus, ALB locus, SHS231 locus, CLYBL locus, Rosa locus, F3 (CD142) locus, MICA locus, MICB locus, LRP1 (CD91) locus, HMGB1 locus, ABO locus, FUT1 locus, and KDM5D locus.

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

16. A pharmaceutical composition for treating a patient having a disease or condition in which a cell-based therapy would be beneficial, comprising a population of the engineered cells according to any one of claims 1 to 14, the pharmaceutical composition comprising administering the population of the engineered cells to the patient.

17. The patient is (i) without a Y chromosome, (ii) not sensitized to the Y chromosome gene, (iii) sensitized to the Y chromosome gene, (iv) previously received cell therapy derived from a donor subject having a Y chromosome or cell therapy in which one or more of the Y chromosome genes are expressed by other means, or (v) a female patient who has previously given birth to a male child The pharmaceutical composition according to claim 16. **Claim 18** A pharmaceutical composition for treating or identifying a patient having a disease or condition in which a cell-based therapy would be beneficial, the composition comprising a population of engineered cells according to any one of claims 1 to 14, wherein: (a) determining whether a biological sample from the patient contains antibodies against one or more Y chromosome genes; (i) obtaining or having obtained a biological sample from the patient; (ii) performing or having performed an assay to determine whether an antibody against protocadherin-11 Y-linked is present in the biological sample; (iii) performing or having performed an assay to determine whether an antibody against neuroligin-4 Y-linked is present in the biological sample; by; (b) administering the population of engineered cells to the patient, (i) if an antibody against protocadherin-11 Y-linked is present in the biological sample, the population of cells comprises reduced expression of protocadherin-11 Y-linked; (ii) if an antibody against neuroligin-4 Y-linked is present in the biological sample, the population of cells comprises reduced expression of neuroligin-4 Y-linked; (iii) if antibodies against both protocadherin-11 Y-linked and neuroligin-4 Y-linked are present in the biological sample, the population of cells comprises reduced expression of protocadherin-11 Y-linked and reduced expression of neuroligin-4 Y-linked; (iv) if neither an antibody against protocadherin-11 Y-linked nor an antibody against neuroligin-4 Y-linked is present in the biological sample, the population of cells does not comprise reduced expression of protocadherin-11 Y-linked nor reduced expression of neuroligin-4 Y-linked; the pharmaceutical composition. **Claim 19** A method for producing engineered cells, comprising reduced expression of one or more Y chromosome genes and MHC class I and / or MHC class II human leukocyte antigen molecules compared to unaltered or unmodified wild-type or control cells, and comprising a first exogenous polynucleotide encoding CD47, the method comprising: (a) obtaining isolated cells; (b) genetically modifying the cell to reduce the expression of one or more Y chromosome genes in the cell; (c) genetically modifying the cell to reduce the expression of MHC class I human leukocyte antigen molecules and / or MHC class II human leukocyte antigen molecules in the cell; (d) introducing a polynucleotide encoding CD47 into the isolated cell, thereby producing the engineered cell. [

20. ] The method according to claim 19, wherein the cell is (i) a CD4 binding factor or a CD8 binding factor, (ii) a polynucleotide encoding a CRISPR / Cas gene editing component targeting the one or more Y chromosome loci, (iii) a polynucleotide encoding a CRISPR / Cas gene editing component targeting the MHC class I and / or the MHC class II human leukocyte antigen locus, and (iv) a first exogenous polynucleotide encoding CD47, contacting the cell with a composition comprising one or more vectors, thereby producing the engineered cell.

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