Disclosed herein are methods and compositions for inactivating TCR and/or HLA genes, using engineered nucleases comprising at least one DNA binding domain and a cleavage domain or cleavage half-domain in conditions able to preserve cell viability. Polynucleotides encoding nucleases, vectors comprising polynucleotides encoding nucleases and cells comprising polynucleotides encoding nucleases and/or cells comprising nucleases are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a divisional application of U.S. Pat. Application No. 16/009,975, filed Jun. 15, 2018, which claims the benefit of U.S. Provisional Application No. 62/521,132, filed Jun. 16, 2017; U.S. Provisional Application 62/542,052, filed Aug. 7, 2017 and U.S. Provisional Application No. 62/573,956, filed Oct. 18, 2017, the disclosures of which are hereby incorporated by reference in their entireties.

SEQUENCE LISTING

[0002] [0001.1] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Mar. 9, 2023, is named 128687-2880.xml and is 248,720 bytes in size.

TECHNICAL FIELD

[0003] The present disclosure is in the field of genome modification of human cells, including lymphocytes and stem cells.

BACKGROUND

[0004] Gene therapy holds enormous potential for a new era of human therapeutics. These methodologies will allow treatment for conditions that have not been addressable by standard medical practice. Gene therapy can include the many variations of genome editing techniques such as disruption (inactivation) or correction of a gene locus, and/or insertion of an expressible transgene that can be controlled either by a specific exogenous promoter operably linked to the transgene, or by the endogenous promoter found at the site of insertion into the genome.

[0005] Delivery and insertion of the transgene are examples of hurdles that must be solved for any real implementation of this technology. For example, although a variety of gene delivery methods are potentially available for therapeutic use, all involve substantial tradeoffs between safety, durability and level of expression. Methods that provide the transgene as an episome (e.g., adenovirus (Ad), adeno-associated virus (AAV) and plasmid-based systems) can yield high initial expression levels, however, these methods lack robust episomal replication, which may limit the duration of expression in mitotically active tissues. In contrast, delivery methods that result in the random integration of the desired transgene (e.g., integrating lentivirus (LV)) provide more durable expression but, due to the untargeted nature of the random insertion, may provoke unregulated growth in the recipient cells, potentially leading to malignancy via activation of oncogenes in the vicinity of the randomly integrated transgene cassette. Moreover, although transgene integration avoids replication-driven loss, it does not prevent eventual silencing of the exogenous promoter fused to the transgene. Over time, such silencing results in reduced transgene expression for the majority of non-specific insertion events. In addition, integration of a transgene rarely occurs in every target cell, which can make it difficult to achieve a high enough expression level of the transgene of interest to achieve the desired therapeutic effect.

[0006] In recent years, a new strategy for genetic modification (e.g., inactivation, correction and/or transgene integration) has been developed that uses cleavage with site-specific nucleases (e.g., zinc finger nucleases (ZFNs), transcription activator-like effector domain nucleases (TALENs), CRISPR/Cas system with an engineered crRNA/tracr RNA (‘single guide RNA’) to guide specific cleavage, etc.) to bias editing at a chosen genomic locus. See, e.g., U.S. Pat. Nos. 9,937,207; 9,255,250; 9,045,763; 9,005,973; 8,956,828; 8,945,868; 8,703,489; 8,586,526; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,067,317; 7,262,054; 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; U.S. Patent Publication Nos. 2017/0211075; 2003/0232410; 2005/0208489; 2005/0026157; 2005/0064474; 2006/0063231; 2008/0159996; 2010/0218264; 2012/0017290; 2011/0265198; 2013/0137104; 2013/0122591; 2013/0177983 and 2013/0177960 and 2015/0056705. Further, targeted nucleases are being developed based on the Argonaute system (e.g., from T. thermophilus, known as ‘TtAgo’, see Swarts, et al. (2014) Nature 507(7491): 258-261), which also may have the potential for uses in genome editing and gene therapy. This nuclease-mediated approach to genetic modification offers the prospect of improved transgene expression, increased safety and expressional durability, as compared to classic integration approaches, since it allows exact transgene positioning for a minimal risk of gene silencing or activation of nearby oncogenes.

[0007] The T cell receptor (TCR) is an essential part of the selective activation of T cells. Bearing some resemblance to an antibody, the antigen recognition part of the TCR is typically made from two chains, α and β, which co-assemble to form a heterodimer. The antibody resemblance lies in the manner in which a single gene encoding a TCR alpha and beta complex is put together. TCR alpha (TCR α) and beta (TCR β) chains are each composed of two regions, a C-terminal constant region and an N-terminal variable region. The genomic loci that encode the TCR alpha and beta chains resemble antibody encoding loci in that the TCR α gene comprises V and J segments, while the β chain locus comprises D segments in addition to V and J segments. For the TCR β locus, there are additionally two different constant regions that are selected from during the selection process. During T cell development, the various segments recombine such that each T cell comprises a unique TCR variable portion in the alpha and beta chains, called the complementarity determining region (CDR), and the body has a large repertoire of T cells which, due to their unique CDRs, are capable of interacting with unique antigens displayed by antigen presenting cells. Once a TCR α or β gene rearrangement has occurred, the expression of the second corresponding TCR α or TCR β is repressed such that each T cell only expresses one unique TCR structure in a process called ‘antigen receptor allelic exclusion’ (see, Brady, et al. (2010) J Immunol 185:3801-3808).

[0008] During T cell activation, the TCR interacts with antigens displayed as peptides on the major histocompatability complex (MHC) of an antigen presenting cell. Recognition of the antigen-MHC complex by the TCR leads to T cell stimulation, which in turn leads to differentiation of both T helper cells (CD4+) and cytotoxic T lymphocytes (CD8+) in memory and effector lymphocytes. These cells then can expand in a clonal manner to give an activated subpopulation within the whole T cell population capable of reacting to one particular antigen.

[0009] MHC proteins are of two classes, I and II. The class I MHC proteins are heterodimers of two proteins, the α chain, which is a transmembrane protein encoded by the MHC1 class I genes, and the β2 microglobulin chain (sometimes referred to as B2M), which is a small extracellular protein that is encoded by a gene that does not lie within the MHC gene cluster. The α chain folds into three globular domains and when the β2 microglobulin chain is associated, the globular structure complex functional and expressed on the cell surface. Peptides are presented on the two most N-terminal domains which are also the most variable. Class II MHC proteins are also heterodimers, but the heterodimers comprise two transmembrane proteins encoded by genes within the MHC complex. The class I MHC:antigen complex interacts with cytotoxic T cells while the class II MHC presents antigens to helper T cells. In addition, class I MHC proteins tend to be expressed in nearly all nucleated cells and platelets (and red blood cells in mice) while class II MHC protein are more selectively expressed. Typically, class II MHC proteins are expressed on B cells, some macrophage and monocytes, Langerhans cells, and dendritic cells.

[0010] In humans, the major histocompatibility complex (MHC) is commonly known as the human leukocyte antigen (HLA). The class I HLA gene cluster in humans comprises three major loci, B, C and A, as well as several minor loci (including E, G and F, all found in the HLA region on chromosome 6). The class II HLA cluster also comprises three major loci, DP, DQ and DR, and both the class I and class II gene clusters are polymorphic, in that there are several different alleles of both the class I and II genes within the population. There are also several accessory proteins that play a role in HLA functioning as well. β-2 microglobulin functions as a chaperon (encoded by B2M, located on chromosome 15) and stabilizes the HLA A, B or C protein expressed on the cell surface and also stabilizes the antigen display groove on the class I structure. It is found in the serum and urine in low amounts normally.

[0011] HLA plays a major role in transplant rejection. The acute phase of transplant rejection can occur within about 1-3 weeks and usually involves the action of host T lymphocytes on donor tissues due to sensitization of the host system to the donor class I and class II HLA molecules. In most cases, the triggering antigens are the class I HLAs. For best success, donors are typed for HLA and matched to the patient recipient as completely as possible. But donation even between family members, which can share a high percentage of HLA identity, is still often not successful. Thus, in order to preserve the graft tissue within the recipient, the patient often must be subjected to profound immunosuppressive therapy to prevent rejection. Such therapy can lead to complications and significant morbidities due to opportunistic infections that the patient may have difficulty overcoming. Regulation of the class I or II genes can be disrupted in the presence of some tumors and such disruption can have consequences on the prognosis of the patients. For example, reduction of B2M expression was found in metastatic colorectal cancers (Shrout, et al. (2008) Br J Canc 98: 1999). Since B2M has a key role in stabilizing the MHC class I complex, loss of B2M in certain solid cancers has been hypothesized to be a mechanism of immune escape from T cell driven immune surveillance. Depressed B2M expression has been shown to be a result of suppression of the normal IFN gamma B2M expressional regulation and/or specific mutations in the B2M coding sequence that result in gene knock-out (Shrout, et al., ibid). Confoundingly, increased B2M is also associated with some types of cancer. Increased B2M levels in the urine serves as a prognosticator for several cancers including prostate, chronic lymphocytic leukemia (CLL) and Non-Hodgkin’s lymphomas.

[0012] Adoptive cell therapy (ACT) is a developing form of cancer therapy based on delivering tumor-specific immune cells to a patient in order for the delivered cells to attack and clear the patient’s cancer. ACT can involve the use of tumor-infiltrating lymphocytes (TILs) which are T-cells that are isolated from a patient’s own tumor masses and expanded ex vivo to re-infuse back into the patient. This approach has been promising in treating metastatic melanoma, where in one study, a long term response rate of >50% was observed (see for example, Rosenberg, et al. (2011) Clin Canc Res 17(13): 4550). TILs are a promising source of cells because they are a mixed set of the patient’s own cells that have T-cell receptors (TCRs) specific for the Tumor associated antigens (TAAs) present on the tumor (Wu, et al. (2012) Cancer J 18(2):160). Other approaches involve editing T cells isolated from a patient’s blood such that they are engineered to be responsive to a tumor in some way (Kalos, et al. (2011) Sci TranslMed 3(95):95ra73).

[0013] Chimeric Antigen Receptors (CARs) are molecules designed to target immune cells to specific molecular targets expressed on cell surfaces. In their most basic form, they are receptors introduced into a cell that couple a specificity domain expressed on the outside of the cell to signaling pathways on the inside of the cell such that when the specificity domain interacts with its target, the cell becomes activated. Often CARs are made from emulating the functional domains of T-cell receptors (TCRs) where an antigen specific domain, such as a scFv or some type of receptor, is fused to the signaling domain, such as ITAMs and other co-stimulatory domains. These constructs are then introduced into a T-cell ex vivo allowing the T-cell to become activated in the presence of a cell expressing the target antigen, resulting in the attack on the targeted cell by the activated T-cell in a non-MHC dependent manner (see Chicaybam, et al. (2011) Int Rev Immunol 30:294-311) when the T-cell is re-introduced into the patient. Thus, adoptive cell therapy using T cells altered ex vivo with an engineered TCR or CAR is a very promising clinical approach for several types of diseases. For example, cancers and their antigens that are being targeted includes follicular lymphoma (CD20 or GD2), neuroblastoma (CD 171), non-Hodgkin lymphoma (CD 19 and CD20), lymphoma (CD19), glioblastoma (IL13Rα2), chronic lymphocytic leukemia or CLL and acute lymphocytic leukemia or ALL (both CD19). Virus specific CARs have also been developed to attack cells harboring virus such as HIV. For example, a clinical trial was initiated using a CAR specific for Gp100 for treatment of HIV (Chicaybam, ibid).

[0014] ACTRs (Antibody-coupled T-cell Receptors) are engineered T cell components that are capable of binding to an exogenously supplied antibody. The binding of the antibody to the ACTR component arms the T cell to interact with the antigen recognized by the antibody, and when that antigen is encountered, the ACTR comprising T cell is triggered to interact with antigen (see U.S. Pat. Publication No. 2015/0139943).

[0015] One of the drawbacks of adoptive cell therapy however is the source of the cell product must be patient specific (autologous) to avoid potential rejection of the transplanted cells. This has led researchers to develop methods of editing a patient’s own T cells to avoid this rejection. For example, a patient’s T cells or hematopoietic stem cells can be manipulated ex vivo with the addition of an engineered CAR, ACTR and/or T cell receptor (TCR), and then further treated with engineered nucleases to knock out T cell check point inhibitors such as PD1 and/or CTLA4 (see International Patent Publication No. WO 2014/059173). For application of this technology to a larger patient population, it would be advantageous to develop a universal population of cells (allogeneic). In addition, knockout of the TCR will result in cells that are unable to mount a graft-versus-host disease (GVHD) response once introduced into a patient.

[0016] Thus, there remains a need for methods and compositions that can be used to modify (e.g., knock out) TCR and/or HLA expression in effector T cells, regulatory T cells, B cells, NK cells or stem cells (e.g., hematopoietic stem cells, induced pluripotent stem cells and embryonic stem cells).

SUMMARY

[0017] Disclosed herein are compositions and methods for partial or complete inactivation or disruption of a TCR and/or B2M gene and compositions and methods for introducing and expressing to desired levels of exogenous transgenes in T lymphocytes, after or simultaneously with the disruption of the endogenous TCR and/or B2M. Also provided herein are methods and compositions for deleting (inactivating) or repressing a TCR and/or B2M gene to produce TCR null T cell or TCR and HLA class I null T cell, B cells, NK cell, stem cell, tissue or whole organism, for example a cell that does not express one or more T cell receptors and/or one or more HLA class I receptors on its surface. Additional genomic modifications may be present in the TCR and/or HLA class I null cells described herein, including, but not limited to genomic modifications to a different gene (e.g., a programmed cell death 1 (PD1) gene, a Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) gene, a CISH gene, a tet2 gene, an human leukocyte antigen (HLA) A gene, an HLA B gene, an HLA C gene, an HLA-DPA gene, an HLA-DQ gene, an HLA-DRA gene, a LMP7 gene, a Transporter associated with Antigen Processing (TAP) 1 gene, a TAP2 gene, a tapasin gene (TAPBP), a class II major histocompatibility complex transactivator (CIITA) gene, a glucocorticoid receptor gene (GR), an IL2RG gene, an RFX5 gene), insertion of transgene (e.g., CAR) into one or more of these or other genes (e.g., safe harbor genes) and any combination of such genomic modifications. In certain embodiments, the TCR null cells and/or HLA class I null cells, or tissues are human cells or tissues that are advantageous for use in transplants. In preferred embodiments, the TCR null T cells and/or HLA class I null cells are prepared for use in adoptive T cell therapy.

[0018] In one aspect, described herein is a zinc finger nuclease comprising: a ZFP from a ZFN designated 68957, 72678, 72732 or 72748; an engineered FokI cleavage domain; and a linker between the FokI cleavage domain and the ZFP. In certain embodiments, the ZFN comprises first and second ZFNs as follows (amino acid and polynucleotide sequences disclosed in the Examples): a ZFN comprising a ZFP from the ZFN designated 72678 and a ZFN comprising a ZFP from the ZFN designated 72732. In certain embodiments the ZFN comprises left and right (first and second) ZFNs as follows: a ZFN designated 57531 and a ZFN designated 72732; a ZFN designated 57531 and a ZFN designated 72748; a ZFN designated 68957 and a ZFN designated 57071; a ZFN designated 68957 and a ZFN designated 72732; a ZFN designated 68957 and a ZFN designated 72748; a ZFN designated 72678 and a ZFN designated 57071; a ZFN designated 72678 and a ZFN designated 72732; and a comprising a ZFP ZFN designated 72678 and a ZFN designated 72748. A zinc finger nuclease (ZFN) comprising left and right (first and second) ZFNs as follows: a ZFN designated 68796 and a ZFN designated 68813; a ZFN designated 68796 and a ZFN designated 68861; a ZFN designated 68812 and a ZFN designated 68813; a ZFN designated 68876 and a ZFN designated 68877; a ZFN designated 68815 and a ZFN designated 55266; a ZFN designated 68879 and a ZFN designated 55266; a ZFN designated 68798 and a ZFN designated 68815; or a ZFN designated 68846 and a ZFN designated 53853. Polynucleotides (e.g., mRNA, plasmids, viral vectors, etc.) encoding a ZFN (including a pair) as disclosed herein are also provided, including a polynucleotide comprising a 2A sequence between the sequences encoding the left and ZFNs. Also disclosed are genetically modified cells (e.g., stem cells, precursor cells, T cells (effector and regulatory), etc.) comprising one or more of the ZFNs and/or polynucleotides disclosed herein and cells descended from these cells (e.g., genetically modified cells that do not comprise the ZFN but include the genetic modification). The genetic modifications include insertions, deletions and combinations thereof in the gene targeted by the ZFN. Additional genomic modifications, for example, modification of a T cell receptor (TCR) gene, modification of an HLA-A gene, modification of an HLA-B gene, modification of an HLA-C gene, modification of a TAP gene, modification of a CTLA-4 gene, modification of a PD1 gene, modification of a CISH gene, modification of a tet-2 gene, and/or insertion of a transgene (e.g., CAR) may be present at the target and/or one or more different loci. Pharmaceutical compositions comprising any of the zinc finger nucleases, polynucleotides, and/or cells as described herein are also provided. Methods of modifying an endogenous beta-2-microglobulin (B2M) and/or TCR gene in a cell are also provided, the method comprising administering a polynucleotide or pharmaceutical composition as described herein to the cell such that the endogenous gene is modified (e.g., deletion, insertion of an exogenous sequence such as a transgene). Methods of using the ZFNs, polynucleotides, cells and/or pharmaceutical compositions as described herein for the treatment and/or prevention of a cancer, an autoimmune disease or graft-versus-host disease are also provided. Kits comprising any of the ZFNs, polynucleotides, cells and/or pharmaceutical compositions as described herein are also provided.

[0019] In other aspects, described herein is an isolated cell (e.g., a eukaryotic cell such as a mammalian cell including a lymphoid cell, a stem cell (e.g., iPSC, embryonic stem cell, MSC or HSC), or a progenitor/precursor cell) in which expression of a TCR gene is modulated by modification of exonic sequences of the TCR gene. In certain embodiments, the modification is to a sequence comprising a sequence of 9-25 (including target sites of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) or more nucleotides (contiguous or non-contiguous) of a sequence as shown in the target sites herein) of a target site as shown in one or more of Tables 1, 2 or 6 (SEQ ID NO: 8-21 and/or 92-103); within 1-5, within 1-10 or within 1-20 base pairs on either side (the flanking genomic sequence) of the target sites shown in Tables 1, 2 or 6(SEQ ID NO:8-21 and/or 92-103); or within AACAGT, AGTGCT, CTCCT, TTGAAA, TGGACTT and AATCCTC or a target site comprising AACAGT, AGTGCT, CTCCT, TTGAAA, TGGACTT and AATCCTC. Alternatively, or in addition, the modifications may also be made to sequences (e.g., genomic sequences) between paired target sites of as described herein (e.g., target sites for the nuclease pairs shown in Table 3, including between the target sites for 55204 and 53759 (between SEQ ID NO:8 and SEQ ID NO:9); between the target sites for 55229 and 53785 (between SEQ ID NO:10 and SEQ ID NO:11); between the target sites for 53810 and 55255 (between SEQ ID NO:12 and SEQ ID NO:13); between the target sites shown for 55248 and 55254/55260 (between SEQ ID NO:14 and SEQ ID NO:13); between the target sites for 55266 and 53853 (between SEQ ID NO:15 and SEQ ID NO:16); between the target sites for 53860 and 53863 (between SEQ ID NO:17 and SEQ ID NO:18); between the target sites for 53856 and 55287 (between SEQ ID NO:21 and SEQ ID NO:18); or between the target sites for 53885 or 52774 and 53909 or 52742 (between SEQ ID NO:19 and SEQ ID NO:20). The modification may be by an exogenous fusion molecule comprising a functional domain (e.g., transcriptional regulatory domain, nuclease domain including any FokI cleavage domain with one or more mutations as compared to wild-type) and a DNA-binding domain, including, but not limited to: (i) a cell comprising an exogenous transcription factor comprising a DNA-binding domain that binds to a target site as shown in any of SEQ ID NO:8-21 and/or 92-103 and a transcriptional regulatory domain in which the transcription factor modifies TRAC gene expression and/or (ii) a cell comprising an insertion and/or a deletion within one or more of the target sites shown herein, including SEQ ID NO:8-21 and/or 92-103; within 1-5, within 1-10 or within 1-20 base pairs on either side (the flanking genomic sequence) of the target sites shown in Tables 1 and 2 (SEQ ID NO: 8-21 and/or 92-103); within AACAGT, AGTGCT, CTCCT, TTGAAA, TGGACTT and AATCCTC; and/or between paired target sites as described herein (e.g., target sites for the nuclease pairs shown in Table 3). Cells comprising these modifications to TCR gene(s) and additional genetic modifications (e.g., B2M gene modification, CTLA, CISH, PD1 and/or tet2 gene modifications, CAR, an antigen-specific TCR (alpha and beta chains), insertions at these or other loci including a transgene encoding an Antibody-coupled T-cell Receptor (ACTR) and/or a transgene encoding an antibody, etc.) are also described.

[0020] In another aspect, described herein is an isolated cell (e.g., a eukaryotic cell such as a mammalian cell including a lymphoid cell, a stem cell (e.g., iPSC, embryonic stem cell, MSC or HSC), or a progenitor/precursor cell) in which expression of a B2M gene is modulated by modification of the B2M gene. In certain embodiments, the modification is to a sequence comprising a sequence of 9-25 (including target sites of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) or more nucleotides (contiguous or non-contiguous) of a sequence as shown in the target sites herein) of a target site as shown in one or more of Tables 5 and 8 (SEQ ID NO: 117, 123, 126 and/or 127); within 1-5, within 1-10 or within 1-20 base pairs on either side (the flanking genomic sequence) of the target sites shown in Tables 5 and 8 (SEQ ID NO:117, 123, 126 and/or 127). Alternatively, or in addition, the modifications may also be made to sequences (e.g., genomic sequences) between paired target sites of as described herein (e.g., target sites for the nuclease pairs shown in Tables 5 and 8, including between the target sites as shown in Table 8 (SEQ ID NO:126 and 127). The modification may be by an exogenous fusion molecule comprising a functional domain (e.g., transcriptional regulatory domain, nuclease domain including any FokI cleavage domain with one or more mutations as compared to wild-type) and a DNA-binding domain (e.g., a ZFP as shown in Table 8 (the ZFP component (designs) of the ZFNs designated 72732; 72748; 68957; or 72678), including, but not limited to: (i) a cell comprising an exogenous transcription factor comprising a DNA-binding domain that binds to a target site as shown in any of Tables 5 or 8 (e.g., SEQ ID NO: 126 or 127) and a transcriptional regulatory domain in which the transcription factor modifies B2M gene expression and/or (ii) a cell comprising an insertion and/or a deletion within one or more of the target sites shown herein, including Tables 5 and 8; within 1-5, within 1-10 or within 1-20 base pairs on either side (the flanking genomic sequence); and/or between paired target sites as described herein (e.g., target sites for the nuclease pairs shown in Table 8). Cells comprising these modifications to B2M genes and additional genetic modifications (e.g., TCR gene modification, CTLA, CISH, PD1 and/or tet2 gene modifications, PD1 modification, a CAR insertion, an antigen-specific TCR (alpha and beta chains), insertions at these or other loci including a transgene encoding an Antibody-coupled T-cell Receptor (ACTR) and/or a transgene encoding an antibody, etc.) are also described.

[0021] The TCR and/or B2M modified cells described herein may include further modifications, for example one or more inactivated T-cell receptor genes in B2M modified cells, additional inactivated TCR genes, PD1 and/or CTLA4 gene and/or a transgene a transgene encoding a chimeric antigen receptor (CAR), a transgene encoding an Antibody-coupled T-cell Receptor (ACTR) and/or a transgene encoding an antibody. Pharmaceutical compositions comprising any cell as described herein are also provided as well as methods of using the cells and pharmaceutical compositions in ex vivo therapies for the treatment of a disorder (e.g., a cancer) in a subject. In certain embodiments, a population of cells comprising one or more modifications (TCR edits, B2M edits, PD1 edits, CISH, tet2 and/or CTLA4 edits, HLA class I gene edits and/or transgene (e.g., CAR) insertions into these or other genes, etc.) as described herein are provided, including a population of cells in which less than 5% (e.g., 0-5% or any value therebetween), preferably less than 3%, even more preferably less than 2% of the cells include any other modifications (e.g., modifications at off-target sites). In certain embodiments, the population of cells includes modifications at off-target sites at background levels (e.g., 2-10-fold less (or any value therebetween)) as compared to cells modified with ZFNs that are not modified as described herein (which unmodified ZFNs are also referred to as “parent” or “parental” ZFNs). The modifications made by the ZFNs are heritable in that, in vivo or in culture, cells descended from (including differentiated cells) cells comprising the ZFNs (and modifications) include the modifications described herein.

[0022] Thus, in one aspect, described herein are cells in which the expression of a TCR gene is modulated (e.g., activated, repressed or inactivated). In preferred embodiments, exonic sequences of a TCR gene are modulated. The modulation may be by an exogenous molecule (e.g., engineered transcription factor comprising a DNA-binding domain and a transcriptional activation or repression domain) that binds to the TCR gene and regulates TCR expression and/or via sequence modification of the TCR gene (e.g., using a nuclease that cleaves the TCR gene and modifies the gene sequence by insertions and/or deletions), including for example a ZFN (e.g., ZFN pair of left and right ZFNs) as shown in Table 6. In some embodiments, cells are described that comprise an engineered nuclease to cause a knockout of a TCR gene. In other embodiments, cells are described that comprise an engineered transcription factor (TF) such that the expression of a TCR gene is modulated. In some embodiments, the cells are T cells. Further described are cells wherein the expression of a TCR gene is modulated and wherein the cells are further engineered to comprise a least one exogenous transgene and/or an additional knock out of at least one endogenous gene (e.g., beta 2 microglobuin (B2M) and/or immunological checkpoint gene such as PD1 and/or CTLA4) or combinations thereof.

[0023] In another aspect, described herein are cells in which the expression of a B2M gene is modulated (e.g., activated, repressed or inactivated). The modulation may be by an exogenous molecule (e.g., engineered transcription factor comprising a DNA-binding domain and a transcriptional activation or repression domain) that binds to the B2M gene and regulates B2M expression and/or via sequence modification of the B2M gene (e.g., using a nuclease that cleaves the B2M gene and modifies the gene sequence by insertions and/or deletions), including for example a ZFN (e.g., ZFN pair of left and right ZFNs) as shown in Table 8 or a ZFN comprising a ZFP having the design (recognition helix region and backbone of ZFPs in ZFNs designated 72732; 72748; 68957; or 72678) described herein (e.g., Table 8) in combination with any FokI domain (wild-type or engineered) and optionally any linker between the FokI domain and the ZFP (e.g., L0, N7a, N7c, etc.). In some embodiments, cells are described that comprise an engineered nuclease to cause a knockout of a B2M gene. In other embodiments, cells are described that comprise an engineered transcription factor (TF) such that the expression of a B2M gene is modulated. In some embodiments, the cells are T cells, including effector T cells and regulatory T cells. Further described are cells wherein the expression of a B2M gene is modulated and wherein the cells are further engineered to comprise a least one exogenous transgene and/or an additional knock out of at least one endogenous gene (e.g., one or more TCR genes and/or immunological checkpoint gene such as PD1 and/or CTLA4) or combinations thereof.

[0024] In any of the cells described herein comprising an exogenous transgene, the exogenous transgene may be integrated into a TCR and/or B2M gene (e.g., when the TCR and/or B2M gene is knocked out) and/or may be integrated into a gene such as a safe harbor gene. In some cases, the exogenous transgene encodes an ACTR, an antigen-specific TCR, and/or a CAR. The transgene construct may be inserted by either HDR- or NHEJ- driven processes. In some aspects the cells with modulated TCR and/or B2M expression comprise at least an exogenous ACTR, an exogenous TCR and an exogenous CAR. Some cells comprising a TCR modulator further comprise a knockout of one or more check point inhibitor genes. In some embodiments, the check point inhibitor is PD1. In other embodiments, the check point inhibitor is CTLA4. In further aspects, the TCR and/or B2M modulated cell comprises a PD1 knockout and a CTLA4 knockout. In some embodiments, the TCR gene modulated is a gene encoding TCR β (TCRB). In some embodiments this is achieved via targeted cleavage of the constant region of this gene (TCR β Constant region, or TRBC). In certain embodiments, the TCR gene modulated is a gene encoding TCR α (TCRA). In further embodiments, insertion is achieved via targeted cleavage of the constant region of a TCR gene, including targeted cleavage of the constant region of a TCR α gene (referred to herein as “TRAC” sequences). In some embodiments, the TCR gene modified cells are further modified at the B2M gene, the HLA-A, -B, -C genes, or the TAP gene, or any combination thereof. In other embodiments, the regulator for HLA class II, CIITA, is also modified.

[0025] In certain embodiments, the cells described herein comprise a modification (e.g., deletion and/or insertion, binding of an engineered TF to repress TCR expression) to a TCRA gene (e.g., modification of exons). In certain embodiments, the modification is within any of the target sites shown in Tables 1, 2 or 6 (SEQ ID NO:8-21 and/or 92-103) and/or between paired target sites (e.g., target sites of nuclease pairs shown in Table 3), including modification by binding to, cleaving, inserting and/or deleting one or more nucleotides within any of these sequences and/or within 1-50 base pairs (including any value therebetween such as 1-5, 1-10 or 1-20 base pairs) of the gene (genomic) sequences flanking these sequences in the TCRA gene. In certain embodiments, the modifications are made using a ZFN (e.g., one or more ZFN pairs) as shown in Table 6. In certain embodiments, the cells comprise a modification (binding to, cleaving, insertions and/or deletions) within one or more of the following sequences: AACAGT, AGTGCT, CTCCT, TTGAAA, TGGACTT and AATCCTC within a TCRA gene (e.g., exons, see FIG. 1B). In certain embodiments, the modification comprises binding of an engineered TF as described herein such that a TCRA gene expression is modulated, for example, repressed or activated.

[0026] In certain embodiments, the cells described herein comprise a modification (e.g., deletion and/or insertion, binding of an engineered TF to repress B2M expression) to a B2M gene. In certain embodiments, the modification is within any of the target sites shown in Tables 5 or 8 and/or between paired target sites (e.g., target sites of nuclease pairs shown in Table 8), including modification by binding to, cleaving, inserting and/or deleting one or more nucleotides within any of these sequences and/or within 1-50 base pairs (including any value therebetween such as 1-5, 1-10 or 1-20 base pairs) of the gene (genomic) sequences flanking these sequences in the B2M gene. In certain embodiments, the modifications are made using a ZFN comprising a ZFP comprising the recognition helix regions and backbone of the ZFP designs of the ZFNs shown in Table 8, a FokI domain (any wild-type or engineered FokI domain) and optionally a linker (any linker between the N- or C-terminal of the FokI domain and the N- or C-terminal of the ZFP designs shown including but not limited to L0, N7a, N7c, etc.). In certain embodiments, the ZFN comprises a ZFN (e.g., a pair of first and second ZFNs) as shown in Table 8. In certain embodiments, the cells comprise a modification (binding to, cleaving, insertions and/or deletions) within one or more of the following sequences: SEQ ID NO: 126 and 127. In certain embodiments, the modification comprises binding of an engineered TF as described herein such that B2M gene expression is modulated, for example, repressed or activated.

[0027] In other embodiments, the modification is a genetic modification (alteration of nucleotide sequence) at or near nuclease(s) binding (target) and/or cleavage site(s), including but not limited to, modifications to sequences within 1-300 (or any number of base pairs therebetween) base pairs upstream, downstream and/or including 1 or more base pairs of the site(s) of cleavage and/or binding site; modifications within 1-100 base pairs (or any number of base pairs therebetween) of including and/or on either side of the binding and/or cleavage site(s); modifications within 1 to 50 base pairs (or any number of base pairs therebetween) including and/or on either side (e.g., 1 to 5, 1 to 10, 1 to 20 or more base pairs) of the binding and/or cleavage site(s); and/or modifications to one or more base pairs within the nuclease binding site and/or cleavage site. In certain embodiments, the modification is at or near (e.g., 1-300 base pairs, 1-50, 1-20, 1-10 or 1-5 or any number of base pairs therebetween) and/or between paired target sites (e.g., Table 3 or 8) of the gene sequence surrounding or between any of the target sites disclosed herein. In certain embodiments, the modification includes modifications of a TCRA and/or B2M gene within one or more of the sequences shown in in the target sites of Tables 1, 2 and 6 (TCRA) and/or Tables 5 and 8 (B2M), for example a modification of 1 or more base pairs to one or more of these sequences. In certain embodiments, the nuclease-mediated genetic modifications are between paired target sites (when a dimer is used to cleave the target). The nuclease-mediated genetic modifications may include insertions and/or deletions of any number of base pairs, including insertions of non-coding sequences of any length and/or transgenes of any length and/or deletions of 1 base pair to over 1000 kb (or any value therebetween including, but not limited to, 1-100 base pairs, 1-50 base pairs, 1-30 base pairs, 1-20 base pairs, 1-10 base pairs or 1-5 base pairs).

[0028] The modified cells of the invention may be a eukaryotic cell, including a non-human mammalian and a human cell such as lymphoid cell (e.g., a T-cell (including an effector T cell (Teff) and a regulatory T cell (Treg)), a B cell or an NK cell), a stem/progenitor cell (e.g., an induced pluripotent stem cell (iPSC), an embryonic stem cell (e.g., human ES), a mesenchymal stem cell (MSC), or a hematopoietic stem cell (HSC). The stem cells may be totipotent or pluripotent (e.g., partially differentiated such as an HSC that is a pluripotent myeloid or lymphoid stem cell). In other embodiments, the invention provides methods for producing cells that have a null genotype for TCR and or HLA expression. Any of the modified stem cells described herein (modified at the TCRA and/or B2M loci) may then be differentiated to generate a differentiated (in vivo or in vitro (culture)) cell descended from a stem cell as described herein with the modifications described herein, including modified TCRA and/or B2M gene expression.

[0029] In another aspect, the compositions (modified cells) and methods described herein can be used, for example, in the treatment or prevention or amelioration of a disorder. The methods typically comprise (a) cleaving or down regulating an endogenous TCR and/or B2M gene in an isolated cell (e.g., T-cell or other lymphocytes) using a nuclease (e.g., ZFN or TALEN) or nuclease system such as CRISPR/Cas with an engineered crRNA/tracr RNA, or using an engineered transcription factor (e.g., ZFP-TF, TALE-TF, Cfp1-TF or Cas9-TF) such that the TCR and/or B2M gene is inactivated or down modulated; and (b) introducing the cell into the subject, thereby treating or preventing the disorder. In some embodiments, the gene encoding TCR β (TCRB) is inactivated or down-modulated. In some embodiments, the gene encoding B2M is inactivated or down-modulated. In some embodiments inactivation is achieved via targeted cleavage of the constant region of this gene (TCR β Constant region, or TRBC). In preferred embodiments, the gene encoding TCR α (TCRA) and/or B2M is inactivated or down modulated. In further preferred embodiments, the disorder is a cancer, an infectious disease or an autoimmune disease. In some embodiments, the modifications are made to induce immune tolerance. In further preferred embodiments inactivation is achieved via targeted cleavage of the constant region of this gene (TCR α Constant region, or abbreviated as TRAC). In some embodiments, a B2M gene is cleaved. In further embodiments, the additional genes (in addition to TCR and/or B2M) are modulated (knocked-out), for example, TCR/B2M double knockouts, additional TCR genes, PD1 and/or CTLA4 and/or one or more therapeutic transgenes are present in the cell (episomal, randomly integrated or integrated via targeted integration such as nuclease-mediated integration). The modified cells may include one or more ZFNs (e.g., ZFN pairs) as described herein, including but not limited to a zinc finger nuclease (ZFN) comprising first and second ZFNs, each ZFN comprising a cleavage domain (e.g., any wild-type or engineered FokI cleavage domain) and a ZFP DNA-binding domain. In certain embodiments, the modifications are made using a ZFN comprising a ZFP (recognition helix regions and backbone) of the “designs” described herein (e.g., Table 6 or Table 8 including the ZFPs of the ZFNs designated 68846, 53853, 72732; 72748; 68957; 55266, 68798, 68879, 68815, 68799 or 72678), a FokI domain (any wild-type or engineered FokI domain) and optionally a linker (any linker between the N- or C-terminal of the FokI domain and the N- or C-terminal of the ZFP designs described herein). In some embodiments the ZFN comprises a pair of ZFNs, in which one ZFN comprises the ZFP of 68846 (SEQ ID NO:177) operably linked to a FokI domain and the other ZFN of the pair comprises the ZFP of 53853 (SEQ ID NO:178) operably linked to a FokI domain. In some embodiments the ZFN comprises a pair of ZFNs, in which one ZFN comprises the ZFP of 72732 (SEQ ID NO: 175) operably linked to a FokI domain and the other ZFN of the pair comprises the ZFP of 72678 (SEQ ID NO:176) operably linked to a FokI domain. In certain embodiments, the ZFN comprises a ZFN (e.g., a pair of first and second (also referred to as left and right) partner ZFNs) described herein as follows: a ZFN designated 68796 and a ZFN designated 68813; a ZFN designated 68796 and a ZFN designated 68861; a ZFN designated 68812 and a ZFN designated 68813; a ZFN designated 68876 and a ZFN designated 68877; a ZFN designated 68815 and a ZFN designated 55266; a ZFN designated 68879 and a ZFN designated 55266; a ZFN designated 68798 and a ZFN designated 68815; or a ZFN designated 68846 and a ZFN designated 53853; a ZFN designated 57531 and a ZFN designated 72732; a ZFN designated 57531 and a ZFN designated 72748; a ZFN designated 68957 and a ZFN designated 57071; a ZFN designated 68957 and a ZFN designated 72732; a ZFN designated 68957 and a ZFN designated 72748; a ZFN designated 72678 and a ZFN designated 57071; a ZFN designated 72678 and a ZFN designated 72732; and a comprising a ZFP ZFN designated 72678 and a ZFN designated 72748. Thus, a ZFN (e.g., each ZFN partner of a paired ZFN) comprises the recognition helix regions and may comprise additional ZFP modifications (e.g., to the backbone regions) described below (e.g., designs shown in Tables 1, 2, 5, 6 and 8) and further comprises any wild-type or engineered FokI cleavage domain (including any combination of the FokI substitution, addition and/or deletion mutants). For example, a ZFN partner may comprise specific zinc finger DNA binding domain fused to any FokI cleavage domain including the cleavage domain (SEQ ID NO: 139) from the wildtype protein or from a mutated sequence (as shown in the Examples, SEQ ID NO: 140-174). A B2M-specific ZFN partner may comprise a B2M-specific zinc finger DNA binding domain (e.g., 72732) fused with a FokI cleavage domain selected from SEQ ID NOs: 139-174. Further, the B2M-specific ZFN partner may comprise a B2M-specific zinc finger DNA binding domain (e.g., 72678) fused to a FokI cleavage domain selected from SEQ ID NOs: 139-174. Similarly, a TRAC-specific ZFN partner may comprise a TRAC-specific zinc finger DNA binding domain (e.g., 68846) fused to a FokI cleavage domain selected from SEQ ID NOs: 139-174, and the TRAC-specific zinc finger DNA binding domain 53853 may be fused to a FokI cleavage domain selected from any of wild-type or engineered FokI cleavage shown, for example a domain as shown in the appended Examples (SEQ ID NOs: 139-174). In some embodiments, the FokI domain is fused at the N-terminal end of the ZFP DNA binding domain while in others, it is fused to the C-terminal end of the ZFP DNA binding domain. Further, any linker can be used to link the DNA-binding domain to the FokI cleavage domain.

[0030] Cells descended from cells modified as described herein (e.g., cells comprising the ZFNs described herein), including but not limited partially or fully differentiated from stem cells modified as described herein, are also provided. These cells typically do not include the ZFNs but do include the genetic modifications made thereby.

[0031] The transcription factor(s) and/or nuclease(s) can be introduced into a cell or the surrounding culture media as mRNA, in protein form and/or as a DNA sequence encoding the nuclease(s). In certain embodiments, the isolated cell introduced into the subject further comprises additional genomic modification, for example, an integrated exogenous sequence (into the cleaved TCR and/or B2M gene or a different gene, for example a safe harbor gene or locus) and/or inactivation (e.g., nuclease-mediated) of additional genes, for example one or more HLA genes, or CTLA-4, CISH, PD1, or tet2 genes. The exogenous sequence (e.g., a CAR or exogenous TCR) or protein may be introduced via a vector (e.g., Ad, AAV, LV), or by using a technique such as electroporation or transient transfection. In some embodiments, the proteins are introduced into the cell by inducing mechanical stress such as cell squeezing (see Kollmannsperger, et al. (2016) Nat Comm 7, 10372 doi:10.1038/ncomms10372). In some aspects, the composition may comprise isolated cell fragments and/or differentiated (partially or fully) cells.

[0032] In some aspects, the modified cells may be used for cell therapy, for example, for adoptive cell transfer. In other embodiments, the cells for use in T cell transplant contain another gene modification of interest. In one aspect, the T cells contain an inserted chimeric antigen receptor (CAR) specific for a marker found on cancer cells. In a further aspect, the inserted CAR is specific for the CD19 marker characteristic of B cells, including B cell malignancies. Such cells would be useful in a therapeutic composition for treating patients without having to match HLA, and so would be able to be used as an “off-the-shelf” therapeutic for any patient in need thereof. In other instances, stem or precursor cells, for example, hematopoietic stem cell or precursor cells (HSC/PC) or induced pluripotent stem cells (iPSC) containing the modifications described herein are expanded prior to introduction. In other aspects, the genetically modified HSC/PCs are given to the subject in a bone marrow transplant wherein the HSC/PC engraft, differentiate and mature in vivo. In some embodiments, the HSC/PC are isolated from the subject following G-CSF-induced mobilization, plerixafor-induced mobilization, and combinations of G-CSF- and plerixafor-induced mobilization, and in others, the cells are isolated from human bone marrow or human umbilical cords. In other embodiments, iPSC are derived from patient or healthy donor cells. In some aspects, the subject is treated to a mild myeloablative procedure prior to introduction of the graft comprising the modified HSC/PC or modified cells derived from iPSC, while in other aspects, the subject is treated with a vigorous myeloablative conditioning regimen. In some embodiments, the methods and compositions of the invention are used to treat or prevent a cancer.

[0033] In another aspect, the TCR- and/or B2M-modulated (modified) T cells contain an inserted Antibody-coupled T-cell Receptor (ACTR) donor sequence. In some embodiments, the ACTR donor sequence is inserted into a TCR gene to disrupt expression of that TCR gene following nuclease induced cleavage. In other embodiments, the donor sequence is inserted into a “safe harbor” locus, such as the AAVS1, HPRT, albumin and CCR5 genes. In some embodiments, the ACTR sequence is inserted via targeted integration where the ACTR donor sequence comprises flanking homology arms that have homology to the sequence flanking the cleavage site of the engineered nuclease. In some embodiments the ACTR donor sequence further comprises a promoter and/or other transcriptional regulatory sequences. In other embodiments, the ACTR donor sequence lacks a promoter. In some embodiments, the ACTR donor is inserted into a TCR β encoding gene (TCRB). In some embodiments insertion is achieved via targeted cleavage of the constant region of this gene (TCR β Constant region, or TRBC). In preferred embodiments, the ACTR donor is inserted into a TCR α encoding gene (TCRA). In further preferred embodiments insertion is achieved via targeted cleavage of the constant region of this gene (TCR α Constant region, abbreviated TRAC). In some embodiments, the donor is inserted into an exon sequence in TCRA, while in others, the donor is inserted into an intronic sequence in TCRA. In still further embodiments, the ACTR donor is inserted into a B2M gene. In some embodiments, the B2M and/or TCR-modulated cells further comprise a CAR. In still further embodiments, the B2M and/or TCR-modulated cells are additionally modulated at an HLA gene or a checkpoint inhibitor gene.

[0034] Also provided are pharmaceutical compositions comprising the modified cells as described herein (e.g., T cells or stem cells with inactivated TCR gene), or pharmaceutical compositions comprising one or more of the TCR and/or B2M gene binding molecules (e.g., engineered transcription factors and/or nucleases) as described herein. In certain embodiments, the pharmaceutical compositions further comprise one or more pharmaceutically acceptable excipients. The modified cells, TCR and/or B2M gene binding molecules (or polynucleotides encoding these molecules) and/or pharmaceutical compositions comprising these cells or molecules are introduced into the subject via methods known in the art, e.g., through intravenous infusion, infusion into a specific vessel such as the hepatic artery, or through direct tissue injection (e.g., muscle). In some embodiments, the subject is an adult human with a disease or condition that can be treated or ameliorated with the composition. In other embodiments, the subject is a pediatric subject where the composition is administered to prevent, treat or ameliorate the disease or condition (e.g., cancer, graft versus host disease, etc.).

[0035] In some aspects, the composition (TCR and/or B2M modulated cells comprising an ACTR) further comprises an exogenous antibody. See, also, U.S. Pat. Publication No. 2017/0196992. In some aspects, the antibody is useful for arming an ACTR-comprising T cell to prevent or treat a condition. In some embodiments, the antibody recognizes an antigen associated with a tumor cell or with cancer associate processes such as EpCAM, CEA, gpA33, mucins, TAG-72, CAIX, PSMA, folate-binding antibodies, CD19, EGFR, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, VEGF, VEGFR, αVβ3 and α5β1 integrins, CD20, CD30, CD33, CD52, CTLA4, and enascin (Scott, et al. (2012) Nat Rev Cancer 12:278). In other embodiments, the antibody recognizes an antigen associated with an infectious disease such as HIV, HCV and the like.

[0036] In another aspect, provided herein are TCR gene DNA-binding domains (e.g., ZFPs, TALEs and sgRNAs) that bind to a target site in a TCR gene. In certain embodiments, the DNA binding domain comprises a ZFP with the recognition helix regions in the order as shown in a single row of Table 1; a TAL-effector domain DNA-binding protein with the RVDs that bind to a target site as shown in the first column of Table 1 or the third column of Table 2; and/or a sgRNA as shown in a single row of Table 2. These DNA-binding proteins can be associated with transcriptional regulatory domains to form engineered transcription factors that modulate TCR expression. Alternatively, these DNA-binding proteins can be associated with one or more nuclease domains to form engineered zinc finger nucleases (ZFNs), TALENs and/or CRISPR/Cas systems that bind to and cleave a TCR gene. In certain embodiments, the ZFNs, TALENs or single guide RNAs (sgRNA) of a CRISPR/Cas system bind to target sites in a human TCR gene. The DNA-binding domain of the transcription factor or nuclease (e.g., ZFP, TALE, sgRNA) may bind to a target site in a TCRA gene comprising 9, 10, 11, 12 or more (e.g., 13, 14, 15, 16, 17, 18, 19, 20 or more) nucleotides of any of the target sites shown herein (e.g., target sites of Table 1 or 2 as shown in SEQ ID NOs:8-21 and/or 92-103). The zinc finger proteins may include 1, 2, 3, 4, 5, 6 or more zinc fingers, each zinc finger having a recognition helix that specifically contacts a target subsite in the target gene. In certain embodiments, the zinc finger proteins comprise 4 or 5 or 6 fingers (designated F1, F2, F3, F4, F5 and F6 and ordered F1 to F4 or F5 or F6 from N-terminus to C-terminus), for example as shown in Table 1. The ZFPs as described herein may also include one or more mutations to phosphate contact residues of the zinc finger protein, for example, the nR-5Qabc mutant described in U.S. Pat. Publication No. 2018/0087072. In other embodiments, the single guide RNAs or TAL-effector DNA-binding domains may bind to a target site as described herein (e.g., target sites of Table 1 or Table 2 or Table 6 as shown in any of SEQ ID NOs:8-21 and/or 92-103) or 12 or more base pairs within any of these target sites or between paired target sites. Exemplary sgRNA target sites are shown in Table 2 (SEQ ID NOs:92-103). sgRNAs that bind to 12 or more nucleotides of the target sites shown in Table 1 or Table 2 are also provided. TALENs may be designed to target sites as described herein (target sites of Table 1 or Table 2 or Table 6) using canonical or non-canonical RVDs as described in U.S. Pat. Nos. 8,586,526 and 9,458,205. The nucleases described herein (comprising a ZFP, a TALE or a sgRNA DNA-binding domain) are capable of making genetic modifications within a TCRA gene comprising any of SEQ ID NO:8-21 and/or 92-103, including modifications (insertions and/or deletions) within any of these sequences (SEQ ID NO:8-21 and/or 92-103) and/or modifications to TCRA gene sequences flanking the target site sequences shown in SEQ ID NO:8-21 and/or 92-103, for instance modifications within exonic sequences of a TCR gene within one or more of the following sequences: AACAGT, AGTGCT, CTCCT, TTGAAA, TGGACTT and AATCCTC.

[0037] In another aspect, provided herein are B2M gene DNA-binding domains (e.g., ZFPs, TALEs and sgRNAs) that bind to a target site in a B2M gene. In certain embodiments, the DNA binding domain comprises a ZFP with the recognition helix regions in the order as shown in a single row of Table 5 or Table 8 (columns labeled “designs”, including the ZFPs of the ZFNs designated 72732; 72748; 68957; or 72678); a TAL-effector domain DNA-binding protein with the RVDs that bind to a target site as shown in the first column of Table 5 or Table 8; and/or a sgRNA that binds to a B2M target site as described herein (Table 5 or Table 8). These DNA-binding proteins can be associated with transcriptional regulatory domains to form engineered transcription factors that modulate B2M expression. Alternatively, these DNA-binding proteins can be associated with one or more nuclease (cleavage) domains to form engineered zinc finger nucleases (ZFNs), TALENs and/or CRISPR/Cas systems that bind to and cleave a B2M gene. In certain embodiments, the ZFNs, TALENs or single guide RNAs (sgRNA) of a CRISPR/Cas system bind to target sites in a human B2M gene. The DNA-binding domain of the transcription factor or nuclease (e.g., ZFP, TALE, sgRNA) may bind to a target site in a B2M gene comprising 9, 10, 11, 12 or more (e.g., 13, 14, 15, 16, 17, 18, 19, 20 or more) nucleotides of any of the target sites shown herein (e.g., Table 5 or Table 8 as shown in SEQ ID NOs: 117, 123, 126 or 127). The zinc finger proteins may include 1, 2, 3, 4, 5, 6 or more zinc fingers, each zinc finger having a recognition helix that specifically contacts a target subsite in the target gene. In certain embodiments, the zinc finger proteins comprise 4 or 5 or 6 fingers (designated F1, F2, F3, F4, F5 and F6 and ordered F1 to F4 or F5 or F6 from N-terminus to C-terminus), for example as shown in Table 5 or Table 8. The ZFPs as described herein may also include one or more mutations to phosphate contact residues of the zinc finger protein, for example, the nR-5Qabc mutant described in U.S. Pat. Publication No. 2018/0087072, including the ZFP designs (recognition helix regions and backbone mutants) of Table 8. In other embodiments, the single guide RNAs or TAL-effector DNA-binding domains may bind to a target site as described herein (e.g., target sites of Tables 5 or 8) or 12 or more base pairs within any of these target sites or between paired target sites. TALE domains may be designed to target sites as described herein (target sites of Tables 5 or 8) using canonical or non-canonical RVDs as described in U.S. Pat. Nos. 8,586,526 and 9,458,205. The nucleases described herein (comprising a ZFP, a TALE or a sgRNA DNA-binding domain) are capable of making genetic modifications within a B2M gene comprising any of the B2M target sites disclosed herein, including modifications (insertions and/or deletions) within any of these sequences and/or modifications to B2M gene sequences flanking the target site sequences shown in Tables 5 and 8 (SEQ ID NO: 117, 123, 126 or 127).

[0038] Any of the nucleases described herein may comprise a DNA-binding domain (e.g., ZFP designs of Table 6 or 8, TALE or sgRNA) as described herein and a cleavage domain and/or a cleavage half-domain (e.g., a wild-type or engineered FokI cleavage half-domain). Thus, in any of the nucleases (e.g., ZFNs, TALENs, CRISPR/Cas systems) described herein, the nuclease domain may comprise a wild-type nuclease domain or nuclease half-domain (e.g., a FokI cleavage half domain). In other embodiments, the nucleases (e.g., ZFNs, TALENs, CRISPR/Cas nucleases) comprise engineered nuclease domains or half-domains, for example engineered FokI cleavage half domains that form obligate heterodimers. See, e.g., U.S. Pat. No. 7,914,796 and 8,034,598. In certain embodiments, one or more FokI endonuclease domains of the nucleases described herein may also comprise phosphate contact mutants (e.g., R416S and/or K525S) as described in U.S. Pat. Publication No. 2018/0087072. Thus, the FokI domain of the nucleases described herein (e.g., ZFNs comprising: (i) ZFP designs as shown in Table 8, including ZFPs of the ZFNs designated 72732; 72748; 68957; or 72678 and (ii) a FokI domain) may include any combination of mutations to the FokI domain (positions numbered relative to full length FokI), including the wildtype FokI catalytic domain sequence, and also, but not limited to, the FokI domains indicated in Table 8, FokI-Sharkey (S418P+K441E); FokI ELD (Q->E at position 486, I->L at 499, N->D at position 496); FokI ELD, Sharkey (Q->E at position 486, I->L at position 499, N->D at position 496, S418P+K441E); FokI ELD, R416E (Q->E at position 486, I->L at position 499, N->D at position 496, R416E); FokI ELD, Sharkey, R416E (Q->E at position 486, I->L at position 499, N->D at position 496, S418P+K441E, R416E); FokI ELD, R416Y (Q->E at position 486, I->L at position 499, N->D at position 496, R416Y); FokI ELD, Sharkey, R416E (Q->E at position 486, I->L at position 499, N->D at position 496, S418P+K441E, R416E); FokI ELD, S418E (Q->E at position 486, I->L at position 499, N->D at position 496, S418E); FokI ELD, Sharkey partial, S418E (Q->E at position 486, I->L at position 499, N->D at position 496, K441E, S418E); FokI ELD, K525S (Q->E at position 486, I->L at position 499, N->D at position 496, K525S); FokI ELD, Sharkey K525S (Q->E at position 486, I->L at position 499, N->D at position 496, S418P+K441E, K525S); FokI ELD, I479T (Q->E at position 486, I->L at position 499, N->D at position 496, I479T); FokI ELD, Sharkey, I479T (Q->E at position 486, I->L at position 499, N->D at position 496, S418P+K441E, I479T); FokI ELD, P478D (Q->E at position 486, I->L at position 499, N->D at position 496, P478D); FokI ELD, Sharkey, P478D (Q->E at position 486, I->L at position 499, N->D at position 496, S418P+K441E, P478D); FokI ELD, Q481D (Q->E at position 486, I->L at position 499, N->D at position 496, Q481D); FokI ELD, Sharkey, Q481D (Q->E at position 486, I->L at position 499, N->D at position 496, S418P+K441E, Q481D); FokI KKR (E->K at position 490, I->K at position 538, H->R at position 537); FokI KKR Sharkey, (E->K at position 490, I->K at position 538, H->R at position 537, S418P+K441E); FokI KKR, Q481E (E->K at position 490, I->K at position 538, H->R at position 537, Q481E); FokI KKR, Sharkey Q481E (E->K at position 490, I->K at position 538, H->R at position 537, S418P+K441E, Q481E); FokI KKR, R416E (E->K at position 490, I->K at position 538, H->R at position 537, R416E); FokI KKR, Sharkey, R416E (E->K at position 490, I->K at position 538, H->R at position 537, S418P+K441E, R416E); FokI KKR, K525S (E->K at position 490, I->K at position 538, H->R at position 537, K525S); FokI KKR, Sharkey, K525S (E->K at position 490, I->K at position 538, H->R at position 537, S418P+K441E, K525S); FokI KKR, R416Y (E->K at position 490, I->K position 538, H->R at position 537, R416Y); FokI KKR, Sharkey, R416Y (E->K at position 490, I->K at position 538, H->R at position 537, S418P+K441E, R416Y); FokI, KKR I479T (E->K at position 490, I->K at position 538, H->R at position 537, I479T); FokI, KKR Sharkey I479T (E->K at position 490, I->K at position 538, H->R at position 537, S418P+K441E, I479T; FokI, KKR P478D(E->K at position 490, I->K at positions 538, H->R at position 537, P478D), FokI KKR Sharkey P478D(E->K at position 490, I->K at position 538, H->R at position 537, P478D); FokI DAD (R->D at position 487, N->D at position 496, I->A at position 499); FokI DAD Sharkey (R->D at position 487, N->D at position 496, I->A at position 499, S418P+K441E); FokI RVR (D->R at position 483, H->R at position 537, I->V at position 538); FokI RVR Sharkey (D->R at position 483, H->R at position 537, I->V at position 538, S418P+K441E). The ZFNs described herein may also include any linker sequence, including but not limited to sequences disclosed in U.S. Pat. No. 7,888,121; 7,914,796; 8,034,598; 8,623,618; 9,567,609; and U.S. Publication No. 2017/0218349, which may be used between the N- or C-terminal of the DNA-binding domain (e.g., ZFP) and N- or C-terminal of the FokI cleavage domain.

[0039] In another aspect, the disclosure provides a polynucleotide encoding any of the proteins, fusion molecules and/or components thereof (e.g., sgRNA or other DNA-binding domain) described herein. The polynucleotide may be part of a viral vector, a non-viral vector (e.g., plasmid) or be in mRNA form. Any of the polynucleotides described herein may also comprise sequences (donor, homology arms or patch sequences) for targeted insertion into the TCR α and/or the TCR β gene. In yet another aspect, a gene delivery vector comprising any of the polynucleotides described herein is provided. In certain embodiments, the vector is an adenoviral vector (e.g., an Ad5/F35 vector) or a lentiviral vector (LV) including integration competent or integration-defective lentiviral vectors or an adeno-associated vector (AAV). Thus, also provided herein are viral vectors comprising a sequence encoding a nuclease (e.g., ZFN or TALEN) and/or a nuclease system (CRISPR/Cas or Ttago) and/or a donor sequence for targeted integration into a target gene. In some embodiments, the donor sequence and the sequences encoding the nuclease are on different vectors. In other embodiments, the nucleases are supplied as polypeptides. In preferred embodiments, the polynucleotides are mRNAs. In some aspects, the mRNA may be chemically modified (See e.g., Kormann, et al. (2011) Nature Biotechnology 29(2):154-157). In other aspects, the mRNA may comprise an ARCA cap (see U.S. Pat. Nos. 7,074,596 and 8,153,773). In some aspects, the mRNA may comprise a cap introduced by enzymatic modification. The enzymatically introduced cap may comprise Cap0, Cap1 or Cap2 (see e.g., Smietanski, et al. (2014) Nature Communications 5:3004). In further aspects, the mRNA may be capped by chemical modification. In further embodiments, the mRNA may comprise a mixture of unmodified and modified nucleotides (see U.S. Patent Publication No. 2012/0195936). In still further embodiments, the mRNA may comprise a WPRE element (see U.S. Patent Publication No. 2016/0326548). In some embodiments, the mRNA is double stranded (See, e.g., Kariko, et al. (2011) Nucl Acid Res 39:e142).

[0040] In yet another aspect, the disclosure provides an isolated cell comprising any of the proteins, polynucleotides and/or vectors described herein. In certain embodiments, the cell is selected from the group consisting of a stem/progenitor cell, or a T-cell (e.g., effective or regulatory T-cell). In a still further aspect, the disclosure provides a cell or cell line which is descended from a cell or line comprising any of the nucleases, transcription factors, polynucleotides and/or vectors described herein, namely a cell or cell line descended (e.g., in culture) from a cell in which TCR and/or B2M has been inactivated by one or more ZFNs and/or in which a donor polynucleotide (e.g., ACTR and/or CAR) has been stably integrated into the genome of the cell. Thus, descendants of cells as described herein may not themselves comprise the molecule, polynucleotides and/or vectors described herein, but, in these cells, a TCR and/or B2M gene is inactivated and/or a donor polynucleotide is integrated into the genome and/or expressed.

[0041] In another aspect, described herein are methods of inactivating a TCR and/or B2M gene in a cell by introducing one or more proteins, polynucleotides and/or vectors into the cell as described herein. In certain embodiments, one or more polynucleotides encoding a ZFN (e.g., ZFN pair) as shown in Table 6 is used to modify the TCR gene in the cell and cells descended from these cells (including differentiated cells) comprise the modification(s). In other embodiments, one or more polynucleotide encoding a ZFN (e.g., ZFN pair) as shown in Table 8 is used to modify the B2M gene in the cell and cells descended from these (including differentiated cells) comprise the modification. In any of the methods described herein the nucleases may induce targeted mutagenesis, deletions of cellular DNA sequences, and/or facilitate targeted recombination at a predetermined chromosomal locus. Thus, in certain embodiments, the nucleases delete and/or insert one or more nucleotides from or into the target gene. In some embodiments a TCR and/or B2M gene is inactivated by nuclease cleavage followed by non-homologous end joining. In other embodiments, a genomic sequence in the target gene (e.g., TCR or B2M) is replaced, for example using a nuclease (or vector encoding said nuclease) as described herein and a “donor” sequence that is inserted into the gene following targeted cleavage with the nuclease. The donor sequence may be present in the nuclease vector, present in a separate vector (e.g., plasmid, linear single or double-stranded DNA, AAV, Ad or LV vector) or, alternatively, may be introduced into the cell using a different nucleic acid delivery mechanism. In some embodiments, the methods further comprise inactivating one or more additional genes (e.g., B2M) and/or integrating one or more transgenes into the genome of the cell, including, but not limited to, integration of one or more transgenes into the inactivated TCR and/or B2M gene and/or into one or more safe harbor genes. In certain embodiments, the methods described herein result in a population of cells in which at least 80-100% (or any value therebetween), including least 90-100% (or any value therebetween) of the cells include the knockout(s) and/or the integrated transgene(s).

[0042] Furthermore, any of the methods described herein can be practiced in vitro, in vivo and/or ex vivo. In certain embodiments, the methods are practiced ex vivo, for example to modify T-cells (effector or regulatory), to make them useful as therapeutics in an allogenic setting to treat a subject (e.g., a subject with cancer or autoimmune disease). Non-limiting examples of cancers that can be treated and/or prevented include lung carcinomas, pancreatic cancers, liver cancers, bone cancers, breast cancers, colorectal cancers, leukemias, ovarian cancers, lymphomas, brain cancers and the like. Non-limiting examples of autoimmune disease include transplant rejection, type 1 diabetes, irritable bowel disease/disorder, multiple sclerosis, lupus, scleroderma, rheumatoid arthritis and the like. The cells may also be used to induce immune tolerance.

[0043] In another aspect, described herein is a method of integrating one or more transgenes into a genome of an isolated cell, the method comprising: introducing, into the cell, (a) one or more donor vectors (e.g., plasmid, linear single or double-stranded DNA, AAVs, plasmids, Ads, mRNAs, etc.) comprising the one or more transgenes and (b) at least one non-naturally occurring nuclease in mRNA form, wherein the at least one nuclease cleaves the genome of the cell such that the one or more transgenes are integrated into the genome of the cell (e.g., into a TCR receptor), wherein the donor vector is introduced into introduced into the electroporation buffer comprising the isolated cell and the mRNA immediately before or immediately after electroporation of the nuclease into the cell. In certain embodiments, the donor vector is introduced into the electroporation buffer after electroporation and prior to transfer of the cells into a culture medium. See, e.g., U.S. Pat. Publication Nos. 2015/0174169 and 2015/0110762. The methods may be used to introduce the transgene(s) into any genomic location, including, but not limited to, a TCR gene, a B2M gene and/or a safe harbor gene (e.g., AAVS1, Rosa, albumin, CCR5, CXCR4, etc.).