WO2023109941A1

WO2023109941A1 - Cell modification

Info

Publication number: WO2023109941A1
Application number: PCT/CN2022/139526
Authority: WO
Inventors: Xinxin Wang; Chunhui Yang; Lianjun SHEN; Wei Cao
Original assignee: Gracell Biotechnologies (Shanghai) Co., Ltd.
Priority date: 2021-12-16
Filing date: 2022-12-16
Publication date: 2023-06-22

Abstract

Described herein are modified cells. Also described herein are methods for modifying the cells or treating a disease or condition with the modified cells.

Description

CELL MODIFICATION

CROSS-REFERENCE

This application claims the benefit of PCT/CN2021/138854, filed December 16, 2021, which application is incorporated herein by reference in its entirety.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein 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. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

BACKGROUND

Cell-based therapy such as cell transplantation or therapeutic delivery has been gaining traction. However, the cells used in cell-based therapy can express cell surface markers that trigger innate immunity. For example, CAR-T cell therapy, while showing promising therapeutic efficacy, has been linked to severe inflammatory side effects in patients. Another example is cell transplantation leading to graft versus host disease (GVHD) . As a result, most current clinical trials rely on autologous cells as the primary source cells for cell-based therapy.

SUMMARY OF THE INVENTION

However, such reliance on autologous cell is expensive and laborious. As such, there remains a need for a source of cells that can be readily expanded or differentiated into cell types suitable for cell-based therapy. There also remains a need for cells to possess knocked-down or knocked-out expression of cell surface molecules such as major histocompatibility complexes (MHCs) so that these cells do not trigger innate immune responses upon transplantation into a subject in need thereof.

Described herein, in some aspects, is a cell comprising at least one genetic modification, where the at least one genetic modification decreases an endogenous expression of SUGT-1 in the cell; and a heterologous polypeptide. In some embodiments, the at least one genetic modification decreases an endogenous expression of a nucleotide-binding oligomerization domain-like receptors (NLR) in the cell. In some embodiments, the NLR comprises a NLRC. In some embodiments, the NLRC comprises a NOD1, NOD2, NLRC3, NLRC4, or NLRC5. In some embodiments, the NLRC comprises the NLRC5. In some embodiments, the at least one genetic modification decreases an endogenous expression of an MHC class II regulatory factor in the cell. In some embodiments, the MHC class II regulatory factor comprises RFX1, RFX2, RFX3, RFX4, or RFX5. In some embodiments, the MHC class II regulatory factor comprises RFX5. In some embodiments, the MHC class II regulatory factor comprises CIITA, CREB, or nuclear transcription factor Y (NF-Y) . In some embodiments, the heterologous polypeptide comprises a heterologous receptor. In some embodiments, the heterologous receptor comprises an antigen binding domain. In some embodiments, the antigen binding domain binds to CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44v6, CD47, CD52, CD56, CD57, CD58, CD70, CD79a, CD79b, CD80, CD81, CD86, CD99, CD117, CD123, CD133, CD135, CD137, CD151, CD171, CD276, BAFF-R, BCMA, B7H4, CEA, CEACM6, Claudin18.2, CLL-1, c-Met, CS-1, CTLA-4, EGFRvIII, GPC2, GPC3, GPRC5, HER2, HER3, HER4/ErbB4, HVEM, MAGE-A, MAGE3, MSLN, MUC-1, MUC-16, NY-ESO-1, OX40, PD-1, PD-L1, PD-L2, PMSA, ROR1, TCRa, TCRb, TLR7, TLR9, VEGFR-2, or WT-1. In some embodiments, the heterologous receptor comprises a transmembrane domain. In some embodiments, the heterologous receptor comprises a signaling domain. In some embodiments, an endogenous expression of MHC-I of the cell is decreased compared to an endogenous expression of MHC-I of a comparable cell that does not have decreased expression of the endogenous gene. In some embodiments, the endogenous expression of MHC-I of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to the endogenous expression of MHC-I or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is not knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is decreased by at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 95%, or at most 99%compared to the endogenous expression of MHC-I or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, an endogenous expression of MHC-II of the cell is decreased compared to an endogenous expression of MHC-II of a comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-II of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to the endogenous expression of MHC-II or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-II of the cell is abrogated. In some embodiments, the cell does not increase NK cell kill activity compared to a comparable cell that does not have an endogenous gene knocked out. In some embodiments, the cell comprises an immune cell or a stem cell. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is a B cell. In some embodiments, the lymphocyte is a T cell. In some embodiments, the T cell is selected from the group consisting of: cytotoxic T cell, natural killer T cell, regulatory T cell, and T helper cell. In some embodiments, the immune cell comprises an innate lymphoid cell (ILC) . In some embodiments, the immune cell is derived from an induced pluripotent stem cell (iPSC) . In some embodiments, the immune cell is an induced pluripotent stem cell derived T cell. In some embodiments, the immune cell is an induced pluripotent stem cell derived natural killer T cell. In some embodiments, the immune cell is an iPSC derived macrophage. In some embodiments, the stem cell is a hematopoietic stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC) .

Described herein, in some aspects, is a cell comprising: at least one genetic modification, where the at least one genetic modification decreases an endogenous expression of NLRC5 in the cell; and a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises a heterologous receptor. In some embodiments, the heterologous receptor comprises an antigen binding domain. In some embodiments, the antigen binding domain binds to CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44v6, CD47, CD52, CD56, CD57, CD58, CD70, CD79a, CD79b, CD80, CD81, CD86, CD99, CD117, CD123, CD133, CD135, CD137, CD151, CD171, CD276, BAFF-R, BCMA, B7H4, CEA, CEACM6, Claudin18.2, CLL-1, c-Met, CS-1, CTLA-4, EGFRvIII, GPC2, GPC3, GPRC5, HER2, HER3, HER4/ErbB4, HVEM, MAGE-A, MAGE3, MSLN, MUC-1, MUC-16, NY-ESO-1, OX40, PD-1, PD-L1, PD-L2, PMSA, ROR1, TCRa, TCRb, TLR7, TLR9, VEGFR-2, or WT-1. In some embodiments, the heterologous receptor comprises a transmembrane domain. In some embodiments, the heterologous receptor comprises a signaling domain. In some embodiments, an endogenous expression of MHC-I of the cell is decreased compared to an endogenous expression of MHC-I of a comparable cell that does not have decreased expression of the endogenous gene. In some embodiments, the endogenous expression of MHC-I of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to the endogenous expression of MHC-I or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is not knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is decreased by at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 95%, or at most 99%compared to the endogenous expression of MHC-I or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, an endogenous expression of MHC-II of the cell is decreased compared to an endogenous expression of MHC-II of a comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-II of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to the endogenous expression of MHC-II or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-II of the cell is abrogated. In some embodiments, the cell does not increase NK cell kill activity compared to a comparable cell that does not have an endogenous gene knocked out. In some embodiments, the cell comprises an immune cell or a stem cell. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is a B cell. In some embodiments, the lymphocyte is a T cell. In some embodiments, the T cell is selected from the group consisting of: cytotoxic T cell, natural killer T cell, regulatory T cell, and T helper cell. In some embodiments, the immune cell comprises an innate lymphoid cell (ILC) . In some embodiments, the immune cell is derived from an induced pluripotent stem cell (iPSC) . In some embodiments, the immune cell is an induced pluripotent stem cell derived T cell. In some embodiments, the immune cell is an induced pluripotent stem cell derived natural killer T cell. In some embodiments, the immune cell is an iPSC derived macrophage. In some embodiments, the stem cell is a hematopoietic stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC) .

Described herein, in some aspects, is a cell comprising: at least one genetic modification, where the at least one genetic modification decreases an endogenous expression of RFX5 in the cell; and a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises a heterologous receptor. In some embodiments, the heterologous receptor comprises an antigen binding domain. In some embodiments, the antigen binding domain binds to CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44v6, CD47, CD52, CD56, CD57, CD58, CD70, CD79a, CD79b, CD80, CD81, CD86, CD99, CD117, CD123, CD133, CD135, CD137, CD151, CD171, CD276, BAFF-R, BCMA, B7H4, CEA, CEACM6, Claudin18.2, CLL-1, c-Met, CS-1, CTLA-4, EGFRvIII, GPC2, GPC3, GPRC5, HER2, HER3, HER4/ErbB4, HVEM, MAGE-A, MAGE3, MSLN, MUC-1, MUC-16, NY-ESO-1, OX40, PD-1, PD-L1, PD-L2, PMSA, ROR1, TCRa, TCRb, TLR7, TLR9, VEGFR-2, or WT-1. In some embodiments, the heterologous receptor comprises a transmembrane domain. In some embodiments, the heterologous receptor comprises a signaling domain. In some embodiments, an endogenous expression of MHC-I of the cell is decreased compared to an endogenous expression of MHC-I of a comparable cell that does not have decreased expression of the endogenous gene. In some embodiments, the endogenous expression of MHC-I of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to the endogenous expression of MHC-I or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is not knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is decreased by at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 95%, or at most 99%compared to the endogenous expression of MHC-I or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, an endogenous expression of MHC-II of the cell is decreased compared to an endogenous expression of MHC-II of a comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-II of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to the endogenous expression of MHC-II or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-II of the cell is abrogated. In some embodiments, the cell does not increase NK cell kill activity compared to a comparable cell that does not have an endogenous gene knocked out. In some embodiments, the cell comprises an immune cell or a stem cell. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is a B cell. In some embodiments, the lymphocyte is a T cell. In some embodiments, the T cell is selected from the group consisting of: cytotoxic T cell, natural killer T cell, regulatory T cell, and T helper cell. In some embodiments, the immune cell comprises an innate lymphoid cell (ILC) . In some embodiments, the immune cell is derived from an induced pluripotent stem cell (iPSC) . In some embodiments, the immune cell is an induced pluripotent stem cell derived T cell. In some embodiments, the immune cell is an induced pluripotent stem cell derived natural killer T cell. In some embodiments, the immune cell is an iPSC derived macrophage. In some embodiments, the stem cell is a hematopoietic stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC) .

Described herein, in some aspects, is a cell comprising: at least one genetic modification, where the at least one genetic modification decreases an endogenous expression of CIITA in the cell; and a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises a heterologous receptor. In some embodiments, the heterologous receptor comprises an antigen binding domain. In some embodiments, the antigen binding domain binds to CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44v6, CD47, CD52, CD56, CD57, CD58, CD70, CD79a, CD79b, CD80, CD81, CD86, CD99, CD117, CD123, CD133, CD135, CD137, CD151, CD171, CD276, BAFF-R, BCMA, B7H4, CEA, CEACM6, Claudin18.2, CLL-1, c-Met, CS-1, CTLA-4, EGFRvIII, GPC2, GPC3, GPRC5, HER2, HER3, HER4/ErbB4, HVEM, MAGE-A, MAGE3, MSLN, MUC-1, MUC-16, NY-ESO-1, OX40, PD-1, PD-L1, PD-L2, PMSA, ROR1, TCRa, TCRb, TLR7, TLR9, VEGFR-2, or WT-1. In some embodiments, the heterologous receptor comprises a transmembrane domain. In some embodiments, the heterologous receptor comprises a signaling domain. In some embodiments, an endogenous expression of MHC-I of the cell is decreased compared to an endogenous expression of MHC-I of a comparable cell that does not have decreased expression of the endogenous gene. In some embodiments, the endogenous expression of MHC-I of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to the endogenous expression of MHC-I or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is not knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is decreased by at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 95%, or at most 99%compared to the endogenous expression of MHC-I or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, an endogenous expression of MHC-II of the cell is decreased compared to an endogenous expression of MHC-II of a comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-II of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to the endogenous expression of MHC-II or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-II of the cell is abrogated. In some embodiments, the cell does not increase NK cell kill activity compared to a comparable cell that does not have an endogenous gene knocked out. In some embodiments, the cell comprises an immune cell or a stem cell. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is a B cell. In some embodiments, the lymphocyte is a T cell. In some embodiments, the T cell is selected from the group consisting of: cytotoxic T cell, natural killer T cell, regulatory T cell, and T helper cell. In some embodiments, the immune cell comprises an innate lymphoid cell (ILC) . In some embodiments, the immune cell is derived from an induced pluripotent stem cell (iPSC) . In some embodiments, the immune cell is an induced pluripotent stem cell derived T cell. In some embodiments, the immune cell is an induced pluripotent stem cell derived natural killer T cell. In some embodiments, the immune cell is an iPSC derived macrophage. In some embodiments, the stem cell is a hematopoietic stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC) .

Described herein, in some embodiments, is a cell line comprising the cell comprising the at least one genetic modification for decreasing SUGT-1, NLR (e.g., NLRC5) , MHC class II regulatory factor (e.g., RFX1, RFX2, RFX3, RFX4, or RFX5) , CIITA, CREB, NF-Y, or any combination thereof.

Described herein, in some embodiments, is a composition comprising the cell comprising the at least one genetic modification for decreasing SUGT-1, NLR (e.g., NLRC5) , MHC class II regulatory factor (e.g., RFX1, RFX2, RFX3, RFX4, or RFX5) , CIITA, CREB, NF-Y, or any combination thereof.

Described herein, in some embodiments, is a pharmaceutical composition comprising the cell comprising the at least one genetic modification for decreasing SUGT-1, NLR (e.g., NLRC5) , MHC class II regulatory factor (e.g., RFX1, RFX2, RFX3, RFX4, or RFX5) , CIITA, CREB, NF-Y, or any combination thereof; the composition described herein; and a pharmaceutically acceptable: excipient, carrier, or diluent. In some embodiments, the pharmaceutical composition comprises a unit dose form. In some embodiments, the pharmaceutical composition is formulated for administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, intratumorally, pulmonarily, endotracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, by inhalation, by inhaled nebulized form, by intraluminal-GI route, or a combination thereof to a subject in need thereof. In some embodiments, the pharmaceutical composition further comprises at least one additional active agent. In some embodiments, the at least one additional active agent comprises a cytokine, a growth factor, a hormone, an enzyme, a small molecule, a compound, or combinations thereof.

Described herein, in some aspects, is a kit comprising: the cell of comprising the cell comprising the at least one genetic modification for decreasing SUGT-1, NLR (e.g., NLRC5) , MHC class II regulatory factor (e.g., RFX1, RFX2, RFX3, RFX4, or RFX5) , CIITA, CREB, NF-Y, or any combination thereof; the composition described herein; or the pharmaceutical composition described herein; and a container.

Described herein, in some aspects, is a method for modifying a cell, comprising: contacting the cell with a gene modifying moiety, where the gene modifying moiety introduces at least one genetic modification in the cell, thereby decreasing an endogenous expression of SUGT-1 in the cell; and expressing a heterologous polypeptide in the cell. In some embodiments, the at least one genetic modification decreases an endogenous expression of a NLR in the cell. In some embodiments, the NLR comprises a NLRC. In some embodiments, the NLRC comprises a NOD1, NOD2, NLRC3, NLRC4, or NLRC5. In some embodiments, the NLRC comprises the NLRC5. In some embodiments, the at least one genetic modification decreases an endogenous expression of a MHC class II regulatory factor in the cell. In some embodiments, the MHC class II regulatory factor comprises RFX1, RFX2, RFX3, RFX4, or RFX5. In some embodiments, the MHC class II regulatory factor comprises RFX5. In some embodiments, the MHC class II regulatory factor comprises CIITA, CREB, or nuclear transcription factor Y (NF-Y) . In some embodiments, the heterologous polypeptide comprises a heterologous receptor. In some embodiments, the heterologous receptor comprises an antigen binding domain. In some embodiments, the antigen binding domain binds to CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44v6, CD47, CD52, CD56, CD57, CD58, CD70, CD79a, CD79b, CD80, CD81, CD86, CD99, CD117, CD123, CD133, CD135, CD137, CD151, CD171, CD276, BAFF-R, BCMA, B7H4, CEA, CEACM6, Claudin18.2, CLL-1, c-Met, CS-1, CTLA-4, EGFRvIII, GPC2, GPC3, GPRC5, HER2, HER3, HER4/ErbB4, HVEM, MAGE-A, MAGE3, MSLN, MUC-1, MUC-16, NY-ESO-1, OX40, PD-1, PD-L1, PD-L2, PMSA, ROR1, TCRa, TCRb, TLR7, TLR9, VEGFR-2, or WT-1. In some embodiments, the heterologous receptor comprises a transmembrane domain. In some embodiments, the heterologous receptor comprises a signaling domain. In some embodiments, an endogenous expression of MHC-I of the cell is decreased compared to an endogenous expression of MHC-I of a comparable cell that does not have decreased expression of the endogenous gene. In some embodiments, the endogenous expression of MHC-I of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to the endogenous expression of MHC-I or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is not knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is decreased by at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 95%, or at most 99%compared to the endogenous expression of MHC-I or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, an endogenous expression of MHC-II of the cell is decreased compared to an endogenous expression of MHC-II of a comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-II of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to the endogenous expression of MHC-II or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-II of the cell is abrogated. In some embodiments, the cell does not increase NK cell kill activity compared to a comparable cell that does not have an endogenous gene knocked out. In some embodiments, the cell comprises an immune cell or a stem cell. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is a B cell. In some embodiments, the lymphocyte is a T cell. In some embodiments, the T cell is selected from the group consisting of: cytotoxic T cell, natural killer T cell, regulatory T cell, and T helper cell. In some embodiments, the immune cell comprises an innate lymphoid cell (ILC) . In some embodiments, the immune cell is derived from an induced pluripotent stem cell (iPSC) . In some embodiments, the immune cell is an induced pluripotent stem cell derived T cell. In some embodiments, the immune cell is an induced pluripotent stem cell derived natural killer T cell. In some embodiments, the immune cell is an iPSC derived macrophage. In some embodiments, the stem cell is a hematopoietic stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC) . In some embodiments, the gene modifying moiety comprises a Cas protein or mRNA. In some embodiments, the gene modifying moiety comprises a Cas/RNP. In some embodiments, the gene modifying moiety comprises a Cas9/RNP. In some embodiments, the gene modifying moiety comprises an inhibitory RNA. In some embodiments, the inhibitory RNA comprises siRNA, miRNA, shRNA or a combination thereof.

Described herein, in some aspects, is a method for modifying a cell, comprising: contacting the cell with a gene modifying moiety, where the gene modifying moiety introduces at least one genetic modification in the cell, thereby decreasing an endogenous expression of NLRC5 in the cell; and expressing a heterologous polypeptide in the cell. In some embodiments, the heterologous polypeptide comprises a heterologous receptor. In some embodiments, the heterologous receptor comprises an antigen binding domain. In some embodiments, the antigen binding domain binds to CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44v6, CD47, CD52, CD56, CD57, CD58, CD70, CD79a, CD79b, CD80, CD81, CD86, CD99, CD117, CD123, CD133, CD135, CD137, CD151, CD171, CD276, BAFF-R, BCMA, B7H4, CEA, CEACM6, Claudin18.2, CLL-1, c-Met, CS-1, CTLA-4, EGFRvIII, GPC2, GPC3, GPRC5, HER2, HER3, HER4/ErbB4, HVEM, MAGE-A, MAGE3, MSLN, MUC-1, MUC-16, NY-ESO-1, OX40, PD-1, PD-L1, PD-L2, PMSA, ROR1, TCRa, TCRb, TLR7, TLR9, VEGFR-2, or WT-1. In some embodiments, the heterologous receptor comprises a transmembrane domain. In some embodiments, the heterologous receptor comprises a signaling domain. In some embodiments, an endogenous expression of MHC-I of the cell is decreased compared to an endogenous expression of MHC-I of a comparable cell that does not have decreased expression of the endogenous gene. In some embodiments, the endogenous expression of MHC-I of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to the endogenous expression of MHC-I or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is not knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is decreased by at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 95%, or at most 99%compared to the endogenous expression of MHC-I or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, an endogenous expression of MHC-II of the cell is decreased compared to an endogenous expression of MHC-II of a comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-II of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% compared to the endogenous expression of MHC-II or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-II of the cell is abrogated. In some embodiments, the cell does not increase NK cell kill activity compared to a comparable cell that does not have an endogenous gene knocked out. In some embodiments, the cell comprises an immune cell or a stem cell. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is a B cell. In some embodiments, the lymphocyte is a T cell. In some embodiments, the T cell is selected from the group consisting of: cytotoxic T cell, natural killer T cell, regulatory T cell, and T helper cell. In some embodiments, the immune cell comprises an innate lymphoid cell (ILC) . In some embodiments, the immune cell is derived from an induced pluripotent stem cell (iPSC) . In some embodiments, the immune cell is an induced pluripotent stem cell derived T cell. In some embodiments, the immune cell is an induced pluripotent stem cell derived natural killer T cell. In some embodiments, the immune cell is an iPSC derived macrophage. In some embodiments, the stem cell is a hematopoietic stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC) . In some embodiments, the gene modifying moiety comprises a Cas protein or mRNA. In some embodiments, the gene modifying moiety comprises a Cas/RNP. In some embodiments, the gene modifying moiety comprises a Cas9/RNP. In some embodiments, the gene modifying moiety comprises an inhibitory RNA. In some embodiments, the inhibitory RNA comprises siRNA, miRNA, shRNA or a combination thereof.

Described herein, in some aspects, is a method for modifying a cell, comprising: contacting the cell with a gene modifying moiety, where the gene modifying moiety introduces at least one genetic modification in the cell, thereby decreasing an endogenous expression of RFX5 in the cell; and expressing a heterologous polypeptide in the cell. In some embodiments, the heterologous polypeptide comprises a heterologous receptor. In some embodiments, the heterologous receptor comprises an antigen binding domain. In some embodiments, the antigen binding domain binds to CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44v6, CD47, CD52, CD56, CD57, CD58, CD70, CD79a, CD79b, CD80, CD81, CD86, CD99, CD117, CD123, CD133, CD135, CD137, CD151, CD171, CD276, BAFF-R, BCMA, B7H4, CEA, CEACM6, Claudin18.2, CLL-1, c-Met, CS-1, CTLA-4, EGFRvIII, GPC2, GPC3, GPRC5, HER2, HER3, HER4/ErbB4, HVEM, MAGE-A, MAGE3, MSLN, MUC-1, MUC-16, NY-ESO-1, OX40, PD-1, PD-L1, PD-L2, PMSA, ROR1, TCRa, TCRb, TLR7, TLR9, VEGFR-2, or WT-1. In some embodiments, the heterologous receptor comprises a transmembrane domain. In some embodiments, the heterologous receptor comprises a signaling domain. In some embodiments, an endogenous expression of MHC-I of the cell is decreased compared to an endogenous expression of MHC-I of a comparable cell that does not have decreased expression of the endogenous gene. In some embodiments, the endogenous expression of MHC-I of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to the endogenous expression of MHC-I or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is not knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is decreased by at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 95%, or at most 99%compared to the endogenous expression of MHC-I or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, an endogenous expression of MHC-II of the cell is decreased compared to an endogenous expression of MHC-II of a comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-II of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to the endogenous expression of MHC-II or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-II of the cell is abrogated. In some embodiments, the cell does not increase NK cell kill activity compared to a comparable cell that does not have an endogenous gene knocked out. In some embodiments, the cell comprises an immune cell or a stem cell. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is a B cell. In some embodiments, the lymphocyte is a T cell. In some embodiments, the T cell is selected from the group consisting of: cytotoxic T cell, natural killer T cell, regulatory T cell, and T helper cell. In some embodiments, the immune cell comprises an innate lymphoid cell (ILC) . In some embodiments, the immune cell is derived from an induced pluripotent stem cell (iPSC) . In some embodiments, the immune cell is an induced pluripotent stem cell derived T cell. In some embodiments, the immune cell is an induced pluripotent stem cell derived natural killer T cell. In some embodiments, the immune cell is an iPSC derived macrophage. In some embodiments, the stem cell is a hematopoietic stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC) . In some embodiments, the gene modifying moiety comprises a Cas protein or mRNA. In some embodiments, the gene modifying moiety comprises a Cas/RNP. In some embodiments, the gene modifying moiety comprises a Cas9/RNP. In some embodiments, the gene modifying moiety comprises an inhibitory RNA. In some embodiments, the inhibitory RNA comprises siRNA, miRNA, shRNA or a combination thereof.

Described herein, in some aspects, is a method for modifying a cell, comprising: contacting the cell with a gene modifying moiety, where the gene modifying moiety introduces at least one genetic modification in the cell, thereby decreasing an endogenous expression of CIITA in the cell; and expressing a heterologous polypeptide in the cell. In some embodiments, the heterologous polypeptide comprises a heterologous receptor. In some embodiments, the heterologous receptor comprises an antigen binding domain. In some embodiments, the antigen binding domain binds to CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44v6, CD47, CD52, CD56, CD57, CD58, CD70, CD79a, CD79b, CD80, CD81, CD86, CD99, CD117, CD123, CD133, CD135, CD137, CD151, CD171, CD276, BAFF-R, BCMA, B7H4, CEA, CEACM6, Claudin18.2, CLL-1, c-Met, CS-1, CTLA-4, EGFRvIII, GPC2, GPC3, GPRC5, HER2, HER3, HER4/ErbB4, HVEM, MAGE-A, MAGE3, MSLN, MUC-1, MUC-16, NY-ESO-1, OX40, PD-1, PD-L1, PD-L2, PMSA, ROR1, TCRa, TCRb, TLR7, TLR9, VEGFR-2, or WT-1. In some embodiments, the heterologous receptor comprises a transmembrane domain. In some embodiments, the heterologous receptor comprises a signaling domain. In some embodiments, an endogenous expression of MHC-I of the cell is decreased compared to an endogenous expression of MHC-I of a comparable cell that does not have decreased expression of the endogenous gene. In some embodiments, the endogenous expression of MHC-I of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to the endogenous expression of MHC-I or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is not knocked out. In some embodiments, the endogenous expression of MHC-I of the cell is decreased by at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 95%, or at most 99%compared to the endogenous expression of MHC-I or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, an endogenous expression of MHC-II of the cell is decreased compared to an endogenous expression of MHC-II of a comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-II of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to the endogenous expression of MHC-II or the comparable cell that does not have an endogenous gene knocked out. In some embodiments, the endogenous expression of MHC-II of the cell is abrogated. In some embodiments, the cell does not increase NK cell kill activity compared to a comparable cell that does not have an endogenous gene knocked out. In some embodiments, the cell comprises an immune cell or a stem cell. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is a B cell. In some embodiments, the lymphocyte is a T cell. In some embodiments, the T cell is selected from the group consisting of: cytotoxic T cell, natural killer T cell, regulatory T cell, and T helper cell. In some embodiments, the immune cell comprises an innate lymphoid cell (ILC) . In some embodiments, the immune cell is derived from an induced pluripotent stem cell (iPSC) . In some embodiments, the immune cell is an induced pluripotent stem cell derived T cell. In some embodiments, the immune cell is an induced pluripotent stem cell derived natural killer T cell. In some embodiments, the immune cell is an iPSC derived macrophage. In some embodiments, the stem cell is a hematopoietic stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC) . In some embodiments, the gene modifying moiety comprises a Cas protein or mRNA. In some embodiments, the gene modifying moiety comprises a Cas/RNP. In some embodiments, the gene modifying moiety comprises a Cas9/RNP. In some embodiments, the gene modifying moiety comprises an inhibitory RNA. In some embodiments, the inhibitory RNA comprises siRNA, miRNA, shRNA or a combination thereof.

Described herein, in some aspects, is a method for treating a disease or condition in a subject in need thereof comprising administering the cell described herein, the composition described herein, or the pharmaceutical composition described herein to the subject in need thereof. In some embodiments, the administering of the cell described herein, the composition described herein, or the pharmaceutical composition described herein to the subject does not increase NK cell kill activity in the subject compared to NK cell kill activity of the subject before administering of the cell described herein, the composition described herein, or the pharmaceutical composition described herein.

Described herein is a method for modifying a cell comprising: contacting the cell with a gene modifying moiety, where the gene modifying moiety introduces at least one genetic modification in the cell, thereby knocking out the at least one endogenous gene. In some embodiments, the knocking out of the at least one endogenous gene decreases endogenous expression of at least one MHC-I and decreases endogenous expression of at least one MHC-II.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 illustrates HLA-I/II expression levels in T cells 8 days after knockout of SUGT1, NLRC5, CIITA, B2M, or RFX5 by CRISPR/Cas9 system. Comparing to HLA non-modified T cells, NLRC5 knockout led to downregulation of HLA-I expression level but had no effect on HLA-II expressions; CIITA knockout led to downregulation of HLA-II expression but had no effect on HLA-I molecules; B2M knockout led to complete downregulation of HLA-I expression but had no effect on HLA-II expressions level; RFX5 or SUGT1 knockout led to downregulation of both HLA-I and HLA-II expression level. HLA-II expression were detected with anti-HLA-DR/DP/DQ antibody. HLA-I expression were detected by anti-b2M antibody.

Fig. 2 illustrates HLA-I/II expression level after knockout of SUGT1, NLRC5, or RFX5 by CRISPR/Cas9 system. SUGT1 knockout was tested with 3 different gRNAs (BD1, BD4, or TF3) . Mean Fluorescent Intensity (MFI) of each sample were labeled to the right side of each graph. HLA-II expression were detected with anti-HLA-DR/DP/DQ antibody. HLA-I expression were detected by anti-b2M antibody or by anti-HLA-A/B/C antibody.

Figs. 3A-3B illustrates modifications on HLA-I and HLA-II molecules modification had no effect on CAR-T cell in vitro function. Fig. 3A illustrates genetic modifications of HLA-I/II related genes did not impact CD7 CAR-T cell mediated killing of T-ALL cell line CCRF-CEM comparing to CD7CAR-T (HLA-I/II non-modified) in 6 hour or 24 hour killing assays. Fig. 3B illustrates genetic modifications of HLA-I/II related genes did not impact CD19 CAR-T cell mediated killing of B-ALL cell line Nalm6 comparing to CD19CAR-T (HLA-I/II non-modified) in 6 hour or 24 hour killing assays. EP mock T: electroporated but non-edited, non-transduced control cells.

Fig. 4A illustrates that different genetic modifications (NLRC5, CIITA, RFX5, SUGT1 or B2M knockout) did not have significant impact on CD7 CAR-T cell mediated killing of primary T cells comparing to HLA-I/II non-modified CAR-T cells.

Fig. 4B illustrates that NLRC5, CIITA, RFX5 or SUGT1 knockout did not have significant impact on CD7 CAR-T cell mediated killing of primary NK cells comparing to HLA-I/II non-modified CAR-T cells. B2M knockout CAR-T cells had reduced killing for primary NK cells at lower (1: 3) E: T ratio.

Fig. 5 illustrates that complete deficiencies of HLA-I (B2M knockout) stimulated the highest killing by NK cells. Knocking out of NLRC5, CIITA, RFX5 or SUGT1 did not have significant impact on killing by NK cells comparing to HLA-I/II non-modified T cells. All the target T cells were TCR (TRAC) knockout with indicated genetic modifications.

Fig. 6A illustrates that alloreactive killing of different genetic modified target T cells by allogeneic CD8+ CTLs. Complete knockout of HLA-I (b2M KO T) or HLA-I/II downregulations (by SUGT1, NLRC5, or RFX5 knockout) mediated reduced killing by allogeneic CD8+ CTLs. CIITA knockout had no significant effect on reduction of killing. All the target T cells were TCR (TRAC) knockout with indicated genetic modifications.

Fig. 6B illustrates that alloreactive killing of different genetic modified target T cells by allogeneic CD4+ CTLs. HLA-I/II downregulations (by SUGT1, RFX5 or CIITA knockout) mediated reduced killing by allogeneic CD4+ CTLs. NLRC5 or B2M knockout had no significant effect on reduction of killing. All the target T cells were TCR (TRAC) knockout with indicated genetic modifications.

Fig. 7A illustrates that HLA-II deficiencies in T cells by knocking out of RFX5 or SUGT1 led to less extent of allogeneic responder T cell activations comparing to CIITA knockout, HLA-I (B2M) knockout, HLA-I deficient (by NLRC5 KO) or HLA-I/II intact (HLA non-modified) cells in Mixed Lymphocyte Reaction (MLR) assay. Responder only: allogeneic responder T cells only, no stimulator cells. All the stimulator cells were TCR (TRAC) knockout with indicated genetic modifications.

Fig. 7B and Fig. 7C illustrate Summary of percentage (Fig. 7B) or total cell number (Fig. 7C) of expanded allogeneic responder T cells from samples in Fig. 7A.

The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments.

DETAILED DESCRIPTION

Overview

Described herein, in some aspects, is a modified cell comprising at least one genetic modification, where the at least one genetic modification decreases gene expression of least one endogenous gene in the cell. In some aspects, the at least one genetic modification knocks out the gene expression of the at least one endogenous gene. In some aspects, the decrease of the gene expression of at least one endogenous gene decreases expression of at least one molecular histocompatibility complex (MHC) of the cell. In some aspects, the decrease of the at least one MHC enables the cell to avoid being targeting by innate immune response such as targeting and degradation by natural killer (NK) cell. In some aspects, the cell with the at least one genetic modification does not need to be further modified to overexpress other MHCs to avoid triggering innate immune response. For example, currently available cells used in cell-based therapy rely on overexpressing human leukocyte antigen (HLA) A or E to compensate for decreasing expression of the MHCs to avoid innate immune response targeting. In some aspects, the cell with decreased MHC described herein can be used for cell transplantation or for delivery of therapeutics. In some aspects, the cell with the decreased MHC expression can also comprise at least one heterologous polypeptide. In some aspects, the at least one heterologous polypeptide comprises at least one heterologous receptor, where the at least one heterologous receptor can exert therapeutic effect such as triggering signaling transduction in the cell or recruiting the cell to a desired location based on the heterologous receptor binding to an antigen or a ligand for the receptor. In some aspects, described herein is a method for modifying the cell described herein. In some embodiments, also described herein is a method for treating a disease or condition in a subject in need thereof with the cell described herein.

In some aspects, described herein is a cell comprising at least one genetic modification for decreasing endogenous expression of SUGT-1; and at least one heterologous polypeptide comprising at least one heterologous receptor. In some embodiments, the decrease of endogenous expression of the SUGT-1 decreases expression of at least one endogenous MHC of the cell. In some embodiments, the cell comprises a different genetic modification where a gene expression of a at least one additional endogenous gene is decreased. In some embodiments, the at least one additional endogenous gene belongs to a nucleotide-binding oligomerization domain-like receptors (NLR) family. In some embodiments, the at least one additional endogenous gene belongs to a MHC class II regulatory factor family. In some embodiments, the decrease endogenous expression of the SUGT-1 in the cell decreases the at least one endogenous MHC of the cell. In some embodiments, the decrease gene expression of both SUGT-1 and either NLR and/or MHC class II regulatory factor decreases the at least one endogenous MHC of the cell. In some embodiments, the decrease of the at one endogenous MHC comprises decreases of MHC-I. In some embodiments, the decrease of the at one endogenous MHC comprises decreases of MHC-II. In some embodiments, the decrease of the at one endogenous MHC comprises decreases of both MHC-I and MHC-II.

In some aspects, described herein is a method for modifying a cell comprising contacting the cell with at least one gene modifying moiety, where the gene modifying moiety introduces the at least one genetic modification into the cell, and where the at least one genetic modification decreases gene expression of any one of the endogenous gene described herein. In some embodiments, the at least one endogenous gene comprises SUGT-1, NLR, MHC class II regulatory factor, or a combination thereof. In some embodiments, the method comprises decreasing the gene expression of the at least one endogenous gene, thereby decreasing at least one MHC described herein. In some embodiments, the method comprises modifying the cell with the decreased gene expression of the at least one endogenous gene to further comprise at least one heterologous receptor. Also described herein is a method for treating a disease or condition in a subject in need thereof comprising administering the cell described herein to the subject in need thereof.

Modified cells

Described herein, ins some embodiments, is a cell modified for decreasing gene expression of at least one endogenous gene described herein. In some aspects, the decreased gene expression of the at least one endogenous gene decreases gene expression of at least one MHC of the cell. In some embodiments, the cell comprises at least genetic modification, where the genetic modification decreases gene expression of the at least one endogenous gene. In some aspects, the at least one genetic modification knocks out the at least one endogenous gene. In some embodiments, the cell with the decreased gene expression of the at least one endogenous gene exhibits a decrease expression of at least one MHC of the cell. In some embodiments, the at least one endogenous gene modified by the at least one genetic modification in the cell comprises SUGT-1, NLR, MHC class II regulatory factor, or a combination thereof. In some embodiments, NLR comprises NLR family CARD domain-containing protein (NLRC) . In some embodiments, the NLRC comprises NOD1 (NLRC1) , NOD2 (NLRC2) , NLRC3, NLRC4, or NLRC5. In some embodiments, the NLRC is NLRC5. In some embodiments, the MHC class II regulatory factor comprises RFX1, RFX2, RFX3, RFX4, or RFX5. In some embodiments, the MHC class II regulatory factor comprises RFX5. In some embodiments, the MHC class II regulatory factor comprises class II comprises transactivator (CIITA) , cAMP Response Element-Binding Protein (CREB) , or nuclear transcription factor Y (NF-Y) .

In some embodiment, the cell with the at least one genetic modification exhibits decreased gene expression of endogenous SUGT-1 by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to endogenous gene expression of SUGT-1 of a comparable cell that does not have the at least one genetic modification. In some embodiments, the gene expression of endogenous SUGT-1 is not knocked out in the cell with the at least one genetic modification. In some embodiments, the endogenous gene expression of SUGT-1 is knocked out in the cell with the at least one genetic modification.

In some embodiment, the cell with the at least one genetic modification exhibits decreased endogenous gene expression of NLRC by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to endogenous gene expression of NLRC of a comparable cell that does not have the at least one genetic modification. In some embodiments, the gene expression of endogenous NLRC is not knocked out in the cell with the at least one genetic modification. In some embodiments, the endogenous gene expression of NLRC is knocked out in the cell with the at least one genetic modification.

In some embodiment, the cell with the at least one genetic modification exhibits decreased endogenous gene expression of NLRC5 by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to endogenous gene expression of NLRC5 of a comparable cell that does not have the at least one genetic modification. In some embodiments, the gene expression of endogenous NLRC5 is not knocked out in the cell with the at least one genetic modification. In some embodiments, the endogenous gene expression of NLRC5 is knocked out in the cell with the at least one genetic modification.

In some embodiment, the cell with the at least one genetic modification exhibits decreased endogenous gene expression of MHC class II regulatory factor by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to endogenous gene expression of MHC class II regulatory factor of a comparable cell that does not have the at least one genetic modification. In some embodiments, the gene expression of endogenous MHC class II regulatory factor is not knocked out in the cell with the at least one genetic modification. In some embodiments, the endogenous gene expression of MHC class II regulatory factor is knocked out in the cell with the at least one genetic modification.

In some embodiment, the cell with the at least one genetic modification exhibits decreased endogenous gene expression of RFX5 by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to endogenous gene expression of RFX5 of a comparable cell that does not have the at least one genetic modification. In some embodiments, the endogenous gene expression of RFX5 is not knocked out in the cell with the at least one genetic modification. In some embodiments, the endogenous gene expression of RFX5 is knocked out in the cell with the at least one genetic modification.

In some embodiment, the cell with the at least one genetic modification exhibits decreased endogenous gene expression of CIITA by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to endogenous gene expression of CIITA of a comparable cell that does not have the at least one genetic modification. In some embodiments, the gene expression of endogenous CIITA is not knocked out in the cell with the at least one genetic modification. In some embodiments, the endogenous gene expression of CIITA is knocked out in the cell with the at least one genetic modification.

In some embodiment, the cell with the at least one genetic modification exhibits decreased endogenous gene expression of SUGT-1 and at least one additional endogenous gene, where the endogenous gene expression of each of the SUGT-1 and the at least one additional endogenous gene is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70 at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to endogenous gene expression of SUGT-1 and the same at least one additional endogenous gene of a comparable cell that does not have the at least one genetic modification. In some embodiments, both gene expression of endogenous SUGT-1 and the at least one additional endogenous gene is not knocked out in the cell with the at least one genetic modification. In some embodiments, both gene expression of endogenous SUGT-1 and the at least one additional gene is knocked out in the cell with the at least one genetic modification. In some embodiments, the at least one additional endogenous gene is NLRC. In some embodiments, the at least one additional endogenous gene is NOD1, NOD2, NLRC3, NLRC4, or NLRC5. In some embodiments, the at least one additional endogenous gene is NLRC5. In some embodiments, the at least one additional endogenous gene is a MHC class II regulatory factor. In some embodiments, the at least one additional endogenous gene is RFX1, RFX2, RFX3, RFX4, or RFX5. In some embodiments, the at least one additional endogenous gene is RFX5. In some embodiments, the at least one additional endogenous gene is CIITA, CREB, nuclear transcription factor Y (NF-Y) , or a combination thereof. In some embodiments, the at least one additional endogenous gene is CIITA.

In some embodiments, the at least one endogenous gene modified by the at least one genetic modification in the cell is SUGT-1. In some embodiments, the at least one endogenous gene modified by the at least one genetic modification in the cell is NLR. In some embodiments, the at least one endogenous gene modified by the at least one genetic modification in the cell is a MHC class II regulatory factor. In some embodiments, the at least one endogenous gene modified by the at least one genetic modification in the cell comprises SUGT-1 and NLR. In some embodiments, the at least one endogenous gene modified by the at least one genetic modification in the cell comprises SUGT-1 and MHC class II regulatory factor. In some embodiments, the at least one endogenous gene modified by the at least one genetic modification in the cell comprises SUGT-1, NLR, and MHC class II regulatory factor. In some embodiments, the at least one endogenous gene modified by the at least one genetic modification in the cell comprises NLR and MHC class II regulatory factor.

In some embodiments, the cell with the at least one endogenous gene modified by the at least one genetic modification exhibits decreased expression of at least one MHC of the cell. In some embodiments, the decreased expression of the at least one MHC comprises decreased expression of MHC-I. In some embodiments, the decreased expression of the at least one MHC comprises decreased expression of MHC-II. In some embodiments, the decreased expression of the at least one MHC comprises decreased expression of MHC-I and MHC-II. For example, Fig. 1 illustrates . HLA-I/II expression levels in T cells 8 days after knockout of SUGT1, NLRC5, CIITA, B2M, or RFX5 by CRISPR/Cas9 system. Comparing to HLA non-modified T cells, NLRC5 knockout led to downregulate of HLA-I expression levels but had no effect on HLA-II expressions; CIITA knockout led to downregulations of HLA-II expressions, but had no effect on HLA-I molecules; B2M knockout led to complete downregulations of HLA-I expressions but had no effect on HLA-II expressions levels; RFX5 or SUGT1 knockout led to downregulations of both HLA-I and HLA-II expression levels. In some embodiments, the decreased expression of the at least one MHC of the cell comprises knock out of the at least one MHC of the cell. In some embodiments, the decreased expression of the at least one MHC of the cell does not knock out the expression of the at least one MHC.

In some embodiment, the cell with the at least one endogenous gene modified by the at least one genetic modification exhibits decreased expression of at least one MHC-I by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC-I of the comparable cell that does not have the at least one genetic modification without directly targeting the HLA gene. In some embodiments, expression of the at least one MHC-I is not knocked out in the cell with the at least one endogenous gene modified by the at least one genetic modification. In some embodiments, expression of the at least one MHC-I is knocked out in the cell with the at least one endogenous gene modified by the at least one genetic modification.

In some embodiment, the cell with the at least one endogenous gene modified by the at least one genetic modification exhibits decreased expression of at least one MHC-II by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC-II of the comparable cell that does not have the at least one genetic modification without directly targeting the HLA gene. In some embodiments, expression of the at least one MHC-II is not knocked out in the cell with the at least one endogenous gene modified by the at least one genetic modification. In some embodiments, expression of the at least one MHC-II is knocked out in the cell with the at least one endogenous gene modified by the at least one genetic modification.

In some embodiment, the cell with the at least one endogenous gene modified by the at least one genetic modification exhibits decreased expression of at least one MHC-I or MHC-II by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC-I or MHC-II of the comparable cell that does not have the at least one genetic modification without targeting the HLA gene. In some embodiments, expression of the at least one MHC-I or MHC-II is not knocked out in the cell with the at least one endogenous gene modified by the at least one genetic modification. In some embodiments, expression of the at least one MHC-I or MHC-II is knocked out in the cell with the at least one endogenous gene modified by the at least one genetic modification.

In some embodiment, the cell with the at least one endogenous gene modified by the at least one genetic modification exhibits decreased expression of at least one MHC-I and MHC-II each by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC-I and MHC-II of the comparable cell that does not have the at least one genetic modification without directly targeting HLA gene. In some embodiments, expression of the at least one MHC-I and MHC-II is not knocked out in the cell with the at least one endogenous gene modified by the at least one genetic modification. In some embodiments, expression of the at least one MHC-I and MHC-II is knocked out in the cell with the at least one endogenous gene modified by the at least one genetic modification. In some embodiments, expression of MHC-I is not knocked out but expression of MHC-II is knocked out in the cell with the at least one endogenous gene modified by the at least one genetic modification. In some embodiments, expression of MHC-I is knocked out but expression of MHC-II is not knocked out in the cell with the at least one endogenous gene modified by the at least one genetic modification. In some embodiments, expression of both MHC-I and MHC-II is not knocked out in the cell with the at least one endogenous gene modified by the at least one genetic modification.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression of endogenous SUGT-1, where the decreased expression of the endogenous SUGT-1 decreases expression of at least one MHC in the cell. In some embodiments, described herein is the cell comprising at least one genetic modification for knocking out gene expression of the endogenous SUGT-1, where the knocking out of the endogenous SUGT-1 decreases expression of at least one MHC in the cell. In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing expression of the endogenous SUGT-1, where the decreased expression of SUGT-1 decreases, but does not knock out, the at least one MHC in the cell. . In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing expression of the endogenous SUGT-1, where the decreased gene expression of SUGT-1 knocks out the at least one MHC in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of the endogenous SUGT-1, where the decreased expression of endogenous SUGT-1 decreases expression of at least one MHC in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC of a comparable cell that does not have the at least genetic modification without directly targeting the HLA gene. In some embodiments, described herein is a cell comprising a knocked out endogenous SUGT-1, where the knocking out of the endogenous SUGT-1 decreases expression of at least one MHC in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC of a comparable cell that does not have the SUGT-1 knock out without directly targeting the HLA gene.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression the endogenous SUGT-1, where the decreased gene expression of the endogenous SUGT-1 decreases expression of at least one MHC-II in the cell. In some embodiments, described herein is the cell comprising a knocked out endogenous SUGT-1, where the knocking out of the endogenous SUGT-1 decreases expression of at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising at least one genetic modification for knocking out gene expression of endogenous SUGT-1, where the knocking out of the endogenous SUGT-1 knocks outs expression of the at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising a knocked out endogenous SUGT-1, where the knocking out of the endogenous SUGT-1 decreases expression, but does not knock out, the at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of endogenous SUGT-1, where the decreased gene expression of the endogenous SUGT-1 decreases expression of at least one MHC-II in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC-II of a comparable cell that does not have the at least one genetic modification without directly targeting the HLA gene.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression the endogenous SUGT-1, where the decreased gene expression of the endogenous SUGT-1 decreases expression of at least one MHC-I in the cell. In some embodiments, described herein is the cell comprising a knocked out endogenous SUGT-1, where the knocking out of the endogenous SUGT-1 decreases expression of at least one MHC-I in the cell. In some embodiments, described herein is a cell comprising at least one genetic modification for knocking out gene expression of endogenous SUGT-1, where the knocking out of the endogenous SUGT-1 knocks outs expression of the at least one MHC-I in the cell. In some embodiments, described herein is a cell comprising a knocked out endogenous SUGT-1, where the knocking out of the endogenous SUGT-1 decreases expression, but does not knock out, the at least one MHC-I in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of endogenous SUGT-1, where the decreased gene expression of the endogenous SUGT-1 decreases expression of at least one MHC-I in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC-I of a comparable cell that does not have the at least one genetic modification without directly targeting the HLA gene.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression of the endogenous SUGT-1, where the decreased gene expression of the endogenous SUGT-1 decreases expression of at least one MHC-I and at least one MCH-II in the cell. In some embodiments, described herein is the cell comprising a knocked out endogenous SUGT-1, where the knocking out of the endogenous SUGT-1 decreases expression of at least one MHC-I and at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising decreased gene expression of the endogenous SUGT-1, where the decreased gene expression of the endogenous SUGT-1 knocks out at least one MHC-1 and at least one MHC-II. In some embodiments, the decreased gene expression of the endogenous SUGT-1 decreases expression, but does not knock out, the at least one MHC-I or the at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of an endogenous SUGT-1, where the decreased gene expression of the endogenous SUGT-1 decreases expression of at least one MHC-I and at least one MHC-II in the cell each by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC-I and MHC-II of a comparable cell that does not have the at least one genetic modification without directly targeting the HLA gene.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression of endogenous NLRC5, where the decreased expression of the endogenous NLRC5 decreases expression of at least one MHC in the cell. In some embodiments, described herein is the cell comprising at least one genetic modification for knocking out gene expression of the endogenous NLRC5, where the knocking out of the endogenous NLRC5 decreases expression of at least one MHC in the cell. In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing expression of the endogenous NLRC5, where the decreased expression of NLRC5 decreases, but does not knock out, the at least one MHC in the cell. . In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing expression of the endogenous NLRC5, where the decreased gene expression of NLRC5 knocks out the at least one MHC in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of the endogenous NLRC5, where the decreased expression of endogenous NLRC5 decreases expression of at least one MHC in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC of a comparable cell that does not have the at least genetic modification without directly targeting HLA gene.. In some embodiments, described herein is a cell comprising a knocked out endogenous NLRC5, where the knocking out of the endogenous NLRC5 decreases expression of at least one MHC in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC of a comparable cell that does not have the NLRC5 knock out without directly targeting HLA gene.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression the endogenous NLRC5, where the decreased gene expression of the endogenous NLRC5 decreases expression of at least one MHC-II in the cell. In some embodiments, described herein is the cell comprising a knocked out endogenous NLRC5, where the knocking out of the endogenous NLRC5 decreases expression of at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising at least one genetic modification for knocking out gene expression of endogenous NLRC5, where the knocking out of the endogenous NLRC5 knocks outs expression of the at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising a knocked out endogenous NLRC5, where the knocking out of the endogenous NLRC5 decreases expression, but does not knock out, the at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of endogenous NLRC5, where the decreased gene expression of the endogenous NLRC5 decreases expression of at least one MHC-II in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC-II of a comparable cell that does not have the at least one genetic modification without directly targeting HLA gene.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression the endogenous NLRC5, where the decreased gene expression of the endogenous NLRC5 decreases expression of at least one MHC-I in the cell. In some embodiments, described herein is the cell comprising a knocked out endogenous NLRC5, where the knocking out of the endogenous NLRC5 decreases expression of at least one MHC-I in the cell. In some embodiments, described herein is a cell comprising at least one genetic modification for knocking out gene expression of endogenous NLRC5, where the knocking out of the endogenous NLRC5 knocks outs expression of the at least one MHC-I in the cell. In some embodiments, described herein is a cell comprising a knocked out endogenous NLRC5, where the knocking out of the endogenous NLRC5 decreases expression, but does not knock out, the at least one MHC-I in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of endogenous NLRC5, where the decreased gene expression of the endogenous NLRC5 decreases expression of at least one MHC-I in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC-I of a comparable cell that does not have the at least one genetic modification without directly targeting HLA gene.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression of the endogenous NLRC5, where the decreased gene expression of the endogenous NLRC5 decreases expression of at least one MHC-I and at least one MCH-II in the cell. In some embodiments, described herein is the cell comprising a knocked out endogenous NLRC5, where the knocking out of the endogenous NLRC5 decreases expression of at least one MHC-I and at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising decreased gene expression of the endogenous NLRC5, where the decreased gene expression of the endogenous NLRC5 knocks out at least one MHC-1 and at least one MHC-II. In some embodiments, the decreased gene expression of the endogenous NLRC5 decreases expression, but does not knock out, the at least one MHC-I or the at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of an endogenous NLRC5, where the decreased gene expression of the endogenous NLRC5 decreases expression of at least one MHC-I and at least one MHC-II in the cell each by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC-I and MHC-II of a comparable cell that does not have the at least one genetic modification without directly targeting HLA gene.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression of endogenous SUGT-1 and NLRC5, where the decreased expression of the endogenous SUGT-1 and NLRC5 decreases expression of at least one MHC in the cell. In some embodiments, described herein is the cell comprising at least one genetic modification for knocking out gene expression of the endogenous SUGT-1 and NLRC5, where the knocking out of the endogenous SUGT-1 and NLRC5 decreases expression of at least one MHC in the cell. In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing expression of the endogenous SUGT-1 and NLRC5, where the decreased expression of SUGT-1 and NLRC5 decreases, but does not knock out, the at least one MHC in the cell. . In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing expression of the endogenous SUGT-1 and NLRC5, where the decreased gene expression of SUGT-1 and NLRC5 knocks out the at least one MHC in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of the endogenous SUGT-1 and NLRC5, where the decreased expression of endogenous SUGT-1 and NLRC5 decreases expression of at least one MHC in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to expression of MHC of a comparable cell that does not have the at least genetic modification. In some embodiments, described herein is a cell comprising a knocked out endogenous SUGT-1 and NLRC5, where the knocking out of the endogenous SUGT-1 and NLRC5 decreases expression of at least one MHC in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC of a comparable cell that does not have the SUGT-1 knock out and NLRC5 knock out without directly targeting HLA gene.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression the endogenous SUGT-1 and NLRC5, where the decreased gene expression of the endogenous SUGT-1 and NLRC5 decreases expression of at least one MHC-II in the cell. In some embodiments, described herein is the cell comprising a knocked out endogenous SUGT-1 and NLRC5, where the knocking out of the endogenous SUGT-1 and NLRC5 decreases expression of at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising at least one genetic modification for knocking out gene expression of endogenous SUGT-1 and NLRC5, where the knocking out of the endogenous SUGT-1 and NLRC5 knocks outs expression of the at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising a knocked out endogenous SUGT-1 and NLRC5, where the knocking out of the endogenous SUGT-1 and NLRC5 decreases expression, but does not knock out, the at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of endogenous SUGT-1 and NLRC5, where the decreased gene expression of the endogenous SUGT-1 and NLRC5 decreases expression of at least one MHC-II in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC-II of a comparable cell that does not have the at least one genetic modification without directly targeting HLA gene.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression the endogenous SUGT-1 and NLRC5, where the decreased gene expression of the endogenous SUGT-1 and NLRC5 decreases expression of at least one MHC-I in the cell. In some embodiments, described herein is the cell comprising a knocked out endogenous SUGT-1 and NLRC5, where the knocking out of the endogenous SUGT-1 and NLRC5 decreases expression of at least one MHC-I in the cell. In some embodiments, described herein is a cell comprising at least one genetic modification for knocking out gene expression of endogenous SUGT-1 and NLRC5, where the knocking out of the endogenous SUGT-1 and NLRC5 knocks outs expression of the at least one MHC-I in the cell. In some embodiments, described herein is a cell comprising a knocked out endogenous SUGT-1 and NLRC5, where the knocking out of the endogenous SUGT-1 and NLRC5 decreases expression, but does not knock out, the at least one MHC-I in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of endogenous SUGT-1 and NLRC5, where the decreased gene expression of the endogenous SUGT-1 and NLRC5 decreases expression of at least one MHC-I in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC-I of a comparable cell that does not have the at least one genetic modification without directly targeting HLA gene.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression of the endogenous SUGT-1 and NLRC5, where the decreased gene expression of the endogenous SUGT-1 and NLRC5 decreases expression of at least one MHC-I and at least one MCH-II in the cell. In some embodiments, described herein is the cell comprising a knocked out endogenous SUGT-1 and NLRC5, where the knocking out of the endogenous SUGT-1 and NLRC5 decreases expression of at least one MHC-I and at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising decreased gene expression of the endogenous SUGT-1 and NLRC5, where the decreased gene expression of the endogenous SUGT-1 and NLRC5 knocks out at least one MHC-1 and at least one MHC-II. In some embodiments, the decreased gene expression of the endogenous SUGT-1 and NLRC5 decreases expression, but does not knock out, the at least one MHC-I or the at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of an endogenous SUGT-1 and NLRC5, where the decreased gene expression of the endogenous SUGT-1 and NLRC5 decreases expression of at least one MHC-I and at least one MHC-II in the cell each by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC-I and MHC-II of a comparable cell that does not have the at least one genetic modification without directly targeting HLA gene.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression of endogenous RFX5, where the decreased expression of the endogenous RFX5 decreases expression of at least one MHC in the cell. In some embodiments, described herein is the cell comprising at least one genetic modification for knocking out gene expression of the endogenous RFX5, where the knocking out of the endogenous RFX5 decreases expression of at least one MHC in the cell. In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing expression of the endogenous RFX5, where the decreased expression of RFX5 decreases, but does not knock out, the at least one MHC in the cell. . In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing expression of the endogenous RFX5, where the decreased gene expression of RFX5 knocks out the at least one MHC in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of the endogenous RFX5, where the decreased expression of endogenous RFX5 decreases expression of at least one MHC in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC of a comparable cell that does not have the at least genetic modification without directly targeting HLA gene. In some embodiments, described herein is a cell comprising a knocked out endogenous RFX5, where the knocking out of the endogenous RFX5 decreases expression of at least one MHC in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC of a comparable cell that does not have the RFX5 knock out without directly targeting HLA gene.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression the endogenous RFX5, where the decreased gene expression of the endogenous RFX5 decreases expression of at least one MHC-II in the cell. In some embodiments, described herein is the cell comprising a knocked out endogenous RFX5, where the knocking out of the endogenous RFX5 decreases expression of at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising at least one genetic modification for knocking out gene expression of endogenous RFX5, where the knocking out of the endogenous RFX5 knocks outs expression of the at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising a knocked out endogenous RFX5, where the knocking out of the endogenous RFX5 decreases expression, but does not knock out, the at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of endogenous RFX5, where the decreased gene expression of the endogenous RFX5 decreases expression of at least one MHC-II in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC-II of a comparable cell that does not have the at least genetic modification without directly targeting HLA gene.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression of endogenous SUGT-1 and RFX5, where the decreased expression of the endogenous SUGT-1 and RFX5 decreases expression of at least one MHC in the cell. In some embodiments, described herein is the cell comprising at least one genetic modification for knocking out gene expression of the endogenous SUGT-1 and RFX5, where the knocking out of the endogenous SUGT-1 and RFX5 decreases expression of at least one MHC in the cell. In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing expression of the endogenous SUGT-1 and RFX5, where the decreased expression of SUGT-1 and RFX5 decreases, but does not knock out, the at least one MHC in the cell. . In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing expression of the endogenous SUGT-1 and RFX5, where the decreased gene expression of SUGT-1 and RFX5 knocks out the at least one MHC in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of the endogenous SUGT-1 and RFX5, where the decreased expression of endogenous SUGT-1 and RFX5 decreases expression of at least one MHC in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC of a comparable cell that does not have the at least genetic modification without directly targeting HLA gene. In some embodiments, described herein is a cell comprising a knocked out endogenous SUGT-1 and RFX5, where the knocking out of the endogenous SUGT-1 and RFX5 decreases expression of at least one MHC in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC of a comparable cell that does not have the SUGT-1 knock out and RFX5 knock out without directly targeting HLA gene.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression the endogenous SUGT-1 and RFX5, where the decreased gene expression of the endogenous SUGT-1 and RFX5 decreases expression of at least one MHC-II in the cell. In some embodiments, described herein is the cell comprising a knocked out endogenous SUGT-1 and RFX5, where the knocking out of the endogenous SUGT-1 and RFX5 decreases expression of at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising at least one genetic modification for knocking out gene expression of endogenous SUGT-1 and RFX5, where the knocking out of the endogenous SUGT-1 and RFX5 knocks outs expression of the at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising a knocked out endogenous SUGT-1 and RFX5, where the knocking out of the endogenous SUGT-1 and RFX5 decreases expression, but does not knock out, the at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of endogenous SUGT-1 and RFX5, where the decreased gene expression of the endogenous SUGT-1 and RFX5 decreases expression of at least one MHC-II in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% compared to expression of MHC-II of a comparable cell that does not have the at least one genetic modification without directly targeting HLA gene.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression the endogenous SUGT-1 and RFX5, where the decreased gene expression of the endogenous SUGT-1 and RFX5 decreases expression of at least one MHC-I in the cell. In some embodiments, described herein is the cell comprising a knocked out endogenous SUGT-1 and RFX5, where the knocking out of the endogenous SUGT-1 and RFX5 decreases expression of at least one MHC-I in the cell. In some embodiments, described herein is a cell comprising at least one genetic modification for knocking out gene expression of endogenous SUGT-1 and RFX5, where the knocking out of the endogenous SUGT-1 and RFX5 knocks outs expression of the at least one MHC-I in the cell. In some embodiments, described herein is a cell comprising a knocked out endogenous SUGT-1 and RFX5, where the knocking out of the endogenous SUGT-1 and RFX5 decreases expression, but does not knock out, the at least one MHC-I in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of endogenous SUGT-1 and RFX5, where the decreased gene expression of the endogenous SUGT-1 and RFX5 decreases expression of at least one MHC-I in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC-I of a comparable cell that does not have the at least one genetic modification without directly targeting HLA gene.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression of the endogenous SUGT-1 and RFX5, where the decreased gene expression of the endogenous SUGT-1 and RFX5 decreases expression of at least one MHC-I and at least one MCH-II in the cell. In some embodiments, described herein is the cell comprising a knocked out endogenous SUGT-1 and RFX5, where the knocking out of the endogenous SUGT-1 and RFX5 decreases expression of at least one MHC-I and at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising decreased gene expression of the endogenous SUGT-1 and RFX5, where the decreased gene expression of the endogenous SUGT-1 and RFX5 knocks out at least one MHC-1 and at least one MHC-II. In some embodiments, the decreased gene expression of the endogenous SUGT-1 and RFX5 decreases expression, but does not knock out, the at least one MHC-I or the at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of an endogenous SUGT-1 and RFX5, where the decreased gene expression of the endogenous SUGT-1 and RFX5 decreases expression of at least one MHC-I and at least one MHC-II in the cell each by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC-I and MHC-II of a comparable cell that does not have the at least one genetic modification without directly targeting HLA gene.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression of endogenous CIITA, where the decreased expression of the endogenous CIITA decreases expression of at least one MHC in the cell. In some embodiments, described herein is the cell comprising at least one genetic modification for knocking out gene expression of the endogenous CIITA, where the knocking out of the endogenous CIITA decreases expression of at least one MHC in the cell. In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing expression of the endogenous CIITA, where the decreased expression of CIITA decreases, but does not knock out, the at least one MHC in the cell. . In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing expression of the endogenous CIITA, where the decreased gene expression of CIITA knocks out the at least one MHC in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of the endogenous CIITA, where the decreased expression of endogenous CIITA decreases expression of at least one MHC in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC of a comparable cell that does not have the at least genetic modification without directly targeting HLA gene. In some embodiments, described herein is a cell comprising a knocked out endogenous CIITA, where the knocking out of the endogenous CIITA decreases expression of at least one MHC in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC of a comparable cell that does not have the CIITA knock out without directly targeting HLA gene.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression the endogenous CIITA, where the decreased gene expression of the endogenous CIITA decreases expression of at least one MHC-II in the cell. In some embodiments, described herein is the cell comprising a knocked out endogenous CIITA, where the knocking out of the endogenous CIITA decreases expression of at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising at least one genetic modification for knocking out gene expression of endogenous CIITA, where the knocking out of the endogenous CIITA knocks outs expression of the at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising a knocked out endogenous CIITA, where the knocking out of the endogenous CIITA decreases expression, but does not knock out, the at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of endogenous CIITA, where the decreased gene expression of the endogenous CIITA decreases expression of at least one MHC-II in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC-II of a comparable cell that does not have the at least one genetic modification without directly targeting HLA gene.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression of endogenous SUGT-1 and CIITA, where the decreased expression of the endogenous SUGT-1 and CIITA decreases expression of at least one MHC in the cell. In some embodiments, described herein is the cell comprising at least one genetic modification for knocking out gene expression of the endogenous SUGT-1 and CIITA, where the knocking out of the endogenous SUGT-1 and CIITA decreases expression of at least one MHC in the cell. In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing expression of the endogenous SUGT-1 and CIITA, where the decreased expression of SUGT-1 and CIITA decreases, but does not knock out, the at least one MHC in the cell. . In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing expression of the endogenous SUGT-1 and CIITA, where the decreased gene expression of SUGT-1 and CIITA knocks out the at least one MHC in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of the endogenous SUGT-1 and CIITA, where the decreased expression of endogenous SUGT-1 and CIITA decreases expression of at least one MHC in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC of a comparable cell that does not have the at least genetic modification without directly targeting HLA gene. In some embodiments, described herein is a cell comprising a knocked out endogenous SUGT-1 and CIITA, where the knocking out of the endogenous SUGT-1 and CIITA decreases expression of at least one MHC in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC of a comparable cell that does not have the SUGT-1 knock out and CIITA knock out without directly targeting HLA gene.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression the endogenous SUGT-1 and CIITA, where the decreased gene expression of the endogenous SUGT-1 and CIITA decreases expression of at least one MHC-II in the cell. In some embodiments, described herein is the cell comprising a knocked out endogenous SUGT-1 and CIITA, where the knocking out of the endogenous SUGT-1 and CIITA decreases expression of at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising at least one genetic modification for knocking out gene expression of endogenous SUGT-1 and CIITA, where the knocking out of the endogenous SUGT-1 and CIITA knocks outs expression of the at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising a knocked out endogenous SUGT-1 and CIITA, where the knocking out of the endogenous SUGT-1 and CIITA decreases expression, but does not knock out, the at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of endogenous SUGT-1 and CIITA, where the decreased gene expression of the endogenous SUGT-1 and CIITA decreases expression of at least one MHC-II in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC-II of a comparable cell that does not have the at least one genetic modification without directly targeting HLA gene.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression the endogenous SUGT-1 and CIITA, where the decreased gene expression of the endogenous SUGT-1 and CIITA decreases expression of at least one MHC-I in the cell. In some embodiments, described herein is the cell comprising a knocked out endogenous SUGT-1 and CIITA, where the knocking out of the endogenous SUGT-1 and CIITA decreases expression of at least one MHC-I in the cell. In some embodiments, described herein is a cell comprising at least one genetic modification for knocking out gene expression of endogenous SUGT-1 and CIITA, where the knocking out of the endogenous SUGT-1 and CIITA knocks outs expression of the at least one MHC-I in the cell. In some embodiments, described herein is a cell comprising a knocked out endogenous SUGT-1 and CIITA, where the knocking out of the endogenous SUGT-1 and CIITA decreases expression, but does not knock out, the at least one MHC-I in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of endogenous SUGT-1 and CIITA, where the decreased gene expression of the endogenous SUGT-1 and CIITA decreases expression of at least one MHC-I in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC-I of a comparable cell that does not have the at least one genetic modification without directly targeting HLA gene.

In some embodiments, described herein is a cell comprising at least one genetic modification for decreasing gene expression of the endogenous SUGT-1 and CIITA, where the decreased gene expression of the endogenous SUGT-1 and CIITA decreases expression of at least one MHC-I and at least one MCH-II in the cell. In some embodiments, described herein is the cell comprising a knocked out endogenous SUGT-1 and CIITA, where the knocking out of the endogenous SUGT-1 and CIITA decreases expression of at least one MHC-I and at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising decreased gene expression of the endogenous SUGT-1 and CIITA, where the decreased gene expression of the endogenous SUGT-1 and CIITA knocks out at least one MHC-1 and at least one MHC-II. In some embodiments, the decreased gene expression of the endogenous SUGT-1 and CIITA decreases expression, but does not knock out, the at least one MHC-I or the at least one MHC-II in the cell. In some embodiments, described herein is a cell comprising a decreased gene expression of an endogenous SUGT-1 and CIITA, where the decreased gene expression of the endogenous SUGT-1 and CIITA decreases expression of at least one MHC-I and at least one MHC-II in the cell each by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%compared to expression of MHC-I and MHC-II of a comparable cell that does not have the at least one genetic modification without directly targeting HLA gene.

In some embodiments, the cell comprising the at least one endogenous gene modified by the at least one genetic modification described herein exhibits decreased expression of at least one MHC-I, where the decreased expression of the at least one MHC-1 allows the cell to not being targeted by innate immunity. For example, the cell comprising decreased expression can be transplanted to a subject, where the cell exhibits a decreased expression of the at least one MHC-1 so that the cell is not targeted by NK cell of the subject. In some embodiments, the cell with the at least one endogenous gene knocked out exhibits decreased expression of at least one MHC-II, where the decreased expression of the at least one MHC-1I allows the cell to not being targeted by innate immunity. In some embodiments, the cell with the at least one endogenous gene knocked out exhibits decreased expression of at least one MHC-I or MHC-II, where the decreased expression of the at least one MHC-I or MHC-1I allows the cell to not being targeted by innate immunity. In some embodiments, the cell with the at least one endogenous gene knocked out exhibits decreased expression of at least one MHC-I and MHC-II, where the decreased expression of the at least one MHC-I and MHC-1I allows the cell to not being targeted by innate immunity.

In some embodiments, the cell comprising the at least one endogenous gene modified by the at least one genetic modification described herein exhibits decreased expression of at least one MHC-I, where the decreased expression of the at least one MHC-1 allows the cell to not cause GVHD in the subject upon the subject being transplanted with the cell as cell-based therapy. In some embodiments, the cell with the at least one endogenous gene knocked out exhibits decreased expression of at least one MHC-II, where the decreased expression of the at least one MHC-1I allows the cell to not cause GVHD in the subject upon the subject being transplanted with the cell as cell-based therapy. In some embodiments, the cell with the at least one endogenous gene knocked out exhibits decreased expression of at least one MHC-I or MHC-II, where the decreased expression of the at least one MHC-I or MHC-1I allows the cell to not cause GVHD in the subject upon the subject being transplanted with the cell as cell-based therapy. In some embodiments, the cell with the at least one endogenous gene knocked out exhibits decreased expression of at least one MHC-I and MHC-II, where the decreased expression of the at least one MHC-I and MHC-1I allows the cell to not cause GVHD in the subject upon the subject being transplanted with the cell as cell-based therapy.

In some aspects, the cell comprising the at least endogenous gene modified by the at least genetic modification described herein does not exhibit decreased cellular function. For example, the cell comprising decreased gene expression of the endogenous gene and decreased expression of the at least one MHC does not exhibit decreased cellular activity. As shown in Fig. 3 and Fig. 4, a T cell comprising the CD7CAR comprising the at least one genetic modification for knocking out endogenous gene (RFX5, B2M, CIITA, NLRC5, or SUGT-1) did not exhibit decreased in vitro cell kill activity compared to control cells (EP Mock T and CD7CAR-T cells) .

In some aspects, the cell comprising the at least endogenous gene modified by the at least genetic modification described herein decreases allograft rejection. Fig. 5 illustrates that complete deficiencies of HLA-I (B2M knockout) stimulated the highest killing by NK cells. Knocking out of NLRC5, CIITA, RFX5 or SUGT1 did not have significant impact on killing by NK cells comparing to HLA-I/II non-modified T cells. All the target T cells were TCR (TRAC) knockout with indicated genetic modifications. Fig. 6A illustrates that alloreactive killing of different genetic modified target T cells by allogeneic CD8+ CTLs. Complete knockout of HLA-I (b2M KO T) or HLA-I/II downregulations (by SUGT1, NLRC5, or RFX5 knockout) mediated reduced killing by allogeneic CD8+ CTLs. CIITA knockout had no significant effect on reduction of killing. All the target T cells were TCR (TRAC) knockout with indicated genetic modifications. Fig. 6B illustrates that alloreactive killing of different genetic modified target T cells by allogeneic CD4+ CTLs. HLA-I/II downregulations (by SUGT1, RFX5 or CIITA knockout) mediated reduced killing by allogeneic CD4+ CTLs. NLRC5 or B2M knockout had no significant effect on reduction of killing. All the target T cells were TCR (TRAC) knockout with indicated genetic modifications. Fig. 7 illustrates that HLA-II deficiencies in T cells by knocking out of RFX5 or SUGT1 led to less extent of allogeneic responder T cell activations comparing to CIITA knockout, HLA-I (B2M) knockout, HLA-I deficient (by NLRC5 KO) or HLA-I/II intact (HLA non-modified) cells in Mixed Lymphocyte Reaction (MLR) assay. Responder only: allogeneic responder T cells only, no stimulator cells. All the stimulator cells were TCR (TRAC) knockout with indicated genetic modifications.

In some aspects, at least one HLA is not overexpressed in the cell with decreased expression of at least one MHC-I or MHC-II for the cell to not being targeted by innate immunity such as being targeted by the NK cell of the transplanted subject or not causing GVHD in the transplanted subject. Non-limiting example of the at least one HLA can include of HLA-A, HLA-E, HLA-DM, HLA-DO, HLA-DR, HLA-DQ, or HLA-DP.

The present disclosure provides a cell to be modified to decrease expression of at least one MHC. The cell (e.g., an immune cell) can be isolated from a sample obtained from a subject. The cell, instead of needing to be obtained from the subject, can be obtained from any other suitable sources.

The cell described herein can be isolated from a sample from a donor who is not the subject in need of a treatment for a disease or condition described herein. The sample can be a bodily fluid or a tissue, including but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some cases, a sample comprises NK cells, NKT cells, T-cells or T-cell progenitor cells. For example, in some cases, the sample is an umbilical cord blood sample, a peripheral blood sample (e.g., a mononuclear cell fraction) or a sample from the subject comprising pluripotent cells. In some embodiments, a sample from the subject can be cultured to generate induced pluripotent stem (iPS) cells and these cells used to produce NK cells, NKT cells or T-cells. Cell samples may be cultured directly from the subject or may be cryopreserved prior to use. In some embodiments, obtaining a cell sample comprises collecting a cell sample. In other aspects, the sample is obtained by a third party. In still further aspects, a sample from a subject can be treated to purify or enrich the T-cells or T-cell progenitors in the sample. For example, the sample can be subjected to gradient purification, cell culture selection and/or cell sorting (e.g., via fluorescence-activated cell sorting (FACS) ) . The cell can be an NK cell. The NK cells can be obtained from peripheral blood, cord-blood, or other sources described herein. The NK cells can be derived from induced pluripotent stem cells. In some embodiments, a cell that can be utilized in a method provided herein can be positive or negative for a given factor. In some aspects, the cell can be prepared from a cell line.

In some embodiments, a cell provided herein can be a CD3+ cell, CD3-cell, a CD5+ cell, CD5-cell, a CD7+ cell, CD7-cell, a CD14+ cell, CD14-cell, CD8+ cell, a CD8-cell, a CD103+ cell, CD103-cell, CD11b+ cell, CD11b-cell, a BDCA1+ cell, a BDCA1-cell, an L-selectin+ cell, an L-selectin-cell, a CD25+, a CD25-cell, a CD27+, a CD27-cell, a CD28+ cell, CD28-cell, a CD44+ cell, a CD44-cell, a CD56+ cell, a CD56-cell, a CD57+ cell, a CD57-cell, a CD62L+ cell, a CD62L-cell, a CD69+ cell, a CD69-cell, a CD45RO+ cell, a CD45RO-cell, a CD127+ cell, a CD127-cell, a CD132+ cell, a CD132-cell, an IL-7+ cell, an IL-7-cell, an IL-15+ cell, an IL-15-cell, a lectin-like receptor G1 positive cell, a lectin-like receptor G1 negative cell, or an differentiated or de-differentiated cell thereof. The examples of factors expressed by cells is not intended to be limiting, and a person having skill in the art will appreciate that a cell may be positive or negative for any factor known in the art. In some embodiments, a cell may be positive for two or more factors. For example, a cell may be CD4+and CD8+.

In some embodiments, a cell may be negative for two or more factors. For example, a cell may be CD25-, CD44-, and CD69-. In some embodiments, a cell may be positive for one or more factors, and negative for one or more factors. For example, a cell may be CD4+ and CD8-. In some embodiments, a cellular marker provided herein can be utilized to select, enrich, or deplete a population of cells. In some embodiments, enriching comprises selecting a monocyte fraction. In some embodiments, enriching comprises sorting a population of immune cells from a monocyte fraction. In some embodiments, the cells may be selected for having or not having one or more given factors (e.g., cells may be separated based on the presence or absence of one or more factors) . In some embodiments, the selected cells can also be transduced and/or expanded in vitro. The selected cells can be expanded in vitro prior to infusion. In some embodiments, selected cells can be transduced with a vector provided herein. It should be understood that cells used in any of the methods disclosed herein may be a mixture (e.g., two or more different cells) of any of the cells disclosed herein. For example, a method of the present disclosure may comprise cells, and the cells are a mixture of CD4+ cells and CD8+ cells. In another example, a method of the present disclosure may comprise cells, and the cells are a mixture of CD4+ cells and

cells. In some cases, a cell can be a stem memory TSCM cell comprised of CD45RO (-) , CCR7 (+) , CD45RA (+) , CD62L+ (L-selectin) , CD27+, CD28+ and IL-7Ra+, stem memory cells can also express CD95, IL-2R13, CXCR3, and LFA-1, and show numerous functional attributes distinctive of memory cells. Cells provided herein can also be central memory TCM cells comprising L-selectin and CCR7, where the central memory cells can secrete, for example, IL-2, but not IFNy or IL-4. Cells can also be effector memory TEM cells comprising L-selectin or CCR7 and produce, for example, effector cytokines such as IFNy and IL-4.

In some aspects, the cell described herein can be an immune cell or stem cell. In some embodiments, the immune cell is a lymphocyte such as a T cell, a B cell, a natural killer (NK) cell, or a macrophage. In some aspects, the T cell is a cytotoxic T cell, a natural killer T cell, a regulatory T cell, or a T helper cell. In some embodiments, the cell to be modified is an immune cell comprising an innate lymphocyte (ILC) . In some aspects, the cell modified by the method described herein is an immune cell derived from induced pluripotent stem cell (iPSC) . In some embodiments, the immune cell is an iPSC derived T cell. In some embodiments, the immune cell is an iPSC derived natural killer T cell. In some embodiments, the cell modified by the method described herein is a stem cell. In some aspects, the stem cell can be a hematopoietic stem cell (HSC) or an induced pluripotent stem cell (iPSC) .

In some embodiments, the cell described herein comprises a cell surface marker. The cell surface marker can be an immune cell antigen. The gene encoding the immune cell antigen of the immune cell used for preparing the cell described herein can be inactivated. Examples of immune cell antigens include, but are not limited to, CD2, CD3, CD4, CDS, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD160, CD161, CD178, CD218, CD226, CD244, CD159a (NKG2A) , CD159c (NKG2C) , NKG2E, CD279, CD314 (NKG2D) , CD305, CD335 (NKP46) , CD337, CD319 (CS1) , TCRa, TCRf3 and SLAMF7. For example, in some cases, the gene encoding CD7 of the immune cell is inactivated.

In some embodiments, the cell described herein exhibits enhanced activity toward tumor cells, but with decreased side effects such as cytokine release syndrome (CRS) , GVHD, and/or host rejection of a graft (HVG) . The modified cell can target a disease-associated antigen (e.g., tumor-associated antigen, or tumor cell marker) and at the same time suppress host immune cells. One or more endogenous genes (e.g., a gene encoding a subunit of a TCR, a gene encoding a subunit of an MHC molecule, or a gene encoding a cell surface marker) of the cell described herein can be inactivated. In some cases, the cell described herein comprises a single CAR. In some cases, the cell described herein comprises a first CAR and a second CAR, each targeting a different antigen. In some cases, the cell described herein comprises a CAR having a first antigen binding domain and a second antigen binding domain. In some cases, the cell described herein comprises a first CAR, a second CAR and a third CAR, each targeting a different antigen. In some cases, the cell described herein comprises a CAR having a first antigen binding domain, a second antigen binding domain and a third antigen binding domain. In some cases, the endogenous antigen of the cell described herein can be inactivated. In some cases, a gene encoding the endogenous antigen can be inactivated (e.g., silenced or knocked out) in the cell. In some cases, a gene encoding endogenous CD7 can be inactivated (e.g., silenced or knocked out) in the cell.

In some cases, the endogenous T cell receptor (TCR) of the cell described herein can be inactivated. In some cases, a gene encoding a subunit of the endogenous TCR of the cell described herein can be inactivated such that the endogenous TCR can be inactivated. The gene encoding the subunit can be TCRa, TCRI3, CD3c, CD3o, CD3y, or CD3. In some cases, the endogenous MHC molecule of the cell described herein can be inactivated. In some cases, the endogenous MHC molecule comprises MHC class I molecule and MHC class II molecule. In some cases, a gene encoding MIIC I molecule can be inactivated. The gene encoding MHC I molecule includes but is not limited to HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G. In some embodiments, the expression of one or more endogenous HLA genes of the cell described herein may be knocked out or partially knocked out. For example, any one of more of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G of the cell described herein may be knocked out or partially knocked out. In some cases, an endogenous HLA-A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G can be knocked out or partially knocked out to decrease T cell killing activity. In some cases, any one of more of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G of the cell described herein can remain intact. In some cases, a subunit of the endogenous MHC molecule in said engineered immune cell can be inactivated such that the endogenous MHC molecule is inactive. In some cases, B2M subunit of the endogenous MHC molecule in said engineered immune cell is inactivated. In some cases, B2M subunit of the endogenous MHC molecule in said engineered immune cell is knocked out or partially knocked out.

In some cases, a killer/phagocyte inhibitor of the cell described herein can be overexpressed. In some other cases, an endogenous HLA can be knocked out with co-expression of killer/phagocyte inhibitor (s) . For example, B2M can be knocked out with co-expression of killer/phagocyte inhibitors. The killer/phagocyte inhibitor may suppress immune response toward the cell described herein . The killer/phagocyte inhibitors include, but are not limited to, HLA-E single chain trimer, HLA-G, CD47, CD24, FASL, PDL1, or functional domains thereof.

In some cases, the cell described herein can exhibit (i) enhanced degree of persistence by remaining viable in vitro while in presence of cells that are heterologous to the cell described herein, including but not limited to heterologous T cells, heterologous NK cells and the mixture of the heterologous T cells and heterologous NK cells, (ii) enhanced degree of expansion, or (iii) enhanced cytotoxicity against a target cell comprising the antigen, compared to an additional engineered immune cell comprising the one or more CARs without the inactivation of the TCR, MHC molecule and/or immune cell antigen. In some cases, the cell described herein can be characterized by exhibiting two or more of (i) enhanced degree of persistence by remaining viable in vitro while in presence of cells that are heterologous to the cell described herein , including but not limited to heterologous T cells, heterologous NK cells and the mixture of the heterologous T cells and heterologous NK cells, (ii) enhanced degree of expansion, and (iii) enhanced cytotoxicity.

In some embodiments, the cell described herein can also comprise an enhancer moiety capable of enhancing one or more activities of the cell described herein. The enhancer moiety can be configured to constitutively upregulate one or more intracellular signaling pathways of the cell described herein . The one or more intracellular signaling pathways can be one or more cytokine signaling pathways. The enhancer moiety can be self-activating through self-oligomerizing. The enhancer moiety can be self-activating through self-dimerizing. The enhancer moiety can be a cytokine or a cytokine receptor. The enhancer moiety can be selected from the group consisting of IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, PD-1, PD-L1, CD122, CSF1R, CTAL-4, TIM-3, CCL21, CCL19, TGFR beta, receptors for the same, functional fragments thereof, functional variants thereof, and combinations thereof.

In some aspects, the cell described herein can further comprise an inducible cell death moiety, which can effect suicide of the cell described herein upon contact with a cell death activator. The inducible cell death moiety can be selected from the group consisting of rapaCasp9, iCasp9, HSV-TK, ACD20, mTMPK, ACD19, RQR8, and EGFRt. In some cases, the inducible cell death moiety is EGFRt, and the cell death activator is an antibody or an antigen binding fragment thereof that binds EGFRt. In some cases, the inducible cell death moiety is HSV-TK, and the cell death activator is GCV. In some cases, the inducible cell death moiety is iCasp9, and the cell death activator is AP1903. The cell death activator can comprise a nucleic acid, a polynucleotide, an amino acid, a polypeptide, lipid, a carbohydrate, a small molecule, an enzyme, a ribosome, a proteasome, a variant thereof, or any combination thereof.

In some aspects, the cell described herein provided herein can comprise a chimeric polypeptide comprising (i) an enhancer moiety capable of enhancing one or more activities of the cell described herein , and (ii) an inducible cell death moiety capable of effecting death of the cell described herein upon contacting the chimeric polypeptide with a cell death activator, wherein the enhancer moiety is linked to the inducible cell death moiety. In some cases, the enhancer moiety and the inducible moiety may be linked by a linker. The linker can be a cleavable linker, for example, a self-cleaving peptide.

In some embodiments, the cell described herein can further comprise at least one heterologous polypeptide comprising at least one heterologous receptor. In some aspects, the heterologous receptor is a chimeric polypeptide receptor (CPR) comprising a binding moiety, wherein the binding moiety comprises (i) a first antigen binding domain, which first antigen binding domain suppresses or decreases a subject's immune response toward the cell described herein when administered into the subject and (ii) a second antigen binding domain capable of binding to a disease-associated antigen. An individual CPR of the one or more CPRs can comprise (i) the first antigen binding domain, (ii) the second antigen binding domain, or (iii) both the first antigen binding domain and the second antigen binding domain. A CPR of the one or more CPRs can further comprise a transmembrane domain and an intracellular signaling region. In some cases, the one or more CPRs in the cell described herein are one or more chimeric antigen receptors (CARs) or engineered T cell receptors (TCRs) . In some cases, the cell described herein s comprise both CARs and engineered TCRs. In some embodiments, the at least one heterologous receptor comprises at least one chimeric antigen receptor (CAR) , where each CAR of the at least one CAR comprise a hinge, a transmembrane domain, a costimulatory, and an intracellular signaling region.

The engineered TCR can be a TCR fusion protein. For example, the TCR fusion protein can comprise a heterologous antigen binding domain fused to one or more subunits of a TCR complex. In some cases, the TCR fusion protein can comprise a TCR subunit comprising at least a portion of a TCR extracellular domain and a TCR intracellular domain; and an antibody domain comprising an antigen binding domain, where the TCR subunit and the antibody domain are linked. The TCR fusion protein can incorporate into a TCR complex when expressed in a T cell. In some cases, the TCR fusion protein can further comprise a TCR transmembrane domain. The TCR extracellular domain, the TCR intracellular domain, or the TCR transmembrane domain can be derived from TCR alpha chain, TCR beta chain, TCR gamma chain, TCR delta chain, CD3 epsilon, CD3 gamma, CD3 delta or CD3 zeta. In some cases, an endogenous TCR of the cell described herein comprising an engineered TCR is inactivated.

In some cases, the cell described herein comprising inactivated endogenous TCR may not cause GVHD. For example, a gene encoding an endogenous TCR subunit can be inactivated. For another example, a gene encoding an endogenous TCR subunit may be mutated such that an endogenous TCR may not be formed.

CARs can comprise an extracellular antigen recognition region, for example, a scFv (single-chain variable fragment) , a transmembrane region, and an intracellular costimulatory signal region. The extracellular domain of CARs can recognize a specific antigen and then transduce the signal through the intracellular domain, causing T cell activation and proliferation, cytolysis toxicity, and secretion of cytokines, thereby eliminating target cells. The patient's autologous T cells (or heterologous donors) can be first isolated, activated and genetically engineered to produce CAR-T cells, which can be then injected into the same patient. In this way, the probability of graft-versus-host disease may be decreased, and the antigen can be recognized by T cells in a non-MHC-restricted manner. In addition, a CAR-T can treat all cancers that express the antigen.

In some aspects, the cell described herein can target both disease-associated antigen (e.g., tumor-associated antigen or tumor cell marker) and immune cell antigen (e.g., CD3, CD7 or CD137) through bispecific or multivalent CAR (s) . For example, the present disclosure provides an engineered immune cell that can target a tumor cell marker and an immune cell antigen such as CD3. The endogenous TCR can be inactivated (e.g., disrupted, inhibited, knocked out or silenced) . The CAR-T of the present disclosure which targets the tumor cell marker and the immune cell antigen can eliminate positive tumor cells and clear host immune cell antigen positive T and NK cells, thereby avoiding host rejection (HVG) . In the present disclosure, the endogenous TCR of the cell described herein can be knocked out, and graft-versus-host disease (GVHD) can be prevented, thereby preparing a general-purpose or universal CAR-T (UCAR-T) cell. The cell described herein can be derived from an autologous T cell or an allogeneic T cell. Moreover, the cell described herein can comprise a cell suicide element (e.g., inducible cell death moiety) , and the CAR-T can be inactivated/cleared at any time to decrease side effects. In some cases, the cell described herein can further comprise an enhancer moiety. The enhancer moiety can regulate one or more activities of the cell described herein when the cell described herein is administered to a subject. For example, the enhancer moiety can be a cytokine (e.g., IL-5 or IL-7) or a cytokine receptor (e.g., IL-5R or IL-7R) . The enhancer moiety can enhance a signaling pathway within the cell described herein , for example, STAT5 signaling pathway. In some embodiments, the cell described herein comprises a bispecific CAR targeting both CD19 and CD3. The cell described herein show in this example can further comprise an inducible cell death moiety such as a truncated epidermal growth factor receptor (EGFRt or tEGFR, which can be used interchangeably herein. The inducible cell death moiety or the enhancer moiety can be introduced in the immune cell via a separate expression vector. In some cases, the inducible cell death moiety and the enhancer moiety may be introduced into the immune cell via an expression vector comprising sequences encoding both moieties. In some cases, the inducible cell death moiety and the enhancer moiety are linked and are expressed as a chimeric polypeptide. The application of the cell described herein can be used for cell-based therapy for treating a disease or condition (e.g., cancer) of a subject, be prepared in large-scale in advance to avoid GVHD and HvG, decrease treatment costs, inactivate CAR-T at any time if necessary, decrease side effects of immunotherapy, and ensure product safety.

Chimeric antigen receptors (CARs)

Described herein, in some aspects, is a cell modified to decrease gene expression of at least one endogenous gene. In some aspects, the decreased gene expression of the at least one endogenous gene decreases expression of MHC-I, MHC II, or a combination thereof. In some aspects, the modified cell expresses at least one heterologous polypeptide comprising at least one heterologous receptor. In some aspects, the heterologous receptor can be CAR or TCR.

In some aspects, the cell provided herein can comprise one or more CARs. The CAR can include an extracellular domain, a transmembrane domain, and an intracellular signaling domain. The extracellular domain can include a target-specific binding element (also known as an antigen binding domain) . The intracellular domain can include a costimulatory signaling region and a zeta chain portion. A costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigens receptors or their ligands that may be needed for an efficient response of lymphocytes to antigen. Between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR, there may be incorporated a spacer domain.

As used herein, the term "spacer domain" generally means any oligo-or polypeptide that functions to link the transmembrane domain to, either the extracellular domain or, the cytoplasmic domain in the polypeptide chain. A spacer domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. With respect to the transmembrane domain, the CAR can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the CAR. In one embodiment, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions of particular use in the present disclosure may be derived from (e.g., comprise at least the transmembrane region (s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or from an immunoglobulin such as IgG4. Alternatively the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo-or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker. The cytoplasmic domain or otherwise the intracellular signaling domain of the CAR of the present disclosure can be responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed in. The term "effector function" refers to a specialized function of a cell.

Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus the term "intracellular signaling domain" refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

Examples of intracellular signaling domains for use in the CAR of the present disclosure include the cytoplasmic sequences of the TCR and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability. Signals generated through the TCR alone may be insufficient for full activation of the T cell and that a secondary or co-stimulatory signal may be included. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences) .

Primary cytoplasmic signaling sequences can regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences that are of particular use in the present disclosure include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. It is particularly preferred that cytoplasmic signaling molecule in the CAR of the present disclosure comprises a cytoplasmic signaling sequence derived from CD3-zeta. In some embodiments, the cytoplasmic domain of the CAR can be designed to comprise the CD3-zeta signaling domain by itself or combined with any other desired cytoplasmic domain (s) useful in the context of the CAR of the present disclosure. For example, the cytoplasmic domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule.

Acostimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that may be needed for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137) , OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1) , CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like. Thus, while the present disclosure is, in some cases, exemplified with 4-1BB as the co-stimulatory signaling element, other costimulatory elements are within the scope of the present disclosure.

The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the present disclosure may be linked to each other in a random or specified order. Optionally, a short oligo-or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage. A glycine-serine doublet provides a particularly suitable linker. In some embodiments, the cytoplasmic domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In another embodiment, the cytoplasmic domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4-1BB. In yet another embodiment, the cytoplasmic domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28 and 4-1BB.

ACAR provided herein can comprise one or more antigen binding domains. In some cases, a CAR provided herein comprises an antigen binding domain that can target both an immune cell antigen (e.g., to inhibit killing activity of a T cell or NK cell) and a disease-associated antigen (e.g., a tumor-associated antigen) . For example, an antigen binding domain targeting both immune cell antigens and cancer antigens include, but not limited to, CD2, CD3, CD4, CD5, CD7, CD8, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD69, CD100, CD122, CD132, CD137, CD161, CD159a, CD159c, CD279, CD314, CD319 (CS1) and TCR. In some cases, a CAR provided herein comprises two antigen binding domains such that one individual CAR is a bispecific CAR, targeting two different antigens. For bispecific CAR, one antigen binding domain can target immune cell antigen, and the other antigen binding domain can target disease-associated antigen. The two antigen binding domains of a bispecific CAR can have a tandem structure, a parallel structure or a loop structure. For example, a CAR can target a tumor cell marker and CD3. The CAR can have a structure as formula I: L-scFyl-I-scFv2-H-TM-C-CD3 (I) , wherein each "-" is independently a linker peptide or a peptide bond; L is optionally a signaling peptide sequence; I is a flexible linker; H is optionally a hinge region; TM is a transmembrane domain; C is a costimulatory domain; CD3 is a cytoplasmic signaling sequence derived from CD3t; one of scFv1 and scFv2 is an antigen binding domain targeting a tumor cell marker, and the other one is an antigen binding domain targeting CD3. The CAR can have a structure as formula II or II': L-VL-scFv-VH-H-TM-C-CD3 (II) , L-VH-scFv-VL-H-TM-C-CD3 (II') , wherein each "-" is independently a linker peptide or a peptide bond; the elements L, H, TM, C and CD3 as described above; scFv is an antigen binding domain targeting a tumor cell marker, VH is an anti-CD3 antibody heavy chain variable region, and VL is an anti-CD3 antibody light chain variable region; or scFv is an antigen binding domain targeting CD-3, VH is an anti-tumor cell marker antibody heavy chain variable region, and VL is an anti-tumor cell marker antibody light chain variable region. In some cases, a CAR can comprise the structure of EGFRt-CD3 scFv-CD19 scFv-Hinge-TM-CD28/41BB-CD3, wherein EGFRt is a truncated EGFR, as a safety switch (e.g., inducible cell death moiety) , CD3 scFv is the svFCv fragment of the heavy and light chain variable regions of the monoclonal antibody OKT3 or UCHT1 linked by a GS linker, and the CD19 scFv fragment is the heavy and light chain variable region of the monoclonal antibody linked by a GS linker. The structure of the CAR can further comprise a hinge, transmembrane regions, costimulatory signaling region of CD28 or 41BB, and/or CD3 intracellular domain. In the present disclosure, the nucleic acid construct of EGFRt-CD3 scFv-CD19 scFv-Hinge-TM-CD28/41BB-CD3 can be inserted into a vector (e.g., a lentiviral vector) . The vector can be packaged in 293T cells. T cells can be sorted from PBMC, and after activation, TCR and PD-1 genes can be knocked out by CRISPR/CAS technology. T cells can then be infected with the vectors to express the CARs. The prepared CAR-T cells can be used to detect the infection efficiency and gene editing efficiency of CAR by flowcytometry.

The immune cell marker, e.g., CD3, of the above examples can be replaced with other immune cell markers such as CD7 and CD137. In some cases, a CAR comprising two antigen binding domains arranged in a tandem form. In some embodiments, the first antigen binding domain and the second antigen binding domain is arranged, from amino terminus to carboxyl terminus, as: (i) VL2-VH2-VL1-VH1; (ii) VL2-VH2-VH1-VL1; (iii) VL1-VH1-VL2-VH2; (iv) VL1-VH1-VH2-VL2; (v) VH2-VL2-VL1-VH1; (vi) VH2-VL2-VH1-VL1; (vii) VH1-VL1-VL2-VH2; or (viii) VH1-VL1-VH2-VL2, wherein VH1 is heavy chain variable domain of the first antigen binding domain, VL1 is light chain variable light domain of the first antigen binding domain, VH2 is heavy chain variable domain of the second antigen binding domain, and VL2 is light chain variable domain of the second antigen binding domain. For example, the CAR can have a structure represented by the following formula IV or IV': L3-scFv1-R-scFv2-H3-TM3-C3-CD3 (IV) ; L3-scFv2-R-scFv1-H3-TM3-C3-CD3 (IV') , wherein each "-" is independently a linker peptide or peptide bond; L3 is an optional signal peptide sequence; scFv1 is an antigen binding domain that targets tumor cell markers; R is a rigid or flexible joint; scFv2 is an antigen binding domain (e.g., an antibody single-chain variable region sequence) that targets T cell and NK cell consensus markers; H3 is an optional hinge region; TM3 is a transmembrane domain; C3 is a costimulatory domain; CD3 is a cytoplasmic signaling sequence derived from CD3. In some cases, a CAR comprising two antigen binding domains arranged in a loop form. In some cases, the first antigen binding domain and the second antigen binding domain is arranged, from amino terminus to carboxyl terminus, as: (i) VL2-VH1-VL1-VH2; (ii) VH2-VL1-VH1-VL2; (iii) VL1-VH2-VL2-VH1; (iv) VH1-VL2-VH2-VL1; (v) VL2-VL1-VH1-VH2; (vi) VH2-VH1-VL1-VL2; (vii) VL1-VL2-VH2-VH1; or (viii) VH1-VH2-VL2-VL1, wherein VH1 is heavy chain variable domain of the first antigen binding domain, VL1 is light chain variable light domain of the first antigen binding domain, VH2 is heavy chain variable domain of the second antigen binding domain, and VL2 is light chain variable domain of the second antigen binding domain. For example, the CAR can have the following formula VI, VI', VI" or VI"' structure: L8-VL1-VH2-I-VL2-VH1-H8-TM8-C8-CD3 (VI) ; L8-VH1-VL2-I-VH2-VL1-H8-TM8-C8-CD3 (VI') ; L8-VL2-VH1-I-VL1-VH2-H8-TM8-C8-CD3 (VI") ; L8-VH2-VL1-I-VH1-VL2-H8-TM8-C8-CD3 (VI'") , wherein each "-" is independently a linker peptide or peptide bond; L8 is an optional signal peptide sequence; VH1 is an anti-tumor cell marker antibody heavy chain variable region, and VL1 is an anti-tumor cell marker antibody light chain variable region; VH2 is an anti-T cell and NK cell consensus marker (such as CD7 or CD2) antibody heavy chain variable region; and VL2 is an anti-T cell and NK cell consensus marker (such as CD7 or CD2) antibody light chain variable region; I is a flexible joint; H8 is an optional hinge region; TM8 is a transmembrane domain; C8 is a costimulatory domain; CD3 is a cytoplasmic signaling sequence derived from CD3.

In some cases, a CAR comprising two antigen binding domains are arranged in a parallel form. The parallel form can comprise a full construct of a first CAR having a first antigen binding domain linked to a full construct of a second CAR having a second antigen binding domain. An example of parallel form can be tEGFR-CD19 scFv-CD28-CD3-CD3 scFv-41BB-CD3. The tEGFR shown here can function as a safety switch, which can be replaced by other safety switches as described in the present disclosure. As described herein, CD19 scFv and CD3 scFv are two examples of antigen binding domains, which may be replaced with various antigen binding domains as described in the present disclosure. CD28 can be an example of transmembrane domain and can be replaced with other transmembrane domains described herein. 41BB can be an example of co-stimulatory domain and can be replaced with other co-stimulatory domains described herein. In some cases, a linker is used to link the first CAR and the second CAR. The linker can be a cleavable linker. The cleavable linker can be self-cleaving peptide such as 2A self-cleaving peptide.

Also contemplated in the present disclosure is a nucleic acid molecule encoding a CAR or a bispecific CAR. The nucleic acid can comprise a first sequence encoding a chimeric antigen receptor (CAR) , wherein the CAR can comprise a binding moiety, which binding moiety comprises (i) a first antigen binding domain, which first antigen binding domain suppresses or decreases a subject's immune response toward the cell described herein when administered into the subject linked to (ii) a second antigen binding domain capable of binding to a disease-associated antigen, and wherein each CAR of the one or more CARs can further comprise a transmembrane domain and an intracellular signaling domain. The first antigen binding domain can target an immune cell antigen selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD160, CD161, CD178, CD218, CD226, CD244, CD159a (NKG2A) , CD159c (NKG2C) , NKG2E, CD279, CD314 (NKG2D) , CD305, CD335 (NKP46) , CD337, CD319 (CS1) , TCRα, TCRβ and SLAMF7. The second antigen binding domain can target a disease-associated antigen such as CD19. Other non-limiting examples of disease-associated antigen includes BCMA, VEGFR2, CD19, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD80, CD86, CD81, CD123, cd171, CD276, B7H4, CD133, EGFR, GPC3; PMSA, CD 3, CEACAM6, c-Met, EGFRvIII, ErbB2, ErbB3 HER-2, HER3, ErbB4 /HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Fltl, KDR , Flt4, CD44V6, CEA, CA125, CD151, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A, Mesothelin, NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, TCRa, TCRp, TLR7, TLR9, PTCH1, WT-1, Robl, Frizzled , OX40, CD79b Claudin 18.2, Folate receptor a, Folate receptor (3, GPC2, CD70, BAFF-R and Notch-1-4.

The nucleic acid molecule can further comprise a second sequence encoding an enhancer moiety, which enhancer moiety can enhance one or more activities of the CAR when expressed in a cell. The enhancer moiety can be selected from the group consisting of IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, PD-1, PD-L1, CD122, CSF1R, CTAL-4, TIM-3, CCL21, CCL19, TGFR beta, receptors for the same, functional fragments thereof, functional variants thereof, and combinations thereof. The nucleic acid molecule can further comprise a second sequence encoding an inducible cell death moiety, which inducible cell death moiety, when expressed in a cell, can effect death of the cell upon contacting the inducible cell death moiety with a cell death activator. The inducible cell death moiety can be selected from the group consisting of rapaCasp9, iCasp9, HSV-TK, ACD20, mTMPK, ACD19, RQR8, and EGFRt.

The nuclei acid molecule can further comprise a third sequence flanked by the first sequence and the second sequence, wherein the third sequence can encode a cleavable linker. The cleavable linker can be a self-cleaving peptide. The nucleic acid molecule can further comprise a regulatory sequence regulating expression of the first sequence and/or the second sequence. Also contemplated in the present disclosure is a kit comprising the nucleic acid molecule described herein. In some cases, the nucleic acid encoding the CAR described herein can be delivered into an immune cell for expression of the CAR to generate an engineered cell.

Methods for modifying cell

Described herein, in some aspects, is a method for modifying a cell with at least genetic modification. In some aspects, the cell is modified to decrease endogenous gene expression of at least one endogenous gene described herein. In some aspects, the cell is modified to knock out endogenous gene expression of at least one endogenous gene described herein. In some aspects, the cell is modified to decrease or knock out endogenous gene expression of SUGT-1, NLRC (e.g., NOD1, NOD2, NLRC3, NLRC4, or NLRC5) , RFX1, RFX2, RFX3, RFX4, RFX5, CIITA, CREB, NF-Y, or a combination thereof. In some aspects, the cell is modified to knock out endogenous gene expression of SUGT-1. In some aspects, the cell is modified to knock out endogenous gene expression of NLRC5. In some aspects, the cell is modified to knock out endogenous gene expression of RFX5. In some aspects, the cell is modified to knock out endogenous gene expression of CIITA. In some aspects, the cell is modified to knock out endogenous gene expression of CIITA. In some aspects, the cell is modified to knock out endogenous gene expression of CIITA. In some aspects, the cell is modified to knock out endogenous gene expression of SUGT-1 and at least one additional gene (e.g., NOD1, NOD2, NLRC3, NLRC4, NLRC5, RFX1, RFX2, RFX3, RFX4, RFX5, CIITA, CREB, NF-Y, or a combination thereof) . In some aspects, the cell is modified to knock out endogenous gene expression of SUGT-1 and at least one additional gene (e.g., NOD1, NOD2, NLRC3, NLRC4, NLRC5, RFX1, RFX2, RFX3, RFX4, RFX5, CIITA, CREB, NF-Y, or a combination thereof) . In some aspects, the decrease or knock out of the at least one endogenous gene decreases expression of at least one MHC. In some aspects, the decrease or knock out of the at least one endogenous gene knocks out expression of at least one MHC.

In some aspects, the method comprises contacting the cell described herein with a gene modifying moiety. In some aspects, the gene modifying moiety is encoded by a heterologous polynucleotide, where upon expression of the heterologous polynucleotide, the gene expression of the at least endogenous gene is decreased or knocked out. In some aspects, the heterologous polynucleotide comprises a vector. In some aspects, the method comprises contacting the cell with a gene modifying moiety. In some aspects, the method comprises expressing a heterologous polypeptide (e.g., a CAR described herein) in the cell comprising the gene modifying moiety.

In some aspects, the gene modifying moiety can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the gene modifying moiety can be transfected into a host cell by physical, chemical, or biological means. In some embodiments, the gene modifying moiety can be delivered into the cell via physical methods such as calcium phosphate precipitation, lipofection, particle bombardment, microinjection, gene gun, electroporation, and the like.

Physical methods for introducing the gene modifying moiety encoding into the cell can include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, gene gun, electroporation, and the like. One method for the introduction of the gene modifying moiety to a host cell is calcium phosphate transfection.

Chemical means for introducing the gene modifying moiety into the cell can include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, spherical nucleic acid (SNA) , liposomes, or lipid nanoparticles. An example colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle) . Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of non-naturally occurring polynucleotide or vector encoding the gene modifying moiety with targeted nanoparticles.

In the case where a non-viral delivery system is utilized, an example delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of gene modifying moiety or vector encoding the gene modifying moiety into a cell (in vitro, ex vivo, or in vivo) . In another aspect, the vector can be associated with a lipid. The vector associated with a lipid can be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the gene modifying moiety or the heterologous polynucleotide encoding the gene modifying moiety, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, in some embodiments, they are present in a bilayer structure, as micelles, or with a “collapsed” structure. Alternately, they are simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which are, in some embodiments, naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use are obtained from commercial sources. Stock solutions of lipids in chloroform or chloroform/methanol are often stored at about -20 ℃. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes are often characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers. However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids, in some embodiments, assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

In some cases, non-viral delivery method comprises lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, exosomes, polycation or lipid: cargo conjugates (or aggregates) , naked polypeptide (e.g., recombinant polypeptides) , naked DNA, artificial virions, and agent-enhanced uptake of polypeptide or DNA.

In some embodiments, the at least one gene modifying moiety can be delivered into the cell via biological methods such as the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors, in some embodiments, are derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. Exemplary viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors (AAV vectors) , pox vectors, parvoviral vectors, baculovirus vectors, measles viral vectors, or herpes simplex virus vectors (HSVs) . In some instances, the retroviral vectors include gamma-retroviral vectors such as vectors derived from the Moloney Murine Keukemia Virus (MoMLV, MMLV, MuLV, or MLV) or the Murine Steam cell Virus (MSCV) genome. In some instances, the retroviral vectors also include lentiviral vectors such as those derived from the human immunodeficiency virus (HIV) genome. In some instances, AAV comprises a serotype, including AAV1 , AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or a combination thereof. Based on these initial serotypes, AAV capsid of each serotype can be engineered to make them better suited for biological functions, tissue or cell selection.

In some aspects, the gene modifying moiety various gene editing methods can be used in the present disclosure to make the cell described herein s, including CRISPR, RNA interference technology, TALENs (transcription activator-like (TAL) effector nucleases) and Zinc finger nucleases (ZFNs) .

In some cases, CRISPR/Cas9 system is used to edit the genes of the immune cells. For example, CRISPR/Cas9 system can be used to knockout endogenous TCRs or cell surface markers (e.g., CS1, CD7, CD137) of the immune cells to generate the cell described herein s for T cell therapy. The CRISPR/Cas9 (clustered regular interspaced short palindromic repeats) /Cas (CRISPR-associated) system is a natural immune system unique to prokaryotes that is resistant to viruses or exogenous plasmids. The Type II CRISPR/Cas system has been applied in many eukaryotic and prokaryotic organisms as a direct genome-directed genome editing tool. The development of the CRISPR/Cas9 system has revolutionized the ability of people to edit DNA sequences and regulate the expression levels of target genes, providing a powerful tool for accurate genome editing of organisms. The simplified CRISPR/Cas9 system can comprise Cas9 protein and gRNA. The principle of action is that gRNA forms a Cas9-gRNA complex with Cas9 protein through its own Cas9 handle, and the base complementary pairing sequence of gRNA in the Cas9-gRNA complex is paired with the target sequence of the target gene by the principle of base complementary pairing. Cas9 uses its own endonuclease activity to cleave the target DNA sequence. Compared to traditional genome editing techniques, the CRISPR/Cas9 system has several distinct advantages: ease of use, simplicity, low cost, programmability, and the ability to edit multiple genes simultaneously. In some embodiments, the gene modifying moiety comprises a gRNA complexed with Cas to form a Cas/RNP. In some aspects, the gene modifying moiety is Cas9/RNP. Fig. 2 is an exemplary experiment showing HLA-I/II expression levels after knockout of SUGT1, NLRC5, or RFX5 by CRISPR/Cas9 system. SUGT1 knockout was tested with 3 different gRNAs (BD1, BD4, or TF3) . Mean Fluorescent Intensity (MFI) of each sample were labeled to the right side of each graph. HLA-II expression were detected with anti-HLA-DR/DP/DQ antibody. HLA-I expression were detected by anti-b2M antibody or by anti-HLA-A/B/C antibody.

Proteins that bind to and are guided by a guide RNA to direct sequence specific cleavage or that, otherwise, bind nucleic acid sequences in a sequence specific way to trigger non-specific cleavage are consistent with the present disclosure. Said proteins include programmable endonucleases, such as programmable Cas endonucleases. Examples of programmable Cas endonucleases consistent with the present disclosure include Cas12a (or Cpf1) , Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas13a, Cas13b, Cas14, Cas9, or others. In some cases, a site-specific endonuclease may include Cas12a (Cpf1) , including any derivative thereof; any variant thereof; and any fragment thereof. Cas12a is classified as a class II, Type V CRISPR/Cas effector protein having about 1, 300 amino acids. Cas12a is smaller than Cas9. Cas12a comprises two major domains such as REC and RuvC domains. Cas12a lacks the HNH endonuclease domain as in Cas9. Cas12a cleaves a double stranded DNA (dsDNA) immediately downstream from T-rich (5′-TTTN-3′) PAM. Cas12a generates a 4-5 nt-long 5’ - overhang 20 nucleotides away from T-rich PAM. In some cases, the sticky ends produced by Cas12a enhance the efficiency of DNA replacement during HR.

In some cases, a site-specific endonuclease may include Cas13a (C2c2) . Alternatively, in some cases, a site-specific endonuclease may include Cas13b. Cas13 is an RNA-targeting endonuclease that exhibits a collateral effect of promiscuous RNAs activity upon target recognition.

In some cases, a site-specific endonuclease such as a Cas polypeptide may be from the organism Streptococcus pyogenes (S. pyogenes) . Alternatively, a number of eubacterial or other microbial organisms may be suitable sources for site specific endonucleases such as Cas polypeptides. Source organisms are selected by any number of criteria, such as GC bias or codon bias of their encoded proteins, optimal growth temperature (which often corresponds to enzyme optimal activity) , availability, presence of regulatory sites, or other relevant criteria. Any of the following non-limiting examples are suitable for use as a source of the site-specific endonuclease: Leptotrichia wadei, Leptotrichia shahii, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinae spiralis, Streptomyces viridochromo genes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius , Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Pseudomonas aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans , Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Leptotrichia shahii, Prevotella, or Francisella novicida. Other source organisms are consistent with the present disclosure herein. In some aspects, the gene modifying moiety described herein comprises a Cas protein, a TALEN, or a ZFN. In some aspects, the gene modifying moiety is a Cas protein.

In some aspects, the gene modifying moiety comprises a nucleic acid (e.g., via RNA interference) for decreasing or knocking out the at least one endogenous gene described herein. In some aspects, the gene modifying moiety comprises inhibitory RNA. In some embodiments, the gene modifying moiety comprises siRNA, miRNA, shRNA or a combination thereof. In some aspects, the gene modifying moiety comprising the inhibitory RNA can be directly delivered into the cell via any of the delivery method described herein. In some aspects, the gene modifying moiety comprising the inhibitor RNA can be expressed by a vector described herein.

In some aspects, the method comprises expressing a heterologous polypeptide comprising a heterologous receptor in the cell comprising the at least one genetic modification. In some aspects, the receptor comprises an antigen binding domain. In some aspects, the antigen binding domain binds to CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44v6, CD47, CD52, CD56, CD57, CD58, CD70, CD79a, CD79b, CD80, CD81, CD86, CD99, CD117, CD123, CD133, CD135, CD137, CD151, CD171, CD276, BAFF-R, BCMA, B7H4, CEA, CEACM6, Claudin18.2, CLL-1, c-Met, CS-1, CTLA-4, EGFRvIII, GPC2, GPC3, GPRC5, HER2, HER3, HER4/ErbB4, HVEM, MAGE-A, MAGE3, MSLN, MUC-1, MUC-16, NY-ESO-1, OX40, PD-1, PD-L1, PD-L2, PMSA, ROR1, TCRa, TCRb, TLR7, TLR9, VEGFR-2, or WT-1. In some embodiment, the antigen binding domain may bind to one or a plurality of antigens described herein. In some aspects, the heterologous receptor comprises a transmembrane domain. In some aspects, the heterologous receptor comprises a signaling domain.

In some embodiments, the method comprises knocking out at least one endogenous genes in a cell, where the cell exhibits decreased expression of at least one MHC-I and where the decreased expression of the at least one MHC-1 allows the cell to not being targeted by innate immunity. For example, the cell comprising decreased expression can be transplanted to a subject, where the cell exhibits a decreased expression of the at least one MHC-1 so that the cell is not targeted by NK cell of the subject. In some embodiments, the cell with the at least one endogenous gene knocked out exhibits decreased expression of at least one MHC-II, where the decreased expression of the at least one MHC-1I allows the cell to not being targeted by innate immunity. In some embodiments, the cell with the at least one endogenous gene knocked out exhibits decreased expression of at least one MHC-I or MHC-II, where the decreased expression of the at least one MHC-I or MHC-1I allows the cell to not being targeted by innate immunity. In some embodiments, the cell with the at least one endogenous gene knocked out exhibits decreased expression of at least one MHC-I and MHC-II, where the decreased expression of the at least one MHC-I and MHC-1I allows the cell to not being targeted by innate immunity.

In some embodiments, the cell with the at least one endogenous gene knocked out exhibits decreased expression of at least one MHC-I, where the decreased expression of the at least one MHC-1 allows the cell to not cause GVHD in the subject upon the subject being transplanted with the cell as cell-based therapy. In some embodiments, the cell with the at least one endogenous gene knocked out exhibits decreased expression of at least one MHC-II, where the decreased expression of the at least one MHC-1I allows the cell to not cause GVHD in the subject upon the subject being transplanted with the cell as cell-based therapy. In some embodiments, the cell with the at least one endogenous gene knocked out exhibits decreased expression of at least one MHC-I or MHC-II, where the decreased expression of the at least one MHC-I or MHC-1I allows the cell to not cause GVHD in the subject upon the subject being transplanted with the cell as cell-based therapy. In some embodiments, the cell with the at least one endogenous gene knocked out exhibits decreased expression of at least one MHC-I and MHC-II, where the decreased expression of the at least one MHC-I and MHC-1I allows the cell to not cause GVHD in the subject upon the subject being transplanted with the cell as cell-based therapy.

In some embodiments, the method comprises contacting the cell with the gene regulating moiety described herein, where the cell comprises an immune cell or a stem cell. In some aspects, the cell is an immune cell described herein (e.g., a lymphocyte, a B cell, or a T cell) In some cases, the T cell can be cytotoxic T cell, alpha beta T cell, a gamma delta T cell, natural killer T cell, regulatory T cell, or T helper cell. In some aspects, the immune cell comprises an ILC. In some aspects, the stem cell is a hematopoietic stem cell or an iPSC. In some aspects, the iPSC can be derived into an immune cell or a T cell.

Without wishing to be bound by theory, introducing a heterologous polypeptide in a cell (e.g., expressing a heterologous receptor, such as a chimeric antigen receptor, in the cell) may not substantially change the expression level of MHC (e.g., MHC I or MHC II) in the cell. For example, a gene encoding the heterologous polypeptide can be introduced to the cell prior to, simultaneously with, or subsequent to the reduced expression level of an endogenous gene (e.g., encoding SUGT-1, NLRC5, CIITA, RFX, etc. ) , and the resulting reduction in expression level of the MHC as a result of the reduced expression level of the endogenous gene would not be effected substantially by expression of the heterologous polypeptide. Alternatively or in addition to, combination of (i) expression of the heterologous polypeptide and (ii) the reduced expression level of the endogenous gene may yield an expression level of the MHC that is lower (e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, etc. ) than that in a cell or a population of cells having (ii) but not (i) .

Methods of treatment

Disclosed herein, in some embodiments, are methods of using the cell described herein. In some embodiments, the methods include treating a disease or condition of a subject by administering a cell or a pharmaceutical composition comprising the cell described herein to the subject. In some embodiments, administration is by any suitable mode of administration, including systemic administration (e.g., intravenous, inhalation, etc. ) . In some embodiments, the subject is human.

In some embodiments, the cell or the pharmaceutical composition is administered at least once during a period of time (e.g., every 2 days, twice a week, once a week, every week, three times per month, two times per month, one time per month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, once a year) . In some embodiments, the composition is administered two or more times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 times) during a period of time.

In some embodiments, the cell or the pharmaceutical composition is administered in a therapeutically-effective amount by various forms and routes including, for example, oral, or topical administration. In some embodiments, a composition may be administered by parenteral, intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intracerebral, subarachnoid, intraocular, intrasternal, ophthalmic, endothelial, local, intranasal, intrapulmonary, rectal, intraarterial, intrathecal, inhalation, intralesional, intradermal, epidural, intracapsular, subcapsular, intracardiac, transtracheal, subcuticular, subarachnoid, or intraspinal administration, e.g., injection or infusion. In some embodiments, a composition may be administered by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa administration) . In some embodiments, the composition is delivered via multiple administration routes.

In some embodiments, the cell or the pharmaceutical composition is administered by intravenous infusion. In some embodiments, the composition is administered by slow continuous infusion over a long period, such as more than 24 hours. In some embodiments, the composition is administered as an intravenous injection or a short infusion.

Acell or a pharmaceutical composition may be administered in a local manner, for example, via injection of the agent directly into an organ, optionally in a depot or sustained release formulation or implant. A composition may be provided in the form of a rapid release formulation, in the form of an extended release formulation, or in the form of an intermediate release formulation. A rapid release form may provide an immediate release. An extended release formulation may provide a controlled release or a sustained delayed release. In some embodiments, a pump may be used for delivery of the composition. In some embodiments, a pen delivery device may be used, for example, for subcutaneous delivery of a composition of the disclosure.

A cell or a pharmaceutical composition provided herein may be administered in conjunction with other therapies, for example, an antiviral therapy, a chemotherapy, an antibiotic, a cell therapy, a cytokine therapy, or an anti-inflammatory agent. In some embodiments, a circular polyribonucleotide or the antibody or the antigen-binding fragment thereof described herein may be used singly or in combination with one or more therapeutic agents as a component of mixtures. In some embodiments, a linear polyribonucleotide or the antibody or the antigen-binding fragment thereof described herein may be used singly or in combination with one or more therapeutic agents as a component of mixtures.

A cell or a pharmaceutical composition may be administered before, during, or after the occurrence of a disease or condition, and the timing of administering the composition containing a therapeutic agent may vary. In some cases, the cell or the pharmaceutical composition may be used as a prophylactic and may be administered continuously to subjects (e.g., the subject for immunization or the subject for treatment) with a susceptibility to an infection by a pathogen or a propensity to a condition or disease associated with the pathogen. Prophylactic administration may lessen a likelihood of the occurrence of the infection, disease or condition, or may decrease the severity of the infection, disease or condition.

A cell or a pharmaceutical composition may be administered to a subject before the onset of the symptoms. The composition may be administered to a subject (e.g., the subject for immunization or the subject for treatment) after (e.g., as soon as possible after) a test result, for example, a test result that provides a diagnosis, a test that shows the presence of a coronavirus in a subject (e.g., the subject for immunization or the subject for treatment) , or a test showing progress of a condition, e.g., a decreased blood oxygen levels. A therapeutic agent may be administered after (e.g., as soon as is practicable after) the onset of a disease or condition is detected or suspected. A therapeutic agent may be administered after (e.g., as soon as is practicable after) a potential exposure to a coronavirus, for example, after a subject (e.g., the subject for immunization or the subject for treatment) has contact with an infected subject, or learns they had contact with an infected subject that may be contagious.

Actual dosage levels of an agent of the disclosure (e.g., the cell or the pharmaceutical composition) may be varied so as to obtain an amount of the agent to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject (e.g., the subject for immunization or the subject for treatment) . The selected dosage level may depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic and/or prophylactic response) . For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally decreased or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects (e.g., the subjects for immunization or the subjects for treatment) ; each unit contains a predetermined quantity of active agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure may be determined by and directly dependent on (a) the unique characteristics of the active agent and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active agent for the treatment of sensitivity in individuals. A dose may be determined by reference to a plasma concentration or a local concentration of the circular polyribonucleotide or antibody or antigen-binding fragment thereof. A dose may be determined by reference to a plasma concentration or a local concentration of the linear polyribonucleotide or antibody or antigen-binding fragment thereof.

A cell or a pharmaceutical composition described herein may be in a unit dosage form suitable for a single administration of a precise dosage. In unit dosage form, the formulation may be divided into unit doses containing appropriate quantities of the compositions. In unit dosage form, the formulation may be divided into unit doses containing appropriate quantities of one or more linear polyribonucleotides, antibodies or the antigen-binding fragments thereof, and/or therapeutic agents. The unit dosage may be in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged injectables, vials, and ampoules. An aqueous suspension composition disclosed herein may be packaged in a single-dose non-reclosable container. Multiple-dose reclosable containers may be used, for example, in combination with or without a preservative. A formulation for injection disclosed herein may be present in a unit dosage form, for example, in ampoules, or in multi dose containers with a preservative.

In some embodiments, a dose may be based on the number of the cells per kilogram of body weight of a subject. In some embodiments, a dose may be is administered in a dosage amount of between about 1000 cells/kg body weight to about 1000000000000 cells/kg body weight. about 1,000 cells/kg body weight to about 1,000,000,000,000 cells/kg body weight. In some embodiments, a dose may be is administered in a dosage amount of between about 1000 cells/kg body weight to about 1000000000000 cells/kg body weight. about 1,000 cells/kg body weight to about 10,000 cells/kg body weight, about 1,000 cells/kg body weight to about 100,000 cells/kg body weight, about 1,000 cells/kg body weight to about 1,000,000 cells/kg body weight, about 1,000 cells/kg body weight to about 10,000,000 cells/kg body weight, about 1,000 cells/kg body weight to about 100,000,000 cells/kg body weight, about 1,000 cells/kg body weight to about 1,000,000,000 cells/kg body weight, about 1,000 cells/kg body weight to about 10,000,000,000 cells/kg body weight, about 1,000 cells/kg body weight to about 100,000,000,000 cells/kg body weight, about 1,000 cells/kg body weight to about 1,000,000,000,000 cells/kg body weight, about 10,000 cells/kg body weight to about 100,000 cells/kg body weight, about 10,000 cells/kg body weight to about 1,000,000 cells/kg body weight, about 10,000 cells/kg body weight to about 10,000,000 cells/kg body weight, about 10,000 cells/kg body weight to about 100,000,000 cells/kg body weight, about 10,000 cells/kg body weight to about 1,000,000,000 cells/kg body weight, about 10,000 cells/kg body weight to about 10,000,000,000 cells/kg body weight, about 10,000 cells/kg body weight to about 100,000,000,000 cells/kg body weight, about 10,000 cells/kg body weight to about 1,000,000,000,000 cells/kg body weight, about 100,000 cells/kg body weight to about 1,000,000 cells/kg body weight, about 100,000 cells/kg body weight to about 10,000,000 cells/kg body weight, about 100,000 cells/kg body weight to about 100,000,000 cells/kg body weight, about 100,000 cells/kg body weight to about 1,000,000,000 cells/kg body weight, about 100,000 cells/kg body weight to about 10,000,000,000 cells/kg body weight, about 100,000 cells/kg body weight to about 100,000,000,000 cells/kg body weight, about 100,000 cells/kg body weight to about 1,000,000,000,000 cells/kg body weight, about 1,000,000 cells/kg body weight to about 10,000,000 cells/kg body weight, about 1,000,000 cells/kg body weight to about 100,000,000 cells/kg body weight, about 1,000,000 cells/kg body weight to about 1,000,000,000 cells/kg body weight, about 1,000,000 cells/kg body weight to about 10,000,000,000 cells/kg body weight, about 1,000,000 cells/kg body weight to about 100,000,000,000 cells/kg body weight, about 1,000,000 cells/kg body weight to about 1,000,000,000,000 cells/kg body weight, about 10,000,000 cells/kg body weight to about 100,000,000 cells/kg body weight, about 10,000,000 cells/kg body weight to about 1,000,000,000 cells/kg body weight, about 10,000,000 cells/kg body weight to about 10,000,000,000 cells/kg body weight, about 10,000,000 cells/kg body weight to about 100,000,000,000 cells/kg body weight, about 10,000,000 cells/kg body weight to about 1,000,000,000,000 cells/kg body weight, about 100,000,000 cells/kg body weight to about 1,000,000,000 cells/kg body weight, about 100,000,000 cells/kg body weight to about 10,000,000,000 cells/kg body weight, about 100,000,000 cells/kg body weight to about 100,000,000,000 cells/kg body weight, about 100,000,000 cells/kg body weight to about 1,000,000,000,000 cells/kg body weight, about 1,000,000,000 cells/kg body weight to about 10,000,000,000 cells/kg body weight, about 1,000,000,000 cells/kg body weight to about 100,000,000,000 cells/kg body weight, about 1,000,000,000 cells/kg body weight to about 1,000,000,000,000 cells/kg body weight, about 10,000,000,000 cells/kg body weight to about 100,000,000,000 cells/kg body weight, about 10,000,000,000 cells/kg body weight to about 1,000,000,000,000 cells/kg body weight, or about 100,000,000,000 cells/kg body weight to about 1,000,000,000,000 cells/kg body weight. In some embodiments, a dose may be is administered in a dosage amount of between about 1000 cells/kg body weight to about 1000000000000 cells/kg body weight. about 1,000 cells/kg body weight, about 10,000 cells/kg body weight, about 100,000 cells/kg body weight, about 1,000,000 cells/kg body weight, about 10,000,000 cells/kg body weight, about 100,000,000 cells/kg body weight, about 1,000,000,000 cells/kg body weight, about 10,000,000,000 cells/kg body weight, about 100,000,000,000 cells/kg body weight, or about 1,000,000,000,000 cells/kg body weight. In some embodiments, a dose may be is administered in a dosage amount of between about 1000 cells/kg body weight to about 1000000000000 cells/kg body weight. at least about 1,000 cells/kg body weight, about 10,000 cells/kg body weight, about 100,000 cells/kg body weight, about 1,000,000 cells/kg body weight, about 10,000,000 cells/kg body weight, about 100,000,000 cells/kg body weight, about 1,000,000,000 cells/kg body weight, about 10,000,000,000 cells/kg body weight, or about 100,000,000,000 cells/kg body weight. In some embodiments, a dose may be is administered in a dosage amount of between about 1000 cells/kg body weight to about 1000000000000 cells/kg body weight. at most about 10,000 cells/kg body weight, about 100,000 cells/kg body weight, about 1,000,000 cells/kg body weight, about 10,000,000 cells/kg body weight, about 100,000,000 cells/kg body weight, about 1,000,000,000 cells/kg body weight, about 10,000,000,000 cells/kg body weight, about 100,000,000,000 cells/kg body weight, or about 1,000,000,000,000 cells/kg body weight. In some embodiments, the cell without the nucleus is administered to the subject twice within at least an hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 1 day, 2 days, a week, 2 weeks, 3 weeks, a month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, a year, 2 years, 3 years, or 4 years.

In some embodiments, the method described herein treats a disease or condition in a subject, there the method comprises administering the cell or the pharmaceutical composition descried herein without triggering innate immune response or GVHD in the subject being treated. In somebodies, the cell or the pharmaceutical composition treats a disease or condition in a subject in need thereof, where the disease or condition is cancer. Non-limiting example of cancer includes Acute Lymphoblastic Leukemia, Acute Lymphocytic Leukemia (ALL) , Acute Myeloid Leukemia (AML) , Adenoid Cystic Carcinoma, Adrenal Gland Cancer, Adrenocortical Carcinoma, Adult Leukemia, AIDS-Related Lymphoma, Amyloidosis, Anal Cancer, Astrocytomas, Ataxia Telangiectasia, Atypical Mole Syndrome, Atypical Teratoid/Rhabdoid Tumor, Basal Cell Carcinoma, Bile Duct Cancer, Birt Hogg Dube Syndrome, Bladder Cancer, Bone Cancer, Brain Tumor, Breast Cancer, Bronchial Tumors, Burkitt Lymphoma, Carcinoid Tumor (Gastrointestinal) , Carcinoma of Unknown Primary, Cardiac (Heart) Tumors, Cervical Cancer, Cholangiocarcinoma, Chordoma, Chronic Lymphocytic Leukemia (CLL) , Chronic Myelogenous Leukemia, Chronic Myeloid Leukemia, Chronic Myeloproliferative Neoplasms, Colorectal Cancer, Craniopharyngioma, Cutaneous T-Cell Lymphoma, Ductal Carcinoma, Embryonal Tumors, Endometrial Cancer, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma, Ewing Sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Eye Cancer, Fallopian Tube Cancer, Fibrous Histiocytoma of Bone, Malignant, and Osteosarcoma, Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carcinoid Tumor, Gastrontestinal Stromal Tumor (GIST) , Germ Cell Tumors, Gestational Trophoblastic Disease, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular Cancer, HER2-Positive Breast Cancer, Histiocytosis, Langerhans Cell, Hodgkin's Lymphoma, Hypopharyngeal Cancer, Intraocular Melanoma, Islet Cell Tumor, Juvenile Polyposis Syndrome, Kaposi Sarcoma, Kidney Cancer, Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukemia, Lip and Oral Cavity Cancer, Liver Cancer, Lobular Carcinoma, Lung Cancer (Non-Small Cell and Small Cell) , Lymphoma, Malignant Fibrous Histiocytoma of Bone and Osteosarcoma, Malignant Glioma, Melanoma, Intraocular Melanoma, Meningioma, Merkel Cell Carcinoma, Mesothelioma, Malignant, Metastatic Cancer, Metastatic Squamous Neck Cancer with Occult Primary, Midline Tract Carcinoma, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma, Plasma Cell Neoplasms, Mycosis Fungoides, Myelodysplastic Syndrome (MDS) , Myeloproliferative Neoplasms, Chronic, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Neuroendocrine Tumor, Non-Hodgkin Lymphoma, Oral Cancer, Lip and Oral Cavity Cancer and Oropharyngeal Cancer, Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, Ovarian Germ Cell Tumors, Pancreatic Cancer, Pancreatic Neuroendocrine Tumors, Papillomatosis, Paraganglioma, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Peritoneal Cancer, Peutz-Jeghers Syndrome, Pharyngeal Cancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Pleuropulmonary Blastoma, Polycythemia Vera, Pregnancy and Breast Cancer, Primary Central Nervous System (CNS) Lymphoma, Primary Peritoneal Cancer, Prostate Cancer, Rectal Cancer, Recurrent Cancer, Renal Cell Carcinoma, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma, Sézary Syndrome, Skin Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Solid tumor, Squamous Cell Carcinoma of the Skin, Squamous Neck Cancer with Occult Primary, Metastatic, Stomach Cancer, T-Cell Lymphoma, Testicular Cancer, Throat Cancer, Thymoma, Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Unusual Cancers of Childhood, Ureter and Renal Pelvis, Transitional Cell Cancer, Urethral Cancer, Uterine (Endometrial) Cancer, Uterine Sarcoma, Vaginal Cancer, Vascular Tumors, Vulvar Cancer, Wilms Tumor, or a combination thereof.

In some embodiments, the cell described herein (e.g., the modified cell with the at least one endogenous gene knocked out for decreasing the expressing of the at least MHC in the cell) can target a cancer cell or a tumor cell for treating the disease or condition in the subject. Non-limiting examples of cancer cell or tumor cell, as used in the present disclosure, may include cell of cancer including Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma, Anal cancer, Anaplastic large cell lymphoma, Anaplastic thyroid cancer, Angioimmunoblastic T-cell lymphoma, Angiomyolipoma, Angiosarcoma, Appendix cancer, Astrocytoma, Atypical teratoid rhabdoid tumor, Basal cell carcinoma, Basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, Biliary tract cancer, Bladder cancer, Blastoma, Bone Cancer, Bone tumor, Brain Stem Glioma, Brain Tumor, Breast Cancer, Brenner tumor, Bronchial Tumor, Bronchioloalveolar carcinoma, Brown tumor, Burkitt's lymphoma, Cancer of Unknown Primary Site, Carcinoid Tumor, Carcinoma, Carcinoma in situ, Carcinoma of the penis, Carcinoma of Unknown Primary Site, Carcinosarcoma, Castleman's Disease, Central Nervous System Embryonal Tumor, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Cholangiocarcinoma, Chondroma, Chondrosarcoma, Chordoma, Choriocarcinoma, Choroid plexus papilloma, Chronic Lymphocytic Leukemia, Chronic monocytic leukemia, Chronic myelogenous leukemia, Chronic Myeloproliferative Disorder, Chronic neutrophilic leukemia, Clear-cell tumor, Colon Cancer, Colorectal cancer, Craniopharyngioma, Cutaneous T-cell lymphoma, Degos disease, Dermatofibrosarcoma protuberans, Dermoid cyst, Desmoplastic small round cell tumor, Diffuse large B cell lymphoma, Dysembryoplastic neuroepithelial tumor, Embryonal carcinoma, Endodermal sinus tumor, Endometrial cancer, Endometrial Uterine Cancer, Endometrioid tumor, Enteropathy-associated T-cell lymphoma, Ependymoblastoma, Ependymoma, Epithelioid sarcoma, Erythroleukemia, Esophageal cancer, Esthesioneuroblastoma, Ewing Family of Tumor, Ewing Family Sarcoma, Ewing's sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Extramammary Paget's disease, Fallopian tube cancer, Fetus in fetu, Fibroma, Fibrosarcoma, Follicular lymphoma, Follicular thyroid cancer, Gallbladder Cancer, Gallbladder cancer, Ganglioglioma, Ganglioneuroma, Gastric Cancer, Gastric lymphoma, Gastrointestinal cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor, Gastrointestinal stromal tumor, Germ cell tumor, Germinoma, Gestational choriocarcinoma, Gestational Trophoblastic Tumor, Giant cell tumor of bone, Glioblastoma multiforme, Glioma, Gliomatosis cerebri, Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell tumor, Hairy Cell Leukemia, Hairy cell leukemia, Head and Neck Cancer, Head and neck cancer, Heart cancer, Hemangioblastoma, Hemangiopericytoma, Hemangiosarcoma, Hematological malignancy, Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary breast-ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin's lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory breast cancer, Intraocular Melanoma, Islet cell carcinoma, Islet Cell Tumor, Juvenile myelomonocytic leukemia, Kaposi Sarcoma, Kaposi's sarcoma, Kidney Cancer, Klatskin tumor, Krukenberg tumor, Laryngeal Cancer, Laryngeal cancer, Lentigo maligna melanoma, Leukemia, Leukemia, Lip and Oral Cavity Cancer, Liposarcoma, Lung cancer, Luteoma, Lymphangioma, Lymphangiosarcoma, Lymphoepithelioma, Lymphoid leukemia, Lymphoma, Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant fibrous histiocytoma, Malignant Fibrous Histiocytoma of Bone, Malignant Glioma, Malignant Mesothelioma, Malignant peripheral nerve sheath tumor, Malignant rhabdoid tumor, Malignant triton tumor, MALT lymphoma, Mantle cell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor, Mediastinal tumor, Medullary thyroid cancer, Medulloblastoma, Medulloblastoma, Medulloepithelioma, Melanoma, Melanoma, Meningioma, Merkel Cell Carcinoma, Mesothelioma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Metastatic urothelial carcinoma, Mixed Mullerian tumor, Monocytic leukemia, Mouth Cancer, Mucinous tumor, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma, Multiple myeloma, Mycosis Fungoides, Mycosis fungoides, Myelodysplastic Disease, Myelodysplastic Syndromes, Myeloid leukemia, Myeloid sarcoma, Myeloproliferative Disease, Myxoma, Nasal Cavity Cancer, Nasopharyngeal Cancer, Nasopharyngeal carcinoma, Neoplasm, Neurinoma, Neuroblastoma, Neuroblastoma, Neurofibroma, Neuroma, Nodular melanoma, Non-Hodgkin Lymphoma, Non-Hodgkin lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Ocular oncology, Oligoastrocytoma, Oligodendroglioma, Oncocytoma, Optic nerve sheath meningioma, Oral Cancer, Oral cancer, Oropharyngeal Cancer, Osteosarcoma, Osteosarcoma, Ovarian Cancer, Ovarian cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Paget's disease of the breast, Pancoast tumor, Pancreatic Cancer, Pancreatic cancer, Papillary thyroid cancer, Papillomatosis, Paraganglioma, Paranasal Sinus Cancer, Parathyroid Cancer, Penile Cancer, Perivascular epithelioid cell tumor, Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumor of Intermediate Differentiation, Pineoblastoma, Pituicytoma, Pituitary adenoma, Pituitary tumor, Plasma Cell Neoplasm, Pleuropulmonary blastoma, Polyembryoma, Precursor T-lymphoblastic lymphoma, Primary central nervous system lymphoma, Primary effusion lymphoma, Primary Hepatocellular Cancer, Primary Liver Cancer, Primary peritoneal cancer, Primitive neuroectodermal tumor, Prostate cancer, Pseudomyxoma peritonei, Rectal Cancer, Renal cell carcinoma, Respiratory Tract Carcinoma Involving the NUT Gene on Chromosome 15, Retinoblastoma, Rhabdomyoma, Rhabdomyosarcoma, Richter's transformation, Sacrococcygeal teratoma, Salivary Gland Cancer, Sarcoma, Schwannomatosis, Sebaceous gland carcinoma, Secondary neoplasm, Seminoma, Serous tumor, Sertoli-Leydig cell tumor, Sex cord-stromal tumor, Sezary Syndrome, Solid tumor, Signet ring cell carcinoma, Skin Cancer, Small blue round cell tumor, Small cell carcinoma, Small Cell Lung Cancer, Small cell lymphoma, Small intestine cancer, Soft tissue sarcoma, Somatostatinoma, Soot wart, Spinal Cord Tumor, Spinal tumor, Splenic marginal zone lymphoma, Squamous cell carcinoma, Stomach cancer, Superficial spreading melanoma, Supratentorial Primitive Neuroectodermal Tumor, Surface epithelial-stromal tumor, Synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocyte leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, Teratoma, Terminal lymphatic cancer, Testicular cancer, Thecoma, Throat Cancer, Thymic Carcinoma, Thymoma, Thyroid cancer, Transitional Cell Cancer of Renal Pelvis and Ureter, Transitional cell carcinoma, Urachal cancer, Urethral cancer, Urogenital neoplasm, Uterine sarcoma, Uveal melanoma, Vaginal Cancer, Verner Morrison syndrome, Verrucous carcinoma, Visual Pathway Glioma, Vulvar Cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms'tumor, and combinations thereof. In some embodiments, the targeted cancer cell represents a subpopulation within a cancer cell population, such as a cancer stem cell. In some embodiments, the cancer is of a hematopoietic lineage, such as a lymphoma.

Pharmaceutical compositions

Described herein is a pharmaceutical composition comprising a therapeutic agent (e.g., the cell described herein) . In some aspects, the cell comprises decreased endogenous gene expression of the at least one gene described herein and decreased expression of at least one MHC. In some aspects, the cell comprises a heterologous polypeptide (e.g., a CAR described herein) . In some aspects, the cell comprises an immune cell or a stem cell such as induced pluripotent stem cell (iPSC) , adult stem cells, mesenchymal stromal cells, embryonic stem cells, fibroblasts, or immortalized cells from a cell line, or a combination thereof. In some aspects, the cell is: obtained from a subject; expanded or modified by the method described herein in an in vitro environment; and administer back to the subject for treating a disease or condition in the subject. In some aspects, the cell is obtained from a source that is not from the subject. In some aspect, the cell is obtained from a cell line.

In some aspects, the pharmaceutical composition comprises a pharmaceutically acceptable: carrier, excipient, or diluent. In some aspects, the pharmaceutical composition described herein includes at least one additional active agent other than the cell described herein. In some aspects, the at least one additional active agent is a chemotherapeutic agent, cytotoxic agent, cytokine, growth-inhibitory agent, anti-hormonal agent, anti-angiogenic agent, or checkpoint inhibitor.

In practicing the methods of treatment or use provided herein, therapeutically effective amount of pharmaceutical composition described herein is administered to a mammal having a disease, disorder, or condition to be treated, e.g., cancer. In some aspects, the mammal is a human. A therapeutically effective amount may vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the therapeutic agent used and other factors. The therapeutic agents, and in some cases, compositions described herein, may be used singly or in combination with one or more therapeutic agents as components of mixtures.

The pharmaceutical composition described herein may be administered to a subject by appropriate administration routes, including but not limited to, intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal administration routes. The composition described herein may include, but not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.

The pharmaceutical composition including a therapeutic agent may be manufactured in a conventional manner, such as, by way of example only, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.

The pharmaceutical composition may include at least an exogenous therapeutic agent as an active ingredient in free-acid or free-base form, or in a pharmaceutically acceptable salt form. In addition, the methods and compositions described herein include the use of N-oxides (if appropriate) , crystalline forms, amorphous phases, as well as active metabolites of these compounds having the same type of activity. In some aspects, therapeutic agents exist in unsolvated form or in solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. The solvated forms of the therapeutic agents are also considered to be disclosed herein.

In certain embodiments, the pharmaceutical composition provided herein includes one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

In some aspects, pharmaceutical composition described herein benefits from antioxidants, metal chelating agents, thiol containing compounds and other general stabilizing agents. Examples of such stabilizing agents, include, but are not limited to: (a) about 0.5%to about 2%w/v glycerol, (b) about 0.1%to about 1%w/v methionine, (c) about 0.1%to about 2%w/v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, I about 0.01%to about 2%w/v ascorbic acid, (f) 0.003%to about 0.02%w/v polysorbate 80, (g) 0.001%to about 0.05%w/v. polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.

The pharmaceutical composition described herein can be formulated into any suitable dosage form, including but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations. In one aspect, a therapeutic agent as discussed herein, e.g., therapeutic agent is formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous, or intravenous injection. In one aspect, formulations suitable for intramuscular, subcutaneous, or intravenous injection include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for rehydration into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (propyleneglycol, polyethylene-glycol, glycerol, cremophor and the like) , suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In some aspects, formulations suitable for subcutaneous injection also contain additives such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the growth of microorganisms may be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. In some cases, it is desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the use of agents delaying absorption, such as aluminum monostearate and gelatin.

For intravenous injections or drips or infusions, a pharmaceutical composition described herein is formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. For other parenteral injections, appropriate formulations include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients.

Parenteral injections may involve bolus injection or continuous infusion. Pharmaceutical composition for injection may be presented in unit dosage form, e.g., in ampoules or in multi dose containers, with an added preservative. The pharmaceutical composition described herein may be in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. In one aspect, the active ingredient is in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

For administration by inhalation, a therapeutic agent is formulated for use as an aerosol, a mist or a powder. Pharmaceutical compositions described herein are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or nebulizers, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, such as, by way of example only, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the therapeutic agent described herein and a suitable powder base such as lactose or starch. Formulations that include a composition are prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. Preferably these compositions and formulations are prepared with suitable nontoxic pharmaceutically acceptable ingredients. The choice of suitable carriers is dependent upon the exact nature of the nasal dosage form desired, e.g., solutions, suspensions, ointments, or gels. Nasal dosage forms generally contain large amounts of water in addition to the active ingredient. Minor amounts of other ingredients such as pH adjusters, emulsifiers or dispersing agents, preservatives, surfactants, gelling agents, or buffering and other stabilizing and solubilizing agents are optionally present. Preferably, the nasal dosage form should be isotonic with nasal secretions.

Pharmaceutical preparation for oral use is obtained by mixing one or more solid excipient with one or more of the compositions described herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. If desired, disintegrating agents are added, such as the cross linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. In some aspects, dyestuffs or pigments are added to the tablets or dragee coatings for identification or to characterize different combinations of active therapeutic agent doses.

In some aspects, the pharmaceutical composition of the exogenous therapeutic agents is in the form of a capsules, including push fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push fit capsules contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active therapeutic agent is dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In some aspects, stabilizers are added. A capsule may be prepared, for example, by placing the bulk blend of the formulation of the therapeutic agent inside of a capsule. In some aspects, the formulations (non-aqueous suspensions and solutions) are placed in a soft gelatin capsule. In other embodiments, the formulations are placed in standard gelatin capsules or non-gelatin capsules such as capsules comprising HPMC. In other embodiments, the formulation is placed in a sprinkle capsule, wherein the capsule is swallowed whole or the capsule is opened and the contents sprinkled on food prior to eating.

Pharmaceutical composition for oral administration can be in dosages suitable for such administration. In one aspect, solid oral dosage forms are prepared by mixing a composition with one or more of the following: antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents. In some aspects, the solid dosage forms disclosed herein are in the form of a tablet, (including a suspension tablet, a fast-melt tablet, a bite-disintegration tablet, a rapid-disintegration tablet, an effervescent tablet, or a caplet) , a pill, a powder, a capsule, solid dispersion, solid solution, bioerodible dosage form, controlled release formulations, pulsatile release dosage forms, multiparticulate dosage forms, beads, pellets, granules. In other embodiments, the composition is in the form of a powder. Compressed tablets are solid dosage forms prepared by compacting the bulk blend of the formulations described above. In various embodiments, tablets will include one or more flavoring agents. In other embodiments, the tablets will include a film surrounding the final compressed tablet. In some aspects, the film coating may provide a delayed release of a therapeutic agent from the formulation. In other embodiments, the film coating aids in patient compliance. Film coatings typically range from about 1%to about 3%of the tablet weight. In some aspects, solid dosage forms, e.g., tablets, effervescent tablets, and capsules, are prepared by mixing particles of a therapeutic agent with one or more pharmaceutical excipients to form a bulk blend composition. The bulk blend is readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. In some aspects, the individual unit dosages include film coatings. These formulations are manufactured by conventional formulation techniques.

In another aspect, dosage forms include microencapsulated formulations. In some aspects, one or more other compatible materials are present in the microencapsulation material. Non-limiting example of materials includes pH modifiers, erosion facilitators, anti-foaming agents, antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.

Liquid formulation dosage forms for oral administration are optionally aqueous suspensions selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups. In addition to therapeutic agent the liquid dosage forms optionally include additives, such as: (a) disintegrating agents; (b) dispersing agents; (c) wetting agents; (d) at least one preservative, (e) viscosity enhancing agents, (f) at least one sweetening agent, and (g) at least one flavoring agent. In some aspects, the aqueous dispersions further include a crystal-forming inhibitor.

In some aspects, the pharmaceutical composition described herein can be self-emulsifying drug delivery systems (SEDDS) . Emulsions are dispersions of one immiscible phase in another, usually in the form of droplets. Generally, emulsions are created by vigorous mechanical dispersion. SEDDS, as opposed to emulsions or microemulsions, spontaneously form emulsions when added to an excess of water without any external mechanical dispersion or agitation. An advantage of SEDDS is that only gentle mixing is required to distribute the droplets throughout the solution. Additionally, water or the aqueous phase is optionally added just prior to administration, which ensures stability of an unstable or hydrophobic active ingredient. Thus, the SEDDS provides an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. In some aspects, SEDDS provides improvements in the bioavailability of hydrophobic active ingredients.

Buccal formulations are administered using a variety of formulations known in the art. In addition, the buccal dosage forms described herein may further include a bioerodible (hydrolysable) polymeric carrier that also serves to adhere the dosage form to the buccal mucosa. For buccal or sublingual administration, the compositions may take the form of tablets, lozenges, or gels formulated in a conventional manner.

For intravenous injections, a pharmaceutical composition is optionally formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. For other parenteral injections, appropriate formulations include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients.

Parenteral injections optionally involve bolus injection or continuous infusion. Formulations for injection are optionally presented in unit dosage form, e.g., in ampoules or in multi dose containers, with an added preservative. In some aspects, a composition described herein is in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents. The compositions for parenteral administration include aqueous solutions of an agent that modulates the activity of a carotid body in water soluble form. Additionally, suspensions of an agent that modulates the activity of a carotid body are optionally prepared as appropriate, e.g., oily injection suspensions.

In some aspects, the pharmaceutical composition can be provided that include particles of a therapeutic agent and at least one dispersing agent or suspending agent for oral administration to a subject. The formulations may be a powder and/or granule for suspension, and upon admixture with water, a substantially uniform suspension is obtained.

Furthermore, the pharmaceutical composition optionally includes one or more pH adjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.

Additionally, the pharmaceutical composition optionally includes one or more salts in an amount required to bring osmolality of the pharmaceutical composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

In one embodiment, the aqueous suspensions and dispersions described herein remain in a homogenous state for at least 4 hours. In one embodiment, an aqueous suspension is re-suspended into a homogenous suspension by physical agitation lasting less than 1 minute. In still another embodiment, no agitation is necessary to maintain a homogeneous aqueous dispersion. Kits

Described herein, in some aspects, are kits for using the cell or for practicing the method described herein. In some aspects, the kits disclosed herein may be used to treat a disease or condition in a subject. In some aspects, the kits comprise an assemblage of materials or components apart from the cell.

In some aspects, the kit comprises the components for assaying the number of units of a biomolecule (e.g., a therapeutic agent) synthesized, and/or released or expressed on the surface by the cell described herein. In some aspects, the kit comprises components for performing assays such as enzyme-linked immunosorbent assay (ELISA) , single-molecular array (Simoa) , PCR, and qPCR. The exact nature of the components configured in the kit depends on its intended purpose. For example, some embodiments are configured for the purpose of treating a disease or condition disclosed herein (e.g., cancer) in a subject. In some aspects, the kit is configured particularly for the purpose of treating mammalian subjects. In some aspects, the kit is configured particularly for the purpose of treating human subjects.

Instructions for use may be included in the kit. In some aspects, the kit comprises instructions for administering the cell to a subject in need thereof. In some aspects, the kit comprises instructions for further engineering the composition to express a biomolecule (e.g., a therapeutic agent) . In some aspects, the kit comprises instructions thawing or otherwise restoring biological activity of the cell, which may have been preserved during storage or transportation. In some aspects, the kit comprises instructions for measure viability of the preserved cell, to ensure efficacy for its intended purpose (e.g., therapeutic efficacy if used for treating a subject) .

Optionally, the kit also contains other useful components, such as, diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandaging materials or other useful paraphernalia. The materials or components assembled in the kit may be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example, the components may be in dissolved, dehydrated, or lyophilized form; they may be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material (s) .

Use of absolute or sequential terms, for example, “will, ” “will not, ” “shall, ” “shall not, ” “must, ” “must not, ” “first, ” “initially, ” “next, ” “subsequently, ” “before, ” “after, ” “lastly, ” and “finally, ” are not meant to limit scope of the present embodiments disclosed herein but as exemplary.

As used herein, the singular forms “a” , “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including” , “includes” , “having” , “has” , “with” , or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising. ”

As used herein, the phrases “at least one” , “one or more” , and “and/or” are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions “at least one of A, B and C” , “at least one of A, B, or C” , “one or more of A, B, and C” , “one or more of A, B, or C” and “A, B, and/or C” means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.

As used herein, “or” may refer to “and” , “or, ” or “and/or” and may be used both exclusively and inclusively. For example, the term “A or B” may refer to “A or B” , “A but not B” , “B but not A” , and “A and B” . In some cases, context may dictate a particular meaning.

Any systems, methods, software, and platforms described herein are modular. Accordingly, terms such as “first” and “second” do not necessarily imply priority, order of importance, or order of acts.

The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error) , and the number or numerical range may vary from, for example, from 1%to 15%of the stated number or numerical range. In examples, the term “about” refers to ±10%of a stated number or value.

The terms “increased” , “increasing” , or “increase” are used herein to generally mean an increase by a statically significant amount. In some aspects, the terms “increased, ” or “increase, ” mean an increase of at least 10%as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%or up to and including a 100%increase or any increase between 10-100%as compared to a reference level, standard, or control. Other examples of “increase” include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level.

The terms “decreased” , “decreasing” , or “decrease” are used herein generally to mean a decrease by a statistically significant amount. In some aspects, “decreased” or “decrease” means a reduction by at least 10%as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%or up to and including a 100%decrease (e.g., absent level or non-detectable level as compared to a reference level) , or any decrease between 10-100%as compared to a reference level. In the context of a marker or symptom, by these terms is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40%or more, and is preferably down to a level accepted as within the range of normal for an individual without a given disease.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

EMBODIMENTS

The following non-limiting embodiments provide illustrative examples of the invention, but do not limit the scope of the invention.

Embodiment 1. A cell comprising: at least one genetic modification, where the at least one genetic modification decreases an endogenous expression of SUGT-1 in the cell; and a heterologous polypeptide.

Embodiment 2. The cell of Embodiment 1, wherein the at least one genetic modification decreases an endogenous expression of a nucleotide-binding oligomerization domain-like receptors (NLR) in the cell.

Embodiment 3. The cell of any one of the preceding Embodiments, wherein the NLR comprises a NLRC.

Embodiment 4. The cell of any one of the preceding Embodiments, wherein the NLRC comprises a NOD1, NOD2, NLRC3, NLRC4, or NLRC5.

Embodiment 5. The cell of any one of the preceding Embodiments, wherein the NLRC comprises the NLRC5.

Embodiment 6. The cell of any one of the preceding Embodiments, wherein the at least one genetic modification decreases an endogenous expression of an MHC class II regulatory factor in the cell.

Embodiment 7. The cell of any one of the preceding Embodiments, wherein the MHC class II regulatory factor comprises RFX1, RFX2, RFX3, RFX4, or RFX5.

Embodiment 8. The cell of any one of the preceding Embodiments, wherein the MHC class II regulatory factor comprises RFX5.

Embodiment 9. The cell of any one of the preceding Embodiments, wherein the MHC class II regulatory factor comprises CIITA, CREB, or nuclear transcription factor Y (NF-Y) .

Embodiment 10. A cell comprising: at least one genetic modification, where the at least one genetic modification decreases an endogenous expression of NLRC5 in the cell; and a heterologous polypeptide.

Embodiment 11. A cell comprising: at least one genetic modification, where the at least one genetic modification decreases an endogenous expression of RFX5 in the cell; and a heterologous polypeptide.

Embodiment 12. A cell comprising: at least one genetic modification, where the at least one genetic modification decreases an endogenous expression of CIITA in the cell; and a heterologous polypeptide.

Embodiment 13. The cell of any one of the preceding Embodiments, wherein the heterologous polypeptide comprises a heterologous receptor.

Embodiment 14. The cell of any one of the preceding Embodiments, wherein the heterologous receptor comprises an antigen binding domain.

Embodiment 15. The cell of any one of the preceding Embodiments, wherein the antigen binding domain binds to CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44v6, CD47, CD52, CD56, CD57, CD58, CD70, CD79a, CD79b, CD80, CD81, CD86, CD99, CD117, CD123, CD133, CD135, CD137, CD151, CD171, CD276, BAFF-R, BCMA, B7H4, CEA, CEACM6, Claudin18.2, CLL-1, c-Met, CS-1, CTLA-4, EGFRvIII, GPC2, GPC3, GPRC5, HER2, HER3, HER4/ErbB4, HVEM, MAGE-A, MAGE3, MSLN, MUC-1, MUC-16, NY-ESO-1, OX40, PD-1, PD-L1, PD-L2, PMSA, ROR1, TCRa, TCRb, TLR7, TLR9, VEGFR-2, or WT-1.

Embodiment 16. The cell of any one of the preceding Embodiments, wherein the heterologous receptor comprises a transmembrane domain.

Embodiment 17. The cell of any one of the preceding Embodiments, wherein the heterologous receptor comprises a signaling domain.

Embodiment 18. The cell of any one of the preceding Embodiments, wherein an endogenous expression of MHC-I of the cell is decreased compared to an endogenous expression of MHC-I of a comparable cell that does not have decreased expression of the endogenous gene.

Embodiment 19. The cell of any one of the preceding Embodiments, wherein the endogenous expression of MHC-I of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to the endogenous expression of MHC-I or the comparable cell that does not have an endogenous gene knocked out.

Embodiment 20. The cell of any one of the preceding Embodiments, wherein the endogenous expression of MHC-I of the cell is knocked out.

Embodiment 21. The cell of any one of the preceding Embodiments, wherein the endogenous expression of MHC-I of the cell is not knocked out.

Embodiment 22. The cell of any one of the preceding Embodiments, wherein the endogenous expression of MHC-I of the cell is decreased by at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 95%, or at most 99%compared to the endogenous expression of MHC-I or the comparable cell that does not have an endogenous gene knocked out.

Embodiment 23. The cell of any one of the preceding Embodiments, wherein an endogenous expression of MHC-II of the cell is decreased compared to an endogenous expression of MHC-II of a comparable cell that does not have an endogenous gene knocked out.

Embodiment 24. The cell of any one of the preceding Embodiments, wherein the endogenous expression of MHC-II of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to the endogenous expression of MHC-II or the comparable cell that does not have an endogenous gene knocked out.

Embodiment 25. The cell of any one of the preceding Embodiments, wherein the endogenous expression of MHC-II of the cell is abrogated.

Embodiment 26. The cell of any one of the preceding Embodiments, wherein the cell does not increase NK cell kill activity compared to a comparable cell that does not have an endogenous gene knocked out.

Embodiment 27. The cell of any one of the preceding Embodiments, wherein the cell comprises an immune cell or a stem cell.

Embodiment 28. The cell of any one of the preceding Embodiments, wherein the immune cell is a lymphocyte.

Embodiment 29. The cell of any one of the preceding Embodiments, wherein the lymphocyte is a B cell.

Embodiment 30. The cell of any one of the preceding Embodiments, wherein the lymphocyte is a T cell.

Embodiment 31. The cell of any one of the preceding Embodiments, wherein the T cell is selected from the group consisting of: cytotoxic T cell, natural killer T cell, regulatory T cell, and T helper cell.

Embodiment 32. The cell of any one of the preceding Embodiments, wherein the immune cell comprises an innate lymphoid cell (ILC) .

Embodiment 33. The cell of any one of the preceding Embodiments, wherein the immune cell is derived from an induced pluripotent stem cell (iPSC) .

Embodiment 34. The cell of any one of the preceding Embodiments, wherein the immune cell is an induced pluripotent stem cell derived T cell.

Embodiment 35. The cell of any one of the preceding Embodiments, wherein the immune cell is an induced pluripotent stem cell derived natural killer T cell.

Embodiment 36. The cell of any one of the preceding Embodiments, wherein the immune cell is an iPSC derived macrophage.

Embodiment 37. The cell of any one of the preceding Embodiments, wherein the stem cell is (i) a hematopoietic stem cell or (ii) an induced pluripotent stem cell (iPSC) .

Embodiment 38. The cell of any one of the preceding Embodiments, wherein:

(i) the heterologous polypeptide is a chimeric antigen receptor (CAR) ; and/or

(ii) the antigen binding domain does not bind to one or more members selected from the group consisting of CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44v6, CD47, CD52, CD56, CD57, CD58, CD70, CD79a, CD79b, CD80, CD81, CD86, CD99, CD117, CD123, CD133, CD135, CD137, CD151, CD171, CD276, BAFF-R, BCMA, B7H4, CEA, CEACM6, Claudin18.2, CLL-1, c-Met, CS-1, CTLA-4, EGFRvIII, GPC2, GPC3, GPRC5, HER2, HER3, HER4/ErbB4, HVEM, MAGE-A, MAGE3, MSLN, MUC-1, MUC-16, NY-ESO-1, OX40, PD-1, PD-L1, PD-L2, PMSA, ROR1, TCRa, TCRb, TLR7, TLR9, VEGFR-2, and WT-1,

optionally wherein the antigen binding domain does not bind to NY-ESO-1; and/or

(iii) the cell does not comprise a genetic modification in a gene encoding beta-2 microglobulin (B2M) .

Embodiment 39. A cell line comprising the cell of any one of the preceding Embodiments.

Embodiment 40. A composition comprising the cell of any one of the preceding Embodiments.

Embodiment 41. A pharmaceutical composition comprising: (i) the cell of any one of the preceding Embodiments, the cell line of Embodiment 39, or the composition of Embodiment 40; and (ii) a pharmaceutically acceptable: excipient, carrier, or diluent.

Embodiment 42. The pharmaceutical composition of Embodiment 41, wherein the pharmaceutical composition comprises a unit dose form.

Embodiment 43. The pharmaceutical composition of any one of the preceding Embodiments, wherein the pharmaceutical composition is formulated for administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, intratumorally, pulmonarily, endotracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, by inhalation, by inhaled nebulized form, by intraluminal-GI route, or a combination thereof to a subject in need thereof.

Embodiment 44. The pharmaceutical composition of any one of the preceding Embodiments, further comprising at least one additional active agent.

Embodiment 45. The pharmaceutical composition of any one of the preceding Embodiments, wherein the at least one additional active agent comprises a cytokine, a growth factor, a hormone, an enzyme, a small molecule, a compound, or combinations thereof.

Embodiment 46. A kit comprising: (i) the cell of any one of the preceding Embodiments, the cell line of Embodiment 39, the composition of Embodiment 40, or the pharmaceutical composition of any one of the preceding Embodiments; and (ii) a container.

Embodiment 47. A method for modifying a cell, comprising: contacting the cell with a gene modifying moiety, where the gene modifying moiety introduces at least one genetic modification in the cell, thereby decreasing an endogenous expression of SUGT-1 in the cell; and expressing a heterologous polypeptide in the cell.

Embodiment 48. The method of Embodiment 47, wherein the at least one genetic modification decreases an endogenous expression of a NLR in the cell.

Embodiment 49. The method of any one of the preceding Embodiments, wherein the NLR comprises a NLRC.

Embodiment 50. The method of any one of the preceding Embodiments, wherein the NLRC comprises a NOD1, NOD2, NLRC3, NLRC4, or NLRC5.

Embodiment 51. The method of any one of the preceding Embodiments, wherein the NLRC comprises the NLRC5.

Embodiment 52. The method of any one of the preceding Embodiments, wherein the at least one genetic modification decreases an endogenous expression of a MHC class II regulatory factor in the cell.

Embodiment 53. The method of any one of the preceding Embodiments, wherein the MHC class II regulatory factor comprises RFX1, RFX2, RFX3, RFX4, or RFX5.

Embodiment 54. The method of any one of the preceding Embodiments, wherein the MHC class II regulatory factor comprises RFX5.

Embodiment 55. The method of any one of the preceding Embodiments, wherein the MHC class II regulatory factor comprises CIITA, CREB, or nuclear transcription factor Y (NF-Y) .

Embodiment 56. A method for modifying a cell, comprising: contacting the cell with a gene modifying moiety, where the gene modifying moiety introduces at least one genetic modification in the cell, thereby decreasing an endogenous expression of NLRC5 in the cell; and expressing a heterologous polypeptide in the cell.

Embodiment 57. A method for modifying a cell, comprising: contacting the cell with a gene modifying moiety, where the gene modifying moiety introduces at least one genetic modification in the cell, thereby decreasing an endogenous expression of RFX5 in the cell; and expressing a heterologous polypeptide in the cell.

Embodiment 58. A method for modifying a cell, comprising: contacting the cell with a gene modifying moiety, where the gene modifying moiety introduces at least one genetic modification in the cell, thereby decreasing an endogenous expression of CIITA in the cell; and expressing a heterologous polypeptide in the cell.

Embodiment 59. The method of any one of any one of the preceding Embodiments, wherein the heterologous polypeptide comprises a heterologous receptor.

Embodiment 60. The method of any one of the preceding Embodiments, wherein the heterologous receptor comprises an antigen binding domain.

Embodiment 61. The method of any one of the preceding Embodiments, wherein the antigen binding domain binds to CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44v6, CD47, CD52, CD56, CD57, CD58, CD70, CD79a, CD79b, CD80, CD81, CD86, CD99, CD117, CD123, CD133, CD135, CD137, CD151, CD171, CD276, BAFF-R, BCMA, B7H4, CEA, CEACAM6, Claudin18.2, CLL-1, c-Met, CS-1, CTLA-4, EGFRvIII, GPC2, GPC3, GPRC5, HER2, HER3, HER4/ErbB4, HVEM, MAGE-A, MAGE3, MSLN, MUC-1, MUC-16, NY-ESO-1, OX40, PD-1, PD-L1, PD-L2, PMSA, ROR1, TCRa, TCRb, TLR7, TLR9, VEGFR-2, or WT-1.

Embodiment 62. The method of any one of the preceding Embodiments, wherein the heterologous receptor comprises a transmembrane domain.

Embodiment 63. The method of any one of the preceding Embodiments, wherein the heterologous receptor comprises a signaling domain.

Embodiment 64. The method of any one of the preceding Embodiments, wherein an endogenous expression of MHC-I of the cell is decreased compared to an endogenous expression of MHC-I of a comparable cell that does not have decreased expression of the endogenous gene.

Embodiment 65. The method of any one of the preceding Embodiments, wherein the endogenous expression of MHC-I of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to the endogenous expression of MHC-I or the comparable cell that does not have decreased expression of the endogenous gene.

Embodiment 66. The method of any one of the preceding Embodiments, wherein the endogenous expression of MHC-I of the cell is knocked out.

Embodiment 67. The method of any one of the preceding Embodiments, wherein the endogenous expression of MHC-I of the cell is not knocked out.

Embodiment 68. The method of any one of the preceding Embodiments, wherein the endogenous expression of MHC-I of the cell is decreased by at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 95%, or at most 99%compared to the endogenous expression of MHC-I of the comparable cell that does not have decreased expression of the endogenous gene.

Embodiment 69. The method of any one of the preceding Embodiments, wherein an endogenous expression of MHC-II of the cell is decreased compared to an endogenous expression of MHC-II of a comparable cell that does not have decreased expression of the endogenous gene.

Embodiment 70. The method of any one of the preceding Embodiments, wherein the endogenous expression of MHC-II of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to the endogenous expression of MHC-II or the comparable cell that does not have decreased expression of the endogenous gene.

Embodiment 71. The method of any one of the preceding Embodiments, wherein the endogenous expression of MHC-II of the cell is abrogated.

Embodiment 72. The method of any one of the preceding Embodiments, wherein the cell does not increase NK cell kill activity compared to a comparable cell that does not have the at least one heterologous polynucleotide.

Embodiment 73. The method of any one of the preceding Embodiments, wherein the cell comprises an immune cell or a stem cell.

Embodiment 74. The method of any one of the preceding Embodiments, wherein the immune cell is a lymphocyte.

Embodiment 75. The method of any one of the preceding Embodiments, wherein the lymphocyte is a B cell.

Embodiment 76. The method of any one of the preceding Embodiments, wherein the lymphocyte is a T cell.

Embodiment 77. The method of any one of the preceding Embodiments, wherein the T cell is selected from the group consisting of: cytotoxic T cell, alpha beta T cell, a gamma delta T cell, natural killer T cell, regulatory T cell, and T helper cell.

Embodiment 78. The method of any one of the preceding Embodiments, wherein the immune cell comprises an ILC.

Embodiment 79. The method of any one of the preceding Embodiments, wherein the stem cell is a hematopoietic stem cell.

Embodiment 80. The method of any one of the preceding Embodiments, wherein the stem cell is an iPSC.

Embodiment 81. The method of any one of the preceding Embodiments, wherein the immune cell is derived from an iPSC.

Embodiment 82. The method of any one of the preceding Embodiments, wherein the immune cell is an iPSC derived T cell.

Embodiment 83. The method of any one of the preceding Embodiments, wherein the immune cell is an iPSC derived natural killer T cell.

Embodiment 84. The method of any one of the preceding Embodiments, wherein the immune cell is an iPSC derived macrophage.

Embodiment 85. The method of any one of the preceding Embodiments, wherein the gene modifying moiety comprises a Cas protein or mRNA.

Embodiment 86. The method of any one of the preceding Embodiments, wherein the gene modifying moiety comprises a Cas/RNP.

Embodiment 87. The method of any one of the preceding Embodiments, wherein the gene modifying moiety comprises a Cas9/RNP.

Embodiment 88. The method of any one of the preceding Embodiments, wherein the gene modifying moiety comprises an inhibitory RNA, optionally wherein the inhibitory RNA comprises siRNA, miRNA, shRNA or a combination thereof.

Embodiment 89. The method of any one of the preceding Embodiments, wherein:

(i) in the cell, the heterologous polypeptide is a chimeric antigen receptor (CAR) ; and/or

(ii) in the cell, the antigen binding domain does not bind to one or more members selected from the group consisting of CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44v6, CD47, CD52, CD56, CD57, CD58, CD70, CD79a, CD79b, CD80, CD81, CD86, CD99, CD117, CD123, CD133, CD135, CD137, CD151, CD171, CD276, BAFF-R, BCMA, B7H4, CEA, CEACM6, Claudin18.2, CLL-1, c-Met, CS-1, CTLA-4, EGFRvIII, GPC2, GPC3, GPRC5, HER2, HER3, HER4/ErbB4, HVEM, MAGE-A, MAGE3, MSLN, MUC-1, MUC-16, NY-ESO-1, OX40, PD-1, PD-L1, PD-L2, PMSA, ROR1, TCRa, TCRb, TLR7, TLR9, VEGFR-2, and WT-1,

optionally wherein the antigen binding domain does not bind to NY-ESO-1; and/or

Embodiment 90. A method for treating a disease or condition in a subject in need thereof comprising administering the cell of any one of the preceding claims, the cell line of Embodiment 39, the composition of Embodiment 40, or the pharmaceutical composition of any one of the preceding claims to the subject in need thereof.

Embodiment 91. The method of Embodiment 90, wherein the administering to the subject does not increase NK cell kill activity in the subject, as compared to NK cell kill activity of the subject before the administering.

Embodiment 92. A method for modifying a cell comprising: contacting the cell with a gene modifying moiety, where the gene modifying moiety introduces at least one genetic modification in the cell, thereby knocking out the at least one endogenous gene, wherein knocking out of the at least one endogenous gene decreases endogenous expression of at least one MHC-I and decreases endogenous expression of at least one MHC-II.

Embodiment 93. A population of cells, wherein a cell of the population comprises:

a reduced expression level of an endogenous gene encoding SUGT-1 in the cell; and

a heterologous polypeptide, wherein:

(i) the heterologous polypeptide is a chimeric antigen receptor (CAR) ; or

(ii) the heterologous polypeptide does not exhibit specific binding to NY-ESO-1.

optionally wherein:

(1) the heterologous polypeptide is CAR; and/or

(2) the heterologous polypeptide does not exhibit specific binding to NY-ESO-1; and/or

(3) the at least at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 85%of the population exhibits reduced expression level of MHC without directly targeting HLA gene,

optionally wherein the at least about 80%of the population exhibits reduced expression level of MHC without directly targeting the HLA gene; and/or

(4) the cell further comprises a reduced expression level of one or more members selected from the group consisting of nucleotide-binding oligomerization domain-like receptors (NLR) , and MHC class II regulatory factor.

Embodiment 94. A population of cells, wherein a cell of the population comprises:

a reduced expression level of an endogenous gene encoding NLR in the cell; and

a heterologous polypeptide, wherein:

(i) the heterologous polypeptide is a chimeric antigen receptor (CAR) ; or

(ii) the heterologous polypeptide does not exhibit specific binding to NY-ESO-1, optionally wherein:

(1) the NLR is a NLR family CARD domain-containing protein (NLRC) , optionally wherein the NLRC comprises a nucleotide binding oligomerization domain (NOD) 1, NOD2, NLRC3, NLRC4, or NLRC5, optionally wherein the NLRC comprises the NLRC5; and/or

(2) the heterologous polypeptide is CAR; and/or

(3) the heterologous polypeptide does not exhibit specific binding to NY-ESO-1; and/or

(4) the at least at least about 40%, at least about 50%, or at least about 60%of the population exhibits reduced expression level of MHC without directly targeting HLA gene,

optionally wherein the at least about 50%of the population exhibits reduced expression level of MHC without directly targeting the HLA gene; and/or

(5) the cell further comprises a reduced expression level of one or more members selected from the group consisting of SUGT-1 and MHC class II regulatory factor.

Embodiment 95. A population of cells, wherein a cell of the population comprises: a reduced expression level of an endogenous gene encoding CIITA in the cell, wherein the cell does not comprise a genetic modification in a gene encoding B2M; and a heterologous polypeptide,

optionally wherein:

(1) the at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%of the population exhibits reduced expression level of MHC without directly targeting HLA gene,

(2) the cell further comprises a reduced expression level of one or more members selected from the group consisting of SUGT-1, NLR, and MHC class II regulatory factor.

Embodiment 96. A population of cells, wherein a cell of the population comprises: a reduced expression level of an endogenous gene encoding an endogenous gene encoding regulatory factor X (RFX) in the cell, wherein the cell does not comprise a genetic modification in a gene encoding B2M or CIITA; and

a heterologous polypeptide,

optionally wherein:

(1) the RFX is RFX1, RFX2, RFX3, RFX4, or RFX5,

optionally wherein the RFX is RFX5; and/or

(2) the cell does not comprise the genetic modification in the gene encoding B2M; and/or

(3) the cell does not comprise the genetic modification in the gene encoding CIITA; and/or

(4) the at least at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%of the population exhibits reduced expression level of MHC without directly targeting HLA gene,

optionally wherein:

(a) the at least about 80%of the population exhibits reduced expression level of MHC without directly targeting the HLA gene; and/or

(b) the at least about 90%of the population exhibits reduced expression level of MHC without directly targeting the HLA gene; and/or

(5) the cell further comprises a reduced expression level of one or more members selected from the group consisting of SUGT-1, NLR, and MHC class II regulatory factor.

Embodiment 97. The population of cells of any one of the Embodiments provided herein, optionally wherein:

(1) the NLR comprises a NLRC,

optionally wherein the NLRC comprises a NOD1, NOD2, NLRC3, NLRC4, or NLRC5,

optionally wherein the NLRC comprises the NLRC5; and/or

(2) the MHC class II regulatory factor comprises RFX1, RFX2, RFX3, RFX4, RFX5, CIITA, CREB, or NF-Y,

optionally wherein:

(a) the MHC class II regulatory factor comprises RFX5; and/or

(b) the MHC class II regulatory factor comprises CIITA; and/or

(3) the MHC comprises MHC-I; and/or

(4) the MHC comprises MHC-II; and/or

(5) the cell does not comprise a genetic modification in HLA gene; and/or

(6) the presence of the reduced expression level of the endogenous gene and the heterologous polypeptide does not substantially reduce cytotoxicity of the cell against a target cell, as compared to a control cell without the reduced expression level of the endogenous gene; and/or

(7) the cell does not increase natural killer (NK) cell kill activity, as compared to a control cell without the reduced expression level of the endogenous gene; and/or

(8) the cell is a stem cell,

optionally wherein the stem cell is a hematopoietic stem cell or an induced pluripotent stem cell (iPSC) ; and/or

(9) wherein the cell comprises an immune cell,

optionally wherein:

(a) the immune cell is a lymphocyte,

optionally wherein the lymphocyte is a B cell or a T cell,

optionally wherein the T cell is selected from the group consisting of cytotoxic T cell, natural killer T cell, regulatory T cell, and T helper cell,

optionally wherein the immune cell comprises an innate lymphoid cell (ILC) , and/or

(b) the immune cell is derived from an induced pluripotent stem cell (iPSC) ; and/or

(10) the heterologous polypeptide comprises a heterologous receptor, optionally wherein:

(a) the heterologous receptor comprises at least one antigen binding domain,

optionally wherein the antigen binding domain binds to cluster of differentiation (CD) 1a, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44v6, CD47, CD52, CD56, CD57, CD58, CD70, CD79a, CD79b, CD80, CD81, CD86, CD99, CD117, CD123, CD133, CD135, CD137, CD151, CD171, CD276, B-cell activating factor receptor (BAFF-R) , B-cell maturation antigen (BCMA) , B7 homolog 4 (B7H4) , Carcinoembryonic antigen (CEA) , CEA cell adhesion molecule 6 (CEACAM6) , Claudin18.2, c-type lectin-like molecule 1 (CLL-1) , c-Met, CS-1, cytotoxic T-lymphocyte-associated antigen (CTLA) -4, epidermal growth factor receptor (EGFR) vIII, glypican family member glypican (GPC) 2, GPC3, G protein coupled receptor class 5 (GPRC5) , human epidermal growth factor receptor (HER) 2, HER3, HER4/ErbB4, herpes virus entry mediator (HVEM) , melanoma antigen gene (MAGE) -A, MAGE3, mesothelin (MSLN) , mucin (MUC) -1, MUC-16, New York esophageal squamous cell carcinoma (NY-ESO) -1, OX40, programmed death (PD) -1, Programmed death-ligand (PD-L) 1, PD-L2, prostate-specific membrane antigen (PMSA) , receptor tyrosine kinase-like orphan receptor 1 (ROR1) , T-cell receptor (TCR) a, TCRb, toll-like receptor (TLR) 7, TLR9, vascular endothelial growth factor receptor (VEGFR) -2, or Wilms tumor (WT) -1; and/or

(b) the heterologous receptor comprises a transmembrane domain; and/or

(c) the heterologous receptor comprises a signaling domain.

Embodiment 98. A method for modifying a population of cells, comprising: reducing expression level of an endogenous gene encoding SUGT-1 in a cell of the population of cells; and

expressing a heterologous polypeptide in the cell, wherein:

(i) the heterologous polypeptide is a chimeric antigen receptor (CAR) ; or

(ii) the heterologous polypeptide does not exhibit specific binding to NY-ESO-1,

optionally wherein:

(1) the heterologous polypeptide is CAR; and/or

Embodiment 99. A method for modifying a population of cells, comprising reducing expression level of an endogenous gene encoding NLR in a cell of the population of cells; and

expressing a heterologous polypeptide in the cell, wherein:

(i) the heterologous polypeptide is a chimeric antigen receptor (CAR) ; or

(1) the NLR is a NLR family CARD domain-containing protein (NLRC) ,

optionally wherein the NLRC comprises a nucleotide binding oligomerization domain (NOD) 1, NOD2, NLRC3, NLRC4, or NLRC5,

optionally wherein the NLRC comprises the NLRC5; and/or

(2) the heterologous polypeptide is CAR; and/or

Embodiment 100. A method for modifying a population of cells, comprising:

reducing expression level of an endogenous gene encoding CIITA in a cell of the population of cells, wherein the cell does not comprise a genetic modification in a gene encoding B2M; and expressing a heterologous polypeptide in the cell,

optionally wherein:

Embodiment 101. A method for modifying a population of cells, comprising: reducing expression level of an endogenous gene encoding RFX in a cell of the population of cells, wherein the cell does not comprise a genetic modification in a gene encoding B2M or CIITA; and

expressing a heterologous polypeptide in the cell,

optionally wherein:

(1) the RFX is RFX1, RFX2, RFX3, RFX4, or RFX5,

optionally wherein the RFX is RFX5; and/or

(4) the at least at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%of the population exhibits reduced expression level of MHC without directly targeting HLA gene, optionally wherein:

Embodiment 102. The method for modifying a population of cells of any one of the Embodiments provided herein,

optionally wherein:

(1) the reducing comprises contacting the cell with a gene modifying moiety that is configured to induce at least one genetic modification in the endogenous gene,

optionally wherein:

(a) the gene modifying moiety comprise a Cas protein,

optionally wherein the Cas protein comprises Cas9 or a modification thereof; and/or

(b) the gene modifying moiety comprises an inhibitory RNA,

optionally wherein the gene modifying moiety comprises an inhibitory RNA,

optionally wherein the inhibitory RNA comprises siRNA, miRNA, or shRNA; and/or

(2) the reducing is performed prior to, simultaneously with, or subsequent to the expressing; and/or

(3) the NLR comprises a NLRC,

optionally wherein the NLRC comprises a NOD1, NOD2, NLRC3, NLRC4, or NLRC5,

optionally wherein the NLRC comprises the NLRC5; and/or

(4) the MHC class II regulatory factor comprises RFX1, RFX2, RFX3, RFX4, RFX5, CIITA, CREB, or NF-Y,

optionally wherein:

(a) the MHC class II regulatory factor comprises RFX5; and/or

(b) the MHC class II regulatory factor comprises CIITA; and/or

(5) the MHC comprises MHC-I; and/or

(6) the MHC comprises MHC-II; and/or

(7) the cell does not comprise a genetic modification in HLA gene; and/or

(8) the presence of the reduced expression level of the endogenous gene and the heterologous polypeptide does not substantially reduce cytotoxicity of the cell against a target cell, as compared to a control cell without the reduced expression level of the endogenous gene; and/or

(9) the cell does not increase natural killer (NK) cell kill activity, as compared to a control cell without the reduced expression level of the endogenous gene; and/or

(10) the cell is a stem cell,

(11) wherein the cell comprises an immune cell,

optionally wherein:

(a) the immune cell is a lymphocyte,

optionally wherein the lymphocyte is a B cell or a T cell,

(12) the heterologous polypeptide comprises a heterologous receptor,

optionally wherein:

(a) the heterologous receptor comprises at least one antigen binding domain,

(b) the heterologous receptor comprises a transmembrane domain; and/or

(c) the heterologous receptor comprises a signaling domain.

Embodiment 103. A method for treating a disease in a subject, comprising:

administrating a population of cells to the subject, wherein a cell of the population comprises:

a heterologous polypeptide, wherein:

(i) the heterologous polypeptide is a chimeric antigen receptor (CAR) ; or

(1) the heterologous polypeptide is CAR; and/or

Embodiment 104. A method for treating a disease in a subject, comprising: administrating a population of cells to the subject, wherein a cell of the population comprises:

a reduced expression level of an endogenous gene encoding NLR in a cell of the population of cells; and

a heterologous polypeptide, wherein:

(i) the heterologous polypeptide is a chimeric antigen receptor (CAR) ; or

(1) the NLR is a NLR family CARD domain-containing protein (NLRC) , optionally wherein the NLRC comprises a nucleotide binding oligomerization domain (NOD) 1, NOD2, NLRC3, NLRC4, or NLRC5,

optionally wherein the NLRC comprises the NLRC5; and/or

(2) the heterologous polypeptide is CAR; and/or

Embodiment 105. A method for treating a disease in a subject, comprising: administrating a population of cells to the subject, wherein a cell of the population comprises:

a reduced expression level of an endogenous gene encoding CIITA in a cell of the population of cells, wherein the cell does not comprise a genetic modification in a gene encoding B2M; and

a heterologous polypeptide,

optionally wherein:

Embodiment 106. A method for treating a disease in a subject, comprising: administrating a population of cells to the subject, wherein a cell of the population comprises:

a reduced expression level of an endogenous gene encoding RFX in a cell of the population of cells, wherein the cell does not comprise a genetic modification in a gene encoding B2M or CIITA; and

a heterologous polypeptide.

optionally wherein:

(1) the RFX is RFX1, RFX2, RFX3, RFX4, or RFX5,

optionally wherein the RFX is RFX5; and/or

optionally wherein:

Embodiment 107. The method for treating a disease in a subject of any one of the Embodiments provided herein,

optionally wherein:

(1) the NLR comprises a NLRC,

optionally wherein the NLRC comprises a NOD1, NOD2, NLRC3, NLRC4, or NLRC5,

optionally wherein the NLRC comprises the NLRC5; and/or

optionally wherein:

(a) the MHC class II regulatory factor comprises RFX5; and/or

(b) the MHC class II regulatory factor comprises CIITA; and/or

(3) the MHC comprises MHC-I; and/or

(4) the MHC comprises MHC-II; and/or

(5) the cell does not comprise a genetic modification in HLA gene; and/or

(8) the cell is a stem cell,

(9) wherein the cell comprises an immune cell,

optionally wherein:

(a) the immune cell is a lymphocyte,

optionally wherein the lymphocyte is a B cell or a T cell,

(10) the heterologous polypeptide comprises a heterologous receptor,

optionally wherein:

(a) the heterologous receptor comprises at least one antigen binding domain,

(b) the heterologous receptor comprises a transmembrane domain; and/or

(c) the heterologous receptor comprises a signaling domain.

EXAMPLES

The following illustrative examples are representative of embodiments of the stimulation, systems, and methods described herein and are not meant to be limiting in any way.

Example 1. Cell modification and functional assay

Example 1 illustrates exemplary experiments for assessing the modified cells in Figs 1-7.

Culture of human cell lines

CCRF-CEM and Nalm-6 were purchased form ATCC. All cells (e.g., CCRF-CEM, Nalm-6, or primary cells such as primary T cells or primary NK cells) were cultured in RMPI-1640 medium (Gibco) supplemented with 10%FBS (Gibco) and 100IU/ml penicillin-streptomycin (Gibco) . Both cell lines were transfected with firefly luciferase-GFP expressing lentivirus.

Lentivirus packaging

Lentivirus stocks were generated by transfection of 293-FT cells with the pFUW-CD19CAR-41bbz or pFUW-CD7CAR-41bbz plasmid together with packaging vectors pMDLg/pRRE, pRSV-Rev and pMD2. G. One day before transfection, 30 million 293FT cells were seeded in 30ml antibiotic free complete DMEM medium (Gibco) in 15cm dish. Culturing medium was replaced 6 hours after transfection. Transfected cells were incubated in 2%FBS DEME medium for another 48-72 hours before harvest virus supernatant.

Peripheral blood mononuclear cells (PBMCs) , T cell and NK cell isolations and T cells expansion

Peripheral blood mononuclear cells were isolated by Ficoll-Paque (GE Healthcare) centrifugation from health donor leukopaks. Pan T cells were isolated form PBMCs by magnetic beads based negative selection using EasySep ^TM Human T Cell Enrichment Kit (STEMCELL, Cat#19051) per manufacturer’s instructions. CD56+ NK cells were isolated form PBMCs by magnetic beads based positive selection using EasySep ^TM Human CD56 Positive Selection Kit (STEMCELL, Cat#17855) per manufacturer’s instructions.

T cell expansion

Primary human T cells were stimulated with Dynabeads ^TM Human T-Activator CD3/CD28 (ThermoFisher Scientific, Cat#11132D) and culture at 37℃ in X-vivo 15 (Lonza) supplemented with 300IU/ml IL-2 (Peprotech) . 2 days post T cell activation, CRISPR/Cas9 based genetic modifications and/or lentivirus transduction were performed followed by additional 8 days of culture for T cell expansion.

Allograft reactive Cytotoxic T lymphocytes (Allo-CTLs) culture and killing of allogeneic T cells

Allo-CTLs were derived from PBMCs (responder) that were stimulated and expanded by T cell receptor deficient (TCR) , HLA-unmatched allogeneic T cells (stimulator) at 37℃ for 12 days with responder to stimulator ratio at 5: 1 (Culture media: X-vivo 15, supplemented with 5%FBS and 60 IU/ml IL-2) . After the 12 days culture, CD4+ or CD8+ allo-CTLs were isolated from expanded PBMC by magnetic beads based positive selection using anti-CD4 microbeads (Miltenyi, Cat#130-045-101) or anti-CD8 microbeads (Miltenyi, Cat#130-045-201) .

Preparation of TCR deficient allogeneic T cells (stimulator)

TCR expression was eliminated from allogeneic T cells using CRISPR/Cas9 system. TCR deficient cells were purified by magnetic beads based negative selection and TCR intact cells were depleted using anti-CD3 microbeads (Miltenyi, Cat#130-050-101) .

Allograft -CTLs killing of HLA-unmatched T cells

For allograft reactive CTLs killing assay, gene modified TCR KO allogenous T cells were used as targeted cells and labeled using CFSE (Sigma) , Target cells were co-cultured with CD4+ or CD8+ allograft reactive CTLs for 1-3 days. Absolute count of remaining living target cells was analyzed using flow cytometry and percent killing was calculated by comparing to control wells (target cells with no allograft CTL) .

Gene disruption by CRISPR/Cas9 system

CRISPR RNP were formed using Cas9 protein (Genscript) and sgRNA (Genscript) at a 1: 1 molar ratio at 37℃ for 15 min and stored frozen at -80℃ until use. CRISPR RNP (50pMol) were nucleofected into pre-activated primary human T cells using Lonza 4D-nucleofactor (Lonza) in P3 primary buffer (Lonza) and EO-115 pulse code. Cells were recovered and cultured in X-vivo 15 media (Lonza) at 37℃ for 6-7 days. Lentivirus were introduced to cells after nucleofection if CAR transgene expression was desired.

Fluorescence-activated cell sorting (FACS) analysis

All FACS analysis was performed on Canto II or Fortessa flow cytometer (BD Biosciences) . Cells were incubated with fluorochrome conjugated antibodies at 4℃ for 30mins, washed 2 times with FACS buffer (DPBS+2%FBS) , and suspended in 250ul FACS buffer. β2M-PE (ThermoFisher) , HLA-A/B/C-APC (eBioscience) , HLA-DR/DP/DQ-FITC (ThermoFisher) , CD3-BV421 (ThermoFisher) , CD4-PE-Cy7 (ThermoFisher) , CD8-APC-Cy7 (ThermoFisher) and live/dead dye 7-AAD or DAPI were used. All analysis was done using the FlowJo v10 software.

Cytotoxicity assays

Luciferase expressing CCRF-CEM (CD7+) or Nalm-6 (CD19+) cells (target) were co-cultured with genetic modified CD7-CAR-T cells or CD19-CAR-T cells (effector) at different effector: target ratios for 6 or 24 hours. Remaining target cells were analyzed by luciferase-based killing and percent of target cell killing by CAR-T cells was calculated by comparing CAR-T containing groups to control groups (target cells with no CAR-T cells) .

For primary human T or NK cell killing assays, primary T or NK cells were used as target cells and labeled with CFSE (Sigma) per manufacturer’ protocol. Target cells were co-cultured with CD7-CAR-T cells or CD19-CAR-T cells at different effecter: target ratios for 6 or 24 hours. Absolute count of remaining living target cells was analyzed using flow cytometry and percent killing was calculated by comparing to control wells (target cells with no CAR-T cells) .

Cytokine analysis

After 24 hours of the co-culture of CAR-T cells and cancer target cells, culture supernatant was collected for cytokines analysis using BD ^TM CBA Human Th1/Th2 Cytokine Kit (BD Biosciences, Cat#551809) .

Mixed lymphocyte reaction (MLR) Assay

MLR assays were used to analyze the extent of allograft response between HLA-unmatched PBMCs and allogeneic T cells. PBMCs were used as responder cells and labeled with VPD450 dye (BD Biosciences) , T cell receptor (TCR) deficient HLA-unmatched allogeneic T cells with/without genetic modifications for HLA-I/II expressions were used as stimulator cells and labeled with CFSE (Sigma) . Stimulator (S) and responder (R) cells were mixed at a S: R=1: 3 ratio and co-cultured for 6-10 days. Cells were stained with CD4-PE-Cy7, CD8-APC-Cy7, TCRαβ-Percp and CD7-PE (Thermo Fisher Scientific) before analysis by flow cytometry. Absolute cell counts were analyzed by flow cytometry and fold of expansion for responder cells were calculated by comparing to responder only control wells (no allogeneic T cells) .

While the foregoing disclosure has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure. For example, all the techniques and apparatus described above can be used in various combinations. All publications, patents, patent applications, and/or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and/or other document were individually and separately indicated to be incorporated by reference for all purposes.

Claims

A cell comprising:

a) at least one genetic modification, where the at least one genetic modification decreases an endogenous expression of SUGT-1 in the cell; and

b) a heterologous polypeptide.
The cell of claim 1, wherein the at least one genetic modification decreases an endogenous expression of a nucleotide-binding oligomerization domain-like receptors (NLR) in the cell.
The cell of claim 2, wherein the NLR comprises a NLRC.
The cell of claim 3, wherein the NLRC comprises a NOD1, NOD2, NLRC3, NLRC4, or NLRC5.
The cell of claim 4, wherein the NLRC comprises the NLRC5.
The cell of claim 1, wherein the at least one genetic modification decreases an endogenous expression of an MHC class II regulatory factor in the cell.
The cell of claim 6, wherein the MHC class II regulatory factor comprises RFX1, RFX2, RFX3, RFX4, or RFX5.
The cell of claim 7, wherein the MHC class II regulatory factor comprises RFX5.
The cell of claim 6 wherein the MHC class II regulatory factor comprises CIITA, CREB, or nuclear transcription factor Y (NF-Y) .
A cell comprising:

a. at least one genetic modification, where the at least one genetic modification decreases an endogenous expression of NLRC5 in the cell; and

b. a heterologous polypeptide.
A cell comprising:

a. at least one genetic modification, where the at least one genetic modification decreases an endogenous expression of RFX5 in the cell; and

b. a heterologous polypeptide.
A cell comprising:

a. at least one genetic modification, where the at least one genetic modification decreases an endogenous expression of CIITA in the cell; and

b. a heterologous polypeptide.
The cell of any one of previous claims, wherein the heterologous polypeptide comprises a heterologous receptor.
The cell of claim 13, wherein the heterologous receptor comprises an antigen binding domain.
The cell of claim 14, wherein the antigen binding domain binds to CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44v6, CD47, CD52, CD56, CD57, CD58, CD70, CD79a, CD79b, CD80, CD81, CD86, CD99, CD117, CD123, CD133, CD135, CD137, CD151, CD171, CD276, BAFF-R, BCMA, B7H4, CEA, CEACM6, Claudin18.2, CLL-1, c-Met, CS-1, CTLA-4, EGFRvIII, GPC2, GPC3, GPRC5, HER2, HER3, HER4/ErbB4, HVEM, MAGE-A, MAGE3, MSLN, MUC-1, MUC-16, NY-ESO-1, OX40, PD-1, PD-L1, PD-L2, PMSA, ROR1, TCRa, TCRb, TLR7, TLR9, VEGFR-2, or WT-1.
The cell of any one of claims 13-15, wherein the heterologous receptor comprises a transmembrane domain.
The cell of any one of claims 13-15, wherein the heterologous receptor comprises a signaling domain.
The cell of any one of previous claims, wherein an endogenous expression of MHC-I of the cell is decreased compared to an endogenous expression of MHC-I of a comparable cell that does not have decreased expression of the endogenous gene.
The cell of claim 18, wherein the endogenous expression of MHC-I of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to the endogenous expression of MHC-I or the comparable cell that does not have an endogenous gene knocked out.
The cell of claim 18, wherein the endogenous expression of MHC-I of the cell is knocked out.
The cell of claim 18, wherein the endogenous expression of MHC-I of the cell is not knocked out.
The cell of claim 18, wherein the endogenous expression of MHC-I of the cell is decreased by at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 95%, or at most 99%compared to the endogenous expression of MHC-I or the comparable cell that does not have an endogenous gene knocked out.
The cell of any one of previous claims, wherein an endogenous expression of MHC-II of the cell is decreased compared to an endogenous expression of MHC-II of a comparable cell that does not have an endogenous gene knocked out.
The cell of claim 23, wherein the endogenous expression of MHC-II of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to the endogenous expression of MHC-II or the comparable cell that does not have an endogenous gene knocked out.
The cell of claim 23, wherein the endogenous expression of MHC-II of the cell is abrogated.
The cell of any one of previous claims, wherein the cell does not increase NK cell kill activity compared to a comparable cell that does not have an endogenous gene knocked out.
The cell of any one of previous claims, wherein the cell comprises an immune cell or a stem cell.
The cell of claim 27, wherein the immune cell is a lymphocyte.
The cell of claim 28, wherein the lymphocyte is a B cell.
The cell of claim 28, wherein the lymphocyte is a T cell.
The cell of claim 30, wherein the T cell is selected from the group consisting of: cytotoxic T cell, natural killer T cell, regulatory T cell, and T helper cell.
The cell of claim 27, wherein the immune cell comprises an innate lymphoid cell (ILC) .
The cell of claim 27, wherein the immune cell is derived from an induced pluripotent stem cell (iPSC) .
The cell of claim 27, wherein the immune cell is an induced pluripotent stem cell derived T cell.
The cell of claim 27, wherein the immune cell is an induced pluripotent stem cell derived natural killer T cell.
The cell of claim 27, wherein the immune cell is an iPSC derived macrophage.
The cell of claim 27, wherein the stem cell is a hematopoietic stem cell.
The cell of claim 27, wherein the stem cell is an induced pluripotent stem cell (iPSC) .
A cell line comprising the cell of any one of previous claims.
A composition comprising the cell of any one of previous claims.
A pharmaceutical composition comprising:

a)the cell of any one of claims 1-39 or the composition of claim 40; and

b)a pharmaceutically acceptable: excipient, carrier, or diluent.
The pharmaceutical composition of claim 41 in a unit dose form.
The pharmaceutical composition of claim 41 or 42, wherein the pharmaceutical composition is formulated for administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, intratumorally, pulmonarily, endotracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, by inhalation, by inhaled nebulized form, by intraluminal-GI route, or a combination thereof to a subject in need thereof.
The pharmaceutical composition of any one of claims 41-43, further comprising at least one additional active agent.
The pharmaceutical composition of claim 44, wherein the at least one additional active agent comprises a cytokine, a growth factor, a hormone, an enzyme, a small molecule, a compound, or combinations thereof.
A kit comprising:

a) the cell of any one of claims 1-39, the composition of claim 40, or the pharmaceutical composition of any one of claims 41-45; and

b) a container.
A method for modifying a cell, comprising:

a) contacting the cell with a gene modifying moiety, where the gene modifying moiety introduces at least one genetic modification in the cell, thereby decreasing an endogenous expression of SUGT-1 in the cell; and

b) expressing a heterologous polypeptide in the cell.
The method of claim 47, wherein the at least one genetic modification decreases an endogenous expression of a NLR in the cell.
The method of claim 48, wherein the NLR comprises a NLRC.
The method of claim 49, wherein the NLRC comprises a NOD1, NOD2, NLRC3, NLRC4, or NLRC5.
The method of claim 50, wherein the NLRC comprises the NLRC5.
The method of claim 47, wherein the at least one genetic modification decreases an endogenous expression of a MHC class II regulatory factor in the cell.
The method of claim 52, wherein the MHC class II regulatory factor comprises RFX1,

RFX2, RFX3, RFX4, or RFX5.
The method of claim 53, wherein the MHC class II regulatory factor comprises RFX5.
The method of claim 52, wherein the MHC class II regulatory factor comprises CIITA, CREB, or nuclear transcription factor Y (NF-Y) .
A method for modifying a cell, comprising:

a) contacting the cell with a gene modifying moiety, where the gene modifying moiety introduces at least one genetic modification in the cell, thereby decreasing an endogenous expression of NLRC5 in the cell; and

b) expressing a heterologous polypeptide in the cell.
A method for modifying a cell, comprising:

a) contacting the cell with a gene modifying moiety, where the gene modifying moiety introduces at least one genetic modification in the cell, thereby decreasing an endogenous expression of RFX5 in the cell; and

b) expressing a heterologous polypeptide in the cell.
A method for modifying a cell, comprising:

a) contacting the cell with a gene modifying moiety, where the gene modifying moiety introduces at least one genetic modification in the cell, thereby decreasing an endogenous expression of CIITA in the cell; and

b) expressing a heterologous polypeptide in the cell.
The method of any one of claims 47-58, wherein the heterologous polypeptide comprises a heterologous receptor.
The method of claim 59, wherein the heterologous receptor comprises an antigen binding domain.
The method of claim 60, wherein the antigen binding domain binds to CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44v6, CD47, CD52, CD56, CD57, CD58, CD70, CD79a, CD79b, CD80, CD81, CD86, CD99, CD117, CD123, CD133, CD135, CD137, CD151, CD171, CD276, BAFF-R, BCMA, B7H4, CEA, CEACM6, Claudin18.2, CLL-1, c-Met, CS-1, CTLA-4, EGFRvIII, GPC2, GPC3, GPRC5, HER2, HER3, HER4/ErbB4, HVEM, MAGE-A, MAGE3, MSLN, MUC-1, MUC-16, NY-ESO-1, OX40, PD-1, PD-L1, PD-L2, PMSA, ROR1, TCRa, TCRb, TLR7, TLR9, VEGFR-2, or WT-1.
The method of claim 59, wherein the heterologous receptor comprises a transmembrane domain.
The method of claim 59, wherein the heterologous receptor comprises a signaling domain.
The method of any one of claims 47-63, wherein an endogenous expression of MHC-I of the cell is decreased compared to an endogenous expression of MHC-I of a comparable cell that does not have decreased expression of the endogenous gene.
The method of claim 64, wherein the endogenous expression of MHC-I of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to the endogenous expression of MHC-I or the comparable cell that does not have decreased expression of the endogenous gene.
The method of claim 64, wherein the endogenous expression of MHC-I of the cell is knocked out.
The method of claim 64, wherein the endogenous expression of MHC-I of the cell is not knocked out.
The method of claim 64, wherein the endogenous expression of MHC-I of the cell is decreased by at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 95%, or at most 99%compared to the endogenous expression of MHC-I of the comparable cell that does not have decreased expression of the endogenous gene.
The method of any one of claims 47-68, wherein an endogenous expression of MHC-II of the cell is decreased compared to an endogenous expression of MHC-II of a comparable cell that does not have decreased expression of the endogenous gene.
The method of claim 69, wherein the endogenous expression of MHC-II of the cell is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%compared to the endogenous expression of MHC-II or the comparable cell that does not have decreased expression of the endogenous gene.
The method of claim 69, wherein the endogenous expression of MHC-II of the cell is abrogated.
The method of any one of claims 47-71, wherein the cell does not increase NK cell kill activity compared to a comparable cell that does not have the at least one heterologous polynucleotide.
The method of any one of claims 47-71, wherein the cell comprises an immune cell or a stem cell.
The method of claim 73, wherein the immune cell is a lymphocyte.
The method of claim 74, wherein the lymphocyte is a B cell.
The method of claim 74, wherein the lymphocyte is a T cell.
The method of claim 76, wherein the T cell is selected from the group consisting of: cytotoxic T cell, alpha beta T cell, a gamma delta T cell, natural killer T cell, regulatory T cell, and T helper cell.
The method of claim 73, wherein the immune cell comprises an ILC.
The method of claim 73, wherein the stem cell is a hematopoietic stem cell.
The method of claim 73, wherein the stem cell is an iPSC.
The method of claim 73, wherein the immune cell is derived from an iPSC.
The method of claim 73, wherein the immune cell is an iPSC derived T cell.
The method of claim 73, wherein the immune cell is an iPSC derived natural killer T cell.
The method of claim 73, wherein the immune cell is an iPSC derived macrophage.
The method of any one of claims 47-58, wherein the gene modifying moiety comprises a Cas protein or mRNA.
The method of claim 85, wherein the gene modifying moiety comprises a Cas/RNP.
The method of claim 86, wherein the gene modifying moiety comprises a Cas9/RNP.
The method of any one of claims 47-58, wherein the gene modifying moiety comprises an inhibitory RNA.
The method of claim 88, wherein the inhibitory RNA comprises siRNA, miRNA, shRNA or a combination thereof.
A method for treating a disease or condition in a subject in need thereof comprising administering the cell of any one of claims 1-39, the composition of claim 40, or the pharmaceutical composition of any one of claims 41-45 to the subject in need thereof.
The method of claim 90, wherein the administering of the cell of any one of claims 1-39, the composition of claim 40, or the pharmaceutical composition of any one of claims 41-45 to the subject does not increase NK cell kill activity in the subject compared to NK cell kill activity of the subject before administering of the cell of any one of claims 1-39, the composition of claim 40, or the pharmaceutical composition of any one of claims 41-45.
A method for modifying a cell comprising:

contacting the cell with a gene modifying moiety, where the gene modifying moiety introduces at least one genetic modification in the cell, thereby knocking out the at least one endogenous gene,

wherein knocking out of the at least one endogenous gene decreases endogenous expression of at least one MHC-I and decreases endogenous expression of at least one MHC-II.