Genetically engineered cells for enhanced immune evasion and methods thereof

Abstract

This disclosure provides compositions and methods of making cells, such as allogenic cells, engineered to evade host immune responses, particularly from T cells and natural killer cells, and populations thereof. The compositions and methods for making engineered cells include making genetic modifications that reduce or eliminate the expression of an NKG2D ligand or integrate sequences encoding HLA molecules, such as HLA-E or HLA-G. Such modifications can enhance the survival of the engineered cells within a host without impeding their ability to differentiate into functional cell types, such as cardiomyocytes.

Description

GENETICALLY ENGINEERED CELLSFOR ENHANCED IMMUNE EVASION AND METHODS THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 645,742, filed May 10, 2024, the entire contents of which are incorporated herein by reference.INCORPORATED BY REFERENCE OF SEQUENCE LISTING

[0002] The Sequence Listing titled 213661-023002_PCT_SL.xml, which was created on May 9, 2025 and is 48,114 bytes in size, is hereby incorporated by reference in its entirety.FIELD OF INVENTION

[0003] This disclosure relates generally to the field of cellular therapies. More particularly, this disclosure relates to genetically modified cells that exhibit enhanced immune evasion properties.BACKGROUND

[0004] Allogenic cell therapies offer remarkable potential for treating various diseases and injuries, including heart failure. Unfortunately, these therapies also face a significant hurdle: the host immune system often rejects transplanted allogenic cells. This immune response, which involves recognizing and destroying foreign cells, severely limits the effectiveness and broad applicability of allogenic cell therapies.

[0005] Recent advancements in genetic engineering provide opportunities to modify cells to evade the host immune system. However, despite advances in genome engineering technologies, significant challenges persist in creating cells with immune evasive capabilities. For example, one concern is whether genetic modifications might adversely affect the functionality of the cell product or the differentiation potential of the engineered cells. This issue is particularly pertinent as the genetic engineering must often be performed on expandable cells, typically undifferentiated stem cells, which poses a risk of compromising their developmental versatility and therapeutic efficacy.SUMMARY

[0006] This disclosure provides compositions and methods for achieving immune evasion in allogenic cell populations. Experimental results disclosed herein show these methods effectively reduce host immune cell cytotoxicity, including from natural killer (NK) and T cells, and ensure the engineered cells can differentiate into specific cell types, like cardiomyocytes, while retaining functionality.

[0007] In addition, this disclosure provides modified Human Leukocyte Antigen (HLA) molecules, particularly modified HLA-E molecules, for reducing T cell activation. In particular, this disclosure provides cells that have been genetically engineered with modified HLA molecules and / or modified p2-microglobulin molecules that effectively reduce T cell activation while also reducing natural killer (NK) cell cytotoxicity. Accordingly, this disclosure addresses a challenge identified by the inventors, z.e., the unexpected finding that induced expression of even minimally polymorphic HLA molecules can trigger robust T cell killing when expressed in B2M knockout cells. The modified HLA and B2M molecules of this disclosure incorporate specific mutations that disrupt T cell receptor (TCR) engagement and / or CD-8 co-receptor binding, while maintaining their ability to inhibit NK cells.

[0008] In a first aspect of the disclosure, provided herein is an engineered cell. In some embodiments, the engineered cell comprises: a genetic modification that reduces or eliminates function of a natural-killer group 2, member D (NKG2D) ligand as compared to a wild-type cell. In some embodiments, the genetic modification is sufficient to reduce or inhibit natural killer (NK) cell-mediated cytotoxicity of the engineered cell, or progeny thereof, as compared to a wild-type cell.

[0009] In some embodiments of this first aspect, the engineered cell further comprises a heterologous nucleic acid sequence encoding at least a functional portion of a human leukocyte antigen (HLA)-E molecule or an HLA-G molecule. In some embodiments, the heterologous nucleic acid is sufficient to further reduce or inhibit NK cell-mediated cytotoxicity of the engineered cell, or the progeny thereof, as compared to a wild-type cell.

[0010] In some embodiments of this first aspect, the engineered cell further comprises one or more genetic modifications that result in reduced T cell-mediated killing as comparedto a wild-type cell. In some embodiments, the one or more genetic modifications that result in reduced T cell-mediated killing as compared to a wild-type cell comprise: (i) a deletion or disruption of a P2 microglobulin (B2M) gene; or (ii) a deletion or disruption of one or more of an HLA-A gene, an HLA-B gene, and an HLA-C gene. In some embodiments, the one or more genetic modifications that result in reduced T cell-mediated killing as compared to a wildtype cell comprise a deletion or disruption of a class II, major histocompatibility complex, transactivator (CIITA) gene.

[0011] In some embodiments of this first aspect, the genetic modification that reduces or eliminates function of a NKG2D ligand as compared to a wild-type cell is a deletion or disruption of a gene encoding the NKG2D ligand. In some embodiments, the deletion or disruption results in reduced or eliminated expression of the gene encoding the NKG2D ligand.

[0012] In some embodiments of this first aspect, the NKG2D ligand comprises MICA, MICB, Raetle, Raetlg, Raetll, Ulbpl, Ulbp2, Ulbp3, or any combination thereof. In some embodiments, the NKG2D ligand comprises MICA.

[0013] In some embodiments of this first aspect, the heterologous nucleic acid sequence encoding at least the functional portion of the HLA-E molecule. In some embodiments, the heterologous nucleic acid sequence encoding at least the functional portion of the HLA-G molecule. In some embodiments, the heterologous nucleic acid sequence encoding at least the functional portion of both the HLA-E molecule and the HLA-G molecule.

[0014] In some embodiments of this first aspect, the HLA-E molecule and / or the HLA-G molecule encoded by the heterologous nucleic acid sequence comprises a binding peptide. In some embodiments, the binding peptide has an amino acid sequence comprising LIL or LFL. In some embodiments, the binding peptide has the amino acid sequence comprising LFL.

[0015] In some embodiments of this first aspect, the HLA-E molecule has an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 6.

[0016] In some embodiments of this first aspect, the heterologous nucleic acid further comprises a sequence encoding at least a functional portion of a P2 microglobulin (B2M). In some embodiments, the functional portion of B2M is linked to the HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of B2M is linked to theHLA-E molecule. In some embodiments, the functional portion of B2M linked to the HLA-E molecule further includes a linker. In some embodiments, the functional portion of B2M linked to the HLA-E molecule further comprises a binding peptide. In some embodiments, the binding peptide has an amino acid sequence comprising LIL or LFL. In some embodiments, the functional portion of B2M linked to the HLA-E molecule has an amino acid sequence that is at least 90% sequence identity to SEQ ID NO: 2 or 4.

[0017] In some embodiments of this first aspect, expression of the heterologous nucleic acid sequence is driven by an endogenous gene promoter in the engineered cell.

[0018] In some embodiments of this first aspect, the heterologous nucleic acid sequence is integrated into a sustained transgene expression locus (STEL) in the engineered cell. In some embodiments, the STEL comprises a locus within a human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene.

[0019] In some embodiments of this first aspect, the engineered cell further comprises a kill switch.

[0020] In some embodiments of this first aspect, the engineered cell comprises a stem cell. In some embodiments, the engineered cell is capable of differentiating into a cardiomyocyte. In some embodiments, the engineered cell comprises an induced pluripotent stem cell, or the progeny thereof.

[0021] In some embodiments of this first aspect the engineered cell comprises a cardiomyocyte, a neural cell, a myeloid cell, a T cell, or a retinal cell.

[0022] In a second aspect of the disclosure, provided herein is a cardiomyocyte differentiated from the engineered cell of any of the engineered cells provided herein. In some embodiments, the cardiomyocyte exhibits reduced natural killer (NK) cell-mediated cytotoxicity compared to a non-engineered cardiomyocyte.

[0023] In a third aspect of the disclosure, provided herein is a method for producing an engineered cardiomyocyte with reduced NK cell-mediated cytotoxicity. In some embodiments, the method comprises: providing the any of the engineered cells described herein; and inducing differentiation of the engineered cell into an engineered cardiomyocyte,wherein the resulting engineered cardiomyocyte exhibits reduced NK cell-mediated cytotoxicity as compared to a wild-type cardiomyocyte.

[0024] In a fourth aspect of the disclosure, provided herein is a population of engineered cells derived from any of the engineered cells provided herein. In some embodiments, the population of engineered cells are at least about a 10% less susceptible to NK cell -mediated cytotoxicity as compared to a population of wild-type cells. In some embodiments, the population of engineered cells are at least about a 20% less susceptible to NK cell-mediated cytotoxicity as compared to a population of wild-type cells.

[0025] In a fifth aspect of the disclosure, provided herein is a pharmaceutical composition comprising any of the populations of engineered cells provided herein, and a pharmaceutical acceptable carrier, excipient, or diluent.

[0026] In a sixth aspect of the disclosure, provided herein is a method of treating a disease or condition of a subject, the method comprising administering any or the population of engineered cells provided herein, or any of the pharmaceutical compositions provided herein. In some embodiments, the disease or condition comprises heart failure, Parkinson’s disease, multiple sclerosis, irritable bowel syndrome, type 1 diabetes, rheumatoid arthritis, Alzheimer’s disease, or neural inflammation. In some embodiments, the subject is a human.

[0027] In a seventh aspect of the disclosure, provided herein is a method for enhancing immune evasion of a cell, or progeny thereof, comprising engineering the cell by: genetically modifying the cell to reduce or eliminate function of a natural-killer group 2, member D (NKG2D) ligand relative to a wild-type cell. In some embodiments, the genetically modifying step enhances the survival probability of the cell when exposed to natural killer cells, as compared to a wild-type cell.

[0028] In some embodiments of this seventh aspect, the engineering the cell further comprises: incorporating a heterologous nucleic acid sequence that encodes at least a functional portion of a human leukocyte antigen (HLA)-E molecule or an HLA-G molecule into the genome of the cell. In some embodiments, the incorporating step further enhances the survival probability of the cell when exposed to natural killer cells, as compared to a wildtype cell.

[0029] In some embodiments of this seventh aspect, engineering the cell further comprises: genetically modifying the cell to achieve reduced T cell-mediated killing as compared to a wild-type cell.

[0030] In some embodiments of this seventh aspect, genetically modifying the cell to achieve reduced T cell-mediated killing as compared to a wild-type cell comprises: (i) introducing a deletion or disruption of the P2 microglobulin (B2M) gene; or (ii) introducing a deletion or disruption of one or more genes selected from HLA-A, HLA-B, and HLA-C. In some embodiments, to achieve reduced T cell-mediated killing as compared to a wild-type cell comprises introducing a deletion or disruption of the class II major histocompatibility complex transactivator (CIITA) gene.

[0031] In some embodiments of this seventh aspect, genetically modifying the cell to reduce or eliminate function of the NKG2D ligand relative to a wild-type cell comprises introducing a deletion or disruption of a gene encoding the NKG2D ligand. In some embodiments, the deletion or disruption results in reduced or eliminated expression of the gene encoding the NKG2D ligand.

[0032] In some embodiments of this seventh aspect, the NKG2D ligand comprises MICA, MICB, Raetle, Raetlg, Raetll, Ulbpl, Ulbp2, Ulbp3, or any combination thereof. In some embodiments, the NKG2D ligand comprises MICA.

[0033] In some embodiments of this seventh aspect, the heterologous nucleic acid sequence encoding at least the functional portion of the HLA-E molecule. In some embodiments of this seventh aspect, the heterologous nucleic acid sequence encoding at least the functional portion of the HLA-G molecule.

[0034] In some embodiments of this seventh aspect, the heterologous nucleic acid sequence encoding at least the functional portion of both the HLA-E molecule and the HLA- G molecule.

[0035] In some embodiments of this seventh aspect, the HLA-E molecule and / or the HLA-G molecule encoded by the heterologous nucleic acid sequence comprises a binding peptide. In some embodiments, the binding peptide has an amino acid sequence comprising LIL or LFL. In some embodiments, the binding peptide has an amino acid sequence comprising LFL.

[0036] In some embodiments of this seventh aspect, the HLA-E molecule has an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 6.

[0037] In some embodiments of this seventh aspect, the heterologous nucleic acid further comprises a sequence encoding at least a functional portion of a P2 microglobulin (B2M). In some embodiments, the functional portion of B2M is linked to the HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of B2M is linked to the HLA-E molecule. In some embodiments, the functional portion of B2M linked to the HLA-E molecule further includes a linker. In some embodiments, the functional portion of B2M linked to the HLA-E molecule further comprises a binding peptide. In some embodiments, the binding peptide has an amino acid sequence comprising LIL or LFL. In some embodiments, the functional portion of B2M linked to the HLA-E molecule has an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 2 or 4.

[0038] In some embodiments of this seventh aspect, the incorporating comprises integrating the heterologous nucleic acid sequence into a genomic locus of the cell. In some embodiments, the genomic locus is a sustained transgene expression locus (STEL). In some embodiments, glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) gene.

[0039] In some embodiments, of this seventh aspect, the method further includes expanding the engineered cell to produce a population of engineered cells.

[0040] In some embodiments, of this seventh aspect, the method further includes differentiating the population of engineered cells into a population of cardiac cells, neural cells, T cells, retinal cells, or myeloid cells.

[0041] In an eight aspect of the disclosure, provided herein is a pharmaceutical composition comprising the population of engineered cells produced by any of the methods for enhancing immune evasion of a cell provided herein, and a pharmaceutical acceptable carrier, excipient, or dilutant.

[0042] In a ninth aspect of the disclosure, provided herein is an engineered cardiomyocyte comprising: a heterologous nucleic acid sequence encoding at least a functional portion of a human leukocyte antigen (HLA)-E molecule or an HLA-G molecule, or a genetic modification that reduces or eliminates function of a natural-killer group 2, member D (NKG2D) ligand as compared to a wild-type cell. In some embodiments, eitherthe heterologous nucleic acid or the genetic modification is sufficient to reduce or inhibit NK cell-mediated cytotoxicity of the engineered cardiomyocyte as compared to a wild-type cardiomyocyte.

[0043] In some embodiments of this ninth aspect, the engineered cardiomyocyte further comprises one or more genetic modifications that result in reduced T cell-mediated killing as compared to a wild-type cardiomyocyte. In some embodiments, the one or more genetic modifications that result in reduced T cell-mediated killing as compared to a wild-type cardiomyocyte comprise: (i) a deletion or disruption of a P2 microglobulin (B2M) gene; or (ii) a deletion or disruption of one or more of an HLA-A gene, an HLA-B gene, and an HLA-C gene. In some embodiments, the one or more genetic modifications that result in reduced T cell-mediated killing as compared to a wild-type cell comprise a deletion or disruption of a class II, major histocompatibility complex, transactivator (CHTA) gene.

[0044] In some embodiments of this ninth aspect, the genetic modification that reduces or eliminates function of a NKG2D ligand as compared to a wild-type cardiomyocyte is a deletion or disruption of a gene encoding the NKG2D ligand. In some embodiments, the deletion or disruption results in reduced or eliminated expression of the gene encoding the NKG2D ligand.

[0045] In some embodiments of this ninth aspect, the NKG2D ligand comprises MICA, MICB, Raetle, Raetlg, Raetll, Ulbpl, Ulbp2, Ulbp3, or any combination thereof. In some embodiments, the NKG2D ligand comprises MICA.

[0046] In some embodiments of this ninth aspect, the heterologous nucleic acid sequence encoding at least the functional portion of the HLA-E molecule.

[0047] In some embodiments of this ninth aspect, the heterologous nucleic acid sequence encoding at least the functional portion of the HLA-G molecule.

[0048] In some embodiments of this ninth aspect, the heterologous nucleic acid sequence encoding at least the functional portion of both the HLA-E molecule and the HLA-G molecule.

[0049] In some embodiments of this ninth aspect, the HLA-E molecule and / or the HLA- G molecule encoded by the heterologous nucleic acid sequence comprises a binding peptide.In some embodiments, the binding peptide has an amino acid sequence comprising LIL or LFL. In some embodiments, the binding peptide has the amino acid sequence comprising LFL.

[0050] In some embodiments of this ninth aspect, the HLA-E molecule has an amino acid sequence that is at least 90% sequence identity to SEQ ID NO: 6.

[0051] In some embodiments of this ninth aspect, the heterologous nucleic acid further comprises a sequence encoding at least a functional portion of a P2 microglobulin (B2M). In some embodiments, the functional portion of B2M is linked to the HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of B2M is linked to the HLA-E molecule. In some embodiments, the functional portion of B2M linked to the HLA-E molecule further includes a linker. In some embodiments, the functional portion of B2M linked to the HLA-E molecule further comprises a binding peptide. In some embodiments, the binding peptide has an amino acid sequence comprising LIL or LFL. In some embodiments, the functional portion of B2M linked to the HLA-E molecule has an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 2 or 4.

[0052] In some embodiments of this ninth aspect, expression of the heterologous nucleic acid sequence is driven by an endogenous gene promoter in the engineered cardiomyocyte.

[0053] In some embodiments of this ninth aspect, the heterologous nucleic acid sequence is integrated into a sustained transgene expression locus (STEL) in the engineered cardiomyocyte. In some embodiments, the STEL comprises a locus within a human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. In some embodiments of this ninth aspect, engineered cell further comprises a kill switch.

[0054] In a tenth aspect of the disclosure, provided herein is a population of any of the engineered cardiomyocytes described herein. In some embodiments, the population of engineered cardiomyocytes is at least about a 10% less susceptible to NK cell-mediated cytotoxicity as compared to a population of wild-type cardiomyocytes. In some embodiments, the population of engineered cardiomyocytes are at least about a 20% less susceptible to NK cell-mediated cytotoxicity as compared to a population of wild-type cardiomyocytes.

[0055] In an eleventh aspect of this disclosure, provided herein is a pharmaceutical composition comprising any of the populations of engineered cardiomyocytes provided herein, and a pharmaceutical acceptable carrier, excipient, or diluent.

[0056] In a twelfth aspect of the disclosure, provided herein is a method of treating a disease or condition of a subject, the method comprising administering the any of the populations of engineered cardiomyocytes provided herein, or any of pharmaceutical composition including a population of cardiomyocytes provided herein. In some embodiments, the disease or condition comprises heart failure. In some embodiments, the subject is a human.

[0057] In a thirteenth aspect of the disclosure, provided herein is a method for producing a cardiomyocyte with enhanced immune evasion, comprising engineering the cardiomyocyte by: (a) generating an engineered stem cell by: (i) incorporating a heterologous nucleic acid sequence that encodes at least a functional portion of a human leukocyte antigen (HLA) molecule into the genome of a stem cell, or (ii) genetically modifying a stem cell to reduce or eliminate function of a natural-killer group 2, member D (NKG2D) ligand relative to a wildtype cell; and (b) differentiating the engineered stem cell into an engineered cardiomyocyte. In some embodiments, either the incorporating step or the genetically modifying step further enhances the survival probability of the cardiomyocyte when exposed to natural killer cells, as compared to a wild-type cardiomyocyte.

[0058] In some embodiments of this thirteenth aspect, generating the stem cell comprises both the incorporating step and the genetically modifying step.

[0059] In some embodiments of this thirteenth aspect, engineering the stem cell further comprises: genetically modifying the stem cell prior to differentiating to achieve reduced T cell-mediated killing as compared to a wild-type cell.

[0060] In some embodiments of this thirteenth aspect, generating the engineered stem cell further comprises: (i) introducing a deletion or disruption of the P2 microglobulin (B2M) gene; or (ii) introducing a deletion or disruption of one or more genes selected from HLA-A, HLA-B, and HLA-C. In some embodiments, genetically modifying the stem cell to achieve reduced T cell-mediated killing as compared to a wild-type cardiomyocyte comprises introducing a deletion or disruption of the class II major histocompatibility complex transactivator (CIITA) gene.

[0061] In some embodiments of this thirteenth aspect, genetically modifying the stem cell to reduce or eliminate function of the NKG2D ligand relative to a wild-type cardiomyocyte comprises introducing a deletion or disruption of a gene encoding the NKG2D ligand. In some embodiments, the deletion or disruption results in reduced or eliminated expression of the gene encoding the NKG2D ligand.

[0062] In some embodiments of this thirteenth aspect, the NKG2D ligand comprises MICA, MICB, Raetle, Raetlg, Raetll, Ulbpl, Ulbp2, Ulbp3, or any combination thereof. In some embodiments, the NKG2D ligand comprises MICA.

[0063] In some embodiments of this thirteenth aspect, the heterologous nucleic acid sequence encoding at least the functional portion of the HLA-E molecule.

[0064] In some embodiments of this thirteenth aspect, the heterologous nucleic acid sequence encoding at least the functional portion of the HLA-G molecule.

[0065] In some embodiments of this thirteenth aspect, the heterologous nucleic acid sequence encoding at least the functional portion of both the HLA-E molecule and the HLA- G molecule.

[0066] In some embodiments of this thirteenth aspect, the HLA-E molecule and / or the HLA-G molecule encoded by the heterologous nucleic acid sequence comprises a binding peptide. In some embodiments, the binding peptide has an amino acid sequence comprising LIL or LFL. In some embodiments, the binding peptide has an amino acid sequence comprising LFL.

[0067] In some embodiments of this thirteenth aspect, the HLA-E molecule has an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 6.

[0068] In some embodiments of this thirteenth aspect, the heterologous nucleic acid further comprises a sequence encoding at least a functional portion of a P2 microglobulin (B2M). In some embodiments, the functional portion of B2M is linked to the HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of B2M is linked to the HLA-E molecule. In some embodiments, the functional portion of B2M linked to the HLA-E molecule further comprises a linker. In some embodiments, the functional portion of B2M linked to the HLA-E molecule further includes a binding peptide. In someembodiments, the binding peptide has an amino acid sequence comprising LIL or LFL. In some embodiments, the functional portion of B2M linked to the HLA-E molecule has an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 2 or 4.

[0069] In some embodiments of this thirteenth aspect, the incorporating comprises integrating the heterologous nucleic acid sequence into a genomic locus of the stem cell.

[0070] In some embodiments of this thirteenth aspect, the genomic locus is a sustained transgene expression locus (STEL). In some embodiments, the STEL comprises a locus within a human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene.

[0071] In some embodiments of this thirteenth aspect, the method further includes expanding the engineered stem cell prior to the differentiating to produce a population of engineered cardiomyocytes.

[0072] In a fourteenth aspect of the disclosure, provided herein is a kit comprising a dosage form suitable for administration to a subject comprising any of the populations of engineered cells described herein, and instructional material for the use of said dosage form.

[0073] In a fifteenth aspect of the disclosure, provided herein is a kit comprising a dosage form suitable for administration to a subject comprising any of the populations of engineered cardiomyocytes described herein, and instructional material for the use of said dosage form.

[0074] In a sixteenth aspect of the disclosure, provided herein is a kit comprising a dosage form suitable for administration to a subject comprising any of the pharmaceutical compositions described herein, and instructional material for the use of said dosage form.

[0075] In another aspect, this disclosure provides an engineered cell, the engineered cell comprising: a genetic modification that reduces or eliminates function of a natural-killer group 2, member D (NKG2D) ligand as compared to a wild-type cell; and a heterologous nucleic acid sequence encoding a fusion protein comprising at least a portion of a human leukocyte antigen (HLA)-E protein covalently linked to at least a portion of a P2- microglobulin (B2M) protein.

[0076] In some embodiments, at least one of the HLA-E protein or the B2M protein comprises a modification that reduces binding affinity to a CD8 co-receptor as compared to acorresponding wild-type protein. In some embodiments, the B2M protein comprises the modification that reduces binding affinity to a CD8 co-receptor.

[0077] In some embodiments, the heterologous nucleic acid sequence encodes an amino acid sequence having at least 95% identity to one of SEQ ID NO. 2, 4, 6, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30. In some embodiments, the amino acid sequence comprises SEQ ID NO: 18.

[0078] In some embodiments, the engineered cell further comprises one or more genetic modifications that result in reduced T cell-mediated killing as compared to a wild-type cell, the one or more genetic modifications comprising: (i) a deletion or disruption of a P2 microglobulin (B2M) gene; (ii) a deletion or disruption of one or more of an HLA-A gene, an HLA-B gene, and an HLA-C gene; or (iii) a deletion or disruption of a class II, major histocompatibility complex, transactivator (CIITA) gene.

[0079] In some embodiments, wherein the genetic modification that reduces or eliminates function of a NKG2D ligand as compared to a wild-type cell is a deletion or disruption of a gene encoding a NKG2D ligand. In some embodiments, the NKG2D ligand comprises MICA, MICB, Raetle, Raetlg, Raetll, Ulbpl, Ulbp2, Ulbp3, or any combination thereof. In some embodiments, the NKG2D ligand comprises MICA.

[0080] In some embodiments, the HLA-E protein encoded by the heterologous nucleic acid sequence comprises a binding peptide. In some embodiments, the binding peptide comprises an amino acid sequence comprising LIL or LFL. In some embodiments, the binding peptide has the amino acid sequence comprising LFL.

[0081] In some embodiments, the HLA-E protein is covalently linked to the B2M protein by a linker.

[0082] In some embodiments, expression of the heterologous nucleic acid sequence is driven by an endogenous gene promoter in the engineered cell. In some embodiments, the heterologous nucleic acid sequence is integrated into a sustained transgene expression locus (STEL) in the engineered cell. In some embodiments, the STEL comprises a locus within a human glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) gene. In some embodiments, the engineered cell further comprises a kill switch.

[0083] In some embodiments, the engineered cell comprises a stem cell. In some embodiments, the engineered cell is capable of differentiating into a cardiomyocyte. In some embodiments, the engineered cell comprises a cardiomyocyte, a neural cell, a myeloid cell, a T cell, or a retinal cell.

[0084] In some embodiments, the reduced binding affinity to the CD8 co-receptor comprises at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% reduction in binding affinity to the CD8 co-receptor relative to a comparable protein.

[0085] In some embodiments, the cardiomyocyte exhibits reduced natural killer (NK) cell-mediated cytotoxicity compared to a non-engineered cardiomyocyte.

[0086] In another aspect, this disclosure provides a method for producing an engineered cardiomyocyte with reduced NK cell-mediated cytotoxicity, the method comprising: providing an engineered cell as described herein; and inducing differentiation of the engineered cell into an engineered cardiomyocyte, wherein the resulting engineered cardiomyocyte exhibits reduced NK cell-mediated cytotoxicity as compared to a wild-type cardiomyocyte.

[0087] In another aspect, this disclosure provides a population of engineered cells derived from an engineered cell as described herein.

[0088] In some embodiments, the population of engineered cells are at least about 10% less susceptible to NK cell-mediated cytotoxicity as compared to a population of wild-type cells.

[0089] In some embodiments, the population of engineered cells are at least about 20% less susceptible to NK cell-mediated cytotoxicity as compared to a population of wild-type cells.

[0090] In another aspect, this disclosure provides a pharmaceutical composition comprising a population of engineered cells as described herein, and a pharmaceutical acceptable carrier, excipient, or diluent.

[0091] In another aspect, this disclosure provides a method comprising administering a population of engineered cells as described herein, or a composition as described herein.

[0092] In some embodiments, the disease or condition comprises heart failure, Parkinson’s disease, multiple sclerosis, irritable bowel syndrome, type 1 diabetes, rheumatoid arthritis, Alzheimer’s disease, or neural inflammation. In some embodiments, the subject is a human.

[0093] In another aspect, this disclosure provides a method for enhancing immune evasion of a cell, or progeny thereof, comprising engineering the cell by: genetically modifying the cell to reduce or eliminate function of a natural-killer group 2, member D (NKG2D) ligand relative to a wild-type cell, incorporating a heterologous nucleic acid sequence that encodes at least a functional portion of a human leukocyte antigen (HLA)-E molecule covalently linked to a p2-microglobulin (B2M) molecule into the genome of the cell, wherein the genetically modifying step enhances the survival probability of the cell when exposed to natural killer cells, as compared to a wild-type cell.

[0094] In some embodiments, at least one of the HLA-E molecule or the B2M molecule comprises a modification that reduces binding affinity to a CD8 co-receptor as compared to a corresponding wild-type molecule. In some embodiments, the B2M molecule comprises the modification that reduces binding affinity to a CD8 co-receptor. In some embodiments, the heterologous nucleic acid encodes an amino acid sequence having at least 95% identity to one of SEQ ID NO. 2, 4, 6, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30. In some embodiments, the amino acid sequence has at least 95% identity to SEQ ID NO. 18.

[0095] In some embodiments, the method further comprises engineering the cell with one or more genetic modifications that result in reduced T cell-mediated killing as compared to a wild-type cell, the one or more genetic modifications comprising: (i) a deletion or disruption of a P2 microglobulin (B2M) gene; (ii) a deletion or disruption of one or more of an HLA-A gene, an HLA-B gene, and an HLA-C gene; or (iii) a deletion or disruption of a class II, major histocompatibility complex, transactivator (CIITA) gene.

[0096] In some embodiments, the genetic modification that reduces or eliminates function of a NKG2D ligand as compared to a wild-type cell is a deletion or disruption of a gene encoding the NKG2D ligand. In some embodiments, the NKG2D ligand comprises MICA, MICB, Raetle, Raetlg, Raetll, Ulbpl, Ulbp2, Ulbp3, or any combination thereof. In some embodiments, the NKG2D ligand comprises MICA.

[0097] In some embodiments, the HLA-E molecule encoded by the heterologous nucleic acid sequence comprises a binding peptide. In some embodiments, the binding peptide comprises an amino acid sequence comprising LIL or LFL. In some embodiments, the binding peptide has the amino acid sequence comprising LFL. In some embodiments, the HLA-E molecule is covalently linked to the B2M molecule by a linker.

[0098] In some embodiments, the incorporating comprises integrating the heterologous nucleic acid sequence into a genomic locus of the cell. In some embodiments, the genomic locus is a sustained transgene expression locus (STEL). In some embodiments, the STEL comprises a locus within a human glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) gene.

[0099] In some embodiments, the method further comprises expanding the engineered cell to produce a population of engineered cells.

[0100] In some embodiments, the method further comprises differentiating the population of engineered cells into a population of cardiac cells, neural cells, T cells, retinal cells, or myeloid cells.

[0101] In another aspect this disclosure provides a pharmaceutical composition comprising a population of engineered cells produced by a method described herein, and a pharmaceutical acceptable carrier, excipient, or dilutant.

[0102] In another aspect, this disclosure provides a kit comprising a dosage form suitable for administration to a subject comprising a population of engineered cells as described herein, and instructional material for the use of said dosage form.

[0103] In another aspect, this disclosure provides an engineered cell, the engineered cell comprising: a genetic modification that reduces or eliminates function of a natural-killer group 2, member D (NKG2D) ligand as compared to a wild-type cell; and an amino acidsequence encoding at least a portion of a human leukocyte antigen (HLA)-E protein covalently linked to at least a portion of a P2-microglobulin (B2M) protein. In some embodiments, at least one of the HLA-E protein or the B2M protein comprises a modification that, when expressed on the surface of the engineered cell, reduces binding affinity to a CD8 co-receptor as compared to a corresponding wild-type protein. In some embodiments, the B2M protein comprises the modification that reduces binding affinity to a CD8 co-receptor. In some embodiments, the amino acid comprises a sequence having at least 95% identity to one of SEQ ID NO. 2, 4, 6, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30. In some embodiments, the amino acid sequence comprises SEQ ID NO: 18.BRIEF DESCRIPTION OF DRAWINGS

[0104] FIG. 1 is a schematic of an exemplary strategy for integrating a heterologous nucleic acid encoding a polypeptide of interest (e.g., a B2M-HLA-E or a B2M-HLA-G molecule having a binding peptide containing LIL or LFL motifs) into a sustained transgene expression locus (STEL; e.g., in-frame to an endogenous GAP DH gene before the STOP codon at the 3’UTR) in the genome of a cell. Expression of the polypeptide of interest can then be driven by an endogenous gene promoter (e.g., a GAPDH promoter).

[0105] FIG. 2 shows exemplary results of natural killer (NK) cell cytotoxicity evaluations of engineered cardiomyocytes. Engineered cardiomyocytes with & B2M knockout (B2M KO) and: 1) a heterologous nucleic acid encoding a B2M-HLA-E molecule including a binding peptide containing a LIL motif (HLA-E (LIL)), 2) a heterologous nucleic acid encoding a B2M-HLA-E molecule including a binding peptide containing a LFL motif (HLA-E (LFL)), 3) a MICA knockout (MICA KO), 4) a heterologous nucleic acid encoding a B2M-HLA-E molecule including a binding peptide containing a LIL motif in combination with MICA knockout (MIC A KO+B2M-HLA-E (LIL)), or a heterologous nucleic acid encoding a B2M-HLA-E molecule including a binding peptide containing a LFL motif in combination with MICA knockout (MIC A KO+HL A-E (LFL)), were challenged with one of seven distinct populations of NK cells (pNKl, pNK2, pNK3, pNK4, pNK5, pNK6 and pNK7). Engineered cardiomyocytes with a B2M knockout ( 2 K0) were used as control cells. Each data point represents an average from different donor NK cells. The flat line represents the median from the cytotoxicity resulting from all donor NK cells. The percentNK cell specific cytotoxicity is identified along the y axis. As used in the figure: NS, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0106] FIGS. 3A-3C are exemplary experimental results of in vivo NK cell killing assays for luciferase-expressing cardiomyocytes with &B2M KO (control cells; FIG. 3A), luciferase-expressing cardiomyocytes engineered with &B2M KO, MICA KO, and a B2M- HLA-E (LIE) knock-in (KI) (FIG. 3B), and luciferase-expressing cardiomyocytes engineered with a B2M KO, MICA KO, and a B2M-HLA-E (LFL) KI (FIG. 3C), alone and in the presence of donor NK cells (donor 1 or donor 2). Luciferase expression is indicated as total photon flux along they axis, while time (days) is indicated along the x axis.

[0107] FIGS. 4A-4C are exemplary luminescence images taken from mice at day 8 of the NK cell killing assays described in Example 3. FIG. 4A shows luminescence images of mice transplanted with control cells (cardiomyocytes with a B2 KO only), control cells transplanted with donor 1 NK cells (DI NK), and control cells transplanted with donor 2 NK cells (D2 NK). FIG. 4B shows luminescence images of mice transplanted with cardiomyocytes engineered with 2Af KO, MICA KO, and B2M-HLA-E (LIL) KI. FIG. 4C shows luminescence images of mice transplanted with cardiomyocytes engineered with B2M KO, MICA KO, and B2M-HLA-E (LFL) KI.

[0108] FIGS. 5A and 5B show exemplary experimental results on the levels of in vivo NK cell-mediated cytotoxicity of cardiomyocytes engineered with a B2M KO, MICA KO, and B2M-HLA-E (LIL) KI, as compared to control cells. FIG. 5A shows exemplary data of luciferase of control cells (luciferase-expressing cardiomyocytes with 2Af KO only) and control cells with Donor 1 NK cells or Donor 2 NK cells of over 8 days. FIG. 5B show exemplary experimental results of luminescence measured from cardiomyocytes engineered with B2M KO, MICA KO, and B2M-HLA-E (LIL) KI (labeled as MICA KO (LIL)) cells alone or in combination with Donor 1 NK or Donor 2 NK cells, as indicated over 8 days. Luciferase (total proton flux) is identified along the y axis. Time is indicated along the x axis

[0109] FIG. 6 shows exemplary experimental results of CD8 T cell proliferation following coculture with either wild type cells or engineered cells. In particular, FIG. 6 shows a comparison of the percentages of proliferating CD8 T cells following coculture across four conditions: wild-type cells (WT), p2-microglobulin knockout cells (B2M KO), B2M knockout cells with HLA-E knock-in (B2M KO HLA-E KI (with LIL bindingpeptide)), and T cells cultured alone. Each bar represents the mean percentage of proliferating CD8 T cells, with datapoints (black triangles) indicating the mean of data from different donor T cells. Error bars indicate standard deviation of mean.

[0110] FIG. 7 shows exemplary experimental results showing CD8 T cell proliferation following coculture with either wild-type or engineered cardiomyocytes (CMs). In particular, FIG. 7 shows a comparison of CD8 T cell proliferation percentages across four test groups: wild-type (WT), B2M knockout (B2M KO), B2M knockout with HLA-E (LIL) KI (B2M KO+HLA-E (LIL)), and T cells cultured alone (Alone). For each test group, two different T cell conditions are indicated: untreated (Nothing, black triangles) and treated with recombinant CD8a (+rCD8, white circles). Each bar represents the mean percentage of proliferating CD8 T cells, with individual datapoints from 1 donor overlaid. Error bars indicate standard deviation of the mean.

[0111] FIG. 8 shows exemplary experimental results showing CD8 T cell proliferation following coculture with engineered cardiomyocytes (CMs). In particular, FIG. 8 shows a comparison of CD8 T cell proliferation percentages across four test groups: wild-type (WT), B2M knockout (B2M KO), B2M KO with beads (+Beads), and T cells cultured alone (Alone). For each test group, four different T cell conditions are indicated: untreated (Nothing, black triangles), treated with recombinant CD8a (+CD8, white circles), treated with isotype control antibody (+Isotype, grey squares), and treated with anti-CD8 clone RPAT8 antibody (+RPAT8 ab, white diamonds). Each bar represents the mean percentage of proliferating CD8 T cells, with individual datapoints from 1 donor overlaid. Error bars indicate standard error of mean.

[0112] FIG. 9 shows a ribbon structure representation of an exemplary HLA-E / P2- microglobulin (B2M) molecule that has been modified according to aspects of this disclosure. The HLA-E molecule is depicted in light gray, while the associated B2M is shown in black. The medium gray segment highlights the locations of specific mutations in the alpha-3 domain of HLA-E (D227K and T228A) that reduce CD8 co-receptor binding.

[0113] FIG. 10 shows a ribbon structure representation of an exemplary HLA-E / B2M molecule that has been modified according to aspects of this disclosure. The HLA-E molecule is depicted in medium gray, while the associated B2M is shown in dark gray. The light gray segment in the HLA-E alpha-3 domain highlights the location of the key mutations(D227K and T228A) that reduce CD8 co-receptor binding. Additionally, this figure illustrates a specific modification in the B2M portion of the molecule to further reduce CD8 co-receptor binding, indicated by the light gray stripe on the B2M structure.

[0114] FIG. 11 shows a schematic representation of an exemplary HLA-E-CD8-B2M molecule modified according to aspects of the disclosure. The construct is depicted as a linear arrangement of its components from left to right. The right most segment represents the HLA-E portion of the construct. The segments labeled “CD8” represents the CD8 portions, e.g., portions encoding CD8a, of the molecule. The segment labeled “B2M” represents the P2-microglobulin portion of the molecule. The segments labeled “L” represent linker sequences. The left most segment labeled “P” represents both the signal peptide and a binding peptide designed to bind the HLA-E peptide-binding groove.

[0115] FIG. 12 shows a ribbon structure representation of an exemplary engineered HLA-E-CD8-B2M molecule. The fusion protein includes a HLA-E segment, with its associated P2-microglobulin (B2M), two CD8a segments (“CD8a dimer”) and a binding polypeptide.DETAILED DESCRIPTION

[0116] Aspects of the present disclosure relate to methods, populations of engineered cells, pharmaceutical compositions, and kits that include an engineered cell that has increased survival when challenged with immune cells, such as, natural killer (NK) cells or T cells. In particular, the methods, populations of engineered cells, pharmaceutical compositions, and kits include an engineered cell that include certain genetic modifications that experimental results herein show reduce NK cell-mediated cytotoxicity.

[0117] The immune response has an important role for the identification and elimination of foreign agents, such as pathogens or foreign antigens. For example, the presence of foreign antigens causes immune cells to mount an immune response against the foreign antigen. Presentation of foreign antigens on Major Histocompatibility Complex (MHC) molecules target a cell for killing by NK cells and cytotoxic T cells, thus clearing the foreign antigen. While NK cell and T cell cytotoxicity functions normally to eliminate pathogen-infected cells, tumor cells, and other deleterious agents, NK cell, and T cells can also targettherapeutic agents such as cell therapies for clearance, thus reducing the therapeutic benefit conceded by these therapies. Thus, designing therapies that can overcome NK cell and T cell cytotoxicity is important to maintain an optimal therapeutic benefit.

[0118] In the embodiments described herein, provided are engineered cells that have increased immune evasion. Said engineered cells can be genetically modified to reduce or eliminate function of specific NKG2D ligands, such as MICA, which are recognized by NK cells. The engineered cells can alternatively or additionally be modified to express an HLA class I molecule (e.g., HLA-E or HLA-G molecules). In some embodiments, the engineered cells can further include one or more modifications that reduced T cell-mediated killing, such as a deletion of a f microglobulin (B2M) gene. In some embodiments, the engineered cells include a B2M knockout (KO), a MICA KO, and an HLA-E molecule knock in (KI). In some embodiments, the HLA-E molecule is linked to at least a portion of an exogenous B2M sequence (B2M-HLA-E). In some embodiments, the B2M-HLA-E molecule further includes a binding peptide. In some embodiments, the binding peptide, in complex with the HLA-E molecule, is capable of binding to an NKG2A (the inhibitory receptor) of NK cells, for instance, the NKG2A / CD94 receptor. Together, these genetic modifications are demonstrated herein to inhibit NK cell and T cell cytotoxicity.

[0119] Accordingly, this disclosure reveals specific genetic modifications that can be performed with induced pluripotent stem cells (iPSCs) to reduce T cell and NK cell cytotoxicity. Experiments show these engineered iPSCs can differentiate into cardiomyocytes, which was previously uncertain due to potential unintended impacts of genetic modifications on differentiation pathways. However, the evidence confirms that the engineered iPSCs, as described herein, not only differentiate into cardiomyocytes but also retain functionality and effectively resist T and NK cell attacks.

[0120] By inhibiting NK cell cytotoxicity, the engineered cells have increased survival after administration into a subject. In some aspects of the embodiments described herein, the engineered cells can also include additional modifications that reduce killing of the cells by T cells, thus further increasing their survival in a subject. The increased survival of the engineered cells described herein lead to higher half-life of the cells when administered to a subject. By increasing the half-life of the engineered cells in a subject, the engineered cells can confer higher therapeutic benefit in the treatment of a disease or disorder in the subject.

[0121] Furthermore, this disclosure provides Human Leukocyte Antigen (HLA) molecules genetically modified for reducing T cell activation while maintaining NK cell inhibition. For example, this disclosure provides engineered HLA-E molecules that include specific mutations to disrupt CD8 T cell binding while reducing NK cell cytotoxicity. The engineered molecules incorporate specific mutations in the alpha-3 domain that disrupt CD8 co-receptor binding while preserving the overall structure and function necessary for NK cell inhibition. This disclosure also provides cells engineered to express these modified HLA (e.g., modified HLA-E) molecules, particularly in the context of P2-microglobulin (B2M) knockout backgrounds, as well as methods for their production and use in cell therapy applications.

[0122] The engineered HLA-E molecules of the disclosure can include various modifications to reduce T cell activation. In some embodiments, these modifications comprise amino acid substitutions at positions 227 and 228 of the HLA-E protein (D227K and T228A), which specifically disrupt the CD8 binding interface. Additional embodiments include modifications to the associated B2M protein, such as a K58E substitution, which reduces CD8 binding while maintaining proper protein folding and NK cell inhibitory function. Alternative embodiments include fusion constructs incorporating CD8 blocking polypeptides strategically positioned to block CD8 co-receptor binding sites, thereby preventing T cell activation through steric hindrance.

[0123] Although the disclosure describes various exemplary alternatives and implementations as provided herein, it should be understood that the various features, aspects, and functionality described in one or more of the individual alternatives are not limited in their applicability to the particular alternative with which they are described. Instead, they can be applied alone or in various combinations to one or more of the other alternatives of the disclosure, whether the alternatives are described or whether the features are presented as a part of the described alternative. The breadth and scope of the present disclosure should not be limited by any exemplary alternatives described or shown herein.I. Definitions

[0124] The following definitions supplement those in the art and are directed to the present disclosure only. The following definitions are not to be imputed to any related orunrelated case, e.g., to any commonly owned patent or patent application. Although some methods and materials similar or equivalent to those described herein can be used to practice features of the disclosure, some preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0125] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0126] It should be understood that the embodiments disclosed herein is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present embodiments, which is defined solely by the claims.

[0127] As used herein, the articles “a,” “an,” and “the” are used herein to refer to one or to more than one (z.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0128] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.

[0129] As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In some instances, the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0130] As used herein, the terms “administration,” “administering” and variants thereof refer to the introduction of a composition or therapeutic agent (e.g., a population of cells) intoa subject. Administration includes concurrent and sequential introduction of the composition or therapeutic agent. Administration of the composition or therapeutic agent (e.g., a population of cells) into a subject is by any suitable route, including orally, pulmonarily, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, surgically, or topically. A suitable route of administration allows the composition or the agent to perform its intended function. Administration also includes self-administration and the administration by another. The administration can also be performed systemic, or it can be local. For instance, a composition or therapeutic agent (e.g., a population of cells) can be administered locally, e.g., by local injection into a tissue.

[0131] As used herein, the term “and / or” should be understood to mean either one, or both of, or any combination of the alternatives.

[0132] As used herein, the terms “P2 microglobulin” and “B2M” are used interchangeably to refer to a polypeptide encoded by the B2M gene. B2M is a component of class I Major Histocompatibility Complexes (MHCs), and forms a complex with MHC class I human leukocyte antigens (HLAs). B2M is also known as IMD43; beta-2-microglobulin; and B2 microglobulin. In some instances, the B2M is a human B2M. An exemplary B2M gene is available as Gene ID: 567 (e.g., available at the website www[dot]ncbi[dot]nlm[dot]nih[dot]gov / gene / 567). An exemplary nucleotide sequence that can be used to generate B2M is available as Transcript ID NM_004048.4 (e.g., available at the website www[dot]ncbi[dot]nlm[dot]nih[dot]gov / nuccore / NM_004048.4).

[0133] As used herein, the term “binding peptide” refers to an amino acid sequence capable of binding to a peptide groove of an HLA molecule, such as an HLA-E molecule, to form a ligand. The ligand can interact with an inhibitory receptor on a NK cell, for example, the NKG2A / CD94 receptor. In some instances, the structure of the binding peptide can be characterized by specific amino acid sequences, such as VMAPRTLIL (SEQ ID NO: 8) (LIL) or VMAPRTLFL (SEQ ID NO: 10) (LFL), which can have important implications for its function. A binding peptide is typically located at the N-terminus of a protein or amino acid molecule, but can also be located at the C-terminus or even internally.

[0134] As used herein, the term “conservative substitution” refers to the replacement of one amino acid for another such that the replacement takes place within a family of aminoacids that are related in their side chains. Alternatively, the term “non-conservative substitution” refers to the replacement of one amino acid residue for another such that the replaced residue is going from one family of amino acids to a different family of residues. Genetically encoded amino acids can be divided into four families: (1) acidic (negatively charged) = Asp (D), Glu (E); (2) basic (positively charged) = Lys (K), Arg (R), His (H); (3) non-polar (hydrophobic) = Cys (C), Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Met(M), Trp (W), Gly (G), Tyr (Y), with non-polar also being subdivided into: (i) strongly hydrophobic = Ala (A), Vai (V), Leu (L), He (I), Met (M), Phe (F); and (ii) moderately hydrophobic = Gly (G), Pro (P), Cys (C), Tyr (Y), Trp (W); and (4) uncharged polar = Asn(N), Gin (Q), Ser (S), Thr (T). In alternative fashion, the amino acid repertoire can be grouped as (1) acidic (negatively charged) = Asp (D), Glu (E); (2) basic (positively charged) = Lys (K), Arg (R), His (H), and (3) aliphatic = Gly (G), Ala (A), Vai (V), Leu (L), He (I), Ser (S), Thr (T), with Ser (S) and Thr (T) optionally being grouped separately as aliphatic- hydroxyl; (4) aromatic = Phe (F), Tyr (Y), Trp (W); (5) amide = Asn (N), Glu (E); and (6) sulfur-containing = Cys (C) and Met (M) (see, for example, Biochemistry, 4th ed., Ed. by L. Stryer, WH Freeman and Co., 1995, which is incorporated by reference herein in its entirety).

[0135] As used herein, the term “deletion” refers to a genetic alteration in which all or part of a gene is removed from the genome of a cell. The deletion can include removal of any number of nucleotides, including up to the entire gene. The deletion can be a complete deletion, in which an entire gene is removed (i.e., a gene knockout), or it can be a partial deletion, in which only part of a gene (e.g., a promoter, one or more exons, a regulatory region, etc.) is removed. The deletion can result in a complete loss of function of a protein encoded by a gene, or a partial loss of function or attenuation of the protein encoded by the gene. The deletion can result in a complete loss of expression of a gene (e.g., eliminate expression), or a partial loss of expression the gene.

[0136] As used herein, the term “disruption” refers to a genetic alteration that is a change in sequence of a gene in the genome of a cell. A disruption can include a change in the sequence of a gene that results in reduced or attenuated function of the encoded protein and / or expression of the gene. A disruption can include, but is not limited to, insertions or substitutions of one or more nucleotides in the sequence of a gene.

[0137] As used herein, the term “cardiac cell” refers to any cell present in the heart that provides a cardiac function, such as heart contraction or blood supply, or otherwise serves tomaintain the structure of the heart. The cardiac cell may be a cell of the epicardium, myocardium or endocardium of the heart. Cardiac cells also include, but are not limited to, cardiac muscle cells or cardiomyocytes, and cells of the cardiac vasculatures, such as cells of a coronary artery or vein. Other non-limiting examples of cardiac cells include epithelial cells, endothelial cells, fibroblasts, cardiac stem or progenitor cells, cardiac conducting cells and cardiac pacemaking cells that constitute the cardiac muscle, blood vessels and cardiac cell supporting structure.

[0138] As used herein, the terms “class II, major histocompatibility complex, transactivator” and “CIITA” are used interchangeably to refer to a polypeptide encoded by the CIITA gene. CIITA is also known as C2TA; CIITAIV; MHC2TA; NLR family, acid domain containing; NLRA; and nucleotide-binding oligomerization domain, leucine rich repeat and acid domain containing. In some instances, the CIITA is a human CIITA. An exemplary CIITA gene is available as Gene ID: 4261 (e.g., available at the website www[dot]ncbi[dot]nlm[dot]nih[dot]gov / gene / 4261). An exemplary nucleotide sequence that can be used to generate CIITA is available as Transcript ID NM 000246.4 (e.g., available at the website www[dot]ncbi[dot]nlm[dot]nih[dot]gov / nuccore / NM_000246.4).

[0139] As used herein, the terms “decrease,” “reduce,” and “inhibit” are interchangeable and refer to any statistically significant reduction in biological activity (e.g., NK cell- mediated toxicity) of a reference protein or cell. For example, an inhibition in biological activity can refer to a reduction of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in biological activity as compared to a control.

[0140] As used herein, the term “differentiation,” and its grammatical equivalents, refers to a process by which a stem cell or progenitor cell alters from one cell type to a more specialized cell type. Each specialized cell type in an organism can express a subset of all the genes that constitute the genome of the cell. Each cell type can be defined by its particular pattern of regulated gene expression. Cell differentiation can thus be described as a transition of a cell from one cell type to another cell type coincident with a switch from one pattern of gene expression to another.

[0141] As used herein, the term “dosage form” refers to a discrete amount of a composition comprising a predetermined amount of the active ingredient (e.g., a population of cells). The amount of the active ingredient is generally equal to the dosage of the activeingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. The relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and any additional ingredients in a pharmaceutical composition will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. The dosage form can further include one or more additional pharmaceutically active agents. In some cases, the dosage form is for administration by injection into a subject.

[0142] As used herein, the term “encoding” refers to the property of specific sequences of nucleotides in a nucleic acid, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (z.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene or at least the exons of a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0143] As used herein, the term “endogenous” refers to a gene, nucleic acid, polypeptide, etc., that is normally present in a particular cell. For example, an endogenous gene may be a gene that is normally present in the genome of a cell.

[0144] As used herein, the term “expression” refers to the transcription and / or translation of a particular nucleotide sequence in a cell.

[0145] As used herein, the terms “enhance” and “increase” are interchangeable and refer to any statistically significant increase in biological activity (e.g., survival) of a reference protein or cell. For example, an increase in biological activity can refer to an increase of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in biological activity as compared to a control.

[0146] As used herein, a “functional portion,” when used in reference to a polypeptide, refers to a subset of the amino acid sequence of said polypeptide that is sufficient to confer a desired biological activity (e.g., reduce or inhibit natural killer (NK) cell-mediated cytotoxicity). The functional portion of the polypeptide can include any subset(s) of aminoacid sequences of the polypeptide that confer the desired biological activity. For instance, the functional portion can include one or more domains necessary for the desired biological activity of the polypeptide.

[0147] As used herein, the term, “fused” refers to at least two sequences that are connected together, such as by a covalent bond (e.g., an amide bond or a phosphodiester bond) and / or by a linker. The covalent bond can be formed by a conjugation (e.g., chemical conjugation or enzymatic conjugation) reaction. Exemplary linkers are further described herein. A fusion protein is a protein created by joining two or more genes, originally coding for separate proteins, into a single gene that is then translated into a single protein.

[0148] As used herein, the term “genetic modification” or “genetic alteration” refers to a change at the DNA level of a cell. A genetic modification includes an insertion, deletion, or substitution, typically within a defined sequence or genomic locus. In some instances, the genetic modification can be a deletion or disruption of a gene that results in a reduction or loss of expression of the encoded gene product. In other instances, the genetic modification includes the integration of a nucleotide sequence heterologous to the genomic locus. The genetic modification can be at a single nucleotide position or at multiple nucleotides, e.g., 2, 3, 4, 5 or more nucleotides, typically in close proximity to each other, e.g., contiguous nucleotides. Depending on the nature of the genetic modification, the expression of a gene that has been genetically modified can be up-regulated or down-regulated.

[0149] As used herein, the term “genetically modified” or “engineered” when used in the context of a cell refers to a cell that includes one or more genetic modifications, and which is not found in nature. The engineered cells can be made by any method known in the art, such as by manipulating the genome of the cell or inserting a new nucleic acid into the cell. For instance, a cell can be modified by integrating a nucleic encoding a gene of interest into a cell using a genome editing technique, such as a CRISPR / Cas system.

[0150] As used herein, the term “heart failure” refers to a disease characterized by the inability of the heart to pump sufficient blood to fulfill the needs of the tissues and organs of the body. Heart failure can be caused by an abnormally low cardiac output.

[0151] As used herein, the term “heterologous,” when used in reference to a nucleic acid and / or polypeptide that is introduced to a host cell, refers a nucleic acid and / or polypeptide that is not naturally found in the host cell or naturally found at a given position in the genomeof the host cells. For example, a construct is heterologous to a host cell if it contains some homologous sequences arranged in a manner not found in the host cell and / or the construct contains some heterologous sequences not found in the host cell.

[0152] As used herein, the terms “human leukocyte antigen” and “HLA” are used interchangeably to refer to a subunit of Major Histocompatibility Complexes (MHCs). MHCs are glycoproteins that presents antigens to immune cells, such as T cells. Class I MHCs are expressed in most nucleated cells and interact with CD8 receptors on the surface of T cells. Class I MHCs are composed of a class I HLA molecule and P2 microglobulin (B2M). Class II MHCs are usually present only in antigen presenting cells and interact with CD4 receptors on the surface of T cells. Class II MHCs are heterodimers of two Class II HLAs.

[0153] As used herein, the terms “human leukocyte antigen-A” and “HLA-A” are used interchangeably to refer to a polypeptide encoded by an HLA-A gene. HLA-A is a class I HLA. HLA-A is also known as HLAA. An exemplary HLA-A gene is available as Gene ID: 3105 (e.g., available at the website www[dot]ncbi[dot]nlm[dot]nih[dot]gov / gene / 3105). There are thousands of known alleles of the HLA-A gene, any one of which is encompassed by the term.

[0154] As used herein, the terms “human leukocyte antigen-B” and “HLA-B” are used interchangeably to refer to a polypeptide encoded by an HLA-B gene. HLA-B is a class I HLA. HLA-B is also known as AS, HLAB; and B-4901. An exemplary HLA-B gene is available as Gene ID: 3106 (e.g., available at the website www[dot]ncbi[dot]nlm[dot]nih[dot]gov / gene / 3106). There are thousands of known alleles of the HLA-B gene, any one of which is encompassed by the term.

[0155] As used herein, the terms “human leukocyte antigen-C” and “HLA-C” are used interchangeably to refer to a polypeptide encoded by an HLA-C gene. HLA-C is a class I HLA. HLA-C is also known as D6S204; HLA-JY3; HLAC; HLC-C; and PSORS1. An exemplary HLA-C gene is available as Gene ID: 3107 (e.g., available at the website www[dot]ncbi[dot]nlm[dot]nih[dot]gov / gene / 3107). There are thousands of known alleles of the HLA-C gene, any one of which is encompassed by the term.

[0156] As used herein, the terms “human leukocyte antigen-E” and “HLA-E” are used interchangeably to refer to a polypeptide encoded by an HLA-E gene. HLA-E is a class I HLA. HLA-E is also known as: Major Histocompatibility Complex, Class I, E; HLA Class IHistocompatibility Antigen, Alpha Chain E; MHC Class I Antigen E; QA1; HLA-6.2; or HLAE. An exemplary HLA-E gene is available as Gene ID: 3133 (e.g., available at the website www[dot]ncbi[dot]nlm[dot]nih[dot]gov / gene / 3133). There are many known alleles of the HLA-E gene, any one of which is encompassed by the term.

[0157] As used herein, the terms “human leukocyte antigen-G” and “HLA-G” are used interchangeably to refer to a polypeptide encoded by an HLA-G gene. HLA-G is a class I HLA. HLA-G is also known as: MHC-G. An exemplary HLA-G gene is available as Gene ID: 3135 (e.g., available at the website www[dot]ncbi[dot]nlm[dot]nih[dot]gov / gene / 3135). There are dozens of known alleles of the HLA-G gene, any one of which is encompassed by the term.

[0158] As used herein, the term “immune cell” refers to a cell of hematopoietic origin functionally involved in the initiation and / or execution of an immune response in an organism. An immune cell can be part of the innate and / or adaptive immune system. Exemplary immune cells include, but are not limited to, cells of the myeloid lineage (e.g., neutrophils, dendritic cells, eosinophils, mast cells, basophils, monocytes, microglia, and precursors thereof), as well as cells of the lymphoid lineage (e.g., T cells, B cells, NK cells, and precursors thereof).

[0159] As used herein, the term “instructional material” refers to a publication, a recording, a diagram, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of using the compositions associated with the publication, recording, diagram or other medium of expression. The instructional material of a kit of the disclosure can, for example, be affixed to a container that contains the population of cells and / or pharmaceutical composition of the disclosure, or be shipped together with a container that contains the population of cells and / or pharmaceutical composition.Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional material and the compositions be used cooperatively by the recipient.

[0160] As used herein, the term “kill switch” refers to a nucleic acid sequence that when expressed under certain conditions in a host cell causes the host cell to die (e.g., undergo apoptosis) or stop proliferating. Exemplary kill switch nucleic acid sequences include, but are not limited to, nucleotide sequence that encode a herpes simplex virus thymidine kinase(HSV-TK), an inducible caspase9 (incasep 9, iCasp9), CD20, and a mutant human thymidylate kinase (mTMPK). In some instances, the kill switch gene is inducible, wherein the kill switch is only activated upon the addition of an activator, e.g., a small molecule drug. In some instances, the kill switch involves depriving a host cell of an agent required for cell growth or proliferation. For example, in some instances a kill switch involves engineering a host cell such that the host cell is dependent upon an external factor (e.g., uridine) to grow and proliferate. Such engineering may involve reducing or eliminating the expression of a gene encoding uridine monophosphate synthetase (UMPS), thereby preventing the host cell from growing or proliferating when the external factor is not contacted with the host cell.

[0161] As used herein, the term “myeloid cell” refers to a cell of the myeloid lineage. Exemplary myeloid cell types include, but are not limited to, monocytes, microglia, macrophages, dendritic cells, basophils, eosinophils, erythrocytes, mast cells or neutrophils, and any precursor cells or any intermediate progenitor thereof.

[0162] As used herein, the terms “MHC class I polypeptide-related sequence A” and “MICA” are used interchangeably to refer to a polypeptide encoded by a MICA gene. MICA is also known as: MIC-A; and PERBI 1.1. An exemplary MICA gene is available as Gene ID: 100507436 (e.g., available at the website www[dot]ncbi[dot]nlm[dot]nih[dot]gov / gene / 100507436).

[0163] As used herein, the terms “MHC class I polypeptide-related sequence B” and “MICB” are used interchangeably to refer to a polypeptide encoded by a. MICB gene. MICB is also known as: PERBI 1.2. An exemplary MICB gene is available as Gene ID: 4277 (e.g., available at the website www[dot]ncbi[dot]nlm[dot]nih[dot]gov / gene / 4277).

[0164] As used herein, the term “natural killer cell” or “NK cell” refers to a type of cytotoxic lymphocyte, part of the innate lymphoid cells, that expresses CD56, also called NCAM1, and / or any of the Natural cytotoxicity triggering receptors, and Killer Cell Lectin Like Receptor Fl, also called NKp80; and lacks expression of CD3. NK cells are generally involved in clearance of cells infected with viruses and other intracellular pathogens, as well as in the removal of tumor cells and senescent cells. “Natural killed cell-mediated cytotoxicity” refers to the killing of cells by NK cells.

[0165] As used herein, the term “a natural-killer group 2, member D ligand” or “NKG2D ligand” refers to a protein that binds and activates signaling through NKG2D. ExemplaryNKG2D ligands include, but are not limited to, MICA, MICB, Raetle, Raetlg, Raetll, Ulbpl, Ulbp2, and Ulbp3.

[0166] As used herein, the term “neural cell” refers to any cells belonging to the nervous system. Exemplary neural cells include, without limitation, neurons and neuron precursor cells (irrespective of any specific neuronal subtype, e.g., including dopaminergic neurons, cortical neurons, spinal or oculomotor neurons, enteric neurons, interneurons, and trigeminal or sensory neurons), microglia and microglia precursor cells, glial cells and glial precursor cells (irrespective of any specific glial subtype, e.g., including oligodendrocytes, astrocytes, dedicated oligodendrocyte precursor cells and bipotent glial precursors, which may give rise to astrocytes and oligodendrocytes), Placode-derived cells, Schwann cells, and satellite cells.

[0167] As used herein, the term “neural inflammation” refers to inflammation of the nervous tissue of the central nervous system, such as the brain or the spinal cord. Neural inflammation can be caused by any means, including but not limited to infection, traumatic brain injury, toxic metabolites, ischemia reperfusion injury or autoimmunity. Neural inflammation can be acute, chronic, or both.

[0168] As used herein, the term “nucleic acid” or “nucleic acid molecule” refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., enantiomeric forms of naturally-occurring nucleotides), or a combination of both. Modified nucleotides can have alterations in sugar moieties and / or in pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like.

[0169] As used herein, the term “Parkinson’s disease” refers to a neurodegenerative disorder characterized by extensive degeneration of dopaminergic neurons in the substantia nigra region in the brain. Parkinson’s disease manifest in changes of both motor and nonmotor functions. Exemplary changes in motor functions that are symptomatic of Parkinson’s disease include, but are not limited to, tremor, hypokinesia, postural instability, abnormal gait and swallowing disturbances. Exemplary changes in non-motor functions that are symptomatic of Parkinson’s disease include, but are not limited to, autonomic and neuropsychiatric disturbances such as anosmia or sleep abnormalities. Parkinson’s disease can be characterized as early Parkinson’s disease, or it can be characterized as advanced Parkinson’s disease, with more severe symptoms in the more advanced stages of the disease.

[0170] As used herein, the terms “patient,” “subject,” “individual,” and the like are used interchangeably and refer to any animal, or cells thereof, whether in vitro or in situ, amenable to the compositions and methods described herein. In some instances, the patient, subject or individual is a human.

[0171] As used herein, the term “pharmaceutically acceptable excipient, carrier or diluent” refers to any material which, when combined with an active ingredient (e.g., a population of cells), allows the ingredient to retain biological activity and is non-reactive with the subject’s immune system. Examples include, but are not limited to, any of the standard pharmaceutical excipients, carriers, or diluents, such as a phosphate buffered saline solution, normal saline, water, emulsions such as oil / water emulsion, and various types of wetting agents.

[0172] As used herein, the terms “polypeptide” and “protein” refer to a polymer of amino acid residues of any length. A polypeptide may include naturally-occurring modifications to one or more of the amino acids in the polymer. For instance, a polypeptide may include disulfide bond formation, glycosylation, lipidation, acetylation, or phosphorylation. A polypeptide may also include amino acid analogs or non-naturally occurring amino acids. Polypeptides may occur as a single chain or as associated chains.

[0173] As used herein, the term “progeny” refers to one or more cells descended from an original cell (e.g., an engineered cell) by cell division and / or differentiation of the original cell. The progeny and the original cell can be the same or different cell type.

[0174] As used herein, the term “promoter,” and its grammatical equivalents, refers to a region of a nucleic acid positioned upstream of a gene where relevant proteins (such as RNA polymerase and transcription factors) bind to initiate transcription of the gene. The promoter can be the promoter of an endogenous house-keeping gene (e.g., GAPDH). The promoter can be a tissue-specific promoter. For example, a promoter of a gene that is turned on or off in certain cell or tissue types. The promoter can be an engineered promoter. For example, the promoter can be engineered to include one or more elements that can enhance or reduce expression of a gene. The one or more elements can include an enhancer.

[0175] As used herein, the terms “retinoic acid early transcript IE” and “Raetle” are used interchangeably to refer to a polypeptide encoded by a Raetle gene. Raetle is also known as: RL-4; LETAL; ULBP4; N2DL-4; NKG2DL4; RAET1E2; and bA350J20.7. An exemplary Raetle gene is available as Gene ID: 135250 (e.g., available at the website www[dot]ncbi[dot]nlm[dot]nih[dot]gov / gene / 135250).

[0176] As used herein, the terms “retinoic acid early transcript 1G” and “Raetlg” are used interchangeably to refer to a polypeptide encoded by a Raetlg gene. Raetlg is also known as: ULBP5. An exemplary Raetlg gene is available as Gene ID: 353091 (e.g., available at the website www[dot]ncbi[dot]nlm[dot]nih[dot]gov / gene / 353091).

[0177] As used herein, the terms “retinoic acid early transcript IL” and “Raetll” are used interchangeably to refer to a polypeptide encoded by a Raetll gene. Raetll is also known as: ULBP6; and RAET1H. An exemplary Raetll gene is available as Gene ID: 154064 (e.g., available at the website www[dot]ncbi[dot]nlm[dot]nih[dot]gov / gene / l 54064).

[0178] Sequence identity, homology or similarity refers to sequence similarity between two polypeptides or between two nucleic acid molecules. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A polypeptide or polypeptide region (or a polynucleotide or polynucleotide region) has a certainpercentage (for example, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of amino acids (or nucleotide bases) are the same in comparing the two sequences. The alignment of two sequences to determine their percent sequence identity can be done using software programs known in the art, such as, for example, those described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999). Preferably, default parameters are used for the alignment. One alignment program well known in the art that can be used is BLAST set to default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the National Center for Biotechnology Information (see also Altschul et al., J. Mol. Biol. 215:403-410 (1990)).

[0179] As used herein, the term “stem cell” refers to a cell with the ability to divide for indefinite periods in culture and to give rise to specialized cells.

[0180] As used herein, the term “substantially” or “essentially” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In some instances, the terms “essentially the same” or “substantially the same” refer a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is about the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0181] As used herein, the term “survival probability” refers to the probability that a cell or population of cells survives following exposure to a given environment or stimuli. For instance, the survival probability can be a function of exposure of the cell or population of cells to NK cell or T cell cytotoxicity.

[0182] As used herein, the term “sustained transgene expression locus” or “STEL” refer to a locus in the genome of an organism that is resistant to silencing of gene expression. For instance, a STEL can be resistant to silencing over time or after changes in cell fate (e.g.,differentiation), such that expression of genes contained in the STEL is sustained. Exemplary STEL include, but are not limited to genes encoding ribosomal subunits, mitochondria proteins, actin proteins, eukaryotic translation factors, and histones. Additional exemplary STEL are described in WO2021072329A1, which is incorporated herein by reference.

[0183] As used herein, the term “T cell” refers to a type of lymphocyte that plays a central role in cell-mediated immunity. T cells may be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. Exemplary T cell types include, but are not limited to, conventional adaptive T cells, which include helper CD4+ T cells (also known as helper T cells), CD8+ T cells (also known as cytotoxic T cells), memory T cells, and regulatory CD4+ T cells (also known as T regs), and innate-like T cells including NK T cells (also known as NKT cells), mucosal associated invariant T cells, and gamma delta T cells. T cells can be naturally occurring or non-natural, e.g., modified T cells, such as CAR-T cells.

[0184] As used herein, the term “treat,” or a grammatical equivalent thereof, refers to a means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0185] As used herein, the terms “ULI 6 binding protein 1” and “Ulbpl” are used interchangeably to refer to a polypeptide encoded by a Ulbpl gene. Ulbpl is also known as: N2DL-1; RAET1I; and NKG2DL1. An exemplary Ulbpl gene is available as Gene ID: 80329 (e.g., available at the website www[dot]ncbi[dot]nlm[dot]nih[dot]gov / gene / 80329).

[0186] As used herein, the terms “ULI 6 binding protein 2” and “Ulbp2” are used interchangeably to refer to a polypeptide encoded by a Ulbp2 gene. Ulbp2 is also known as: N2DL2; RAET1H; RAET1L; NKG2DL2; and ALCAN-alpha. An exemplary Ulbp2 gene is available as Gene ID: 80328 (e.g., available at the website www[dot]ncbi[dot]nlm[dot]nih[dot]gov / gene / 80328).

[0187] As used herein, the terms “ULI 6 binding protein 3” and “Ulbp3” are used interchangeably to refer to a polypeptide encoded by a Ulbp3 gene. Ulbp3 is also known as: N2DL-3; RAET1N; and NKG2DL3. An exemplary Ulbp3 gene is available as Gene ID: 79465 (e.g., available at the website www[dot]ncbi[dot]nlm[dot]nih[dot]gov / gene / 79465).

[0188] As used herein, the term “variant” when used in the context of a polypeptide, protein, nucleic acid, or polynucleotide refers to a polypeptide, protein, nucleic acid, or polynucleotide that includes at least one alteration (e.g., a substitution, a deletion, or an addition of one or more amino acid or nucleotide) relative to the amino acid sequence of a parent polypeptide or protein (e.g., endogenous polypeptide) or the nucleotide sequence of a parent nucleic acid or polynucleotide (e.g., endogenous gene), but the resulting variant polypeptide, protein, nucleic acid, or polynucleotide retains substantially the same function as the parent a polypeptide, protein, nucleic acid, or polynucleotide. The parent sequence of amino acids or nucleic acids can be, for example, a wild-type sequence or a homolog thereof, or a variant of a wild-type sequence or homolog thereof.

[0189] Throughout this disclosure, various aspects of the embodiments can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the embodiments. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example,I, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.II. Engineered cells

[0190] Certain aspects of the disclosure provide for engineered cells having enhanced immune evasion. In some embodiments, the engineered cell includes a genetic modification that reduces or eliminates function of a natural-killer group 2, member D (NKG2D) ligand as compared to a wild-type cell, or a heterologous nucleic acid sequence encoding at least a functional portion of a human leukocyte antigen (HLA) molecule. In some embodiments, the engineered cell includes the genetic modification. In some embodiments, the engineered cell includes the heterologous nucleic acid. In some embodiments, the engineered cell includes both the genetic modification and the heterologous nucleic acid.

[0191] In some embodiments, either the genetic modification or the heterologous nucleic acid is sufficient to reduce or inhibit natural killer (NK) cell-mediated cytotoxicity of theengineered cell, or progeny thereof, as compared to a wild-type cell. In some embodiments, either the genetic modification or the heterologous nucleic acid is sufficient to reduce or inhibit NK cell-mediated cytotoxicity of the engineered cell, or progeny thereof, 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%, or at least 90% as compared to a wild-type cell. In some embodiments, either the genetic modification or the heterologous nucleic acid is sufficient to reduce or inhibit NK cell-mediated cytotoxicity of the engineered cell, or progeny thereof, by at least 10% as compared to a wild-type cell. In some embodiments, either the genetic modification or the heterologous nucleic acid is sufficient to reduce or inhibit NK cell-mediated cytotoxicity of the engineered cell, or progeny thereof, by at least 20% as compared to a wild-type cell. In some embodiments, either the genetic modification or the heterologous nucleic acid is sufficient to reduce or inhibit NK cell-mediated cytotoxicity of the engineered cell, or progeny thereof, by at least 30% as compared to a wild-type cell. In some embodiments, either the genetic modification or the heterologous nucleic acid is sufficient to reduce or inhibit NK cell-mediated cytotoxicity of the engineered cell, or the progeny thereof, by at least 50% as compared to a wild-type cell. In some embodiments, either the genetic modification or the heterologous nucleic acid is sufficient to reduce or inhibit NK cell- mediated cytotoxicity of the engineered cell, or progeny thereof, by at least 80% as compared to a wild-type cell.

[0192] In some embodiments, the genetic modification that reduces or eliminates function of the NKG2D ligand as compared to a wild-type cell is a deletion or disruption of a gene encoding the NKG2D ligand. In some embodiments, the genetic modification that reduces or eliminates function of the NKG2D ligand as compared to a wild-type cell is the deletion of the gene encoding the NKG2D ligand. In some embodiments, the genetic modification that reduces or eliminates function of the NKG2D ligand as compared to a wildtype cell is the disruption of the gene encoding the NKG2D ligand. In some embodiments, the genetic modification that reduces or eliminates function of the NKG2D ligand as compared to a wild-type cell includes a deletion or disruption that results in reduced or eliminated expression of the gene encoding the NKG2D ligand.

[0193] In some embodiments, the NKG2D ligand includes MICA, MICB, Raetle, Raetlg, Raetll, Ulbpl, Ulbp2, Ulbp3, or any combination thereof. In some embodiments, the NKG2D ligand includes MICA. In some embodiments, the NKG2D ligand includes MICB.In some embodiments, the NKG2D ligand includes Raetle. In some embodiments, the NKG2D ligand includes Raetlg. In some embodiments, the NKG2D ligand includes Raetll. In some embodiments, the NKG2D ligand includes Ulbpl. In some embodiments, the NKG2D ligand includes Ulbp2. In some embodiments, the NKG2D ligand includes Ulbp3.

[0194] In some embodiments, the heterologous nucleic acid encodes at least a functional portion of an HLA-E molecule or an HLA-G molecule. In some embodiments, the heterologous nucleic acid sequence encodes at least the functional portion of the HLA-E molecule. In some embodiments, the heterologous nucleic acid sequence encodes at least the functional portion of the HLA-G molecule. In some embodiments, the heterologous nucleic acid sequence encodes both at least the functional portion of both the HLA-E molecule and the HLA-G molecule. In some embodiments, the functional portion of the HLA-E molecule or the HLA-G molecule includes at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or 300 amino acids of the full-length sequence of the HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of the HLA-E molecule or the HLA- G molecule includes at least 50 amino acids of the full-length sequence of the HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of the HLA- E molecule or the HLA-G molecule includes at least 100 amino acids of the full-length sequence of the HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of the HLA-E molecule or the HLA-G molecule includes at least 200 amino acids of the full-length sequence of the HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of the HLA-E molecule or the HLA-G molecule includes at least 300 amino acids of the full-length sequence of the HLA-E molecule or the HLA-G molecule.

[0195] In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 75% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 85% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 90% identity to SEQ IDNO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 95% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 98% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 99% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a portion of an HLA-E molecule that is identical to SEQ ID NO: 6.

[0196] In some embodiments, the heterologous nucleic acid encodes at least a functional portion of an HLA molecule that includes a binding peptide. Exemplary binding peptides include, but are not limited to, binding peptides having the amino acid sequence VMAPRTLFL (SEQ ID NO: 10) or VMAPRTLIL (SEQ ID NO: 8). Other exemplary binding peptides are described in, for example, Michaelsson, J., et al. J Exp Med. 2002 Dec2; 196(11): 1403-14; Miller, J. D., et al. J Immunol 1 August 2003; 171 (3): 1369-1375; and Lee, N., et al. J Immunol 15 May 1998; 160 (10): 4951-4960. In some embodiments, the binding peptide has an amino acid sequence that includes LIL or LFL. Exemplary binding peptides that include LIL or LFL include, but are not limited to, binding peptides having the amino acid sequence VMAPRTLIL (SEQ ID NO: 8) or VMAPRTLFL (SEQ ID NO: 10). In some embodiments, the binding peptide has an amino acid sequence corresponding to any one of SEQ ID NOs. 8 or 10. In some embodiments, the HLA molecule that includes the binding peptide is an HLA-E molecule or an HLA-G molecule.

[0197] In some embodiments, the heterologous nucleic acid further includes a sequence encoding at least a functional portion of a P2 microglobulin (B2M). In some embodiments, the functional portion of the B2M includes at least 10, 20, 30, 40, 50, 60, 70, 90 or 100 amino acids of the full-length sequence of the B2M. In some embodiments, the functional portion of the B2M includes at least 10 amino acids of the full-length sequence of the B2M. In some embodiments, the functional portion of the B2M includes at least 30 amino acids of the full- length sequence of the B2M. In some embodiments, the functional portion of the B2M includes at least 50 amino acids of the full-length sequence of the B2M. In some embodiments, the functional portion of the B2M includes at least 90 amino acids of the full- length sequence of the B2M. In some embodiments, the functional portion of the B2M includes an extracellular domain of the B2M. In some embodiments, the functional portion ofB2M is linked to the HLA-E molecule or the HLA-G molecule encoded by the heterologous nucleic acid. In some embodiments, the functional portion of B2M is linked to the HLA-E molecule. In some embodiments, the functional portion of B2M is linked to the HLA-G molecule. In some embodiments, the functional portion of B2M is linked to the N-terminus of the HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of B2M is linked to the C-terminus of the HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of B2M is linked to the HLA-E molecule or the HLA-G molecule through a linker. Any suitable linker that can be used to link two or more functional polypeptides known in the art may be used. In some embodiments, the functional portion of B2M linked to the HLA-E molecule further includes a binding peptide. In some embodiments, the binding peptide has an amino acid sequence including LIL or LFL.

[0198] In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least 75% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least 85% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 90% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least 95% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least 98% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least 99% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that is identical to SEQ ID NO: 2 or 4.

[0199] In some embodiments, the heterologous nucleic acid sequence is integrated into a genomic locus. In some embodiments, the heterologous nucleic acid sequence is integrated into a sustained transgene expression locus (STEL) in the genome of the engineered cell. In some embodiments, the STEL is a gene locus that encodes a protein involved in one or moreof: ribonucleoprotein complex formation, focal adhesion, cell-substrate adherens junction, cell-substrate junction, cell anchoring, extracellular exosome, extracellular vesicle, intracellular organelle, anchoring junction, RNA binding, nucleic acid binding (e.g., rRNA or mRNA binding), and protein binding. In some embodiments, the STEL is a glyceraldehyde 3- phosphate dehydrogenase (GAPD ) gene. In some embodiments, the STEL is a ribosomal protein gene locus, such as an RPL or RPS gene locus. Examples of RPL genes are RPL10, RPL13, RPS18, RPL3, RPLP1, RPL13A, RPL15, RPL41, RPL11, RPL32, RPL18A, RPL19, RPL28, RPL29, RPL9, RPL8, RPL6, RPL 18, RPL7, RPL7A, RPL21, RPL37A, RPL 12, RPL5, RPL34, RPL35A, RPL30, RPL24, RPL39, RPL37, RPL 14, RPL27A, RPLP2, RPLPO, RPL23A, RPL26, RPL36, RPL35, RPL23, RPL4, and RPL22. Examples of RPS genes are RPS2, RPS 19, RPS 14, RPS3A, RPS 12, RPS3, RPS6, RPS23, RPS27A, RPS8, RPS4X, RPS7, RPS24, RPS27, RPS15A, RPS9, RPS28, RPS 13, RPSA, RPS5, RPS 16, RPS25, RPS15, RPS20, and RPSII. In some embodiments, the STEL is a gene locus encoding a mitochondrial protein, such as MT-Col, MT-C02, MT-ND4, MT-ND1, and MT-ND2. In some embodiments, the STEL is a gene locus encoding an actin protein, such as ACTG1 and ACTB. In some embodiments, the STEL is a gene locus encoding a eukaryotic translation elongation factor, such as EEF1A1 and EEF2, or a eukaryotic translation initiation factor such as EIEI. In some embodiments, the STEL is a gene locus encoding a histone, such as H3F3 A and H3F3B. In some embodiments, the STEL is a gene locus selected from FTL, FTH1, TPT1, IMSB10, GAPDH, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2, and SRP14.

[0200] Advantageously, by integrating the at least one heterologous nucleic acid encoding at least the functional portion of the HLA molecule into a STEL in the genome of the cells, the polypeptide encoded by the heterologous nucleic acid can be governed by an endogenous gene promoter, such as a GAPDH promoter. Consequently, the expression of at least the functional portion of the HLA molecule can be linked to an endogenous gene’s expression. The continued activity of the endogenous gene in the engineered cells will then imply that the expression of at least the functional portion of the HLA molecule will remain sustained and constitutive. By leveraging the inherent regulatory mechanisms of the endogenous gene, the disclosure offers a useful mechanism to maintain expression of at least the functional portion of the HLA molecule at a level sufficient to reduce or inhibit NK cell- mediated cytotoxicity.

[0201] In some embodiments, the at least one heterologous nucleic acid encoding at least the functional portion of the HLA molecule is integrated into a sustained transcriptionally active payload region (STAPLR), z.e., an intergenic region in the mammalian genome that allows consistent levels of expression of transgenes integrated therein, including as the cell undergoes changes in its differentiation state. For more information on STAPLR, see international application PCT / US2023 / 066396, which is incorporated herein by reference.

[0202] In some embodiments, the engineered cell further includes one or more genetic modifications that result in reduced T cell-mediated killing of the engineered cell, or progeny thereof, as compared to a wild-type cell. In some embodiments, the engineered cell further includes one or more genetic modifications that result in at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% reduced T cell-mediated killing of the engineered cell, or progeny thereof, as compared to a wild-type cell. In some embodiments, the engineered cell further includes one or more genetic modifications that result in at least 20% reduced T cell-mediated killing of the engineered cell, or progeny thereof, as compared to a wild-type cell. In some embodiments, the engineered cell further includes one or more genetic modifications that result in at least 50% reduced T cell-mediated killing of the engineered cell, or progeny thereof, as compared to a wild-type cell. In some embodiments, the engineered cell further includes one or more genetic modifications that result in at least 80% reduced T cell-mediated killing of the engineered cell, or progeny thereof, as compared to a wild-type cell.

[0203] In some embodiments, the one or more genetic modifications that results in reduced T cell-mediated killing of the engineered cell, or progeny thereof, as compared to a wild-type cell includes a deletion or disruption of a [>2 microglobulin (B2M) gene. In some embodiments, the one or more genetic modifications that result in reduced T cell-mediated killing of the engineered cell, or progeny thereof, as compared to a wild-type cell includes a deletion or disruption of one or more of an HLA-A gene, an HLA-B gene, and an HLA-C gene.

[0204] In some embodiments, the engineered cell further includes a deletion or disruption of the CIITA gene. Accordingly, in some embodiments, the engineered cell includes one or more genetic modification that result in reduced T cell-mediated killing of the engineered cell, or progeny thereof, as compared to a wild-type cell as described herein (e.g., a deletion or disruption of &B2M, HLA-A, HLA-B, and / or HLA-C) and a deletion or disruption of the CIITA gene.

[0205] In some embodiments, the engineered cell further includes a kill switch. The inclusion of a kill switch can be used to increase the safety of the engineered cell, or progeny thereof. For instance, activation of the kill switch can remove the engineered cells, or progeny thereof, without interfering with a subject’s own cells. In some embodiments, the kill switch is a gene that encodes a herpes simplex virus thymidine kinase (HSV-TK), an inducible caspase9 (also known as incasep 9, and iCasp9), CD20, or a mutant human thymidylate kinase (mTMPK). In some embodiments, the kill switch is under the control of an inducible promoter. In some embodiments, the kill switch is encoded by a heterologous nucleic acid. In some embodiments, the at least one heterologous nucleic acid encoding the kill switch is integrated into the genome of the engineered cell.

[0206] In some embodiments, the engineered cell further includes at least one selectable marker gene. The selectable marker can be used for detection or selection of the engineered cell, or progeny thereof, and its expression can be detected by any suitable method known in the art, such as for example, FACS or culturing in a selection media. Non-limiting examples of selectable marker genes include, but are not limited to, fluorescent proteins (such as green fluorescent protein (GFP), blue fluorescent protein (EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent protein (ECFP, Cerulean, CyPet, mTurquoise2), and yellow fluorescent protein derivatives (YFP, Citrine, Venus, Ypet, EYFP)), b-galactosidase (LacZ), chloramphenicol acetyltransferase (cat), neomycin phosphotransferase (neo), enzymes (such as oxidases and peroxidases), and antigenic molecules. In some embodiments, the selectable marker gene can be driven by an endogenous gene promoter.

[0207] In some embodiments, the engineered cell includes an engineered stem cell. In some embodiments, the engineered stem cell includes a pluripotent stem cell. In some embodiments, the pluripotent stem cell includes an embryonic stem cell (ESC) or induced pluripotent stem cell (iPSC). In some embodiments, the engineered cell includes the iPSC. In some embodiments, the engineered stem cell includes a multipotent stem cell.

[0208] In some embodiments, the engineered cell includes a cardiac cell, a neural cell, a myeloid cell, a T cell (e.g., a regulator T cell), or a retinal cell. In some embodiments, the engineered cell includes the cardiac cell. In some embodiments, the cardiac cell is a cell of the epicardium, the myocardium, or the endocardium of the heart. In some embodiments, the cardiac cell is a cardiomyocyte, a cardiac fibroblast, a cardiac smooth muscle cell, an epicardium cells, a cardiac endothelial cell, a Purkinje fiber, or a pacemaker cell. In someembodiments, the engineered cell includes the cardiomyocyte. In some embodiments, the cardiomyocyte is an immature cardiomyocyte. In some embodiments, the cardiomyocyte is a mature cardiomyocyte. In some embodiments, the engineered cell includes a neural cell. In some embodiments, the neural cell is a neuron or a glial cell. In some embodiments, the engineered cell includes a myeloid cell. In some embodiments, the myeloid cell is a monocyte, a microglia, a macrophage, a dendritic cell, a basophil, an eosinophil, an erythrocyte, a mast cell, a neutrophil, a megakaryocyte, or a platelet, or any precursor progenitor cell thereof. In some embodiments, the cardiac cell, the neural cell, the myeloid cell, the T cell (e.g., a regulator T cell), or the retinal cell are derived from an engineered stem cell (e.g., an engineered stem cell described herein).

[0209] In some embodiments, the engineered cell is capable of differentiating into another cell type, such as a cardiac cell, a neural cell, a myeloid cell, a T cell (e.g., a regulator T cell), or a retinal cell. In some embodiments, the engineered cell is capable of differentiating into a cardiac cell. In some embodiments, the engineered cell is capable of differentiating into a cardiomyocyte.

[0210] In some embodiments, the progeny of the engineered cell is the result of cell division of the engineered cell. In some embodiments, the progeny of the engineered cell is the result of differentiation of the engineered cell. In some embodiments, the engineered cell and progeny thereof are the same cell type. In some embodiments, the engineered cell and the progeny thereof are a different cell type.

[0211] In some embodiments, the engineered cell is an iPSC, and progeny thereof is a cardiac cell, a neural cell, a myeloid cell, a T cell, or a retinal cell. In some embodiments, the engineered cell is an iPSC, and progeny thereof is the cardiac cell. In some embodiments, the engineered cell is an iPSC, and progeny thereof is a cardiomyocyte.

[0212] In some embodiments, the engineered cell is a human engineered cell.Population of cells

[0213] In some aspects of the disclosure, provided herein as populations of engineered cells (e.g., a population of the engineered cells described herein).

[0214] In some embodiments, the population of engineered cells, or progeny thereof, is less susceptible to NK cell-mediated cytotoxicity as compared to a population of cells not engineered to have the at least one heterologous nucleic acid sequence. The susceptibility of the population of engineered cells, or progeny thereof, to NK cell mediate cytotoxicity can be measured by any suitable method known in the art. For instance, the susceptibility to NK cell mediate cytotoxicity may be measured by a NK cell cytotoxicity assay, e.g., a NK cell cytotoxicity assay as described in Example 2. In some embodiments, the population of engineered cells, or progeny thereof, is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% less susceptible to NK cell-mediated cytotoxicity as compared to a population of cells not engineered to have the at least one heterologous nucleic acid sequence. In some embodiments, the population of engineered cells, or progeny thereof, is at least about 10% less susceptible to NK cell-mediated cytotoxicity as compared to a population of cells not engineered to have the at least one heterologous nucleic acid sequence. In some embodiments, the population of engineered cells, or progeny thereof, is at least about 20% less susceptible to NK cell-mediated cytotoxicity as compared to a population of cells not engineered to have the at least one heterologous nucleic acid sequence. In some embodiments, the population of engineered cells, or progeny thereof, is at least about 30% less susceptible to NK cell-mediated cytotoxicity as compared to a population of cells not engineered to have the at least one heterologous nucleic acid sequence. In some embodiments, the population of engineered cells, or progeny thereof, is at least about 50% less susceptible to NK cell-mediated cytotoxicity as compared to a population of cells not engineered to have the at least one heterologous nucleic acid sequence.

[0215] In some embodiments, the population of engineered cells is a population of engineered cardiac cells. In some embodiments, the population of engineered cells is a population of engineered cardiomyocytes. In some embodiments, the population of cells is a population of engineered cells capable of differentiating into a cardiac cell. In some embodiments, the population of cells is a population of engineered cells capable of differentiating into a cardiomyocyte.III. Pharmaceutical compositions

[0216] Certain aspects of the disclosure provide for pharmaceutical compositions that include a population of engineered cells (e.g., a population of engineered cell described herein), and a pharmaceutically acceptable excipient, carrier or diluent.

[0217] In some embodiments, the pharmaceutical composition includes a population of engineered cells as described herein, which can include a genetic modification that reduces or eliminates function of a natural-killer group 2, member D (NKG2D) ligand as compared to a wild-type cell, or a heterologous nucleic acid sequence encoding at least a functional portion of a human leukocyte antigen (HLA) molecule. In some embodiments, pharmaceutical composition includes a population of engineered cells that include the genetic modification. In some embodiments, pharmaceutical composition includes a population of engineered cells that include the heterologous nucleic acid. In some embodiments, pharmaceutical composition includes a population of engineered cells that include both the genetic modification and the heterologous nucleic acid.

[0218] In some embodiments, the pharmaceutical composition includes a population of engineered cells as described herein, which can include a genetic modification that reduces or eliminates function of a NKG2D ligand as compared to a wild-type cell, or a heterologous nucleic acid sequence encoding at least a functional portion of a HLA molecule, wherein either the genetic modification or the heterologous nucleic acid is sufficient to reduce or inhibit natural killer (NK) cell-mediated cytotoxicity of the population of engineered cells, or progeny thereof, as compared to a population of wild-type cell. In some embodiments, either the genetic modification or the heterologous nucleic acid is sufficient to reduce or inhibit NK cell-mediated cytotoxicity of the population of engineered cells, or progeny thereof, 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%, or at least 90% as compared to a population of wild-type cells. In some embodiments, either the genetic modification or the heterologous nucleic acid is sufficient to reduce or inhibit NK cell-mediated cytotoxicity of the population of engineered cells, or progeny thereof, by at least 10% as compared to a population of wild-type cells. In some embodiments, either the genetic modification or the heterologous nucleic acid is sufficient to reduce or inhibit NK cell-mediated cytotoxicity of the population of engineered cells, or progeny thereof, by at least 20% as compared to a population of wild-type cells. In some embodiments, either the genetic modification or the heterologous nucleic acid is sufficient to reduce or inhibit NK cell-mediated cytotoxicity of the population of engineered cells, orprogeny thereof, by at least 30% as compared to a population of wild-type cells. In some embodiments, either the genetic modification or the heterologous nucleic acid is sufficient to reduce or inhibit NK cell-mediated cytotoxicity of the population of engineered cells, or progeny thereof, by at least 50% as compared to a population of wild-type cells. In some embodiments, either the genetic modification or the heterologous nucleic acid is sufficient to reduce or inhibit NK cell-mediated cytotoxicity of the population of engineered cells, or progeny thereof, by at least 80% as compared to a population of wild-type cells.

[0219] In some embodiments, the pharmaceutical composition includes a population of engineered cells as described herein, which can include a genetic modification that reduces or eliminates function of a NKG2D ligand as compared to a wild-type cell, wherein the genetic modification that reduces or eliminates function of the NKG2D ligand as compared to a wildtype cell is a deletion or disruption of a gene encoding the NKG2D ligand. In some embodiments, the genetic modification that reduces or eliminates function of the NKG2D ligand as compared to a wild-type cell is the deletion of the gene encoding the NKG2D ligand. In some embodiments, the genetic modification that reduces or eliminates function of the NKG2D ligand as compared to a wild-type cell is the disruption of the gene encoding the NKG2D ligand. In some embodiments, the genetic modification that reduces or eliminates function of the NKG2D ligand as compared to a wild-type cell includes a deletion or disruption that results in reduced or eliminated expression of the gene encoding the NKG2D ligand.

[0220] In some embodiments, the pharmaceutical composition includes a population of engineered cells as described herein, which can include a genetic modification that reduces or eliminates function of a NKG2D ligand as compared to a wild-type cell, wherein the NKG2D ligand includes MICA, MICB, Raetle, Raetlg, Raetll, Ulbpl, Ulbp2, Ulbp3, or any combination thereof. In some embodiments, the NKG2D ligand includes MICA. In some embodiments, the NKG2D ligand includes MICB. In some embodiments, the NKG2D ligand includes Raetle. In some embodiments, the NKG2D ligand includes Raetlg. In some embodiments, the NKG2D ligand includes Raetll. In some embodiments, the NKG2D ligand includes Ulbpl. In some embodiments, the NKG2D ligand includes Ulbp2. In some embodiments, the NKG2D ligand includes Ulbp3.

[0221] In some embodiments, the pharmaceutical composition includes a population of engineered cells as described herein, which can include a heterologous nucleic acid sequenceencoding at least a functional portion of a HLA molecule, wherein the heterologous nucleic acid encodes at least a functional portion of an HLA-E molecule or an HLA-G molecule. In some embodiments, the heterologous nucleic acid sequence encodes at least the functional portion of the HLA-E molecule. In some embodiments, the heterologous nucleic acid sequence encodes at least the functional portion of the HLA-G molecule. In some embodiments, the heterologous nucleic acid sequence encodes both at least the functional portion of both the HLA-E molecule and the HLA-G molecule. In some embodiments, the functional portion of the HLA-E molecule or the HLA-G molecule includes at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or 300 amino acids of the full-length sequence of the HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of the HLA-E molecule or the HLA-G molecule includes at least 50 amino acids of the full-length sequence of the HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of the HLA-E molecule or the HLA-G molecule includes at least 100 amino acids of the full-length sequence of the HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of the HLA-E molecule or the HLA-G molecule includes at least 200 amino acids of the full-length sequence of the HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of the HLA-E molecule or the HLA-G molecule includes at least 300 amino acids of the full- length sequence of the HLA-E molecule or the HLA-G molecule.

[0222] In some embodiments, the pharmaceutical composition includes a population of engineered cells as described herein, which can include a heterologous nucleic acid sequence encoding at least a functional portion of a HLA molecule, wherein the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 75% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 85% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 90% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 95% identity to SEQ ID NO: 6. In someembodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 98% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 99% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that is identical to SEQ ID NO: 6.

[0223] In some embodiments, the pharmaceutical composition includes a population of engineered cells as described herein, which can include a heterologous nucleic acid sequence encoding at least a functional portion of a HLA molecule, wherein the heterologous nucleic acid encodes at least a functional portion of an HLA molecule that includes a binding peptide. In some embodiments, the binding peptide has an amino acid sequence that includes LIL or LFL. In some embodiments, the binding peptide has an amino acid sequence corresponding to any one of SEQ ID NOS: 8 or 10. In some embodiments, the HLA molecule that includes the binding peptide is an HLA-E molecule or an HLA-G molecule.

[0224] In some embodiments, the pharmaceutical composition includes a population of engineered cells as described herein, which can include a heterologous nucleic acid further including a sequence encoding at least a functional portion of a P2 microglobulin (B2M). In some embodiments, the functional portion of the B2M includes at least 10, 20, 30, 40, 50, 60, 70, 90 or 100 amino acids of the full-length sequence of the B2M. In some embodiments, the functional portion of the B2M includes at least 10 amino acids of the full-length sequence of the B2M. In some embodiments, the functional portion of the B2M includes at least 30 amino acids of the full-length sequence of the B2M. In some embodiments, the functional portion of the B2M includes at least 50 amino acids of the full-length sequence of the B2M. In some embodiments, the functional portion of the B2M includes at least 90 amino acids of the full-length sequence of the B2M. In some embodiments, the functional portion of B2M is linked to the HLA-E molecule or the HLA-G molecule encoded by the heterologous nucleic acid. In some embodiments, the functional portion of B2M is linked to the HLA-E molecule. In some embodiments, the functional portion of B2M is linked to the HLA-G molecule. In some embodiments, the functional portion of B2M is linked to the N-terminus of HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of B2M is linked to the C-terminus of HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of B2M is linked to the HLA-E molecule or the HLA-G moleculethrough a linker as described herein. In some embodiments, the functional portion of B2M linked to the HLA-E molecule further includes a binding peptide. In some embodiments, the binding peptide has an amino acid sequence including LIL or LFL.

[0225] In some embodiments, the pharmaceutical composition includes a population of engineered cells as described herein, which can include a heterologous nucleic acid encoding a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least 75% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least 85% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 90% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least 95% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least 98% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least 99% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that is identical to SEQ ID NO: 2 or 4.

[0226] In some embodiments, the pharmaceutical composition includes a population of engineered cells as described herein, which can include a heterologous nucleic acid sequence encoding at least a functional portion of a HLA molecule, wherein the heterologous nucleic acid sequence is integrated into a genomic locus. In some embodiments, the heterologous nucleic acid sequence is integrated into a sustained transgene expression locus (STEL) in the genome of the engineered cell. In some embodiments, the STEL is a gene locus that encodes a protein involved in one or more of: ribonucleoprotein complex formation, focal adhesion, cell-substrate adherens junction, cell-substrate junction, cell anchoring, extracellular exosome, extracellular vesicle, intracellular organelle, anchoring junction, RNA binding, nucleic acid binding (e.g., rRNA or mRNA binding), and protein binding. In some embodiments, the STEL is a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. Insome embodiments, the STEL is a ribosomal protein gene locus, such as an RPL or RPS gene locus. Examples of RPL genes are RPL10, RPL13, RPS18, RPL3, RPLP1, RPL13A, RPL15, RPL41, RPL11, RPL32, RPL18A, RPL19, RPL28, RPL29, RPL9, RPL8, RPL6, RPL 18, RPL7, RPL7A, RPL21, RPL37A, RPL 12, RPL5, RPL34, RPL35A, RPL30, RPL24, RPL39, RPL37, RPL 14, RPL27A, RPLP2, RPLPO, RPL23A, RPL26, RPL36, RPL35, RPL23, RPL4, andRPL22. Examples of RPS genes are RPS2, RPS 19, RPS 14, RPS3A, RPS 12, RPS3, RPS6, RPS23, RPS27A, RPS8, RPS4X, RPS7, RPS24, RPS27, RPS15A, RPS9, RPS28, RPS 13, RPSA, RPS5, RPS16, RPS25, RPS15, RPS20, and RPSIL In some embodiments, the STEL is a gene locus encoding a mitochondrial protein, such as MT-C01, MT-C02, MT-ND4, MT-ND1, and MT-ND2. In some embodiments, the STEL is a gene locus encoding an actin protein, such as ACTG1 and ACTB. In some embodiments, the STEL is a gene locus encoding a eukaryotic translation elongation factor, such as EEF1A1 and EEF2, or a eukaryotic translation initiation factor such as EIEI. In some embodiments, the STEL is a gene locus encoding a histone, such as H3F3 A and H3F3B. In some embodiments, the STEL is a gene locus selected from FTL, FTH1, TPT1, IMSB10, GAPDH, PLMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2, and SRP14.

[0227] In some embodiments, the pharmaceutical composition includes a population of engineered cells as described herein. Such a population of engineered cells can include one or more genetic modifications that result in reduced T cell-mediated killing of the population of engineered cells, or progeny thereof, as compared to a population of wild-type cells. In some embodiments, the pharmaceutical composition includes a population of engineered cells that further include one or more genetic modifications that result in at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% reduced T cell-mediated killing of the population of engineered cells as, or progeny thereof, compared to a population of wild-type cells. In some embodiments, the pharmaceutical composition includes a population of engineered cells that further include includes one or more genetic modifications that result in at least 20% reduced T cell-mediated killing of the population of engineered cells, or progeny thereof, as compared to a population of wild-type cells. In some embodiments, the pharmaceutical composition includes a population of engineered cells that further include one or more genetic modifications that result in at least 50% reduced T cell-mediated killing of the population of engineered cells, or progeny thereof, as compared to a population of wild-type cells. In some embodiments, the pharmaceutical composition includes a population of engineered cells that further include oneor more genetic modifications that result in at least 80% reduced T cell-mediated killing of the population of engineered cells, or progeny thereof, as compared to a population of wildtype cells. In some embodiments, the one or more genetic modifications that results in reduced T cell-mediated killing of the population of engineered cells, or progeny thereof, as compared to a wild-type cell includes a deletion or disruption of a [>2 microglobulin (B2M) gene. In some embodiments, the one or more genetic modifications that results in reduced T cell-mediated killing of the population of engineered cells, or progeny thereof, as compared to a wild-type cell includes a deletion or disruption of one or more of an HLA-A gene, an HLA-B gene, and an HLA-C gene.

[0228] In some embodiments, the pharmaceutical composition includes a population of engineered cells described herein, which can further include a deletion or disruption of the CIITA gene. Accordingly, in some embodiments, the pharmaceutical composition includes a population of engineered cells that included a one or more genetic modifications that result in reduced T cell-mediated killing of the population of engineered cells, or progeny thereof, as compared to a population of wild-type cells as described herein (e.g., a deletion or disruption of a B2M, HLA-A, HLA-B, and / or HLA-C) and a deletion or disruption of the CIITA gene.

[0229] In some embodiments, the pharmaceutical composition includes a population of engineered cells described herein, which can further include a kill switch. In some embodiments, the kill switch is a gene that encodes a herpes simplex virus thymidine kinase (HSV-TK), an inducible caspase9 (also known as incasep 9, and iCasp9), CD20, or a mutant human thymidylate kinase (mTMPK). In some embodiments, the kill switch is under the control of an inducible promoter. In some embodiments, the kill switch is encoded by a heterologous nucleic acid. In some embodiments, the at least one heterologous nucleic acid encoding the kill switch is integrated into the genome of each cell in the population of engineered cells.

[0230] In some embodiments, the pharmaceutical composition includes a population of engineered cells described herein, which can further include at least one selectable marker gene. Non-limiting examples of selectable marker genes include, but are not limited to, fluorescent proteins (such as green fluorescent protein (GFP), blue fluorescent protein (EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent protein (ECFP, Cerulean, CyPet, mTurquoise2), and yellow fluorescent protein derivatives (YFP, Citrine, Venus, YPet, EYFP)), b-galactosidase (LacZ), chloramphenicol acetyltransferase (cat), neomycinphosphotransferase (neo), enzymes (such as oxidases and peroxidases), and antigenic molecules. In some embodiments, the selectable marker gene can be driven by an endogenous gene promoter.

[0231] In some embodiments, the pharmaceutical composition includes a population of engineered cells described herein, which can include an engineered stem cell. In some embodiments, the engineered stem cell includes a pluripotent stem cell. In some embodiments, the pluripotent stem cell includes an embryonic stem cell (ESC) or induced pluripotent stem cell (iPSC). In some embodiments, the engineered stem cell includes a multipotent stem cell.

[0232] In some embodiments, the pharmaceutical composition includes a population of engineered cells described herein, which can include a cardiac cell, a neural cell, a myeloid cell, a T cell (e.g., a regulator T cell), or a retinal cell. In some embodiments, the pharmaceutical composition includes a population of engineered cells that include the cardiac cell. In some embodiments, the cardiac cell is a cell of the epicardium, the myocardium, or the endocardium of the heart. In some embodiments, the cardiac cell is a cardiomyocyte, a cardiac fibroblast, a cardiac smooth muscle cell, an epicardium cells, a cardiac endothelial cell, a Purkinje fiber, or a pacemaker cell. In some embodiments, the pharmaceutical composition includes a population of engineered cells that include the cardiomyocyte. In some embodiments, the pharmaceutical composition includes a population of cells that include immature cardiomyocytes. In some embodiments, the pharmaceutical composition includes a population of cells that include mature cardiomyocytes. In some embodiments, the pharmaceutical composition includes a population of engineered cells that include a neural cell. In some embodiments, the neural cell is a neuron or a glial cell. In some embodiments, the pharmaceutical composition includes a population of engineered cells that include a myeloid cell. In some embodiments, the myeloid cell is a monocyte, a microglia, a macrophage, a dendritic cell, a basophil, an eosinophil, an erythrocyte, a mast cell, a neutrophil, a megakaryocyte, or a platelet, or any precursor progenitor cell thereof. In some embodiments, the cardiac cell, the neural cell, the myeloid cell, the T cell (e.g., a regulator T cell), or the retinal cell in the population of engineered cells in the pharmaceutical composition are derived from an engineered stem cell (e.g., an engineered stem cell described herein).

[0233] In some embodiments, the pharmaceutical composition includes a population of engineered cells described herein, which can include an engineered cell capable of differentiating into includes a cardiac cell, a neural cell, a myeloid cell, a T cell (e.g., a regulator T cell), or a retinal cell. In some embodiments, the pharmaceutical composition includes a population of engineered cells that include an engineered cell capable of differentiating into a cardiac cell. In some embodiments, the pharmaceutical composition includes a population of engineered cells that include an engineered cell capable of differentiating into a cardiomyocyte.

[0234] In some embodiments, the pharmaceutical composition includes a population of human engineered cells.

[0235] In some embodiments, the pharmaceutical composition is formulated for administration to a subject. The pharmaceutical composition be formulated for any suitable means of administration, such as for administration by local injection into a tissue.

[0236] The pharmaceutically acceptable excipient, carrier, or diluent can be any excipient, carrier or diluent known in the art. For instance, the pharmaceutically acceptable excipient, carrier, or diluent can be a cell culture medium (e.g., one that optionally lacks any animal-derived component), sterilized water, physiological saline, general buffers (e.g., phosphoric acid, citric acid, other organic acids, etc.), stabilizers, salts, anti-oxidants, surfactants, suspensions, isotonic agents, and / or preservatives can be included in a pharmaceutical composition described herein. The specific excipient, carrier, or diluent will depend on the route of administration intended for the pharmaceutical composition. In some embodiments, the pharmaceutically acceptable excipient, carrier, or diluent is an excipient, carrier, or diluent suitable for administration of the pharmaceutical composition by injection to a subject.

[0237] The pharmaceutical compositions described herein can be used for the treatment of a disease or disorder in a subject. In some embodiments, the pharmaceutical composition is for use in treating heart failure, Parkinson’s disease, multiple sclerosis, irritable bowel syndrome, type 1 diabetes, rheumatoid arthritis, or neural inflammation. In some embodiments, pharmaceutical composition is for use in treating heart failure. In some embodiments, pharmaceutical composition is for use in treating Parkinson’s disease. In some embodiments, pharmaceutical composition is for use in treating multiple sclerosis. In someembodiments, pharmaceutical composition is for use in treating irritable bowel syndrome. In some embodiments, pharmaceutical composition is for use in treating type 1 diabetes. In some embodiments, pharmaceutical composition is for use in treating rheumatoid arthritis. In some embodiments, pharmaceutical composition is for use in treating neural inflammation.

[0238] In some embodiments, the pharmaceutical composition includes a population of engineered cells allogenic to a subject that is administered the pharmaceutical composition. In some embodiments, the pharmaceutical composition includes a population of engineered cells autologous to a subject that is administered the pharmaceutical composition.IV. Methods of Producing Cells

[0239] Certain aspects of the disclosure provide for methods of producing engineered cells with enhanced immune evasion (e.g., an engineered cell described herein). In some embodiments, the method for producing an engineered cell with enhanced immune evasion includes genetically modifying the cell to reduce or eliminate function of a natural-killer group 2, member D (NKG2D) ligand relative to a wild-type cell; or incorporating a heterologous nucleic acid sequence that encodes at least a functional portion of a human leukocyte antigen (HL A) molecule into the genome of the cell.

[0240] In some embodiments, either the genetically modifying step or the incorporating step enhances the survival probability of the engineered cell, or progeny thereof, when exposed to NK cells, as compared to a wild-type cell. In some embodiments, either the genetically modifying step or the incorporating step enhances the survival probability of the engineered cell, or progeny thereof, when exposed to NK cells 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%, or at least 90% as compared to a wild-type cell. In some embodiments, either the genetically modifying step or the incorporating step enhances the survival probability of the engineered cell, or progeny thereof, when exposed to NK cells by at least 10% as compared to a wild-type cell. In some embodiments, either the genetically modifying step or the incorporating step enhances the survival probability of the engineered cell, or progeny thereof, when exposed to NK cells by at least 20% as compared to a wild-type cell. In some embodiments, either the genetically modifying step or the incorporating step enhances the survival probability of the engineered cell, or progeny thereof, when exposed to NK cells by at least 30% as comparedto a wild-type cell. In some embodiments, either the genetically modifying step or the incorporating step enhances the survival probability of the engineered cell, or progeny thereof, when exposed to NK cells by at least 50% as compared to a wild-type cell. In some embodiments, e either the genetically modifying step or the incorporating step enhances the survival probability of the engineered cell, or progeny thereof, when exposed to NK cells by at least 80% as compared to a wild-type cell.

[0241] In some embodiments, the method for producing an engineered cell with enhanced immune evasion includes the genetically modifying step as described herein. In some embodiments, the method for preparing an engineered cell described herein includes the incorporating step as described herein. In some embodiments, the method for producing an engineered cell with enhanced immune evasion includes both the genetically modifying step and the incorporating step as described herein.

[0242] In some embodiments, genetically modifying the cell to reduce or eliminate function of the NKG2D ligand relative to a wild-type cell includes introducing a deletion or disruption of a gene encoding the NKG2D ligand. In some embodiments, NKG2D ligand includes MICA, MICB, Raetle, Raetlg, Raetll, Ulbpl, Ulbp2, Ulbp3, or any combination thereof. In some embodiments, the NKG2D ligand includes MICA.

[0243] Any suitable technique for genetically modifying a cell can be used to genetically modify the cell to reduce or eliminate function of NKG2D ligand. For instance, the genetic modification can be introduced using, for example, known gene editing systems such as those utilizing genome-targeting elements, including a DNA-binding domain (e.g., zinc finger DNA-binding protein or a TALE DNA-binding domain), guide RNA elements (e.g., CRISPR guide RNA), and guide DNA elements (e.g., NgAgo guide DNA). In some embodiments, genetically modifying the cell to reduce or eliminate function of NKG2D ligand includes using the CRISPR / Cas system, e.g., a CRISPR / Cas9 system or a CRISPR / Casl2 system.

[0244] In some embodiments, the incorporating of the heterologous nucleic acid encoding at least the functional portion of the HLA molecule includes integrating the heterologous nucleic acid into a genomic locus of the cell. Any suitable technique for integrating nucleic acids into the genome of a host cell can be used to integrate the heterologous nucleic acid encoding at least the functional portion of the HLA molecule. An exemplary strategy for integrating a heterologous nucleic acid encoding a polypeptide ofinterest (e.g., an HLA molecule) into the genome of a cell is shown in FIG. 1. The heterologous nucleic acid can be integrated into the genome of the cell using, for example, known gene editing systems such as those utilizing genome-targeting elements, including a DNA-binding domain (e.g., zinc finger DNA-binding protein or a TALE DNA-binding domain), guide RNA elements (e.g., CRISPR guide RNA), and guide DNA elements (e.g., NgAgo guide DNA). Programmable gene-targeting and nuclease elements enable precise targeted integration of the heterologous nucleic acid. In some instances, the at least one heterologous nucleic acid is integrated into the genome of the cell using a meganuclease based system, a zinc finger nuclease (ZFN) based system, a Transcription Activator-Like Effector-based Nuclease (TALEN) based system, a CRISPR-based system, or NgAgo-based system.

[0245] In some embodiments, the heterologous nucleic acid encoding at least the functional portion of the HLA molecule is integrated into the genome of the cell using a CRISPR / Cas system. The CRISPR / Cas system has been used for introducing genetic modifications and gene regulation in various species. Without being limited by theory, a target nucleic acid can be modified by the interaction of the CRISPR / Cas system and a sequence present in the target nucleic acid, for example, to cause cleavage (e.g., hydrolysis of one or more phosphodiester bonds) of the target nucleic acid and introduce the genetic modification. In some embodiments, the heterologous nucleic acid encoding at least the functional portion of the HLA molecule is integrated into the genome of the cell using a CRISPR / Cas9 system or a CRISPR / Casl2 system.

[0246] In some embodiments, the heterologous nucleic acid encodes at least a functional portion of an HLA-E molecule or an HLA-G molecule. In some embodiments, the heterologous nucleic acid sequence encodes at least the functional portion of the HLA-E molecule. In some embodiments, the heterologous nucleic acid sequence encodes at least the functional portion of the HLA-G molecule. In some embodiments, the heterologous nucleic acid sequence encodes both at least the functional portion of both the HLA-E molecule and the HLA-G molecule. In some embodiments, the functional portion of the HLA-E molecule or the HLA-G molecule includes at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or 300 amino acids of the full-length sequence of the HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of the HLA-E molecule or the HLA- G molecule includes at least 50 amino acids of the full-length sequence of the HLA-Emolecule or the HLA-G molecule. In some embodiments, the functional portion of the HLA- E molecule or the HLA-G molecule includes at least 100 amino acids of the full-length sequence of the HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of the HLA-E molecule or the HLA-G molecule includes at least 200 amino acids of the full-length sequence of the HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of the HLA-E molecule or the HLA-G molecule includes at least 300 amino acids of the full-length sequence of the HLA-E molecule or the HLA-G molecule.

[0247] In some embodiments, the method for preparing an engineered cell described herein includes introducing a heterologous nucleic acid nucleic acid that encodes at least a functional portion of an HLA-E molecule that has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 75% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 85% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 90% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 95% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 98% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 99% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that is identical to SEQ ID NO: 6.

[0248] In some embodiments, the heterologous nucleic acid encodes at least a functional portion of an HLA molecule that includes a binding peptide. In some embodiments, the binding peptide has an amino acid sequence that includes LIL or LFL. In some embodiments, the binding peptide has an amino acid sequence corresponding to any one of SEQ ID NOS: 8 or 10. In some embodiments, the HLA molecule that includes the binding peptide is an HLA-E molecule or an HLA-G molecule.

[0249] In some embodiments, the method for preparing an engineered cell described herein includes introducing a heterologous nucleic acid further including a sequence encoding at least a functional portion of a P2 microglobulin (B2M). In some embodiments, the functional portion of the B2M includes at least 10, 20, 30, 40, 50, 60, 70, 90 or 100 amino acids of the full-length sequence of the B2M. In some embodiments, the functional portion of the B2M includes at least 10 amino acids of the full-length sequence of the B2M. In some embodiments, the functional portion of the B2M includes at least 30 amino acids of the full- length sequence of the B2M. In some embodiments, the functional portion of the B2M includes at least 50 amino acids of the full-length sequence of the B2M. In some embodiments, the functional portion of the B2M includes at least 90 amino acids of the full- length sequence of the B2M. In some embodiments, the functional portion of B2M is linked to the HLA-E molecule or the HLA-G molecule encoded by the heterologous nucleic acid. In some embodiments, the functional portion of B2M is linked to the HLA-E molecule. In some embodiments, the functional portion of B2M is linked to the HLA-G molecule. In some embodiments, the functional portion of B2M is linked to the N-terminus of HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of B2M is linked to the C-terminus of HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of B2M is linked to the HLA-E molecule or the HLA-G molecule through a linker as described herein. In some embodiments, the functional portion of B2M linked to the HLA-E molecule further includes a binding peptide. In some embodiments, the binding peptide has an amino acid sequence including LIL or LFL.

[0250] In some embodiments, the method for preparing an engineered cell described herein includes introducing a heterologous nucleic acid encoding a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least 75% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least 85% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 90% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least95% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least 98% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least 99% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that is identical to SEQ ID NO: 2 or 4.

[0251] In some embodiments, the heterologous nucleic acid encoding at least the functional portion of the HLA molecule is integrated into a STEL in the genome of the cell. In some embodiments, the STEL is a gene locus that encodes a protein involved in one or more of: ribonucleoprotein complex formation, focal adhesion, cell-substrate adherens junction, cell-substrate junction, cell anchoring, extracellular exosome, extracellular vesicle, intracellular organelle, anchoring junction, RNA binding, nucleic acid binding (e.g., rRNA or mRNA binding), and protein binding. In some embodiments, the STEL is a glyceraldehyde 3- phosphate dehydrogenase (GAPDEL) gene. In some embodiments, the STEL is a ribosomal protein gene locus, such as an RPL or RPS gene locus. Examples of RPL genes are RPL10, RPL13, RPS18, RPL3, RPLP1, RPL13A, RPL15, RPL41, RPL11, RPL32, RPL18A, RPL19, RPL28, RPL29, RPL9, RPL8, RPL6, RPL 18, RPL7, RPL7A, RPL21, RPL37A, RPL 12, RPL5, RPL34, RPL35A, RPL30, RPL24, RPL39, RPL37, RPL 14, RPL27A, RPLP2, RPLPO, RPL23A, RPL26, RPL36, RPL35, RPL23, RPL4, and RPL22. Examples of RPS genes are RPS2, RPS 19, RPS 14, RPS3A, RPS 12, RPS3, RPS6, RPS23, RPS27A, RPS8, RPS4X, RPS7, RPS24, RPS27, RPS15A, RPS9, RPS28, RPS 13, RPSA, RPS5, RPS 16, RPS25, RPS15, RPS20, and RPSII. In some embodiments, the STEL is a gene locus encoding a mitochondrial protein, such as MT-CO1, MT-C02, MT-ND4, MT-ND1, and MT-ND2. In some embodiments, the STEL is a gene locus encoding an actin protein, such as ACTG1 and ACTB. In some embodiments, the STEL is a gene locus encoding a eukaryotic translation elongation factor, such as EEF1A1 and EEF2, or a eukaryotic translation initiation factor such as EIEI. In some embodiments, the STEL is a gene locus encoding a histone, such as H3F3 A and H3F3B. In some embodiments, the STEL is a gene locus selected from FTL, FTH1, TPT1, IMSB10, GAPDH, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2, and SRP14.

[0252] In some embodiments, the heterologous nucleic acid encoding at least the functional portion of the HLA molecule further includes one or more regulatory elements. Such a regulatory element can include a regulatory sequence, which is any DNA sequence responsible for the regulation of gene expression, such as promoters and operators. The regulatory element can be a segment of a nucleic acid molecule, which is able to increase or decrease the expression of specific genes within an organism.

[0253] In some embodiments, the regulatory element is a promoter. A promoter is a nucleotide sequence that directs the transcription of a structural gene. In some alternatives, a promoter is in the 5’ non-coding region of a gene, proximal to the transcriptional start site of a structural gene. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. Without being limiting, these promoter elements can include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation-specific elements (DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993);), cyclic AMP response elements (CREs), serum response elements (SREs; Treisman et al., Seminars in Cancer Biol. 1 :47 (1990); incorporated by reference in its entirety), glucocorticoid response elements (GREs), and binding sites for other transcription factors, such as CRE / ATF (O’Reilly et al., J. Biol. Chem. 267: 19938 (1992); incorporated by reference in its entirety), AP2 (Ye et al., J. Biol. Chem. 269:25728 (1994); incorporated by reference in its entirety), SPI, cAMP response element binding protein (CREB; Loeken et al., Gene Expr. 3:253 (1993); hereby expressly incorporated by reference in its entirety) and octamer factors (see, in general, Watson et al., eds., Molecular Biology of the Gene, 4th ed. (The Benjamin / Cummings Publishing Company, Inc. 1987; incorporated by reference in its entirety)), and Lemaigre and Rousseau, Biochem. J. 303: 1 (1994); incorporated by reference in its entirety).

[0254] In some alternatives, promoters used herein can be inducible or constitutive promoters. Without being limiting, inducible promoters can include, for example, a tamoxifen inducible promoter, tetracycline inducible promoter, or a doxocycline inducible promoter (e.g., tre) promoter. Constitutive promoters can include, for example, SV40, CMV, UBC, EFlalpha, PGK, or CAGG. Any suitable promoter known in the art for expression of a gene in a population of engineered cells as described herein can be used. In some embodiments, the heterologous nucleic acid encoding at least the functional portion of the HLA moleculeincludes a promoter. In some embodiments, expression of the HLA molecule is driven by an endogenous gene promoter (e.g., a GAPDH gene promoter).

[0255] The heterologous nucleic acid encoding at least the functional portion of the HLA molecule can be introduced to a cell by any suitable method known in the art. For instance, the at least one heterologous nucleic acid can be introduced by electroporation, sonoporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, viral vector systems (e.g., retroviral, lentivirus, adenoviral, adeno-associated, vaccinia and herpes simplex virus vectors) and agent-enhanced uptake of DNA.

[0256] In some embodiments, the heterologous nucleic acid encoding at least the functional portion of the HLA molecule is introduced into the cell in a vector. The vector can be a plasmid, a virus, or another vector designed for introducing a nucleic acid of interest into a cell. Viral vectors include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Exemplary viral vectors that can be used include an adeno- associated virus, a lentivirus, a retrovirus, a herpes simplex virus, vaccinia, or an adenovirus. The vector is used to introduce a gene of interest into a host cell in which the vector will interact with polymerases in the cell to express the protein encoded in the vector. The vector can exist in the cell extra-chromosomally or integrated into the genome of the host cell. In some embodiments, use of a viral vector will lead to integration of the heterologous nucleic acid into the genome of the cell.

[0257] In some embodiments, the method for preparing an engineered cells described herein includes introducing a heterologous nucleic acid encoding a kill switch into the cell. In some embodiments, the kill switch is a gene that encodes a herpes simplex virus thymidine kinase (HSV-TK), an inducible caspase9 (also known as incasep 9, and iCasp9), CD20, or a mutant human thymidylate kinase (mTMPK). In some embodiments, the kill switch is under the control of an inducible promoter.

[0258] In some embodiments, the nucleic acid encoding the kill switch is integrated into the genome of the cell using the CRISPR / Cas system. The nucleic acid encoding the kill switch can be integrated into the same or a different locus as the heterologous nucleic acid encoding at least the functional portion of the HLA molecule.

[0259] In some embodiments, the nucleic acid encoding the kill switch is introduced into the cell in a vector. The nucleic acid encoding the kill switch can be introduced into the cell in the same vector or a different vector as the heterologous nucleic acid encoding at least the functional portion of the HLA molecule.

[0260] In some embodiments, the method for producing an engineered cell with enhanced immune evasion further includes genetically modifying the cells to achieve reduced T cell- mediated killing of the engineered cell, or progeny thereof, as compared to a wild-type cell. In some embodiments, the e genetic modification results in at least 20% reduced T cell- mediated killing of the engineered cell, or progeny thereof, as compared to a wild-type cell. In some embodiments, the e genetic modification results in at least 50% reduced T cell- mediated killing of the engineered cell, or progeny thereof, as compared to a wild-type cell. In some embodiments, the e genetic modification results in at least 80% reduced T cell- mediated killing of the engineered cell, or progeny thereof, as compared to a wild-type cell.

[0261] In some embodiments, the genetic modification that results in reduced T cell- mediated killing of the engineered cell, or progeny thereof, as compared to a wild-type cell includes a deletion or disruption of a fJ>2 microglobulin (B2M) gene. In some embodiments, the genetic modification that reduces T cell-mediated killing of the engineered cell, or progeny thereof, as compared to a wild-type cell includes a deletion or disruption of one or more of an HLA-A gene, an HLA-B gene, and an HLA-C gene.

[0262] In some embodiments, the method for producing an engineered cell with enhanced immune evasion further includes introducing a deletion or disruption of the CIITA gene. Accordingly, in some embodiments, the method includes preparing an engineered cell that includes a genetic modification that results in reduced T cell-mediated killing of the engineered cell, or progeny thereof, as compared to a wild-type cell as described herein (e.g., a deletion or disruption of a B2M, HLA-A, HLA-B, and / or HLA-C) and a deletion or disruption of the CIITA gene.

[0263] In some embodiments, the method for producing an engineered cell with enhanced immune evasion includes genetically modifying a stem cell. In some embodiments, the stem cell is a pluripotent stem cell. In some embodiments, the pluripotent stem cell is an induce pluripotent stem cell (iPSC). Methods for obtaining iPSCs for use in the method for preparing an engineered cells as described herein are known in the art. For example, the iPSCs can beprepared by inducing expression of one or more genes, e.g., POU5F1 / OCT4 combined with, but not restricted to, S0X2, KLF, c-MYC, NANOG, and / or LIN28ILIN28A. Reprogramming factors can be delivered by various means (e.g., viral, non-viral, RNA, DNA, or protein delivery). Alternatively, endogenous genes can be activated by using, e.g., a CRISPR / Cas system to reprogram non-pluripotent cells into iPSCs.

[0264] In some embodiments, the method for producing an engineered cell with enhanced immune evasion includes genetically modifying an iPSC, and progeny thereof is a cardiac cell, a neural cell, a myeloid cell, a T cell, or a retinal cell. In some embodiments, the method for producing an engineered cell with enhanced immune evasion includes genetically modifying an iPSC, and progeny thereof is the cardiac cell. In some embodiments, the method for producing an engineered cell with enhanced immune evasion includes genetically modifying an iPSC, and progeny thereof is a cardiomyocyte.

[0265] In some embodiments, the method for producing an engineered cell with enhanced immune evasion includes genetically modifying a stem cell and differentiating the stem cell into the engineered cell. Methods for inducing differentiation of stem cell into cells of various lineages are well known in the art. For example, methods for inducing differentiation of stem cells into myeloid cells or neural cells are described, for instance, in U.S. Patent No. 11,525,119, B2 and U.S. Patent No. 10,260,044 Bl, and in Slukvin et al., J Imm. (2006) 176:2924-32; and Su et ah, Clin Cancer Res. (2008) 14(19):6207-17; WO2021072329 Al; and Tseng et al., Regen Med. (2009) 4(4):513-26, the disclosures of which are incorporated herein by reference. In some embodiments, differentiating the stem cell into the engineered cell includes contacting the stem cell with one or more differentiation factors. The specific combination of differentiation factors used will depend on the desired cell type(s). In some embodiments, differentiating the stem cell into the engineered cell includes contacting the iPSCs with one or more differentiation factors that drive the commitment and / or differentiation into myeloid progenitor cells.

[0266] In some embodiments, the method for producing an engineered cell with enhanced immune evasion includes genetically modifying a stem cell and differentiating the genetically modified stem cell into a cardiac cell. In some embodiments, the cardiac cell is a cardiomyocyte. In some embodiments, the cardiomyocyte is an immature cardiomyocyte. In some embodiments, the cardiomyocyte is a mature cardiomyocyte. In some embodiments, the stem cell is a pluripotent stem cell. Any suitable method known in the art fordifferentiating stem cells into cardiac cells can be used in connection with the methods of the disclosure. Numerous methods exist for differentiating stem cells into cardiac cells are described, in for example, Kattman et al., Cell Stem Cell (2011) 8(2):228-40, WO2016131137, WO2018098597, U.S. Pat. 9,453,201, WO2020227232, WO2020227232A2; and WO2021072329, the disclosures of which are incorporated herein by reference.

[0267] In some embodiments, differentiating the stem cell into a cardiac cell includes incubating the stem cell in one or more cardiac differentiation media. For example, the cardiac differentiation media can contain varying concentrations of bone-morphogenetic protein (BMP; such as BMP4) and activin (such as activin A). Titration of differentiation factor concentration can be performed to determine the optimal concentration necessary for achieving differentiation into the desired cardiac cell type. For instance, the differentiation factors can be selected to direct differentiation into cardiomyocytes. In some embodiments, differentiating the stem cells into cardiac cells involves modulating Wnt / p-catenin signaling under fully defined conditions. For example, in some embodiments, modulating of Wnt signaling can be achieved by contacting the stem cells with a WNT activator (e.g., CHIR) followed by deactivation with a small molecule, IWR1. For further information for differentiation into cardiomyocytes has been described in Lian, Xiaojun, et al. “Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt / p- catenin signaling under fully defined conditions.” Nature protocols 8.1 (2013): 162-175; and Lee, Jee Hoon, et al. “Human pluripotent stem cell-derived atrial and ventricular cardiomyocytes develop from distinct mesoderm populations.” Cell stem cell 21.2 (2017): 179-194, each of which are incorporated by reference. In some embodiments, differentiating the stem cell into a cardiac cell, including a cardiomyocyte, includes culturing the stem cell in RPMI+B27, without insulin, for 7 days. During this time the cultured cells are treated with CHIR99021 during day 1 and day 2. Subsequently, the cultured cells are treated with IWR1 for 2 days. For example, see the methods described in Ho et al, “Integrating Human-Induced Pluripotent Stem Cell Expansion Capability and Cardiomyocyte Differentiation Potential in a Microcarrier Suspension Culture”, Bioreactors in Stem Cell Biology: Methods and Protocols (2022), which is incorporated by reference. After 7 days, the cells are cultured in RPMI+B27 with insulin for another 7 days.

[0268] In some embodiments, the method for producing an engineered cell with enhanced immune evasion includes genetically modifying a stem cell and differentiating the genetically modified stem cell into a neural cell. In some embodiments, the stem cell is a pluripotent stem cell. Any suitable method known in the art for differentiating stem cells into neural cells can be used in connection with the methods of the disclosure. Exemplary methods for differentiating stem cells into neural cells are described, in for example, Nat Biotechnol; 27(3): 275-280; Cell Stem Cell, 28(2): 343-355; WO2016196661A1; and W02010096496A2, the disclosures of which are incorporated herein by reference.

[0269] For many neural cell types, differentiation from the stem cell can be first directed to adopt a primitive neural cell fate through a neural induction process involving dual SMAD inhibition (Chambers et al., Nat Biotechnol. (2009) 27(3):275- 80). Primitive neural cells adopt anterior characteristics, so the lack of other signals will provide anterior / forebrain cortical cells. Caudalizing signals can be blocked to prevent paracrine signals that might otherwise generate cultures with more posterior character (for example, XAV939 can block WNT and SU5402 can block FGF signals). Dorsal cortical neurons can be made by blocking SHH activation, while ventral cortical neurons can be made through SHH activation. More caudal cell types, such as serotonergic neurons or spinal motor neurons can be made by caudalizing cultures through the addition of FGF and / or WNT signals. For some neural cell types, retinoic acid (another caudalizing agent) can be added to posteriorize cultures. The production of glial cell types can generally follow the same patterning of primitive neural cells before extended culture in FGF2 and / or EGF containing medium. Peripheral nervous system cell types can follow the same general principles but with a timely WNT signal early in the differentiation process.

[0270] In some embodiments, the method for producing an engineered cell with enhanced immune evasion includes genetically modifying a stem cell and differentiating the genetically modified stem cell into a myeloid cell. In some embodiments, the stem cell is a pluripotent stem cell. Any suitable method known in the art for differentiating stem cells into myeloid cells can be used in connection with the methods of the disclosure. Exemplary methods for differentiating stem cells into myeloid cells are described, in for example, Muffat et.al, Nat Med. 2016 Nov; 22(11): 1358-1367, Panday a et. al, Nat Neurosci. 2017 May; 20(5): 753- 759, Abud et. al, Neuron 2017 Apr 19;94(2):278-293, Douvaras et. al, Stem Cell Reports, Volume 8, Issue 6, P1516-1524, June 06, 2017, Van Wilgenburg PLOS ONE,https: / / doi.org / 10.1371 / journal.pone.0071098 — Aug 2013, Haenseler et.al, Stem Cell Reports 2017 Jun 6;8(6):1727-1742 and Takata et.al, Immunity 2017 Jul 18;47(1): 183-198, the disclosures of which are incorporated herein by reference.

[0271] In some embodiments, differentiating the genetically modified stem cell into a myeloid cell includes culturing the stem cell under conditions that induce myeloid differentiation, leading to the generation of a myeloid cell. In some embodiments, the stem cell is cultured under conditions that induce myeloid differentiation, leading to the generation of a CX3CR1+ myeloid cell. In some embodiments, the stem cell is cultured under conditions that induce myeloid differentiation, leading to the generation of a CD45+ myeloid cell. In some embodiments, the stem cell is cultured under conditions that induce myeloid differentiation, leading to the generation of a a CD45+ / CD14+ / CX3CR1+ myeloid cell.

[0272] In some embodiments, the stem cell is cultured in a bioreactor. In some embodiments, the stem cell is cultured in a cell factory under active gassing. By “active gassing” is meant exerting or applying a gas mixture pressure gradient in the cell factory or cell factories.

[0273] In some embodiments, the stem cell is differentiated into the myeloid cell using a multi-step process is used in which the stem cell is cultured with different combinations of cytokines and tissue culture media at each stage. For instance, in some embodiments, the differentiating the stem cell into a myeloid cell includes performing one or more of the following steps: First, contacting a cell culture with a first composition that includes BMP4 in a culture medium, when the cell culture is initially contacted with the first composition the cell culture includes the stem cell. A small molecule able to activate the same pathway as BMP4 can be used; Second, contacting the cell culture with a second composition that includes one or more of SCF, and VEGF, and optionally bFGF, (for example each of SCF, and VEGF, with or without bFGF) in a hematopoietic cell medium; Third, contacting the cell culture with a third composition that includes one or more of SCF, IL-3, TPO, M-CSF, and FLT3 ligand (for example each of SCF, IL-3, TPO, M-CSF, and FLT3 ligand) in a hematopoietic cell medium; and fourth, contacting the cell culture with a fourth composition that includes one or more of M-CSF, FLT3 ligand, and GM-CSF (for example each of M- CSF, FLT3 ligand, and GM-CSF) in a hematopoietic cell medium. In some embodiments all of the above four steps are performed in order. In some of such embodiments, the medium used for any of these four steps is a serum free medium. In some of such embodiments, themedium used for any of these four steps is a chemically defined medium. In the first of the above four steps, in some embodiments, a tissue culture medium suitable for maintenance of stem cells is used, while in other embodiments, a tissue culture medium suitable for differentiation of stem cells is used. In the last three of the above four steps, any suitable hematopoietic cell medium can be used.

[0274] In some embodiments, the method for producing an engineered cell with enhanced immune evasion includes genetically modifying a stem cell and differentiating the genetically modified stem cell into a retinal cell. In some embodiments, the stem cell is a pluripotent stem cell. Any suitable method known in the art for differentiating stem cells into retinal cells can be used in connection with the methods of the disclosure. Methods for differentiating stem cells into retinal cells are described in, for example, WO 2019 / 204817 and WO 2011 / 043591A2.

[0275] In some embodiments, the method for producing an engineered cell with enhanced immune evasion is a method for producing an engineered cardiomyocyte with enhanced immune evasion. In some embodiments, the method for producing a cardiomyocyte with enhanced immune evasion includes genetically modifying a stem cell and differentiating the genetically modified stem cell into a cardiomyocyte. In some embodiments, the stem cell is a pluripotent stem cell. In some embodiments, the resulting engineered cardiomyocyte exhibits reduced NK cell-mediated cytotoxicity as compared to a wild-type cardiomyocyte.

[0276] In some embodiments, the method for producing an engineered cell with enhanced immune evasion further includes expanding the engineered cell to produce a population of engineered cells. Expanding the engineered cell can include culturing or contacting the engineered cells with a medium having a cytokine and / or a growth factor mixture permissive for expansion of the engineered cell.

[0277] In some embodiments, the method for producing an engineered cell with enhanced immune evasion further includes preserving the population of engineered cells after the expansion. For instance, the population of engineered cells can be cryopreserved. The cryopreserved population of engineered cells can be thawed at a later time and can be diluted for downstream applications.In some embodiments, the population of engineered cells produced by the methods described herein is used in preparing a composition for treating a disease or condition in a subject.V. Methods of Treatment

[0278] Certain aspects of the disclosure provide for methods of treating a disease or condition that include administering a population of engineered cells (e.g., a population of engineered cell described herein), or a pharmaceutical composition as described herein.

[0279] In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells that include a genetic modification that reduces or eliminates function of a natural-killer group 2, member D (NKG2D) ligand as compared to a wild-type cell, or a heterologous nucleic acid sequence encoding at least a functional portion of a human leukocyte antigen (HLA) molecule. In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells that include the genetic modification. In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells that include the heterologous nucleic acid. In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells that include both the genetic modification and the heterologous nucleic acid.

[0280] In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells that include a genetic modification that reduces or eliminates function of a NKG2D ligand as compared to a wild-type cell, or a heterologous nucleic acid sequence encoding at least a functional portion of a HLA molecule, wherein either the genetic modification or the heterologous nucleic acid is sufficient to reduce or inhibit natural killer (NK) cell-mediated cytotoxicity of the population of engineered cells, or progeny thereof, as compared to a population of wild-type cell. In some embodiments, either the genetic modification or the heterologous nucleic acid is sufficient to reduce or inhibit NK cell-mediated cytotoxicity of the population of engineered cells, or progeny thereof, 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%, or at least 90% as compared to a population of wild-type cells. In some embodiments, either the genetic modification or the heterologous nucleic acid is sufficient to reduce or inhibit NK cell-mediated cytotoxicity of the population of engineered cells, or progeny thereof, by at least 10% as compared to a population of wild-type cells. In some embodiments, either the genetic modification or the heterologous nucleic acid issufficient to reduce or inhibit NK cell-mediated cytotoxicity of the population of engineered cells, or progeny thereof, by at least 20% as compared to a population of wild-type cells. In some embodiments, either the genetic modification or the heterologous nucleic acid is sufficient to reduce or inhibit NK cell-mediated cytotoxicity of the population of engineered cells, or progeny thereof, by at least 30% as compared to a population of wild-type cells. In some embodiments, either the genetic modification or the heterologous nucleic acid is sufficient to reduce or inhibit NK cell-mediated cytotoxicity of the population of engineered cells, or progeny thereof, by at least 50% as compared to a population of wild-type cells. In some embodiments, either the genetic modification or the heterologous nucleic acid is sufficient to reduce or inhibit NK cell-mediated cytotoxicity of the population of engineered cells, or progeny thereof, by at least 80% as compared to a population of wild-type cells.

[0281] In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells that include a genetic modification that reduces or eliminates function of a NKG2D ligand as compared to a wild-type cell, wherein the genetic modification that reduces or eliminates function of the NKG2D ligand as compared to a wild-type cell is a deletion or disruption of a gene encoding the NKG2D ligand. In some embodiments, the genetic modification that reduces or eliminates function of the NKG2D ligand as compared to a wild-type cell is the deletion of the gene encoding the NKG2D ligand. In some embodiments, the genetic modification that reduces or eliminates function of the NKG2D ligand as compared to a wild-type cell is the disruption of the gene encoding the NKG2D ligand. In some embodiments, the genetic modification that reduces or eliminates function of the NKG2D ligand as compared to a wild-type cell includes a deletion or disruption that results in reduced or eliminated expression of the gene encoding the NKG2D ligand.

[0282] In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells that include a genetic modification that reduces or eliminates function of a NKG2D ligand as compared to a wild-type cell, wherein the NKG2D ligand includes MICA, MICB, Raetle, Raetlg, Raetll, Ulbpl, Ulbp2, Ulbp3, or any combination thereof. In some embodiments, the NKG2D ligand includes MICA. In some embodiments, the NKG2D ligand includes MICB. In some embodiments, the NKG2D ligand includes Raetle. In some embodiments, the NKG2D ligand includes Raetlg. In some embodiments, the NKG2D ligand includes Raetll. In some embodiments, the NKG2D ligandincludes Ulbpl. In some embodiments, the NKG2D ligand includes Ulbp2. In some embodiments, the NKG2D ligand includes Ulbp3.

[0283] In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells that include a heterologous nucleic acid sequence encoding at least a functional portion of a HLA molecule, wherein the heterologous nucleic acid encodes at least a functional portion of an HLA-E molecule or an HLA-G molecule. In some embodiments, the heterologous nucleic acid sequence encodes at least the functional portion of the HLA-E molecule. In some embodiments, the heterologous nucleic acid sequence encodes at least the functional portion of the HLA-G molecule. In some embodiments, the heterologous nucleic acid sequence encodes both at least the functional portion of both the HLA-E molecule and the HLA-G molecule. In some embodiments, the functional portion of the HLA-E molecule or the HLA-G molecule includes at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or 300 amino acids of the full-length sequence of the HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of the HLA-E molecule or the HLA-G molecule includes at least 50 amino acids of the full-length sequence of the HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of the HLA-E molecule or the HLA-G molecule includes at least 100 amino acids of the full-length sequence of the HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of the HLA-E molecule or the HLA-G molecule includes at least 200 amino acids of the full-length sequence of the HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of the HLA-E molecule or the HLA-G molecule includes at least 300 amino acids of the full- length sequence of the HLA-E molecule or the HLA-G molecule.

[0284] In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells that include a heterologous nucleic acid sequence encoding at least a functional portion of a HLA molecule, wherein the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 75% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portionof an HLA-E molecule that has at least 85% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 90% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 95% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 98% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 99% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that is identical to SEQ ID NO: 6.

[0285] In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells that include a heterologous nucleic acid sequence encoding at least a functional portion of a HLA molecule, wherein the heterologous nucleic acid encodes at least a functional portion of an HLA molecule that includes a binding peptide. In some embodiments, the binding peptide has an amino acid sequence that includes LIL or LFL. In some embodiments, the binding peptide has an amino acid sequence corresponding to any one of SEQ ID NOS: 8 or 10.

[0286] In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells that include a heterologous nucleic acid further including a sequence encoding at least a functional portion of a P2 microglobulin (B2M). In some embodiments, the functional portion of the B2M includes at least 10, 20, 30, 40, 50, 60, 70, 90 or 100 amino acids of the full-length sequence of the B2M. In some embodiments, the functional portion of the B2M includes at least 10 amino acids of the full-length sequence of the B2M. In some embodiments, the functional portion of the B2M includes at least 30 amino acids of the full-length sequence of the B2M. In some embodiments, the functional portion of the B2M includes at least 50 amino acids of the full-length sequence of the B2M. In some embodiments, the functional portion of the B2M includes at least 90 amino acids of the full-length sequence of the B2M. In some embodiments, the functional portion of B2M is linked to the HLA-E molecule or the HLA-G molecule encoded by the heterologous nucleic acid. In some embodiments, the functional portion of B2M is linked to the HLA-E molecule. In some embodiments, the functional portion of B2M is linked to the HLA-G molecule. Insome embodiments, the functional portion of B2M is linked to the N-terminus of HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of B2M is linked to the C-terminus of HLA-E molecule or the HLA-G molecule. In some embodiments, the functional portion of B2M is linked to the HLA-E molecule or the HLA-G molecule through a linker as described herein. In some embodiments, the functional portion of B2M linked to the HLA-E molecule further includes a binding peptide. In some embodiments, the binding peptide has an amino acid sequence including LIL or LFL.

[0287] In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells that include a heterologous nucleic acid encoding a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least 75% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least 85% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid nucleic acid encodes at least a functional portion of an HLA-E molecule that has at least 90% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least 95% identity to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least 98% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that has at least 99% identity to SEQ ID NO: 2 or 4. In some embodiments, the heterologous nucleic acid encodes a B2M linked to a HLA-E molecule that has an amino acid sequence that is identical to SEQ ID NO: 2 or 4.

[0288] In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells that include a heterologous nucleic acid sequence encoding at least a functional portion of a HLA molecule, wherein the heterologous nucleic acid sequence encoding at least a functional portion of the HLA molecule is integrated into a genomic locus. In some embodiments, the heterologous nucleic acid sequence is integrated into a sustained transgene expression locus (STEL) in the genome of each of the engineered cells in the population of engineered cells. In some embodiments, theSTEL is a gene locus that encodes a protein involved in one or more of: ribonucleoprotein complex formation, focal adhesion, cell-substrate adherens junction, cell-substrate junction, cell anchoring, extracellular exosome, extracellular vesicle, intracellular organelle, anchoring junction, RNA binding, nucleic acid binding (e.g., rRNA or mRNA binding), and protein binding. In some embodiments, the STEL is a glyceraldehyde 3-phosphate dehydrogenase (GAPDEL) gene. In some embodiments, the STEL is a ribosomal protein gene locus, such as an RPL or RPS gene locus. Examples of RPL genes are RPL10, RPL13, RPS18, RPL3, RPLP1, RPL13A, RPL15, RPL41, RPL11, RPL32, RPL18A, RPL19, RPL28, RPL29, RPL9, RPL8, RPL6, RPL 18, RPL7, RPL7A, RPL21, RPL37A, RPL12, RPL5, RPL34, RPL35A, RPL30, RPL24, RPL39, RPL37, RPL 14, RPL27A, RPLP2, RPLPO, RPL23A, RPL26, RPL36, RPL35, RPL23, RPL4, and RPL22. Examples of RPS genes are RPS2, RPS 19, RPS 14, RPS3A, RPS 12, RPS3, RPS6, RPS23, RPS27A, RPS8, RPS4X, RPS7, RPS24, RPS27, RPS15A, RPS9, RPS28, RPS 13, RPSA, RPS5, RPS16, RPS25, RPS15, RPS20, and RPSIL In some embodiments, the STEL is a gene locus encoding a mitochondrial protein, such as MT- CO1, MT-C02, MT-ND4, MT-ND1, and MT-ND2. In some embodiments, the STEL is a gene locus encoding an actin protein, such as ACTG1 and ACTB. In some embodiments, the STEL is a gene locus encoding a eukaryotic translation elongation factor, such as EEF1A1 and EEF2, or a eukaryotic translation initiation factor such as EIEI. In some embodiments, the STEL is a gene locus encoding a histone, such as H3F3 A and H3F3B. In some embodiments, the STEL is a gene locus selected from FTL, FTH1, TPT1, IMSB10, GAPDH, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2, and SRP14.

[0289] In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells described herein that further include one or more genetic modifications that result in reduced T cell-mediated killing of the population of engineered cells, or progeny thereof, as compared to a population of wild-type cells. In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells that further include one or more genetic modifications that result in at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% reduced T cell-mediated killing of the population of engineered cells, or progeny thereof, as compared to a population of wild-type cells. In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells that further include includes one or more genetic modifications that result in at least 20% reduced T cell-mediated killing of the population of engineeredcells, or progeny thereof, as compared to a population of wild-type cells. In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells that further include one or more genetic modifications that result in at least 50% reduced T cell-mediated killing of the population of engineered cells, or progeny thereof, as compared to a population of wild-type cells. In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells that further include one or more genetic modifications that result in at least 80% reduced T cell-mediated killing of the population of engineered cells, or progeny thereof, as compared to a population of wild-type cells.

[0290] In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells described herein that further include one or more genetic modifications that result in reduced T cell-mediated killing of the population of engineered cells, or progeny thereof, as compared to a population of wild-type cells, wherein the one or more genetic modifications that results in reduced T cell-mediated killing of the population of engineered cells as compared to a population of wild-type cells include a deletion or disruption of a 12 microglobulin (B2M) gene. In some embodiments, the one or more genetic modifications that results in reduced T cell-mediated killing of the population of engineered cells, or progeny thereof, as compared to a population of wild-type cells include a deletion or disruption of one or more of an HLA-A gene, an HLA-B gene, and an HLA-C gene.

[0291] In some embodiments, method for treating a disease or condition includes administering composition includes a population of engineered cells that further include a deletion or disruption of the CIITA gene. Accordingly, in some embodiments, the method for treating a disease or condition includes administering a population of engineered cells described that include one or more genetic modifications that result in reduced T cell- mediated killing of the population of engineered cells, or progeny thereof, as compared to a population of wild-type cells as described herein (e.g., a deletion or disruption of & B2M, HLA-A, HLA-B, and / or HLA-C) and a deletion or disruption of the CIITA gene.

[0292] In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells that further include a kill switch. In some embodiments, the kill switch is a gene that encodes a herpes simplex virus thymidine kinase (HSV-TK), an inducible caspase9 (also known as incasep 9, and iCasp9), CD20, or a mutant human thymidylate kinase (mTMPK). In some embodiments, the kill switch is under thecontrol of an inducible promoter. In some embodiments, the kill switch is encoded by a heterologous nucleic acid. In some embodiments, the at least one heterologous nucleic acid encoding the kill switch is integrated into the genome of each engineered cell in the population of engineered cells.

[0293] In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells described herein that further include at least one selectable marker gene. Non-limiting examples of selectable marker genes include, but are not limited to, fluorescent proteins (such as green fluorescent protein (GFP), blue fluorescent protein (EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent protein (ECFP, Cerulean, CyPet, mTurquoise2), and yellow fluorescent protein derivatives (YFP, Citrine, Venus, YPet, EYFP)), b-galactosidase (LacZ), chloramphenicol acetyltransferase (cat), neomycin phosphotransferase (neo), enzymes (such as oxidases and peroxidases), and antigenic molecules. In some embodiments, the selectable marker gene can be driven by an endogenous gene promoter.

[0294] In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells described herein that include an engineered stem cell. In some embodiments, the engineered stem cell includes a pluripotent stem cell. In some embodiments, the pluripotent stem cell includes an embryonic stem cell (ESC) or induced pluripotent stem cell (iPSC). In some embodiments, the engineered stem cell includes a multipotent stem cell.

[0295] In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells described herein that include a cardiac cell, a neural cell, a myeloid cell, a T cell (e.g., a regulator T cell), or a retinal cell. In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells that include the cardiac cell. In some embodiments, the cardiac cell is a cell of the epicardium, the myocardium, or the endocardium of the heart. In some embodiments, the cardiac cell is a cardiomyocyte, a cardiac fibroblast, a cardiac smooth muscle cell, an epicardium cells, a cardiac endothelial cell, a Purkinje fiber, or a pacemaker cell. In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells that include the cardiomyocyte. In some embodiments, the method for treating a disease or condition includes administering that include the neural cell. In some embodiments, the neural cell is a neuron or a glial cell. Insome embodiments, the method for treating a disease or condition includes administering a population of engineered cells that include the myeloid cell. In some embodiments, the myeloid cell is a monocyte, a microglia, a macrophage, a dendritic cell, a basophil, an eosinophil, an erythrocyte, a mast cell, a neutrophil, a megakaryocyte, or a platelet, or any precursor progenitor cell thereof. In some embodiments, the cardiac cell, the neural cell, the myeloid cell, the T cell (e.g., a regulator T cell), or the retinal cell in the population of engineered cells for use in the methods for treating a disease or condition described herein are derived from an engineered stem cell (e.g., an engineered stem cell described herein).

[0296] In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells described herein that includes an engineered cell capable of differentiating into another cell type, such as a cardiac cell, a neural cell, a myeloid cell, a T cell (e.g., a regulator T cell), or a retinal cell. In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells that include an engineered cell capable of differentiating into a cardiac cell. In some embodiments, the method for treating a disease or condition includes administering a population of engineered cells that include an engineered cell capable of differentiating into a cardiomyocyte.

[0297] In some embodiments, the method for treating a disease or condition includes administering a population of human engineered cells to the subject. In some embodiments, the subject is a human. In some embodiments, the population of engineered cells is allogenic to the subject. In some embodiments, the population of engineered cells is autologous to the subject.

[0298] In some embodiments, the disease or condition to be treated by the methods described herein is heart failure, Parkinson’s disease, multiple sclerosis, irritable bowel syndrome, type 1 diabetes, rheumatoid arthritis, or neural inflammation. In some embodiments, the method is for treating heart failure. In some embodiments, the method is for treating Parkinson’s disease. In some embodiments, the method is for treating multiple sclerosis. In some embodiments, the method is for treating irritable bowel syndrome. In some embodiments, the method is for treating type 1 diabetes. In some embodiments, the method is for treating rheumatoid arthritis. In some embodiments, the method is for treating neural inflammation.

[0299] In some embodiments, the administration of the population of engineered cells results in improvement of at least one symptom associated with the disease or disorder, e.g., as determined by responsiveness / non-responsiveness, or indicators known in the art. The choice of technologies and methods, and the time and frequency of examination can be determined and / or adjusted by a person skilled in the art based on the subject’s specific condition.

[0300] In some embodiments, the method further includes examining the subject for responsiveness to the population of engineered cells. The responsiveness of the subject can be determined as an improvement of at least one parameter of disease progression. The subject can have partial response or complete response to a treatment. The response to a treatment can be determined based on methods known in the art. A person skilled in the art can determine the proper methods based on the type of diseases being evaluated.

[0301] In some embodiments, the method further includes administering a kill switch activator after administration of the population of engineered cells. The kill switch activator can be used for removal of the population of engineered cells after a period of time. The kill switch activator can be administered any time following administration of the population of engineered cells, such as for example, after the subject has achieved a certain level of responsiveness as measured by improvement of at least one symptom associated with the disease or disorder. In some embodiments, the method includes administering an activator of a kill switch selected from a gene that encodes a herpes simplex virus thymidine kinase (HSV-TK), an inducible caspase9 (also known as incasep 9, and iCasp9), CD20, or a mutant human thymidylate kinase (mTMPK).

[0302] The population of engineered cells of the methods described herein can be administered to a subject by any suitable route of administration. In some embodiments, the methods include administering the engineered cells surgically, e.g., during open heart surgery. In some embodiments, the methods include by injection. In some embodiments, the method include intracerebroventricular (ICV) injection. In some embodiments, the method includes administering the population of engineered cells orally, pulmonarily, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, or topically. In some embodiments, the population of engineered cells is administered by directly injecting into a tissue. For instance, in some embodiments, the population of engineered cells is administered by direct injection into a subject’s heart by, forexample, intracoronary administration, intramyocardial administration, or transendocardial administration. The administration of the population of engineered cells to a subject can be performed by any suitable means, such as by injection, by catheter, by an implantable device, or by any other device suitable for administration. By way of example, the population of engineered cells can be introduced to the heart by using a catheter inserted via the femoral, subclavian, jugular or axillary vein, or by endocardial transplantation into the ventricle or atrium region. The population of engineered cells also can be transplanted into the ventricle or atrium region by an epicardial approach, using a needle inserted through the chest.

[0303] The population of engineered cells can be administered to the subject at one time or over a series of administrations and can be administered to the patient at any time from diagnosis of the disease or disorder onwards. For instance, the population of engineered cells can be administered as one or more doses over the course of a treatment. The doses can be administered using the same or a different route of administration, and can contain the same amount or a different amount of the population of engineered cells.

[0304] The duration of administration can depend on the route of administration of the population of engineered cells.

[0305] In some embodiments, the methods further include administering an additional therapy to the subject. The additional therapy will depend on the disease or disorder being treated by the methods described herein.VI. Methods for Enhancing Immune Evasion

[0306] In certain aspects, the disclosure provides for methods of enhancing immune evasion of a cell, or progeny thereof. In some embodiments, the method comprises engineering the cell by genetically modifying the cell to reduce or eliminate function of a natural-killer group 2, member D (NKG2D) ligand relative to a wild-type cell, and incorporating a heterologous nucleic acid sequence that encodes at least a functional portion of a human leukocyte antigen (HLA)-E molecule covalently linked to a P2-microglobulin (B2M) molecule disclosed herein into the genome of the cell, wherein the genetically modifying step enhances the survival probability of the cell when exposed to natural killer cells, as compared to a wild-type cell.

[0307] In some embodiments of the methods for enhancing immune evasion of a cell, at least one of the HLA-E protein or the B2M protein comprises a modification that reduces binding affinity to a CD8 co-receptor as compared to a corresponding wild-type protein. In certain embodiments, the B2M molecule comprises the modification that reduces binding affinity to a CD8 co-receptor. In certain embodiments, the heterologous nucleic acid encodes an amino acid sequence having at least 95% identity to one of SEQ ID NO. 2, 4, 6, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30. In certain embodiments, the amino acid sequence has at least 95% identity to SEQ ID NO. 18.

[0308] In some embodiments, the method further comprises engineering the cell with one or more genetic modifications that result in reduced T cell-mediated killing as compared to a wild-type cell, the one or more genetic modifications comprising: (i) a deletion or disruption of a P2 microglobulin (B2M) gene; (ii) a deletion or disruption of one or more of an HLA-A gene, an HLA-B gene, and an HLA-C gene; or (iii) a deletion or disruption of a class II, major histocompatibility complex, transactivator (CHTA) gene.

[0309] In certain embodiments of the methods, the genetic modification that reduces or eliminates function of a NKG2D ligand as compared to a wild-type cell is a deletion or disruption of a gene encoding the NKG2D ligand. The NKG2D ligand can comprise MICA, MICB, Raetle, Raetlg, Raetll, Ulbpl, Ulbp2, Ulbp3, or any combination thereof.

[0310] In certain embodiments of the methods, the HLA-E molecule encoded by the heterologous nucleic acid sequence comprises a binding peptide. The binding peptide can comprise an amino acid sequence comprising LIL or LFL.

[0311] In some embodiments of the methods, the HLA-E molecule is covalently linked to the B2M molecule by a linker described herein.

[0312] In some embodiments of the methods, the incorporating step comprises integrating the heterologous nucleic acid sequence into a genomic locus of the cell. The genomic locus can be a sustained transgene expression locus (STEL), such as a locus within a human glyceraldehyde 3-phosphate dehydrogenase (GAPDBT) gene.

[0313] In some embodiments, the method further comprises expanding the engineered cell to produce a population of engineered cells. In some embodiments, the method furthercomprises differentiating the population of engineered cells into a population of cardiac cells, neural cells, T cells, retinal cells, or myeloid cells.VII. Kits and Dosage Forms

[0314] Certain aspects of the disclosure provide for kits that include a population of engineered cells or a pharmaceutical composition that includes population of engineered cells, including any of the population of engineered cells or the pharmaceutical compositions described herein.

[0315] In some embodiments, the kit includes instructional material for the use of said population of engineered cells or the pharmaceutical composition. In some embodiments, the instructional material includes instructions of preparing the pharmaceutical composition or the population of engineered cells for administration into a subject. Such instructions can include, but are not limited to, instructions for preparing or storing the population of engineered cells or the pharmaceutical composition, adding additives to the treatment, or combining the population of cells or the pharmaceutical composition with an additional therapeutic agent. In some embodiments, the instructional material includes instructions for administering the population of engineered cells or the pharmaceutical composition to a subject.

[0316] In some embodiments, the kit further includes an applicator for administering a population of engineered cells or a pharmaceutical composition that includes a population of engineered cells described herein. The applicator can be any device suitable for administration of the population of engineered cells or composition described herein to a subject, including, but not limited to, a hypodermic syringe, a needle, a balloon-dilating catheter, a pipette, and the like. The applicator can be a single-use or multiple-use administration device, and can be included in the kit as a pre-filled delivery system with, e.g., a pharmaceutical composition that includes the population of cells.

[0317] Certain aspects of the disclosure also provide for dosage forms of a pharmaceutical composition that includes a population of engineered cells suitable for administration to a subject. The dosage form can be in any form suitable for administration to a subject by any suitable route, including orally, pulmonarily, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally,intralymphatically, intracranial, intracerebral or topically. In some embodiments, the dosage form is suitable for administration by injection into a subjectVIII. Engineered Human Leukocyte Antigen (HLA) and B2M molecules

[0318] A Human Leukocyte Antigen-E molecule described herein refers to a cell surface protein that interact with immune cells thereby modulating immune activity. The immune response has an important role for the identification and elimination of foreign agents, such as pathogens or foreign antigens. For example, the presence of foreign antigens causes immune cells to mount an immune response against the foreign antigen. Presentation of foreign antigens on Major Histocompatibility Complex (MHC) molecules target a cell for killing by natural killer cells and cytotoxic T cells, thus clearing the foreign antigen. While natural killer cell and T cell cytotoxicity functions normally to eliminate pathogen-infected cells, tumor cells, and other deleterious agents, natural killer cell, and T cells can also target therapeutic agents such as cell therapies for clearance, thus reducing the therapeutic benefit conceded by these therapies. Thus, designing therapies that can overcome natural killer cell and T cell cytotoxicity is key to maintain an optimal therapeutic benefit.

[0319] Accordingly, it is an insight of the present disclosure that modifying an MHC’s interaction deleterious immune cells can modulate the immune activity of said immune cells. An HLA-E molecule described herein can interact with receptors on natural killer (NK) cells and certain T cells (e.g., CD8+ T cells) and inhibit NK cell and / or T cell activity. Certain aspects of the disclosure provide an engineered HLA-E that is a variant of a reference HLA-E (e.g., a WT HLA-E). Such an engineered HLA-E comprises one or more altered activity relative to a reference HLA-E (e.g., a WT HLA-E). Specifically, an HLA-E molecule described herein can interact with receptors on natural killer (NK) cells and certain T cells (e.g., CD8+ T cells) and exhibit an enhanced immune evasion activity relative to a reference HLA-E (e.g., a WT HLA-E), and / or a maintained structural stability relative to the reference HLA-E (e.g., a WT HLA-E). In some embodiments, an engineered HLA-E provided herein comprises: a reduced binding affinity to a CD8 co-receptor relative to a reference HLA-E (e.g., a WT HLA-E), a binding affinity to a NK cell relative to a reference HLA-E (e.g., a WT HLA-E), an inhibitory activity to a NK cell relative to a reference HLA-E (e.g., a WT HLA- E), a folding activity relative to a reference HLA-E (e.g., a WT HLA-E), a stable surfaceexpression relative to a reference HLA-E (e.g., a WT HLA-E), a reduced CD8+ T cell proliferation relative to a reference HLA-E (e.g., a WT HLA-E), a reduced CD8+ T cell activation relative to a reference HLA-E (e.g., a WT HLA-E), or any combination thereof.

[0320] In some embodiments, an enhanced immune evasion activity relative to a reference HLA-E (e.g., a WT HLA-E) comprises a reduced binding affinity to a CD8 coreceptor relative to a reference HLA-E (e.g., a WT HLA-E), a binding affinity to a NK cell relative to a reference HLA-E (e.g., a WT HLA-E), an inhibitory activity to a NK cell relative to a reference HLA-E (e.g., a WT HLA-E), a reduced CD8+ T cell proliferation relative to a reference HLA-E (e.g., a WT HLA-E), a reduced CD8+ T cell activation relative to a reference HLA-E (e.g., a WT HLA-E), or any combination thereof. In some embodiments, a maintained structural stability relative to the reference HLA-E (e.g., a WT HLA-E) comprises a folding activity relative to a reference HLA-E (e.g., a WT HLA-E), a stable surface expression relative to a reference HLA-E (e.g., a WT HLA-E), or both.

[0321] In some embodiments, an enhanced immune evasion activity relative to a reference HLA-E (e.g., a WT HLA-E) comprises a reduced binding affinity to a CD8 coreceptor relative to a reference HLA-E (e.g., a WT HLA-E). In some embodiments, a reduced binding affinity to a CD8 co-receptor comprises at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 97%, at least about 98%, at least about 99%, or about 100% reduction in binding affinity to the CD8 co-receptor relative to a reference HLA-E (e.g., a WT HLA-E).

[0322] In some embodiments, an enhanced immune evasion activity relative to a reference HLA-E (e.g., a WT HLA-E) comprises a binding affinity to a NK cell relative to a reference HLA-E (e.g., a WT HLA-E). In some embodiments, a binding affinity to the NK cell comprises binding to one or more inhibitory receptors on the NK cells. In some embodiments, a binding affinity to a NK cell relative to a reference HLA-E (e.g., a WT HLA- E) comprises an increased binding to one or more inhibitor receptors on the NK cell. In some embodiments, an increased binding to one or more inhibitor receptors on the NK cell relative to a reference HLA-E (e.g., a WT HLA-E) comprises at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 97%, at least about 98%, at least about 99%, or about 100%, or greater increased binding to one or more inhibitor receptors on the NK cell relative to a reference HLA-E (e.g., a WT HLA-E). In some embodiments, an inhibitory receptor is: Natural Killer Group 2 member A. (NKG2A).

[0323] In some embodiments, an enhanced immune evasion activity relative to a reference HLA-E (e.g., a WT HLA-E) comprises an inhibitory activity to a NK cell relative to a reference HLA-E (e.g., a WT HLA-E). In some embodiments, an inhibitory activity to a NK comprises decreased cytotoxicity activity of the NK cell relative to a reference HLA-E (e.g., a WT HLA-E). In some embodiments, a decreased cytotoxicity activity of an NK cell comprises at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 97%, at least about 98%, at least about 99%, or about 100% decrease in cytotoxicity activity relative to a reference HLA- E (e.g, a WT HLA-E).

[0324] In some embodiments, an enhanced immune evasion activity relative to a reference HLA-E (e.g., a WT HLA-E) comprises a reduced CD8+ T cell proliferation relative to a reference HLA-E (e.g., a WT HLA-E). In some embodiments, a reduced CD8+ T cell proliferation comprises at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 97%, at least about 98%, at least about 99%, or about 100% decrease of CD8+ T cell proliferation relative to a reference HLA-E (e.g., a WT HLA-E).

[0325] In some embodiments, an enhanced immune evasion activity relative to a reference HLA-E (e.g., a WT HLA-E) comprises a reduced CD8+ T cell activation relative to a reference HLA-E (e.g., a WT HLA-E). In some embodiments, a reduced CD8+ T cellactivation comprises at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 97%, at least about 98%, at least about 99%, or about 100% decrease of CD8+ T cell activation relative to a reference HLA-E (e.g., a WT HLA-E).

[0326] In some embodiments, a maintained structural stability relative to the reference HLA-E (e.g., a WT HLA-E) comprises a folding activity relative to a reference HLA-E (e.g., a WT HLA-E).

[0327] In some embodiments, a maintained structural stability relative to the reference HLA-E (e.g., a WT HLA-E) comprises a stable surface expression relative to a reference HLA-E (e.g., a WT HLA-E).

[0328] In some embodiments, an engineered HLA-E includes one or more amino acid alterations at one or more positions e.g., one or more amino acid alterations at one or more positions as compared to wild-type HLA-E), a combination of alterations, one or more non- naturally occurring polypeptides, one or more amino acid alterations at one or more positions in the one or more non-naturally occurring polypeptides, a combination of alterations in the one or more non-naturally occurring polypeptides, one or more linkers, or any combination thereof, relative to a reference HLA-E (e.g., a WT HLA-E) and comprises one or more enhanced immune evasion activity and / or maintained structural stability relative to a reference HLA-E (e.g., a WT HLA-E).Variant HLA-E molecules

[0329] In some embodiments, an engineered HLA-E provided herein is a variant of a reference HLA-E, wherein the reference HLA-E has an amino acid sequence of SEQ ID NO: 6. In some embodiments, an engineered HLA-E provided herein is a variant of a reference HLA-E, wherein the reference HLA-E has an amino acid sequence of SEQ ID NO: 6, and the engineered HLA-E has one or more amino acid alterations. In some embodiments, an engineered HLA-E provided herein includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid alterations.

[0330] In some embodiments, the one or more amino acid alterations comprises one or more amino acid deletions, additions, substitutions, or a combination thereof. In some embodiments, an engineered HLA-E provided herein includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid deletions, additions, substitutions, or a combination thereof. In some embodiments, one or more amino acid substitutions can be one or more conservative and / or non-conservative amino acid substitutions. In some embodiments, an engineered HLA-E provided herein includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions and / or non-conservative amino acid substitutions.

[0331] In some embodiments, an engineered HLA-E provided herein is a variant of a reference HLA-E, wherein the reference HLA-E has an amino acid sequence of SEQ ID NO: 6, and the engineered HLA-E has one or more amino acid alterations. The one or more amino acid alterations may be located at one or more positions corresponding to one or more positions in SEQ ID NO: 6. As used herein, the phrase “a residue corresponding to position X in SEQ ID NO: Y” refers to a residue at a corresponding position following an alignment of two sequences. For example, the residue in SEQ ID NO: 6 corresponding to position 227 is the residue at position 227 in SEQ ID NO: 12.

[0332] In some embodiments, an engineered HLA-E provided herein is a variant of a reference HLA-E, wherein the reference HLA-E has an amino acid sequence of SEQ ID NO: 6, and the engineered HLA-E has one or more amino acid alterations at a residue corresponding to position 227, 228, or both relative to SEQ ID NO: 6. In some embodiments, an engineered HLA-E provided herein includes one or more amino acid alterations at a residue corresponding to position 227 and 228 relative to SEQ ID NO: 6.

[0333] As a non-limiting example, a conservative amino acid substitution relative to the T228 residue in SEQ ID NO: 6 may include substitution of T for another aliphatic amino acid (e.g., Gly (G), Ala (A), Vai (V), Leu (L), He (I), or Ser (S)). As a non-limiting example, a non-conservative amino acid substitution relative to the D227 residue in SEQ ID NO: 6 may include substitution of D, an acidic residue, for basic amino acid (e.g., Lys (K), Arg (R), or His (H)).

[0334] For example, in some embodiments, the one or more amino acid alterations result in an engineered HLA-E having an: K at a residue corresponding to position 227 in SEQ IDNO: 6; A at a residue corresponding to position 228 in SEQ ID NO: 6; or both. In some embodiments, the one or more amino acid alterations comprises D227K, T228A, or both.

[0335] In some embodiments, other than the one or more engineered HLA-E amino acid alterations, the engineered HLA-E comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 6. In some embodiments, other than the one or more engineered HLA-E amino acid alterations, the engineered HLA-E comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 6. In some embodiments, other than the one or more engineered HLA-E amino acid alterations, the engineered HLA-E comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, other than the one or more engineered HLA-E amino acid alterations, the engineered HLA-E comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 6. In some embodiments, other than the one or more engineered HLA-E amino acid alterations, the engineered HLA-E comprises an amino acid sequence that has at least 96% sequence identity to SEQ ID NO: 6. In some embodiments, other than the one or more engineered HLA-E amino acid alterations, the engineered HLA-E comprises an amino acid sequence that has at least 97% sequence identity to SEQ ID NO: 6. In some embodiments, other than the one or more engineered HLA-E amino acid alterations, the engineered HLA-E comprises an amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 6. In some embodiments, other than the one or more engineered HLA-E amino acid alterations, the engineered HLA-E comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 6. In some embodiments, other than the one or more engineered HLA-E amino acid alterations, the engineered HLA-E comprises the amino acid sequence of SEQ ID NO: 6.

[0336] In some embodiments, the engineered HLA-E comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 12. In some embodiments, the engineered HLA-E comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 12. In some embodiments, the engineered HLA-E comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 12. In some embodiments, the engineered HLA-E comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 12. In some embodiments, the engineered HLA-E comprises an amino acid sequence that has at least 96% sequence identity to SEQ ID NO: 12. In some embodiments, the engineered HLA-E comprises an amino acid sequence that has at least 97%sequence identity to SEQ ID NO: 12. In some embodiments, the engineered HLA-E comprises an amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 12. In some embodiments, the engineered HLA-E comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 12. In some embodiments, the engineered HLA- E comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the engineered HLA-E comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 14. In some embodiments, the engineered HLA-E comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 14. In some embodiments, the engineered HLA-E comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 14. In some embodiments, the engineered HLA-E comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 14. In some embodiments, the engineered HLA-E comprises an amino acid sequence that has at least 96% sequence identity to SEQ ID NO: 14. In some embodiments, the engineered HLA-E comprises an amino acid sequence that has at least 97% sequence identity to SEQ ID NO: 14. In some embodiments, the engineered HLA-E comprises an amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 14. In some embodiments, the engineered HLA- E comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 14. In some embodiments, the engineered HLA-E comprises the amino acid sequence of SEQ ID NO: 14.

[0337] In some embodiments, an engineered HLA-E provided herein includes has one or more non-naturally occurring polypeptides. In some embodiments, wherein an engineered HLA-E comprises one or more non-naturally occurring peptides, the engineered HLA-E is fused or conjugated to the one or more non-naturally occurring polypeptides. In some embodiments, wherein more than one non-naturally occurring polypeptide is present, each non-naturally occurring polypeptide can be fused or conjugated to an engineered HLA-E provided herein or to another non-naturally occurring polypeptide. In embodiments wherein the engineered HLA-E provided herein is fused or otherwise linked to one or more non- naturally occurring polypeptide, such a molecule can be referred to as a fusion protein herein. Methods of attaching or fusing engineered HLA-E molecules to non-naturally occurring polypeptides are further described herein. It is understood that when referring to engineered HLA-E molecules, reference is also made to fusion proteins, such as those described herein.Fusion proteins

[0338] Provided herein are fusion proteins or a use thereof. A fusion protein described herein comprises an engineered HLA-E fused to at least one non-naturally occurring polypeptide. In some embodiments, fusion proteins are multimeric proteins. In some embodiments, a multimeric protein is a homomeric protein. In some embodiments, a multimeric protein is a heteromeric protein.

[0339] In some embodiments, a non-naturally occurring polypeptide can be attached to the N termini of the engineered HLA-E. In some embodiments, a non-naturally occurring polypeptide can be attached to the C termini of the engineered HLA-E. In some embodiments, a non-naturally occurring polypeptide can be attached to the engineered HLA- E molecule in the following formats, reading from the N-termini to the C-termini from the left to the right:[non-naturally occurring polypeptide]-[engineered HLA-E][engineered HLA-E]-[ non-naturally occurring polypeptide] wherein represents an attachment (e.g., by a linker or through conjugation), and wherein each of the engineered HLA-E and / or non-naturally occurring polypeptide can further comprise attachments to one or more non-naturally occurring polypeptides, such as those described herein.

[0340] In some embodiments, one or more non-naturally occurring polypeptides comprises one or more of: a B2M polypeptide, a CD8 blocking polypeptide, a binding polypeptide, a peptide linker, or any combination thereof. In some embodiments, a fusion protein provided herein comprises 1, 2, 3, 4, 5, 6, 7, 8, or more non-naturally occurring polypeptides.CD8 Blocking Polypeptides

[0341] In some embodiments, a fusion protein provided herein comprises a non-naturally occurring CD8 blocking polypeptide. In some embodiments, a non-naturally occurring CD8 blocking polypeptide is a portion of a protein that causes steric hinderance to a CD8 binding site on an HLA-E protein, wherein the CD8 blocking polypeptide is not found in nature to be on the same amino acid strand as an HLA-E protein. In some embodiments, an engineered HLA-E provided herein comprises 1, 2, 3, 4, 5, 6, 7, 8, or more CD8 blocking polypeptides.In some embodiments, an engineered HLA-E provided herein comprises 1 or 2 CD8 blocking polypeptides.

[0342] In some embodiments, a CD8 blocking polypeptide comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 20. In some embodiments, a CD8 blocking polypeptide comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 20. In some embodiments, a CD8 blocking polypeptide comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 20. In some embodiments, a CD8 blocking polypeptide comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 20. In some embodiments, a CD8 blocking polypeptide comprises an amino acid sequence that has at least 96% sequence identity to SEQ ID NO: 20. In some embodiments, a CD8 blocking polypeptide comprises an amino acid sequence that has at least 97% sequence identity to SEQ ID NO: 20. In some embodiments, a CD8 blocking polypeptide comprises an amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 20. In some embodiments, a CD8 blocking polypeptide comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 20. In some embodiments, a CD8 blocking polypeptide comprises the amino acid sequence of SEQ ID NO: 20. In embodiments wherein more than one CD8 blocking polypeptide is present, the CD8 blocking polypeptides are identical, non-identical, or any combination thereof. In embodiments wherein more than one CD8 blocking polypeptide is present, each CD8 blocking polypeptide individually comprises an amino acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to SEQ ID NO: 20. In some embodiments, a CD8 blocking polypeptide comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 22. In some embodiments, a CD8 blocking polypeptide comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 22. In some embodiments, a CD8 blocking polypeptide comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 22. In some embodiments, a CD8 blocking polypeptide comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 22. In some embodiments, a CD8 blocking polypeptide comprises an amino acid sequence that has at least 96% sequence identity to SEQ ID NO: 22. In some embodiments, a CD8 blocking polypeptide comprises an amino acid sequence that has at least 97% sequence identity to SEQ ID NO: 22. In some embodiments, a CD8 blocking polypeptide comprises an amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 22. In someembodiments, a CD8 blocking polypeptide comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 22. In some embodiments, a CD8 blocking polypeptide comprises the amino acid sequence of SEQ ID NO: 20. In embodiments wherein more than one CD8 blocking polypeptide is present, the CD8 blocking polypeptides are identical, non-identical, or any combination thereof. In embodiments wherein more than one CD8 blocking polypeptide is present, each CD8 blocking polypeptide individually comprises an amino acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to SEQ ID NO: 22.

[0343] In some embodiments, the one or more amino acid alterations comprises one or more amino acid deletions, additions, substitutions, or a combination thereof. In some embodiments, a CD8 blocking polypeptide provided herein includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid deletions, additions, substitutions, or a combination thereof. In some embodiments, one or more amino acid substitutions can be one or more conservative and / or non-conservative amino acid substitutions. In some embodiments, a CD8 blocking polypeptide provided herein includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions and / or non-conservative amino acid substitutions.

[0344] In some embodiments, a CD8 blocking polypeptide can be attached to the N termini of the engineered HLA-E. In some embodiments, a CD8 blocking polypeptide can be attached to the C termini of the engineered HLA-E. In some embodiments, a CD8 blocking polypeptide can be attached to the N termini of a non-naturally occurring polypeptide (e.g., a second CD8 blocking polypeptide). In some embodiments, a CD8 blocking polypeptide can be attached to the C termini of a non-naturally occurring polypeptide (e.g., a second CD8 blocking polypeptide or a B2M polypeptide). In some embodiments, CD8 blocking polypeptide present in the fusion protein in the following formats, reading from the N-termini to the C-termini from the left to the right:[CD8 Blocking Polypeptide]-[engineered HLA-E][engineered HLA-E]-[CD8 Blocking Polypeptide][CD8 Blocking Polypeptide]-[ non-naturally occurring polypeptide][non-naturally occurring polypeptide]-[CD8 Blocking Polypeptide][CD8 Blocking Polypeptide]-[non-naturally occurring polypeptide]-[engineered HLA-E][non-naturally occurring polypeptide]-[CD8 Blocking Polypeptide]-[engineered HLA-E][engineered HLA-E] -[non-naturally occurring polypeptide]-[CD8 Blocking Polypeptide][engineered HLA-E]-[CD8 Blocking Polypeptide]-[non-naturally occurring polypeptide][CD8 Blocking Polypeptide]-[engineered HLA-E] -[non-naturally occurring polypeptide][non-naturally occurring polypeptide]-[engineered HLA-E]-[CD8 Blocking Polypeptide][first CD8 Blocking Polypeptide]-[second CD8 Blocking Polypeptide]-[engineered HLA-E][engineered HLA-E]-[first CD8 Blocking Polypeptide]-[second CD8 Blocking Polypeptide][first CD8 Blocking Polypeptide]-[engineered HLA-E] -[second CD8 Blocking Polypeptide] wherein represents an attachment (e.g., by a linker or through conjugation), and wherein each of the engineered HLA-E, non-naturally occurring polypeptide and / or CD8 Blocking Polypeptide can further comprise attachments to one or more non-naturally occurring polypeptides, such as those described herein.B2M Polypeptides

[0345] In some embodiments, a fusion protein provided herein comprises a non-naturally occurring B2M polypeptide. In nature, a WT B2M protein exists as a separate protein from a WT HLA-E and complexes with an HLA-E molecule to form a stable heterodimer. In some embodiments, a non-naturally occurring B2M polypeptide refers to a portion of a B2Mprotein that is engineered to exist on the same amino acid strand as an engineered HLA-E molecule described herein. In some embodiments, a non-naturally occurring B2M polypeptide is a variant of a corresponding equal length portion of a WT B2M protein (e.g., SEQ ID NO: 16). In some embodiments, an engineered HLA-E comprising a non-naturally occurring B2M polypeptide provided herein complexes to the non-naturally occurring B2M polypeptide when expressed to form a stable heterodimer. In some embodiments, an engineered HLA-E comprising a non-naturally occurring B2M polypeptide provided herein can have an enhanced immune evasion activity as described herein relative to a reference HLA-E (e.g., a WT HLA-E) complexed with a WT B2M protein. In some embodiments, an engineered HLA-E comprising a non-naturally occurring B2M polypeptide described herein can maintain structural stability (e.g., proper folding and / or stability) relative to a reference HLA-E (e.g., a WT HLA-E) complexed with a WT B2M protein. In some embodiments, a non-naturally occurring B2M polypeptide described herein can comprise a beta sheet structure, one or more conserved cysteine residues to form disulfide bonds, and / or one or more hydrophobic interactions between the B2M polypeptide and an Alpha chain (e.g., an Alpha-3 polypeptide described herein) thereby maintaining structural stability (e.g., proper folding and / or stability). In some embodiments, an engineered HLA-E provided herein comprises 1, 2, 3, 4, 5, 6, 7, 8, or more B2M polypeptides.

[0346] In some embodiments, a B2M polypeptide provided herein is a variant of a reference B2M polypeptide, wherein the reference B2M polypeptide has an amino acid sequence of SEQ ID NO: 16. In some embodiments, a B2M polypeptide provided herein is a variant of a reference B2M polypeptide, wherein the reference B2M polypeptide has an amino acid sequence of SEQ ID NO: 16, and the variant B2M polypeptide has one or more amino acid alterations. In some embodiments, a variant B2M polypeptide provided herein includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid alterations.

[0347] In some embodiments, the one or more amino acid alterations comprises one or more amino acid deletions, additions, substitutions, or a combination thereof. In some embodiments, a B2M polypeptide provided herein includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid deletions, additions, substitutions, or a combination thereof. In some embodiments, one or more amino acid substitutions can be one or more conservative and / or non-conservative amino acid substitutions. In some embodiments, a B2M polypeptideprovided herein includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions and / or non-conservative amino acid substitutions.

[0348] In some embodiments, a B2M polypeptide provided herein is a variant of a reference B2M polypeptide, wherein the reference B2M polypeptide has an amino acid sequence of SEQ ID NO: 16, and the variant B2M polypeptide has one or more amino acid alterations. The one or more amino acid alterations may be located at one or more positions corresponding to one or more positions in SEQ ID NO: 16. In some embodiments, a reference sequence is a B2M polypeptide that is not SEQ ID NO: 16.

[0349] In some embodiments, a B2M polypeptide provided herein is a variant of a reference B2M polypeptide, wherein the reference B2M polypeptide has an amino acid sequence of SEQ ID NO: 16, and the B2M polypeptide has one or more amino acid alterations at a position relative to SEQ ID NO: 16. Accordingly, in some embodiments, a B2M polypeptide provided herein includes one or more amino acid alterations at a residue corresponding to position 58 relative to SEQ ID NO: 16.

[0350] In some embodiments, a B2M polypeptide provided herein includes one or more conservative amino acid substitutions relative to a wild-type B2M polypeptide. As a nonlimiting example, a conservative amino acid substitution relative to the K58 residue in SEQ ID NO: 16 may include substitution of K for another basic (positively charged) residue (e.g., Arg (R) or His (H)). As a non-limiting example, a non-conservative amino acid substitution relative to the K58 residue in SEQ ID NO: 16 may include substitution of K, a basic residue, for an acidic amino acid (negatively charged) (e.g., Asp (D) or Glu (E)). For example, in some embodiments, the one or more amino acid alterations result in a B2M polypeptide having an: E at a residue corresponding to position 58 in SEQ ID NO: 16. In some embodiments, the one or more amino acid alterations comprises K58E.

[0351] In some embodiments, other than the one or more B2M polypeptide amino acid alterations, the B2M polypeptide comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 16. In some embodiments, other than the one or more B2M polypeptide amino acid alterations, the B2M polypeptide comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 16. In some embodiments, other than the one or more B2M polypeptide amino acid alterations, the B2M polypeptide comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 16. In someembodiments, other than the one or more B2M polypeptide amino acid alterations, the B2M polypeptide comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 16. In some embodiments, other than the one or more B2M polypeptide amino acid alterations, the B2M polypeptide comprises an amino acid sequence that has at least 96% sequence identity to SEQ ID NO: 16. In some embodiments, other than the one or more B2M polypeptide amino acid alterations, the B2M polypeptide comprises an amino acid sequence that has at least 97% sequence identity to SEQ ID NO: 16. In some embodiments, other than the one or more B2M polypeptide amino acid alterations, the B2M polypeptide comprises an amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 16. In some embodiments, other than the one or more B2M polypeptide amino acid alterations, the B2M polypeptide comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 16. In some embodiments, other than the one or more B2M polypeptide amino acid alterations, the B2M polypeptide comprises the amino acid sequence of SEQ ID NO: 16.

[0352] In some embodiments, the B2M polypeptide comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 18. In some embodiments, the B2M polypeptide comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 18. In some embodiments, the B2M polypeptide comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 18. In some embodiments, the B2M polypeptide comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 18. In some embodiments, the B2M polypeptide comprises an amino acid sequence that has at least 96% sequence identity to SEQ ID NO: 18. In some embodiments, the B2M polypeptide comprises an amino acid sequence that has at least 97% sequence identity to SEQ ID NO: 18. In some embodiments, the B2M polypeptide comprises an amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 18. In some embodiments, the B2M polypeptide comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 18. In some embodiments, the B2M polypeptide comprises the amino acid sequence of SEQ ID NO: 18.

[0353] In some embodiments, a B2M polypeptide can be attached to the N termini of the engineered HLA-E. In some embodiments, a B2M polypeptide can be attached to the C termini of the engineered HLA-E. In some embodiments, a B2M polypeptide can be attached to the N termini of a non-naturally occurring polypeptide (e.g., a second B2M polypeptide).In some embodiments, a B2M polypeptide can be attached to the C termini of a non-naturally occurring polypeptide (e.g., a binding polypeptide). In some embodiments, B2M polypeptide can present in the fusion protein in the following formats, reading from the N-termini to the C-termini from the left to the right:[B2M Polypeptide]-[engineered HLA-E][engineered HLA-E]-[B2M Polypeptide][B2M Polypeptide]-[non-naturally occurring polypeptide][non-naturally occurring polypeptide]-[B2M Polypeptide] wherein represents an attachment (e.g., by a linker or through conjugation), and wherein each of the engineered HLA-E, non-naturally occurring polypeptide and / or B2M Polypeptide can further comprise attachments to one or more non-naturally occurring polypeptides, such as those described herein, and / or the engineered HLA described herein.Binding Polypeptide

[0354] In some embodiments, a fusion protein provided herein comprises a non-naturally occurring binding polypeptide. A binding polypeptide can be any peptide, polypeptide, or protein that binds to a HLA-E molecule, such as an engineered HLA-E molecule described herein. In some embodiments, a non-naturally occurring binding polypeptide is not found in nature to be on the same amino acid strand as an engineered HLA-E protein described herein. In some embodiments, a fusion protein provided herein comprises 1, 2, 3, 4, 5, 6, 7, 8, or more binding polypeptides.

[0355] In some embodiments, a binding polypeptide comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 8. In some embodiments, a binding polypeptide comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 8. In some embodiments, a binding polypeptide comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 8. In some embodiments, a binding polypeptide comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 8. In some embodiments, a binding polypeptide comprises an amino acid sequence that has at least 96% sequence identity to SEQ ID NO: 8. In some embodiments, a binding polypeptide comprises an amino acid sequence that has at least 97%sequence identity to SEQ ID NO: 8. In some embodiments, a binding polypeptide comprises an amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 8. In some embodiments, a binding polypeptide comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 8. In some embodiments, a binding polypeptide comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, a binding polypeptide comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 10. In some embodiments, a binding polypeptide comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 10. In some embodiments, a binding polypeptide comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 10. In some embodiments, a binding polypeptide comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 10. In some embodiments, a binding polypeptide comprises an amino acid sequence that has at least 96% sequence identity to SEQ ID NO: 10. In some embodiments, a binding polypeptide comprises an amino acid sequence that has at least 97% sequence identity to SEQ ID NO: 10. In some embodiments, a binding polypeptide comprises an amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 10. In some embodiments, a binding polypeptide comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 10. In some embodiments, a binding polypeptide comprises the amino acid sequence of SEQ ID NO: 10.

[0356] A binding polypeptide can be attached to the N termini of the engineered HLA-E.A binding polypeptide can be attached to the N termini of the fusion protein.

[0357] In some embodiments, a binding polypeptide can be attached to the N termini of the engineered HLA-E. In some embodiments, a binding polypeptide can be attached to the C termini of the engineered HLA-E. In some embodiments, a binding polypeptide can be attached to the N termini of a non-naturally occurring polypeptide (e.g., a B2M polypeptide). In some embodiments, a binding polypeptide can be attached to the C termini of a non- naturally occurring polypeptide (e.g., a B2M polypeptide). In some embodiments, binding polypeptide can be present in the fusion molecule in the following formats, reading from the N-termini to the C-termini from the left to the right:[Binding Polypeptide]-[engineered HLA-E][engineered HLA-E] -[Binding Polypeptide][Binding Polypeptide]-[non-naturally occurring polypeptide][non-naturally occurring polypeptide]-[Binding Polypeptide] wherein represents an attachment (e.g., by a linker or through conjugation), and wherein each of the engineered HLA-E, non-naturally occurring polypeptide and / or binding polypeptide can further comprise attachments to one or more non-naturally occurring polypeptides, such as those described herein, and / or an engineered HLA-E.Linkers

[0358] In some embodiments, a fusion protein provided herein comprises a linker. In some embodiments, a fusion protein provided herein comprises 1, 2, 3, 4, 5, 6, 7, 8, or more linkers. In some embodiments, a linker comprises a bond or molecule that links a first polypeptide to a second polypeptide. A linking bond can be a covalent bond. A linking molecule can be one or more chemical groups and / or one or more amino acids. A linker comprised of one or more amino acid can also be described as a peptide linker wherein a peptide linker comprises at least two amino acids linked by an amide bond. In general, a linker connects a terminus of a first polypeptide to a terminus of a second polypeptide. In some embodiments, carboxy terminus of a first polypeptide is linked to the amino terminus of a second polypeptide. In some embodiments, a terminus of a first polypeptide is linked to a terminus of a second polypeptide through an amide bond or a peptide bond. In some embodiments, a first polypeptide and a second polypeptide are directly linked by a covalent bond.

[0359] In some embodiments, the linker is from 1 to 100 amino acids in length. In some embodiments, the linker is more 100 amino acids in length. In some embodiments, the linker is from 1 to 30, from 1 to 25, from 1 to 20, from 1 to 15, from 1 to 10, from 1 to 50, from 1 to 25, or from 1 to 10 amino acids in length. In some embodiments, a linker includes glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, GSGGSn (SEQ ID NO: 31), GGSGGSn (SEQ ID NO: 32), and GGGSn (SEQ ID NO: 33), where n is an integer of at least one), glycine-alanine polymers, and alanine-serine polymers. An ordinarily skilled artisan will recognize that a linker can be designed to impart degrees of flexibility, such as by comprising small amino acids (e.g., glycine and alanine) or degrees of rigidity, such as by comprising a beta sheet or an alpha helix) based on the desired structure. A linker can be a cleavable linker, including a self-immolative linker.

[0360] In some embodiments, a fusion protein provided herein comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 24. In some embodiments, a fusion protein provided herein comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 24. In some embodiments, a fusion protein provided herein comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 24. In some embodiments, a fusion protein provided herein comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 24. In some embodiments, a fusion protein provided herein comprises an amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 24. In some embodiments, a fusion protein provided herein comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 24. In some embodiments, a fusion protein provided herein comprises an amino acid sequence that is identical to SEQ ID NO: 24. In some embodiments, a fusion protein provided herein comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 26. In some embodiments, a fusion protein provided herein comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 26. In some embodiments, a fusion protein provided herein comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 26. In some embodiments, a fusion protein provided herein comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 26. In some embodiments, a fusion protein provided herein comprises an amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 26. In some embodiments, a fusion protein provided herein comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 26. In some embodiments, a fusion protein provided herein comprises an amino acid sequence that is identical to SEQ ID NO: 26. In some embodiments, a fusion protein provided herein comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 28. In some embodiments, a fusion protein provided herein comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 28. In some embodiments, a fusion protein provided herein comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 28. In some embodiments, a fusion protein provided herein comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 28. In some embodiments, a fusion protein provided herein comprises an amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 28. In some embodiments, a fusion protein provided herein comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 28. In some embodiments, a fusionprotein provided herein comprises an amino acid sequence that is identical to SEQ ID NO: 28.

[0361] Methods of generating and assaying an engineered HLA-E or fusion protein thereof described herein are well known to one of skill in the art. Examples of such methods are described in the Examples provided herein. Any of a variety of methods can be used to generate an engineered HLA-E or fusion protein thereof disclosed herein. Such methods include, but are not limited to, site-directed mutagenesis, random mutagenesis, combinatorial libraries, and other mutagenesis methods described herein (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999); Gillman et al., Directed Evolution Library Creation: Methods and Protocols (Methods in Molecular Biology) Springer, 2nd ed (2014)). One non-limiting example of a method for preparing an engineered HLA-E or fusion protein thereof is to express heterologous nucleic acids (e.g., recombinant nucleic acids) encoding an engineered HLA-E in a suitable engineered cell, such as a bacterial cell, a yeast cell, or other suitable cell, using methods well known in the art.

[0362] In some embodiments, an engineered HLA-E provided herein is an isolated HLA- E. In some embodiments, a fusion protein provided herein is an isolated fusion protein. An isolated engineered HLA-E and / or fusion protein thereof as provided herein can be isolated by a variety of methods well-known in the art, for example, recombinant expression systems, precipitation, gel filtration, ion-exchange, reverse-phase and affinity chromatography, and the like. Other well-known methods are described in Deutscher et al., Guide to Protein Purification: Methods in Enzymology, Vol. 182, (Academic Press, (1990)). Alternatively, the isolated polypeptides of the present disclosure can be obtained using well-known recombinant methods (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999)). The methods and conditions for biochemical purification of a polypeptide described herein can be chosen by those skilled in the art, and purification monitored, for example, by a functional assay.Heterologous Nucleic Acids

[0363] In some embodiments, provided herein is at least one heterologous nucleic acid that has a nucleotide sequence encoding an engineered HLA-E or a fusion protein comprising the same as described herein. Accordingly, in some embodiments, provided herein is a heterologous nucleic acid selected from (a) a nucleic acid molecule encoding an engineered HLA-E that is a variant of a reference HLA-E (SEQ ID NO: 6), and, in some embodiments, a combination of alterations thereof; (b) a heterologous nucleic acid that hybridizes to an isolated nucleic acid of (a) under highly stringent hybridization conditions; and (c) a heterologous nucleic acid that is complementary to (a) or (b). In further embodiments, provided herein is a heterologous nucleic acid selected from (a) a nucleic acid molecule encoding a fusion protein comprising an engineered HLA-E that is a variant of a reference HLA-E (SEQ ID NO: 6), and, in some embodiments, a combination of alterations thereof, fused to one or more non-naturally occurring polypeptides; (b) a heterologous nucleic acid that hybridizes to an isolated nucleic acid of (a) under highly stringent hybridization conditions; and (c) a heterologous nucleic acid that is complementary to (a) or (b).Encoded Variant HLA-E molecules

[0364] In some embodiments, provided herein is a heterologous nucleic acid encoding an engineered HLA-E provided herein is a variant of a reference HLA-E, wherein the reference HLA-E has an amino acid sequence of SEQ ID NO: 6, and the engineered HLA-E has one or more amino acid alterations at a position described herein relative to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid encodes an engineered HLA-E that includes one or more amino acid alterations at a residue corresponding to position 227, 228, or both relative to SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid encodes an engineered HLA-E that includes one or more amino acid alterations at a residue corresponding to position 227 and 228 relative to SEQ ID NO: 6. In some embodiments, the one or more amino acid alterations result in an engineered HLA-E, encoded by the heterologous nucleic acid provided herein, having an: K at a residue corresponding to position 227 in SEQ ID NO: 6; A at a residue corresponding to position 228 in SEQ ID NO: 6; or both. In some embodiments, the one or more amino acid alterations comprises D227K, T228A, or both.

[0365] In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E wherein, other than the one or more engineered HLA-E amino acid alterations, the engineered HLA-E comprises an amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 6. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E wherein, other than the one or more engineered HLA-E amino acid alterations, the engineered HLA-E comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 6. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E wherein, other than the one or more engineered HLA-E amino acid alterations, the engineered HLA-E comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E wherein, other than the one or more engineered HLA-E amino acid alterations, the engineered HLA-E comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 6. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E wherein, other than the one or more engineered HLA-E amino acid alterations, the engineered HLA-E comprises an amino acid sequence that has at least 96% sequence identity to SEQ ID NO: 6. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E wherein, other than the one or more engineered HLA-E amino acid alterations, the engineered HLA-E comprises an amino acid sequence that has at least 97% sequence identity to SEQ ID NO: 6. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E wherein, other than the one or more engineered HLA-E amino acid alterations, the engineered HLA-E comprises an amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 6. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E wherein, other than the one or more engineered HLA-E amino acid alterations, the engineered HLA-E comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 6. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E wherein, other than the one or more engineered HLA-E amino acid alterations, the engineered HLA-E comprises the amino acid sequence of SEQ ID NO: 6.

[0366] In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E that comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 12. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E that comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 12. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E that comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 12. In some embodiments, a heterologousnucleic acid provided herein encodes an engineered HLA-E that comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 12. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E that comprises an amino acid sequence that has at least 96% sequence identity to SEQ ID NO: 12. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E that comprises an amino acid sequence that has at least 97% sequence identity to SEQ ID NO: 12. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E that comprises an amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 12. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E that comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 12. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E that comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E that comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 14. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E that comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 14. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E that comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 14. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E that comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 14. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E that comprises an amino acid sequence that has at least 96% sequence identity to SEQ ID NO: 14. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E that comprises an amino acid sequence that has at least 97% sequence identity to SEQ ID NO: 14. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E that comprises an amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 14. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E that comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 14. In some embodiments, a heterologous nucleic acid provided herein encodes an engineered HLA-E that comprises the amino acid sequence of SEQ ID NO: 14.Encoded Fusion Proteins

[0367] In some embodiments, provided herein is a heterologous nucleic acid, wherein the heterologous nucleic acid encodes one or more non-naturally occurring polypeptides. In some embodiments, provided herein is a heterologous nucleic acid, wherein the heterologous nucleic acid encodes an engineered HLA-E described herein that includes one or more non- naturally occurring polypeptides. In some embodiments, provided herein is a heterologous nucleic acid, wherein the heterologous nucleic acid encodes an engineered HLA-E described herein that is fused or conjugated to one or more non-naturally occurring polypeptides. In some embodiments, provided herein is a heterologous nucleic acid, wherein the heterologous nucleic acid encodes a fusion protein comprising an engineered HLA-E described herein fused to one or more non-naturally occurring polypeptides.

[0368] In some embodiments, provided herein is a heterologous nucleic acid encoding one or more non-naturally occurring polypeptides, wherein the one or more non-naturally occurring polypeptides comprise one or more of: a B2M polypeptide, a CD8 blocking polypeptide, a binding polypeptide, a peptide linker, or any combination thereof. In some embodiments, a heterologous nucleic acid described herein encodes a fusion protein comprising 1, 2, 3, 4, 5, 6, 7, 8, or more non-naturally occurring polypeptides.Encoded CD8 Blocking Polypeptides

[0369] In some embodiments, a heterologous nucleic acid described herein encodes a non-naturally occurring CD8 blocking polypeptide. In some embodiments, a heterologous nucleic acid described herein encodes 1, 2, 3, 4, 5, 6, 7, 8, or more CD8 blocking polypeptides. In some embodiments, a heterologous nucleic acid described herein encodes 1 or 2 CD8 blocking polypeptides.

[0370] In some embodiments, a heterologous nucleic acid provided herein encodes a CD8 blocking polypeptide, wherein the CD8 blocking polypeptide comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 20. In some embodiments, a heterologous nucleic acid provided herein encodes a CD8 blocking polypeptide, wherein the CD8 blocking polypeptide comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 20. In some embodiments, a heterologous nucleic acid provided herein encodes a CD8 blocking polypeptide, wherein the CD8 blocking polypeptide comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 20. In some embodiments, a heterologous nucleic acid provided herein encodes a CD8 blockingpolypeptide, wherein the CD8 blocking polypeptide comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 20. In some embodiments, a heterologous nucleic acid provided herein encodes a CD8 blocking polypeptide, wherein the CD8 blocking polypeptide comprises an amino acid sequence that has at least 96% sequence identity to SEQ ID NO: 20. In some embodiments, a heterologous nucleic acid provided herein encodes a CD8 blocking polypeptide, wherein the CD8 blocking polypeptide comprises an amino acid sequence that has at least 97% sequence identity to SEQ ID NO: 20. In some embodiments, a heterologous nucleic acid provided herein encodes a CD8 blocking polypeptide, wherein the CD8 blocking polypeptide comprises an amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 20. In some embodiments, a heterologous nucleic acid provided herein encodes a CD8 blocking polypeptide, wherein the CD8 blocking polypeptide comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 20. In some embodiments, a heterologous nucleic acid provided herein encodes a CD8 blocking polypeptide, wherein the CD8 blocking polypeptide comprises the amino acid sequence of SEQ ID NO: 20. In embodiments, a heterologous nucleic acid provided herein encodes more than one CD8 blocking polypeptide, wherein where more than one CD8 blocking polypeptide is present, the CD8 blocking polypeptides are identical, non-identical, or any combination thereof. In embodiments, a heterologous nucleic acid provided herein encodes more than one CD8 blocking polypeptide, wherein where more than one CD8 blocking polypeptide is present, each CD8 blocking polypeptide individually comprises an amino acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to SEQ ID NO: 20.

[0371] In some embodiments, a heterologous nucleic acid provided herein encodes a CD8 blocking polypeptide, wherein the CD8 blocking polypeptide provided herein is a variant of a reference CD8 blocking polypeptide, wherein the reference CD8 blocking polypeptide has an amino acid sequence of SEQ ID NO: 20. In some embodiments, a heterologous nucleic acid provided herein encodes a CD8 blocking polypeptide, wherein the CD8 blocking polypeptide provided herein is a variant of a reference CD8 blocking polypeptide that has an amino acid sequence of SEQ ID NO: 20, and the variant CD8 blocking polypeptide has one or more amino acid alterations. In some embodiments, a heterologous nucleic acid provided herein encodes a variant CD8 blocking polypeptide that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid alterations.

[0372] In some embodiments, a heterologous nucleic acid provided herein encodes a variant CD8 blocking polypeptide, wherein the variant CD8 blocking polypeptide comprises one or more amino acid alterations comprising one or more amino acid deletions, additions, substitutions, or a combination thereof. In some embodiments, a heterologous nucleic acid provided herein encodes a CD8 blocking polypeptide, wherein the CD8 blocking polypeptide includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid deletions, additions, substitutions, or a combination thereof. In some embodiments, one or more amino acid substitutions can be one or more conservative and / or non-conservative amino acid substitutions. In some embodiments, a heterologous nucleic acid provided herein encodes a variant CD8 blocking polypeptide, wherein the variant CD8 blocking polypeptide includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions and / or non-conservative amino acid substitutions.Encoded B2M Polypeptides

[0373] In some embodiments, a heterologous nucleic acid provided herein encodes a non- naturally occurring B2M polypeptide. In some embodiments, a heterologous nucleic acid provided herein encodes a non-naturally occurring B2M polypeptide that is a variant of a corresponding equal length portion of a WT B2M protein (e.g., SEQ ID NO: 16). In some embodiments, a heterologous nucleic acid provided herein encodes 1, 2, 3, 4, 5, 6, 7, 8, or more B2M polypeptides.

[0374] In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide that is a variant of a reference B2M polypeptide, wherein the reference B2M polypeptide has an amino acid sequence of SEQ ID NO: 16. In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide that is a variant of a reference B2M polypeptide, wherein the reference B2M polypeptide has an amino acid sequence of SEQ ID NO: 16, and the variant B2M polypeptide has one or more amino acid alterations. In some embodiments, a heterologous nucleic acid provided herein encodes a variant B2M polypeptide that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid alterations.

[0375] In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide that comprises one or more amino acid alterations, where the one or more amino acid alterations comprise one or more amino acid deletions, additions, substitutions, ora combination thereof. In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid deletions, additions, substitutions, or a combination thereof. In some embodiments, one or more amino acid substitutions can be one or more conservative and / or non-conservative amino acid substitutions. In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions and / or non-conservative amino acid substitutions.

[0376] In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide that is a variant of a reference B2M polypeptide, wherein the reference B2M polypeptide has an amino acid sequence of SEQ ID NO: 16, and the variant B2M polypeptide has one or more amino acid alterations. In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide that comprises one or more amino acid alterations wherein the one or more amino acid alterations may be located at one or more positions corresponding to the one or more positions described in TABLE 3. In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide that comprises one or more amino acid alterations wherein the one or more amino acid alterations may be located at one or more positions corresponding to one or more positions in SEQ ID NO: 16. In some embodiments, a reference sequence is a B2M polypeptide that is not SEQ ID NO: 16.

[0377] In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide that is a variant of a reference B2M polypeptide, wherein the reference B2M polypeptide has an amino acid sequence of SEQ ID NO: 16. Accordingly, in some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide that includes one or more amino acid alterations at a residue corresponding to position 58 relative to SEQ ID NO: 16.

[0378] In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide that includes one or more amino acid alterations, wherein each of the one or more amino acid alterations are conservative amino acid substitutions or non-conservative amino acid substitutions. In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide that includes one or more conservative amino acid substitutions relative to a wild-type B2M polypeptide. For example, in some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide that comprises one or more aminoacid alterations, wherein the one or more amino acid alterations result in a B2M polypeptide having an: E at a residue corresponding to position 58 in SEQ ID NO: 16. In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide that comprises one or more amino acid alterations, wherein the one or more amino acid alterations comprises K58E.

[0379] In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide, wherein, other than the one or more B2M polypeptide amino acid alterations, the B2M polypeptide comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 16. In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide, wherein, other than the one or more B2M polypeptide amino acid alterations, the B2M polypeptide comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 16. In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide, wherein, other than the one or more B2M polypeptide amino acid alterations, the B2M polypeptide comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 16. In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide, wherein, other than the one or more B2M polypeptide amino acid alterations, the B2M polypeptide comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 16. In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide, wherein, other than the one or more B2M polypeptide amino acid alterations, the B2M polypeptide comprises an amino acid sequence that has at least 96% sequence identity to SEQ ID NO: 16. In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide, wherein, other than the one or more B2M polypeptide amino acid alterations, the B2M polypeptide comprises an amino acid sequence that has at least 97% sequence identity to SEQ ID NO: 16. In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide, wherein, other than the one or more B2M polypeptide amino acid alterations, the B2M polypeptide comprises an amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 16. In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide, wherein, other than the one or more B2M polypeptide amino acid alterations, the B2M polypeptide comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 16. In some embodiments, a heterologous nucleic acid provided herein encodesa B2M polypeptide, wherein, other than the one or more B2M polypeptide amino acid alterations, the B2M polypeptide comprises the amino acid sequence of SEQ ID NO: 16.

[0380] In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide, wherein the B2M polypeptide comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 18. In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide, wherein the B2M polypeptide comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 18. In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide, wherein the B2M polypeptide comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 18. In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide, wherein the B2M polypeptide comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 18. In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide, wherein the B2M polypeptide comprises an amino acid sequence that has at least 96% sequence identity to SEQ ID NO: 18. In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide, wherein the B2M polypeptide comprises an amino acid sequence that has at least 97% sequence identity to SEQ ID NO: 18. In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide, wherein the B2M polypeptide comprises an amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 18. In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide, wherein the B2M polypeptide comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 18. In some embodiments, a heterologous nucleic acid provided herein encodes a B2M polypeptide, wherein the B2M polypeptide comprises the amino acid sequence of SEQ ID NO: 18.Encoded Binding Polypeptides

[0381] In some embodiments, a heterologous nucleic acid provided herein encodes a non- naturally occurring binding polypeptide. In some embodiments, a heterologous nucleic acid provided herein encodes 1, 2, 3, 4, 5, 6, 7, 8, or more binding polypeptides.

[0382] In some embodiments, a heterologous nucleic acid provided herein encodes a binding polypeptide that comprises an amino acid sequence that has at least 80% sequenceidentity to SEQ ID NO: 8. In some embodiments, a heterologous nucleic acid provided herein encodes a binding polypeptide that comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 8. In some embodiments, a heterologous nucleic acid provided herein encodes a binding polypeptide that comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 8. In some embodiments, a heterologous nucleic acid provided herein encodes a binding polypeptide that comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 8. In some embodiments, a heterologous nucleic acid provided herein encodes a binding polypeptide that comprises an amino acid sequence that has at least 96% sequence identity to SEQ ID NO: 8. In some embodiments, a heterologous nucleic acid provided herein encodes a binding polypeptide that comprises an amino acid sequence that has at least 97% sequence identity to SEQ ID NO: 8. In some embodiments, a heterologous nucleic acid provided herein encodes a binding polypeptide that comprises an amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 8. In some embodiments, a heterologous nucleic acid provided herein encodes a binding polypeptide that comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 8. In some embodiments, a heterologous nucleic acid provided herein encodes a binding polypeptide that comprises the amino acid sequence of SEQ ID NO: 8.Encoded Linkers

[0383] In some embodiments, a heterologous nucleic acid provided herein encodes a linker. In some embodiments, a heterologous nucleic acid provided herein encodes 1, 2, 3, 4, 5, 6, 7, 8, or more linkers. In some embodiments, a heterologous nucleic acid provided herein encodes a linker comprising a bond or molecule that links a first polypeptide to a second polypeptide, such as one or more: covalent bonds, chemical groups, and / or amino acids. In some embodiments, a heterologous nucleic acid provided herein encodes a peptide linker. In some embodiments, a heterologous nucleic acid provided herein encodes 1, 2, 3, 4 or more peptide linkers.

[0384] In some embodiments, a heterologous nucleic acid provided herein encodes a linker that is from 1 to 100 amino acids in length. In some embodiments, a heterologous nucleic acid provided herein encodes a linker that is more 100 amino acids in length. In some embodiments, a heterologous nucleic acid provided herein encodes a linker that is from 1 to 30, from 1 to 25, from 1 to 20, from 1 to 15, from 1 to 10, from 1 to 50, from 1 to 25, or from 1 to 10 amino acids in length. In some embodiments, a heterologous nucleic acid providedherein encodes a linker that includes glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, GSGGSn (SEQ ID NO: 31), GGSGGSn (SEQ ID NO: 32), and GGGSn (SEQ ID NO: 33), where n is an integer of at least one), glycine-alanine polymers, and alanine-serine polymers. In some embodiments, a heterologous nucleic acid provided herein encodes a linker that is a flexible linker, a rigid linker, or any partial structure in between, a cleavable linker, including a self-immolative linker, and / or any one of the linkers set forth in TABLE 4.

[0385] In some embodiments, provided herein is a heterologous nucleic acid that includes a nucleotide sequence encoding an engineered HLA-E and / or fusion protein thereof as described herein that is operatively linked to a promoter. Such a promoter can express the engineered HLA-E and / or fusion protein thereof in an engineered cell as described herein.

[0386] A heterologous nucleic acid encoding an engineered HLA-E and / or fusion protein thereof as described herein also includes a nucleic acid that hybridizes to a nucleic acid disclosed herein or a nucleic acid that hybridizes to a nucleic acid that encodes an amino acid sequence disclosed. Hybridization conditions can include highly stringent, moderately stringent, or low stringency hybridization conditions that are well known to one of skill in the art such as those described herein. Similarly, a heterologous nucleic acid that can be used in the compositions and methods described herein can be described as having a certain percent sequence identity to a nucleic acid disclosed herein or a nucleic acid that hybridizes to a nucleic acid molecule that encodes an amino acid sequence disclosed herein. For example, the nucleic acid can have at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or be identical, to a nucleotide described herein.

[0387] Stringent hybridization refers to conditions under which hybridized polynucleotides are stable. As known to those of skill in the art, the stability of hybridized polynucleotides is reflected in the melting temperature (Tm) of the hybrids. In general, the stability of hybridized polynucleotides is a function of the salt concentration, for example, the sodium ion concentration, and temperature. A hybridization reaction can be performed under conditions of lower stringency, followed by washes of varying, but higher, stringency. Reference to hybridization stringency relates to such washing conditions. Highly stringent hybridization includes conditions that permit hybridization of only those nucleotide sequences that form stable hybridized polynucleotides in 0.018M NaCl at 65°C, for example,if a hybrid is not stable in 0.018M NaCl at 65°C, it will not be stable under high stringency conditions, as contemplated herein. High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0.1X SSPE, and 0.1% SDS at 65°C. Hybridization conditions other than highly stringent hybridization conditions can also be used to describe the nucleotide sequences disclosed herein. For example, the phrase moderately stringent hybridization refers to conditions equivalent to hybridization in 50% formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0.2X SSPE, 0.2% SDS, at 42°C. The phrase low stringency hybridization refers to conditions equivalent to hybridization in 10% formamide, 5X Denhart's solution, 6X SSPE, 0.2% SDS at 22°C, followed by washing in IX SSPE, 0.2% SDS, at 37°C. Denhart's solution contains 1% Ficoll, 1% polyvinylpyrolidone, and 1% bovine serum albumin (BSA). 20X SSPE (sodium chloride, sodium phosphate, ethylene diamine tetraacetic acid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025 M (EDTA). Other suitable low, moderate and high stringency hybridization buffers and conditions are well known to those of skill in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999).

[0388] A heterologous nucleic acid encoding an engineered HLA-E and / or fusion protein thereof as described herein can have at least a certain sequence identity to a nucleotide sequence disclosed herein. Accordingly, in some aspects described herein, a heterologous nucleic acid encoding an engineered HLA-E and / or fusion protein thereof has a nucleotide sequence of at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, or is identical, to a nucleic acid disclosed herein or a nucleic acid that hybridizes to a nucleic acid that encodes an amino acid sequence disclosed herein.

[0389] One skilled in the art will readily understand the meaning of a wild-type sequence based on what is well known in the art. It is further understood that such a heterologous nucleic acid described herein can exclude a nucleotide sequence encoding a naturally occurring amino acid sequence as found in nature. Similarly, an engineered HLA-E and / orfusion protein thereof as described herein can exclude an amino acid sequence as found in nature. Thus, in some embodiments, the heterologous nucleic acid or engineered HLA-E and / or fusion protein thereof as described herein is as set forth herein, with the proviso that the encoded amino acid sequence is not the wild-type parental sequence or a naturally occurring amino acid sequence and / or that the nucleotide sequence is not a wild-type or naturally occurring nucleotide sequence. A naturally occurring amino acid or nucleotide sequence is understood by those skilled in the art as relating to a sequence that is found in a naturally occurring organism as found in nature. Thus, a nucleotide or amino acid sequence that is not found in the same state or having the same nucleotide or encoded amino acid sequence as in a naturally occurring organism is included within the meaning of a heterologous nucleotide and / or amino acid sequence described herein. For example, a nucleotide or amino acid sequence that has been altered at one or more nucleotide or amino acid positions from a parent sequence, including variants as described herein, are included within the meaning of a nucleotide or amino acid sequence described herein that is not naturally occurring. A heterologous nucleic acid described herein excludes a naturally occurring chromosome that contains the nucleotide sequence, and can further exclude other molecules, as found in a naturally occurring cell, such as DNA binding proteins, for example, proteins such as histones that bind to chromosomes within a eukaryotic cell.

[0390] Thus, a heterologous nucleic acid described here has physical and chemical differences compared to a naturally occurring nucleic acid. A heterologous or non-naturally occurring nucleic acid described herein does not contain or does not necessarily have some or all of the chemical bonds, either covalent or non-covalent bonds, of a naturally occurring nucleic acid as found in nature. A heterologous nucleic acid described herein thus differs from a naturally occurring nucleic acid, for example, by having a different chemical structure than a naturally occurring nucleic acid as found in a chromosome. A different chemical structure can occur, for example, by cleavage of phosphodiester bonds that release a heterologous nucleic acid from a naturally occurring chromosome. A heterologous nucleic acid described herein can also differ from a naturally occurring nucleic acid by isolating or separating the nucleic acid from proteins that bind to chromosomal DNA in either prokaryotic or eukaryotic cells, thereby differing from a naturally occurring nucleic acid by different non-covalent bonds. With respect to nucleic acids of prokaryotic origin, a non-naturally occurring nucleic acid described herein does not necessarily have some or all of the naturally occurring chemical bonds of a chromosome, for example, binding to DNA binding proteins such aspolymerases or chromosome structural proteins, or is not in a higher order structure such as being supercoiled. With respect to nucleic acids of eukaryotic origin, a non-naturally occurring nucleic acid described herein also does not contain the same internal nucleic acid chemical bonds or chemical bonds with structural proteins as found in chromatin. For example, a non-naturally occurring nucleic acid described herein is not chemically bonded to histones or scaffold proteins and is not contained in a centromere or telomere. Thus, the non- naturally occurring nucleic acids described herein are chemically distinct from a naturally occurring nucleic acid because they either lack or contain different van der Waals interactions, hydrogen bonds, ionic or electrostatic bonds, and / or covalent bonds from a nucleic acid as found in nature. Such differences in bonds can occur either internally within separate regions of the nucleic acid (that is cis) or such difference in bonds can occur in trans, for example, interactions with chromosomal proteins. In the case of a nucleic acid of eukaryotic origin, a cDNA is considered to be a heterologous or non-naturally occurring nucleic acid since the chemical bonds within a cDNA differ from the covalent bonds, that is the sequence, of a gene on chromosomal DNA. Thus, it is understood by those skilled in the art that heterologous or non-naturally occurring nucleic acid is distinct from a naturally occurring nucleic acid.Expressing Heterologous Nucleic Acids

[0391] In some embodiments, a heterologous nucleic acid encoding an engineered HLA- E and / or fusion protein described herein further includes one or more regulatory elements. Such a regulatory element can include a regulatory sequence, which is any DNA sequence responsible for the regulation of gene expression, such as promoters and operators. The regulatory element can be a segment of a nucleic acid molecule, which is able to increase or decreasing the expression of specific genes within an organism.

[0392] In some embodiments, the regulatory element is a promoter. A promoter is a nucleotide sequence that directs the transcription of a structural gene. In some alternatives, a promoter is in the 5’ non-coding region of a gene, proximal to the transcriptional start site of a structural gene. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. Without being limiting, these promoter elements can include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation-specific elements (DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993);), cyclic AMP response elements (CREs), serum response elements(SREs; Treisman et al., Seminars in Cancer Biol. 1 :47 (1990); incorporated by reference in its entirety), glucocorticoid response elements (GREs), and binding sites for other transcription factors, such as CRE / ATF (O’Reilly et al., J. Biol. Chem. 267: 19938 (1992); incorporated by reference in its entirety), AP2 (Ye et al., J. Biol. Chem. 269:25728 (1994); incorporated by reference in its entirety), SPI, cAMP response element binding protein (CREB; Loeken et al., Gene Expr. 3:253 (1993); hereby expressly incorporated by reference in its entirety) and octamer factors (see, in general, Watson et al., eds., Molecular Biology of the Gene, 4th ed. (The Benjamin / Cummings Publishing Company, Inc. 1987; incorporated by reference in its entirety)), and Lemaigre and Rousseau, Biochem. J. 303: 1 (1994); incorporated by reference in its entirety).

[0393] In some alternatives, promoters used herein can be inducible or constitutive promoters. Without being limiting, inducible promoters can include, for example, a tamoxifen inducible promoter, tetracycline inducible promoter, or a doxycycline inducible promoter (e.g., tre) promoter. Constitutive promoters can include, for example, SV40, CMV, UBC, EFlalpha, PGK, or CAGG. Any suitable promoter known in the art for expression of a gene in a population of engineered cells as described herein can be used. In some embodiments, a heterologous nucleic acid encoding an engineered HLA-E and / or fusion protein described herein includes a promoter. In some embodiments, expression of the engineered HLA-E and / or fusion protein described herein is driven by an endogenous gene promoter.

[0394] A heterologous nucleic acid encoding an engineered HLA-E and / or a fusion protein described herein can be introduced to a cell by any suitable method known in the art. For instance, the at least one heterologous nucleic acid can be introduced by electroporation, sonoporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, viral vector systems (e.g., retroviral, lentivirus, adenoviral, adeno-associated, vaccinia and herpes simplex virus vectors) and agent-enhanced uptake of DNA. In some embodiments, the at least one heterologous nucleic acid encoding an engineered HLA-E and / or a fusion protein described herein can be introduced into the cell as RNA, for example, mRNA or selfreplicating RNA.

[0395] In some embodiments, a heterologous nucleic acid encoding an engineered HLA- E and / or a fusion protein described herein is introduced into the cell in a vector. In some embodiments, a heterologous nucleic acid is a recombinant nucleic acid. In someembodiments, the at least one heterologous nucleic acid is a recombinant nucleic acid and introduced into the cell in a vector. Methods of generating recombinant nucleic acids are described herein.

[0396] The vector can be a plasmid, a virus, or another vector designed for introducing a nucleic acid of interest into a cell. Viral vectors include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Exemplary viral vectors that can be used include an adeno-associated virus, a lentivirus, a retrovirus, a herpes simplex virus, vaccinia, or an adenovirus. The vector is used to introduce a gene of interest into a host cell in which the vector will interact with polymerases in the cell to express the protein encoded in the vector. The vector can exist in the cell extra-chromosomally or integrated into the genome of the host cell. In some embodiments, use of a viral vector will lead to integration of the heterologous nucleic acid into the genome of the cell. In some embodiments, provided herein is a vector containing a heterologous nucleic acid described herein. In some embodiments, the vector is an expression vector. In some embodiments, the vector comprises double stranded DNA.

[0397] In some embodiments, also provided herein is a heterologous nucleic acid encoding a kill switch into an engineered cell. In some embodiments, the kill switch is a gene that encodes a herpes simplex virus thymidine kinase (HSV-TK), an inducible caspase9 (also known as incasep 9, and iCasp9), CD20, or a mutant human thymidylate kinase (mTMPK). In some embodiments, the kill switch is under the control of an inducible promoter.

[0398] In some embodiments, the nucleic acid encoding the kill switch is integrated into the genome of the cell using the CRISPR / Cas system. The nucleic acid encoding the kill switch can be integrated into the same or a different locus as a heterologous nucleic acid encoding an engineered HLA-E and / or a fusion protein described herein.

[0399] In some embodiments, the nucleic acid encoding the kill switch is introduced into the cell in a vector. The nucleic acid encoding the kill switch can be introduced into the cell in the same vector or a different vector as a heterologous nucleic acid encoding an engineered HLA-E and / or a ...

Claims

CLAIMSWhat is claimed is:

1. An engineered cell, the engineered cell comprising: a genetic modification that reduces or eliminates function of a natural-killer group 2, member D (NKG2D) ligand as compared to a wild-type cell; and a heterologous nucleic acid sequence encoding a fusion protein comprising at least a portion of a human leukocyte antigen (HLA)-E protein covalently linked to at least a portion of a P2-microglobulin (B2M) protein.

2. The engineered cell of claim 1, wherein at least one of the HLA-E protein or the B2M protein comprises a modification that reduces binding affinity to a CD8 co-receptor as compared to a corresponding wild-type protein.

3. The engineered cell of claim 2, wherein the B2M protein comprises the modification that reduces binding affinity to a CD8 co-receptor.

4. The engineered cell of claim 2 or claim 3, wherein the heterologous nucleic acid sequence encodes an amino acid sequence having at least 95% identity to one of SEQ ID NO. 2, 4, 6, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30.

5. The engineered cell of claim 3, wherein the amino acid sequence comprises SEQ ID NO: 18.

6. The engineered cell of any of claims 1 to 5, wherein the engineered cell further comprises one or more genetic modifications that result in reduced T cell-mediated killing as compared to a wild-type cell, the one or more genetic modifications comprising:(i) a deletion or disruption of a P2 microglobulin (B2M) gene;(ii) a deletion or disruption of one or more of an HLA-A gene, an HLA-B gene, and an HLA-C gene; or(iii) a deletion or disruption of a class II, major histocompatibility complex, transactivator (CIITA) gene.

7. The engineered cell of any one of claims 1 to 6, wherein the genetic modification that reduces or eliminates function of a NKG2D ligand as compared to a wild-type cell is a deletion or disruption of a gene encoding a NKG2D ligand.

8. The engineered cell of any of claims 1 to 7, wherein the NKG2D ligand comprises MICA, MICB, Raetle, Raetlg, Raetll, Ulbpl, Ulbp2, Ulbp3, or any combination thereof.

9. The engineered cell of any one of claims 1 to 8, wherein the NKG2D ligand comprises MICA.

10. The engineered cell of any one of claims 1 to 9, wherein the HLA-E protein encoded by the heterologous nucleic acid sequence comprises a binding peptide.

11. The engineered cell of claim 10, wherein the binding peptide comprises an amino acid sequence comprising LIL or LFL.

12. The engineered cell of claim 11, wherein the binding peptide has the amino acid sequence comprising LFL.

13. The engineered cell of any of claims 1 to 12, wherein the HLA-E protein is covalently linked to the B2M protein by a linker.

14. The engineered cell of any one of claims 1 to 13, wherein expression of the heterologous nucleic acid sequence is driven by an endogenous gene promoter in the engineered cell.

15. The engineered cell of any one of claims 1 to 14, wherein the heterologous nucleic acid sequence is integrated into a sustained transgene expression locus (STEL) in the engineered cell.

16. The engineered cell of claim 15, wherein the STEL comprises a locus within a human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene.The engineered cell of any one of claims 1 to 16, wherein the engineered cell further comprises a kill switch.

18. The engineered cell of any one of claims 1 to 17, wherein the engineered cell comprises a stem cell.

19. The engineered cell of any one of claims 1 to 18, wherein the engineered cell is capable of differentiating into a cardiomyocyte.

20. The engineered cell of any one of claims 1 to 17, wherein the engineered cell comprises a cardiomyocyte, a neural cell, a myeloid cell, a T cell, or a retinal cell.

21. The engineered cell of any of claims 2 to 20, wherein the reduced binding affinity to the CD8 co-receptor comprises at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% reduction in binding affinity to the CD8 co-receptor relative to a comparable protein.

22. A cardiomyocyte differentiated from the engineered cell of any of claims 1 to 21, wherein the cardiomyocyte exhibits reduced natural killer (NK) cell-mediated cytotoxicity compared to a non-engineered cardiomyocyte.

23. A method for producing an engineered cardiomyocyte with reduced NK cell-mediated cytotoxicity, the method comprising: providing the engineered cell of any of claims 1 to 19; and inducing differentiation of the engineered cell into an engineered cardiomyocyte, wherein the resulting engineered cardiomyocyte exhibits reduced NK cell-mediated cytotoxicity as compared to a wild-type cardiomyocyte.

24. A population of engineered cells derived from the engineered cell of any one of claims 1-20.

25. The population of engineered cells of claim 24, wherein the population of engineered cells are at least about 10% less susceptible to NK cell-mediated cytotoxicity as compared to a population of wild-type cells.

26. The population of engineered cells of claim 24 or 25, wherein the population of engineered cells are at least about 20% less susceptible to NK cell-mediated cytotoxicity as compared to a population of wild-type cells.

27. A pharmaceutical composition comprising the population of engineered cells of any one of claims 24 to 26, and a pharmaceutical acceptable carrier, excipient, or diluent.

28. A method of treating a disease or condition of a subject, the method comprising administering the population of engineered cells of any one of claims 24 to 26, or the composition of claim 27.

29. The method of claim 28, wherein the disease or condition comprises heart failure, Parkinson’s disease, multiple sclerosis, irritable bowel syndrome, type 1 diabetes, rheumatoid arthritis, Alzheimer’s disease, or neural inflammation.

30. The method of claim 28 or 29, wherein the subject is a human.

31. A method for enhancing immune evasion of a cell, or progeny thereof, comprising engineering the cell by: genetically modifying the cell to reduce or eliminate function of a natural-killer group 2, member D (NKG2D) ligand relative to a wild-type cell, and incorporating a heterologous nucleic acid sequence that encodes at least a functional portion of a human leukocyte antigen (HLA)-E molecule covalently linked to a P2- microglobulin (B2M) molecule into the genome of the cell, wherein the genetically modifying step enhances the survival probability of the cell when exposed to natural killer cells, as compared to a wild-type cell.

32. The method of claim 31, wherein at least one of the HLA-E molecule or the B2M molecule comprises a modification that reduces binding affinity to a CD8 co-receptor as compared to a corresponding wild-type molecule.

33. The method of claim 32, wherein the B2M molecule comprises the modification that reduces binding affinity to a CD8 co-receptor.

34. The method of claim 32 or claim 33, wherein the heterologous nucleic acid encodes an amino acid sequence having at least 95% identity to one of SEQ ID NO. 2, 4, 6, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30.

35. The method of claim 33, wherein the amino acid sequence has at least 95% identity to SEQ ID NO. 18.

36. The method of any of claims 30 to 35, further comprising engineering the cell with one or more genetic modifications that result in reduced T cell-mediated killing as compared to a wild-type cell, the one or more genetic modifications comprising:(i) a deletion or disruption of a P2 microglobulin (B2M) gene;(ii) a deletion or disruption of one or more of an HLA-A gene, an HLA-B gene, and an HLA-C gene; or(iii) a deletion or disruption of a class II, major histocompatibility complex, transactivator (CIITA) gene.

37. The method of any one of claims 30 to 36, wherein the genetic modification that reduces or eliminates function of a NKG2D ligand as compared to a wild-type cell is a deletion or disruption of a gene encoding the NKG2D ligand.

38. The method of claim 37, wherein the NKG2D ligand comprises MICA, MICB, Raetle, Raetlg, Raetll, Ulbpl, Ulbp2, Ulbp3, or any combination thereof.

39. The method of any of claims 30 to 38, wherein the NKG2D ligand comprises MICA.

40. The method of any one of claims 30 to 39, wherein the HLA-E molecule encoded by the heterologous nucleic acid sequence comprises a binding peptide.

41. The method of claim 40, wherein the binding peptide comprises an amino acid sequence comprising LIL or LFL.

42. The method of claim 41, wherein the binding peptide has the amino acid sequence comprising LFL.

43. The method of any of claims 30 to 42, wherein the HLA-E molecule is covalently linked to the B2M molecule by a linker.

44. The method of any one of claims 30 to 43, wherein the incorporating comprises integrating the heterologous nucleic acid sequence into a genomic locus of the cell.

45. The method of claim 44, wherein the genomic locus is a sustained transgene expression locus (STEL).

46. The method of claim 45, wherein the STEL comprises a locus within a human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene.

47. The method of any one of claims 30 to 46, further comprising expanding the engineered cell to produce a population of engineered cells.

48. The method of claim 47, further comprising differentiating the population of engineered cells into a population of cardiac cells, neural cells, T cells, retinal cells, or myeloid cells.

49. A pharmaceutical composition comprising the population of engineered cells produced by the method of claim 47 or 48, and a pharmaceutical acceptable carrier, excipient, or dilutant.

50. A kit comprising a dosage form suitable for administration to a subject comprising the population of engineered cells of any one of claims 24 to 26, and instructional material for the use of said dosage form.

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