Genetically-modified immune cells comprising a microRNA-adapted shRNA (shRNAmiR)

Abstract

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Description

This is a divisional application of Australian patent application No. 2020253538, the 5 entire contents of which are incorporated herein by reference. FIELD OF THE INVENTION The invention relates to the field of oncology, cancer immunotherapy, molecular biology and recombinant nucleic acid technology. In particular, the invention relates to genetically-modified immune cells comprising a microRNA-adapted shRNA (shRNAmiR) molecule that 10 enables stable knockdown of a particular target gene. The invention further relates to the use of such genetically-modified immune cells for reducing the expression of an endogenous protein and treating a disease, including cancer, in a subject. REFERENCE TO A SEQUENCE LISTING SUBMITTED AS 15                         A TEXT FILE VIA EFS-WEB The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 3, 2020 is named PBIO-037WO_Seq_List_4-20, and is 58,911 bytes in size. 20                    BACKGROUND OF THE INVENTION T cell adoptive immunotherapy is a promising approach for cancer treatment. The immunotherapy treatment methods disclosed herein utilize isolated human T cells that have been genetically-modified to enhance their specificity for a specific tumor associated antigen. Genetic modification may involve the expression of a chimeric antigen receptor or an exogenous T cell 25 receptor to graft antigen specificity onto the T cell. By contrast to exogenous T cell receptors, chimeric antigen receptors derive their specificity from the variable domains of a monoclonal antibody. Thus, T cells expressing chimeric antigen receptors (CAR T cells) induce tumor immunoreactivity in a major histocompatibility complex non-restricted manner. T cell adoptive immunotherapy has been utilized as a clinical therapy for a number of cancers, including B cell 30 malignancies (e.g., acute lymphoblastic leukemia, B cell non-Hodgkin lymphoma, acute myeloid 2026205474   09 Jul 2026 leukemia, and chronic lymphocytic leukemia), multiple myeloma, neuroblastoma, glioblastoma, advanced gliomas, ovarian cancer, mesothelioma, melanoma, prostate cancer, pancreatic cancer, and others. Despite its potential usefulness as a cancer treatment, adoptive immunotherapy with CAR 5 T cells has been limited, in part, by expression of the endogenous T cell receptor on the cell surface. CAR T cells expressing an endogenous T cell receptor may recognize major and minor histocompatibility antigens following administration to an allogeneic patient, which can lead to the development of graft-versus-host-disease (GVHD). As a result, clinical trials have largely focused on the use of autologous CAR T cells, wherein a patient’s T cells are isolated, 10 genetically-modified to incorporate a chimeric antigen receptor, and then re-infused into the same patient. An autologous approach provides immune tolerance to the administered CAR T cells; however, this approach is constrained by both the time and expense necessary to produce patient-specific CAR T cells after a patient’s cancer has been diagnosed. Thus, it would be advantageous to develop “off the shelf” CAR T cells, prepared using T 15 cells from a third party, healthy donor, that have reduced expression, or have no detectable cellsurface expression, of an endogenous T cell receptor (e.g., an alpha / beta T cell receptor) and do not initiate GVHD upon administration. Such products could be generated and validated in advance of diagnosis and could be made available to patients as soon as necessary. Therefore, a need exists for the development of allogeneic CAR T cells that lack an endogenous T cell 20 receptor in order to prevent the occurrence of GVHD. To this end, engineered meganucleases having specificity for the beta-2 microglobulin gene have been generated in order to fully knockout the expression of the beta-2 microglobulin (B2M) protein expression (see, for example, International Publication No. WO 2017 / 112859). B2M is a component of the major histocompatibility complex (MHC) class I molecule, which 25 will not assemble on the cell surface without B2M present. Thus, knockout of B2M is a means for eliminating MHC class I molecules which should reduce GVHD when CAR T cells are administered to allogeneic patients. A consequence of fully eliminating B2M and MHC class I molecule expression on the cell surface of CAR T cells, however, is that they become more susceptible to targeting by 30 natural killer (NK) cells which see them as non-self. In view of this phenomenon, a knockdown approach for CAR T cells was developed in order to produce an incomplete knockdown of B2M 2026205474   09 Jul 2026 (see, for example, International Publication No. WO 2018 / 208837). Essentially, a cassette comprising a B2M-targeted shRNA-coding sequence was introduced into the T cell receptor alpha constant region gene of the T cell by nuclease-mediated targeted insertion. The shRNA-coding sequence was included on a cassette that also comprised a CAR coding sequence, 5 allowing for the production of CAR T cells that were TCR-negative, CAR-positive, and had a partial knockdown of cell-surface B2M. The data in this project demonstrated that these CAR T cells were indeed less susceptible to NK cell killing than CAR T cells that exhibited a complete knockout of B2M. As described herein, however, further experiments showed that the cassette comprising 10 the shRNA-coding sequence was not stable, and that B2M knockdown was transient. Ultimately, the cell removed the shRNA-coding sequence from the genome, causing a return of B2M expression. Therefore, a need remains for the production of CAR T cells that can maintain a stable knockdown of endogenous proteins, such as B2M. In searching for an answer to this problem in the art, a technology was discovered herein that can be used to produce genetic 15 knockdown of various degrees of proteins of interest in immune cells. SUMMARY OF THE INVENTION The present invention provides genetically-modified immune cells (and populations thereof) expressing a microRNA-adapted shRNA (shRNAmiR) that reduces the expression of a 20 target protein. Using shRNAmiRs for knocking down the expression of a target protein allows for stable knockdown of protein expression, which is ideal for those target proteins for which knockdown, and not knockout, is preferred. For example, immune cells that express beta-2 microglobulin (B2M) at reduced levels via expression of B2M-targeted shRNAmiRs are less sensitive to cytolysis by natural killer (NK) cells than those cells in which B2M expression has 25 been knocked out by gene inactivation. Thus, further provided are methods for reducing the expression of an endogenous protein in an immune cell by introducing a template nucleic acid comprising a nucleic acid sequence encoding a shRNAmiR that is inserted into the cell’s genome and expressed in order to reduce the expression of the endogenous protein. Thus, in one aspect, the invention provides a genetically-modified immune cell 30 comprising in its genome a nucleic acid sequence encoding a microRNA-adapted shRNA (shRNAmiR). The shRNAmiR is expressed in the genetically-modified immune cell and 2026205474   09 Jul 2026 reduces expression of a target protein in the genetically-modified immune cell. A reduction in target protein expression is mediated by the binding of the shRNAmiR guide sequence to mRNA encoding the target protein. In some embodiments, the genetically-modified immune cell is a genetically-modified T 5 cell, or a cell derived therefrom. In certain embodiments, the genetically-modified immune cell is a genetically-modified natural killer (NK) cell, or a cell derived therefrom. In other embodiments, the genetically-modified immune cell is a genetically-modified B cell, or a cell derived therefrom. In various embodiments, the genetically-modified immune cell is a genetically-modified monocyte or macrophage, or a cell derived therefrom. 10          In some embodiments, the shRNAmiR comprises, from 5' to 3': (a) a 5' miR scaffold domain; (b) a 5' miR basal stem domain; (c) a passenger strand; (d) a miR loop domain; (e) a guide strand; (f) a 3' miR basal stem domain; and (g) a 3' miR scaffold domain. In some embodiments, the miR loop domain is a miR-30a loop domain, a miR-15 loop domain, a miR-16 loop domain, a miR-155 loop domain, a miR-22 loop domain, a miR-103 loop 15 domain, or a miR-107 loop domain. In particular embodiments, the miR loop domain is a miR-30a loop domain. In certain embodiments, the miR-30a loop domain comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 3. In particular embodiments, the miR-30a loop domain comprises a nucleic acid 20 sequence of SEQ ID NO: 3. In some embodiments, the shRNAmiR comprises a microRNA-E (miR-E) scaffold, a miR-30 (e.g., miR-30a) scaffold, a miR-15 scaffold, a miR-16 scaffold, a miR-155 scaffold, a miR-22 scaffold, a miR-103 scaffold, or a miR-107 scaffold. In certain embodiments, the shRNAmiR comprises a miR-E scaffold. 25          In some embodiments, the shRNAmiR comprises a structure wherein: (a) the 5' miR scaffold domain comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 1; (b) the 5' miR basal stem domain comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 2; (c) the 3' miR basal stem domain 30 comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 4; and / or (d) the 3' miR scaffold domain comprises a 2026205474   09 Jul 2026 nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 5. In certain embodiments, the shRNAmiR comprises a structure wherein: (a) the 5' miR scaffold domain comprises a nucleic acid sequence of SEQ ID NO: 1; (b) the 5' miR basal stem 5 domain comprises a nucleic acid sequence of SEQ ID NO: 2; (c) the 3' miR basal stem domain comprises a nucleic acid sequence of SEQ ID NO: 4; and (d) the 3' miR scaffold domain comprises a nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the genetically-modified immune cell comprises in its genome a nucleic acid sequence encoding a chimeric antigen receptor (CAR) or an exogenous T cell 10 receptor (TCR), wherein the CAR or the exogenous TCR is expressed by the genetically-modified immune cell. In some embodiments, the genetically-modified immune cell comprises in its genome a nucleic acid sequence encoding an HLA class I histocompatibility antigen, alpha chain E (HLA-E) fusion protein. In some embodiments, the HLA-E fusion protein comprises an amino acid 15 sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 66. In some embodiments, the HLA-E fusion protein comprises an amino acid sequence of SEQ ID NO: 66. In some embodiments, the nucleic acid sequence encoding the shRNAmiR is located within a different gene than the nucleic acid sequence encoding the CAR or the exogenous TCR. 20 In certain embodiments, the nucleic acid sequence encoding the shRNAmiR, or the nucleic acid sequence encoding the CAR or the exogenous TCR, is located within a TCR alpha gene or a TCR alpha constant region gene. In particular embodiments, the nucleic acid sequence encoding the shRNAmiR, or the nucleic acid sequence encoding the CAR or the exogenous TCR, is located within a TCR alpha constant region gene within a sequence comprising SEQ ID NO: 58. 25          In some embodiments, the nucleic acid sequence encoding the shRNAmiR is located within the same gene as the nucleic acid sequence encoding the CAR or the exogenous TCR. In certain embodiments, the gene is a TCR alpha gene or TCR alpha constant region gene. In particular embodiments, the nucleic acid sequence encoding the shRNAmiR and the nucleic acid sequence encoding the CAR or the exogenous TCR is located within a TCR alpha constant 30 region gene within a sequence comprising SEQ ID NO: 58. In certain embodiments, the nucleic acid sequence encoding the shRNAmiR and the nucleic acid encoding the CAR or the exogenous 2026205474   09 Jul 2026 TCR are within a cassette in the gene. In some such embodiments, the nucleic acid sequence encoding the shRNAmiR and the nucleic acid sequence encoding the CAR or the exogenous TCR are operably linked to a same promoter. In some such embodiments, the genetically-modified immune cell comprises in its genome a cassette comprising, from 5' to 3': (a) the 5 nucleic acid sequence encoding the CAR or the exogenous TCR; and (b) the nucleic acid sequence encoding the shRNAmiR. In other such embodiments, the genetically-modified immune cell comprises in its genome a cassette comprising, from 5' to 3': (a) the nucleic acid sequence encoding the shRNAmiR; and (b) the nucleic acid sequence encoding the CAR or the exogenous TCR. In some such embodiments, the nucleic acid sequence encoding the CAR or 10 the exogenous TCR and the nucleic acid sequence encoding the shRNAmiR are separated by a 2A or IRES sequence. In certain such embodiments, the nucleic acid sequence encoding the shRNAmiR is in the same orientation as the nucleic acid sequence encoding the CAR or the exogenous TCR. In other such embodiments, the nucleic acid sequence encoding the shRNAmiR is in a reverse orientation as the nucleic acid sequence encoding the CAR or the 15 exogenous TCR. In some such embodiments, an intron sequence is positioned within the nucleic acid sequence encoding the CAR or the exogenous TCR, and the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence. In some such embodiments, the cassette comprises a promoter that is operably linked to the nucleic acid sequence encoding the shRNAmiR and the nucleic acid sequence encoding the CAR or the exogenous TCR. In some 20 such embodiments, the cassette comprises a termination signal. In certain embodiments, the nucleic acid sequence encoding the shRNAmiR is located within the same gene as the nucleic acid sequence encoding the HLA-E fusion protein. In some embodiments, the gene is a TCR alpha gene or a TCR alpha constant region gene. In some embodiments, the nucleic acid sequence encoding the shRNAmiR and the nucleic acid sequence 25 encoding the HLA-E fusion protein are within a cassette in the gene. In some such embodiments, the nucleic acid sequence encoding the shRNAmiR and nucleic acid sequence encoding the HLA-E fusion protein are operably linked to a same promoter. In some such embodiments, the genetically-modified immune cell comprises in its genome a cassette comprising, from 5' to 3': (a) the nucleic acid sequence encoding the HLA-E fusion protein; and 30   (b) the nucleic acid sequence encoding the shRNAmiR. In some such embodiments, the genetically-modified immune cell comprises in its genome a cassette comprising, from 5' to 3': 2026205474   09 Jul 2026 (a) the nucleic acid sequence encoding the shRNAmiR; and (b) the nucleic acid sequence encoding the HLA-E fusion protein. In some such embodiments, the nucleic acid sequence encoding the HLA-E fusion protein and the nucleic acid sequence encoding the shRNAmiR are separated by a 2A or IRES sequence. In certain such embodiments, an intron sequence is 5 positioned within the nucleic acid sequence encoding the HLA-E fusion protein, wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence. In some such embodiments, the cassette comprises a promoter, wherein the nucleic acid sequence encoding the shRNAmiR and nucleic acid sequence encoding the HLA-E fusion protein are operably linked to the promoter. In some such embodiments, the cassette comprises a 10 termination signal. In some embodiments, the nucleic acid sequence encoding the shRNAmiR, the nucleic acid sequence encoding the CAR or the exogenous TCR, and the nucleic acid sequence encoding the HLA-E fusion protein are located within the same gene. In some embodiments, the gene is a TCR alpha gene or a TCR alpha constant region gene. In some embodiments, the nucleic acid 15 sequence encoding the shRNAmiR, the nucleic acid sequence encoding the CAR or the exogenous TCR, and the nucleic acid sequence encoding the HLA-E fusion protein are within a cassette in the gene. In some such embodiments, the nucleic acid sequence encoding the shRNAmiR, the nucleic acid sequence encoding the CAR or the exogenous TCR, and the nucleic acid sequence encoding the HLA-E fusion protein are operably linked to a same promoter. In 20 some such embodiments, the genetically-modified immune cell comprises within its genome a cassette comprising: (a) the nucleic acid sequence encoding the CAR or the exogenous TCR; (b) a 2A or IRES sequence; (c) the nucleic acid sequence encoding the HLA-E fusion protein; and (d) the nucleic acid sequence encoding the shRNAmiR. In some such embodiments, an intron sequence is positioned within the nucleic acid sequence encoding the CAR or the exogenous 25 TCR, wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence. In other such embodiments, an intron sequence is positioned within the nucleic acid sequence encoding the HLA-E fusion protein, wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence. In some such embodiments, the cassette comprises a promoter that is operably linked to the nucleic acid sequence encoding the CAR or 30 the exogenous TCR, the nucleic acid sequence encoding the HLA-E fusion protein, and the 2026205474   09 Jul 2026 nucleic acid sequence encoding the shRNAmiR. In some such embodiments, the cassette comprises a termination signal. In some such embodiments, the genetically-modified immune cell comprises within its genome a cassette comprising, from 5' to 3': (a) a promoter; (b) the nucleic acid sequence 5 encoding the CAR or the exogenous TCR; (c) a 2A or IRES sequence; (d) the nucleic acid sequence encoding the HLA-E fusion protein, wherein an intron sequence is positioned within the nucleic acid sequence encoding the HLA-E fusion protein, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence; and (e) optionally a termination signal; wherein the nucleic acid sequence encoding the CAR or the exogenous TCR, 10 the nucleic acid sequence encoding the HLA-E fusion protein, and the nucleic acid sequence encoding the shRNAmiR are operably linked to the promoter. In some such embodiments, the genetically-modified immune cell comprises within its genome a cassette comprising, from 5' to 3': (a) a promoter; (b) the nucleic acid sequence encoding the HLA-E fusion protein, wherein an intron sequence is positioned within the nucleic 15 acid sequence encoding the HLA-E fusion protein, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence; (c) a 2A or IRES sequence; (d) the nucleic acid sequence encoding the CAR or the exogenous TCR; and (e) optionally a termination signal; wherein the nucleic acid sequence encoding the CAR or the exogenous TCR, the nucleic acid sequence encoding the HLA-E fusion protein, and the nucleic acid sequence 20 encoding the shRNAmiR are operably linked to the promoter. In some such embodiments, the genetically-modified immune cell comprises within its genome a cassette comprising, from 5' to 3': (a) a promoter; (b) the nucleic acid sequence encoding the CAR or the exogenous TCR, wherein an intron sequence is positioned within the nucleic acid sequence encoding the CAR or the exogenous TCR, and wherein the nucleic acid 25 sequence encoding the shRNAmiR is positioned within the intron sequence; (c) a 2A or IRES sequence; (d) the nucleic acid sequence encoding the HLA-E fusion protein; and (e) optionally a termination signal; wherein the nucleic acid sequence encoding the CAR or the exogenous TCR, the nucleic acid sequence encoding the HLA-E fusion protein, and the nucleic acid sequence encoding the shRNAmiR are operably linked to the promoter. 30          In some such embodiments, the genetically-modified immune cell comprises within its genome a cassette comprising, from 5' to 3': (a) a promoter; (b) the nucleic acid sequence 2026205474   09 Jul 2026 encoding the HLA-E fusion protein; (c) a 2A or IRES sequence; (d) the nucleic acid sequence encoding the CAR or the exogenous TCR, wherein an intron sequence is positioned within the nucleic acid sequence encoding the CAR or the exogenous TCR, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence; and (e) optionally a 5 termination signal; wherein the nucleic acid sequence encoding the CAR or the exogenous TCR, the nucleic acid sequence encoding the HLA-E fusion protein, and the nucleic acid sequence encoding the shRNAmiR are operably linked to the promoter. In some embodiments described above, the intron sequence is a synthetic intron sequence. In certain embodiments, the intron sequence comprises a nucleic acid sequence 10 having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 69. In particular embodiments, the intron sequence comprises a nucleic acid sequence of SEQ ID NO: 69. In some embodiments described above, the termination signal is a polyA sequence or a bovine growth hormone (BGH) termination signal. In certain embodiments, the polyA sequence 15 comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 68. In particular embodiments, the polyA sequence comprises a nucleic acid sequence of SEQ ID NO: 68. In certain embodiments, the BGH termination signal comprises a nucleic acid sequence having least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 71. In particular embodiments, 20 the BGH termination signal comprises a nucleic acid sequence of SEQ ID NO: 71. In some embodiments described above, the promoter comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 67. In particular embodiments, the promoter comprises a nucleic acid sequence of SEQ ID NO: 67. 25          In some embodiments described above, the 2A sequence is a P2A / furin site comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%,at least 95%, or more, sequence identity to SEQ ID NO: 70. In particular embodiments, the 2A sequence is a P2A / furin site comprising a nucleic acid sequence of SEQ ID NO: 70. In some embodiments described above, the CAR comprises a signal peptide comprising 30 an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, 2026205474   09 Jul 2026 sequence identity to SEQ ID NO: 73. In particular embodiments, the CAR comprises a signal peptide comprising an amino acid sequence of SEQ ID NO: 73. In particular embodiments, the genetically-modified immune cell comprises in its genome a cassette comprising, from 5' to 3': (a) a promoter comprising a nucleic acid sequence 5 having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 67; (b) the nucleic acid sequence encoding the CAR, wherein the CAR comprises a signal peptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 73; (c) a P2A / furin site comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, 10 sequence identity to SEQ ID NO: 70; (d) the nucleic acid sequence encoding the HLA-E fusion protein, wherein the HLA-E fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 66, and wherein an intron sequence is positioned within the nucleic acid sequence encoding the HLA-E fusion protein, wherein the intron sequence comprises a nucleic acid sequence having at least 15   80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 69, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence; and (e) optionally a termination signal comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 68; wherein the nucleic acid sequence encoding the CAR, the nucleic acid sequence encoding the 20 HLA-E fusion protein, and the nucleic acid sequence encoding the shRNAmiR are operably linked to the promoter. In particular embodiments, the genetically-modified immune cell comprises in its genome a cassette comprising, from 5' to 3': (a) a promoter comprising a nucleic acid sequence of SEQ ID NO: 67; (b) the nucleic acid sequence encoding the CAR, wherein the CAR 25 comprises a signal peptide comprising an amino acid sequence of SEQ ID NO: 73; (c) a P2A / furin site comprising a nucleic acid sequence of SEQ ID NO: 70; (d) the nucleic acid sequence encoding the HLA-E fusion protein, wherein the HLA-E fusion protein comprises an amino acid sequence of SEQ ID NO: 66, and wherein an intron sequence is positioned within the nucleic acid sequence encoding the HLA-E fusion protein, wherein the intron sequence 30 comprises a nucleic acid sequence of SEQ ID NO: 69, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence; and (e) optionally a 2026205474   09 Jul 2026 termination signal comprising a nucleic acid sequence of SEQ ID NO: 68; wherein the nucleic acid sequence encoding the CAR, the nucleic acid sequence encoding the HLA-E fusion protein, and the nucleic acid sequence encoding the shRNAmiR are operably linked to the promoter. In particular embodiments, the genetically-modified immune cell comprises in its 5 genome a cassette comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 74, wherein the cassette is positioned in the genome within a TCR alpha constant region gene. In particular embodiments, the genetically-modified immune cell comprises in its genome a cassette comprising a nucleic acid sequence of SEQ ID NO: 74, wherein the cassette is positioned in the genome within a TCR 10 alpha constant region gene. In some embodiments described above, the cassette comprises two or more nucleic acids encoding shRNAmiRs. In certain embodiments, the two or more nucleic acids can encode the same shRNAmiR. In some embodiments, the two or more nucleic acids can encode different shRNAmiRs that reduce the expression of the same target protein. In other embodiments, the 15 two or more nucleic acids encode different shRNAmiRs that reduce the expression of different target proteins. In certain embodiments, the cassette can comprise two or more nucleic acids encoding different shRNAmiRs described herein. In particular embodiments, the cassette can comprise a nucleic acid sequence encoding a shRNAmiR that reduces the expression of B2M, and a nucleic acid sequence encoding a shRNAmiR that reduces the expression of CD52. 20         In some embodiments, the nucleic acid sequence encoding the shRNAmiR and the nucleic acid sequence encoding the CAR or the exogenous TCR are located in the same gene and are operably linked to different promoters. In some such embodiments, the genetically-modified immune cell comprises in its genome a cassette comprising, from 5' to 3': (a) a first promoter; (b) the nucleic acid sequence encoding the CAR or exogenous TCR which is operably linked to the 25 first promoter; (c) a second promoter; and (d) the nucleic acid sequence encoding the shRNAmiR which is operably linked to the second promoter. In other such embodiments, the genetically-modified immune cell comprises in its genome a cassette comprising, from 5' to 3': (a) a first promoter; (b) the nucleic acid sequence encoding the shRNAmiR which is operably linked to the first promoter; (c) a second promoter; and (d) the nucleic acid sequence encoding the CAR or 30 exogenous TCR which is operably linked to the second promoter. In some such embodiments, the nucleic acid sequence encoding the shRNAmiR is in the same orientation as the nucleic acid 2026205474   09 Jul 2026 sequence encoding the CAR or exogenous TCR. In other such embodiments, the nucleic acid sequence encoding the shRNAmiR is in a reverse orientation as the nucleic acid sequence encoding the CAR or exogenous TCR. In certain such embodiments, the first promoter and the second promoter are identical. In other embodiments, the first promoter and the second promoter 5 are different. In some such embodiments, the cassette comprises one or more termination signals. In some embodiments, the nucleic acid sequence encoding the shRNAmiR and the nucleic acid sequence encoding the HLA-E fusion protein are operably linked to different promoters. In some such embodiments, the genetically-modified immune cell comprises in its 10 genome a cassette comprising, from 5' to 3': (a) a first promoter; (b) the nucleic acid sequence encoding the HLA-E fusion protein which is operably linked to the first promoter; (c) a second promoter; and (d) the nucleic acid sequence encoding the shRNAmiR which is operably linked to the second promoter. In other such embodiments, the genetically-modified immune cell comprises in its genome a cassette comprising, from 5' to 3': (a) a first promoter; (b) the nucleic 15 acid sequence encoding the shRNAmiR which is operably linked to the first promoter; (c) a second promoter; and (d) the nucleic acid sequence encoding the HLA-E fusion protein which is operably linked to the second promoter. In certain such embodiments, the first promoter and the second promoter are identical. In other such embodiments, the first promoter and the second promoter are different. In some such embodiments, the cassette comprises one or more 20 termination signals. In some embodiments, the genetically-modified immune cell comprises within its genome a cassette comprising: (a) the nucleic acid sequence encoding the CAR or the exogenous TCR; (b) the nucleic acid sequence encoding the HLA-E fusion protein; and (c) the nucleic acid sequence encoding the shRNAmiR; wherein the nucleic acid sequence encoding the CAR or the 25 exogenous TCR is operably linked to a first promoter, and wherein the nucleic acid sequence encoding the HLA-E fusion protein and the nucleic acid sequence encoding the shRNAmiR are operably linked to a second promoter. In some such embodiments, an intron sequence is positioned within the nucleic acid sequence encoding the HLA-E fusion protein, wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence. In 30 some embodiments, the genetically-modified immune cell comprises within its genome a cassette comprising: (a) the nucleic acid sequence encoding the CAR or the exogenous TCR; (b) 2026205474   09 Jul 2026 the nucleic acid sequence encoding the HLA-E fusion protein; and (c) the nucleic acid sequence encoding the shRNAmiR; wherein the nucleic acid sequence encoding the CAR or the exogenous TCR and the nucleic acid sequence encoding the shRNAmiR are operably linked to a first promoter, and wherein the nucleic acid sequence encoding the HLA-E fusion protein is 5 operably linked to a second promoter. In some such embodiments, an intron sequence is positioned within the nucleic acid sequence encoding the CAR or the exogenous TCR, wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence. In some such embodiments, the cassette comprises a first termination signal capable of terminating transcription of the CAR or the exogenous TCR, and a second termination signal capable of 10 terminating transcription of the HLA-E fusion protein. In other such embodiments, the cassette comprises a first termination signal capable of terminating transcription of the HLA-E fusion protein, and a second termination signal capable of terminating transcription of the CAR or the exogenous TCR. In some such embodiments, the genetically-modified immune cell comprises within its 15 genome a cassette comprising, from 5' to 3': (a) a first promoter; (b) the nucleic acid sequence encoding the CAR or the exogenous TCR; (c) optionally a first termination signal; (d) a second promoter; (e) the nucleic acid sequence encoding the HLA-E fusion protein, wherein an intron sequence is positioned within the nucleic acid sequence encoding the HLA-E fusion protein, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron 20 sequence; and (f) optionally a second termination signal; wherein the nucleic acid sequence encoding the CAR or the exogenous TCR is operably linked to the first promoter, and wherein the nucleic acid sequence encoding the HLA-E fusion protein and the nucleic acid sequence encoding the shRNAmiR are operably linked to the second promoter. In some such embodiments, the genetically-modified immune cell comprises within its 25 genome a cassette comprising, from 5' to 3': (a) a first promoter; (b) the nucleic acid sequence encoding the HLA-E fusion protein, wherein an intron sequence is positioned within the nucleic acid sequence encoding the HLA-E fusion protein, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence; (c) optionally a first termination signal; (d) a second promoter; (e) the nucleic acid sequence encoding the CAR or the 30 exogenous TCR; and (f) optionally a second termination signal; wherein the nucleic acid sequence encoding the HLA-E fusion protein and the nucleic acid sequence encoding the 2026205474   09 Jul 2026 shRNAmiR are operably linked to the first promoter, and wherein the nucleic acid sequence encoding the CAR or the exogenous TCR is operably linked to the second promoter. In some such embodiments, the genetically-modified immune cell comprises within its genome a cassette comprising, from 5' to 3': (a) a first promoter; (b) the nucleic acid sequence 5 encoding the CAR or the exogenous TCR, wherein an intron sequence is positioned within the nucleic acid sequence encoding the CAR or the exogenous TCR, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence; (c) optionally a first termination signal; (d) a second promoter; (e) the nucleic acid sequence encoding the HLA-E fusion protein; and (f) optionally a second termination signal; wherein the nucleic acid sequence 10 encoding the CAR or the exogenous TCR and the nucleic acid sequence encoding the shRNAmiR are operably linked to the first promoter, and wherein the nucleic acid sequence encoding the HLA-E fusion protein is operably linked to the second promoter. In some such embodiments, the genetically-modified immune cell comprises within its genome a cassette comprising, from 5' to 3': (a) a first promoter; (b) the nucleic acid sequence 15 encoding the HLA-E fusion protein; (c) optionally a first termination signal; (d) a second promoter; (e) the nucleic acid sequence encoding the CAR or the exogenous TCR, wherein an intron sequence is positioned within the nucleic acid sequence encoding the CAR or the exogenous TCR, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence; and (f) optionally a second termination signal; wherein the nucleic 20 acid sequence encoding the HLA-E fusion protein is operably linked to the first promoter, and wherein the nucleic acid sequence encoding the CAR or the exogenous TCR and the nucleic acid sequence encoding the shRNAmiR are operably linked to the second promoter. In some embodiments described above, the intron sequence is a synthetic intron sequence. In certain embodiments, the intron sequence comprises a nucleic acid sequence 25 having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 69. In particular embodiments, the intron sequence comprises a nucleic acid sequence of SEQ ID NO: 69. In some embodiments described above, the one or more termination signals is a polyA sequence or a BGH termination signal. 2026205474   09 Jul 2026 In some embodiments described above, the first termination signal is identical to the second termination signal. In some such embodiments, the first termination signal and the second termination signal are a polyA sequence or a BGH termination signal. In some embodiments described above, the first termination signal is different from the 5 second termination signal. In some embodiments, the first termination signal is a polyA sequence and the second termination signal is a BGH termination signal. In some embodiments, the first termination signal is a BGH termination signal and the second termination signal is a polyA sequence. In some embodiments described above, the polyA sequence comprises a nucleic acid 10 sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 68. In certain embodiments, the polyA sequence comprises a nucleic acid sequence of SEQ ID NO: 68. In some embodiments, the BGH termination signal comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 71. In certain embodiments, the BGH termination signal 15 comprises a nucleic acid sequence of SEQ ID NO: 71. In some embodiments described above, the first promoter and the second promoter are identical. In some such embodiments, the first promoter and the second promoter are a JeT promoter or an EF1 alpha core promoter. In some embodiments described above, the first promoter is different from the second 20 promoter. In certain embodiments, the first promoter is a JeT promoter, and the second promoter is an EF1 alpha core promoter. In certain embodiments, the first promoter is an EF1 alpha core promoter, and the second promoter is a JeT promoter. In certain embodiments described above, the JeT promoter comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence 25 identity to SEQ ID NO: 67. In particular embodiments, the JeT promoter comprises a nucleic acid sequence of SEQ ID NO: 67. In certain embodiments, the EF1 alpha core promoter comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 72. In some embodiments, the EF1 alpha core promoter comprises a nucleic acid sequence of SEQ ID NO: 72. 30          In some embodiments described above, the CAR comprises a signal peptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, 2026205474   09 Jul 2026 sequence identity to SEQ ID NO: 73. In particular embodiments, the CAR comprises a signal peptide comprising an amino acid sequence of SEQ ID NO: 73. In some embodiments, the genetically-modified immune cell comprises in its genome a cassette comprising, from 5' to 3': (a) a first promoter comprising a nucleic acid sequence having 5 at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 67; (b) the nucleic acid sequence encoding the CAR, wherein the CAR comprises a signal peptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 73; (c) optionally a first termination signal comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, 10 or more, sequence identity to SEQ ID NO: 70; (d) a second promoter comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 72; (e) the nucleic acid sequence encoding the HLA-E fusion protein, wherein the HLA-E fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 66, and wherein 15 an intron sequence is positioned within the nucleic acid sequence encoding the HLA-E fusion protein, wherein the intron sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 69, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence; and (f) optionally a second termination signal comprising a nucleic acid sequence having at least 20   80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 71; wherein the nucleic acid sequence encoding the CAR is operably linked to the first promoter, and wherein the nucleic acid sequence encoding the HLA-E fusion protein and the nucleic acid sequence encoding the shRNAmiR are operably linked to the second promoter. In particular embodiments, the genetically-modified immune cell comprises in its 25 genome a cassette comprising, from 5' to 3': (a) a first promoter comprising a nucleic acid sequence of SEQ ID NO: 67; (b) the nucleic acid sequence encoding the CAR, wherein the CAR comprises a signal peptide comprising an amino acid sequence of SEQ ID NO: 73; (c) optionally a first termination signal comprising a nucleic acid sequence of SEQ ID NO: 70; (d) a second promoter comprising a nucleic acid sequence of SEQ ID NO: 72; (e) the nucleic acid sequence 30 encoding the HLA-E fusion protein, wherein the HLA-E fusion protein comprises an amino acid sequence of SEQ ID NO: 66, and wherein an intron sequence is positioned within the nucleic 2026205474   09 Jul 2026 acid sequence encoding the HLA-E fusion protein, wherein the intron sequence comprises a nucleic acid sequence of SEQ ID NO: 69, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence; and (f) optionally a second termination signal comprising a nucleic acid sequence of SEQ ID NO: 71; wherein the nucleic acid sequence 5 encoding the CAR is operably linked to the first promoter, and wherein the nucleic acid sequence encoding the HLA-E fusion protein and the nucleic acid sequence encoding the shRNAmiR are operably linked to the second promoter. In particular embodiments, the genetically-modified immune cell comprises in its genome a cassette comprising a nucleic acid sequence having at least 80%, at least 85%, at least 10   90%, at least 95%, or more, sequence identity to SEQ ID NO: 75, wherein the cassette is positioned in the genome within a TCR alpha constant region gene. In particular embodiments, the genetically-modified immune cell comprises in its genome a cassette comprising a nucleic acid sequence of SEQ ID NO: 75, wherein the cassette is positioned in the genome within a TCR alpha constant region gene. 15          In some embodiments described above, the cassette comprises two or more nucleic acids encoding shRNAmiRs. In certain embodiments, the two or more nucleic acids can encode the same shRNAmiR. In some embodiments, the two or more nucleic acids can encode different shRNAmiRs that reduce the expression of the same target protein. In other embodiments, the two or more nucleic acids encode different shRNAmiRs that reduce the expression of different 20 target proteins. In certain embodiments, the cassette can comprise two or more nucleic acids encoding different shRNAmiRs described herein. In particular embodiments, the cassette can comprise a nucleic acid sequence encoding a shRNAmiR that reduces the expression of B2M, and a nucleic acid sequence encoding a shRNAmiR that reduces the expression of CD52. In some embodiments, expression of the target protein is reduced by at least about 10%, 25 about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell. In some embodiments, the target protein is beta-2 microglobulin, CS1, transforming growth factor-beta receptor 2 (TGFBR2), Cbl proto-oncogene B (CBL-B), CD52, a TCR alpha 30 gene, a TCR alpha constant region gene, CD7, glucocorticoid receptor (GR), deoxycytidine 2026205474   09 Jul 2026 kinase (DCK), nuclear receptor subfamily 2 group F member 6 (NR2F6), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), or C-C chemokine receptor type 5 (CCR5). In some embodiments, the target protein is beta-2 microglobulin. In some such embodiments, cell surface expression of beta-2 microglobulin is reduced by at least about 10%, 5 about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell. In further embodiments, expression of MHC class I molecules is reduced on the cell surface by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up 10 to about 99% compared to a control cell. In some such embodiments, the genetically-modified immune cell has reduced allogenicity compared to a control cell. In some such embodiments, the shRNAmiR has a structure wherein: (a) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 17 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 18; (b) the passenger strand comprises a nucleic acid 15 sequence of SEQ ID NO: 7 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 8; (c) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 9 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 10; (d) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 11 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 12; (e) the passenger strand comprises a nucleic acid sequence of 20 SEQ ID NO: 13 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 14; or (f) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 15 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 16. In certain such embodiments, the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 17 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 18. In particular such embodiments, the 25 nucleic acid sequence encoding the shRNAmiR comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 46. In further such embodiments, the nucleic acid sequence encoding the shRNAmiR comprises the sequence of SEQ ID NO: 46. In some embodiments, the target protein is CS1. In some such embodiments, cell surface 30 expression of CS1 is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 2026205474   09 Jul 2026 90%, about 95%, or up to about 99% compared to a control cell. In some such embodiments, the genetically-modified immune cell expresses a CAR having specificity for CS1. In further such embodiments, the genetically-modified immune cell is less susceptible to fratricide by a genetically-modified immune cell expressing a CAR having specificity for CS1 compared to a 5 control cell. In some such embodiments, the shRNAmiR has a structure wherein: (a) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 21 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 22; (b) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 23 and the guide strand comprises a nucleic acid sequence of SEQ ID 10 NO: 24; or (c) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 25 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 26. In certain such embodiments, the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 25 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 26. In particular such embodiments, the shRNAmiR comprises a sequence having at least 80%, at least 85%, at least 15   90%, at least 95%, or more, sequence identity to SEQ ID NO: 50. In further such embodiments, the shRNAmiR comprises the sequence of SEQ ID NO: 50. In some embodiments, the target protein is TGFBR2. In some such embodiments, the cell surface expression of TGFBR2 is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, 20 about 85%, about 90%, about 95%, or up to about 99% compared to a control cell. In further embodiments, the genetically-modified immune cell is less susceptible to immunosuppression by transforming growth factor B1 (TGFB1) compared to a control cell. In some such embodiments, the shRNAmiR has a structure wherein: (a) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 27 and the guide strand comprises a 25 nucleic acid sequence of SEQ ID NO: 28; (b) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 29 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 30; (c) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 31 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 32; (d) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 33 and the guide strand comprises a nucleic 30 acid sequence of SEQ ID NO: 34; or (e) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 35 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 36. 2026205474   09 Jul 2026 In certain such embodiments, the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 31 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 32. In particular such embodiments, the nucleic acid sequence encoding the shRNAmiR comprises a sequence having at least 80%, at least 95%, at least 90%, at least 95%, or more, sequence identity to SEQ 5 ID NO: 53. In further such embodiments, the nucleic acid sequence encoding the shRNAmiR comprises the sequence of SEQ ID NO: 53. In some embodiments, the target protein is CBL-B. In some such embodiments, cell surface expression of CBL-B is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 10   85%, about 90%, about 95%, or up to about 99% compared to a control cell. In further such embodiments, the immune cell is less susceptible to suppression of T cell receptor (TCR) signaling by degradation of downstream signaling proteins compared to a control cell. In some embodiments, the target protein is CD52. In some such embodiments, cell surface expression of CD52 is reduced by at least about 10%, about 20%, about 30%, about 40%, 15 about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell. In further embodiments, the genetically-modified immune cell is less susceptible to CD52 antibody-induced cell death. In some such embodiments, the shRNAmiR has a structure wherein: (a) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 37 and the guide strand comprises a 20 nucleic acid sequence of SEQ ID NO: 38; or (b) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 39 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 40. In certain such embodiments, the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 37 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 38. In particular such embodiments, the nucleic acid sequence encoding the shRNAmiR comprises a 25 sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 56. In further such embodiments, the nucleic acid sequence encoding the shRNAmiR comprises the sequence of SEQ ID NO: 56. In some embodiments, the target protein is DCK. In some such embodiments, cell surface expression of DCK is reduced by at least about 10%, about 20%, about 30%, about 40%, 30 about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell. In further such 2026205474   09 Jul 2026 embodiments, the genetically-modified immune cell is less susceptible to effects of purine nucleoside analogs (e.g., fludarabine) on cell proliferation. In some such embodiments, the shRNAmiR has a structure wherein: (a) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 76 and the guide strand comprises a 5 nucleic acid sequence of SEQ ID NO: 77; (b) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 78 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 79; (c) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 80 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 81; (d) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 82 and the guide strand comprises a nucleic 10 acid sequence of SEQ ID NO: 83; or (e) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 84 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 85. In particular such embodiments, the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 76 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 77. In particular such embodiments, the nucleic acid sequence encoding the shRNAmiR comprises a 15 sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 86. In further such embodiments, the nucleic acid sequence encoding the shRNAmiR comprises the sequence of SEQ ID NO: 86. In some embodiments, the target protein is GR. In some such embodiments, cell surface expression of GR is reduced by at least about 10%, about 20%, about 30%, about 40%, about 20   50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell. In further such embodiments, the genetically-modified immune cell is less susceptible to effects of glucocorticoids (e.g., dexamethasone), such as reduced proliferation. In some such embodiments, the shRNAmiR has a structure wherein: (a) the passenger 25 strand comprises a nucleic acid sequence of SEQ ID NO: 91 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 92; (b) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 93 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 94; (c) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 95 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 96; (d) the passenger strand 30 comprises a nucleic acid sequence of SEQ ID NO: 97 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 98; (e) the passenger strand comprises a nucleic acid sequence of 2026205474   09 Jul 2026 SEQ ID NO: 99 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 100; (f) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 101 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 102; (g) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 103 and the guide strand comprises a nucleic acid 5 sequence of SEQ ID NO: 104; (h) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 105 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 106; or (i) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 107 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 108. In particular such embodiments, the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 95 and the guide strand 10 comprises a nucleic acid sequence of SEQ ID NO: 96. In particular such embodiments, the nucleic acid sequence encoding the shRNAmiR comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 111. In further such embodiments, the nucleic acid sequence encoding the shRNAmiR comprises the sequence of SEQ ID NO: 111. 15          In another aspect, the invention provides a method for reducing the expression of an endogenous protein in an immune cell, the method comprising introducing into the immune cell a template nucleic acid comprising a nucleic acid sequence encoding a shRNAmiR, wherein the template nucleic acid is inserted into the genome of the immune cell. The shRNAmiR is expressed in the immune cell and reduces expression of an endogenous target protein in the 20 immune cell. A reduction in target protein expression is mediated by the binding of the shRNAmiR guide sequence to mRNA encoding the target protein. In some embodiments of the method, the immune cell is a T cell, or a cell derived therefrom. In certain embodiments, the immune cell is a natural killer (NK) cell, or a cell derived therefrom. In other embodiments, the immune cell is a B cell, or a cell derived 25 therefrom. In various embodiments, the immune cell is a monocyte or macrophage, or a cell derived therefrom. In some embodiments of the method, the template nucleic acid is inserted into the genome of the immune cell by random integration. In some such embodiments of the method, the template nucleic acid is introduced into the immune cell using a viral vector (i.e., a 30 recombinant virus), such as a lentiviral vector (i.e., a recombinant lentivirus). 2026205474   09 Jul 2026 In some embodiments of the method, the immune cell expresses a CAR or exogenous TCR. In some embodiments, the method further comprises introducing into the immune cell a second nucleic acid encoding an engineered nuclease having specificity for a recognition 5 sequence in the genome of the immune cell. The engineered nuclease is expressed in the immune cell and generates a cleavage site at the recognition sequence. The template nucleic acid comprising a nucleic acid sequence encoding the shRNAmiR is inserted into the genome of the immune cell at the cleavage site. In some such embodiments of the method, the template nucleic acid is flanked by homology arms having homology to sequences flanking the recognition 10 sequence, and the template nucleic acid is inserted at the cleavage site by homologous recombination. In some such embodiments of the method, the template nucleic acid is introduced into the immune cell using a viral vector (i.e., a recombinant virus). In some such embodiments, the viral vector is a recombinant AAV vector (i.e., a recombinant AAV). In particular embodiments, the AAV vector has a serotype of AAV2 or AAV6. In some such 15 embodiments of the method, the recognition sequence is within a target gene. In some such embodiments of the method, the expression of a protein encoded by the target gene is disrupted in the immune cell. In certain such embodiments of the method, the target gene is a TCR alpha gene or a TCR alpha constant region gene, and the immune cell does not have detectable cellsurface expression of an endogenous TCR (e.g., an alpha / beta TCR). In some such embodiments 20 of the method, the engineered nuclease is an engineered meganuclease, a zinc finger nuclease, a TALEN, a compact TALEN, a CRISPR system nuclease, or a megaTAL. In particular such embodiments of the method, the engineered nuclease is an engineered meganuclease. In certain such embodiments of the method, the second nucleic acid encoding the engineered nuclease is introduced using an mRNA. 25          In some embodiments of the method, the immune cell into which the template nucleic acid is introduced further comprises in its genome a nucleic acid sequence encoding a CAR or exogenous TCR. In certain embodiments of the method, the immune cell into which the template nucleic acid is introduced further comprises in its genome a nucleic acid sequence encoding an HLA-E fusion protein. 30          In some embodiments of the method, the template nucleic acid further comprises a nucleic acid sequence encoding a CAR or an exogenous TCR, wherein the CAR or the 2026205474   09 Jul 2026 exogenous TCR is expressed by the immune cell. In some such embodiments, the nucleic acid sequence encoding the shRNAmiR and the nucleic acid sequence encoding the CAR or the exogenous TCR are operably linked to a same promoter in the immune cell following introduction of the template nucleic acid at the cleavage site. In some such embodiments of the 5 method, the template nucleic acid comprises, from 5' to 3': (a) the nucleic acid sequence encoding the CAR or the exogenous TCR; and (b) the nucleic acid sequence encoding the shRNAmiR. In other such embodiments of the method, the template nucleic acid comprises, from 5' to 3': (a) the nucleic acid sequence encoding the shRNAmiR; and (b) the nucleic acid sequence encoding the CAR or the exogenous TCR. In certain such embodiments of the method, 10 the nucleic acid sequence encoding the CAR or the exogenous TCR and the nucleic acid sequence encoding the shRNAmiR are separated by a 2A or IRES sequence. In some such embodiments of the method, the nucleic acid sequence encoding the shRNAmiR is in the same orientation as the nucleic acid sequence encoding the CAR or the exogenous TCR. In other such embodiments of the method, the nucleic acid sequence encoding the shRNAmiR is in a reverse 15 orientation as the nucleic acid sequence encoding the CAR or the exogenous TCR. In some such embodiments of the method, an intron sequence is positioned within the nucleic acid sequence encoding the CAR or the exogenous TCR, wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence. In certain such embodiments of the method, the template nucleic acid comprises a promoter, wherein the promoter is operably linked 20 to the nucleic acid sequence encoding the CAR or the exogenous TCR and to the nucleic acid sequence encoding the shRNAmiR. In some such embodiments of the method, the template nucleic acid comprises a termination signal. In some embodiments of the method, the template nucleic acid comprises a nucleic acid sequence encoding an HLA-E fusion protein, wherein the HLA-E fusion protein is expressed by 25 the immune cell. In some such embodiments of the method, the shRNAmiR and the nucleic acid sequence encoding the HLA-E fusion protein are operably linked to a same promoter in the immune cell following introduction of the template nucleic acid at the cleavage site. In certain such embodiments of the method, the template nucleic acid comprises, from 5' to 3': (a) the nucleic acid sequence encoding the HLA-E fusion protein; and (b) the nucleic acid sequence 30 encoding the shRNAmiR. In other such embodiments of the method, the template nucleic acid comprises, from 5' to 3': (a) the nucleic acid sequence encoding the shRNAmiR; and (b) the 2026205474   09 Jul 2026 nucleic acid sequence encoding the HLA-E fusion protein. In some such embodiments of the method, the nucleic acid sequence encoding the HLA-E fusion protein and the nucleic acid sequence encoding the shRNAmiR are separated by a 2A or IRES sequence. In certain such embodiments, of the method, an intron sequence is positioned within the nucleic acid sequence 5 encoding the HLA-E fusion protein, wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence. In some such embodiments of the method, the template nucleic acid comprises a promoter, wherein the promoter is operably linked to the nucleic acid sequence encoding the HLA-E fusion protein and to the nucleic acid sequence encoding the shRNAmiR. In certain such embodiments of the method, the template nucleic acid comprises a 10 termination signal. In some embodiments of the method, the template nucleic acid comprises a nucleic acid sequence encoding a CAR or an exogenous TCR and a nucleic acid sequence encoding an HLA-E fusion protein, wherein the CAR or the exogenous TCR and the HLA-E fusion protein are expressed by the immune cell. In some embodiments of the method, the nucleic acid sequence 15 encoding the shRNAmiR, the nucleic acid sequence encoding the CAR or the exogenous TCR, and the nucleic acid sequence encoding the HLA-E fusion protein are operably linked to a same promoter following introduction of the template nucleic acid at the cleavage site. In some such embodiments of the method, the template nucleic acid comprises: (a) the nucleic acid sequence encoding the CAR or the exogenous TCR; (b) a 2A or IRES sequence; (c) the nucleic acid 20 sequence encoding the HLA-E fusion protein; and (d) the nucleic acid sequence encoding the shRNAmiR. In certain such embodiments of the method, an intron sequence is positioned within the nucleic acid sequence encoding the HLA-E fusion protein, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence. In other such embodiments of the method, an intron sequence is positioned within the nucleic acid sequence 25 encoding the CAR or the exogenous TCR, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence. In some such embodiments of the method, the template nucleic acid comprises a promoter that is operably linked to the nucleic acid sequence encoding the CAR or the exogenous TCR, the nucleic acid sequence encoding the HLA-E fusion protein, and the nucleic acid sequence encoding the shRNAmiR. In certain such 30 embodiments of the method, the template nucleic acid comprises a termination signal. 2026205474   09 Jul 2026 In some such embodiments of the method, the template nucleic acid comprises, from 5' to 3': (a) a promoter; (b) the nucleic acid sequence encoding the CAR or the exogenous TCR; (c) a 2A or IRES sequence; (d) the nucleic acid sequence encoding the HLA-E fusion protein, wherein an intron sequence is positioned within the nucleic acid sequence encoding the HLA-E fusion 5 protein, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence; and (e) optionally a termination signal; wherein the nucleic acid sequence encoding the CAR or the exogenous TCR, the nucleic acid sequence encoding the HLA-E fusion protein, and the nucleic acid sequence encoding the shRNAmiR are operably linked to the promoter. 10          In some such embodiments of the method, the template nucleic acid comprises, from 5' to 3': (a) a promoter; (b) the nucleic acid sequence encoding the HLA-E fusion protein, wherein an intron sequence is positioned within the nucleic acid sequence encoding the HLA-E fusion protein, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence; (c) a 2A or IRES sequence; (d) the nucleic acid sequence encoding the CAR or 15 the exogenous TCR; and (e) optionally a termination signal; wherein the nucleic acid sequence encoding the CAR or the exogenous TCR, the nucleic acid sequence encoding the HLA-E fusion protein, and the nucleic acid sequence encoding the shRNAmiR are operably linked to the promoter. In some such embodiments of the method, the template nucleic acid comprises, from 5' to 20   3': (a) a promoter; (b) the nucleic acid sequence encoding the CAR or the exogenous TCR, wherein an intron sequence is positioned within the nucleic acid sequence encoding the CAR or the exogenous TCR, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence; (c) a 2A or IRES sequence; (d) the nucleic acid sequence encoding the HLA-E fusion protein; and (e) optionally a termination signal; wherein the nucleic 25 acid sequence encoding the CAR or the exogenous TCR, the nucleic acid sequence encoding the HLA-E fusion protein, and the nucleic acid sequence encoding the shRNAmiR are operably linked to the promoter. In some such embodiments of the method, the template nucleic acid comprises, from 5' to 3': (a) a promoter; (b) the nucleic acid sequence encoding the HLA-E fusion protein; (c) a 2A or 30 IRES sequence; (d) the nucleic acid sequence encoding the CAR or the exogenous TCR, wherein an intron sequence is positioned within the nucleic acid sequence encoding the CAR or the 2026205474   09 Jul 2026 exogenous TCR, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence; and (e) optionally a termination signal; wherein the nucleic acid sequence encoding the CAR or the exogenous TCR, the nucleic acid sequence encoding the HLA-E fusion protein, and the nucleic acid sequence encoding the shRNAmiR are operably 5 linked to the promoter. In some embodiments of the method described above, the intron sequence is a synthetic intron sequence. In certain embodiments of the method, the intron sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 69. In particular embodiments of the method, the intron sequence 10 comprises a nucleic acid sequence of SEQ ID NO: 69. In some embodiments of the method described above, the termination signal is a polyA sequence or a bovine growth hormone (BGH) termination signal. In certain embodiments of the method, the polyA sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 68. In particular 15 embodiments of the method, the polyA sequence comprises a nucleic acid sequence of SEQ ID NO: 68. In certain embodiments of the method, the BGH termination signal comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 71. In particular embodiments of the method, the BGH termination signal comprises a nucleic acid sequence of SEQ ID NO: 71. 20          In certain embodiments of the method described above, the promoter comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 67. In particular embodiments of the method, the promoter comprises a nucleic acid sequence of SEQ ID NO: 67. In certain embodiments of the method described above, the 2A sequence is a P2A / furin 25 site comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 70. In particular embodiments of the method, the 2A sequence is a P2A / furin site comprising a nucleic acid sequence of SEQ ID NO: 70. In certain embodiments of the method described above, the HLA-E fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, 30 or more, sequence identity to SEQ ID NO: 66. In particular embodiments of the method, the HLA-E fusion protein comprises an amino acid sequence of SEQ ID NO: 66. 2026205474   09 Jul 2026 In certain embodiments of the method described above, the CAR comprises a signal peptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 73. In particular embodiments of the method, the CAR comprises a signal peptide comprising an amino acid sequence of SEQ ID NO: 5    73. In particular embodiments of the method, the template nucleic acid comprises, from 5' to 3': (a) a promoter comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 67; (b) the nucleic acid sequence encoding the CAR, wherein the CAR comprises a signal peptide comprising an amino acid 10 sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 73; (c) a P2A / furin site comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 70; (d) the nucleic acid sequence encoding the HLA-E fusion protein, wherein the HLA-E fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at 15 least 95%, or more, sequence identity to SEQ ID NO: 66, and wherein an intron sequence is positioned within the nucleic acid sequence encoding the HLA-E fusion protein, wherein the intron sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 69, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence; and (e) optionally a 20 termination signal comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 68; wherein the nucleic acid sequence encoding the CAR, the nucleic acid sequence encoding the HLA-E fusion protein, and the nucleic acid sequence encoding the shRNAmiR are operably linked to the promoter. In particular embodiments of the method, the template nucleic acid comprises, from 5' to 25   3': (a) a promoter comprising a nucleic acid sequence of SEQ ID NO: 67; (b) the nucleic acid sequence encoding the CAR, wherein the CAR comprises a signal peptide comprising an amino acid sequence of SEQ ID NO: 73; (c) a P2A / furin site comprising a nucleic acid sequence of SEQ ID NO: 70; (d) the nucleic acid sequence encoding the HLA-E fusion protein, wherein the HLA-E fusion protein comprises an amino acid sequence of SEQ ID NO: 66, and wherein an 30 intron sequence is positioned within the nucleic acid sequence encoding the HLA-E fusion protein, wherein the intron sequence comprises a nucleic acid sequence of SEQ ID NO: 69, and 2026205474   09 Jul 2026 wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence; and (e) optionally a termination signal comprising a nucleic acid sequence of SEQ ID NO: 68; wherein the nucleic acid sequence encoding the CAR, the nucleic acid sequence encoding the HLA-E fusion protein, and the nucleic acid sequence encoding the shRNAmiR are 5 operably linked to the promoter. In particular embodiments of the method, the template nucleic acid comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 74, wherein the template nucleic acid is inserted in the genome within a TCR alpha constant region gene. In particular embodiments of the method, the template nucleic 10 acid comprises a nucleic acid sequence of SEQ ID NO: 74, wherein the template nucleic acid is inserted in the genome within a TCR alpha constant region gene. In some embodiments of the method described above, the template nucleic acid comprises two or more nucleic acids encoding shRNAmiRs. In certain embodiments of the method, the two or more nucleic acids can encode the same shRNAmiR. In some embodiments 15 of the method, the two or more nucleic acids can encode different shRNAmiRs that reduce the expression of the same target protein. In other embodiments of the method, the two or more nucleic acids encode different shRNAmiRs that reduce the expression of different target proteins. In certain embodiments of the method, the template nucleic acid can comprise two or more nucleic acids encoding different shRNAmiRs described herein. In particular embodiments 20 of the method, the template nucleic acid can comprise a nucleic acid sequence encoding a shRNAmiR that reduces the expression of B2M, and a nucleic acid sequence encoding a shRNAmiR that reduces the expression of CD52. In some embodiments of the method, the nucleic acid sequence encoding the shRNAmiR and the nucleic acid sequence encoding the CAR or the exogenous TCR are operably linked to 25 different promoters in the immune cell following introduction of the template nucleic acid at the cleavage site. In some such embodiments of the method, the template nucleic acid comprises, from 5' to 3': (a) a first promoter; (b) the nucleic acid sequence encoding the CAR or exogenous TCR which is operably linked to the first promoter; (c) a second promoter; and (d) the nucleic acid sequence encoding the shRNAmiR which is operably linked to the second promoter. In 30 other such embodiments of the method, the template nucleic acid comprises, from 5' to 3': (a) a first promoter; (b) the nucleic acid sequence encoding the shRNAmiR which is operably linked 2026205474   09 Jul 2026 to the first promoter; (c) a second promoter; and (d) the nucleic acid sequence encoding the CAR or exogenous TCR which is operably linked to the second promoter. In certain such embodiments of the method, the nucleic acid sequence encoding the shRNAmiR is in the same orientation as the nucleic acid sequence encoding the CAR or exogenous TCR. In other such 5 embodiments of the method, the nucleic acid sequence encoding the shRNAmiR is in a reverse orientation as the nucleic acid sequence encoding the CAR or exogenous TCR. In particular such embodiments of the method, the first promoter and the second promoter are identical. In other such embodiments of the method, the first promoter and the second promoter are different. In some such embodiments of the method, the template nucleic acid comprises one or more 10 termination signals. In some embodiments of the method, the nucleic acid sequence encoding the shRNAmiR and the nucleic acid sequence encoding the HLA-E fusion protein are operably linked to different promoters in the immune cell following introduction of the template nucleic acid at the cleavage site. In certain such embodiments of the method, the template nucleic acid comprises, 15 from 5' to 3': (a) a first promoter; (b) the nucleic acid sequence encoding the HLA-E fusion protein which is operably linked to the first promoter; (c) a second promoter; and (d) the nucleic acid sequence encoding the shRNAmiR which is operably linked to the second promoter. In some such embodiments of the method, the template nucleic acid comprises, from 5' to 3': (a) a first promoter; (b) the nucleic acid sequence encoding the shRNAmiR which is operably linked 20 to the first promoter; (c) a second promoter; and (d) the nucleic acid sequence encoding the HLA-E fusion protein which is operably linked to the second promoter. In certain such embodiments of the method, the first promoter and the second promoter are identical. In other such embodiments of the method, the first promoter and the second promoter are different. In some such embodiments of the method, the template nucleic acid comprises one or more 25 termination signals. In some embodiments of the method, the template nucleic acid comprises: (a) the nucleic acid sequence encoding the CAR or the exogenous TCR; (b) the nucleic acid sequence encoding the HLA-E fusion protein; and (c) the nucleic acid sequence encoding the shRNAmiR; wherein the nucleic acid sequence encoding the CAR or the exogenous TCR is operably linked to a first 30 promoter, and wherein the nucleic acid sequence encoding the HLA-E fusion protein and the nucleic acid sequence encoding the shRNAmiR are operably linked to a second promoter. In 2026205474   09 Jul 2026 some embodiments of the method, an intron sequence is positioned within the nucleic acid sequence encoding the HLA-E fusion protein, wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence. In some embodiments of the method, the template nucleic acid comprises: (a) the nucleic acid sequence encoding the CAR or the 5 exogenous TCR; (b) the nucleic acid sequence encoding the HLA-E fusion protein; and (c) the nucleic acid sequence encoding the shRNAmiR; wherein the nucleic acid sequence encoding the CAR or the exogenous TCR and the nucleic acid sequence encoding the shRNAmiR are operably linked to a first promoter, and wherein the nucleic acid sequence encoding the HLA-E fusion protein is operably linked to a second promoter. In some embodiments of the method, an 10 intron sequence is positioned within the nucleic acid sequence encoding the CAR or the exogenous TCR, wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence. In some such embodiments of the method, the template nucleic acid comprises a first termination signal capable of terminating transcription of the CAR or the exogenous TCR, and a 15 second termination signal capable of terminating transcription of the HLA-E fusion protein. In some such embodiments of the method, the template nucleic acid comprises a first termination signal capable of terminating transcription of the HLA-E fusion protein, and a second termination signal capable of terminating transcription of the CAR or the exogenous TCR. In some such embodiments of the method, the template nucleic acid comprises, from 5' to 20   3': (a) a first promoter; (b) the nucleic acid sequence encoding the CAR or the exogenous TCR; (c) optionally a first termination signal; (d) a second promoter; (e) the nucleic acid sequence encoding the HLA-E fusion protein, wherein an intron sequence is positioned within the nucleic acid sequence encoding the HLA-E fusion protein, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence; and (f) optionally a second 25 termination signal; wherein the nucleic acid sequence encoding the CAR or the exogenous TCR is operably linked to the first promoter, and wherein the nucleic acid sequence encoding the HLA-E fusion protein and the nucleic acid sequence encoding the shRNAmiR are operably linked to the second promoter. In some such embodiments of the method, the template nucleic acid comprises, from 5' to 30   3': (a) a first promoter; (b) the nucleic acid sequence encoding the HLA-E fusion protein, wherein an intron sequence is positioned within the nucleic acid sequence encoding the HLA-E 2026205474   09 Jul 2026 fusion protein, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence; (c) optionally a first termination signal; (d) a second promoter; (e) the nucleic acid sequence encoding the CAR or the exogenous TCR; and (f) optionally a second termination signal; wherein the nucleic acid sequence encoding the HLA-E fusion protein and 5 the nucleic acid sequence encoding the shRNAmiR are operably linked to the first promoter, and wherein the nucleic acid sequence encoding the CAR or the exogenous TCR is operably linked to the second promoter. In some such embodiments of the method, the template nucleic acid comprises, from 5' to 3': (a) a first promoter; (b) the nucleic acid sequence encoding the CAR or the exogenous TCR, 10 wherein an intron sequence is positioned within the nucleic acid sequence encoding the CAR or the exogenous TCR, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence; (c) optionally a first termination signal; (d) a second promoter; (e) the nucleic acid sequence encoding the HLA-E fusion protein; and (f) optionally a second termination signal; wherein the nucleic acid sequence encoding the CAR or the 15 exogenous TCR and the nucleic acid sequence encoding the shRNAmiR are operably linked to the first promoter, and wherein the nucleic acid sequence encoding the HLA-E fusion protein is operably linked to the second promoter. In some such embodiments of the method, the template nucleic acid comprises, from 5' to 3': (a) a first promoter; (b) the nucleic acid sequence encoding the HLA-E fusion protein; (c) 20 optionally a first termination signal; (d) a second promoter; (e) the nucleic acid sequence encoding the CAR or the exogenous TCR, wherein an intron sequence is positioned within the nucleic acid sequence encoding the CAR or the exogenous TCR, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence; and (f) optionally a second termination signal; wherein the nucleic acid sequence encoding the HLA-E fusion protein 25 is operably linked to the first promoter, and wherein the nucleic acid sequence encoding the CAR or the exogenous TCR and the nucleic acid sequence encoding the shRNAmiR are operably linked to the second promoter. In some embodiments of the method described above, the intron sequence is a synthetic intron sequence. In certain embodiments of the method, the intron sequence comprises a nucleic 30 acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence 2026205474   09 Jul 2026 identity to SEQ ID NO: 69. In particular embodiments of the method, the intron sequence comprises a nucleic acid sequence of SEQ ID NO: 69. In some embodiments of the method described above, the one or more termination signals are a polyA sequence or a BGH termination signal. 5          In some embodiments of the method described above, the first termination signal is identical to the second termination signal. In other embodiments of the method, the first termination signal is different from the second termination signal. In certain embodiments of the method, the first termination signal is a polyA sequence and the second termination signal is a BGH termination signal. In certain embodiments of the method, the first termination signal is a 10 BGH termination signal and the second termination signal is a polyA sequence. In certain embodiments of the method described above, the polyA sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 68. In particular embodiments of the method, the polyA sequence comprises a nucleic acid sequence of SEQ ID NO: 68. In certain embodiments of the 15 method, the BGH termination signal comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 71. In particular embodiments of the method, the BGH termination signal comprises a nucleic acid sequence of SEQ ID NO: 71. In some embodiments of the method described above, the first promoter and the second 20 promoter are identical. In other embodiments of the method, the first promoter is different from the second promoter. In certain embodiments of the method, the first promoter is a JeT promoter, and the second promoter is an EF1 alpha core promoter. In certain embodiments of the method, the first promoter is an EF1 alpha core promoter, and the second promoter is a JeT promoter. 25          In certain embodiments of the method described above, the JeT promoter comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 67. In particular embodiments of the method, the JeT promoter comprises a nucleic acid sequence of SEQ ID NO: 67. In certain embodiments of the method, the EF1 alpha core promoter comprises a nucleic acid sequence having at least 80%, at 30 least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 72. In 2026205474   09 Jul 2026 particular embodiments, the EF1 alpha core promoter comprises a nucleic acid sequence of SEQ ID NO: 72. In some embodiments of the method described above, the HLA-E fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, 5 or more, sequence identity to SEQ ID NO: 66. In particular embodiments of the method, the HLA-E fusion protein comprises an amino acid sequence of SEQ ID NO: 66. In some embodiments of the method described above, the CAR comprises a signal peptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 73. In particular embodiments of the 10 method, the CAR comprises a signal peptide comprising an amino acid sequence of SEQ ID NO: 73. In particular embodiments of the method, the template nucleic acid comprises, from 5' to 3': (a) a first promoter comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 67; (b) the nucleic acid 15 sequence encoding the CAR, wherein the CAR comprises a signal peptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 73; (c) optionally a first termination signal comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 70; (d) a second promoter comprising a nucleic acid sequence having at 20 least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 72; (e) the nucleic acid sequence encoding the HLA-E fusion protein, wherein the HLA-E fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 66, and wherein an intron sequence is positioned within the nucleic acid sequence encoding the HLA-E fusion protein, wherein the 25 intron sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 69, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence; and (f) optionally a second termination signal comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 71; wherein the nucleic 30 acid sequence encoding the CAR is operably linked to the first promoter, and wherein the nucleic 2026205474   09 Jul 2026 acid sequence encoding the HLA-E fusion protein and the nucleic acid sequence encoding the shRNAmiR are operably linked to the second promoter. In particular embodiments of the method, the template nucleic acid comprises, from 5' to 3': (a) a first promoter comprising a nucleic acid sequence of SEQ ID NO: 67; (b) the nucleic 5 acid sequence encoding the CAR, wherein the CAR comprises a signal peptide comprising an amino acid sequence of SEQ ID NO: 73; (c) optionally a first termination signal comprising a nucleic acid sequence of SEQ ID NO: 70; (d) a second promoter comprising a nucleic acid sequence of SEQ ID NO: 72; (e) the nucleic acid sequence encoding the HLA-E fusion protein, wherein the HLA-E fusion protein comprises an amino acid sequence of SEQ ID NO: 66, and 10 wherein an intron sequence is positioned within the nucleic acid sequence encoding the HLA-E fusion protein, wherein the intron sequence comprises a nucleic acid sequence of SEQ ID NO: 69, and wherein the nucleic acid sequence encoding the shRNAmiR is positioned within the intron sequence; and (f) optionally a second termination signal comprising a nucleic acid sequence of SEQ ID NO: 71; wherein the nucleic acid sequence encoding the CAR is operably 15 linked to the first promoter, and wherein the nucleic acid sequence encoding the HLA-E fusion protein and the nucleic acid sequence encoding the shRNAmiR are operably linked to the second promoter. In particular embodiments of the method, the template nucleic acid comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence 20 identity to SEQ ID NO: 75, wherein the cassette is inserted in the genome within a TCR alpha constant region gene. In particular embodiments of the method, the template nucleic acid comprises a nucleic acid sequence of SEQ ID NO: 75, wherein the cassette is inserted in the genome within a TCR alpha constant region gene. In some embodiments of the method described above, the template nucleic acid 25 comprises two or more nucleic acids encoding shRNAmiRs. In certain embodiments of the method, the two or more nucleic acids can encode the same shRNAmiR. In some embodiments of the method, the two or more nucleic acids can encode different shRNAmiRs that reduce the expression of the same target protein. In other embodiments of the method, the two or more nucleic acids encode different shRNAmiRs that reduce the expression of different target 30 proteins. In certain embodiments of the method, the template nucleic acid can comprise two or more nucleic acids encoding different shRNAmiRs described herein. In particular embodiments 2026205474   09 Jul 2026 of the method, the template nucleic acid can comprise a nucleic acid sequence encoding a shRNAmiR that reduces the expression of B2M, and a nucleic acid sequence encoding a shRNAmiR that reduces the expression of CD52. In some embodiments of the method, the shRNAmiR comprises, from 5' to 3': (a) a 5' 5 miR scaffold domain; (b) a 5' miR basal stem domain; (c) a passenger strand; (d) a miR loop domain; (e) a guide strand; (f) a 3' miR basal stem domain; and (g) a 3' miR scaffold domain. In some embodiments of the method, the miR loop domain is a miR-30a loop domain, a miR-15 loop domain, a miR-16 loop domain, a miR-155 loop domain, a miR-22 loop domain, a miR-103 loop domain, or a miR-107 loop domain. In particular embodiments of the method, the 10 miR loop domain is a miR-30a loop domain. In certain embodiments of the method, the miR-30a loop domain comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 3. In particular embodiments of the method, the miR-30a loop domain comprises a nucleic acid sequence of SEQ ID NO: 3. 15         In some embodiments of the method, the shRNAmiR comprises a microRNA-E (miR-E) scaffold, a miR-30 (e.g., miR-30a) scaffold, a miR-15 scaffold, a miR-16 scaffold, a miR-155 scaffold, a miR-22 scaffold, a miR-103 scaffold, or a miR-107 scaffold. In certain embodiments of the method, the shRNAmiR comprises a miR-E scaffold. In some embodiments of the method, the shRNAmiR comprises a structure wherein: (a) 20 the 5' miR scaffold domain comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 1; (b) the 5' miR basal stem domain comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 2; (c) the 3' miR basal stem domain comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, 25 or more, sequence identity to SEQ ID NO: 4; and / or (d) the 3' miR scaffold domain comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 5. In certain embodiments of the method, the shRNAmiR comprises a structure wherein: (a) the 5' miR scaffold domain comprises a nucleic acid sequence of SEQ ID NO: 1; (b) the 5' miR 30 basal stem domain comprises a nucleic acid sequence of SEQ ID NO: 2; (c) the 3' miR basal 2026205474   09 Jul 2026 stem domain comprises a nucleic acid sequence of SEQ ID NO: 4; and (d) the 3' miR scaffold domain comprises a nucleic acid sequence of SEQ ID NO: 5. In some embodiments of the method, expression of the target protein is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 5   65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell. In some embodiments of the method, the target protein is beta-2 microglobulin, CS1, transforming growth factor-beta receptor 2 (TGFBR2), Cbl proto-oncogene B (CBL-B), CD52, a TCR alpha gene, a TCR alpha constant region gene, CD7, glucocorticoid receptor (GR), 10 deoxycytidine kinase (DCK), nuclear receptor subfamily 2 group F member 6 (NR2F6), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), or C-C chemokine receptor type 5 (CCR5). In some embodiments of the method, the target protein is beta-2 microglobulin. In some such embodiments of the method, cell surface expression of beta-2 microglobulin is reduced by 15 at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell. In further such embodiments of the method, expression of MHC class I molecules is reduced on the cell surface by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, 20 about 85%, about 90%, about 95%, or up to about 99% compared to a control cell. In some such embodiments of the method, the immune cell has reduced allogenicity compared to a control cell. In some such embodiments of the method, the shRNAmiR has a structure wherein: (a) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 17 and the guide strand 25 comprises a nucleic acid sequence of SEQ ID NO: 18; (b) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 7 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 8; (c) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 9 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 10; (d) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 11 and the guide strand comprises a 30 nucleic acid sequence of SEQ ID NO: 12; (e) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 13 and the guide strand comprises a nucleic acid sequence of SEQ ID 2026205474   09 Jul 2026 NO: 14; or (f) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 15 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 16. In certain such embodiments of the method, the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 17 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 18. In particular such 5 embodiments of the method, the nucleic acid sequence encoding the shRNAmiR comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 46. In further such embodiments of the method, the nucleic acid sequence encoding the shRNAmiR comprises the sequence of SEQ ID NO: 46. In some embodiments of the method, the target protein is CS1. In some such 10 embodiments of the method, cell surface expression of CS1 is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell. In some such embodiments of the method, the immune cell expresses a CAR having specificity for CS1. In further such embodiments of the method, the immune cell is less 15 susceptible to fratricide by a immune cell expressing a CAR having specificity for CS1 compared to a control cell. In some such embodiments of the method, the shRNAmiR has a structure wherein: (a) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 21 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 22; (b) the passenger strand comprises a 20 nucleic acid sequence of SEQ ID NO: 23 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 24; or (c) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 25 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 26. In certain such embodiments of the method, the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 25 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 26. In 25 particular such embodiments of the method, the shRNAmiR comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 50. In further such embodiments of the method, the shRNAmiR comprises the sequence of SEQ ID NO: 50. In some embodiments of the method, the target protein is TGFBR2. In some such 30 embodiments of the method, the cell surface expression of TGFBR2 is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 2026205474   09 Jul 2026 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell. In further such embodiments of the method, the immune cell is less susceptible to immunosuppression by transforming growth factor B1 (TGFB1) compared to a control cell. In some such embodiments of the method, the shRNAmiR has a structure wherein: (a) 5 the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 27 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 28; (b) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 29 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 30; (c) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 31 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 32; (d) the passenger 10 strand comprises a nucleic acid sequence of SEQ ID NO: 33 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 34; or (e) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 35 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 36. In certain such embodiments of the method, the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 31 and the guide strand comprises a nucleic acid sequence of SEQ 15 ID NO: 32. In particular such embodiments of the method, the nucleic acid sequence encoding the shRNAmiR comprises a sequence having at least 80%, at least 95%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 53. In further such embodiments of the method, the nucleic acid sequence encoding the shRNAmiR comprises the sequence of SEQ ID NO: 53. In some embodiments of the method, the target protein is CBL-B. In some such 20 embodiments of the method, cell surface expression of CBL-B is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell. In further such embodiments of the method, the immune cell is less susceptible to suppression of T cell receptor (TCR) signaling by degradation of downstream signaling proteins 25 compared to a control cell. In some embodiments of the method, the target protein is CD52. In some such embodiments of the method, cell surface expression of CD52 is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a 30 control cell. In further such embodiments of the method, the immune cell is less susceptible to CD52 antibody-induced cell death. 2026205474   09 Jul 2026 In some such embodiments of the method, the shRNAmiR has a structure wherein: (a) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 37 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 38; or (b) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 39 and the guide strand comprises a nucleic acid sequence 5 of SEQ ID NO: 40. In certain such embodiments of the method, the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 37 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 38. In particular such embodiments of the method, the nucleic acid sequence encoding the shRNAmiR comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 56. In further such embodiments of the 10 method, the nucleic acid sequence encoding the shRNAmiR comprises the sequence of SEQ ID NO: 56. In some embodiments of the method, the target protein is DCK. In some such embodiments, cell surface expression of DCK is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, 15 about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell. In further such embodiments of the method, the immune cell is less susceptible to effects of purine nucleoside analogs (e.g., fludarabine) on cell proliferation. In some such embodiments of the method, the shRNAmiR has a structure wherein: (a) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 76 and the guide strand 20 comprises a nucleic acid sequence of SEQ ID NO: 77; (b) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 78 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 79; (c) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 80 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 81; (d) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 82 and the guide strand comprises a 25 nucleic acid sequence of SEQ ID NO: 83; or (e) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 84 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 85. In particular such embodiments of the method, the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 76 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 77. In particular such embodiments of the method, the nucleic acid sequence encoding 30 the shRNAmiR comprises a sequence having at least 80%, at least 85%, at least 90%, at least 2026205474   09 Jul 2026 95%, or more, sequence identity to SEQ ID NO: 86. In further such embodiments of the method, the nucleic acid sequence encoding the shRNAmiR comprises the sequence of SEQ ID NO: 86. In some embodiments of the method, the target protein is GR. In some such embodiments of the method, cell surface expression of GR is reduced by at least about 10%, 5 about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell. In further such embodiments, the immune cell is less susceptible to effects of glucocorticoids (e.g., dexamethasone), such as reduced proliferation. In some such embodiments of the method, the shRNAmiR has a structure wherein: (a) 10 the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 91 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 92; (b) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 93 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 94; (c) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 95 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 96; (d) the passenger 15 strand comprises a nucleic acid sequence of SEQ ID NO: 97 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 98; (e) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 99 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 100; (f) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 101 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 102; (g) the passenger strand 20 comprises a nucleic acid sequence of SEQ ID NO: 103 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 104; (h) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 105 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 106; or (i) the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 107 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 108. In particular such embodiments 25 of the method, the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 95 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 96. In particular such embodiments of the method, the nucleic acid sequence encoding the shRNAmiR comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to SEQ ID NO: 111. In further such embodiments of the method, the nucleic acid 30 sequence encoding the shRNAmiR comprises the sequence of SEQ ID NO: 111. 2026205474   09 Jul 2026 In another aspect, the invention provides an immune cell made by any of the methods described herein. In some embodiments, the target protein is beta-2 microglobulin and the immune cell made by the method has reduced cell-surface expression of beta-2 microglobulin and / or MHC class I proteins. In some embodiments, the target protein is CS1 and the immune 5 cell made by the method has reduced cell-surface expression of CS1. In some embodiments, the target protein is TGFRB2, and the immune cell made by the method has reduced expression of TGFBR2. In some embodiments, the target protein is CBL-B, and the immune cell made by the method has reduced expression of CBL-B. In some embodiments, the target protein is CD52, and the immune cell made by the method has reduced cell-surface expression of CD52. In some 10 embodiments, the target protein is DCK, and the immune cell made by the method has reduced expression of DCK. In some embodiments, the target protein is GR, and the immune cell made by the method has reduced expression of GR. In another aspect, the invention provides a population of cells comprising a plurality of the genetically-modified immune cells, or a plurality of the immune cells, described herein. In 15 some embodiments, at least about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to 100% of cells in the population are the genetically-modified immune cells described herein, or the immune cells described herein. In another aspect, the invention provides a pharmaceutical composition comprising a 20 pharmaceutically-acceptable carrier and a plurality of genetically-modified immune cells described herein, or a plurality of the immune cells described herein. In some embodiments, the pharmaceutical composition comprises a population of cells described herein. In another aspect, the invention provides a method of immunotherapy for treating a disease in a subject in need thereof, the method comprising administering to the subject an 25 effective amount of a pharmaceutical composition described herein. In some embodiments, the method is an immunotherapy for the treatment of a cancer in a subject in need thereof, wherein the genetically-modified immune cell, or immune cell, is a genetically-modified human T cell, or a cell derived therefrom, or a genetically-modified NK cell, or a cell derived therefrom, and wherein the genetically-modified immune cell, or immune cell, comprises a CAR or exogenous 30 TCR, wherein the CAR or the exogenous TCR comprises an extracellular ligand-binding domain having specificity for a tumor-specific antigen. In some embodiments of the method, the 2026205474   09 Jul 2026 genetically-modified immune cell or the immune cell comprises an inactivated TCR alpha gene or an inactivated TCR alpha constant region gene. In further embodiments of the method, the genetically-modified immune cell, or the immune cell, has no detectable cell-surface expression of an endogenous TCR (e.g., an alpha / beta TCR). In some embodiments of the method, the 5 cancer is selected from the group consisting of a cancer of carcinoma, lymphoma, sarcoma, blastomas, and leukemia. In certain embodiments of the method, the cancer is selected from the group consisting of a cancer of B cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin lymphoma. In particular 10 embodiments of the method, the cancer of B cell origin is selected from the group consisting of B-lineage acute lymphoblastic leukemia, B cell chronic lymphocytic leukemia, B cell nonHodgkin lymphoma, and multiple myeloma. In particular embodiments of the method, the subject can be a mammal, such as a human. In another aspect, the invention provides a method for treating a disease, such as cancer, 15 in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a population of any genetically-modified immune cells described herein (e.g., a genetically-modified human T cell or NK cell expressing a CAR or exogenous TCR) that comprise in their genome a nucleic acid sequence encoding a shRNAmiR that reduces the expression of endogenous deoxycytidine kinase (DCK), wherein the population 20 of genetically-modified immune cells is administered to the subject before, during, or after administration of a purine nucleoside. Reduction of DCK expression by the shRNAmiR reduces the effect of the purine nucleoside on proliferation or in vivo persistence of the genetically-modified immune cells. In particular embodiments of the method, the population of genetically-modified immune 25 cells and the purine nucleoside are administered such that the genetically-modified immune cells are present in the subject (i.e., have not been eliminated by the host) when the purine nucleoside is administered, or while the purine nucleoside is present in the subject at an effective level. In some embodiments of the method, the purine nucleoside is fludarabine. In some such embodiments of the method, fludarabine is administered alone or in combination with another 30 chemotherapeutic compound as part of a lymphodepletion regimen for immunotherapy. 2026205474   09 Jul 2026 In particular embodiments of the method, the genetically-modified immune cells are genetically-modified human T cells or genetically-modified NK cells expressing a CAR or exogenous TCR having specificity for an antigen on the targeted cancer cells. In another aspect, the invention provides a method for treating a disease, such as cancer, 5 in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a population of any genetically-modified immune cells described herein (e.g., a genetically-modified human T cell or NK cell expressing a CAR or exogenous TCR) that comprise in their genome a nucleic acid sequence encoding a shRNAmiR that reduces the expression of endogenous glucocorticoid receptor (GR), wherein the population 10 of genetically-modified immune cells is administered to the subject before, during, or after administration of a corticosteroid. Reduction of GR expression by the shRNAmiR reduces the effect of the corticosteroid on proliferation or in vivo persistence of the genetically-modified immune cells. In particular embodiments of the method, the population of genetically-modified immune 15 cells and the corticosteroid are administered such that the genetically-modified immune cells are present in the subject (i.e., have not been eliminated by the host) when the corticosteroid is administered, or while the corticosteroid is present in the subject at an effective level. In some embodiments of the method, the corticosteroid is dexamethasone or methylprednisolone. In some such embodiments of the method, the corticosteroid is administered alone or in 20 combination with another compound as part of a treatment for reducing cytokine release syndrome during immunotherapy. In particular embodiments of the method, the genetically-modified immune cells are genetically-modified human T cells or genetically-modified NK cells expressing a CAR or exogenous TCR having specificity for an antigen on the targeted cancer cells. 25          In another aspect, the invention provides a genetically-modified immune cell or a population thereof, as described herein, or an immune cell or a population thereof, as described herein, for use as a medicament. The invention further provides the use of a genetically-modified immune cell or a population thereof, as described herein, or an immune cell or a population thereof, as described herein, in the manufacture of a medicament for treating a disease 30 in a subject in need thereof. In one such aspect, the medicament is useful in the treatment of a cancer. 2026205474   09 Jul 2026 In another aspect, the invention provides a genetically-modified cell or population thereof, as described herein, or an immune cell or a population thereof, as described herein, for use in treatment of a disease, and preferably in the treatment of a cancer. 5                     BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows beta-2 microglobulin expression or HLA-A, B, and C expression (i.e., MHC class I molecule expression) on T cells transduced with an AAV comprising construct 7056 which expresses a single copy of the shRNA472 in a 3' to 5' head-to-tail configuration with the CAR. Figure 1A shows the B2M surface levels in CD3- / CAR+ cells compared to 10 meganuclease-edited cells expressing no shRNA from a control culture. Figure 1B shows B2M levels on CD3- / CAR+ versus CD3+ / CAR- populations in the same culture. Figure 1C shows HLA-ABC (i.e., MHC class I molecule) surface levels in CD3- / CAR+ cells compared to meganuclease-edited cells expressing no shRNA from a control culture. Figure 1D shows HLA-ABC levels on CD3- / CAR+ versus CD3+ / CAR- populations in the same culture. 15         Figure 2 shows the frequency of CD3- / CAR+ cells, and the knockdown of B2M, in cultures produced with AAV 7056. Figure 2A shows the frequency of CD3- / CAR+ cells at day 4. Figure 2B shows knockdown of B2M at day 4. Figure 2C shows the frequency of CD3- / CAR+ cells at day 7. Figure 2D shows knockdown of B2M at day 7. Figure 2E shows the frequency of CD3- / CAR+ cells at day 10. Figure 2F shows knockdown of B2M at day 10. 20         Figure 3 shows the frequency of CD3- / CAR+ cells, and the knockdown of B2M, in cultures produced with AAV 7206, AAV 7056, or AAV 7282 three days post-transduction. Figure 3A shows the frequency of CD3- / CAR+ cells in 7206-tranduced cells. Figure 3B shows knockdown of B2M in 7206-tranduced cells. Figure 3C shows the frequency of CD3- / CAR+ cells in 7282-tranduced cells. Figure 3D shows knockdown of B2M in 7282-tranduced cells. 25 Figure 3E shows the frequency of CD3- / CAR+ cells in 7056-tranduced cells. Figure 3F shows knockdown of B2M in 7056-tranduced cells. Figure 4 shows the frequency of CD3- / CAR+ cells, and the knockdown of B2M, in cultures produced with AAV 7206, AAV 7056, or AAV 7282 seven days post-transduction. Figure 4A shows the frequency of CD3- / CAR+ cells in 7206-tranduced cells. Figure 4B shows 30 knockdown of B2M in 7206-tranduced cells. Figure 4C shows the frequency of CD3- / CAR+ cells in 7282-tranduced cells. Figure 4D shows knockdown of B2M in 7282-tranduced cells. 2026205474   09 Jul 2026 Figure 4E shows the frequency of CD3- / CAR+ cells in 7056-tranduced cells. Figure 4F shows knockdown of B2M in 7056-tranduced cells. Figure 5 shows the frequency of CD3- / CAR+ cells, and the knockdown of B2M, in cultures produced with AAV 7206, AAV 7056, or AAV 7282 eleven days post-transduction. 5 Figure 5A shows the frequency of CD3- / CAR+ cells in 7206-tranduced cells. Figure 5B shows knockdown of B2M in 7206-tranduced cells. Figure 5C shows the frequency of CD3- / CAR+ cells in 7282-tranduced cells. Figure 5D shows knockdown of B2M in 7282-tranduced cells. Figure 5E shows the frequency of CD3- / CAR+ cells in 7056-tranduced cells. Figure 5F shows knockdown of B2M in 7056-tranduced cells. 10         Figure 6 shows the effects of B2M knockout or knockdown on the sensitivity of CAR T cells to cytolysis by alloantigen-specific cytotoxic lymphocytes (CTLs) or NK cells. Figure 6A shows the cytolytic activity of primed alloantigen-specific CTLs against B2M knockout and B2M knockdown CAR T cell populations. Figure 6B shows the cytolytic activity of NK cells against B2M knockout and B2M knockdown CAR T cell populations. 15         Figure 7 shows knockout of B2M using an engineered meganuclease and targeted insertion of a donor template comprising a coding sequence for an HLA-E polypeptide. Figure 7A shows B2M knockout without targeted insertion. Figure 7B shows B2M knockout and targeted insertion of the donor template using AAV7346. Figure 8 shows purification of T cell populations with B2M knockout and cell surface 20 expression of HLA-E encoded by the inserted donor template. Figure 8A shows the cell population that is B2M-negative. Figure 8B shows the purified B2M-negative population. Figure 8C shows the cell population that is HLA-E-positive. Figure 8D shows the purified HLA-E-positive cell population. Figure 9 shows CAR T cell killing by alloantigen-primed CTLs and cell killing by natural 25 killer (NK) cells. Figure 9A shows killing of B2M-positive, B2M-knockout, and B2M-knockout / HLA-E knock in, CAR T cells by alloantigen-primed CTLs at increasing Effector:Target ratios. Figure 9B shows NK cell killing of B2M-positive, B2M-knockout, and B2M-knockout / HLA-E knock in, CAR T cells at increasing Effector:Target ratios. Figure 10 shows in vivo efficacy and stability of shRNAmiR-induced knockdown of 30 B2M in CAR T cells. Figure 10A shows bioluminescence imaging of total flux over time in mice engrafted with NALM-6 cells and treated with vehicle (shown as black line with triangles), 2026205474   09 Jul 2026 mice treated with CD19-directed CAR T cells (shown as dark gray line with circles), or mice treated with CD19-directed CAR T cells with an integrated B2M-targeting shRNAmiR (shown as light gray line with triangles). Figure 10B shows flow cytometry staining for B2M expression on human CD45+ cells 14 days after administration of either CD19-directed CAR T cells with an 5 integrated B2M-targeting shRNAmiR (shown as light gray histogram) or control CD19-directed CAR T cells (shown as dark gray histogram). Figure 11 shows shRNAmiR-induced stable knockdown of CS1 in CAR T cells. Three candidate guide and passenger strand sequences for a CS1 / SLAMF7 shRNAmiR were built into a miR-E scaffold and positioned after the stop codon of a BCMA-specific CAR. Constructs were 10 designated AAV72101-72103 and were used for transduction of donor T cells. Figure 11A shows CAR and CS1 staining seven days after transduction with an AAV encoding a BCMA CAR but no shRNAmiR construct. Figure 11B shows CAR and CS1 staining seven days after transduction with AAV 72101. Figure 11C shows CAR and CS1 staining seven days after transduction with AAV 72102. Figure 11D shows CAR and CS1 staining seven days after 15 transduction with AAV 72103. Figure 12 shows shRNAmiR-induced stable knockdown of TGFRB2 in CAR T cells. Multiple candidate guide and passenger strand sequences for a TGFBR2 shRNAmiR were built into a miR-E scaffold and positioned after the stop codon of a CD19-specific CAR. Constructs were designated AAV 72110-72114 and were used for transduction of donor T cells. Figure 12A 20 shows CAR and TGFBR2 staining after mock transduction. Figure 12B shows CAR and TGFBR2 staining at day 14 post-transduction with AAV 72110. Figure 12C shows CAR and TGFBR2 staining at day 14 post-transduction with AAV 72111. Figure 12D shows CAR and TGFBR2 staining at day 14 post-transduction with AAV 72112. Figure 12E shows CAR and TGFBR2 staining at day 14 post-transduction with AAV 72113. Figure 12F shows CAR and 25 TGFBR2 staining at day 14 post-transduction with AAV 72114. Figure 13 shows flow cytometry detecting knockout of TGFBR2 in T cells using two engineered meganucleases. Figure 13A shows a negative staining control. Figure 13B shows mock-transfected T cells. Figure 13C shows T cells transfected with mRNA encoding the TGF 1-2x.5 meganuclease. Figure 13D shows T cells transfected with mRNA encoding the TGF 1 30   2L.296 meganuclease. 2026205474   09 Jul 2026 Figure 14 shows flow cytometry staining for phosphorylated SMAD 2 / 3 in TGFBR2-positive and negative T cells treated with TGFB1. Figure 15 shows flow cytometry for phosphorylated SMAD 2 / 3 in TGFBR2-positive CAR T cells, CAR T cells expressing an anti-TGFBR2 shRNAmiR to knockdown protein 5 expression, and T cells treated with an engineered meganuclease to knockout TGFBR2 expression. Figure 16 shows flow cytometry for phosphorylated SMAD 2 / 3 in untreated control BCMA CAR T cells (A), BCMA CAR T cells treated with TGF01 (B), BCMA CAR T cells treated with TGF01 having either TGFBR2 knocked out with the TGF 1-2L.296 meganuclease 10   (C) or TGFBR2 knocked down with the 72112 TGFBR2 shRNAmiR (D). Figure 17 shows BCMA CAR T cell numbers over time following co-culture with normal K562 cells, K562 cells transfected to stably express BCMA (KBCMA), or K562 cells stably expressing BCMA and constitutively secreting active TGF01 (KBCMA-TGF). BCMA CAR T cells were modified to knock down TGFBR2 using a shRNAmiR (TGFBRKD) or modified to 15 knock down B2M with a shRNAmiR (CtrlKD). Figure 18 shows target cell numbers over time following co-culture of BCMA CAR T cells with K562 cells transfected to stably express BCMA (KBCMA), or K562 cells stably expressing BCMA and constitutively secreting active TGF01 (KBCMA-TGF). BCMA CAR T cells were modified to knock down TGFBR2 using a shRNAmiR (TGFBRKD) or modified to 20 knock down B2M with a shRNAmiR (CtrlKD). Figure 19 shows BCMA CAR T cell numbers over time following co-culture with normal K562 cells, K562 cells transfected to stably express BCMA, and K562 cells stably expressing BCMA and constitutively secreting active TGF01. BCMA CAR T cells were modified to knock down TGFBR2 using a shRNAmiR (72154), modified to knock down B2M with a shRNAmiR 25   (72155), or modified to knockout TGFBR2 with an engineered meganuclease (dKO). Figure 20 shows the CD4:CD8 ratio of BCMA CAR T cells over time following coculture with K562 cells transfected to stably express BCMA, and K562 cells stably expressing BCMA and constitutively secreting active TGF01. BCMA CAR T cells were modified to knock down TGFBR2 using a shRNAmiR (72154), modified to knock down B2M with a shRNAmiR 30   (72155), or modified to knockout TGFBR2 with an engineered meganuclease (dKO). 2026205474   09 Jul 2026 Figure 21 shows BCMA CAR T cell numbers over time following co-culture with normal U266 cells, or U266 cells constitutively secreting active TGF01. BCMA CAR T cells were modified to knock down TGFBR2 using a shRNAmiR (72154), modified to knock down B2M with a shRNAmiR (72155), or modified to knockout TGFBR2 with an engineered meganuclease 5 (dKO). Figure 22 shows BCMA CAR T cell numbers over time following co-culture with U266 cells, or U266 cells constitutively secreting active TGF01. BCMA CAR T cells were modified to knock down TGFBR2 using a shRNAmiR (72154), modified to knock down B2M with a shRNAmiR (72155), or modified to knockout TGFBR2 with an engineered meganuclease 10 (dKO). Figure 23 shows flow cytometry plots of the number of CD4+ CAR T cells at different time points in co-culture with U266 cells. Figure 23A shows CAR T cells incorporating the 72154 construct. Figure 23B shows CAR T cells incorporating the 72155 construct. Figure 23C shows CAR T cells having an HLA-E fusion protein knocked into the B2M gene. 15          Figure 24 shows flow cytometry plots of live versus dead U266 cells after co-culture with BCMA CAR T cell variants for 16 days. Figure 24A shows co-culture of U266 cells with BCMA CAR T cells modified to knock down TGFBR2 using a shRNAmiR (TGFbRKD). Figure 24B shows co-culture of U266 cells with BCMA CAR T cells modified to knock down B2M using a shRNAmiR (Ctrl KD). Figure 24C shows co-culture of U266 cells with BCMA 20 CAR T cells modified to knockout TGFBR2 with an engineered meganuclease (TGFbR KO). Figure 24D shows co-culture of U266 cells secreting active TGF01 with BCMA CAR T cells modified to knock down TGFBR2 using a shRNAmiR (TGFbRKD). Figure 24E shows coculture of U266 cells secreting active TGF01 with BCMA CAR T cells modified to knock down B2M using a shRNAmiR (Ctrl KD). Figure 24F shows co-culture of U266 cells secreting active 25 TGF01 with BCMA CAR T cells modified to knockout TGFBR2 with an engineered meganuclease (TGFbR KO). Figure 25 shows shRNAmiR-induced stable knockdown of CD52 in CAR T cells. Multiple candidate guide and passenger strand sequences for a CD52 shRNAmiR were built into a miR-E scaffold and positioned after the stop codon of a CD19-specific CAR. Constructs were 30 designated AAV 72123 and AAV 72124 and were used for transduction of donor T cells. Figure 25A shows staining of CD3- / CAR+ T cell populations following AAV transduction. Figure 25B 2026205474   09 Jul 2026 shows knockdown of CD52 in CD3- / CAR+ T cells 10 days after transduction with AAV 72123. Figure 25C shows knockdown of CD52 in CD3- / CAR+ T cells 10 days after transduction with AAV 72124. Figure 26 shows flow cytometry staining for B2M and CD52 in T cells expressing a 5 B2M-targeting shRNAmiR and a CD52-targeting shRNAmiR. Figure 27 shows the percentage of CAR+ T cells recovered following CD52 depletion of CAR T populations expressing a B2M-targeting shRNAmiR and a CD52-targeting shRNAmiR. Figure 28 shows diagrams of constructs 73161, 73162, 73163, and 73164. Figure 28A shows construct 73161 which comprises a JeT promoter, a CD19 CAR gene, P2A / furin site, an 10 HLA-E gene comprising a synthetic intron which comprises a B2M-targeting shRNAmiR, and an SV40 bi-directional polyA sequence. Figure 28B shows construct 73162 which comprises a JeT promoter, a CD19 CAR gene comprising a synthetic intron which comprises a B2M-targeting shRNAmiR, an SV40 bi-directional polyA sequence, a second JeT promoter, an HLA-E gene, and a bovine growth hormone (BGH) termination signal. Figure 28C shows 15 construct 73163 which comprises a JeT promoter, a CD19 CAR gene, an SV40 bi-directional polyA sequence, an EF1 alpha core promoter, an HLA-E gene comprising a synthetic intron which comprises a B2M-targeting shRNAmiR, and a BGH termination signal. Figure 28D shows the 73164 construct which comprises a JeT promoter, a CD19 CAR gene, an SV40 bidirectional polyA sequence, a second JeT promoter, an HLA-E gene comprising a synthetic 20 intron which comprises a B2M-targeting shRNAmiR, and a BGH termination signal. Figure 29 shows a table summarizing the CAR phenotype of T cells in which the identified constructs were introduced by AAV and inserted into the TRAC locus only (7206 and 73161-73164), or cells in which a CAR gene was inserted in the TRAC locus and an HLA-E gene was inserted in the B2M locus (dKO dKI). The table provides the percentage of cells that 25 were CD3- / CAR+, percentage of CD3 knockout cells that had a CAR knock-in, mean fluorescence intensity (MFI) of the expressed CAR, and comparison of the MFI of each CAR when compared to the CAR introduced using the 7206 construct. Figure 30 shows a table summarizing the HLA-ABC and HLA-E phenotypes of T cells in which the identified constructs were introduced by AAV and inserted into the TRAC locus only 30   (7206 and 73161-73164), or cells in which a CAR gene was inserted in the TRAC locus and an HLA-E gene was inserted in the B2M locus (dKO dKI). The table provides the percentage of 2026205474   09 Jul 2026 HLA-ABC expression compared to wild-type in the CD3- / CAR+ population, the percentage of HLA-ABC knockdown with the wild-type gated out, the percentage of cells expressing HLA-E in the CD3- / CAR+ population, the MFI of HLA-E expression in such cells, and the percentage of cells in the CAR- population that were HLA-E+. 5          Figure 31 shows a table summarizing characteristics of CAR T cells in which the identified constructs were introduced by AAV and inserted into the TRAC locus only (7206 and 73161-73164), or cells in which a CAR gene was inserted in the TRAC locus and an HLA-E gene was inserted in the B2M locus (dKO dKI). Figure 32 shows cytolysis of CD19 CAR T variants prepared with T cells from a first 10 donor (HC6366) when co-cultured with alloantigen-primed CTLs from two different donors (K3212 or K2916). Figure 32A shows killing of CAR T cells by K3212 alloantigen-primed CTLs. Figure 32B shows killing of CAR T cells by K2916 alloantigen-primed CTLs. Figure 33 shows natural killer (NK) cell cytolysis of CD19 CAR T variants in co-culture at multiple time points at a 1:1 ratio. Figure 33A shows cytolysis at 24 hours. Figure 33B shows 15 cytolysis at 48 hours. Figure 33C shows cytolysis at 120 hours. Figure 34 shows luminescence measurements demonstrating in vivo efficacy of CD19 CAR T variants in immunodeficient mice engrafted with NALM / 6 leukemia cells. Figure 35 shows survival of immunodeficient mice engrafted with NALM / 6 leukemia cells treated with CD19 CAR T variants. 20         Figure 36 shows changes in the percent knock in of a CD19 CAR sequence, with or without a DCK shRNAmiR, in CD3 knockout T cells following incubation with different concentrations of fludarabine. Figure 37 shows events / uL of CD3- / CAR+ population versus the concentration of fludarabine incubated with CD19 CAR T cell variants that include, or do not include, a DCK 25 shRNAmiR. Figure 38 shows the number of viable cells / mL on day 4 following post-treatment of different CD19 CAR T cell variants that include, or do not include, a DCK shRNAmiR, with different concentrations of fludarabine. Figure 39 shows the number of viable cells / mL on day 8 following post-treatment of 30 different CD19 CAR T cell variants that include, or do not include, a DCK shRNAmiR, with different concentrations of fludarabine. 2026205474   09 Jul 2026 Figure 40 shows the percent cytolysis of CD19-expressing HEK293 cells (targets) cocultured with CD19 CAR T cells (effectors) lacking a DCK shRNAmiR in the presence of different concentrations of fludarabine. Cells were co-cultured at E:T ratios of 2:1, 1:2, and 1:4. Figure 41 shows the percent cytolysis of CD19-expressing HEK293 cells (targets) co- 5 cultured with CD19 CAR T cells (effectors) comprising a DCK shRNAmiR (72138) in the presence of different concentrations of fludarabine. Cells were co-cultured at E:T ratios of 2:1, 1:2, and 1:4. Figure 42 shows the percent cytolysis of CD19-expressing HEK293 cells (targets) cocultured with CD19 CAR T cells (effectors) comprising a DCK shRNAmiR (72136) in the 10 presence of different concentrations of fludarabine. Cells were co-cultured at E:T ratios of 2:1, 1:2, and 1:4. Figure 43 shows changes in the percent knock in of a CD19 CAR sequence, with or without a GR shRNAmiR, in CD3 knockout T cells following incubation with different concentrations of dexamethasone. 15          Figure 44 shows events / uL of CD3- / CAR+ population versus the concentration of dexamethasone incubated with CD19 CAR T cell variants that include, or do not include, a GR shRNAmiR. BRIEF DESCRIPTION OF THE SEQUENCES 20          SEQ ID NO: 1 sets forth the nucleic acid sequence of the 5' miR-E scaffold domain coding sequence. SEQ ID NO: 2 sets forth the nucleic acid sequence of the 5' mir-E basal stem domain coding sequence. SEQ ID NO: 3 sets forth the nucleic acid sequence of the miR-30a loop domain coding 25 sequence. SEQ ID NO: 4 sets forth the nucleic acid sequence of the 3' miR-E basal stem domain coding sequence. SEQ ID NO: 5 sets forth the nucleic acid sequence of the 3' miR-E scaffold domain coding sequence. 30          SEQ ID NO: 6 sets forth the nucleic acid sequence encoding the shRNA 472. 2026205474   09 Jul 2026 SEQ ID NO: 7 sets forth the nucleic acid sequence encoding the passenger strand of the 7282 beta-2 microglobulin (B2M) shRNAmiR. SEQ ID NO: 8 sets forth the nucleic acid sequence encoding the guide strand of the 7282 B2M shRNAmiR. 5          SEQ ID NO: 9 sets forth the nucleic acid sequence encoding the passenger strand of the 7285 B2M shRNAmiR. SEQ ID NO: 10 sets forth the nucleic acid sequence encoding the guide strand of the 7285 B2M shRNAmiR. SEQ ID NO: 11 sets forth the nucleic acid sequence encoding the passenger strand of the 10  7286 B2M shRNAmiR. SEQ ID NO: 12 sets forth the nucleic acid sequence encoding the guide strand of the 7286 B2M shRNAmiR. SEQ ID NO: 13 sets forth the nucleic acid sequence encoding the passenger strand of the 7287 B2M shRNAmiR. 15          SEQ ID NO: 14 sets forth the nucleic acid sequence encoding the guide strand of the 7287 B2M shRNAmiR. SEQ ID NO: 15 sets forth the nucleic acid sequence encoding the passenger strand of the 7288 B2M shRNAmiR. SEQ ID NO: 16 sets forth the nucleic acid sequence encoding the guide strand of the 20  7288 B2M shRNAmiR. SEQ ID NO: 17 sets forth the nucleic acid sequence encoding the passenger strand of the 7289 B2M shRNAmiR. SEQ ID NO: 18 sets forth the nucleic acid sequence encoding the guide strand of the 7289 B2M shRNAmiR. 25          SEQ ID NO: 19 sets forth the nucleic acid sequence encoding the passenger strand of the 7290 B2M shRNAmiR. SEQ ID NO: 20 sets forth the nucleic acid sequence encoding the guide strand of the 7290 B2M shRNAmiR. SEQ ID NO: 21 sets forth the nucleic acid sequence encoding the passenger strand of the 30   72101 CS1 shRNAmiR. 2026205474   09 Jul 2026 SEQ ID NO: 22 sets forth the nucleic acid sequence encoding the guide strand of the 72101 CS1 shRNAmiR. SEQ ID NO: 23 sets for the nucleic acid sequence encoding the passenger strand of the 72102 CS1 shRNAmiR. 5          SEQ ID NO: 24 sets for the nucleic acid sequence encoding the guide strand of the 72102 CS1 shRNAmiR. SEQ ID NO: 25 sets for the nucleic acid sequence encoding the passenger strand of the 72103 CS1 shRNAmiR. SEQ ID NO: 26 sets forth the nucleic acid sequence encoding the guide strand of the 10   72103 CS1 shRNAmiR. SEQ ID NO: 27 sets forth the nucleic acid sequence encoding the passenger strand of the 72110 transforming growth factor beta receptor 2 (TGFBR2) shRNAmiR. SEQ ID NO: 28 sets forth the nucleic acid sequence encoding the guide strand of the 72110 TGFBR2 shRNAmiR. 15          SEQ ID NO: 29 sets forth the nucleic acid sequence encoding the passenger strand of the 72111 TGFBR2 shRNAmiR. SEQ ID NO: 30 sets forth the nucleic acid sequence encoding the guide strand of the 72111 TGFBR2 shRNAmiR. SEQ ID NO: 31 sets forth the nucleic acid sequence encoding the passenger strand of the 20  72112 TGFBR2 shRNAmiR. SEQ ID NO: 32 sets forth the nucleic acid sequence encoding the guide strand of the 72112 TGFBR2 shRNAmiR. SEQ ID NO: 33 sets forth the nucleic acid sequence encoding the passenger strand of the 72113 TGFBR2 shRNAmiR. 25          SEQ ID NO: 34 sets forth the nucleic acid sequence encoding the guide strand of the 72113 TGFBR2 shRNAmiR. SEQ ID NO: 35 sets forth the nucleic acid sequence encoding the passenger strand of the 72114 TGFBR2 shRNAmiR. SEQ ID NO: 36 sets forth the nucleic acid sequence encoding the guide strand of the 30  72114 TGFBR2 shRNAmiR. 2026205474   09 Jul 2026 SEQ ID NO: 37 sets forth the nucleic acid sequence encoding the passenger strand of the 72123 CD52 shRNAmiR. SEQ ID NO: 38 sets forth the nucleic acid sequence encoding the guide strand of the 72123 CD52 shRNAmiR. 5          SEQ ID NO: 39 sets forth the nucleic acid sequence encoding the passenger strand of the 72124 CD52 shRNAmiR. SEQ ID NO: 40 sets forth the nucleic acid sequence encoding the guide strand of the 72124 CD52 shRNAmiR. SEQ ID NO: 41 sets forth the nucleic acid sequence encoding the 7282 B2M shRNAmiR. 10         SEQ ID NO: 42 sets forth the nucleic acid sequence encoding the 7285 B2M shRNAmiR. SEQ ID NO: 43 sets forth the nucleic acid sequence encoding the 7286 B2M shRNAmiR. SEQ ID NO: 44 sets forth the nucleic acid sequence encoding the 7287 B2M shRNAmiR. SEQ ID NO: 45 sets forth the nucleic acid sequence encoding the 7288 B2M shRNAmiR. SEQ ID NO: 46 sets forth the nucleic acid sequence encoding the 7289 B2M shRNAmiR. 15         SEQ ID NO: 47 sets forth the nucleic acid sequence encoding the 7290 B2M shRNAmiR. SEQ ID NO: 48 sets forth the nucleic acid sequence encoding the 72101 CS1 shRNAmiR. SEQ ID NO: 49 sets forth the nucleic acid sequence encoding the 72102 CS1 shRNAmiR. 20          SEQ ID NO: 50 sets forth the nucleic acid sequence encoding the 72103 CS1 shRNAmiR. SEQ ID NO: 51 sets forth the nucleic acid sequence encoding the 72110 TGFBR2 shRNAmiR. SEQ ID NO: 52 sets forth the nucleic acid sequence encoding the 72111 TGFBR2 25 shRNAmiR. SEQ ID NO: 53 sets forth the nucleic acid sequence encoding the 72112 TGFBR2 shRNAmiR. SEQ ID NO: 54 sets forth the nucleic acid sequence encoding the 72113 TGFBR2 shRNAmiR. 30         SEQ ID NO: 55 sets forth the nucleic acid sequence encoding the 72114 TGFBR2 shRNAmiR. 2026205474   09 Jul 2026 SEQ ID NO: 56 sets forth the nucleic acid sequence encoding the 72123 CD52 shRNAmiR. SEQ ID NO: 57 sets forth the nucleic acid sequence encoding the 72124 CD52 shRNAmiR. 5          SEQ ID NO: 58 sets forth the nucleic acid sequence of the TRC 1-2 recognition sequence (sense). SEQ ID NO: 59 sets forth the nucleic acid sequence of the TRC 1-2 recognition sequence (antisense). SEQ ID NO: 60 sets forth the nucleic acid sequence of the B2M 13-14 recognition 10 sequence (sense). SEQ ID NO: 61 sets forth the nucleic acid sequence of the B2M 13-14 recognition sequence (antisense). SEQ ID NO: 62 sets forth the nucleic acid sequence of the TGF 1-2 recognition sequence (sense). 15          SEQ ID NO: 63 sets forth the nucleic acid sequence of the TGF 1-2 recognition sequence (antisense). SEQ ID NO: 64 sets forth the amino acid sequence of the TGF 1-2x.5 meganuclease. SEQ ID NO: 65 sets forth the amino acid sequence of the TGF 1-2L.296 meganuclease. SEQ ID NO: 66 sets forth the amino acid sequence of an HLA class I histocompatibility 20 antigen, alpha chain E (HLA-E) fusion polypeptide. SEQ ID NO: 67 sets forth the nucleic acid sequence of a JeT promoter. SEQ ID NO: 68 sets forth the nucleic acid sequence of a bidirectional SV40 polyA signal. SEQ ID NO: 69 sets forth the nucleic acid sequence of a synthetic intron. 25          SEQ ID NO: 70 sets forth the nucleic acid sequence of a P2A / furin site. SEQ ID NO: 71 sets forth the nucleic acid sequence of a bovine growth hormone termination signal. SEQ ID NO: 72 sets forth the nucleic acid sequence of an EF1 alpha core promoter. SEQ ID NO: 73 sets forth the amino acid sequence of a signal peptide. 30          SEQ ID NO: 74 sets forth the nucleic acid sequence of a cassette comprised by construct 73161. 2026205474   09 Jul 2026 SEQ ID NO: 75 sets forth the nucleic acid sequence of a cassette comprised by construct 73163. SEQ ID NO: 76 sets forth the nucleic acid sequence encoding the passenger strand of the 72136 DCK shRNAmiR. 5          SEQ ID NO: 77 sets forth the nucleic acid sequence encoding the guide strand of the 72136 DCK shRNAmiR. SEQ ID NO: 78 sets forth the nucleic acid sequence encoding the passenger strand of the 72137 DCK shRNAmiR. SEQ ID NO: 79 sets forth the nucleic acid sequence encoding the guide strand of the 10  72137 DCK shRNAmiR. SEQ ID NO: 80 sets forth the nucleic acid sequence encoding the passenger strand of the 72138 DCK shRNAmiR. SEQ ID NO: 81 sets forth the nucleic acid sequence encoding the guide strand of the 72138 DCK shRNAmiR. 15          SEQ ID NO: 82 sets forth the nucleic acid sequence encoding the passenger strand of the 72139 DCK shRNAmiR. SEQ ID NO: 83 sets forth the nucleic acid sequence encoding the guide strand of the 72139 DCK shRNAmiR. SEQ ID NO: 84 sets forth the nucleic acid sequence encoding the passenger strand of the 20  72140 DCK shRNAmiR. SEQ ID NO: 85 sets forth the nucleic acid sequence encoding the guide strand of the 72140 DCK shRNAmiR. SEQ ID NO: 86 sets forth the nucleic acid sequence encoding the 72136 DCK shRNAmiR. 25         SEQ ID NO: 87 sets forth the nucleic acid sequence encoding the 72137 DCK shRNAmiR. SEQ ID NO: 88 sets forth the nucleic acid sequence encoding the 72138 DCK shRNAmiR. SEQ ID NO: 89 sets forth the nucleic acid sequence encoding the 72139 DCK 30 shRNAmiR. 2026205474   09 Jul 2026 SEQ ID NO: 90 sets forth the nucleic acid sequence encoding the 72140 DCK shRNAmiR. SEQ ID NO: 91 sets forth the nucleic acid sequence encoding the passenger strand of the 72142 GR shRNAmiR. 5          SEQ ID NO: 92 sets forth the nucleic acid sequence encoding the guide strand of the 72142 GR shRNAmiR. SEQ ID NO: 93 sets forth the nucleic acid sequence encoding the passenger strand of the 72143 GR shRNAmiR. SEQ ID NO: 94 sets forth the nucleic acid sequence encoding the guide strand of the 10  72143 GR shRNAmiR. SEQ ID NO: 95 sets forth the nucleic acid sequence encoding the passenger strand of the 72145 GR shRNAmiR. SEQ ID NO: 96 sets forth the nucleic acid sequence encoding the guide strand of the 72145 GR shRNAmiR. 15          SEQ ID NO: 97 sets forth the nucleic acid sequence encoding the passenger strand of the 72146 GR shRNAmiR. SEQ ID NO: 98 sets forth the nucleic acid sequence encoding the guide strand of the 72146 GR shRNAmiR. SEQ ID NO: 99 sets forth the nucleic acid sequence encoding the passenger strand of the 20  72148 GR shRNAmiR. SEQ ID NO: 100 sets forth the nucleic acid sequence encoding the guide strand of the 72148 GR shRNAmiR. SEQ ID NO: 101 sets forth the nucleic acid sequence encoding the passenger strand of the 72149 GR shRNAmiR. 25          SEQ ID NO: 102 sets forth the nucleic acid sequence encoding the guide strand of the 72149 GR shRNAmiR. SEQ ID NO: 103 sets forth the nucleic acid sequence encoding the passenger strand of the 72150 GR shRNAmiR. SEQ ID NO: 104 sets forth the nucleic acid sequence encoding the guide strand of the 30  72150 GR shRNAmiR. 2026205474   09 Jul 2026 SEQ ID NO: 105 sets forth the nucleic acid sequence encoding the passenger strand of the 72151 GR shRNAmiR. SEQ ID NO: 106 sets forth the nucleic acid sequence encoding the guide strand of the 72151 GR shRNAmiR. 5          SEQ ID NO: 107 sets forth the nucleic acid sequence encoding the passenger strand of the 72152 GR shRNAmiR. SEQ ID NO: 108 sets forth the nucleic acid sequence encoding the guide strand of the 72152 GR shRNAmiR. SEQ ID NO: 109 sets forth the nucleic acid sequence encoding the 72142 GR 10 shRNAmiR. SEQ ID NO: 110 sets forth the nucleic acid sequence encoding the 72143 GR shRNAmiR. SEQ ID NO: 111 sets forth the nucleic acid sequence encoding the 72145 GR shRNAmiR. 15          SEQ ID NO: 112 sets forth the nucleic acid sequence encoding the 72146 GR shRNAmiR. SEQ ID NO: 113 sets forth the nucleic acid sequence encoding the 72148 GR shRNAmiR. SEQ ID NO: 114 sets forth the nucleic acid sequence encoding the 72149 GR 20 shRNAmiR. SEQ ID NO: 115 sets forth the nucleic acid sequence encoding the 72150 GR shRNAmiR. SEQ ID NO: 116 sets forth the nucleic acid sequence encoding the 72151 GR shRNAmiR. 25          SEQ ID NO: 117 sets forth the nucleic acid sequence encoding the 72152 GR shRNAmiR. SEQ ID NO: 118 sets forth the amino acid sequence of an HLA-E-01:03 protein. SEQ ID NO: 119 sets forth the amino acid sequence of a beta-2 microglobulin protein. SEQ ID NO: 120 sets forth the amino acid sequence of an HLA-G leader peptide. 30          SEQ ID NO: 121 sets forth the amino acid sequence of a (GGGGS)3 linker peptide. SEQ ID NO: 122 sets forth the amino acid sequence of a (GGGGS)4 linker peptide. 2026205474   09 Jul 2026 SEQ ID NO: 123 sets forth the amino acid sequence of a wild-type I-CreI homing endonuclease from Chlamydomonas reinhardtii. DETAILED DESCRIPTION OF THE INVENTION 5 1.1 References and Definitions The patent and scientific literature referred to herein establishes knowledge that is available to those of skill in the art. The issued US patents, allowed applications, published foreign applications, and references, including GenBank database sequences, which are cited 10 herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. The present invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to 15 those skilled in the art. For example, features illustrated with respect to one embodiment can be incorporated into other embodiments, and features illustrated with respect to a particular embodiment can be deleted from that embodiment. In addition, numerous variations and additions to the embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention. 20          Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, and other references mentioned herein are 25 incorporated by reference herein in their entirety. As used herein, “a,” “an,” or “the” can mean one or more than one. For example, “a” cell can mean a single cell or a multiplicity of cells. As used herein, unless specifically indicated otherwise, the word “or” is used in the inclusive sense of “and / or” and not the exclusive sense of “either / or.” 2026205474   09 Jul 2026 As used herein, the terms “exogenous” or “heterologous” in reference to a nucleotide sequence or amino acid sequence are intended to mean a sequence that is purely synthetic, that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. 5          As used herein, the term “endogenous” in reference to a nucleotide sequence or protein is intended to mean a sequence or protein that is naturally comprised within or expressed by a cell. As used herein, the terms “nuclease” and “endonuclease” are used interchangeably to refer to naturally-occurring or engineered enzymes which cleave a phosphodiester bond within a polynucleotide chain. 10          As used herein, the term “shRNA” or “short hairpin RNA” refers to an artificial RNA molecule comprising a hairpin that can be used to silence gene expression via RNA interference. As used herein, the term “miRNA” or “microRNA” or “miR” refers to mature microRNAs (miRNAs) that are endogenously encoded ~22 nt long RNAs that post-transcriptionally reduce the expression of target genes. miRNAs are found in plants, animals, 15 and some viruses and are generally expressed in a highly tissue- or developmental-stage-specific fashion. A "stem-loop structure" refers to a nucleic acid having a secondary structure that includes a region of nucleotides which are known or predicted to form a double strand (stem portion) that is linked on one side by a region of predominantly single-stranded nucleotides (loop portion), In 20 some cases, the loop may also be very short and thereby not be recognized by Dicer, leading to Dicer-independent shRNAs (comparable to the endogenous miR0431). The term “hairpin” is also used herein to refer to stem-loop structures. The actual primary sequence of nucleotides within the stem-loop structure is not critical to the practice of the description as long as the secondary structure is present. As is known in the art, the secondary structure does not require 25 exact base-pairing. Thus, the stem may include one or more base mismatches. Alternatively, the base-pairing may be exact (i.e., not include any mismatches). As used herein, the terms “shRNAmiR” and “microRNA-adapted shRNA” refer to shRNA sequences embedded within a microRNA scaffold. A shRNAmiR molecule mimics naturally-occurring pri-miRNA molecules in that they comprise a hairpin flanked by sequences 30 necessary for efficient processing and can be processed by the Drosha enzyme into pre-miRNAs, exported into the cytoplasm, and cleaved by Dicer, after which the mature miRNA can enter the 2026205474   09 Jul 2026 RISC. The microRNA scaffold can be derived from naturally-occurring microRNA, premiRNAs, or pri-miRNAs or variants thereof. In some embodiments, the shRNA sequences which the shRNAmiR is based upon is of a different length from miRNAs (which are 22 nucleotides long) and the miRNA scaffold must therefore be modified in order to accommodate 5 the longer or shorter shRNA sequence length. As used herein, the term “microRNA flanking sequences” refers to nucleotide sequences comprising microRNA processing elements. MicroRNA processing elements are the minimal nucleic acid sequences which contribute to the production of mature microRNA from primary microRNA or precursor microRNA. Often these elements are located within a 40 nucleotide 10 sequence that flanks a microRNA stem-loop structure. In some instances, the microRNA processing elements are found within a stretch of nucleotide sequences of between 5 and 4,000 nucleotides in length that flank a microRNA stem-loop structure. MicroRNA flanking sequences used in the shRNAmiR molecules can be naturally-occurring sequences flanking naturally-occurring microRNA or can be variants thereof. MicroRNA flanking sequences include miR 15 scaffold domains and miR basal stem domains. shRNAmiR molecules used in the presently disclosed compositions and methods can comprise in the 5' to 3' direction: (a) a 5' miR scaffold domain; (b) a 5' miR basal stem domain; (c) a passenger strand; (d) a miR loop domain; (e) a guide strand; (f) a 3' miR basal stem domain; and (g) a 3' miR scaffold domain. 20          As used herein, the term “miR scaffold domain” as it relates to a shRNAmiR refers to a nucleotide sequence that can flank either the 5' or 3' end of a microRNA or shRNA in a shRNAmiR molecule and can be derived from a naturally-occurring microRNA flanking sequence or a variant thereof. In general, the miR basal stem domain sequence separates the shRNA sequence (passenger and guide strand, and miR loop domain) and the scaffold domains. 25 The 5' miR scaffold domain can comprise a restriction enzyme (e.g., type IIS restriction enzyme) recognition sequence at or near its 3' end and the 3' miR scaffold domain can comprise a restriction enzyme recognition sequence at or near its 5' end, thus facilitating the insertion of a shRNA sequence. In some embodiments, the secondary structure of the miR scaffold domain is more important than the actual sequence thereof. 30          As used herein, the term “miR basal stem domain” as it relates to a shRNAmiR refers to sequences immediately flanking the passenger and guide strand sequences that comprise the base 2026205474   09 Jul 2026 of the hairpin stem below the passenger:guide duplex. Thus, the 5' and 3' miR basal stem domains are complementary (fully or partially) in sequence to one another. In some embodiments, the 5' and 3' miR basal stem domains comprise sequences that when hybridized together, form two mismatch bubbles, each comprising one or two mismatched base pairs. 5          As used herein, the term “passenger strand” as it relates to a shRNAmiR refers to the sequence of the shRNAmiR, which is complementary (fully or partially) to the guide sequence. As used herein, the term “guide strand” as it relates to a shRNAmiR refers to the sequence of the shRNAmiR that has complementarity (full or partial) with the target mRNA sequence for which a reduction in expression is desired. 10          As used herein, a “miR loop domain” as it relates to a shRNAmiR refers to the single stranded loop sequence at one end of the passenger:guide duplex of the shRNAmiR. The miR loop domain can be derived from a naturally-occurring pre-microRNA loop sequence or a variant thereof. As used herein, the terms “cleave” or “cleavage” refer to the hydrolysis of phosphodiester 15 bonds within the backbone of a recognition sequence within a target sequence that results in a double-stranded break within the target sequence, referred to herein as a “cleavage site”. As used herein, the term “meganuclease” refers to an endonuclease that binds doublestranded DNA at a recognition sequence that is greater than 12 base pairs. In some embodiments, the recognition sequence for a meganuclease of the present disclosure is 22 base pairs. A 20 meganuclease can be an endonuclease that is derived from I-CreI (SEQ ID NO: 123), and can refer to an engineered variant of I-CreI that has been modified relative to natural I-CreI with respect to, for example, DNA-binding specificity, DNA cleavage activity, DNA-binding affinity, or dimerization properties. Methods for producing such modified variants of I-CreI are known in the art (e.g., WO 2007 / 047859, incorporated by reference in its entirety). A meganuclease as 25 used herein binds to double-stranded DNA as a heterodimer. A meganuclease may also be a “single-chain meganuclease” in which a pair of DNA-binding domains is joined into a single polypeptide using a peptide linker. The term “homing endonuclease” is synonymous with the term “meganuclease.” Meganucleases of the present disclosure are substantially non-toxic when expressed in cells, particularly in human immune cells, such that cells can be transfected and 30 maintained at 37oC without observing deleterious effects on cell viability or significant 2026205474   09 Jul 2026 reductions in meganuclease cleavage activity when measured using the methods described herein. As used herein, the term “single-chain meganuclease” refers to a polypeptide comprising a pair of nuclease subunits joined by a linker. A single-chain meganuclease has the organization: 5 N-terminal subunit - Linker - C-terminal subunit. The two meganuclease subunits will generally be non-identical in amino acid sequence and will bind non-identical DNA sequences. Thus, single-chain meganucleases typically cleave pseudo-palindromic or non-palindromic recognition sequences. A single-chain meganuclease may be referred to as a “single-chain heterodimer” or “single-chain heterodimeric meganuclease” although it is not, in fact, dimeric. 10 For clarity, unless otherwise specified, the term “meganuclease” can refer to a dimeric or singlechain meganuclease. As used herein, the term “linker” refers to an exogenous peptide sequence used to join two nuclease subunits into a single polypeptide. A linker may have a sequence that is found in natural proteins or may be an artificial sequence that is not found in any natural protein. A linker 15 may be flexible and lacking in secondary structure or may have a propensity to form a specific three-dimensional structure under physiological conditions. A linker can include, without limitation, those encompassed by U.S. Patent Nos. 8,445,251, 9,340,777, 9,434,931, and 10,041,053, each of which is incorporated by reference in its entirety. In some embodiments, a linker may have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, 20 at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to residues 154-195 of SEQ ID NO: 65. In some embodiments, a linker may have an amino acid sequence comprising residues 154-195 of SEQ ID NO: 65. As used herein, the term “TALEN” refers to an endonuclease comprising a DNA-binding domain comprising a plurality of TAL domain repeats fused to a nuclease domain or an active 25 portion thereof from an endonuclease or exonuclease, including but not limited to a restriction endonuclease, homing endonuclease, S1 nuclease, mung bean nuclease, pancreatic DNAse I, micrococcal nuclease, and yeast HO endonuclease. See, for example, Christian et al. (2010) Genetics 186:757-761, which is incorporated by reference in its entirety. Nuclease domains useful for the design of TALENs include those from a Type IIs restriction endonuclease, 30 including but not limited to FokI, FoM, StsI, HhaI, HindIII, Nod, BbvCI, EcoRI, BglI, and AlwI. Additional Type IIs restriction endonucleases are described in International Publication No. WO 2026205474   09 Jul 2026 2007 / 014275, which is incorporated by reference in its entirety. In some embodiments, the nuclease domain of the TALEN is a FokI nuclease domain or an active portion thereof. TAL domain repeats can be derived from the TALE (transcription activator-like effector) family of proteins used in the infection process by plant pathogens of the Xanthomonas genus. TAL 5 domain repeats are 33-34 amino acid sequences with divergent 12th and 13th amino acids. These two positions, referred to as the repeat variable dipeptide (RVD), are highly variable and show a strong correlation with specific nucleotide recognition. Each base pair in the DNA target sequence is contacted by a single TAL repeat with the specificity resulting from the RVD. In some embodiments, the TALEN comprises 16-22 TAL domain repeats. DNA cleavage by a 10 TALEN requires two DNA recognition regions (i.e., “half-sites”) flanking a nonspecific central region (i.e., the “spacer”). The term “spacer” in reference to a TALEN refers to the nucleic acid sequence that separates the two nucleic acid sequences recognized and bound by each monomer constituting a TALEN. The TAL domain repeats can be native sequences from a naturally-occurring TALE protein or can be redesigned through rational or experimental means to produce 15 a protein that binds to a pre-determined DNA sequence (see, for example, Boch et al. (2009) Science 326(5959):1509-1512 and Moscou and Bogdanove (2009) Science 326(5959):1501, each of which is incorporated by reference in its entirety). See also, U.S. Publication No. 20110145940 and International Publication No. WO 2010 / 079430 for methods for engineering a TALEN to recognize and bind a specific sequence and examples of RVDs and their 20 corresponding target nucleotides. In some embodiments, each nuclease (e.g., FokI) monomer can be fused to a TAL effector sequence that recognizes and binds a different DNA sequence, and only when the two recognition sites are in close proximity do the inactive monomers come together to create a functional enzyme. It is understood that the term “TALEN” can refer to a single TALEN protein or, alternatively, a pair of TALEN proteins (i.e., a left TALEN protein 25 and a right TALEN protein) which bind to the upstream and downstream half-sites adjacent to the TALEN spacer sequence and work in concert to generate a cleavage site within the spacer sequence. Given a predetermined DNA locus or spacer sequence, upstream and downstream half-sites can be identified using a number of programs known in the art (Kornel Labun; Tessa G. Montague; James A. Gagnon; Summer B. Thyme; Eivind Valen. (2016). CHOPCHOP v2: a 30 web tool for the next generation of CRISPR genome engineering. Nucleic Acids Research; doi:10.1093 / nar / gkw398; Tessa G. Montague; Jose M. Cruz; James A. Gagnon; George M. 2026205474   09 Jul 2026 Church; Eivind Valen. (2014). CHOPCHOP: a CRISPR / Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 42. W401-W407). It is also understood that a TALEN recognition sequence can be defined as the DNA binding sequence (i.e., half-site) of a single TALEN protein or, alternatively, a DNA sequence comprising the upstream half-site, the spacer sequence, and 5 the downstream half-site. As used herein, the term “compact TALEN” refers to an endonuclease comprising a DNA-binding domain with one or more TAL domain repeats fused in any orientation to any portion of the I-TevI homing endonuclease or any of the endonucleases listed in Table 2 in U.S. Application No. 20130117869 (which is incorporated by reference in its entirety), including but 10 not limited to MmeI, EndA, End1, I-BasI, I-TevII, I-TevIII, I-TwoI, MspI, MvaI, NucA, and NucM. Compact TALENs do not require dimerization for DNA processing activity, alleviating the need for dual target sites with intervening DNA spacers. In some embodiments, the compact TALEN comprises 16-22 TAL domain repeats. As used herein, the term “megaTAL” refers to a single-chain endonuclease comprising a 15 transcription activator-like effector (TALE) DNA binding domain with an engineered, sequencespecific homing endonuclease. As used herein, the terms “zinc finger nuclease” or “ZFN” refers to a chimeric protein comprising a zinc finger DNA-binding domain fused to a nuclease domain from an endonuclease or exonuclease, including but not limited to a restriction endonuclease, homing endonuclease, S1 20 nuclease, mung bean nuclease, pancreatic DNAse I, micrococcal nuclease, and yeast HO endonuclease. Nuclease domains useful for the design of zinc finger nucleases include those from a Type IIs restriction endonuclease, including but not limited to FokI, FoM, and StsI restriction enzyme. Additional Type IIs restriction endonucleases are described in International Publication No. WO 2007 / 014275, which is incorporated by reference in its entirety. The 25 structure of a zinc finger domain is stabilized through coordination of a zinc ion. DNA binding proteins comprising one or more zinc finger domains bind DNA in a sequence-specific manner. The zinc finger domain can be a native sequence or can be redesigned through rational or experimental means to produce a protein which binds to a pre-determined DNA sequence ~18 basepairs in length, comprising a pair of nine basepair half-sites separated by 2-10 basepairs. 30 See, for example, U.S. Pat. Nos. 5,789,538, 5,925,523, 6,007,988, 6,013,453, 6,200,759, and International Publication Nos. WO 95 / 19431, WO 96 / 06166, WO 98 / 53057, WO 98 / 54311, WO 2026205474   09 Jul 2026 00 / 27878, WO 01 / 60970, WO 01 / 88197, and WO 02 / 099084, each of which is incorporated by reference in its entirety. By fusing this engineered protein domain to a nuclease domain, such as FokI nuclease, it is possible to target DNA breaks with genome-level specificity. The selection of target sites, zinc finger proteins and methods for design and construction of zinc finger 5 nucleases are known to those of skill in the art and are described in detail in U.S. Publications Nos. 20030232410, 20050208489, 2005064474, 20050026157, 20060188987 and International Publication No. WO 07 / 014275, each of which is incorporated by reference in its entirety. In the case of a zinc finger, the DNA binding domains typically recognize an 18-bp recognition sequence comprising a pair of nine basepair “half-sites” separated by a 2-10 basepair “spacer 10 sequence”, and cleavage by the nuclease creates a blunt end or a 5' overhang of variable length (frequently four basepairs). It is understood that the term “zinc finger nuclease” can refer to a single zinc finger protein or, alternatively, a pair of zinc finger proteins (i.e., a left ZFN protein and a right ZFN protein) that bind to the upstream and downstream half-sites adjacent to the zinc finger nuclease spacer sequence and work in concert to generate a cleavage site within the spacer 15 sequence. Given a predetermined DNA locus or spacer sequence, upstream and downstream half-sites can be identified using a number of programs known in the art (Mandell JG, Barbas CF 3rd. Zinc Finger Tools: custom DNA-binding domains for transcription factors and nucleases. Nucleic Acids Res. 2006 Jul 1;34 (Web Server issue):W516-23). It is also understood that a zinc finger nuclease recognition sequence can be defined as the DNA binding sequence (i.e., half-site) 20 of a single zinc finger nuclease protein or, alternatively, a DNA sequence comprising the upstream half-site, the spacer sequence, and the downstream half-site. As used herein, the terms “CRISPR nuclease” or “CRISPR system nuclease” refers to a CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) endonuclease or a variant thereof, such as Cas9, that associates with a guide RNA that directs 25 nucleic acid cleavage by the associated endonuclease by hybridizing to a recognition site in a polynucleotide. In certain embodiments, the CRISPR nuclease is a class 2 CRISPR enzyme. In some of these embodiments, the CRISPR nuclease is a class 2, type II enzyme, such as Cas9. In other embodiments, the CRISPR nuclease is a class 2, type V enzyme, such as Cpf1. The guide RNA comprises a direct repeat and a guide sequence (often referred to as a spacer in the context 30 of an endogenous CRISPR system), which is complementary to the target recognition site. In certain embodiments, the CRISPR system further comprises a tracrRNA (trans-activating 2026205474   09 Jul 2026 CRISPR RNA) that is complementary (fully or partially) to the direct repeat sequence (sometimes referred to as a tracr-mate sequence) present on the guide RNA. In particular embodiments, the CRISPR nuclease can be mutated with respect to a corresponding wild-type enzyme such that the enzyme lacks the ability to cleave one strand of a target polynucleotide, 5 functioning as a nickase, cleaving only a single strand of the target DNA. Non-limiting examples of CRISPR enzymes that function as a nickase include Cas9 enzymes with a D10A mutation within the RuvC I catalytic domain, or with a H840A, N854A, or N863A mutation. Given a predetermined DNA locus, recognition sequences can be identified using a number of programs known in the art (Kornel Labun; Tessa G. Montague; James A. Gagnon; Summer B. 10 Thyme; Eivind Valen. (2016). CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Research; doi:10.1093 / nar / gkw398; Tessa G. Montague; Jose M. Cruz; James A. Gagnon; George M. Church; Eivind Valen. (2014). CHOPCHOP: a CRISPR / Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 42. W401-W407). As used herein, a “template nucleic acid” refers to a nucleic acid (i.e., a polynucleotide) 15 that is desired to be inserted into a cleavage site within a cell’s genome. As used herein, the terms “recombinant” or “engineered,” with respect to a protein, means having an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids that encode the protein and cells or organisms that express the protein. With respect to a nucleic acid, the term “recombinant” or “engineered” means having an 20 altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation, and other gene transfer technologies; homologous recombination; site-directed mutagenesis; and gene fusion. In accordance with this definition, a protein having an amino acid sequence identical to a naturally-occurring protein, but produced 25 by cloning and expression in a heterologous host, is not considered recombinant or engineered. As used herein, the term “wild-type” refers to the most common naturally occurring allele (i.e., polynucleotide sequence) in the allele population of the same type of gene, wherein a polypeptide encoded by the wild-type allele has its original functions. The term “wild-type” also refers to a polypeptide encoded by a wild-type allele. Wild-type alleles (i.e., polynucleotides) 30 and polypeptides are distinguishable from mutant or variant alleles and polypeptides, which comprise one or more mutations and / or substitutions relative to the wild-type sequence(s). 2026205474   09 Jul 2026 Whereas a wild-type allele or polypeptide can confer a normal phenotype in an organism, a mutant or variant allele or polypeptide can, in some instances, confer an altered phenotype. Wild-type nucleases are distinguishable from recombinant or non-naturally-occurring nucleases. The term “wild-type” can also refer to a cell, an organism, and / or a subject which possesses a 5 wild-type allele of a particular gene, or a cell, an organism, and / or a subject used for comparative purposes. As used herein, the term “genetically-modified” refers to a cell or organism in which, or in an ancestor of which, a genomic DNA sequence has been deliberately modified by recombinant technology. As used herein, the term “genetically-modified” encompasses the term 10 “transgenic.” As used herein with respect to recombinant proteins, the term “modification” means any insertion, deletion, or substitution of an amino acid residue in the recombinant sequence relative to a reference sequence (e.g., a wild-type or a native sequence). As used herein, the terms “recognition sequence” or “recognition site” refers to a DNA 15 sequence that is bound and cleaved by a nuclease. In the case of a meganuclease, a recognition sequence comprises a pair of inverted, 9 basepair “half sites” which are separated by four basepairs. In the case of a single-chain meganuclease, the N-terminal domain of the protein contacts a first half-site and the C-terminal domain of the protein contacts a second half-site. Cleavage by a meganuclease produces four basepair 3' overhangs. “Overhangs,” or “sticky 20 ends” are short, single-stranded DNA segments that can be produced by endonuclease cleavage of a double-stranded DNA sequence. In the case of meganucleases and single-chain meganucleases derived from I-CreI, the overhang comprises bases 10-13 of the 22 basepair recognition sequence. In the case of a compact TALEN, the recognition sequence comprises a first CNNNGN sequence that is recognized by the I-TevI domain, followed by a non-specific 25 spacer 4-16 basepairs in length, followed by a second sequence 16-22 bp in length that is recognized by the TAL-effector domain (this sequence typically has a 5' T base). Cleavage by a compact TALEN produces two basepair 3' overhangs. In the case of a CRISPR nuclease, the recognition sequence is the sequence, typically 16-24 basepairs, to which the guide RNA binds to direct cleavage. Full complementarity between the guide sequence and the recognition 30 sequence is not necessarily required to effect cleavage. Cleavage by a CRISPR nuclease can produce blunt ends (such as by a class 2, type II CRISPR nuclease) or overhanging ends (such as 2026205474   09 Jul 2026 by a class 2, type V CRISPR nuclease), depending on the CRISPR nuclease. In those embodiments wherein a CpfI CRISPR nuclease is utilized, cleavage by the CRISPR complex comprising the same will result in 5' overhangs and in certain embodiments, 5 nucleotide 5' overhangs. Each CRISPR nuclease enzyme also requires the recognition of a PAM (protospacer 5 adjacent motif) sequence that is near the recognition sequence complementary to the guide RNA. The precise sequence, length requirements for the PAM, and distance from the target sequence differ depending on the CRISPR nuclease enzyme, but PAMs are typically 2-5 base pair sequences adjacent to the target / recognition sequence. PAM sequences for particular CRISPR nuclease enzymes are known in the art (see, for example, U.S. Patent No. 8,697,359 and U.S. 10 Publication No. 20160208243, each of which is incorporated by reference in its entirety) and PAM sequences for novel or engineered CRISPR nuclease enzymes can be identified using methods known in the art, such as a PAM depletion assay (see, for example, Karvelis et al. (2017) Methods 121-122:3-8, which is incorporated herein in its entirety). In the case of a zinc finger, the DNA binding domains typically recognize an 18-bp recognition sequence comprising 15 a pair of nine basepair “half-sites” separated by 2-10 basepairs and cleavage by the nuclease creates a blunt end or a 5' overhang of variable length (frequently four basepairs). As used herein, the term “target site” or “target sequence” refers to a region of the chromosomal DNA of a cell comprising a recognition sequence for a nuclease. As used herein, the terms “DNA-binding affinity” or “binding affinity” means the 20 tendency of a nuclease to non-covalently associate with a reference DNA molecule (e.g., a recognition sequence or an arbitrary sequence). Binding affinity is measured by a dissociation constant, Kd. As used herein, a nuclease has “altered” binding affinity if the Kd of the nuclease for a reference recognition sequence is increased or decreased by a statistically significant percent change relative to a reference nuclease. 25          As used herein, the term “specificity” means the ability of a nuclease to bind and cleave double-stranded DNA molecules only at a particular sequence of base pairs referred to as the recognition sequence, or only at a particular set of recognition sequences. The set of recognition sequences will share certain conserved positions or sequence motifs but may be degenerate at one or more positions. A highly-specific nuclease is capable of cleaving only one or a very few 30 recognition sequences. Specificity can be determined by any method known in the art. 2026205474   09 Jul 2026 As used herein, the term “homologous recombination” or “HR” refers to the natural, cellular process in which a double-stranded DNA-break is repaired using a homologous DNA sequence as the repair template (see, e.g. Cahill et al. (2006), Front. Biosci. 11:1958-1976). The homologous DNA sequence may be an endogenous chromosomal sequence or an exogenous 5 nucleic acid that was delivered to the cell. As used herein, the term “non-homologous end-joining” or “NHEJ” refers to the natural, cellular process in which a double-stranded DNA-break is repaired by the direct joining of two non-homologous DNA segments (see, e.g. Cahill et al. (2006), Front. Biosci. 11:1958-1976). DNA repair by non-homologous end-joining is error-prone and frequently results in the 10 untemplated addition or deletion of DNA sequences at the site of repair. In some instances, cleavage at a target recognition sequence results in NHEJ at a target recognition site. Nuclease-induced cleavage of a target site in the coding sequence of a gene followed by DNA repair by NHEJ can introduce mutations into the coding sequence, such as frameshift mutations, that disrupt gene function. Thus, engineered nucleases can be used to effectively knock-out a gene in 15 a population of cells. As used herein, the term “disrupted” or “disrupts” or “disrupts expression” or “disrupting a target sequence” refers to the introduction of a mutation (e.g., frameshift mutation) that interferes with the gene function and prevents expression and / or function of the polypeptide / expression product encoded thereby. For example, nuclease-mediated disruption of 20 a gene can result in the expression of a truncated protein and / or expression of a protein that does not retain its wild-type function. Additionally, introduction of a template nucleic acid into a gene can result in no expression of an encoded protein, expression of a truncated protein, and / or expression of a protein that does not retain its wild-type function. As used herein, the term “chimeric antigen receptor” or “CAR” refers to an engineered 25 receptor that confers or grafts specificity for an antigen onto an immune effector cell (e.g., a human T cell). A chimeric antigen receptor comprises at least an extracellular ligand-binding domain or moiety, a transmembrane domain, and an intracellular domain that comprises one or more signaling domains and / or co-stimulatory domains. In some embodiments, the extracellular ligand-binding domain or moiety is an antibody, 30 or antibody fragment. In this context, the term “antibody fragment” can refer to at least one portion of an antibody, that retains the ability to specifically interact with (e.g., by binding, steric 2026205474   09 Jul 2026 hindrance, stabilizing / destabilizing, spatial distribution) an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab‘)2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid 5 VHH domains, multi-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody. An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and 10 Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies). In some embodiments, the extracellular ligand-binding domain or moiety is in the form of a single-chain variable fragment (scFv) derived from a monoclonal antibody, which provides 15 specificity for a particular epitope or antigen (e.g., an epitope or antigen preferentially present on the surface of a cell, such as a cancer cell or other disease-causing cell or particle). In some embodiments, the scFv is attached via a linker sequence. In some embodiments, the scFv is murine, humanized, or fully human. The extracellular ligand-binding domain of a chimeric antigen receptor can also comprise 20 an autoantigen (see, Payne et al. (2016), Science 353 (6295): 179-184), that can be recognized by autoantigen-specific B cell receptors on B lymphocytes, thus directing T cells to specifically target and kill autoreactive B lymphocytes in antibody-mediated autoimmune diseases. Such CARs can be referred to as chimeric autoantibody receptors (CAARs), and their use is encompassed by the invention. The extracellular ligand-binding domain of a chimeric antigen 25 receptor can also comprise a naturally-occurring ligand for an antigen of interest, or a fragment of a naturally-occurring ligand which retains the ability to bind the antigen of interest. The intracellular stimulatory domain can include one or more cytoplasmic signaling domains that transmit an activation signal to the T cell following antigen binding. Such cytoplasmic signaling domains can include, without limitation, a CD3 zeta signaling domain. 30          The intracellular stimulatory domain can also include one or more intracellular co stimulatory domains that transmit a proliferative and / or cell-survival signal after ligand binding. 2026205474   09 Jul 2026 Such intracellular co-stimulatory domains can be any of those known in the art and can include, without limitation, those co-stimulatory domains disclosed in WO 2018 / 067697 including, for example, Novel 6. Further examples of co-stimulatory domains can include 4-1BB (CD137), CD27, CD28, CD8, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-5   1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, or any combination thereof. A chimeric antigen receptor further includes additional structural elements, including a transmembrane domain that is attached to the extracellular ligand-binding domain via a hinge or spacer sequence. The transmembrane domain can be derived from any membrane-bound or 10 transmembrane protein. For example, the transmembrane polypeptide can be a subunit of the T-cell receptor (e.g., an a, 0, y or Z, polypeptide constituting CD3 complex), IL2 receptor p55 (a chain), p75 (0 chain) or y chain, subunit chain of Fc receptors (e.g., Fcy receptor III) or CD proteins such as the CD8 alpha chain. In certain examples, the transmembrane domain is a CD8 alpha domain. Alternatively, the transmembrane domain can be synthetic and can comprise 15 predominantly hydrophobic residues such as leucine and valine. The hinge region refers to any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. For example, a hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. Hinge regions may be derived from all or part of naturally occurring molecules, 20 such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region. Alternatively, the hinge region may be a synthetic sequence that corresponds to a naturally occurring hinge sequence or may be an entirely synthetic hinge sequence. In particular examples, a hinge domain can comprise a part of a human CD8 alpha chain, FcyRllla receptor or IgGl. In certain examples, the hinge region can be a CD8 alpha 25 domain. As used herein, the terms “exogenous T cell receptor” or “exogenous TCR” refer to a TCR whose sequence is introduced into the genome of an immune cell (e.g., a human T cell) that may or may not endogenously express the TCR. Expression of an exogenous TCR on an immune cell can confer specificity for a specific epitope or antigen (e.g., an epitope or antigen 30 preferentially present on the surface of a cancer cell or other disease-causing cell or particle). Such exogenous T cell receptors can comprise alpha and beta chains or, alternatively, may 2026205474   09 Jul 2026 comprise gamma and delta chains. Exogenous TCRs useful in the invention may have specificity to any antigen or epitope of interest. As used herein, the term “HLA class I histocompatibility antigen, alpha chain E fusion protein” or “HLA-E fusion protein” refers to a protein comprising an HLA-E protein fused to at 5 least one additional protein that enables expression of the HLA-E protein on the cell-surface. HLA-E proteins can include, for example, an HLA-E-01:01 or HLA-E-01:03 protein (e.g., SEQ ID NO: 118). An HLA-E fusion protein can comprise, for example, an HLA-E protein fused to a beta-2 microglobulin protein (e.g., SEQ ID NO: 119) that enables expression of the HLA-E protein on the cell-surface. In further examples, the HLA-E fusion protein can comprise an 10 HLA-E protein fused to both a beta-2 microglobulin protein and an additional protein that is loaded into the HLA-E protein for presentation such as, for example, an HLA-G leader peptide (e.g., SEQ ID NO: 120) and others known in the art. The proteins of the HLA-E fusion protein can be fused by polypeptide linkers such as, for example, a linker comprising SEQ ID NO: 121 (i.e., a (GGGGS)3 linker) or SEQ ID NO: 122 (i.e., a (GGGGS)4 linker). 15           As used herein, the term “reduced expression” in reference to a target protein (i.e., an endogenously expressed protein) refers to any reduction in the expression of the endogenous protein by a genetically-modified cell when compared to a control cell. The term reduced can also refer to a reduction in the percentage of cells in a population of cells that express wild-type levels of an endogenous protein targeted by a shRNAmiR of the disclosure when compared to a 20 population of control cells. Such a reduction in the percentage of cells in a population that fully express the targeted endogenous protein may be up to 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or up to 100%. It is understood in the context of this disclosure that the term “reduced” encompasses a partial or incomplete knockdown of a target or endogenous protein, and is distinguished from a complete knockdown, such as that 25 achieved by gene inactivation by a nuclease. As used herein, the term with respect to both amino acid sequences and nucleic acid sequences, the terms “percent identity,” “sequence identity,” “percentage similarity,” “sequence similarity” and the like refer to a measure of the degree of similarity of two sequences based upon an alignment of the sequences that maximizes similarity between aligned amino acid 30 residues or nucleotides, and which is a function of the number of identical or similar residues or nucleotides, the number of total residues or nucleotides, and the presence and length of gaps in 2026205474   09 Jul 2026 the sequence alignment. A variety of algorithms and computer programs are available for determining sequence similarity using standard parameters. As used herein, sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available through the National Center for 5 Biotechnology Information (www.ncbi.nlm.nih.gov / ), and are described in, for example, Altschul et al. (1990), J. Mol. Biol. 215:403-410; Gish and States (1993), Nature Genet. 3:266272; Madden et al. (1996), Meth. Enzymol.266:131-141; Altschul et al. (1997), Nucleic Acids Res. 25:33 89-3402); Zhang et al. (2000), J. Comput. Biol. 7(1-2):203-14. As used herein, percent similarity of two amino acid sequences is the score based upon the following parameters 10 for the BLASTp algorithm: word size=3; gap opening penalty=-11; gap extension penalty=-1; and scoring matrix=BLOSUM62. As used herein, percent similarity of two nucleic acid sequences is the score based upon the following parameters for the BLASTn algorithm: word size=11; gap opening penalty=-5; gap extension penalty=-2; match reward=1; and mismatch penalty=-3. 15          As used herein, the term “corresponding to” with respect to modifications of two proteins or amino acid sequences is used to indicate that a specified modification in the first protein is a substitution of the same amino acid residue as in the modification in the second protein, and that the amino acid position of the modification in the first protein corresponds to or aligns with the amino acid position of the modification in the second protein when the two proteins are 20 subjected to standard sequence alignments (e.g., using the BLASTp program). Thus, the modification of residue “X” to amino acid “A” in the first protein will correspond to the modification of residue “Y” to amino acid “A” in the second protein if residues X and Y correspond to each other in a sequence alignment and despite the fact that X and Y may be different numbers. 25          As used herein, the term “T cell receptor alpha gene” or “TCR alpha gene” refer to the locus in a T cell which encodes the T cell receptor alpha subunit. The T cell receptor alpha gene can refer to NCBI Gene ID number 6955, before or after rearrangement. Following rearrangement, the T cell receptor alpha gene comprises an endogenous promoter, rearranged V and J segments, the endogenous splice donor site, an intron, the endogenous splice acceptor site, 30 and the T cell receptor alpha constant region locus, which comprises the subunit coding exons. 2026205474   09 Jul 2026 As used herein, the term “T cell receptor alpha constant region” or “TCR alpha constant region” refers to the coding sequence of the T cell receptor alpha gene. The TCR alpha constant region includes the wild-type sequence, and functional variants thereof, identified by NCBI Gene ID NO. 28755. 5          As used herein, the term “T cell receptor beta gene” or “TCR beta gene” refers to the locus in a T cell which encodes the T cell receptor beta subunit. The T cell receptor beta gene can refer to NCBI Gene ID number 6957. As used herein, the term “recombinant DNA construct,” “recombinant construct,” “cassette,” “expression cassette,” “expression construct,” “chimeric construct,” “construct,” and 10 “recombinant DNA fragment” are used interchangeably herein and are single or double-stranded polynucleotides. A recombinant construct comprises an artificial combination of nucleic acid fragments, including, without limitation, regulatory and coding sequences that are not found together in nature. For example, a recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences 15 and coding sequences derived from the same source and arranged in a manner different than that found in nature. Such a construct may be used by itself or may be used in conjunction with a vector. As used herein, the term “vector” or “recombinant DNA vector” may be a construct that includes a replication system and sequences that are capable of transcription and translation of a 20 polypeptide-encoding sequence in a given host cell. If a vector is used, then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art. Vectors can include, without limitation, plasmid vectors and recombinant AAV vectors, or any other vector known in the art suitable for delivering a gene to a target cell. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to 25 successfully transform, select and propagate host cells comprising any of the isolated nucleotides or nucleic acid sequences of the invention. As used herein, a “vector” can also refer to a viral vector (i.e., a recombinant virus). Viral vectors can include, without limitation, retroviral vectors (i.e., recombinant retroviruses), lentiviral vectors (i.e., recombinant lentiviruses), adenoviral vectors (i.e., recombinant 30 adenoviruses), and adeno-associated viral vectors (AAV) (i.e., recombinant AAVs). 2026205474   09 Jul 2026 As used herein, the term “immune cell” refers to any cell that is part of the immune system (innate and / or adaptive) and is of hematopoietic origin. Non-limiting examples of immune cells include lymphocytes, B cells, T cells, monocytes, macrophages, dendritic cells, granulocytes, megakaryocytes, monocytes, macrophages, natural killer cells, myeloid-derived 5 suppressor cells, innate lymphoid cells, platelets, red blood cells, thymocytes, leukocytes, neutrophils, mast cells, eosinophils, basophils, and granulocytes. As used herein, a “human T cell” or “T cell” or “isolated human T cell” refers to a T cell isolated from a donor, particularly a human donor. T cells, and cells derived therefrom, include isolated T cells that have not been passaged in culture, T cells that have been passaged and 10 maintained under cell culture conditions without immortalization, and T cells that have been immortalized and can be maintained under cell culture conditions indefinitely. As used herein, a “human NK cell” or “NK cell” refers to a NK cell isolated from a donor, particularly a human donor. NK cells, and cells derived therefrom, include isolated NK cells that have not been passaged in culture, NK cells that have been passaged and maintained 15 under cell culture conditions without immortalization, and NK cells that have been immortalized and can be maintained under cell culture conditions indefinitely. As used herein, a “human B cell” or “B cell” refers to a B cell isolated from a donor, particularly a human donor. B cells, and cells derived therefrom, include isolated T cells that have not been passaged in culture, B cells that have been passaged and maintained under cell 20 culture conditions without immortalization, and B cells that have been immortalized and can be maintained under cell culture conditions indefinitely. As used herein, the term “a control” or “a control cell” refers to a cell that provides a reference point for measuring changes in genotype or phenotype of a genetically-modified cell. A control cell may comprise, for example: (a) a wild-type cell, i.e., of the same genotype as the 25 starting material for the genetic alteration which resulted in the genetically-modified cell; (b) a cell of the same genotype as the genetically-modified cell but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest); or, (c) a cell genetically identical to the genetically-modified cell but which is not exposed to conditions or stimuli or further genetic modifications that would induce expression of altered genotype or 30 phenotype. 2026205474   09 Jul 2026 As used herein, the terms “treatment” or “treating a subject” refers to the administration of a genetically-modified immune cell or population of genetically-modified immune cells of the invention to a subject having a disease. For example, the subject can have a disease such as cancer, and treatment can represent immunotherapy for the treatment of the disease. Desirable 5 effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some aspects, a genetically-modified immune cell or population of genetically-modified immune cells described herein is 10 administered during treatment in the form of a pharmaceutical composition of the invention. The term “effective amount” or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results. The therapeutically effective amount will vary depending on the formulation or composition used, the disease and its severity and the age, weight, physical condition and responsiveness of the subject to be treated. 15 In specific embodiments, an effective amount of a genetically-modified immune cell or population of genetically-modified immune cells of the invention, or pharmaceutical compositions disclosed herein, reduces at least one symptom of a disease in a subject. In those embodiments wherein the disease is a cancer, an effective amount of the pharmaceutical compositions disclosed herein reduces the level of proliferation or metastasis of cancer, causes a 20 partial or full response or remission of cancer, or reduces at least one symptom of cancer in a subject. As used herein, the term “cancer” should be understood to encompass any neoplastic disease (whether invasive or metastatic) which is characterized by abnormal and uncontrolled cell division causing malignant growth or tumor. 25          As used herein, the term “carcinoma” refers to a malignant growth made up of epithelial cells. As used herein, the term “leukemia” refers to malignancies of the hematopoietic organs / systems and is generally characterized by an abnormal proliferation and development of leukocytes and their precursors in the blood and bone marrow. 2026205474   09 Jul 2026 As used herein, the term “sarcoma” refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillary, heterogeneous, or homogeneous substance. As used herein, the term “melanoma” refers to a tumor arising from the melanocytic 5 system of the skin and other organs. As used herein, the term “lymphoma” refers to a group of blood cell tumors that develop from lymphocytes. As used herein, the term “blastoma” refers to a type of cancer that is caused by malignancies in precursor cells or blasts (immature or embryonic tissue). 10          As used herein, the recitation of a numerical range for a variable is intended to convey that the invention may be practiced with the variable equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable can be equal to any integer value within the numerical range, including the end-points of the range. Similarly, for a variable which is inherently continuous, the variable can be equal to any real value within the numerical range, 15 including the end-points of the range. As an example, and without limitation, a variable which is described as having values between 0 and 2 can take the values 0, 1 or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other real values =0 and ^2 if the variable is inherently continuous. 20   2.1 Principle of the Invention The present invention is based, in part, on the discovery that microRNA-adapted shRNA (shRNAmiR) molecules can be used to generate genetically-modified immune cells having a stable reduction in expression of an endogenous protein. It is demonstrated herein that the insertion of a nucleic acid sequence encoding a shRNAmiR into the genome of a T cell provides 25 stable knockdown of a spectrum of endogenous proteins including, for example, the stable and effective knockdown of beta-2 microglobulin (B2M), CS1, transforming growth factor beta receptor II (TGFBR2), Cbl proto-oncogene B (CBL-B), deoxycytidine kinase (DCK), glucocorticoid (GR), and cluster of differentiation 52 (CD52). Knockdown of endogenous proteins can confer properties that, in some instances, can be advantageous compared to 30 knockout by gene inactivation. For example, B2M knockdown by shRNAmiR produces CAR T cells that are less allogeneic and less susceptible to natural killer (NK) cell killing than CAR T 2026205474   09 Jul 2026 cells exhibiting a complete knockout of B2M. Further, it is demonstrated that the incorporation of a shRNAmiR molecule into the genome of an immune cell solves the stability and toxicity problems observed with the insertion of a cassette encoding an shRNA molecule. Given the demonstration that shRNAmiR molecules can be used to reduce the expression 5 of multiple endogenous proteins, the presently disclosed compositions and methods can be used to stably knockdown the expression at various degrees of not only the B2M protein, but any endogenous protein of interest, within an immune cell. 2.2 MicroRNA-Adapted shRNA (shRNAmiR) 10          RNA interference (RNAi) or RNA silencing refers to a process by which gene expression is negatively regulated by non-coding RNAs such as microRNAs. The negative regulation can result from one or more of three possible mechanisms: (1) by repressing the translation of target mRNAs, (2) through deadenylation and destabilization of transcripts, and (3) through cleavage and degradation of mRNAs. RNAi is normally triggered by double-stranded RNA (dsRNA) or 15 endogenous microRNA precursors (pri-miRNAs / pre-miRNAs). The production of endogenous microRNA molecules begins with the transcription of a primary miRNA (pri-mRNA) from an RNA polymerase II (Pol II) promoter. Each pri-miRNA can contain from one to six pre-miRNAs. Pre-miRNAs are hairpin loop structures composed of about 70 nucleotides, with each hairpin being flanked by sequences necessary for efficient 20 processing. The enzyme Drosha liberates hairpins from the pri-miRNAs by cleaving RNA about 11 nucleotides from the hairpin base. Pre-miRNAs that are generated by Drosha cleavage comprise a 2 nucleotide overhang at the 3' end. This 2 nucleotide overhang is bound by the Exportin-5 protein, which mediates export of the pre-miRNA from the nucleus into the cytoplasm. In the cytoplasm, pre-miRNA hairpins are cleaved by Dicer through interactions 25 with the 5’ and 3’ ends of the hairpin. Dicer cleaves the pre-miRNA hairpin in the loop region to produce an imperfect miRNA:miRNA duplex, which is about 22 nucleotides in length. A single strand of the miRNA:miRNA duplex (mature miRNA) is incorporated into the RNA-induced silencing complex (RISC) where the miRNA and its mRNA target interact. Since its discovery, RNAi has emerged as a powerful genetic tool for suppressing gene 30 expression in mammalian cells. Gene knockdown can be achieved by expression of synthetic short hairpin RNAs (shRNAs) that mimic pre-miRNAs and are processed by Dicer and fed into 2026205474   09 Jul 2026 the RISC. However, as described herein, shRNAs may not allow for prolonged reduction of protein expression in immune cells. In contrast, expression of the microRNA-adapted shRNA (shRNAmiR) molecules of the present invention result in persistent reduction of protein expression and reduced toxicity effects. The shRNAmiR molecules mimic pri-miRNA 5 molecules in that they comprise a hairpin flanked by sequences necessary for efficient processing, and can be processed by the Drosha enzyme into pri-miRNAs, exported into the cytoplasm, and cleaved by Dicer, after which the mature miRNA can enter the RISC. The present invention provides genetically-modified immune cells expressing a shRNAmiR molecule that reduces the abundance of an endogenous protein. 10          The shRNAmiR molecule can comprise a microRNA scaffold in that the structure of the shRNAmiR molecule can mimic that of a naturally-occurring microRNA (or pri-miRNA or premiRNA) or a variant thereof. Sequences of microRNAs (and pri-miRNAs and pre-miRNAs) are known in the art. Non-limiting examples of suitable miR scaffolds for the presently disclosed shRNAmiRs include miR-E, miR-30 (e.g., miR-30a), miR-15, miR-16, miR-155, miR-22, miR- 15   103, and miR-107. In particular embodiments, the shRNAmiR used in the presently disclosed compositions and methods comprises a mir-E scaffold. The mir-E scaffold is a synthetically-derived variant of miR-30a and its genesis is described in International Publication No. WO 2014 / 117050, which is incorporated by reference in its entirety. The presently disclosed shRNAmiR molecules can comprise the following domains in 20 the 5' to 3' direction: (a) a 5' miR scaffold domain; (b) a 5' miR basal stem domain; (c) a passenger strand; (d) a miR loop domain; (e) a guide strand; (f) a 3' miR basal stem domain; and (g) a 3' miR scaffold domain. The miR scaffold domains and basal stem domains flank the miRNA stem-loop and are referred to herein as microRNA flanking sequences that comprise the microRNA processing elements (the minimal nucleic acid sequences which contribute to the 25   production of mature microRNA from primary microRNA or precursor microRNA). Often these elements are located within a 40 nucleotide sequence that flanks a microRNA stem-loop structure. In some instances, the microRNA processing elements are found within a stretch of nucleotide sequences of between 5 and 4,000 nucleotides in length that flank a microRNA stemloop structure. 30          In some embodiments, the miRNA flanking sequences are about 3 to about 4,000 nt in length and can be present on either or both the 5' and 3' ends of the shRNAmiR molecule. In 2026205474   09 Jul 2026 other embodiments, the minimal length of the microRNA flanking sequence of the shRNAmiR molecule is about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 5   140, about 150, about 200, and any integer therein between. In other embodiments the maximal length of the microRNA flanking sequence of the shRNAmiR molecule is about 2,000, about 2,100, about 2,200, about 2,300, about 2,400, about 2,500, about 2,600, about 2,700, about 2,800, about 2,900, about 3,000, about 3,100, about 3,200, about 3,300, about 3,400, about 3,500, about 3,600, about 3,700, about 3,800, about 3,900, about 4,000, and any integer therein 10 between. The microRNA flanking sequences may be native microRNA flanking sequences or artificial microRNA flanking sequences. A native microRNA flanking sequence is a nucleotide sequence that is ordinarily comprised within naturally existing systems with microRNA sequences (i.e., these sequences are found within the genomic sequences surrounding the 15 minimal microRNA hairpin in vivo). Artificial microRNA flanking sequences are nucleotides sequences that are not found to be flanking microRNA sequences in naturally existing systems. The artificial microRNA flanking sequences may be flanking sequences found naturally in the context of other microRNA sequences. Alternatively, they may be composed of minimal microRNA processing elements which are found within naturally occurring flanking sequences 20 and inserted into other random nucleic acid sequences that do not naturally occur as flanking sequences or only partially occur as natural flanking sequences. In some embodiments, the 5' miR scaffold domain is about 10 to about 150 nucleotides in length, including but not limited to about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, and about 25   150 nucleotides long. In some of these embodiments, the 5' miR scaffold domain is about 111 nucleotides in length. The 5' miR scaffold domain may comprise a 3' sequence that is a recognition sequence for a type IIS restriction enzyme. In some of these embodiments, the 5' miR scaffold domain comprises a XhoI recognition sequence on its 3' end. In particular embodiments, the 5' miR scaffold domain has at least about 50%, at least about 55%, at least 30 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 91%, at least about 92%, at least about 93%, 2026205474   09 Jul 2026 at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the sequence set forth as SEQ ID NO: 1. In certain embodiments, the 5' miR scaffold domain has the sequence set forth as SEQ ID NO: 1. The 5' miR basal stem domain of the shRNAmiR can be about 5 to about 30 nucleotides 5 in length in some embodiments, including but not limited to about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, and about 30 nucleotides long. In some of these embodiments, the 5' miR basal stem domain is about 20 nucleotides in length. In particular embodiments, the 5' miR basal stem 10 domain has 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 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the sequence set forth as SEQ ID NO: 2. In certain embodiments, the 5' 15 miR basal stem domain has the sequence set forth as SEQ ID NO: 2. The shRNAmiR molecules of the presently disclosed compositions and methods comprise a stem-loop structure, wherein the stem is comprised of the hybridized passenger and guide strands and the loop is single-stranded. The miR loop domain can be derived from a naturally-occurring pre-microRNA or pri-microRNA loop sequence or a variant thereof. In 20 some embodiments, the miR loop domain has the sequence of a loop domain from any one of miR-30 (e.g., miR-30a), miR-15, miR-16, miR-155, miR-22, miR-103, and miR-107. In particular embodiments, the shRNAmiR comprises a miR-30a loop domain, the sequence of which is set forth as SEQ ID NO: 3. In certain embodiments, the miR loop domain is about 5 to about 30 nucleotides in 25 length, including but not limited to about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, and about 30 nucleotides long. In some of these embodiments, the miR loop domain is about 15 nucleotides in length. In particular embodiments, the miR loop domain has at least about 50%, 30 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 91%, at least 2026205474   09 Jul 2026 about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the sequence set forth as SEQ ID NO: 3. In certain embodiments, the miR loop domain has the sequence set forth as SEQ ID NO: 3. 5         The 3' miR basal stem domain of the shRNAmiR can be about 5 to about 30 nucleotides in length in some embodiments, including but not limited to about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, and about 30 nucleotides long. In some of these embodiments, the 3' miR basal 10 stem domain is about 18 nucleotides in length. In particular embodiments, the 3' miR basal stem domain has 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 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more 15 sequence identity to the sequence set forth as SEQ ID NO: 4. In certain embodiments, the 3' miR basal stem domain has the sequence set forth as SEQ ID NO: 4. In some embodiments, the 3' miR scaffold domain is about 50 to about 150 nucleotides in length, including but not limited to about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, or about 20   150 nucleotides long. In some of these embodiments, the 3' miR scaffold domain is about 116 nucleotides in length. In particular embodiments, the 3' miR scaffold domain has 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 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, 25 at least about 97%, at least about 98%, at least about 99% or more sequence identity to the sequence set forth as SEQ ID NO: 5. In certain embodiments, the 3' miR scaffold domain has the sequence set forth as SEQ ID NO: 5. The guide strand of the shRNAmiR is the sequence that targets the mRNA, leading to reduction in abundance of the protein encoded by the mRNA. After the guide strand binds to its 30 target mRNA, RISC either degrades the target transcript and / or prevents the target transcript from being loaded into the ribsome for translation. The guide strand is of sufficient 2026205474   09 Jul 2026 complementarity with the target mRNA in order to lead to reduced expression of the target mRNA. In some embodiments, the guide strand is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99% or 100% complementary to the target mRNA sequence. 5 In certain embodiments, the guide strand hybridizes with the target mRNA within a coding sequence. The guide strand can comprise 1, 2, 3, 4, 5, or more mismatching nucleotides with the target mRNA sequence. In other embodiments, the guide strand hybridizes with the target mRNA in a non-coding region, such as a 5' or 3' untranslated region (UTR). In some embodiments, the guide strand is about 15 to about 25 nucleotides in length, including but not 10 limited to about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, and about 25 nucleotides long. In some of these embodiments, the guide strand is about 22 nucleotides in length. In particular embodiments wherein the shRNA sequence from which the shRNAmiR is derived is less than 22 nucleotides in length, which is the length of most naturally-occurring microRNAs, an additional nucleotide is added to the shRNA sequence and in 15 certain embodiments, this additional nucleotide is one that is complementary with the corresponding position within the target mRNA. The passenger strand of the shRNAmiR is the sequence that is fully or partially complementary with the guide strand sequence. In some embodiments, the passenger strand is about 15 to about 25 nucleotides in length, including but not limited to about 15 to about 25 20 nucleotides in length, including but not limited to about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, and about 25 nucleotides long. In some of these embodiments, the passenger strand is about 22 nucleotides in length. The passenger strand can be at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99% or 25   100% complementary to the guide strand sequence. The passenger strand can comprise 1, 2, 3, 4, 5, or more mismatching nucleotides with the guide strand. In certain embodiments, however, the guide:passenger strand duplex does not comprise any mismatching nucleotides. In general, guide / passenger strand sequences should be selected that do not form any secondary structures within themselves. Further, the use of guide / passenger strand sequences that target sites within 30 an mRNA that comprise single-nucleotide polymorphisms should be avoided. Guide / passenger 2026205474   09 Jul 2026 strand sequences that are specific for the target mRNA are preferred to avoid any off-target effects (i.e., reduction in expression of non-target mRNAs). In order to aid in the selection of suitable shRNAmiR guide / passenger strands, or sequences for other shRNAmiR domains, any program known in the art that models the 5 predicted secondary structure of a RNA molecule can be used, including but not limited to Mfold, RNAfold, and UNAFold. Any program known in the art that can predict the efficiency of a shRNA or miRNA guide / passenger sequence to target a particular mRNA can be used to select suitable guide / passenger strand sequences, including but not limited to those disclosed in Agarwal et al. (2015) eLife 4:e05005; and Knott et al. (2014) Mol Cell 56(6):796-807, each of 10 which is incorporated herein in its entirety. 2.3 Genetically-Modified Immune Cells The invention provides genetically-modified immune cells and populations thereof and methods for producing the same. In some embodiments, the genetically-modified immune cells 15 of the presently disclosed compositions and methods are human immune cells. In some embodiments, the immune cells are T cells, or cells derived therefrom. In other embodiments, the immune cells are natural killer (NK) cells, or cells derived therefrom. In still other embodiments, the immune cells are B cells, or cells derived therefrom. In yet other embodiments, the immune cells are monocyte or macrophage cells or cells derived therefrom. 20          Immune cells (e.g., T cells) can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present disclosure, any number of T cell lines, NK cell lines, B cell lines, monocyte cells lines, or macrophage cell lines available in the art may be used. In some 25 embodiments of the present disclosure, immune cells (e.g., T cells) are obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. Immune cells of the invention can also be induced pluripotent stem cell (iPSC)-derived cells that have been differentiated into functional immune cells (e.g., T cells, NK cell, B cells). 2026205474   09 Jul 2026 The genetically-modified immune cells of the presently disclosed compositions and methods comprise in the cells’ genome a nucleic acid sequence encoding a shRNAmiR, leading to the reduction of expression of a target protein. In some of those embodiments wherein the expression of an endogenous protein is 5 reduced by a shRNAmiR, the expression of the endogenous protein is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell (e.g., a cell not expressing a shRNAmiR). Any method known in the art can be used to determine the expression level of an endogenous protein targeted by a shRNAmiR, 10 including but not limited to, ELISA, flow cytometry, Western blot, immunocytochemistry, and immunoprecipitation. Expressing a shRNAmiR by cells within a population can lead to a reduction in the percentage of cells in the population of cells that fully express the endogenous protein to which the shRNAmiR is targeted when compared to a population of control cells. Such a reduction 15 may be up to 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or up to 100% of cells in the population. A nucleic acid sequence encoding a shRNAmiR can be present in the genome of the genetically-modified immune cell, for example, in a cassette. Such cassettes can be inserted into the genome, for example, by introducing a template nucleic acid of the invention either by 20 random integration (e.g., lentiviral transduction) or by targeted insertion into a selected site (e.g., by nuclease-mediated targeted insertion). Cassettes comprising the shRNAmiR-encoding sequence can include, for example, nucleic acid sequences encoding additional proteins, such as those described herein (e.g., CARs, exogenous TCRs, fusion proteins), and may also include control sequences such as promoters and termination sequences. The nucleic acid sequence 25 encoding the shRNAmiR can be positioned at any number of locations within the cassette or template nucleic acid that allow for expression of the shRNAmiR. In some examples, a nucleic acid sequence encoding a shRNAmiR is positioned between the stop codon of another transgene (e.g., a nucleic acid sequence encoding a CAR, exogenous TCR, or fusion protein) and a termination signal. In other examples, another transgene present in the cassette or template 30 nucleic acid (e.g., a nucleic acid sequence encoding a CAR, exogenous TCR, or fusion protein) comprises an intron that is positioned within the transgene sequence. Here, “positioned” is 2026205474   09 Jul 2026 intended to mean that the intron sequence is inserted into the transgene sequence, such that the resulting sequence comprises a 5' portion of the transgene, the intron sequence, and a 3' portion of the transgene. In some such examples, the nucleic acid sequence encoding the shRNAmiR can be positioned within such an intron. Here, “positioned” is intended to mean that the shRNAmiR- 5 encoding sequence is inserted into the intron sequence, such that the resulting sequence comprises a 5' portion of the intron sequence, the shRNAmiR-encoding sequence, and a 3' portion of the intron sequence. In such cases, the shRNAmiR is expressed and the intron sequence is spliced out by the cell when the transgene is expressed. Introns that can be included in this manner can be naturally-occurring introns or, alternatively, synthetic introns. A particular 10 example of a synthetic intron useful for the invention is encoded by SEQ ID NO: 69. In certain embodiments, the genetically-modified immune cell can further comprise in its genome a nucleic acid sequence encoding a CAR or an exogenous TCR. In some embodiments, the genetically-modified immune cell can further comprise in its genome a nucleic acid sequence encoding an HLA-E fusion protein capable of being expressed on the immune cell surface. 15          The CAR / TCR-encoding nucleic acid sequence and / or the nucleic acid sequence encoding the HLA-E fusion protein can be located within the same gene as the shRNAmiR-encoding sequence. Alternatively, the CAR / TCR-encoding nucleic acid sequence and / or the nucleic acid sequence encoding the HLA-E fusion protein can be located within a different gene as the shRNAmiR-encoding sequence. 20          Each of the coding sequences can be operably linked to different promoters. In other embodiments, the shRNAmiR-encoding sequence is operably linked to the same promoter as the nucleic acid sequence encoding the CAR or exogenous TCR and / or the nucleic acid sequence encoding the HLA-E fusion protein. In some specific examples, where a nucleic acid sequence encoding a shRNAmiR, a nucleic acid sequence encoding a CAR or exogenous TCR, and a 25 nucleic acid sequence encoding an HLA-E fusion protein are all located within the same gene, two of the nucleic acid sequences are operably linked to a first promoter, and the third nucleic acid sequence is operably linked to a second promoter. In other specific examples, where a nucleic acid sequence encoding a shRNAmiR, a nucleic acid sequence encoding a CAR or exogenous TCR, and a nucleic acid sequence encoding an HLA-E fusion protein are all located 30 within the same gene, all three of the nucleic acid sequences are operably linked to the same promoter. In various embodiments, the nucleic acid sequences can be operably linked to an 2026205474   09 Jul 2026 endogenous promoter following insertion into the genome. In some such cases, the cassettes or template nucleic acids of the invention may not require an exogenous promoter in order for the encoded sequences to be expressed. Further, in such cases, the cassettes or template nucleic acids may comprise elements (e.g., splice acceptor sequences, 2A or IRES sequences, and the 5 like) necessary for the nucleic acids to be operably linked to the endogenous promoter. In other embodiments, the cassettes or template nucleic acids of the invention comprise one or more exogenous promoters that are operably linked to the nucleic acid sequences and drive expression of the shRNAmiR, CAR or exogenous TCR, and / or HLA-E fusion protein. Each of the coding sequences can be present in the genome in the same orientation or in 10 different orientations from each other. For example, one coding sequence can be on the plus strand of the double-stranded DNA and another coding sequence on the minus strand. In some embodiments, the shRNAmiR-encoding nucleic acid sequence is 3' downstream of the nucleic acid sequence encoding the CAR or exogenous TCR and / or the nucleic acid sequence encoding the HLA-E fusion protein. In alternative embodiments, the shRNAmiR-encoding sequence is 5' 15 upstream of the CAR / TCR-encoding sequence and / or the nucleic acid sequence encoding the HLA-E fusion protein. In certain embodiments, nucleic acid sequences, such as those encoding a CAR or exogenous TCR, a shRNAmiR, and / or an HLA-E fusion protein, are operably linked to the same promoter and are separated by any element known in the art to allow for the translation of two or 20 more genes (i.e., cistrons) from the same nucleic acid molecule. Such elements can include, but are not limited to, an IRES element, a T2A element, a P2A element (e.g., P2A / furin), an E2A element, and an F2A element. In certain embodiments, the genetically-modified immune cell comprises a nucleic acid sequence encoding a cell-surface protein that protects the immune cell from NK cell killing. In 25 some examples, the nucleic acid sequence encodes a non-classical MHC I protein. Non-classical MHC class I proteins can include, without limitation, HLA-E, HLA-F, HLA-G, and HLA-H. In particular examples, the nucleic acid sequence encodes an HLA-E protein. Examples of HLA-E proteins include, without limitation, an HLA-E-01:01 protein or an HLA-E-01:03 protein (e.g., SEQ ID NO: 118). In particular examples of the invention, the nucleic acid sequence encodes a 30 fusion protein comprising a non-classical MHC class I protein (e.g., HLA-E) and at least one additional protein that enables expression of the MHC class I protein on the cell-surface of the 2026205474   09 Jul 2026 immune cell. The fusion protein can comprise, for example, an MHC class I protein (e.g., HLA-E) fused to a beta-2 microglobulin protein (e.g., SEQ ID NO: 119), that enables expression of the MHC class I protein on the cell-surface. In order to inhibit expression of endogenous beta-2 microglobulin, and not expression of a fusion protein that comprises beta-2 microglobulin, the 5 beta-2 microglobulin coding sequence in the fusion protein can be altered (e.g., codon optimized) such that the shRNAmiR does not have specificity for the altered sequence. In further examples, the fusion protein can comprise a non-classical MHC class I protein (e.g., HLA-E) fused to both a beta-2 microglobulin protein and an additional protein that is presented extracellularly by the non-classical MHC. Such additional proteins can include, for example, an HLA-G leader 10 peptide (e.g., SEQ ID NO: 120). The individual proteins of the fusion protein can be fused by polypeptide linkers such as, for example, a linker comprising SEQ ID NO: 121 (i.e., a (GGGGS)3 linker) or SEQ ID NO: 122 (i.e., a (GGGGS)4 linker). In specific embodiments, the fusion protein is an HLA-E fusion protein comprising an HLA-E protein, a beta-2 microglobulin protein, and an HLA-G leader peptide. In some such 15 embodiments, the HLA-E protein is an HLA-E-01:01 protein or an HLA-E-01:03 protein, and in particular embodiments, the HLA-E protein is an HLA-E-01:03 protein having an amino acid sequence of SEQ ID NO: 118. In some such embodiments, the beta-2 microglobulin protein has an amino acid sequence of SEQ ID NO: 119, and the HLA-G leader peptide has an amino acid sequence of SEQ ID NO: 120. In further such embodiments, the HLA-E protein, the beta-2 20 microglobulin protein, and the HLA-G leader protein are fused by polypeptide linkers comprising SEQ ID NO: 121 (i.e., a (GGGGS)3 linker) or SEQ ID NO: 122 (i.e., a (GGGGS)4 linker). In a particular embodiment, the fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or up to at least 99% sequence identity to 25 SEQ ID NO: 66. In a specific embodiment, the fusion protein comprises an amino acid sequence of SEQ ID NO: 66. In certain embodiments, the genetically-modified immune cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR). Generally, a CAR of the present disclosure will comprise at least an extracellular domain, a transmembrane domain, and an 30 intracellular domain. In some embodiments, the extracellular domain comprises a target-specific binding element otherwise referred to as an extracellular ligand-binding domain or moiety. In 2026205474   09 Jul 2026 some embodiments, the intracellular domain, or cytoplasmic domain, comprises at least one costimulatory domain and one or more signaling domains. In some embodiments, a CAR useful in the invention comprises an extracellular ligandbinding domain. The choice of ligand-binding domain depends upon the type and number of 5 ligands that define the surface of a target cell. For example, the ligand-binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Thus, some examples of cell surface markers that may act as ligands for the ligand-binding domain in a CAR can include those associated with viruses, bacterial and parasitic infections, autoimmune disease, and cancer cells. In some embodiments, a CAR is 10 engineered to target a cancer-specific antigen of interest by way of engineering a desired ligandbinding moiety that specifically binds to an antigen on a cancer (i.e., tumor) cell. In the context of the present disclosure, “cancer antigen,” tumor antigen,” “cancer-specific antigen,” or “tumorspecific antigen” refer to antigens that are common to specific hyperproliferative disorders such as cancer. 15          In some embodiments, the extracellular ligand-binding domain of the CAR is specific for any antigen or epitope of interest, particularly any tumor antigen or epitope of interest. As nonlimiting examples, in some embodiments the antigen of the target is a tumor-associated surface antigen, such as ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), 20   CD19, CD20, CD22, CD30, CD40, CD79B, IL1RAP, glypican 3 (GPC3), CLL-1, disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, B-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostase specific antigen 25 (PSA), PAP, NY-ESO-1, LAGA-la, p53, prostein, PSMA, surviving and telomerase, prostatecarcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGFl)-l, IGF-II, IGFI receptor, mesothelin, a major histocompatibility complex (MHC) molecule presenting a tumor-specific peptide epitope, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigens, the extra domain A (EDA) and extra domain B (EDB) of fibronectin and 30 the Al domain of tenascin-C (TnC Al) and fibroblast associated protein (fap); a lineage-specific or tissue specific antigen such as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD38, CD123, 2026205474   09 Jul 2026 CD133, CD138, CTLA-4, B7- 1 (CD80), B7-2 (CD86), endoglin, a major histocompatibility complex (MHC) molecule, BCMA (CD269, TNFRSF 17), CS1, or a virus-specific surface antigen such as an HIV-specific antigen (such as HIV gpl20); an EBV-specific antigen, a CMV-specific antigen, a HPV-specific antigen such as the E6 or E7 oncoproteins, a Lasse Virus- 5 specific antigen, an Influenza Virus-specific antigen, as well as any derivate or variant of these surface markers. In some examples, the extracellular ligand-binding domain or moiety is an antibody, or antibody fragment. An antibody fragment can, for example, be at least one portion of an antibody, that retains the ability to specifically interact with (e.g., by binding, steric hindrance, 10 stabilizing / destabilizing, spatial distribution) an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab‘)2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, multi-specific antibodies formed from antibody fragments such as a bivalent fragment 15 comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody. An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen binding fragments can also be grafted into 20 scaffolds based on polypeptides such as a fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies). In some embodiments, the extracellular ligand-binding domain or moiety is in the form of a single-chain variable fragment (scFv) derived from a monoclonal antibody, which provides specificity for a particular epitope or antigen (e.g., an epitope or antigen preferentially present on 25 the surface of a cell, such as a cancer cell or other disease-causing cell or particle). In some such embodiments, the scFv can comprise a heavy chain variable (VH) domain and a light chain variable (VL) domain from a monoclonal antibody having specificity for an antigen. In some embodiments, the scFv is attached via a linker sequence. In some embodiments, the scFv is murine, humanized, or fully human. 30          The extracellular ligand-binding domain of a chimeric antigen receptor can also comprise an autoantigen (see, Payne et al. (2016), Science 353 (6295): 179-184), that can be recognized 2026205474   09 Jul 2026 by autoantigen-specific B cell receptors on B lymphocytes, thus directing T cells to specifically target and kill autoreactive B lymphocytes in antibody-mediated autoimmune diseases. Such CARs can be referred to as chimeric autoantibody receptors (CAARs), and their use is encompassed by the invention. 5          In some embodiments, the extracellular domain of a chimeric antigen receptor can comprise a naturally-occurring ligand for an antigen of interest, or a fragment of a naturally-occurring ligand which retains the ability to bind the antigen of interest. A CAR can comprise a transmembrane domain which links the extracellular ligandbinding domain with the intracellular signaling and co-stimulatory domains via a hinge region or 10 spacer sequence. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. For example, the transmembrane polypeptide can be a subunit of the T-cell receptor (e.g., an a, 0, y or Z, polypeptide constituting CD3 complex), IL2 receptor p55 (a chain), p75 (0 chain) or y chain, subunit chain of Fc receptors (e.g., Fcy receptor III) or CD proteins such as the CD8 alpha chain. In certain examples, the transmembrane domain is a CD8 15 alpha domain. Alternatively, the transmembrane domain can be synthetic and can comprise predominantly hydrophobic residues such as leucine and valine. The hinge region refers to any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. For example, a hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 20   50 amino acids. Hinge regions may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region. Alternatively, the hinge region may be a synthetic sequence that corresponds to a naturally occurring hinge sequence or may be an entirely synthetic hinge sequence. In particular examples, a hinge domain can comprise a part of a human CD8 alpha 25 chain, FcyRllla receptor or IgGl. In certain examples, the hinge region can be a CD8 alpha domain. Intracellular signaling domains of a CAR are responsible for activation of at least one of the normal effector functions of the cell in which the CAR has been placed and / or activation of proliferative and cell survival pathways. The term “effector function” refers to a specialized 30 function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. The intracellular stimulatory domain can include 2026205474   09 Jul 2026 one or more cytoplasmic signaling domains that transmit an activation signal to the T cell following antigen binding. Such cytoplasmic signaling domains can include, without limitation, a CD3 zeta signaling domain. The intracellular stimulatory domain can also include one or more intracellular co- 5 stimulatory domains that transmit a proliferative and / or cell-survival signal after ligand binding. In some cases, the co-stimulatory domain can comprise one or more TRAF-binding domains. Such intracellular co-stimulatory domains can be any of those known in the art and can include, without limitation, those co-stimulatory domains disclosed in WO 2018 / 067697 including, for example, Novel 6 (“N6”). Further examples of co-stimulatory domains can include 4-1BB 10   (CD137), CD27, CD28, CD8, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function- associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, or any combination thereof. In a particular embodiment, the co-stimulatory domain is an N6 domain. In another particular embodiment, the co-stimulatory domain is a 4-1BB co-stimulatory domain. 15          In other embodiments, the genetically-modified immune cell comprises a nucleic acid sequence encoding an exogenous T cell receptor (TCR). Such exogenous T cell receptors can comprise alpha and beta chains or, alternatively, may comprise gamma and delta chains. Exogenous TCRs useful in the invention may have specificity to any antigen or epitope of interest such as, without limitation, any antigen or epitope disclosed herein. 20          In particular embodiments, the CAR or the exogenous TCR can be specific for any type of cancer cell. Such cancers can include, without limitation, carcinoma, lymphoma, sarcoma, blastomas, leukemia, cancers of B cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin lymphoma. In specific 25 embodiments, cancers and disorders include but are not limited to pre-B ALL (pediatric indication), adult ALL, mantle cell lymphoma, diffuse large B cell lymphoma, salvage post allogenic bone marrow transplantation, and the like. These cancers can be treated using a combination of CARs that target, for example, CD19, CD20, CD22, and / or ROR1. In some nonlimiting examples, a genetically-modified immune cell or population thereof of the present 30 disclosure targets carcinomas, lymphomas, sarcomas, melanomas, blastomas, leukemias, and germ cell tumors, including but not limited to cancers of B-cell origin, neuroblastoma, 2026205474   09 Jul 2026 osteosarcoma, prostate cancer, renal cell carcinoma, liver cancer, gastric cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, breast cancer, lung cancer, cutaneous or intraocular malignant melanoma, renal cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, 5 uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, non-Hodgkin lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, lymphocytic 10 lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, environmentally induced cancers including those induced by asbestos, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphomas, acute myeloid lymphoma, 15 chronic myelogenous leukemia, chronic lymphoid leukemia, immunoblastic large cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, and T-cell lymphoma, and any combinations of said cancers. In certain embodiments, cancers of B-cell origin include, without limitation, B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell lymphoma, diffuse large B cell lymphoma, pre-B ALL (pediatric 20 indication), mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, Burkitt’s lymphoma, multiple myeloma, and B-cell non-Hodgkin lymphoma. In some examples, cancers can include, without limitation, cancers of B cell origin or multiple myeloma. In some examples, the cancer of B cell origin is acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), or non-Hodgkin lymphoma (NHL). In 25 some examples, the cancer of B cell origin is mantle cell lymphoma (MCL) or diffuse large B cell lymphoma (DLBCL). In some embodiments, genetically-modified immune cells of the invention comprise an inactivated TCR alpha gene and / or an inactivated TCR beta gene. Inactivation of the TCR alpha gene and / or TCR beta gene to generate the genetically-modified cells of the present invention 30 occurs in at least one or both alleles where the TCR alpha gene and / or TCR beta gene is being expressed. Accordingly, inactivation of one or both genes prevents expression of the 2026205474   09 Jul 2026 endogenous TCR alpha chain or the endogenous TCR beta chain protein. Expression of these proteins is required for assembly of the endogenous alpha / beta TCR on the cell surface. Thus, inactivation of the TCR alpha gene and / or the TCR beta gene results in genetically-modified immune that have no detectable cell surface expression of the endogenous alpha / beta TCR. The 5 endogenous alpha / beta TCR incorporates CD3. Therefore, cells with an inactivated TCR alpha gene and / or TCR beta chain can have no detectable cell surface expression of CD3. In particular embodiments, the inactivated gene is a TCR alpha constant region (TRAC) gene. In some examples, the TCR alpha gene, the TRAC gene, or the TCR beta gene is inactivated by insertion of a template nucleic acid into a cleavage site in the gene. Insertion of 10 the template nucleic acid disrupts expression of the endogenous TCR alpha chain or TCR beta chain and, therefore, prevents assembly of an endogenous alpha / beta TCR on the T cell surface. In some examples, the template nucleic acid is inserted into the TRAC gene. In a particular example, a template nucleic acid is inserted into the TRAC gene at an engineered meganuclease recognition sequence comprising SEQ ID NO: 58 (i.e., the TRC 1-2 recognition sequence). In 15 particular examples, the CAR transgene is inserted into SEQ ID NO: 58 between nucleotide positions 13 and 14. In some of those embodiments wherein the genetically-modified immune cell expresses a CAR or exogenous TCR, such cells have no detectable cell-surface expression of an endogenous T cell receptor (e.g., an alpha / beta T cell receptor). Thus, the invention further provides a 20 population of genetically-modified immune cells that express a shRNAmiR and have no detectable cell-surface expression of an endogenous T cell receptor (e.g., an alpha / beta T cell receptor), and in some embodiments also express a CAR or exogenous TCR. For example, the population can include a plurality of genetically-modified immune cells of the invention which express a CAR (i.e., are CAR+), or an exogenous T cell receptor (i.e., exoTCR+), and have no 25 cell-surface expression of an endogenous T cell receptor (i.e., are TCR-). As used herein, “detectable cell-surface expression of an endogenous TCR” refers to the ability to detect one or more components of the TCR complex (e.g., an alpha / beta TCR complex) on the cell surface of an immune cell using standard experimental methods. Such methods can include, for example, immunostaining and / or flow cytometry specific for components of the 30 TCR itself, such as a TCR alpha or TCR beta chain, or for components of the assembled cellsurface TCR complex, such as CD3. Methods for detecting cell-surface expression of an 2026205474   09 Jul 2026 endogenous TCR (e.g., an alpha / beta TCR) on an immune cell include those described in the examples herein, and, for example, those described in MacLeod et al. (2017) Molecular Therapy 25(4): 949-961. 5   2.4 shRNAmiR Target Proteins The genetically-modified immune cells of the presently disclosed compositions and methods can comprise and express a shRNAmiR that reduces the expression of any endogenous protein. Non-limiting examples of endogenous proteins whose expression can be reduced with a shRNAmiR include beta-2 microglobulin (B2M), transforming growth factor beta receptor 2 10 (TGFBR2), Cbl proto-oncogene B (CBL-B), CS1, CD52, deoxycytidine kinase (DCK), glucocorticoid receptor (GR), a T cell receptor alpha gene, and a T cell receptor alpha constant region gene. A. Beta-2 Microglobulin 15          In some embodiments, the endogenous protein with reduced expression levels as the result of the expression of a shRNAmiR molecule is B2M. B2M is a component of the major histocompatibility complex (MHC) class I molecule, which will not assemble on the cell surface without B2M present. MHC class I molecules are comprised of al, a2, and a3 proteins, in addition to B2M. Within MHC class I molecules, the B2M protein is situated beside the a3 20 protein and below the al protein on the cell surface. B2M lacks a transmembrane region and is necessary for the stability of the peptide-binding groove of MHC class I molecules. The shRNAmiR molecule may target any region of a B2M mRNA. Representative B2M mRNA and protein sequences are known in the art. A non-limiting example of a B2M mRNA sequence is NCBI Acc. No. NM_004048.3 and a B2M protein sequence is NCBI Acc. No. 25   NP_004039.1. In some of those embodiments wherein the expression of B2M is reduced by a shRNAmiR, the cell surface expression of B2M is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell 30   (e.g., a cell not expressing a B2M-targeted shRNAmiR). 2026205474   09 Jul 2026 Given that B2M is necessary for the assembly of the MHC class I molecule on the cell surface, cells with reduced expression of B2M also exhibit a reduction in MHC class I molecules on the cell surface. In some of these embodiments, the expression of MHC class I molecules is reduced on the cell surface by at least about 10%, about 20%, about 30%, about 40%, about 5   50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell (e.g., a cell not expressing a B2M-targeted shRNAmiR). shRNAmiR molecules that target B2M may comprise any passenger and corresponding guide sequence that is complementary (fully or partially) to a sequence within the B2M gene. In 10 some embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 17 and 18, respectively (e.g., B2M 7289 shRNAmiR). In particular embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 7 and 8, respectively (e.g., B2M 7282 shRNAmiR). In other embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID 15 NO: 9 and 10, respectively (e.g., B2M 7285 shRNAmiR). In still other embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 11 and 12, respectively (e.g., B2M 7286 shRNAmiR). In yet other embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 13 and 14, respectively (e.g., B2M 7287 shRNAmiR). In particular embodiments, the 20 passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 15 and 16, respectively (e.g., B2M 7288 shRNAmiR). In certain embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 19 and 20, respectively (e.g., B2M 7290 shRNAmiR). The B2M-targeted shRNAmiR may comprise a sequence having at least 50%, at least 25   55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NOs: 41-47. In particular embodiments, the shRNAmiR comprises the sequence set forth in any one of SEQ ID NOs: 41-47. In some embodiments, the shRNAmiR 30 comprises the sequence set forth in SEQ ID NO: 41. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 42. In some embodiments, the shRNAmiR 2026205474   09 Jul 2026 comprises the sequence set forth in SEQ ID NO: 43. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 44. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 45. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 46. In some embodiments, the shRNAmiR 5 comprises the sequence set forth in SEQ ID NO: 47. Cells with reduced levels of B2M and MHC class I molecules can exhibit reduced allogenicity compared to a control cell (e.g., a cell not expressing a B2M-targeted shRNAmiR). As used herein, the term “allogenicity” refers to the ability of a cell to be recognized and acted upon by the immune system as “other” or not autologous. Allogenicity can be measured using 10 any method known in the art, including those methods described elsewhere herein wherein the percentage of living cells were quantitated after incubation with primed alloantigen-specific CTLs or NK cells. B.    CS1 15          In some embodiments, the endogenous protein with reduced expression levels as the result of the expression of a shRNAmiR molecule is CS1 (also known as CCND3 subset 1, CRACC, CD319, and SLAMF7). CS1 is a member of the signaling lymphocyte activatingmolecule (SLAM)-related receptor family and is expressed on the surface of normal NK cells, B cells, T cells, dendritic cells, NK-T cells, and monocytes. CS1 is overexpressed by multiple 20 myeloma cells and can serve as an immunotherapeutic target for multiple myeloma. The shRNAmiR molecule may target any region of a CS1 mRNA. Representative CS1 mRNA and protein sequences are known in the art. A non-limiting example of a CS1 mRNA sequence is NCBI Acc. No. NM_021181 and a CS1 protein sequence is NCBI Acc. No. NP_067004.3. 25          In some of those embodiments wherein the expression of CS1 is reduced by a shRNAmiR, the cell surface expression of CS1 is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell (e.g., a cell not expressing a CS1-targeted shRNAmiR). 30         shRNAmiR molecules that target CS1 may comprise any passenger and corresponding guide sequence that is complementary (fully or partially) to a sequence within the CS1 gene. In 2026205474   09 Jul 2026 some embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 21 and 22, respectively. In some embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 23 and 24, respectively. In some embodiments, the passenger and guide sequence of the shRNAmiR 5 comprise the sequences set forth as SEQ ID NO: 25 and 26, respectively. The CS1-targeted shRNAmiR may comprise a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to the nucleic acid sequence set forth 10 in any one of SEQ ID NOs: 48-50. In particular embodiments, the shRNAmiR comprises the sequence set forth in any one of SEQ ID NOs: 48-50. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 48. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 49. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 50. 15          In some of the embodiments wherein the genetically-modified cell expresses a shRNAmiR that reduces the expression of CS1, the genetically-modified immune cell comprises a CAR having specificity for CS1. Non-limiting examples of CARs having specificity for CS1 include, without limitation, those described in WO 2014 / 179759, WO2015121454, and WO 2016 / 090369. 20         Cells having the expression of CS1 knocked down via shRNAmiR expression can be less susceptible to fratricide by a genetically-modified immune cell expressing a CAR having specificity for CS1 as compared to a control cell (e.g., a cell not expressing a CS1-targeted shRNAmiR). This is useful when using CAR-expressing cells with specificity for CS1 for the treatment of a disease, such as multiple myeloma, wherein prolonged presence of the CAR- 25 expressing cell and thus, killing of the diseased cells (e.g., multiple myeloma cell) is desired. As used herein, the term “fratricide” refers to the killing of cells by cells of like genotype and / or phenotype. Fratricide by a genetically-modified immune cell expressing a CS1-specific CAR can be measured using any method known in the art, including but not limited to incubation of immune cells expressing the CS1-specific shRNAmiR with immune cells expressing a CS1- 30 specific CAR and quantitating the number of living shRNAmiR-expressing cells. 2026205474   09 Jul 2026 C.   TGFBR2 In some embodiments, the endogenous protein with reduced expression levels as the result of the expression of a shRNAmiR molecule is transforming growth factor beta receptor 2 (TGFBR2). TGFBR2 is a transmembrane receptor that binds transforming growth factor-beta 5 (TGFB). TGFBR2 comprises a serine / threonine protein kinase domain and heterodimerizes with other TGFB receptors. The shRNAmiR molecule may target any region of a TGFBR2 mRNA. Representative B2M mRNA and protein sequences are known in the art. A non-limiting example of a TGFBR2 mRNA sequence is NM_001024847.2 and a TGFBR2 protein sequence is NCBI Acc. No. 10   NP_001020018.1. _ In some of those embodiments wherein the expression of TGFBR2 is reduced by a shRNAmiR, the cell surface expression of TGFBR2 is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control 15 cell (e.g., a cell not expressing a TGFBR2-targeted shRNAmiR). shRNAmiR molecules that target TGFBR2 may comprise any passenger and corresponding guide sequence that is complementary (fully or partially) to a sequence within the TGFBR2 gene. In some embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 27 and 28, respectively (e.g., TGFBR2 721110 20 shRNAmiR). In other embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 29 and 30, respectively (e.g., TGFBR2 721111 shRNAmiR). In still other embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 31 and 32, respectively (e.g., TGFBR2 721112 shRNAmiR). In yet other embodiments, the passenger and guide sequence of the shRNAmiR 25 comprise the sequences set forth as SEQ ID NO: 33 and 34, respectively (e.g., TGFBR2 721113 shRNAmiR). In particular embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 35 and 36, respectively (e.g., TGFBR2 721114 shRNAmiR). The TGFBR2-targeted shRNAmiR may comprise a sequence having at least 50%, at 30 least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at 2026205474   09 Jul 2026 least 97%, at least 98%, at least 99%, or more sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NOs: 51-55. In particular embodiments, the shRNAmiR comprises the sequence set forth in any one of SEQ ID NOs: 51-55. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 51. In some embodiments, the shRNAmiR 5 comprises the sequence set forth in SEQ ID NO: 52. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 53. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 54. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 55. In some of the embodiments wherein the genetically-modified immune cell expresses a 10 shRNAmiR that reduces the expression of TGFBR2, the genetically-modified immune cell is less susceptible to immunosuppression by transforming growth factor B1 (TGFB1) compared to a control cell (e.g., an immune cell not expressing a TGFBR2-targeted shRNAmiR). As used herein, the term “immunosuppression” refers to the reduction of the activation or efficacy of the immune system. Immunosuppression by TGFB1 can be measured using any method known in 15 the art, including but not limited to measuring the effects of TGFB1 on T cell differentiation and / or cytokine production. D.    CBL-B In some embodiments, the endogenous protein with reduced expression levels as the 20 result of the expression of a shRNAmiR molecule is Cbl proto-oncogene B (CBL-B). CBL-B is an E3 ubiquitin ligase that catalyzes the attachment of ubiquitin to a protein, thus targeting the protein for degradation. The shRNAmiR molecule may target any region of a CBL-B mRNA. Representative CBL-B mRNA and protein sequences are known in the art. A non-limiting example of a CBL-B 25 mRNA sequence is NCBI Acc. No. NM_170662.5 and a CBL-B protein sequence is NCBI Acc. No. NP_733762.2. In some of those embodiments wherein the expression of CBL-B is reduced by a shRNAmiR, the cell surface expression of CBL-B is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, 30 about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell (e.g., a cell not expressing a CBL-B-targeted shRNAmiR). 2026205474   09 Jul 2026 shRNAmiR molecules that target CBL-B may comprise any passenger and corresponding guide sequence that is complementary (fully or partially) to a sequence within the CBL-B gene. In some of the embodiments wherein the genetically-modified immune cell expresses a shRNAmiR that reduces the expression of CBL-B, the genetically-modified immune cell is less 5 susceptible to suppression of T cell receptor (TCR) signaling by degradation of downstream signaling proteins compared to a control cell (e.g., an immune cell not expressing a CBL-B-targeted shRNAmiR). CBL proteins regulates the turnover of p85 (a regulatory subunit of PI3K), phospholipase C-g, and tyrosine kinases, such as Lck, Fyn, and ZAP70, all of which are involved in TCR signaling (Lee et al. (2003) Science 302:1218-1222). Susceptibility to suppression of 10 TCR signaling by degradation of downstream signaling proteins can be measured using any method known in the art, including but not limited to ELISA, flow cytometry, Western blot, immunocytochemistry, and immunoprecipitation. E.    CD52 15          In some embodiments, the endogenous protein with reduced expression levels as the result of the expression of a shRNAmiR molecule is CD52 (cluster of differentiation 52), which is also known as CAmPATH-1 antigen. CD52 is a glycoprotein present on the surface of mature lymphocytes and on monocytes and dendritic cells. Soluble CD52 molecules interact with sialic acid-binding immunoglobulin-like lectin 10 (Siglec10) to inhibit T cell proliferation and 20 activation (Zhao et al. (2017) Inflamm Res 66(7):571-578). The shRNAmiR molecule may target any region of a CD52 mRNA. Representative CD52 mRNA and protein sequences are known in the art. A non-limiting example of a CD52 mRNA sequence is NCBI Acc. No. NM_001803.3 and a CD52 protein sequence is NCBI Acc. No. NP_001794.2. 25         In some of those embodiments wherein the expression of CD52 is reduced by a shRNAmiR, the cell surface expression of CD52 is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell (e.g., a cell not expressing a CD52-targeted shRNAmiR). 30         shRNAmiR molecules that target CD52 may comprise any passenger and corresponding guide sequence that is complementary (fully or partially) to a sequence within the CD52 gene. 2026205474   09 Jul 2026 In some embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 37 and 38, respectively (e.g., CD52 72123 shRNAmiR). In other embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 39 and 40, respectively (e.g., CD52 72124 shRNAmiR). 5         The CD52-targeted shRNAmiR may comprise a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to the nucleic acid sequence set forth as SEQ ID NO: 56 or 57. In particular embodiments, the shRNAmiR comprises the sequence set 10 forth as SEQ ID NO: 56 or 57. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 56. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 57. In some of the embodiments wherein the genetically-modified immune cell expresses a shRNAmiR that reduces the expression of CD52, the genetically-modified immune cell is less 15 susceptible to CD52 antibody-induced cell death. F.    Deoxycytidine kinase (DCK) In some embodiments, the endogenous protein with reduced expression levels as the result of the expression of a shRNAmiR molecule is deoxycytidine kinase (DCK). DCK 20 predominantly phosphorylates deoxycytidine and converts it into deoxycytidine monophosphate. The shRNAmiR molecule may target any region of a DCK mRNA. Representative DCK mRNA and protein sequences are known in the art. A non-limiting example of a DCK mRNA sequence is NCBI Acc. No. NM_000788.3 and a DCK protein sequence is NCBI Acc. No. NP_000779.1. 25         In some of those embodiments wherein the expression of DCK is reduced by a shRNAmiR, the cell surface expression of DCK is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell (e.g., a cell not expressing a DCK-targeted shRNAmiR). 30         shRNAmiR molecules that target DCK may comprise any passenger and corresponding guide sequence that is complementary (fully or partially) to a sequence within the DCK gene. In 2026205474   09 Jul 2026 some embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 76 and 77, respectively (e.g., DCK 72136 shRNAmiR). In other embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 78 and 79, respectively (e.g., DCK 72137 shRNAmiR). In other 5 embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 80 and 81, respectively (e.g., DCK 72138 shRNAmiR). In other embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 82 and 83, respectively (e.g., DCK 72139 shRNAmiR). In other embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set 10 forth as SEQ ID NO: 84 and 85, respectively (e.g., DCK 72140 shRNAmiR). The DCK-targeted shRNAmiR may comprise a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to the nucleic acid sequence set forth 15 in any one of SEQ ID NOs: 86-90. In particular embodiments, the shRNAmiR comprises the sequence set forth in any one of SEQ ID NOs: 86-90. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 86. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 87. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 88. In some embodiments, the shRNAmiR 20 comprises the sequence set forth in SEQ ID NO: 89. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 90. In some of the embodiments wherein the genetically-modified immune cell expresses a shRNAmiR that reduces the expression of DCK, the genetically-modified immune cell is less susceptible to effects of purine nucleoside analogs (e.g., fludarabine) on cell proliferation. 25 Indeed, genetically-modified immune cells having reduced expression of DCK can be enriched by incubation of a cell population with a purine nucleoside analog such as fludarabine. Further, genetically-modified immune cells (e.g., CAR T cells) having reduced expression of DCK may have greater persistence in vivo during immunotherapy when a purine nucleoside analog such as fludarabine is administered during the course of therapy. 30 2026205474   09 Jul 2026 G. Glucocorticoid receptor (GR) In some embodiments, the endogenous protein with reduced expression levels as the result of the expression of a shRNAmiR molecule is glucocorticoid receptor (GR). Binding of glucocorticoids, such as cortisol or dexamethasone, can induce the release of protein, such as 5 heat shock proteins, that can lead to transactivation or transrepression in the cell. The shRNAmiR molecule may target any region of a GR mRNA. Representative GR mRNA and protein sequences are known in the art. A non-limiting example of a GR mRNA sequence is NCBI Acc. No. AM183262.1. In some of those embodiments wherein the expression of GR is reduced by a shRNAmiR, 10 the expression of GR is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell (e.g., a cell not expressing a GR-targeted shRNAmiR). shRNAmiR molecules that target GR may comprise any passenger and corresponding 15 guide sequence that is complementary (fully or partially) to a sequence within the GR gene. In some embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 91 and 92, respectively (e.g., GR 72142 shRNAmiR). In other embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 93 and 94, respectively (e.g., GR 72143 shRNAmiR). In other 20 embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 95 and 96, respectively (e.g., GR 72145 shRNAmiR). In other embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 97 and 98, respectively (e.g., GR 72146 shRNAmiR). In other embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set 25 forth as SEQ ID NO: 99 and 100, respectively (e.g., GR 72148 shRNAmiR). In other embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 101 and 102, respectively (e.g., GR 72149 shRNAmiR). In other embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 103 and 104, respectively (e.g., GR 72150 shRNAmiR). In other 30 embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 105 and 106, respectively (e.g., GR 72151 shRNAmiR). In other 2026205474   09 Jul 2026 embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 107 and 108, respectively (e.g., GR 72152 shRNAmiR). The GR-targeted shRNAmiR may comprise a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at 5 least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NOs: 109-117. In particular embodiments, the shRNAmiR comprises the sequence set forth in any one of SEQ ID NOs: 109-117. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 109. In some embodiments, the shRNAmiR 10 comprises the sequence set forth in SEQ ID NO: 110. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 111. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 112. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 113. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 114. In some embodiments, the shRNAmiR 15 comprises the sequence set forth in SEQ ID NO: 115. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 116. In some embodiments, the shRNAmiR comprises the sequence set forth in SEQ ID NO: 117. In some of the embodiments wherein the genetically-modified immune cell expresses a shRNAmiR that reduces the expression of GR, the genetically-modified immune cell is less 20 susceptible to effects of glucocorticoids, such as cortisol or dexamethasone, such as reduced proliferation. Indeed, genetically-modified immune cells having reduced expression of GR can be enriched by incubation of a cell population with a glucocorticoid. Further, genetically-modified immune cells (e.g., CAR T cells) having reduced expression of GR may have greater persistence in vivo during immunotherapy when a glucocorticoid (e.g., steroid) is administered 25 during the course of therapy. 2.5 Methods for Reducing Expression of Endogenous Proteins The present invention provides methods for reducing the expression of an endogenous protein in an immune cell by introducing into the cell a template nucleic acid comprising a 30 nucleic acid sequence encoding a shRNAmiR, whereby the template nucleic acid is inserted into the genome and expressed. 2026205474   09 Jul 2026 The template nucleic acid can be inserted into the genome of the immune cell by random integration. Alternatively, the template nucleic acid can be inserted into a target gene by nuclease-mediated targeted insertion, wherein an engineered nuclease has specificity for a recognition sequence in the genome of the immune cell and generates a cleavage site at the 5 recognition sequence, allowing for the insertion of the template nucleic acid into the genome of the immune cell at the cleavage site. Any engineered nuclease can be used for targeted insertion of the template nucleic acid, including an engineered meganuclease, a zinc finger nuclease, a TALEN, a compact TALEN, a CRISPR system nuclease, or a megaTAL. 10          For example, zinc-finger nucleases (ZFNs) can be engineered to recognize and cut pre determined sites in a genome. ZFNs are chimeric proteins comprising a zinc finger DNA-binding domain fused to a nuclease domain from an endonuclease or exonuclease (e.g., Type IIs restriction endonuclease, such as the FokI restriction enzyme). The zinc finger domain can be a native sequence or can be redesigned through rational or experimental means to produce a 15 protein which binds to a pre-determined DNA sequence ~18 basepairs in length. By fusing this engineered protein domain to the nuclease domain, it is possible to target DNA breaks with genome-level specificity. ZFNs have been used extensively to target gene addition, removal, and substitution in a wide range of eukaryotic organisms (reviewed in S. Durai et al., Nucleic Acids Res 33, 5978 (2005)). 20          Likewise, TAL-effector nucleases (TALENs) can be generated to cleave specific sites in genomic DNA. Like a ZFN, a TALEN comprises an engineered, site-specific DNA-binding domain fused to an endonuclease or exonuclease (e.g., Type IIs restriction endonuclease, such as the FokI restriction enzyme) (reviewed in Mak, et al. (2013) Curr Opin Struct Biol. 23:93-9). In this case, however, the DNA binding domain comprises a tandem array of TAL-effector 25 domains, each of which specifically recognizes a single DNA basepair. Compact TALENs are an alternative endonuclease architecture that avoids the need for dimerization (Beurdeley, et al. (2013) Nat Commun. 4:1762). A Compact TALEN comprises an engineered, site-specific TAL-effector DNA-binding domain fused to the nuclease domain from the I-TevI homing endonuclease or any of the endonucleases listed in Table 2 in U.S. 30 Application No. 20130117869. Compact TALENs do not require dimerization for DNA processing activity, so a Compact TALEN is functional as a monomer. 2026205474   09 Jul 2026 Engineered endonucleases based on the CRISPR / Cas system are also known in the art (Ran, et al. (2013) Nat Protoc. 8:2281-2308; Mali et al. (2013) Nat Methods. 10:957-63). A CRISPR system comprises two components: (1) a CRISPR nuclease; and (2) a short “guide RNA” comprising a ~20 nucleotide targeting sequence that directs the nuclease to a location of 5 interest in the genome. The CRISPR system may also comprise a tracrRNA. By expressing multiple guide RNAs in the same cell, each having a different targeting sequence, it is possible to target DNA breaks simultaneously to multiple sites in the genome. Engineered meganucleases that bind double-stranded DNA at a recognition sequence that is greater than 12 base pairs can be used for the presently disclosed methods. A meganuclease 10 can be an endonuclease that is derived from I-CreI and can refer to an engineered variant of I-CreI that has been modified relative to natural I-CreI with respect to, for example, DNA-binding specificity, DNA cleavage activity, DNA-binding affinity, or dimerization properties. Methods for producing such modified variants of I-CreI are known in the art (e.g. WO 2007 / 047859, incorporated by reference in its entirety). A meganuclease as used herein binds to double- 15 stranded DNA as a heterodimer. A meganuclease may also be a “single-chain meganuclease” in which a pair of DNA-binding domains is joined into a single polypeptide using a peptide linker. Nucleases referred to as megaTALs are single-chain endonucleases comprising a transcription activator-like effector (TALE) DNA binding domain with an engineered, sequencespecific homing endonuclease. 20          In particular embodiments, the recognition sequence of the engineered nuclease is within a target gene. The target gene can be any gene in which the sequence is desired to be altered (e.g., addition or subtraction of nucleotide, substitution of nucleotide, or insertion of a heterologous or exogenous sequence). For example, knockout of a target gene by genetic inactivation may be desired. In some embodiments, the target gene is a TCR alpha gene or a 25 TCR beta gene. In particular embodiments, the target gene can be the TCR alpha constant region (TRAC) gene. In some specific embodiments, the target gene is the TRAC gene and the recognition sequence is the TRC 1-2 recognition sequence set forth in SEQ ID NO: 58. In some of these embodiments, the insertion of the template nucleic acid into the target gene leads to disruption of expression of the full-length endogenous protein encoded by the 30 target gene. Thus, in some of those embodiments wherein the target gene is a TCR alpha gene, TRAC gene, or TCR beta gene, the genetically-modified immune cell does not have detectable 2026205474   09 Jul 2026 cell-surface expression of an endogenous TCR, such as an alpha / beta TCR, because the endogenous TCR will not properly assemble at the cell surface in the absence of the endogenous proteins encoded by these genes. In particular embodiments in which the genetically-modified immune cell does not have 5 detectable cell-surface expression of an endogenous TCR (e.g., an alpha / beta TCR) due to inactivation of a gene encoding a component of an alpha / beta TCR, the genetically-modified immune cell further expresses a CAR or exogenous TCR and / or an HLA-E fusion protein. The CAR or exogenous TCR and / or the HLA-E fusion protein can be encoded by sequences comprised within the template nucleic acid. In some of these embodiments, the CAR / TCR- 10 encoding sequence and / or the HLA-E fusion protein-encoding sequence is operably linked to a different promoter than the shRNAmiR-encoding sequence. In alternative embodiments, the CAR / TCR-encoding sequence and / or the HLA-E fusion protein-encoding sequence is operably linked to the same promoter, or to a different promoter, as the shRNAmiR-encoding sequence. The CAR / TCR-encoding sequence and / or the HLA-E fusion protein-encoding sequence can be 15   5' or 3' of the shRNAmiR-encoding sequence, and the coding sequences can be in the same or different orientation, such as 5' to 3' or 3' to 5'. Further, the coding sequences may be separated by an element known in the art to allow for the translation of two or more genes (i.e., cistrons) from the same nucleic acid molecule including, but not limited to, an IRES element, a T2A element, a P2A element (e.g., a P2A / furin), an E2A element, and an F2A element. 20          The use of nucleases for disrupting expression of an endogenous TCR has been disclosed, including the use of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), megaTALs, and CRISPR systems (e.g., Osborn et al. (2016), Molecular Therapy 24(3): 570-581; Eyquem et al. (2017), Nature 543: 113-117; U.S. Patent No. 8,956,828; U.S. Publication No. US2014 / 0301990; U.S. Publication No. US2012 / 0321667). The specific use of 25 engineered meganucleases for cleaving DNA targets in the human TRAC gene has also been previously disclosed. For example, International Publication No. WO 2014 / 191527, which disclosed variants of the I-OnuI meganuclease that were engineered to target a recognition sequence within exon 1 of the TCR alpha constant region gene. Moreover, in International Publication Nos. WO 2017 / 062439 and WO 2017 / 062451, 30 Applicants disclosed engineered meganucleases which have specificity for recognition sequences in exon 1 of the TCR alpha constant region (TRAC) gene. These included “TRC 1-2 2026205474   09 Jul 2026 meganucleases” which have specificity for the TRC 1-2 recognition sequence (SEQ ID NO: 58) in exon 1 of the TRAC gene. The ‘439 and ‘451 publications also disclosed methods for targeted insertion of a CAR coding sequence or an exogenous TCR coding sequence into the TCR 1-2 meganuclease cleavage site. 5          In particular embodiments, the nucleases used to practice the invention are single-chain meganucleases. A single-chain meganuclease comprises an N-terminal subunit and a C-terminal subunit joined by a linker peptide. Each of the two domains recognizes half of the recognition sequence (i.e., a recognition half-site) and the site of DNA cleavage is at the middle of the recognition sequence near the interface of the two subunits. DNA strand breaks are offset by 10 four base pairs such that DNA cleavage by a meganuclease generates a pair of four base pair, 3' single-strand overhangs. Engineered nucleases can be delivered into a cell in the form of protein or, preferably, as a nucleic acid encoding the engineered nuclease. Such nucleic acids can be DNA (e.g., circular or linearized plasmid DNA or PCR products) or RNA (e.g., mRNA). For embodiments in which 15 the engineered nuclease coding sequence is delivered in DNA form, it should be operably linked to a promoter to facilitate transcription of the nuclease gene. Mammalian promoters suitable for the invention include constitutive promoters such as the cytomegalovirus early (CMV) promoter (Thomsen et al. (1984), Proc Natl Acad Sci USA. 81(3):659-63) or the SV40 early promoter (Benoist and Chambon (1981), Nature. 290(5804):304-10) as well as inducible promoters such 20 as the tetracycline-inducible promoter (Dingermann et al. (1992), Mol Cell Biol. 12(9):4038-45). A nucleic acid encoding an engineered nuclease can also be operably linked to a synthetic promoter. Synthetic promoters can include, without limitation, the JeT promoter (WO 2002 / 012514). In certain embodiments, a nucleic acid sequence encoding an engineered nuclease is 25 delivered on a recombinant DNA construct or expression cassette. For example, the recombinant DNA construct can comprise an expression cassette (i.e., “cassette”) comprising a promoter and a nucleic acid sequence encoding an engineered nuclease described herein. In some embodiments, mRNA encoding the engineered nuclease is delivered to the cell because this reduces the likelihood that the gene encoding the engineered nuclease will integrate 30 into the genome of the cell. 2026205474   09 Jul 2026 The mRNA encoding an engineered nuclease can be produced using methods known in the art such as in vitro transcription. In some embodiments, the mRNA comprises a modified 5' cap. Such modified 5' caps are known in the art and can include, without limitation, an antireverse cap analogs (ARCA) (US7074596), 7-methyl-guanosine, CleanCap® analogs, such as 5 Cap 1 analogs (Trilink; San Diego, CA), or enzymatically capped using, for example, a vaccinia capping enzyme or the like. In some embodiments, the mRNA may be polyadenylated. The mRNA may contain various 5' and 3' untranslated sequence elements to enhance expression of the encoded engineered nuclease and / or stability of the mRNA itself. Such elements can include, for example, posttranslational regulatory elements such as a woodchuck hepatitis virus 10 posttranslational regulatory element. The mRNA may contain nucleoside analogs or naturally-occurring nucleosides, such as pseudouridine, 5-methylcytidine, N6-methyladenosine, 5-methyluridine, or 2-thiouridine. Additional nucleoside analogs include, for example, those described in US 8,278,036. In another particular embodiment, a nucleic acid encoding an engineered nuclease can be 15 introduced into the cell using a single-stranded DNA template. The single-stranded DNA can further comprise a 5' and / or a 3' AAV inverted terminal repeat (ITR) upstream and / or downstream of the sequence encoding the engineered nuclease. In other embodiments, the single-stranded DNA can further comprise a 5' and / or a 3' homology arm upstream and / or downstream of the sequence encoding the engineered nuclease. 20          In another particular embodiment, genes encoding a nuclease can be introduced into a cell using a linearized DNA template. In some examples, a plasmid DNA encoding a nuclease can be digested by one or more restriction enzymes such that the circular plasmid DNA is linearized prior to being introduced into a cell. Purified nuclease proteins can be delivered into cells to cleave genomic DNA, which 25 allows for homologous recombination or non-homologous end-joining at the cleavage site with a sequence of interest, by a variety of different mechanisms known in the art, including those further detailed herein below. In some embodiments, nuclease proteins, or DNA / mRNA encoding the nuclease, are coupled to a cell penetrating peptide or targeting ligand to facilitate cellular uptake. Examples of 30 cell penetrating peptides known in the art include poly-arginine (Jearawiriyapaisarn, et al. (2008) Mol Ther. 16:1624-9), TAT peptide from the HIV virus (Hudecz et al. (2005), Med. Res. Rev. 2026205474   09 Jul 2026 25: 679-736), MPG (Simeoni, et al. (2003) Nucleic Acids Res. 31:2717-2724), Pep-1 (Deshayes et al. (2004) Biochemistry 43: 7698-7706), and HSV-1 VP-22 (Deshayes et al. (2005) Cell Mol Life Sci. 62:1839-49). In an alternative embodiment, nuclease proteins, or DNA / mRNA encoding nucleases, are coupled covalently or non-covalently to an antibody that recognizes a 5 specific cell-surface receptor expressed on target cells such that the nuclease protein / DNA / mRNA binds to and is internalized by the target cells. Alternatively, nuclease protein / DNA / mRNA can be coupled covalently or non-covalently to the natural ligand (or a portion of the natural ligand) for such a cell-surface receptor. (McCall, et al. (2014) Tissue Barriers. 2(4):e944449; Dinda, et al. (2013) Curr Pharm Biotechnol. 14:1264-74; Kang, et al. 10   (2014) Curr Pharm Biotechnol. 15(3):220-30; Qian et al. (2014) Expert Opin Drug Metab Toxicol. 10(11):1491-508). In some embodiments, nuclease proteins, or DNA / mRNA encoding nucleases, are coupled covalently or, preferably, non-covalently to a nanoparticle or encapsulated within such a nanoparticle using methods known in the art (Sharma, et al. (2014) Biomed Res Int. 2014). A 15 nanoparticle is a nanoscale delivery system whose length scale is <1 pm, preferably <100 nm. Such nanoparticles may be designed using a core composed of metal, lipid, polymer, or biological macromolecule, and multiple copies of the nuclease proteins, mRNA, or DNA can be attached to or encapsulated with the nanoparticle core. This increases the copy number of the protein / mRNA / DNA that is delivered to each cell and, so, increases the intracellular expression 20 of each nuclease to maximize the likelihood that the target recognition sequences will be cut. The surface of such nanoparticles may be further modified with polymers or lipids (e.g., chitosan, cationic polymers, or cationic lipids) to form a core-shell nanoparticle whose surface confers additional functionalities to enhance cellular delivery and uptake of the payload (Jian et al. (2012) Biomaterials. 33(30): 7621-30). Nanoparticles may additionally be advantageously 25 coupled to targeting molecules to direct the nanoparticle to the appropriate cell type and / or increase the likelihood of cellular uptake. Examples of such targeting molecules include antibodies specific for cell-surface receptors and the natural ligands (or portions of the natural ligands) for cell surface receptors. In some embodiments, the nuclease proteins or DNA / mRNA encoding the nucleases are 30 encapsulated within liposomes or complexed using cationic lipids (see, e.g., Lipofectamine™, Life Technologies Corp., Carlsbad, CA; Zuris et al. (2015) Nat Biotechnol. 33: 73-80; Mishra et 2026205474   09 Jul 2026 al. (2011) J Drug Deliv. 2011:863734). The liposome and lipoplex formulations can protect the payload from degradation, and facilitate cellular uptake and delivery efficiency through fusion with and / or disruption of the cellular membranes of the target cells. In some embodiments, nuclease proteins, or DNA / mRNA encoding nucleases, are 5 encapsulated within polymeric scaffolds (e.g., PLGA) or complexed using cationic polymers (e.g., PEI, PLL) (Tamboli et al. (2011) Ther Deliv. 2(4): 523-536). Polymeric carriers can be designed to provide tunable drug release rates through control of polymer erosion and drug diffusion, and high drug encapsulation efficiencies can offer protection of the therapeutic payload until intracellular delivery to the desired target cell population. 10         In some embodiments, nuclease proteins, or DNA / mRNA encoding recombinant nucleases, are combined with amphiphilic molecules that self-assemble into micelles (Tong et al. (2007) J Gene Med. 9(11): 956-66). Polymeric micelles may include a micellar shell formed with a hydrophilic polymer (e.g., polyethyleneglycol) that can prevent aggregation, mask charge interactions, and reduce nonspecific interactions. 15         In some embodiments, nuclease proteins, or DNA / mRNA encoding meganucleases, are formulated into an emulsion or a nanoemulsion (i.e., having an average particle diameter of < 1 nm) for administration and / or delivery to the target cell. The term “emulsion” refers to, without limitation, any oil-in-water, water-in-oil, water-in-oil-in-water, or oil-in-water-in-oil dispersions or droplets, including lipid structures that can form as a result of hydrophobic forces that drive 20 apolar residues (e.g., long hydrocarbon chains) away from water and polar head groups toward water, when a water immiscible phase is mixed with an aqueous phase. These other lipid structures include, but are not limited to, unilamellar, paucilamellar, and multilamellar lipid vesicles, micelles, and lamellar phases. Emulsions are composed of an aqueous phase and a lipophilic phase (typically containing an oil and an organic solvent). Emulsions also frequently 25 contain one or more surfactants. Nanoemulsion formulations are well known, e.g., as described in US Patent Application Nos. 2002 / 0045667 and 2004 / 0043041, and US Pat. Nos. 6,015,832, 6,506,803, 6,635,676, and 6,559,189, each of which is incorporated herein by reference in its entirety. In some embodiments, nuclease proteins, or DNA / mRNA encoding nucleases, are 30 covalently attached to, or non-covalently associated with, multifunctional polymer conjugates, DNA dendrimers, and polymeric dendrimers (Mastorakos et al. (2015) Nanoscale. 7(9): 3845- 2026205474   09 Jul 2026 56; Cheng et al. (2008) J Pharm Sci. 97(1): 123-43). The dendrimer generation can control the payload capacity and size, and can provide a high drug payload capacity. Moreover, display of multiple surface groups can be leveraged to improve stability, reduce nonspecific interactions, and enhance cell-specific targeting and drug release. 5          In some embodiments, genes encoding a nuclease are delivered using a viral vector (i.e., a recombinant virus). Such vectors are known in the art and include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated virus (AAV) vectors (reviewed in Vannucci, et al. (2013 New Microbiol. 36:1-22). Recombinant AAV vectors useful in the invention can have any serotype that allows for transduction of the virus into the cell and insertion of the nuclease 10 gene into the cell genome. In particular embodiments, recombinant AAV vectors have a serotype of AAV2 or AAV6. AAV vectors can also be self-complementary such that they do not require second-strand DNA synthesis in the host cell (McCarty, et al. (2001) Gene Ther. 8:124854). Polynucleotides delivered by recombinant AAV vectors, including those that deliver a template nucleic acid disclosed herein, can include left (5') and right (3') inverted terminal 15 repeats. If the nuclease genes are delivered in DNA form (e.g. plasmid) and / or via a viral vector (e.g. AAV) they must be operably linked to a promoter. In some embodiments, this can be a viral promoter such as endogenous promoters from the viral vector (e.g. the LTR of a lentiviral vector) or the well-known cytomegalovirus- or SV40 virus-early promoters. In a preferred 20 embodiment, nuclease genes are operably linked to a promoter that drives gene expression preferentially in the target cell (e.g., a T cell). The CAR / TCR coding sequence and / or the HLA-E fusion protein coding sequence can further comprise additional control sequences. For example, the sequence can include homologous recombination enhancer sequences, Kozak sequences, polyadenylation sequences, 25 transcriptional termination sequences, selectable marker sequences (e.g., antibiotic resistance genes), origins of replication, and the like. Sequences encoding engineered nucleases can also include at least one nuclear localization signal. Examples of nuclear localization signals are known in the art (see, e.g., Lange et al., J. Biol. Chem., 2007, 282:5101-5105). The invention further provides for the introduction of a template nucleic acid into a target 30 gene. In some embodiments, the template nucleic acid comprises a 5' homology arm and a 3' homology arm flanking the elements of the insert. Such homology arms have sequence 2026205474   09 Jul 2026 homology to corresponding sequences 5' upstream and 3' downstream of the nuclease recognition sequence where a cleavage site is produced. In general, homology arms can have a length of at least 50 base pairs, preferably at least 100 base pairs, and up to 2000 base pairs or more, and can have at least 90%, preferably at least 95%, or more, sequence homology to their 5 corresponding sequences in the genome. A template nucleic acid disclosed herein (e.g., encoding a shRNAmiR, a nucleic acid encoding a CAR or exogenous TCR, and / or an HLA-E fusion protein), can be introduced into the cell by any of the means previously discussed. In a particular embodiment, the template nucleic acid is introduced by way of a viral vector (i.e., a recombinant virus), such as a 10 recombinant lentivirus , a recombinant retrovirus, a recombinant adenovirus, or preferably a recombinant AAV vector (i.e., a recombinant AAV). Recombinant AAV vectors useful for introducing an exogenous nucleic acid (e.g., a template nucleic acid) can have any serotype that allows for transduction of the virus into the cell and insertion of the exogenous nucleic acid sequence into the cell genome. In particular embodiments, the recombinant AAV vectors have a 15 serotype of AAV2 or AAV6. The recombinant AAV vectors can also be self-complementary such that they do not require second-strand DNA synthesis in the host cell. In another particular embodiment, the template nucleic acid disclosed herein (e.g., encoding a shRNAmiR, a nucleic acid encoding a CAR or exogenous TCR, and / or an HLA-E fusion protein), can be introduced into the cell using a single-stranded DNA template. The 20 single-stranded DNA can comprise the exogenous sequence of interest and, in preferred embodiments, can comprise 5' and 3' homology arms to promote insertion of the nucleic acid sequence into the meganuclease cleavage site by homologous recombination. The singlestranded DNA can further comprise a 5' AAV inverted terminal repeat (ITR) sequence 5' upstream of the 5' homology arm, and a 3' AAV ITR sequence 3' downstream of the 3' homology 25 arm. In another particular embodiment, the template nucleic acid disclosed herein (e.g., encoding a shRNAmiR, a nucleic acid encoding a CAR or exogenous TCR, and / or an HLA-E fusion protein) can be introduced into the cell by transfection with a linearized DNA template. In some examples, a plasmid DNA can be digested by one or more restriction enzymes such that 30 the circular plasmid DNA is linearized prior to transfection into the cell. 2026205474   09 Jul 2026 Immune cells (e.g., T cells) modified by the present invention may require activation prior to introduction of a nuclease and / or an exogenous sequence of interest. For example, T cells can be contacted with anti-CD3 and anti-CD28 antibodies that are soluble or conjugated to a support (i.e., beads) for a period of time sufficient to activate the cells. 5          Genetically-modified immune cells of the invention can be further modified to express one or more inducible suicide genes, the induction of which provokes cell death and allows for selective destruction of the cells in vitro or in vivo. In some examples, a suicide gene can encode a cytotoxic polypeptide, a polypeptide that has the ability to convert a non-toxic pro-drug into a cytotoxic drug, and / or a polypeptide that activates a cytotoxic gene pathway within the cell. 10 That is, a suicide gene is a nucleic acid that encodes a product that causes cell death by itself or in the presence of other compounds. A representative example of such a suicide gene is one that encodes thymidine kinase of herpes simplex virus. Additional examples are genes that encode thymidine kinase of varicella zoster virus and the bacterial gene cytosine deaminase that can convert 5-fluorocytos...

Claims

1. A genetically-modified immune cell comprising in its genome a nucleic acidsequence encoding a microRNA-adapted shRNA (shRNAmiR), wherein said shRNAmiR is expressed in said genetically-modified immune cell, and wherein said shRNAmiR reduces expression of a target protein in said genetically-modified immune cell.

2. The genetically-modified immune cell of claim 1, wherein said genetically-modified immune cell is a genetically-modified T cell, or a cell derived therefrom.

3. The genetically-modified immune cell of claim 1, wherein said genetically-modified immune cell is a genetically-modified natural killer (NK) cell, or a cell derived therefrom.

4. The genetically-modified immune cell of claim 1, wherein said genetically-modified immune cell is a genetically-modified B cell, or a cell derived therefrom.

5. The genetically-modified immune cell of claim 1, wherein said genetically-modified immune cell is a genetically-modified monocyte or macrophage, or a cell derived therefrom.

6. The genetically-modified immune cell of any one of claims 1-5, wherein saidshRNAmiR comprises, from 5' to 3':(a) a 5' miR scaffold domain;(b)    a 5' miR basal stem domain;(c)    a passenger strand;(d)    a miR loop domain;(e)     a guide strand;(f)    a 3' miR basal stem domain; and(g) a 3' miR scaffold domain.2026205474   09 Jul 20267.     The genetically-modified immune cell of claim 6, wherein said miR loop domainis a miR-30a loop domain.

8. The genetically-modified immune cell of claim 7, wherein said miR-30a loopdomain comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 3.

9. The genetically-modified immune cell of claim 8, wherein said miR-30a loopdomain comprises a nucleic acid sequence of SEQ ID NO: 3.

10. The genetically-modified immune cell of any one of claims 6-9, wherein said shRNAmiR comprises a microRNA-E (miR-E) scaffold, a miR-30 (e.g., miR-30a) scaffold, a miR-15 scaffold, a miR-16 scaffold, a miR-155 scaffold, a miR-22 scaffold, a miR-103 scaffold, or a miR-107 scaffold.

11. The genetically-modified immune cell of claim 10, wherein said shRNAmiR comprises a miR-E scaffold.

12. The genetically-modified immune cell of any one of claims 6-11, wherein:(a) said 5' miR scaffold domain comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 1;(b) said 5' miR basal stem domain comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 2;(c) said 3' miR basal stem domain comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 4; and(d) said 3' miR scaffold domain comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 5.

13. The genetically-modified immune cell of claim 12, wherein:(a) said 5' miR scaffold domain comprises a nucleic acid sequence of SEQ ID NO: 1;2026205474   09 Jul 2026(b) said 5' miR basal stem domain comprises a nucleic acid sequence of SEQ ID NO: 2;(c) said 3' miR basal stem domain comprises a nucleic acid sequence of SEQ ID NO: 4; and(d) said 3' miR scaffold domain comprises a nucleic acid sequence of SEQ ID NO: 5.

14. The genetically-modified immune cell of any one of claims 1-13, wherein genetically-modified immune cell comprises in its genome a nucleic acid sequence encoding a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR), wherein said CAR or said exogenous TCR is expressed by said genetically-modified immune cell.

15. The genetically-modified immune cell of any one of claims 1-14, wherein said genetically-modified immune cell comprises in its genome a nucleic acid sequence encoding an HLA class I histocompatibility antigen, alpha chain E (HLA-E) fusion protein.

16. The genetically-modified immune cell of claim 15, wherein said HLA-E fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 66.

17. The genetically-modified immune cell of claim 15 or claim 16, wherein said HLA-E fusion protein comprises an amino acid sequence of SEQ ID NO: 66.

18. The genetically-modified immune cell of any one of claims 14-17, wherein said nucleic acid sequence encoding said shRNAmiR is located within a different gene than said nucleic acid sequence encoding said CAR or said exogenous TCR.

19. The genetically-modified immune cell of claim 18, wherein said nucleic acid sequence encoding said shRNAmiR, or said nucleic acid sequence encoding said CAR or said exogenous TCR, is located within a TCR alpha gene or a TCR alpha constant region gene.2026205474   09 Jul 202620. The genetically-modified immune cell of any one of claims 14-17, wherein said nucleic acid sequence encoding said shRNAmiR is located within the same gene as said nucleic acid sequence encoding said CAR or said exogenous TCR.

21. The genetically-modified immune cell of claim 20, wherein said gene is a TCR alpha gene or a TCR alpha constant region gene.

22. The genetically-modified immune cell of claim 20 or claim 21, wherein said nucleic acid sequence encoding said shRNAmiR and said nucleic acid sequence encoding said CAR or said exogenous TCR are within a cassette in said gene.

23. The genetically-modified immune cell of any one of claims 20-22, wherein said nucleic acid sequence encoding said shRNAmiR and said nucleic acid sequence encoding said CAR or said exogenous TCR are operably linked to a same promoter.

24. The genetically-modified immune cell of any one of claims 20-23, wherein said genetically-modified immune cell comprises in its genome a cassette comprising, from 5' to 3':(a) said nucleic acid sequence encoding said CAR or said exogenous TCR; and(b) said nucleic acid sequence encoding said shRNAmiR.

25. The genetically-modified immune cell of any one of claims 20-23, wherein said genetically-modified immune cell comprises in its genome a cassette comprising, from 5' to 3':(a)    said nucleic acid sequence encoding said shRNAmiR; and(b)    said nucleic acid sequence encoding said CAR or said exogenous TCR.

26. The genetically-modified immune cell of any one of claims 20-25, wherein said nucleic acid sequence encoding said CAR or said exogenous TCR and said nucleic acid sequence encoding said shRNAmiR are separated by a 2A or IRES sequence.2026205474   09 Jul 202627. The genetically-modified immune cell of any one of claims 20-26, wherein said nucleic acid sequence encoding said shRNAmiR is in the same orientation as said nucleic acid sequence encoding said CAR or said exogenous TCR.

28. The genetically-modified immune cell of any one of claims 20-26, wherein said nucleic acid sequence encoding said shRNAmiR is in a reverse orientation as said nucleic acid sequence encoding said CAR or said exogenous TCR.

29. The genetically-modified immune cell of any one of claims 20-23, wherein an intron sequence is positioned within said nucleic acid sequence encoding said CAR or said exogenous TCR, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence.

30. The genetically-modified immune cell of any one of claims 22-29, wherein said cassette comprises a promoter that is operably linked to said nucleic acid sequence encoding said shRNAmiR and said nucleic acid sequence encoding said CAR or said exogenous TCR.

31. The genetically-modified immune cell of any one of claims 22-30, wherein said cassette comprises a termination signal.

32. The genetically-modified immune cell of any one of claims 15-17, wherein said nucleic acid sequence encoding said shRNAmiR is located within the same gene as said nucleic acid sequence encoding said HLA-E fusion protein.

33. The genetically-modified immune cell of claim 32, wherein said gene is a TCR alpha gene or a TCR alpha constant region gene.

34. The genetically-modified immune cell of claim 32 or claim 33, wherein said nucleic acid sequence encoding said shRNAmiR and said nucleic acid sequence encoding said HLA-E fusion protein are within a cassette in said gene.2026205474   09 Jul 202635. The genetically-modified immune cell of any one of claims 32-34, wherein said nucleic acid sequence encoding said shRNAmiR and nucleic acid sequence encoding said HLA-E fusion protein are operably linked to a same promoter.

36. The genetically-modified immune cell of any one of claims 32-35, wherein said genetically-modified immune cell comprises in its genome a cassette comprising, from 5' to 3':(a)    said nucleic acid sequence encoding said HLA-E fusion protein; and(b)    said nucleic acid sequence encoding said shRNAmiR.

37. The genetically-modified immune cell of any one of claims 32-35, wherein said genetically-modified immune cell comprises in its genome a cassette comprising, from 5' to 3':(a)    said nucleic acid sequence encoding said shRNAmiR; and(b)    said nucleic acid sequence encoding said HLA-E fusion protein.

38. The genetically-modified immune cell of any one of claims 32-37, wherein said nucleic acid sequence encoding said HLA-E fusion protein and said nucleic acid sequence encoding said shRNAmiR are separated by a 2A or IRES sequence.

39. The genetically-modified immune cell of any one of claims 32-35, wherein an intron sequence is positioned within said nucleic acid sequence encoding said HLA-E fusion protein, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence.

40. The genetically-modified immune cell of any one of claims 34-39, wherein said cassette comprises a promoter, wherein said nucleic acid sequence encoding said shRNAmiR and nucleic acid sequence encoding said HLA-E fusion protein are operably linked to said promoter.

41. The genetically-modified immune cell of any one of claims 34-40, wherein said cassette comprises a termination signal.2026205474   09 Jul 202642. The genetically-modified immune cell of any one of claims 15-17, wherein said nucleic acid sequence encoding said shRNAmiR, said nucleic acid sequence encoding said CAR or said exogenous TCR, and said nucleic acid sequence encoding said HLA-E fusion protein are located within the same gene.

43. The genetically-modified immune cell of claim 42, wherein said gene is a TCR alpha gene or a TCR alpha constant region gene.

44. The genetically-modified immune cell of claim 42 or claim 43, wherein said nucleic acid sequence encoding said shRNAmiR, said nucleic acid sequence encoding said CAR or said exogenous TCR, and said nucleic acid sequence encoding said HLA-E fusion protein are within a cassette in said gene.

45. The genetically-modified immune cell of any one of claims 42-44, wherein said nucleic acid sequence encoding said shRNAmiR, said nucleic acid sequence encoding said CAR or said exogenous TCR, and said nucleic acid sequence encoding said HLA-E fusion protein are operably linked to a same promoter.

46. The genetically-modified immune cell of any one of claims 42-45, wherein said genetically-modified immune cell comprises within its genome a cassette comprising:(a) said nucleic acid sequence encoding said CAR or said exogenous TCR;(b) a 2A or IRES sequence;(c)    said nucleic acid sequence encoding said HLA-E fusion protein; and(d)    said nucleic acid sequence encoding said shRNAmiR.

47. The genetically-modified immune cell of claim 42-46, wherein an intron sequence is positioned within said nucleic acid sequence encoding said CAR or said exogenous TCR, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence.2026205474   09 Jul 202648. The genetically-modified immune cell of any one of claims 42-46, wherein an intron sequence is positioned within said nucleic acid sequence encoding said HLA-E fusion protein, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence.

49. The genetically-modified immune cell of any one of claims 44-48, wherein said cassette comprises a promoter that is operably linked to said nucleic acid sequence encoding said CAR or said exogenous TCR, said nucleic acid sequence encoding said HLA-E fusion protein, and said nucleic acid sequence encoding said shRNAmiR.

50. The genetically-modified immune cell of any one of claims 44-49, wherein said cassette comprises a termination signal.

51. The genetically-modified immune cell of claim 45, wherein said genetically-modified immune cell comprises within its genome a cassette comprising, from 5' to 3':(a)    a promoter;(b) said nucleic acid sequence encoding said CAR or said exogenous TCR;(c)    a 2A or IRES sequence;(d) said nucleic acid sequence encoding said HLA-E fusion protein, wherein an intron sequence is positioned within said nucleic acid sequence encoding said HLA-E fusion protein, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence; and(e) optionally a termination signal;wherein said nucleic acid sequence encoding said CAR or said exogenous TCR, said nucleic acid sequence encoding said HLA-E fusion protein, and said nucleic acid sequence encoding said shRNAmiR are operably linked to said promoter.

52. The genetically-modified immune cell of claim 45, wherein said genetically-modified immune cell comprises within its genome a cassette comprising, from 5' to 3':(a) a promoter;2026205474   09 Jul 2026(b) said nucleic acid sequence encoding said HLA-E fusion protein, wherein an intron sequence is positioned within said nucleic acid sequence encoding said HLA-E fusion protein, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence;(c)    a 2A or IRES sequence;(d) said nucleic acid sequence encoding said CAR or said exogenous TCR; and(e)    optionally a termination signal;wherein said nucleic acid sequence encoding said CAR or said exogenous TCR, said nucleic acid sequence encoding said HLA-E fusion protein, and said nucleic acid sequence encoding said shRNAmiR are operably linked to said promoter.

53. The genetically-modified immune cell of claim 45, wherein said genetically-modified immune cell comprises within its genome a cassette comprising, from 5' to 3':(a) a promoter;(b) said nucleic acid sequence encoding said CAR or said exogenous TCR, wherein an intron sequence is positioned within said nucleic acid sequence encoding said CAR or said exogenous TCR, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence;(c)    a 2A or IRES sequence;(d) said nucleic acid sequence encoding said HLA-E fusion protein; and(e)    optionally a termination signal;wherein said nucleic acid sequence encoding said CAR or said exogenous TCR, said nucleic acid sequence encoding said HLA-E fusion protein, and said nucleic acid sequence encoding said shRNAmiR are operably linked to said promoter.

54. The genetically-modified immune cell of claim 45, wherein said genetically-modified immune cell comprises within its genome a cassette comprising, from 5' to 3':(a)    a promoter;(b) said nucleic acid sequence encoding said HLA-E fusion protein;(c)    a 2A or IRES sequence;2026205474   09 Jul 2026(d) said nucleic acid sequence encoding said CAR or said exogenous TCR, wherein an intron sequence is positioned within said nucleic acid sequence encoding said CAR or said exogenous TCR, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence; and(e) optionally a termination signal;wherein said nucleic acid sequence encoding said CAR or said exogenous TCR, said nucleic acid sequence encoding said HLA-E fusion protein, and said nucleic acid sequence encoding said shRNAmiR are operably linked to said promoter.

55. The genetically-modified immune cell of any one of claims 29-31, 39-41, 47-50, or 51-54, wherein said intron sequence is a synthetic intron sequence.

56. The genetically-modified immune cell of any one of claims 29-31, 39-41, 47-50, or 51-54, wherein said intron sequence comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 69.

57. The genetically-modified immune cell of any one of claims 29-31, 39-41, 47-50, or 51-54, wherein said intron sequence comprises a nucleic acid sequence of SEQ ID NO: 69.

58. The genetically-modified immune cell of any one of claims 31, 41, 50, or 51-57, wherein said termination signal is a polyA sequence or a bovine growth hormone (BGH) termination signal.

59. The genetically-modified immune cell of claim 58, wherein said polyA sequence comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 68.

60. The genetically-modified immune cell of claim 58, wherein said polyA sequence comprises a nucleic acid sequence of SEQ ID NO: 68.2026205474   09 Jul 202661. The genetically-modified immune cell of claim 58, wherein said BGH termination signal comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 71.

62. The genetically-modified immune cell of claim 58, wherein said BGH termination signal comprises a nucleic acid sequence of SEQ ID NO: 71.

63. The genetically-modified immune cell of any one of claims 23-31, 35-41, or 4562, wherein said promoter comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 67.

64. The genetically-modified immune cell of any one of claims 23-31, 35-41, or 4562, wherein said promoter comprises a nucleic acid sequence of SEQ ID NO: 67.

65. The genetically-modified immune cell of any one of claims 26-28, 30, 31, 38, 40, 41, or 46-64, wherein said 2A sequence is a P2A / furin site comprising a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 70.

66. The genetically-modified immune cell of any one of claims 26-28, 30, 31, 38, 40, 41, or 46-64, wherein said 2A sequence is a P2A / furin site comprising a nucleic acid sequence of SEQ ID NO: 70.

67. The genetically-modified immune cell of any one of claims 14-66, wherein said CAR comprises a signal peptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 73.

68. The genetically-modified immune cell of any one of claims 14-66, wherein said CAR comprises a signal peptide comprising an amino acid sequence of SEQ ID NO: 73.

69. The genetically-modified immune cell of claim 45, wherein said genetically-modified immune cell comprises in its genome a cassette comprising, from 5' to 3':2026205474   09 Jul 2026(a) a promoter comprising a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 67;(b) said nucleic acid sequence encoding said CAR, wherein said CAR comprises a signal peptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 73;(c) a P2A / furin site comprising a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 70;(d) said nucleic acid sequence encoding said HLA-E fusion protein, wherein said HLA-E fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 66, and wherein an intron sequence is positioned within said nucleic acid sequence encoding said HLA-E fusion protein, wherein said intron sequence comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 69, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence; and(e) optionally a termination signal comprising a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 68;wherein said nucleic acid sequence encoding said CAR, said nucleic acid sequence encoding said HLA-E fusion protein, and said nucleic acid sequence encoding said shRNAmiR are operably linked to said promoter.

70. The genetically-modified immune cell of claim 45, wherein said genetically-modified immune cell comprises in its genome a cassette comprising, from 5' to 3':(a) a promoter comprising a nucleic acid sequence of SEQ ID NO: 67;(b) said nucleic acid sequence encoding said CAR, wherein said CAR comprises a signal peptide comprising an amino acid sequence of SEQ ID NO: 73;(c) a P2A / furin site comprising a nucleic acid sequence of SEQ ID NO: 70;(d) said nucleic acid sequence encoding said HLA-E fusion protein, wherein said HLA-E fusion protein comprises an amino acid sequence of SEQ ID NO: 66, and wherein an intron sequence is positioned within said nucleic acid sequence encoding said HLA-E fusion protein, wherein said intron sequence comprises a nucleic acid sequence of SEQ ID NO: 69, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence; and2026205474   09 Jul 2026(e) optionally a termination signal comprising a nucleic acid sequence of SEQ ID NO: 68;wherein said nucleic acid sequence encoding said CAR, said nucleic acid sequence encoding said HLA-E fusion protein, and said nucleic acid sequence encoding said shRNAmiR are operably linked to said promoter.

71. The genetically-modified immune cell of claim 69 or claim 70, wherein said genetically-modified immune cell comprises in its genome a cassette comprising a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 74, wherein said cassette is positioned in the genome within a TCR alpha constant region gene.

72. The genetically-modified immune cell of claim 69 or claim 70, wherein said genetically-modified immune cell comprises in its genome a cassette comprising a nucleic acid sequence of SEQ ID NO: 74, wherein said cassette is positioned in the genome within a TCR alpha constant region gene.

73. The genetically-modified immune cell of any one of claims 20-22, wherein said nucleic acid sequence encoding said shRNAmiR and said nucleic acid sequence encoding said CAR or said exogenous TCR are operably linked to different promoters.

74. The genetically-modified immune cell of claim 73, wherein said genetically-modified immune cell comprises in its genome a cassette comprising, from 5' to 3':(a) a first promoter;(b) said nucleic acid sequence encoding said CAR or exogenous TCR which is operably linked to said first promoter;(c) a second promoter; and(d) said nucleic acid sequence encoding said shRNAmiR which is operably linked to said second promoter.

75. The genetically-modified immune cell of claim 73, wherein said genetically-modified immune cell comprises in its genome a cassette comprising, from 5' to 3':2026205474   09 Jul 2026(a) a first promoter;(b) said nucleic acid sequence encoding said shRNAmiR which is operably linked to said first promoter;(c) a second promoter; and(d) said nucleic acid sequence encoding said CAR or exogenous TCR which is operably linked to said second promoter.

76. The genetically-modified immune cell of any one of claims 73-75, wherein said nucleic acid sequence encoding said shRNAmiR is in the same orientation as said nucleic acid sequence encoding said CAR or exogenous TCR.

77. The genetically-modified immune cell of any one of claims 83-85, wherein said nucleic acid sequence encoding said shRNAmiR is in a reverse orientation as said nucleic acid sequence encoding said CAR or exogenous TCR.

78. The genetically-modified immune cell of any one of claims 74-77, wherein said first promoter and said second promoter are identical.

79. The genetically-modified immune cell of any one of claims 74-77, wherein said first promoter and said second promoter are different.

80. The genetically-modified immune cell of any one of claims 74-79, wherein said cassette comprises one or more termination signals.

81. The genetically-modified immune cell of any one of claims 20-22, wherein said nucleic acid sequence encoding said shRNAmiR and said nucleic acid sequence encoding said HLA-E fusion protein are operably linked to different promoters.

82. The genetically-modified immune cell of claim 81, wherein said genetically-modified immune cell comprises in its genome a cassette comprising, from 5' to 3':(a) a first promoter;2026205474   09 Jul 2026(b) said nucleic acid sequence encoding said HLA-E fusion protein which is operably linked to said first promoter;(c) a second promoter; and(d) said nucleic acid sequence encoding said shRNAmiR which is operably linked to said second promoter.

83. The genetically-modified immune cell of claim 81, wherein said genetically-modified immune cell comprises in its genome a cassette comprising, from 5' to 3':(a) a first promoter;(b) said nucleic acid sequence encoding said shRNAmiR which is operably linked to said first promoter;(c) a second promoter; and(d) said nucleic acid sequence encoding said HLA-E fusion protein which is operably linked to said second promoter.

84. The genetically-modified immune cell of any one of claims 80-82, wherein said first promoter and said second promoter are identical.

85. The genetically-modified immune cell of any one of claims 80-82, wherein said first promoter and said second promoter are different.

86. The genetically-modified immune cell of any one of claims 82-85, wherein said cassette comprises one or more termination signals.

87. The genetically-modified immune cell of any one of claims 42-44, wherein said genetically-modified immune cell comprises within its genome a cassette comprising:(a)    said nucleic acid sequence encoding said CAR or said exogenous TCR;(b)    said nucleic acid sequence encoding said HLA-E fusion protein; and(c)    said nucleic acid sequence encoding said shRNAmiR;wherein said nucleic acid sequence encoding said CAR or said exogenous TCR is operably linked to a first promoter,2026205474   09 Jul 2026and wherein said nucleic acid sequence encoding said HLA-E fusion protein and said nucleic acid sequence encoding said shRNAmiR are operably linked to a second promoter.

88. The genetically-modified immune cell of any one of claims 42-44, wherein said genetically-modified immune cell comprises within its genome a cassette comprising:(a)    said nucleic acid sequence encoding said CAR or said exogenous TCR;(b)    said nucleic acid sequence encoding said HLA-E fusion protein; and(c)    said nucleic acid sequence encoding said shRNAmiR;wherein said nucleic acid sequence encoding said CAR or said exogenous TCR and said nucleic acid sequence encoding said shRNAmiR are operably linked to a first promoter,and wherein said nucleic acid sequence encoding said HLA-E fusion protein is operably linked to a second promoter.

89. The genetically-modified immune cell of claim 87, wherein an intron sequence is positioned within said nucleic acid sequence encoding said HLA-E fusion protein, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence.

90. The genetically-modified immune cell of claim 88, wherein an intron sequence is positioned within said nucleic acid sequence encoding said CAR or said exogenous TCR, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence.

91. The genetically-modified immune cell of any one of claims 87-90, wherein said cassette comprises a first termination signal capable of terminating transcription of said CAR or said exogenous TCR, and a second termination signal capable of terminating transcription of said HLA-E fusion protein.

92. The genetically-modified immune cell of any one of claims 87-90, wherein said cassette comprises a first termination signal capable of terminating transcription of said HLA-E fusion protein, and a second termination signal capable of terminating transcription of said CAR or said exogenous TCR.2026205474   09 Jul 202693. The genetically-modified immune cell of any one of claims 42-44, wherein said genetically-modified immune cell comprises within its genome a cassette comprising, from 5' to 3':(a)     a first promoter;(b)    said nucleic acid sequence encoding said CAR or said exogenous TCR;(c)     optionally a first termination signal;(d)    a second promoter;(e)    said nucleic acid sequence encoding said HLA-E fusion protein, wherein an intronsequence is positioned within said nucleic acid sequence encoding said HLA-E fusion protein, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence; and(f) optionally a second termination signal;wherein said nucleic acid sequence encoding said CAR or said exogenous TCR is operably linked to said first promoter, and wherein said nucleic acid sequence encoding said HLA-E fusion protein and said nucleic acid sequence encoding said shRNAmiR are operably linked to said second promoter.

94. The genetically-modified immune cell of any one of claims 42-44, wherein said genetically-modified immune cell comprises within its genome a cassette comprising, from 5' to 3':(a) a first promoter;(b) said nucleic acid sequence encoding said HLA-E fusion protein, wherein an intron sequence is positioned within said nucleic acid sequence encoding said HLA-E fusion protein, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence;(c)     optionally a first termination signal;(d)    a second promoter;(e) said nucleic acid sequence encoding said CAR or said exogenous TCR; and(f)     optionally a second termination signal;2026205474   09 Jul 2026wherein said nucleic acid sequence encoding said HLA-E fusion protein and said nucleic acid sequence encoding said shRNAmiR are operably linked to said first promoter, and wherein said nucleic acid sequence encoding said CAR or said exogenous TCR is operably linked to said second promoter.

95. The genetically-modified immune cell of any one of claims 42-442, wherein said genetically-modified immune cell comprises within its genome a cassette comprising, from 5' to 3':(a) a first promoter;(b) said nucleic acid sequence encoding said CAR or said exogenous TCR, wherein an intron sequence is positioned within said nucleic acid sequence encoding said CAR or said exogenous TCR, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence;(c)     optionally a first termination signal;(d)    a second promoter;(e) said nucleic acid sequence encoding said HLA-E fusion protein; and(f)     optionally a second termination signal;wherein said nucleic acid sequence encoding said CAR or said exogenous TCR and said nucleic acid sequence encoding said shRNAmiR are operably linked to said first promoter, and wherein said nucleic acid sequence encoding said HLA-E fusion protein is operably linked to said second promoter.

96. The genetically-modified immune cell of any one of claims 42-44, wherein said genetically-modified immune cell comprises within its genome a cassette comprising, from 5' to 3':(a)     a first promoter;(b)    said nucleic acid sequence encoding said HLA-E fusion protein;(c)     optionally a first termination signal;(d)    a second promoter;(e) said nucleic acid sequence encoding said CAR or said exogenous TCR, wherein an intron sequence is positioned within said nucleic acid sequence encoding said CAR or said2026205474   09 Jul 2026exogenous TCR, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence; and(f) optionally a second termination signal;wherein said nucleic acid sequence encoding said HLA-E fusion protein is operably linked to said first promoter, and wherein said nucleic acid sequence encoding said CAR or said exogenous TCR and said nucleic acid sequence encoding said shRNAmiR are operably linked to said second promoter.

97. The genetically-modified immune cell of any one of claims 89-96, wherein said intron sequence is a synthetic intron sequence.

98. The genetically-modified immune cell of any one of claims 89-96, wherein said intron sequence comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 69.

99. The genetically-modified immune cell of any one of claims 89-96, wherein said intron sequence comprises a nucleic acid sequence of SEQ ID NO: 69.

100. The genetically-modified immune cell of claim 80 or claim 86, wherein said one or more termination signals is a polyA sequence or a BGH termination signal.

101. The genetically-modified immune cell of any one of claims 91-99, wherein said first termination signal is identical to said second termination signal.

102. The genetically-modified immune cell of claim 101, wherein said first termination signal and said second termination signal are a polyA sequence or a BGH termination signal.

103. The genetically-modified immune cell of any one of claims 91-99, wherein said first termination signal is different from said second termination signal.2026205474   09 Jul 2026104. The genetically-modified immune cell of claim 103, wherein said first termination signal is a polyA sequence and said second termination signal is a BGH termination signal.

105. The genetically-modified immune cell of claim 103, wherein said first termination signal is a BGH termination signal and said second termination signal is a polyA sequence.

106. The genetically-modified immune cell of any one of claims 100, 102, 104, or 105, wherein said polyA sequence comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 68.

107. The genetically-modified immune cell of any one of claims 100, 102, 104, or 105, wherein said polyA sequence comprises a nucleic acid sequence of SEQ ID NO: 68.

108. The genetically-modified immune cell of any one of claims 100, 102, or 104-107, wherein said BGH termination signal comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 71.

109. The genetically-modified immune cell of any one of claims 100, 102, or 104-107, wherein said BGH termination signal comprises a nucleic acid sequence of SEQ ID NO: 71.

110. The genetically-modified immune cell of any one of claims 74-80 or 82-109, wherein said first promoter and said second promoter are identical.

111. The genetically-modified immune cell of claim 110, wherein said first promoter and said second promoter are a JeT promoter or an EF1 alpha core promoter.

112. The genetically-modified immune cell of any one of claims 74-80 or 82-109, wherein said first promoter is different from said second promoter.

113. The genetically-modified immune cell of claim 112, wherein said first promoter is a JeT promoter, and wherein said second promoter is an EF1 alpha core promoter.2026205474   09 Jul 2026114. The genetically-modified immune cell of claim 112, wherein said first promoter is an EF1 alpha core promoter, and wherein said second promoter is a JeT promoter.

115. The genetically-modified immune cell of any one of claims 111, 113, or 114, wherein said JeT promoter comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 67.

116. The genetically-modified immune cell of any one of claims 111, 113, or 114, wherein said JeT promoter comprises a nucleic acid sequence of SEQ ID NO: 67.

117. The genetically-modified immune cell of any one of claims 111 or 113-116, wherein said EF1 alpha core promoter comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 72.

118. The genetically-modified immune cell of any one of claims 111 or 113-116, wherein said EF1 alpha core promoter comprises a nucleic acid sequence of SEQ ID NO: 72.

119. The genetically-modified immune cell of any one of claims 73-118, wherein said CAR comprises a signal peptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 73.

120. The genetically-modified immune cell of any one of claims 73-118, wherein said CAR comprises a signal peptide comprising an amino acid sequence of SEQ ID NO: 73.

121. The genetically-modified immune cell of any one of claims 42-44, wherein said genetically-modified immune cell comprises in its genome a cassette comprising, from 5' to 3':(a) a first promoter comprising a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 67;2026205474   09 Jul 2026(b) said nucleic acid sequence encoding said CAR, wherein said CAR comprises a signal peptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 73;(c) optionally a first termination signal comprising a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 70;(d) a second promoter comprising a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 72;(e) said nucleic acid sequence encoding said HLA-E fusion protein, wherein said HLA-E fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 66, and wherein an intron sequence is positioned within said nucleic acid sequence encoding said HLA-E fusion protein, wherein said intron sequence comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 69, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence; and(f) optionally a second termination signal comprising a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 71;wherein said nucleic acid sequence encoding said CAR is operably linked to said first promoter, and wherein said nucleic acid sequence encoding said HLA-E fusion protein and said nucleic acid sequence encoding said shRNAmiR are operably linked to said second promoter.

122. The genetically-modified immune cell of any one of claims 42-44, wherein said genetically-modified immune cell comprises in its genome a cassette comprising, from 5' to 3':(a) a first promoter comprising a nucleic acid sequence of SEQ ID NO: 67;(b) said nucleic acid sequence encoding said CAR, wherein said CAR comprises a signal peptide comprising an amino acid sequence of SEQ ID NO: 73;(c) optionally a first termination signal comprising a nucleic acid sequence of SEQ ID NO: 70;(d) a second promoter comprising a nucleic acid sequence of SEQ ID NO: 72;(e) said nucleic acid sequence encoding said HLA-E fusion protein, wherein said HLA-E fusion protein comprises an amino acid sequence of SEQ ID NO: 66, and wherein an intron sequence is positioned within said nucleic acid sequence encoding said HLA-E fusion protein, wherein said intron sequence comprises a nucleic acid sequence of SEQ ID NO: 69, and2026205474   09 Jul 2026wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence; and(f) optionally a second termination signal comprising a nucleic acid sequence of SEQ ID NO: 71;wherein said nucleic acid sequence encoding said CAR is operably linked to said first promoter, and wherein said nucleic acid sequence encoding said HLA-E fusion protein and said nucleic acid sequence encoding said shRNAmiR are operably linked to said second promoter.

123. The genetically-modified immune cell of claim 121 or claim 122, wherein said genetically-modified immune cell comprises in its genome a cassette comprising a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 75, wherein said cassette is positioned in the genome within a TCR alpha constant region gene.

124. The genetically-modified immune cell of claim 121 or claim 122, wherein said genetically-modified immune cell comprises in its genome a cassette comprising a nucleic acid sequence of SEQ ID NO: 75, wherein said cassette is positioned in the genome within a TCR alpha constant region gene.

125. The genetically-modified immune cell of any one of claims 1-124, wherein expression of said target protein is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell.

126. The genetically-modified immune cell of any one of claims 1-125, wherein said target protein is beta-2 microglobulin, CS1, transforming growth factor-beta receptor 2 (TGFBR2), Cbl proto-oncogene B (CBL-B), CD52, a TCR alpha gene, a TCR alpha constant region gene, CD7, glucocorticoid receptor (GR), deoxycytidine kinase (DCK), nuclear receptor subfamily 2 group F member 6 (NR2F6), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), or C-C chemokine receptor type 5 (CCR5).2026205474   09 Jul 2026127. The genetically-modified immune cell of claim 126, wherein said target protein is beta-2 microglobulin.

128. The genetically-modified immune cell of claim 127, wherein cell surface expression of beta-2 microglobulin is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell.

129. The genetically-modified immune cell of claim 127 or claim 128, wherein expression of MHC class I molecules is reduced on the cell surface by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell.

130. The genetically-modified immune cell of any one of claims 127-129, wherein said genetically-modified immune cell has reduced allogenicity compared to a control cell.

131. The genetically-modified immune cell of any one of claims 127-130, wherein:(a) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 17 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 18;(b) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 7 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 8;(c) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 9 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 10;(d) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 11 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 12;(e) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:13 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 14; or(f) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:15 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 16.2026205474   09 Jul 2026132. The genetically-modified immune cell of any one of claims 127-131, wherein said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 17 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 18.

133. The genetically-modified immune cell of any one of claims 127-132, wherein said nucleic acid sequence encoding said shRNAmiR comprises a sequence having at least 80% sequence identity to SEQ ID NO: 46.

134. The genetically-modified immune cell of claim 133, wherein said nucleic acid sequence encoding said shRNAmiR comprises the sequence of SEQ ID NO: 46.

135. The genetically-modified immune cell of claim 126, wherein said target protein is CS1.

136. The genetically-modified immune cell of claim 135, wherein cell surface expression of CS1 is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell.

137. The genetically-modified immune cell of claim 135 or claim 136, wherein said genetically-modified immune cell expresses a CAR having specificity for CS1.

138. The genetically-modified immune cell of any one of claims 135-137, wherein said genetically-modified immune cell is less susceptible to fratricide by a genetically-modified immune cell expressing a CAR having specificity for CS1 compared to a control cell.

139. The genetically-modified immune cell of any one of claims 135-138, wherein:(a) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:21 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 22;(b) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:23 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 24; or2026205474   09 Jul 2026(c) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:25 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 26.

140. The genetically-modified immune cell of any one of claims 135-139, wherein said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 25 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 26.

141. The genetically-modified immune cell of any one of claims 135-140, wherein said nucleic acid sequence encoding said shRNAmiR comprises a sequence having at least 80% sequence identity to SEQ ID NO: 50.

142. The genetically-modified immune cell of claim 141, wherein said nucleic acid sequence encoding said shRNAmiR comprises the sequence of SEQ ID NO: 50.

143. The genetically-modified immune cell of claim 126, wherein said target protein is TGFBR2.

144. The genetically-modified immune cell of claim 143, wherein cell surface expression of TGFBR2 is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell.

145. The genetically-modified immune cell of claim 143 or claim 144, wherein said genetically-modified immune cell is less susceptible to immunosuppression by transforming growth factor B1 (TGFB1) compared to a control cell.

146. The genetically-modified immune cell of any one of claims 143-145, wherein:(a) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:27 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 28;(b) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:29 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 30;2026205474   09 Jul 2026(c) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:31 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 32;(d) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:33 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 34; or(e) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:35 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 36.

147. The genetically-modified immune cell of any one of claims 143-146, wherein said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 31 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 32.

148. The genetically-modified immune cell of any one of claims 143-147, wherein said nucleic acid sequence encoding said shRNAmiR comprises a sequence having at least 80% sequence identity to SEQ ID NO: 53.

149. The genetically-modified immune cell of claim 148, wherein said nucleic acid sequence encoding said shRNAmiR comprises the sequence of SEQ ID NO: 53.

150. The genetically-modified immune cell of claim 126, wherein said target protein is CBL-B.

151. The genetically-modified immune cell of claim 150, wherein cell surface expression of CBL-B is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell.

152. The genetically-modified immune cell of claim 150 or claim 151, wherein said genetically-modified immune cell is less susceptible to suppression of T cell receptor (TCR) signaling by degradation of downstream signaling proteins compared to a control cell.2026205474   09 Jul 2026153. The genetically-modified immune cell of claim 126, wherein said target protein is CD52.

154. The genetically-modified immune cell of claim 153, wherein cell surface expression of CD52 is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell.

155. The genetically-modified immune cell of claim 153 or claim 154, wherein said genetically-modified immune cell is less susceptible to CD52 antibody-induced cell death.

156. The genetically-modified immune cell of any one of claims 153-155, wherein:(a) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:37 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 38; or(b) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:39 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 40.

157. The genetically-modified immune cell of any one of claims 153-156, wherein said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 37 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 38.

158. The genetically-modified immune cell of any one of claims 153-157, wherein said nucleic acid sequence encoding said shRNAmiR comprises a sequence having at least 80% sequence identity to SEQ ID NO: 56.

159. The genetically-modified immune cell of claim 158, wherein said nucleic acid sequence encoding said shRNAmiR comprises the sequence of SEQ ID NO: 56.

160. The genetically-modified immune cell of claim 126, wherein said target protein is DCK.2026205474   09 Jul 2026161. The genetically-modified immune cell of claim 160, wherein cell surface expression of DCK is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell.

162. The genetically-modified immune cell of claim 160 or claim 161, wherein said genetically-modified immune cell is less susceptible to effects of purine nucleoside analogs on cell proliferation.

163. The genetically-modified immune cell of any one of claims 160-162, wherein:(a) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:76 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 77;(b) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:78 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 79;(c) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:80 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 81;(d) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:82 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 83; or(e) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 84 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 85.

164. The genetically-modified immune cell of any one of claims 160-163, wherein said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 76 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 77.

165. The genetically-modified immune cell of any one of claims 160-164, wherein said nucleic acid sequence encoding said shRNAmiR comprises a sequence having at least 80% sequence identity to SEQ ID NO: 86.

166. The genetically-modified immune cell of claim 165, wherein said nucleic acid sequence encoding said shRNAmiR comprises the sequence of SEQ ID NO: 86.2026205474   09 Jul 2026167. The genetically-modified immune cell of claim 126, wherein said target protein is GR.

168. The genetically-modified immune cell of claim 167, wherein cell surface expression of GR is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell.

169. The genetically-modified immune cell of claim 167 or claim 168, wherein said genetically-modified immune cell is less susceptible to effects of glucocorticoids on cell proliferation.

170. The genetically-modified immune cell of any one of claims 167-169, wherein:(a) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 91 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 92;(b) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 93 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 94;(c) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 95 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 96;(d) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 97 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 98;(e) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 99 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 100;(f) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 101 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 102;(g) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 103 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 104;(h) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 105 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 106; or2026205474   09 Jul 2026(i)    said passenger strand comprises a nucleic acid sequence of SEQ ID NO:107 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 108.

171. The genetically-modified immune cell of any one of claims 167-170, wherein said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 95 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 96.

172. The genetically-modified immune cell of any one of claims 167-171, wherein said nucleic acid sequence encoding said shRNAmiR comprises a sequence having at least 80% sequence identity to SEQ ID NO: 111.

173. The genetically-modified immune cell of claim 172, wherein said nucleic acid sequence encoding said shRNAmiR comprises the sequence of SEQ ID NO: 111.

174. A method for reducing the expression of an endogenous protein in an immune cell, said method comprising introducing into said immune cell a template nucleic acid comprising a nucleic acid sequence encoding a shRNAmiR, wherein said template nucleic acid is inserted into the genome of said immune cell and said shRNAmiR is expressed, and wherein said shRNAmiR reduces expression of an endogenous target protein in said immune cell.

175. The method of claim 174, wherein said immune cell is a T cell, or a cell derived therefrom.

176. The method of claim 174, wherein said immune cell is a natural killer (NK) cell, or a cell derived therefrom.

177. The method of claim 174, wherein said immune cell is a B cell, or a cell derived therefrom.

178. The method of claim 174, wherein said immune cell is a monocyte or macrophage, or a cell derived therefrom.2026205474   09 Jul 2026179.   The method of any one of claims 174-178, wherein said template nucleic acid isinserted into the genome of said immune cell by random integration.

180. The method of any one of claims 174-179, wherein said template nucleic acid is introduced into said immune cell using a lentiviral vector.

181. The method of any one of claims 174-180, wherein said immune cell expresses aCAR or exogenous TCR.

182. The method of any one of claims 174-178, wherein said method further comprises introducing into said immune cell a second nucleic acid encoding an engineered nuclease having specificity for a recognition sequence in the genome of said immune cell, wherein said engineered nuclease is expressed in said immune cell and generates a cleavage site at said recognition sequence, and wherein said template nucleic acid is inserted into the genome of said immune cell at said cleavage site.

183. The method of claim 182, wherein said template nucleic acid is flanked by homology arms having homology to sequences flanking said recognition sequence, and wherein said template nucleic acid is inserted at said cleavage site by homologous recombination.

184. The method of claim 182 or claim 183, wherein said template nucleic acid is introduced into said immune cell by a viral vector.

185. The method of claim 184, wherein said viral vector is a recombinant AAV vector.

186. The method of claim 185, wherein said recombinant AAV vector has a serotypeof AAV2 or AAV6.

187. The method of any one of claims 182-186, wherein said recognition sequence is within a target gene.2026205474   09 Jul 2026188. The method of claim 187, wherein expression of a protein encoded by said target gene is disrupted in said immune cell.

189. The method of claim 187 or claim 188, wherein said target gene is selected from the group consisting of a TCR alpha gene and a TCR alpha constant region gene.

190. The method of claim 189, wherein said target gene is a TCR alpha constant region gene, and wherein said immune cell does not have detectable cell-surface expression of an endogenous TCR.

191. The method of any one of claims 182-190, wherein said engineered nuclease is an engineered meganuclease, a zinc finger nuclease, a TALEN, a compact TALEN, a CRISPR system nuclease, or a megaTAL.

192. The method of any one of claims 182-191, wherein said engineered nuclease is an engineered meganuclease.

193. The method of any one of claims 182-192, wherein said second nucleic acid encoding said engineered nuclease is introduced using an mRNA.

194. The method of any one of claims 174-193, wherein said immune cell comprises in its genome a nucleic acid sequence encoding a CAR or exogenous TCR.

195. The method of any one of claims 174-194, wherein said immune cell comprises in its genome a nucleic acid sequence encoding an HLA-E fusion protein.

196. The method of any one of claims 174-193 or 195, wherein said template nucleic acid comprises a nucleic acid sequence encoding a CAR or an exogenous TCR, wherein said CAR or said exogenous TCR is expressed by said immune cell.2026205474   09 Jul 2026197. The method of claim 196, wherein said nucleic acid sequence encoding said shRNAmiR and said nucleic acid sequence encoding said CAR or said exogenous TCR are operably linked to a same promoter in said immune cell following introduction of said template nucleic acid at said cleavage site.

198. The method of claim 196 or claim 197, wherein said template nucleic acid comprises, from 5' to 3':(a)    said nucleic acid sequence encoding said CAR or said exogenous TCR; and(b)    said nucleic acid sequence encoding said shRNAmiR.

199. The method of any one of claims 196 or claim 197, wherein said template nucleic acid comprises, from 5' to 3':(a)    said nucleic acid sequence encoding said shRNAmiR; and(b)    said nucleic acid sequence encoding said CAR or said exogenous TCR.

200. The method of any one of claims 196-199, wherein said nucleic acid sequence encoding said CAR or said exogenous TCR and said nucleic acid sequence encoding said shRNAmiR are separated by a 2A or IRES sequence.

201. The method of any one of claims 196-200, wherein said nucleic acid sequence encoding said shRNAmiR is in the same orientation as said nucleic acid sequence encoding said CAR or said exogenous TCR.

202. The method of any one of claims 196-200, wherein said nucleic acid sequence encoding said shRNAmiR is in a reverse orientation as said nucleic acid sequence encoding said CAR or said exogenous TCR.

203. The method of claim 196 or claim 197, wherein an intron sequence is positioned within said nucleic acid sequence encoding said CAR or said exogenous TCR, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence.2026205474   09 Jul 2026204. The method of any one of claims 196-203, wherein said template nucleic acid comprises a promoter, wherein said promoter is operably linked to said nucleic acid sequence encoding said CAR or said exogenous TCR and to said nucleic acid sequence encoding said shRNAmiR.

205. The method of any one of claims 196-204, wherein said template nucleic acid comprises a termination signal.

206. The method of any one of claims 174-194, wherein said template nucleic acid comprises a nucleic acid sequence encoding an HLA-E fusion protein, wherein said HLA-E fusion protein is expressed by said immune cell.

207. The method of claim 206, wherein said nucleic acid sequence encoding said shRNAmiR and said nucleic acid sequence encoding said HLA-E fusion protein are operably linked to a same promoter in said immune cell following introduction of said template nucleic acid at said cleavage site.

208. The method of claim 206 or claim 207, wherein said template nucleic acid comprises, from 5' to 3':(a)    said nucleic acid sequence encoding said HLA-E fusion protein; and(b)    said nucleic acid sequence encoding said shRNAmiR.

209. The method of any one of claims 206 or claim 207, wherein said template nucleic acid comprises, from 5' to 3':(a)    said nucleic acid sequence encoding said shRNAmiR; and(b)    said nucleic acid sequence encoding said HLA-E fusion protein.

210. The method of any one of claims 206-209, wherein said nucleic acid sequence encoding said HLA-E fusion protein and said nucleic acid sequence encoding said shRNAmiR are separated by a 2A or IRES sequence.2026205474   09 Jul 2026211. The method of claim 206 or claim 207, wherein an intron sequence is positioned within said nucleic acid sequence encoding said HLA-E fusion protein, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence.

212. The method of any one of claims 206-211, wherein said template nucleic acid comprises a promoter, wherein said promoter is operably linked to said nucleic acid sequence encoding said HLA-E fusion protein and to said nucleic acid sequence encoding said shRNAmiR.

213. The method of any one of claims 206-212, wherein said template nucleic acid comprises a termination signal.

214. The method of any one of claims 174-193, wherein said template nucleic acid comprises a nucleic acid sequence encoding a CAR or an exogenous TCR and a nucleic acid sequence encoding an HLA-E fusion protein, wherein said CAR or said exogenous TCR and said HLA-E fusion protein are expressed by said immune cell.

215. The method of claim 214, wherein said nucleic acid sequence encoding said shRNAmiR, said nucleic acid sequence encoding said CAR or said exogenous TCR, and said nucleic acid sequence encoding said HLA-E fusion protein are operably linked to a same promoter following introduction of said template nucleic acid at said cleavage site.

216. The method of claim 215, wherein said template nucleic acid comprises:(a) said nucleic acid sequence encoding said CAR or said exogenous TCR;(b) a 2A or IRES sequence;(c)    said nucleic acid sequence encoding said HLA-E fusion protein; and(d)    said nucleic acid sequence encoding said shRNAmiR.

217. The method of claim 216, wherein an intron sequence is positioned within said nucleic acid sequence encoding said HLA-E fusion protein, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence.2026205474   09 Jul 2026218. The method of claim 216, wherein an intron sequence is positioned within said nucleic acid sequence encoding said CAR or said exogenous TCR, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence.

219. The method of claim 216, wherein said template nucleic acid comprises a promoter that is operably linked to said nucleic acid sequence encoding said CAR or said exogenous TCR, said nucleic acid sequence encoding said HLA-E fusion protein, and said nucleic acid sequence encoding said shRNAmiR.

220. The method of any one of claims 215-219, wherein said template nucleic acid comprises a termination signal.

221. The method of claim 215, wherein said template nucleic acid comprises, from 5' to 3':(a)    a promoter;(b) said nucleic acid sequence encoding said CAR or said exogenous TCR;(c)    a 2A or IRES sequence;(d) said nucleic acid sequence encoding said HLA-E fusion protein, wherein an intron sequence is positioned within said nucleic acid sequence encoding said HLA-E fusion protein, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence; and(e) optionally a termination signal;wherein said nucleic acid sequence encoding said CAR or said exogenous TCR, said nucleic acid sequence encoding said HLA-E fusion protein, and said nucleic acid sequence encoding said shRNAmiR are operably linked to said promoter.

222. The method of claim 215, wherein said template nucleic acid comprises, from 5' to 3':(a) a promoter;2026205474   09 Jul 2026(b) said nucleic acid sequence encoding said HLA-E fusion protein, wherein an intron sequence is positioned within said nucleic acid sequence encoding said HLA-E fusion protein, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence;(c)    a 2A or IRES sequence;(d) said nucleic acid sequence encoding said CAR or said exogenous TCR; and(e)    optionally a termination signal;wherein said nucleic acid sequence encoding said CAR or said exogenous TCR, said nucleic acid sequence encoding said HLA-E fusion protein, and said nucleic acid sequence encoding said shRNAmiR are operably linked to said promoter.

223. The method of claim 215, wherein said template nucleic acid comprises, from 5' to 3':(a) a promoter;(b) said nucleic acid sequence encoding said CAR or said exogenous TCR, wherein an intron sequence is positioned within said nucleic acid sequence encoding said CAR or said exogenous TCR, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence;(c)    a 2A or IRES sequence;(d) said nucleic acid sequence encoding said HLA-E fusion protein; and(e)    optionally a termination signal;wherein said nucleic acid sequence encoding said CAR or said exogenous TCR, said nucleic acid sequence encoding said HLA-E fusion protein, and said nucleic acid sequence encoding said shRNAmiR are operably linked to said promoter.

224. The method of claim 215, wherein said template nucleic acid comprises, from 5' to 3':(a)    a promoter;(b) said nucleic acid sequence encoding said HLA-E fusion protein;(c)    a 2A or IRES sequence;2026205474   09 Jul 2026(d) said nucleic acid sequence encoding said CAR or said exogenous TCR, wherein an intron sequence is positioned within said nucleic acid sequence encoding said CAR or said exogenous TCR, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence; and(e) optionally a termination signal;wherein said nucleic acid sequence encoding said CAR or said exogenous TCR, said nucleic acid sequence encoding said HLA-E fusion protein, and said nucleic acid sequence encoding said shRNAmiR are operably linked to said promoter.

225. The method of any one of claims 203-205, 211-213, or 217-224, wherein said intron sequence is a synthetic intron sequence.

226. The method of any one of claims 203-205, 211-213, or 217-224, wherein said intron sequence comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 69.

227. The method of any one of claims 203-205, 211-213, or 217-224, wherein said intron sequence comprises a nucleic acid sequence of SEQ ID NO: 69.

228. The method of any one of claims 205, 213, or 220-227, wherein said termination signal is a polyA sequence or a bovine growth hormone (BGH) termination signal.

229. The method of claim 228, wherein said polyA sequence comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 68.

230. The method of claim 228, wherein said polyA sequence comprises a nucleic acid sequence of SEQ ID NO: 68.

231. The method of claim 228, wherein said BGH termination signal comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 71.2026205474   09 Jul 2026232. The method of claim 228, wherein said BGH termination signal comprises a nucleic acid sequence of SEQ ID NO: 71.

233. The method of any one of claims 197-205, 207-213, or 215-232, wherein said promoter comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 67.

234. The method of any one of claims 197-205, 207-213, or 215-232, wherein said promoter comprises a nucleic acid sequence of SEQ ID NO: 67.

235. The method of any one of claims 200-202, 204, 205, 210, 212, 213, or 216-234, wherein said 2A sequence is a P2A / furin site comprising a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 70.

236. The method of any one of claims 200-202, 204, 205, 210, 212, 213, or 216-234, wherein said 2A sequence is a P2A / furin site comprising a nucleic acid sequence of SEQ ID NO: 70.

237. The method of any one of claims 206-236, wherein said HLA-E fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 66.

238. The method of any one of claims 206-236, wherein said HLA-E fusion protein comprises an amino acid sequence of SEQ ID NO: 66.

239. The method of any one of claims 181 or 194-238, wherein said CAR comprises a signal peptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 73.

240. The method of any one of claims 181 or 194-238, wherein said CAR comprises a signal peptide comprising an amino acid sequence of SEQ ID NO: 73.2026205474   09 Jul 2026241. The method of claim 215, wherein said template nucleic acid comprises, from 5' to 3':(a) a promoter comprising a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 67;(b) said nucleic acid sequence encoding said CAR, wherein said CAR comprises a signal peptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 73;(c) a P2A / furin site comprising a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 70;(d) said nucleic acid sequence encoding said HLA-E fusion protein, wherein said HLA-E fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 66, and wherein an intron sequence is positioned within said nucleic acid sequence encoding said HLA-E fusion protein, wherein said intron sequence comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 69, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence; and(e) optionally a termination signal comprising a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 68;wherein said nucleic acid sequence encoding said CAR, said nucleic acid sequence encoding said HLA-E fusion protein, and said nucleic acid sequence encoding said shRNAmiR are operably linked to said promoter.

242. The method of claim 215, wherein said template nucleic acid comprises, from 5' to 3':(a) a promoter comprising a nucleic acid sequence of SEQ ID NO: 67;(b) said nucleic acid sequence encoding said CAR, wherein said CAR comprises a signal peptide comprising an amino acid sequence of SEQ ID NO: 73;(c) a P2A / furin site comprising a nucleic acid sequence of SEQ ID NO: 70;(d) said nucleic acid sequence encoding said HLA-E fusion protein, wherein said HLA-E fusion protein comprises an amino acid sequence of SEQ ID NO: 66, and wherein an intron sequence is positioned within said nucleic acid sequence encoding said HLA-E fusion protein, wherein said intron sequence comprises a nucleic acid sequence of SEQ ID NO: 69, and2026205474   09 Jul 2026wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence; and(e) optionally a termination signal comprising a nucleic acid sequence of SEQ ID NO: 68;wherein said nucleic acid sequence encoding said CAR, said nucleic acid sequence encoding said HLA-E fusion protein, and said nucleic acid sequence encoding said shRNAmiR are operably linked to said promoter.

243. The method of claim 241 or claim 242, wherein said template nucleic acid comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 74, wherein said template nucleic acid is inserted in the genome within a TCR alpha constant region gene.

244. The method of claim 241 or claim 242, wherein said template nucleic acid comprises a nucleic acid sequence of SEQ ID NO: 74, wherein said template nucleic acid is inserted in the genome within a TCR alpha constant region gene.

245. The method of claim 196, wherein said nucleic acid sequence encoding said shRNAmiR and said nucleic acid sequence encoding said CAR or said exogenous TCR are operably linked to different promoters in said immune cell following introduction of said template nucleic acid at said cleavage site.

246. The method of claim 245, wherein said template nucleic acid comprises, from 5' to 3':(a) a first promoter;(b) said nucleic acid sequence encoding said CAR or exogenous TCR which is operably linked to said first promoter;(c) a second promoter; and(d) said nucleic acid sequence encoding said shRNAmiR which is operably linked to said second promoter.2026205474   09 Jul 2026247. The method of claim 245, wherein said template nucleic acid comprises, from 5' to 3':(a) a first promoter;(b) said nucleic acid sequence encoding said shRNAmiR which is operably linked to said first promoter;(c) a second promoter; and(d) said nucleic acid sequence encoding said CAR or exogenous TCR which is operably linked to said second promoter.

248. The method of any one of claims 245-247, wherein said nucleic acid sequence encoding said shRNAmiR is in the same orientation as said nucleic acid sequence encoding said CAR or exogenous TCR.

249. The method of any one of claims 245-247, wherein said nucleic acid sequence encoding said shRNAmiR is in a reverse orientation as said nucleic acid sequence encoding said CAR or exogenous TCR.

250. The method of any one of claims 245-249, wherein said first promoter and said second promoter are identical.

251. The method of any one of claims 245-249, wherein said first promoter and said second promoter are different.

252. The method of any one of claims 245-251, wherein said template nucleic acid comprises one or more termination signals.

253. The method of claim 206, wherein said nucleic acid sequence encoding said shRNAmiR and said nucleic acid sequence encoding said HLA-E fusion protein are operably linked to different promoters in said immune cell following introduction of said template nucleic acid at said cleavage site.2026205474   09 Jul 2026254. The method of claim 253, wherein said template nucleic acid comprises, from 5' to 3':(a) a first promoter;(b) said nucleic acid sequence encoding said HLA-E fusion protein which is operably linked to said first promoter;(c) a second promoter; and(d) said nucleic acid sequence encoding said shRNAmiR which is operably linked to said second promoter.

255. The method of claim 253, wherein said template nucleic acid comprises, from 5' to 3':(a) a first promoter;(b) said nucleic acid sequence encoding said shRNAmiR which is operably linked to said first promoter;(c) a second promoter; and(d) said nucleic acid sequence encoding said HLA-E fusion protein which is operably linked to said second promoter.

256. The method of any one of claims 253-255, wherein said first promoter and said second promoter are identical.

257. The method of any one of claims 253-255, wherein said first promoter and said second promoter are different.

258. The method of any one of claims 253-257, wherein said template nucleic acid comprises one or more termination signals.2026205474   09 Jul 2026259.   The method of claim 214, wherein said template nucleic acid comprises:(a)    said nucleic acid sequence encoding said CAR or said exogenous TCR;(b)    said nucleic acid sequence encoding said HLA-E fusion protein; and(c)    said nucleic acid sequence encoding said shRNAmiR;wherein said nucleic acid sequence encoding said CAR or said exogenous TCR is operably linked to a first promoter,and wherein said nucleic acid sequence encoding said HLA-E fusion protein and said nucleic acid sequence encoding said shRNAmiR are operably linked to a second promoter.

260. The method of claim 214, wherein said template nucleic acid comprises:(a)    said nucleic acid sequence encoding said CAR or said exogenous TCR;(b)    said nucleic acid sequence encoding said HLA-E fusion protein; and(c)    said nucleic acid sequence encoding said shRNAmiR;wherein said nucleic acid sequence encoding said CAR or said exogenous TCR and said nucleic acid sequence encoding said shRNAmiR are operably linked to a first promoter,and wherein said nucleic acid sequence encoding said HLA-E fusion protein is operably linked to a second promoter.

261. The method of claim 259, wherein an intron sequence is positioned within said nucleic acid sequence encoding said HLA-E fusion protein, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence.

262. The method of claim 260, wherein an intron sequence is positioned within said nucleic acid sequence encoding said CAR or said exogenous TCR, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence.

263. The method of any one of claims 259-262, wherein said template nucleic acid comprises a first termination signal capable of terminating transcription of said CAR or said exogenous TCR, and a second termination signal capable of terminating transcription of said HLA-E fusion protein.2026205474   09 Jul 2026264. The method of any one of claims 259-262, wherein said template nucleic acid comprises a first termination signal capable of terminating transcription of said HLA-E fusion protein, and a second termination signal capable of terminating transcription of said CAR or said exogenous TCR.

265. The method of claim 214, wherein said template nucleic acid comprises, from 5' to 3':(a)     a first promoter;(b)    said nucleic acid sequence encoding said CAR or said exogenous TCR;(c)     optionally a first termination signal;(d)    a second promoter;(e)    said nucleic acid sequence encoding said HLA-E fusion protein, wherein an intronsequence is positioned within said nucleic acid sequence encoding said HLA-E fusion protein, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence; and(f) optionally a second termination signal;wherein said nucleic acid sequence encoding said CAR or said exogenous TCR is operably linked to said first promoter, and wherein said nucleic acid sequence encoding said HLA-E fusion protein and said nucleic acid sequence encoding said shRNAmiR are operably linked to said second promoter.

266. The method of claim 214, wherein said template nucleic acid comprises, from 5' to 3':(a) a first promoter;(b) said nucleic acid sequence encoding said HLA-E fusion protein, wherein an intron sequence is positioned within said nucleic acid sequence encoding said HLA-E fusion protein, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence;(c)     optionally a first termination signal;(d)    a second promoter;(e) said nucleic acid sequence encoding said CAR or said exogenous TCR; and2026205474   09 Jul 2026(f) optionally a second termination signal;wherein said nucleic acid sequence encoding said HLA-E fusion protein and said nucleic acid sequence encoding said shRNAmiR are operably linked to said first promoter, and wherein said nucleic acid sequence encoding said CAR or said exogenous TCR is operably linked to said second promoter.

267. The method of claim 214, wherein said template nucleic acid comprises, from 5' to 3':(a) a first promoter;(b) said nucleic acid sequence encoding said CAR or said exogenous TCR, wherein an intron sequence is positioned within said nucleic acid sequence encoding said CAR or said exogenous TCR, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence;(c)     optionally a first termination signal;(d)    a second promoter;(e) said nucleic acid sequence encoding said HLA-E fusion protein; and(f)     optionally a second termination signal;wherein said nucleic acid sequence encoding said CAR or said exogenous TCR and said nucleic acid sequence encoding said shRNAmiR are operably linked to said first promoter, and wherein said nucleic acid sequence encoding said HLA-E fusion protein is operably linked to said second promoter.

268. The method of claim 214, wherein said template nucleic acid comprises, from 5' to 3':(a)     a first promoter;(b)    said nucleic acid sequence encoding said HLA-E fusion protein;(c)     optionally a first termination signal;(d)    a second promoter;(e) said nucleic acid sequence encoding said CAR or said exogenous TCR, wherein an intron sequence is positioned within said nucleic acid sequence encoding said CAR or said2026205474   09 Jul 2026exogenous TCR, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence; and(f) optionally a second termination signal;wherein said nucleic acid sequence encoding said HLA-E fusion protein is operably linked to said first promoter, and wherein said nucleic acid sequence encoding said CAR or said exogenous TCR and said nucleic acid sequence encoding said shRNAmiR are operably linked to said second promoter.

269. The method of any one of claims 261-268, wherein said intron sequence is a synthetic intron sequence.

270. The method of any one of claims 261-268, wherein said intron sequence comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 69.

271. The method of any one of claims 261-268, wherein said intron sequence comprises a nucleic acid sequence of SEQ ID NO: 69.

272. The method of claim 252 or claim 258, wherein said one or more termination signals are a polyA sequence or a BGH termination signal.

273. The method of any one of claims 263-271, wherein said first termination signal is identical to said second termination signal.

274. The method of any one of claims 263-271, wherein said first termination signal is different from said second termination signal.

275. The method of claim 274, wherein said first termination signal is a polyA sequence and said second termination signal is a BGH termination signal.

276. The method of claim 274, wherein said first termination signal is a BGH termination signal and said second termination signal is a polyA sequence.2026205474   09 Jul 2026277. The method of any one of claims 272, 275, or 276, wherein said polyA sequence comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 68.

278. The method of any one of claims 272, 275, or 276, wherein said polyA sequence comprises a nucleic acid sequence of SEQ ID NO: 68.

279. The method of any one of claims 272, 275, or 276, wherein said BGH termination signal comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 71.

280. The method of any one of claims 272, 275, or 276, wherein said BGH termination signal comprises a nucleic acid sequence of SEQ ID NO: 71.

281. The method of any one of claims 246-252 or 254-280, wherein said first promoter and said second promoter are identical.

282. The method of any one of claims 246-252 or 254-280, wherein said first promoter is different from said second promoter.

283. The method of claim 282, wherein said first promoter is a JeT promoter, and wherein said second promoter is an EF1 alpha core promoter.

284. The method of claim 282, wherein said first promoter is an EF1 alpha core promoter, and wherein said second promoter is a JeT promoter.

285. The method of claim 283 or claim 284, wherein said JeT promoter comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 67.

286. The method of claim 283 or claim 284, wherein said JeT promoter comprises a nucleic acid sequence of SEQ ID NO: 67.2026205474   09 Jul 2026287. The method of any one of claims 283-286, wherein said EF1 alpha core promoter comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 72.

288. The method of any one of claims 283-286, wherein said EF1 alpha core promoter comprises a nucleic acid sequence of SEQ ID NO: 72.

289. The method of any one of claims 253-288, wherein said HLA-E fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 66.

290. The method of any one of claims 253-288, wherein said HLA-E fusion protein comprises an amino acid sequence of SEQ ID NO: 66.

291. The method of any one of claims 245-290, wherein said CAR comprises a signal peptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 73.

292. The method of any one of claims 245-290, wherein said CAR comprises a signal peptide comprising an amino acid sequence of SEQ ID NO: 73.

293. The method of claim 214, wherein said template nucleic acid comprises, from 5' to 3':(a) a first promoter comprising a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 67;(b) said nucleic acid sequence encoding said CAR, wherein said CAR comprises a signal peptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 73;(c) optionally a first termination signal comprising a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 70;(d) a second promoter comprising a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 72;2026205474   09 Jul 2026(e) said nucleic acid sequence encoding said HLA-E fusion protein, wherein said HLA-E fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 66, and wherein an intron sequence is positioned within said nucleic acid sequence encoding said HLA-E fusion protein, wherein said intron sequence comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 69, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence; and(f) optionally a second termination signal comprising a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 71;wherein said nucleic acid sequence encoding said CAR is operably linked to said first promoter, and wherein said nucleic acid sequence encoding said HLA-E fusion protein and said nucleic acid sequence encoding said shRNAmiR are operably linked to said second promoter.

294. The method of claim 214, wherein said template nucleic acid comprises, from 5' to 3':(a) a first promoter comprising a nucleic acid sequence of SEQ ID NO: 67;(b) said nucleic acid sequence encoding said CAR, wherein said CAR comprises a signal peptide comprising an amino acid sequence of SEQ ID NO: 73;(c) optionally a first termination signal comprising a nucleic acid sequence of SEQ ID NO: 70;(d) a second promoter comprising a nucleic acid sequence of SEQ ID NO: 72;(e) said nucleic acid sequence encoding said HLA-E fusion protein, wherein said HLA-E fusion protein comprises an amino acid sequence of SEQ ID NO: 66, and wherein an intron sequence is positioned within said nucleic acid sequence encoding said HLA-E fusion protein, wherein said intron sequence comprises a nucleic acid sequence of SEQ ID NO: 69, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence; and(f) optionally a second termination signal comprising a nucleic acid sequence of SEQ ID NO: 71;wherein said nucleic acid sequence encoding said CAR is operably linked to said first promoter, and wherein said nucleic acid sequence encoding said HLA-E fusion protein and said nucleic acid sequence encoding said shRNAmiR are operably linked to said second promoter.2026205474   09 Jul 2026295. The method of claim 293 or claim 294, wherein said template nucleic acid comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 75, wherein said cassette is inserted in the genome within a TCR alpha constant region gene.

296. The method of claim 293 or claim 294, wherein said template nucleic acid comprises a nucleic acid sequence of SEQ ID NO: 75, wherein said cassette is inserted in the genome within a TCR alpha constant region gene.

297. The method of any one of claims 174-296, wherein said shRNAmiR comprises, from 5' to 3':(a) a 5' miR scaffold domain;(b)    a 5' miR basal stem domain;(c)    a passenger strand;(d)    a miR loop domain;(e)     a guide strand;(f)    a 3' miR basal stem domain; and(g) a 3' miR scaffold domain.

298. The method of claim 297, wherein said miR loop domain is a miR-30a loop domain.

299. The method of claim 298, wherein said nucleic acid sequence encoding said shRNAmiR comprises a miR-30a loop domain coding sequence having at least 80% sequence identity to SEQ ID NO: 3.

300. The method of claim 298, wherein said nucleic acid sequence encoding said shRNAmiR comprises a miR-30a loop domain coding sequence of SEQ ID NO: 3.2026205474   09 Jul 2026301. The method of any one of claims 174-300, wherein said shRNAmiR comprises a microRNA-E (miR-E) scaffold, a miR-30 (e.g., miR-30a) scaffold, a miR-15 scaffold, a miR-16 scaffold, a miR-155 scaffold, a miR-22 scaffold, a miR-103 scaffold, or a miR-107 scaffold.

302. The method of claim 301, wherein said shRNAmiR comprises a miR-E scaffold.

303. The method of any one of claims 297-302, wherein:(a) said 5' miR scaffold domain comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 1;(b) said 5' miR basal stem domain comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 2;(c) said 3' miR basal stem domain comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 4; and(d) said 3' miR scaffold domain comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 5.

304. The method of any one of claims 297-303, wherein:(a) said 5' miR scaffold domain comprises a nucleic acid sequence of SEQ ID NO: 1;(b) said 5' miR basal stem domain comprises a nucleic acid sequence of SEQ ID NO: 2;(c) said 3' miR basal stem domain comprises a nucleic acid sequence of SEQ ID NO: 4; and(d) said 3' miR scaffold domain comprises a nucleic acid sequence of SEQ ID NO: 5.

305. The method of any one of claims 174-304, wherein expression of said target protein is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell.2026205474   09 Jul 2026306. The method of any one of claims 174-305, wherein said target protein is beta-2 microglobulin, CS1, transforming growth factor receptor 2 (TGFBR2), Cbl proto-oncogene B (CBL-B), CD52, a TCR alpha gene, a TCR alpha constant region gene, CD7, glucocorticoid receptor (GR), deoxycytidine kinase (DCK), nuclear receptor subfamily 2 group F member 6 (NR2F6), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), or C-C chemokine receptor type 5 (CCR5).

307. The method of claim 306, wherein said target protein is beta-2 microglobulin.

308. The method of claim 307, wherein cell-surface expression of beta-2microglobulin is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell.

309. The method of claim 307 or claim 308, wherein expression of MHC class I molecules is reduced on the cell surface by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell.

310. The method of any one of claims 307-309, wherein said immune cell has reduced allogenicity compared to a control cell.

311. The method of any one of claims 307-310, wherein:(a) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 17 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 18;(b) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 7 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 8;(c) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 9 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 10;(d) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 11 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 12;2026205474   09 Jul 2026(e) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:13 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 14; or(f) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:15 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 16.

312. The method of any one of claims 307-311, wherein said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 17 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 18.

313. The method of any one of claims 307-312, wherein said nucleic acid sequence encoding said shRNAmiR comprises a sequence having at least 80% sequence identity to SEQ ID NO: 46.

314. The method of any one of claims 307-313, wherein said nucleic acid sequence encoding said shRNAmiR comprises the sequence of SEQ ID NO: 46.

315. The method of claim 306, wherein said target protein is CS1.

316. The method of claim 315, wherein cell surface expression of CS1 is reduced by atleast about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell.

317. The method of claim 315 or claim 316, wherein said immune cell expresses a CAR having specificity for CS1.

318. The method of any one of claims 315-317, wherein said immune cell is less susceptible to fratricide by an immune cell expressing a CAR having specificity for CS1 compared to a control cell.

319. The method of any one of claims 315-318, wherein:2026205474   09 Jul 2026(a) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 21 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 22;(b) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 23 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 24; or(c) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 25 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 26.

320. The method of any one of claims 315-319, wherein said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 25 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 26.

321. The method of any one of claims 315-320, wherein said nucleic acid sequence encoding said shRNAmiR comprises a sequence having at least 80% sequence identity to SEQ ID NO: 50.

322. The method of claim 315-321, wherein said nucleic acid sequence encoding said shRNAmiR comprises the sequence of SEQ ID NO: 50.

323. The method of claim 306, wherein said target protein is TGFBR2.

324. The method of claim 323, wherein cell surface expression of TGFBR2 is reducedby at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell.

325. The method of claim 323 or claim 324, wherein said immune cell is less susceptible to immunosuppression by transforming growth factor B1 (TGFB1) compared to a control cell.

326. The method of any one of claims 323-325, wherein:2026205474   09 Jul 2026(a) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 27 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 28;(b) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 29 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 30;(c) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 31 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 32;(d) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 33 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 34; or(e) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 35 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 36.

327. The method of any one of claims 323-326, wherein said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 31 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 32.

328. The method of any one of claims 323-327, wherein said nucleic acid sequence encoding said shRNAmiR comprises a sequence having at least 80% sequence identity to SEQ ID NO: 53.

329. The method of any one of claims 323-328, wherein said nucleic acid sequence encoding said shRNAmiR comprises the sequence of SEQ ID NO: 53.

330. The method of claim 306, wherein said target protein is CBL-B.

331. The method of claim 330, wherein cell surface expression of CBL-B is reducedby at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell.2026205474   09 Jul 2026332. The method of claim 330 or claim 331, wherein said immune cell is less susceptible to suppression of T cell receptor (TCR) signaling by degradation of downstream signaling proteins compared to a control cell.

333. The method of claim 306, wherein said target protein is CD52.

334. The method of claim 333, wherein cell surface expression of CD52 is reduced byat least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell.

335. The method of claim 333 or claim 334, wherein said immune cell is less susceptible to CD52 antibody-induced cell death.

336. The method of any one of claims 333-335, wherein:(a) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:37 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 38; or(b) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:39 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 40.

337. The method of any one of claims 333-336, wherein said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 37 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 38.

338. The method of any one of claims 333-337, wherein said nucleic acid sequence encoding said shRNAmiR comprises a sequence having at least 80% sequence identity to SEQ ID NO: 56.

339. The method of any one of claims 333-338, wherein said nucleic acid sequence encoding said shRNAmiR comprises the sequence of SEQ ID NO: 56.2026205474   09 Jul 2026340.   The method of claim 306, wherein said target protein is DCK.

341. The method of claim 340, wherein cell surface expression of DCK is reduced byat least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell.

342. The method of claim 340 or claim 341, wherein said immune cell is less susceptible to effects of purine nucleoside analogs on cell proliferation.

343. The method of any one of claims 340-342, wherein:(a) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:76 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 77;(b) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:78 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 79;(c) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:80 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 81;(d) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:82 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 83; or(e) said passenger strand comprises a nucleic acid sequence of SEQ ID NO:84 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 85.

344. The method of any one of claims 340-343, wherein said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 76 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 77.

345. The method of any one of claims 340-344, wherein said nucleic acid sequence encoding said shRNAmiR comprises a sequence having at least 80% sequence identity to SEQ ID NO: 86.2026205474   09 Jul 2026346. The method of any one of claims 340-345, wherein said nucleic acid sequence encoding said shRNAmiR comprises the sequence of SEQ ID NO: 86.

347. The method of claim 306, wherein said target protein is GR.

348. The method of claim 347, wherein cell surface expression of GR is reduced by atleast about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell.

349. The method of claim 347 or claim 348, wherein said immune cell is less susceptible to effects of glucocorticoids on cell proliferation.

350. The method of any one of claims 347-349, wherein:(a) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 91 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 92;(b) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 93 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 94;(c) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 95 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 96;(d) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 97 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 98;(e) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 99 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 100;(f) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 101 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 102;(g) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 103 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 104;(h) said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 105 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 106; or2026205474   09 Jul 2026(i)    said passenger strand comprises a nucleic acid sequence of SEQ ID NO:107 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 108.

351. The method of any one of claims 347-350, wherein said passenger strand comprises a nucleic acid sequence of SEQ ID NO: 95 and said guide strand comprises a nucleic acid sequence of SEQ ID NO: 96.

352. The method of any one of claims 347-351, wherein said nucleic acid sequence encoding said shRNAmiR comprises a sequence having at least 80% sequence identity to SEQ ID NO: 111.

353. The method of any one of claims 347-352, wherein said nucleic acid sequence encoding said shRNAmiR comprises the sequence of SEQ ID NO: 111.

354. An immune cell made by the method of any one of claims 174-306.

355. An immune cell made by the method of any one of claims 307-314.

356. An immune cell made by the method of any one of claims 315-322.

357. An immune cell made by the method of any one of claims 323-329.

358. An immune cell made by the method of any one of claims 330-332.

359. An immune cell made by the method of any one of claims 333-339.

360. An immune cell made by the method of any one of claims 340-346.

361. An immune cell made by the method of any one of claims 347-353.2026205474   09 Jul 2026362. A population of cells comprising a plurality of said genetically-modified immune cell of any one of claims 1-173 or a plurality of said immune cell of any one of claims 354-361.

363. The population of cells of claim 362, wherein at least about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to 100% of cells in said population are said genetically-modified immune cell of any one of claims 1-173 or said immune cell of any one of claims 354361.

364. A pharmaceutical composition comprising a pharmaceutically-acceptable carrier and said genetically-modified immune cell of any one of claims 1-173 or said immune cell of any one of claims 354-361.

365. The pharmaceutical composition of claim 326, wherein said pharmaceutical composition comprises a pharmaceutically-acceptable carrier and said population of cells of claim 362 or claim 363.

366. A method of immunotherapy for treating a disease in a subject in need thereof, said method comprising administering to said subject an effective amount of said pharmaceutical composition of claim 364 or claim 365.

367. The method of claim 366, wherein said method is an immunotherapy for the treatment of a cancer in a subject in need thereof, and wherein said genetically-modified immune cell or said immune cell is a genetically-modified human T cell, or a cell derived therefrom, or a genetically-modified NK cell, or a cell derived therefrom, and wherein said genetically-modified immune cell or said immune cell comprises a CAR or an exogenous TCR, wherein said CAR or said exogenous TCR comprises an extracellular ligand-binding domain having specificity for a tumor-specific antigen.2026205474   09 Jul 2026368. The method of claim 366 or claim 367, wherein said genetically-modified immune cell or said immune cell comprises an inactivated TCR alpha gene or an inactivated TCR alpha constant region gene.

369. The method of any one of claims 366-368, wherein said genetically-modified immune cell or said immune cell has no detectable cell-surface expression of an endogenous alpha / beta TCR.

370. The method of any one of claims 367-369, wherein said cancer is selected from the group consisting of a cancer of carcinoma, lymphoma, sarcoma, blastomas, and leukemia.

371. The method of any one of claims 367-370, wherein said cancer is selected from the group consisting of a cancer of B cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin’s lymphoma.

372. The method of claim 371, wherein said cancer of B cell origin is selected from the group consisting of B lineage acute lymphoblastic leukemia, B cell chronic lymphocytic leukemia, B cell non-Hodgkin lymphoma, and multiple myeloma.

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