Engineered T cells

Abstract

The present disclosure relates to T cells engineered to include a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and an insertion of a sequence(s) encoding a regulatory T cell-promoting molecule, and compositions and uses thereof.

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. This ASCII copy was created on December 28, 2021, is named 12793_0030-00304_SL.txt, and is 120,061 bytes in size.

[0002] Related Applications This application incorporates by reference U.S. Provisional Application No. 63 / 131,987, filed December 30, 2020, the entire contents of which are expressly incorporated by reference herein. [Background technology]

[0003] Adaptive immunity is a defense mechanism by which the body can eliminate foreign pathogens. T cells are the immune cells capable of mediating this immune response. The T cell receptor (TCR) is a protein complex on the surface of T cells that can recognize antigens. T cell diversity results from rearrangements of the TCR alpha and beta loci.

[0004] One of the hallmarks of adaptive immunity is the ability to distinguish between "self" and "non-self" antigens. Autoimmune and autoinflammatory disorders are characterized by pathological immune responses to "self" antigens. Rearrangements of some TCR alpha and beta loci generate autoreactive T cells. Owen et al., Regulatory T Cell Development in the Thymus, J Immunol 203(8)(2019). Many autoreactive T cells are eliminated by clonal deletion in the thymus, but other cells can escape clonal deletion and induce harmful immune responses. Ibid. A specialized type of T cell called regulatory T cells (Tregs) is important for "self" tolerance. Ibid. Tregs can suppress excessive immune responses (e.g., in graft-versus-host disease), autoimmune responses, and unwanted immune responses. Ibid. Dysregulation of Tregs (e.g., when there are insufficient numbers of Tregs or when Tregs are not functioning properly) can contribute to autoimmune responses. Ibid.

[0005] Current therapies for treating autoimmune disorders aim at suppressing adaptive immune processes or immune cell activation. While these therapies may suppress harmful immune responses (e.g., autoimmune responses), they may also suppress beneficial immune responses. Treg therapy has been used to suppress antigen-specific immune responses in a variety of diseases, including graft-versus-host disease (GvHD), in which donor cells mediate immune attack on host tissues after hematopoietic stem cell transplantation. Pierini et al., T Cells Expressing Chimeric Antigen Receptor Promoter Immune Tolerance, JCI Insight 2(20)(2017). However, "significant challenges remain in the clinical implementation of Treg-based therapies." Ibid. Thus, there remains a need for effective T cell therapies (including Treg therapy) to suppress immune response(s), including inflammation and autoimmunity. Summary of the Invention

[0006] The present disclosure provides T cells or populations of T cells engineered to contain a heterologous nucleic acid encoding a regulatory T cell promoting molecule under the control of a promoter sequence, and a modification in an endogenous nucleic acid sequence encoding interferon-gamma (IFNG) that knocks down expression of IFNG, and a modification in an endogenous nucleic acid sequence encoding tumor necrosis factor alpha (TNFA) that knocks down expression of TNFA, as well as compositions and uses thereof, for example, to suppress immune response(s), including inflammation and autoimmunity. In some embodiments, the regulatory T cell promoting molecule is selected from interleukin-10 (IL10), cytotoxic T lymphocyte associated protein 4 (CTLA4), transforming growth factor beta 1 (TGFB1), indoleamine 2,3-dioxygenase 1 (IDO1), ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), 5'-nucleotidase ecto (NT5E), interleukin-22 (IL-22), amphiregulin (AREG), interleukin-35 (IL35), GARP, CD274 molecule (CD274), forkhead box P3 (FOXP3), IKAROS family zinc finger 2 (IKZF2), familial eosinophilia (EOS), interferon regulatory factor 4 (IRF4), lymphoid enhancer binding factor 1 (LEF1), and BTB domain and CNC homolog 2 (BACH2).

[0007] In some embodiments, a T cell or population of T cells is engineered to contain a heterologous nucleic acid encoding IL10 under the control of a promoter sequence, a modification in an endogenous nucleic acid sequence encoding IFNG that knocks down expression of IFNG, and a modification in an endogenous nucleic acid sequence encoding TNFA that knocks down expression of TNFA.

[0008] In some embodiments, a T cell or population of T cells is engineered to contain a heterologous nucleic acid encoding CTLA4 under the control of a promoter sequence, a modification in an endogenous nucleic acid sequence encoding IFNG that knocks down expression of IFNG, and a modification in an endogenous nucleic acid sequence encoding TNFA that knocks down expression of TNFA.

[0009] In some embodiments, a T cell or population of T cells is engineered to contain heterologous nucleic acid sequences encoding IL10 and CTLA4, each under the control of a promoter sequence, a modification in an endogenous nucleic acid sequence encoding IFNG that knocks down expression of IFNG, and a modification in an endogenous nucleic acid sequence encoding TNFA that knocks down expression of TNFA.

[0010] In some embodiments, the T cell or population of T cells is further engineered to include a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding interleukin 17A (IL17A), interleukin 2 (IL2), interleukin 6 (IL6), perforin 1 (PRF1), granzyme A (GZMA), or granzyme B (GZMB).

[0011] In some embodiments, the T cell or population of T cells is further engineered to contain a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding an endogenous T cell receptor (TCR).

[0012] In some embodiments, the T cells or population of T cells are further engineered to contain a heterologous coding sequence for a targeting receptor under the control of a promoter sequence. In some embodiments, the targeting receptor comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the targeted receptor is targeted to a ligand selected from mucosal vascular addressin cell adhesion molecule 1 (MADCAM1), tumor necrosis factor alpha (TNFA), CEA cell adhesion molecule 6 (CEACAM6), vascular cell adhesion molecule 1 (VCAM1), citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNF receptor superfamily member 17 (TNFRSF17), dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), desmoglein 3 (DSG3), and MHC class I HLA-A (HLA-A*02).

[0013] In some embodiments, at least 30%, 35%, preferably at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the population of T cells comprise an insertion of a sequence encoding a regulatory T cell-promoting molecule, e.g., as assessed by sequencing (e.g., NGS). In some embodiments, at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, 80%, 85%, 90%, or 95% of the population of T cells comprise a modification (e.g., knockdown) in the IFNG sequence, e.g., as assessed by sequencing (e.g., NGS). In some embodiments, at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, 80%, 85%, 90%, or 95% of the population of T cells comprise a modification (e.g., knockdown) in the TNFA sequence, e.g., as assessed by sequencing (e.g., NGS). In some embodiments, at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, 80%, 85%, 90%, or 95% of the population of T cells comprise a modification (e.g., knockdown) in the TCR sequence, e.g., as assessed by sequencing (e.g., NGS). In some embodiments, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the population of T cells contain an insertion of a sequence encoding a targeting receptor (e.g., a CAR), e.g., as assessed by sequencing (e.g., NGS).

[0014] The modifications described herein for knocking down the expression of a gene can include one or more of insertion, deletion, or substitution. The heterologous sequences described herein can be incorporated into an expression construct(s). Multiple heterologous sequences can be incorporated into one expression construct or into separate expression constructs. The heterologous sequences described herein can be incorporated into an episomal expression construct(s). The heterologous sequences described herein can be inserted into a genome, for example, as a targeted or non-targeted insertion. In some embodiments, the targeted insertion is an insertion into a site selected from the TCR gene locus, the TNF gene locus, the IFNG gene locus, the IL17A gene locus, the IL6 gene locus, the IL2 gene locus, the adeno-associated virus integration site 1 (AAVS1) locus.

[0015] Pharmaceutical compositions and uses of the engineered T cells are also provided herein. In some embodiments, the engineered T cells and pharmaceutical compositions thereof can be administered to a subject in need of immunosuppression. In some embodiments, the engineered T cells and pharmaceutical compositions thereof can be useful in treating immune disorders or autoimmune diseases, such as ulcerative colitis, Crohn's disease, rheumatoid arthritis, psoriasis, multiple sclerosis, systemic lupus erythematosus, type 1 diabetes, and graft-versus-host disease (GvHD).

[0016] In some embodiments, the insertion of the sequence(s) described herein or the modification (e.g., knockdown) of the sequence(s) can be mediated by a guide RNA in combination with an RNA-guided DNA binding agent (e.g., Cas nuclease). In some embodiments, the insertion of the sequence(s) described herein or the knockdown of the sequence(s) can be mediated by another suitable gene editing system (e.g., a zinc finger nuclease (ZFN) system or a transcription activator-like effector nuclease (TALEN) system). [Brief description of the drawings]

[0017] [Figure 1A]1 shows histograms of flow cytometry data showing the intensity of fluorescent staining of CD3+CD4+ cells untransduced or transduced with the indicated coding sequence insertions, or CD3+CD4+CD25+nTregs. 2 shows histograms of the fluorescent intensity of CTLA expression in transduced T cells or the indicated controls. [Figure 1B] 4 shows histograms of flow cytometry data showing the intensity of fluorescent staining of CD3+CD4+ cells untransduced or transduced with the indicated coding sequence insertions, or CD3+CD4+CD25+nTregs. 5 shows histograms of the fluorescent intensity of IL10 expression in transduced T cells or the indicated controls. [Figure 1C] 4 shows histograms of flow cytometry data showing the intensity of fluorescent staining of CD3+CD4+ cells untransduced or transduced with the indicated coding sequence insertions, or CD3+CD4+CD25+nTregs. 5 shows histograms of the fluorescent intensity of Foxp3 expression in transduced T cells or the indicated controls. [Figure 1D] 4 shows histograms of flow cytometry data showing the intensity of fluorescent staining of CD3+CD4+ cells untransduced or transduced with the indicated coding sequence insertions, or CD3+CD4+CD25+nTregs. 5 shows histograms of the fluorescent intensity of Helios expression in transduced T cells or the indicated controls. [Figure 1E] 4 shows histograms of flow cytometry data showing the intensity of fluorescent staining of CD3+CD4+ cells untransduced or transduced with the indicated coding sequence insertions, or CD3+CD4+CD25+nTregs. 5 shows histograms of the fluorescent intensity of Eos expression in transduced T cells or the indicated controls. [Diagram 2]Graphs showing results from a mouse model of GvHD. A, Survival curves showing days of survival after injection of CD3+CD4+ cells, CD4+CD25+ Tregs, PBMCs transduced with the indicated coding sequence or control insertions, or no cells (irradiation only). B, quantification of human lymphocytes isolated from the spleens of mice at sacrifice after injection of transduced CD3+CD4+ cells or controls as indicated. [Figure 3A] 1 is a graph showing the results of in vitro cytokine profile analysis of stimulated CD3+CD4+ cells transduced with the indicated coding sequence or the indicated control insertion, showing in vitro IL6 production by transduced T cells or the indicated control upon cell stimulation. [Figure 3B] 1 is a graph showing the results of in vitro cytokine profile analysis of stimulated CD3+CD4+ cells transduced with the indicated coding sequence or the indicated control insert, showing in vitro TNF-alpha production by transduced T cells or the indicated control upon cell stimulation. [Figure 3C] 1 is a graph showing the results of in vitro cytokine profile analysis of stimulated CD3+CD4+ cells transduced with the indicated coding sequence or the indicated control insertion, showing in vitro IL10 production by transduced T cells or the indicated control upon cell stimulation. [Figure 3D] 1 is a graph showing the results of in vitro cytokine profile analysis of stimulated CD3+CD4+ cells transduced with the indicated coding sequence or the indicated control insert, showing in vitro IL13 production by transduced T cells or the indicated control upon cell stimulation. [Figure 3E] 1 is a graph showing the results of in vitro cytokine profile analysis of stimulated CD3+CD4+ cells transduced with the indicated coding sequence or the indicated control insertion, showing in vitro IL2 production by transduced T cells or the indicated control upon cell stimulation. [Figure 3F] Graph showing the results of in vitro cytokine profile analysis of stimulated CD3+CD4+ cells transduced with the indicated coding sequence or the indicated control insert, showing in vitro IFN-gamma production by transduced T cells or the indicated control upon cell stimulation. [Figure 4] Graph showing percent inhibition of cell proliferation by transduced T cells, as measured by CTV dilution, in a mixed lymphocyte reaction assay in which CTV-labeled T cells and CD3-depleted PBMCs were mixed with CD3+CD4+ cells transduced with the indicated ratios of insertions of the indicated coding sequences or the indicated controls. [Figure 5A] 1 is a histogram of flow cytometry data showing the intensity of fluorescent staining of CD3+CD4+ cells or CD3+CD4+CD25+ nTregs that are untransduced or transduced with the insertion of coding sequences for IL10 and CTLA4 alone (no editing) or in combination with knockout (KO) of either or both of IFNG and TNFA. 2 is a histogram of the fluorescent intensity of CTLA4 expression in transduced T cells or the indicated controls. [Figure 5B] 1 is a histogram of flow cytometry data showing the intensity of fluorescent staining of CD3+CD4+ cells or CD3+CD4+CD25+ nTregs that are untransduced or transduced with the insertion of the coding sequences for IL10 and CTLA4 alone (no editing) or in combination with knockout (KO) of either or both of IFNG and TNFA. 2 is a histogram of the fluorescent intensity of IL10 expression in transduced T cells or the indicated controls. [Figure 5C] 4 is a histogram of flow cytometry data showing the intensity of fluorescent staining of CD3+CD4+ cells or CD3+CD4+CD25+ nTregs that are untransduced or transduced with the insertion of coding sequences for IL10 and CTLA4 alone (no editing) or in combination with knockout (KO) of either or both of IFNG and TNFA. 5 is a histogram of the fluorescent intensity of FOXP3 expression in transduced T cells or the indicated controls. [Figure 5D] 1 is a histogram of flow cytometry data showing the intensity of fluorescent staining of CD3+CD4+ cells or CD3+CD4+CD25+nTregs that are untransduced or transduced with the insertion of coding sequences for IL10 and CTLA4 alone (no editing) or in combination with knockout (KO) of either or both of IFNG and TNFA. 2 is a histogram of the fluorescent intensity of Helios expression in transduced T cells or the indicated controls. [Figure 5E] 1 is a histogram of flow cytometry data showing the intensity of fluorescent staining of CD3+CD4+ cells or CD3+CD4+CD25+ nTregs that are untransduced or transduced with the insertion of coding sequences for IL10 and CTLA4 alone (no editing) or in combination with knockout (KO) of either or both of IFNG and TNFA. 2 is a histogram of the fluorescent intensity of Eos expression in transduced T cells or the indicated controls. [Figure 6A] Graph showing results from a mouse model of GvHD. Survival curves showing days of survival after injection of mice with PBMC, CD3+CD4+ cells untransduced or transduced with only insertions of coding sequences for IL10 and CTLA4 (no editing) or in combination with editing of either or both of IFNG and TNFA (KO), CD3+CD4+CD25+nTregs, or no cells (irradiation only). [Figure 6B] Graph showing results from a mouse model of GvHD. Graph showing quantification of human lymphocytes isolated from the spleens of mice at sacrifice after injection with transduced CD3+CD4+ cells or control cells as indicated. [Figure 7A]Graph showing in vitro cytokine profile analysis of stimulated CD3+CD4+ cells or CD4+CD25+nTregs that are untransduced or transduced with insertions of IL10 and CTLA4 coding sequences alone (no editing) or in combination with editing of either IFNG or TNFA or both (KO). In vitro IL6 production by transduced T cells or the indicated controls upon cell stimulation is shown. [Figure 7B] Graph showing the results of in vitro cytokine profile analysis of stimulated CD3+CD4+ cells or CD4+CD25+ nTregs that are untransduced or transduced with insertions of the coding sequences for IL10 and CTLA4 alone (no editing) or in combination with editing of either IFNG or TNFA or both (KO). Showing in vitro TNF-alpha production by transduced T cells or the indicated controls upon cell stimulation. [Figure 7C] Graph showing the results of in vitro cytokine profile analysis of stimulated CD3+CD4+ cells or CD4+CD25+ nTregs that were untransduced or transduced with insertion of IL10 and CTLA4 coding sequences alone (no editing) or in combination with editing of either IFNG or TNFA or both (KO). IL10 production by transduced T cells in vitro upon cell stimulation is shown. [Figure 7D] Graph showing the results of in vitro cytokine profile analysis of stimulated CD3+CD4+ cells or CD4+CD25+ nTregs that were untransduced or transduced with insertion of IL10 and CTLA4 coding sequences alone (no editing) or in combination with editing of either IFNG or TNFA or both (KO). IL13 production by transduced T cells in vitro upon cell stimulation is shown. [Figure 7E]Graph showing the results of in vitro cytokine profile analysis of stimulated CD3+CD4+ cells or CD4+CD25+ nTregs that were untransduced or transduced with insertion of IL10 and CTLA4 coding sequences alone (no editing) or in combination with editing of either IFNG or TNFA or both (KO). IL2 production by transduced T cells in vitro upon cell stimulation is shown. [Figure 7F] Graph showing the results of in vitro cytokine profile analysis in stimulated CD3+CD4+ cells or CD4+CD25+ nTregs that are untransduced or transduced with insertions of IL10 and CTLA4 coding sequences alone (no editing) or in combination with editing of either IFNG or TNFA or both (KO). IFN-gamma production by engineered cells in vitro upon cell stimulation is shown. [Figure 8] Graph showing percent inhibition of cell proliferation by engineered T cells, as measured by CTV dilution, in a mixed lymphocyte reaction assay in which CTV-labeled T cells and CD3-depleted PBMCs were mixed with CD3+CD4+ cells transduced with the indicated coding sequences or the indicated controls at the indicated ratios. [Figure 9] Graphs showing results from a mouse model of GvHD. A, Survival curves showing days of survival after mice were injected with PBMCs, CD3+CD4+ cells untransduced or transduced with insertions of IL10 and CTLA4 coding sequences (wild type (wt) or high affinity (HA)) in combination with editing (KO) of both IFNG and TNFA as indicated, CD3+CD4+CD25+nTregs, or no cells were injected (vehicle). B, quantification of human lymphocytes isolated from the spleens of mice at sacrifice after injection with transduced CD3+CD4+ cells or control cells as indicated. [Figure 10]Graphs showing percent proliferation inhibition measured by CTV dilution in a mixed lymphocyte assay. A shows the inhibition of proliferation with or without inflammatory preconditioning. B shows the inhibition of proliferation with or without inflammatory preconditioning in the presence of the additional inflammatory cytokines indicated. Respective p values ​​shown are *p<0.05, **p<0.01, and ***p<0.001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Reference will now be made in detail to certain specific embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention will be described in conjunction with the illustrated embodiments, it will be understood that they are not intended to limit the invention to these embodiments. Instead, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the invention as defined by the accompanying embodiments.

[0019] The section headings used herein are for organizational purposes only and should not be construed as limiting the desired subject matter in any manner. In the event that any material incorporated by reference conflicts with any term defined herein or any other express content of this specification, the present specification shall control.

[0020] I. Definition Before describing the teachings of the present invention in detail, it should be understood that the present disclosure is not limited to specific compositions or process steps, which may vary. As used herein and in the accompanying embodiments, it should be noted that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "conjugate" includes a plurality of conjugates, reference to a "cell" includes a plurality of cells or cell populations, etc. As used herein, the term "include" and grammatical variations thereof are intended to be non-limiting, and the recitation of items in a list does not exclude other similar items that may be substituted or added to the recited items.

[0021] Numerical ranges are inclusive of the numbers defining the range. Measurements and measurables are understood to be approximations taking into account significant digits and error associated with measurement. Additionally, the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" are not intended to be limiting. It should be understood that the foregoing general description and detailed description are exemplary and explanatory only and are not intended to limit the present teachings.

[0022] Unless otherwise noted herein, embodiments described herein as "comprising" various components are also contemplated as "consisting" or "consisting essentially" of the described components, and embodiments described herein as "consisting" of various components are also contemplated as "comprising" or "consisting essentially" of the described components. Also, embodiments described herein as "consisting essentially" of various components are also contemplated as "consisting" of or "including" the described components (this interchangeability does not apply to the use of these terms in the claims).

[0023] The term "or" is used in its inclusive sense, ie, equivalent to "and / or," unless the context clearly dictates otherwise.

[0024] The term "about", when used before a list or range, modifies each member of the list or each endpoint of the range. The term "about" or "approximately" refers to an acceptable error for a particular value as determined by one of ordinary skill in the art, which error depends in part on how the value is measured or determined. The term "about" is used herein to mean within a range of typical tolerances in the art. For example, "about" can be understood as about 2 standard deviations from the mean. In certain embodiments, about means +10%. In certain embodiments, about means +5%.

[0025] The term "at least" before a number or a series of numbers is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that may be logically included, as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 17 nucleotides of a 20 nucleotide nucleic acid molecule" means that 17, 18, 19, or 20 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers in the series or range.

[0026] As used herein, "less than" or "less than" is understood to mean the expression and the logical lower zero or adjacent integer value that is logical from the context. For example, a duplex region of "2 nucleotide base pairs or less" has 2, 1, or 0 nucleotide base pairs. When "less than" or "less than" is present before a series of numbers or ranges, it is understood that each of the series of numbers or ranges is modified.

[0027] As used herein, ranges include both upper and lower limits.

[0028] As used herein, when a value is expressed as a maximum amount of 100% (e.g., 100% inhibition or 100% encapsulation), it is understood that the value is limited by the detection method.For example, 100% inhibition is understood to be inhibition to a level below the detection level of the assay, and 100% encapsulation is understood to be that the substance intended to be encapsulated cannot be detected outside the vesicle.

[0029] Unless otherwise stated, the following terms and expressions are intended to have the following meanings as used herein:

[0030] As used herein, "knockdown" refers to reducing the expression of a particular gene product (e.g., full-length or wild-type mRNA, protein, or both) in, for example, a cell, cell population, tissue, or organ, by gene editing. In some embodiments, gene editing can be assessed by sequencing, for example, next generation sequencing (NGS). Expression can be reduced, for example, by at least 70%, 75%, 80%, 85%, 90%, 95%, or to below the detection level of the assay, compared to a suitable control in which the gene sequence is not modified. Protein knockdown can be measured by detecting the amount of protein from a tissue, cell population, or liquid of interest. Methods for measuring mRNA knockdown are known, including sequencing of mRNA isolated from a tissue or cell population of interest. Flow cytometry analysis is a known method for measuring knockdown of protein expression. For secreted proteins, knockdown can be assessed in tissue culture medium or blood, or liquids derived therefrom, such as serum or plasma. In some embodiments, "knockdown" may refer to some loss of expression of a particular gene product, for example, a reduction in the amount of full-length wild-type mRNA that is transcribed or translated into a full-length protein, or a reduction in the amount of protein expressed by a population of cells. It is well understood what changes in the sequence of an mRNA result in reduced expression of wild-type or full-length protein. In some embodiments, "knockdown" may refer to some loss of expression of a particular gene product (e.g., IFNG or TNFA gene product in body fluids or tissue culture medium). Modification of the nucleic acid sequence that codes for an endogenous nucleic acid sequence (e.g., IFNG or TNFA) may result in knockdown.

[0031] As used herein, "T cell receptor" or "TCR" refers to a receptor in a T cell. In general, a TCR is a heterodimeric receptor molecule that includes two TCR polypeptide chains, an α chain and a β chain. After antigen binding, the α chain and the β chain TCR polypeptide can complex with various CD3 molecules and induce immune response(s), including inflammation and autoimmunity. As used herein, knockdown of a TCR refers to partial or total knockdown of any TCR gene (e.g., deletion of a portion of the TRBC1 gene, alone or in combination with partial or total knockdown of other TCR gene(s)).

[0032] "TRAC" is used to refer to the T cell receptor alpha chain. The human wild type TRAC sequence is available at NCBI Gene ID: 28755; Ensembl: ENSG00000277734. T cell receptor alpha constant, TCRA, IMD7, TRCA, and TRA are gene synonyms of TRAC.

[0033] "TRBC" refers to the T cell receptor β chain (e.g., TRBC1 and TRBC2). "TRBC1" and "TRBC2" refer to two homologous genes that encode the T cell receptor β chain, which is the gene product of the TRBC1 or TRBC2 gene.

[0034] The human wild type TRBC1 sequence is available at NCBI Gene ID: 28639; Ensembl: ENSG00000211751. T cell receptor beta constant, V_segment translation product, BV05S1J2.2, TCRBC1, and TCRB are gene synonyms of TRBC1.

[0035] The human wild type TRBC2 sequence is available at NCBI Gene ID: 28638; Ensembl: ENSG00000211772. T cell receptor beta constant, V_segment translation product, and TCRBC2 are gene synonyms of TRBC2.

[0036] As used herein, "immune response" refers to one or more immune system reaction(s), e.g., increased production or activity of immune system cells (e.g., but not limited to, T cells, B cells, natural killer cells, monocytes, neutrophils, eosinophils, basophils, mast cells, erythrocytes, dendritic cells, antigen-presenting cells, macrophages, or phagocytes) compared to an unstimulated control immune system. An immune response can be triggered when the immune system is exposed to an antigen, e.g., a foreign antigen or a self-antigen (e.g., but not limited to, a pathogen (microbe, virus, prion, fungus, etc.), an allergen (dust, pollen, dust mite, etc.), a toxin (chemical, drug, etc.), or a physiological change (hypercholesterolemia, obesity, organ transplant, etc.). An immune response can also include a response in GvHD in which donor cells mediate an immune attack of host tissues after hematopoietic stem cell transplantation. An immune response can result in inflammation. An immune response may target, attack, eliminate, or neutralize an antigen (e.g., foreign or self). An immune response may be desirable or undesirable. An immune response may be acute or chronic. An immune response may damage the cells, tissues, or organs against which the immune response is initiated.

[0037] As used herein, "autoimmune response" refers to one or more immune system reaction(s) against self-antigens (e.g., autoantigens presented by the subject's own cells, tissues, or organs). An autoimmune response may result in increased production or activity of immune system cells (e.g., but not limited to, T cells, B cells, natural killer cells, monocytes, neutrophils, eosinophils, basophils, mast cells, erythrocytes, dendritic cells, antigen-presenting cells, macrophages, or phagocytes) compared to a suitable control (e.g., a healthy control). An autoimmune response may result in inflammation (e.g., persistent inflammation) or lead to an autoimmune disease. An autoimmune response may target, attack, eliminate, or neutralize self-antigens produced by the subject's own cells, tissues, or organs, which may lead to an autoimmune disease.

[0038] As used herein, "suppressing an immune response(s)" refers to reducing or inhibiting the level of one or more immune system reaction(s) (e.g., production or activity of immune system cells) compared to a suitable control (e.g., a control not treated or prior to treatment with the engineered T cells described herein). "Suppressing" an immune response(s) can refer to reducing the production or activity of immune system cells compared to a suitable control (e.g., a control not treated or prior to treatment with the engineered T cells described herein). "Suppressing" an immune response can refer to increasing immune tolerance. For example, immune system cell production or activity can be measured by cell counts (e.g., lymphocyte or spleen cell counts), cell activity (e.g., T cell assays), or gene or protein expression (e.g., biomarker expression), and production or activity is reduced by 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or below the level of detection of the assay, as compared to a suitable control (e.g., a control not treated or prior to treatment with the engineered T cells described herein).

[0039] As used herein, "autoimmune disease" or "autoimmune disorder" refers to a condition characterized by a pathological immune response against a subject's own antigens, cells, tissues, or organs. Examples of autoimmune diseases and disorders include, but are not limited to, ulcerative colitis, Crohn's disease, rheumatoid arthritis, psoriasis, multiple sclerosis, systemic lupus erythematosus, and type 1 diabetes. In some embodiments, engineered T cells are used autologous or allogeneic.

[0040] As used herein, "immune disorder" is understood as a disease or condition characterized by pathological or undesired immune response in a subject.In certain embodiments, the immune disorder is an autoimmune disease.In certain embodiments, the immune disorder is GvHD.In certain embodiments, the subject with immune disorder needs suppression of immune response.In certain embodiments, the subject with immune disorder needs increased immune tolerance.

[0041] "T cells" play a central role in immune responses following exposure to an antigen. T cells can be naturally occurring or non-naturally occurring, e.g., where T cells are generated by engineering (e.g., from stem cells) or by transdifferentiation (e.g., reprogramming of somatic cells). T cells can be distinguished from other lymphocytes by the presence of T cell receptors on the cell surface. Included in this definition are conventional and adaptive T cells (including helper CD4+ T cells, cytotoxic CD8+ T cells, memory T cells, and regulatory CD4+ T cells), as well as innate-like T cells (including natural killer T cells, mucosal-associated invariant T cells, and gamma delta T cells). In some embodiments, T cells are CD4+. In some embodiments, T cells are CD3+ / CD4+.

[0042] "Regulatory T cells" or "Tregs" refer to specialized T cells that play a central role in self-tolerance by suppressing excessive immune response(s), including inflammation and autoimmunity. Tregs can be naturally occurring or non-naturally occurring, e.g., where Tregs are engineered (e.g., by modifying (e.g., knocking down) endogenous nucleic acid sequences encoding IFNG and TNFA and inserting at least one sequence(s) encoding a regulatory T cell-promoting molecule). Naturally occurring Tregs, or natural Tregs, or nTregs, are specialized T cells that typically develop in the thymus and function to promote self-tolerance by suppressing excessive immune response(s). In some embodiments, cells (e.g., conventional T cells or populations of conventional T cells, e.g., populations of T cells not enriched for the presence of nTreg cells) can be engineered to exhibit phenotypic characteristics and suppressive functions of regulatory T cells by modifying endogenous nucleic acid sequences encoding TNFA and IFNG, e.g., knocking down nucleic acid sequences encoding TNFA and IFNG, and inserting sequence(s) encoding a regulatory T cell-promoting molecule into the cells, which may be referred to as transduced or "engineered" T cells. In some embodiments, engineered T cells include a modification of an endogenous nucleic acid sequence encoding IFNG, a modification of an endogenous nucleic acid sequence encoding TNFA, and an insertion of a heterologous regulatory T cell-promoting molecule (e.g., IL10 or CTLA4). The modification of the endogenous nucleic acid sequence (e.g., a modification that knocks down expression of an endogenous gene) can include or consist of one or more indel or substitution mutations in the genomic sequence.

[0043] As used herein, "regulatory T cell-promoting molecules" refer to molecules that promote the conversion of conventional T cells to regulatory T cells, including immunosuppressive molecules and Treg transcription factors. Additionally, regulatory T cell-promoting molecules also refer to molecules that confer regulatory activity to conventional T cells, including Treg-associated immunosuppressive molecules and transcription factors. Examples of immunosuppressive molecules include, but are not limited to, interleukin-10 (IL10), cytotoxic T lymphocyte-associated protein 4 (CTLA4), transforming growth factor beta 1 (TGFB1), indoleamine 2,3-dioxygenase 1 (IDO1), ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), 5'-nucleotidase ecto (NT5E), interleukin-22 (IL22), amphiregulin (AR1), and IL1. EG), interleukin-35 (IL35), leucine-rich repeat containing 32 (GARP), CD274 molecule (CD274), forkhead box P3 (FOXP3), IKAROS family zinc finger 2 (IKZF2), familial eosinophilia (EOS), interferon regulatory factor 4 (IRF4), lymphoid enhancer binding factor 1 (LEF1), and BTB domain and CNC homolog 2 (BACH2). In some embodiments, regulatory T cell promoting molecules can be used in specific combinations (e.g., IL10 and CTLA4, ENTPD1 and NT5E, and IL22 and AREG). In particular, a combination of IL10 and CTLA4 is provided herein. In some embodiments, the expression of immunosuppressive molecules can be promoted by the expression of transcription factors such as FoxP3, Helios, Eos, IRF4, Lef1, or BACH2.

[0044] In some embodiments, conventional T cells can be engineered to modify, insert, or delete sequences in the genome, with the "engineered" T cells exhibiting one or more phenotypic characteristics and suppressive functions of natural regulatory T cells. For example, the "engineered" T cells can exhibit suppressive activity in a mixed lymphocyte reaction assay, such as provided in Examples 2 and 3 below, or can suppress graft-versus-host disease, preferably statistically significantly, in a mouse model, such as those presented in Examples 2 and 3 below (see also, e.g., Parmar et al., Ex vivo fucosylation of third-party human regulatory T cells enhances anti-graft-versus-host disease potency in vivo, Blood 125(9) (2015)). In some embodiments, the "engineered" T cells are conventional T cells that have been modified by the insertion of coding sequences for regulatory T cell promoting molecules and by modification (e.g., knockdown) of expression of pro-inflammatory cytokines (e.g., both IFNG and TNFA). In some embodiments, the starting T cell population for engineering is not enriched for the presence of natural Tregs, for example the starting T cell population has less than 20% natural Tregs.

[0045] As used herein, "pro-inflammatory" molecules (e.g., cytokines) increase immune responses as described herein and, for example, reduce the efficacy of Tregs in a dose-responsive manner in the mouse model of graft-versus-host disease presented in Examples 2 and 3. Examples of pro-inflammatory molecules include, but are not limited to, IFNG, TNFA, IL17A, IL6, IL2, These include perforin 1 (PRF1), granzyme A (GZMA), and granzyme B (GZMB).

[0046] As used herein, a "targeting receptor" refers to a receptor present on the surface of a cell (e.g., a T cell) that enables the cell to bind to a target site (e.g., a particular cell or tissue within an organism). Targeting receptors include, but are not limited to, chimeric antigen receptors (CARs), T cell receptors (TCRs), and receptors for cell surface molecules operably linked via at least a transmembrane domain within an internal signaling domain capable of activating a T cell upon binding of an extracellular receptor portion of the protein, such as mucosal addressin cell adhesion molecule-1 (MadCAM-1), TNFA, CEA cell adhesion molecule 6 (CEACAM6), vascular cell adhesion molecule 1 (VCAM1), citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNF receptor superfamily member 17 (TNFRSF17), solute carrier family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), desmoglein 3 (DSG3), and MHC class I. HLA-A (HLA-A*02) is one example.

[0047] As used herein, "chimeric antigen receptor" refers to an extracellular antigen recognition domain (e.g., scFv, VHH, nanobody) operably linked to an intracellular signaling domain that activates T cells upon antigen binding. CARs are composed of four regions: an antigen recognition domain, an extracellular hinge region, a transmembrane domain, and an intracellular T cell signaling domain. Such receptors are well known in the art (see, e.g., WO2020092057, WO2019191114, WO2019147805, WO2018208837, the contents of each of which are incorporated herein by reference in their entirety). Also contemplated are reverse universal CARs that facilitate the binding of immune cells to target cells via adaptor molecules (see, e.g., WO2019238722, the contents of which are incorporated herein in their entirety). CARs can be targeted against any antigen for which antibodies can be developed, and are typically directed to molecules displayed on the surface of the targeted cells or tissues. In some embodiments, the CAR can target the engineered T cells to the gastrointestinal tract, e.g., the CAR targets MAdCAM-1. In some embodiments, the CAR can target the engineered T cells to tissues that contain endothelial cells, e.g., the CAR targets VCAM-1, e.g., to suppress immune responses in disorders such as Crohn's disease and multiple sclerosis. In some embodiments, the CAR can target the engineered T cells to endothelial cells, e.g., the CAR targets CEACAM6, e.g., to suppress immune responses in disorders such as Crohn's disease. In some embodiments, the CAR can target the engineered T cells to pre-B cells, e.g., the CAR targets CD19, e.g., to suppress immune responses in disorders such as multiple sclerosis and systemic lupus erythematosus. In some embodiments, the CAR can target the engineered T cells to B lymphocytes, e.g., the CAR targets CD20, e.g., to suppress immune responses in disorders such as multiple sclerosis and systemic lupus erythematosus.In some embodiments, the CAR can target engineered T cells to inflamed tissues, for example, the CAR targets TNFA, for example, to suppress immune responses in disorders such as rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, or Crohn's disease. In some embodiments, the CAR can target engineered T cells to inflamed tissues, for example, the CAR targets TGF-b1, for example, to suppress immune responses in disorders such as inflammatory bowel disease, ulcerative colitis, or Crohn's disease. In some embodiments, the CAR can target engineered T cells to neural tissues, for example, the CAR targets MBP, MOG, or PLP1, for example, to suppress immune responses in disorders such as multiple sclerosis. In some embodiments, the CAR can target engineered T cells to tissues containing mature B lymphocytes, for example, the CAR targets TNFRSF17, for example, to suppress immune responses in disorders such as systemic lupus erythematosus. In some embodiments, the CAR can target engineered T cells to synovial tissue, for example, the CAR targets citrullinated vimentin to suppress immune responses in disorders such as rheumatoid arthritis. In some embodiments, the CAR targets dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), demoglein 3 (DSG3), or MHC class I HLA-A (HLA-A*02). Additional CAR targets (e.g., inflammatory antigens) are known in the art. See, for example, WO2020092057A1, the contents of which are incorporated herein in their entirety.

[0048] As used herein, "treatment" refers to any administration or application of a therapeutic agent for a disease or disorder in a subject, including inhibiting the disease, arresting its occurrence, alleviating one or more symptoms of the disease, curing the disease, preventing one or more symptoms of the disease, or preventing the recurrence of one or more symptoms of the disease. Treating an autoimmune or inflammatory response or disorder can include alleviating inflammation associated with a particular disorder, thereby alleviating symptoms specific to the disease. Treatment with the engineered T cells described herein can be used before, after, or in combination with additional therapeutic agents, such as anti-inflammatory agents, immunosuppressants, or biologics for the treatment of autoimmune disorders (e.g., Remicade, Humira).

[0049] "Promoter" refers to a regulatory region that controls the expression of a gene to which it is linked.

[0050] "Polynucleotide" and "nucleic acid" are used herein to refer to polymeric compounds that include nucleosides or nucleoside analogs with nitrogen-containing heterocyclic bases or base analogs linked together along a backbone, and include polymers of traditional RNA, DNA, mixed RNA-DNA, and analogs thereof. The nucleic acid "backbone" can be composed of a variety of linkages, including sugar-phosphodiester linkages, peptide-nucleic acid linkages ("peptide nucleic acid" or PNA; PCT No. WO95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or one or more of these combinations. The sugar moiety of the nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions (e.g., 2' methoxy or 2' halide substitutions). The nitrogenous bases include the conventional bases (A, G, C, T, U), their analogs (e.g., modified uridines, such as 5-methoxyuridine, pseudouridine, or N1-methylpseudouridine, etc.); inosine; derivatives of purines or pyrimidines (e.g., N 4-methyldeoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substituents at the 5- or 6-position (e.g., 5-methylcytosine), purine bases with substituents at the 2-, 6-, or 8-position, 2-amino-6-methylaminopurine, O 6 -methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine, and O 4 -alkyl-pyrimidines; U.S. Pat. No. 5,378,825 and PCT Publication No. WO 93 / 13121. For a general review, see The Biochemistry of the Nucleic Acids 5-36 (Adams et al., ed., 11 th ed., 1992). Nucleic acids can contain one or more "abasic" residues in which the backbone does not contain a nitrogenous base at the position(s) of the polymer (U.S. Pat. No. 5,585,481). Nucleic acids can contain only conventional RNA or DNA sugars, bases, and linkages, or can contain both conventional building blocks and substitutions (e.g., polymers containing conventional bases with 2' methoxy linkages, or both conventional bases and one or more base analogs). Nucleic acids include "locked nucleic acids" (LNAs), which are analogs that contain one or more LNA nucleotide monomers with a bicyclic furanose unit locked into an RNA-mimicking sugar conformation, enhancing hybridization affinity to complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). RNA and DNA can have different sugar moieties and can be distinguished by the presence of uracil or its analogs in RNA and thymine or its analogs in DNA.

[0051] "Guide RNA", "gRNA", or simply "guide" are used interchangeably herein to refer to a guide comprising a guide sequence, e.g., either a crRNA (also known as CRISPR RNA) or a combination of a crRNA and a trRNA (also known as tracrRNA). The crRNA and trRNA can be associated as a single RNA molecule (single guide RNA, sgRNA) or, e.g., in two separate RNA molecules (dual guide RNA, dgRNA). "Guide RNA" or "gRNA" refer to each type. The trRNA can be a naturally occurring sequence or a trRNA sequence that has a modification or variation compared to the naturally occurring sequence. The guide RNA (e.g., sgRNA or dgRNA) can include modified RNA as described herein.

[0052] As used herein, a "guide sequence" refers to a sequence in a guide RNA that is complementary to a target sequence and functions to guide the guide RNA to the target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA-binding agent. A "guide sequence" may also be referred to as a "targeting sequence" or a "spacer sequence." A guide sequence may be, for example, 20 base pairs in length for Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologs / orthologues. Shorter or longer sequences (e.g., 15, 16, 17, 18, 19, 21, 22, 23, 24, or 25 nucleotides in length) can also be used as a guide. For example, in some embodiments, the guide sequence comprises at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence that is complementary to the target. In some embodiments, the target sequence is, for example, within a gene or on a chromosome, and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between a guide sequence and its corresponding target sequence can be about 75%, 80%, 85%, 90%, 95%, or 100%. For example, in some embodiments, the guide sequence comprises a sequence having about 75%, 80%, 85%, 90%, 95%, or 100% identity for at least 15, 16, 17, 18, 19, or 20 consecutive nucleotides of the sequence. In some embodiments, the guide sequence and the target region can be 100% complementary or identical. In other embodiments, the guide sequence and the target region can include at least one mismatch. For example, the guide sequence and the target sequence can include one, two, three, or four mismatches, where the total length of the target sequence is at least 17, 18, 19, 20, or more base pairs. In some embodiments, the guide sequence and the target region may contain one to four mismatches or may be fully complementary, where the guide sequence comprises at least 17, 18, 19, 20, or more nucleotides. In some embodiments, the guide sequence and the target region may contain one, two, three, or four mismatches, where the guide sequence comprises 20 nucleotides.

[0053] Since the nucleic acid substrate of the RNA-guided DNA binder is a double-stranded nucleic acid, the target sequence of the RNA-guided DNA binder includes both the plus and minus strands of genomic DNA (i.e., the given sequence and the reverse complement of the sequence). Thus, when a guide sequence is described as being "complementary to a target sequence," it should be understood that the guide sequence can guide the guide RNA to bind to the reverse complement of the target sequence. Thus, in some embodiments, when the guide sequence binds to the reverse complement of the target sequence, the guide sequence is identical to a certain nucleotide in the target sequence (e.g., the target sequence without PAM), except that T is replaced by U in the guide sequence.

[0054] As used herein, "RNA-guided DNA binding agent" refers to a polypeptide or complex of polypeptides having RNA and DNA binding activity, or a DNA-binding subunit of such a complex, where the DNA-binding activity is sequence-specific and dependent on the sequence of the RNA. The term RNA-guided DNA binding agent also includes nucleic acids encoding such polypeptides. Exemplary RNA-guided DNA binding agents include Cas cleavase / nickases. Exemplary RNA-guided DNA binding agents can include inactivated forms thereof ("dCas DNA binding agents") (e.g., where these agents have been modified to allow DNA cleavage, e.g., via fusion with a FokI cleavase domain). "Cas nuclease" as used herein encompasses Cas cleavase and Cas nickase. Cas cleavase and Cas nickase include the Csm or Cmr complex of type III CRISPR systems, Cas10, its Csm1 or Cmr2 subunits, the Cascade complex of type I CRISPR systems, its Cas3 subunits, and class 2 Cas nucleases. As used herein, a "Class 2 Cas nuclease" is a single-chain polypeptide with RNA-guided DNA binding activity. Class 2 Cas nucleases include Class 2 Cas cleavase / nickases (e.g., H840A, D10A, or N863A variants), which further have RNA-guided DNA cleavase or nickase activity, and Class 2 dCas DNA binders in which the cleavase / nickase activity has been inactivated (e.g., when these binders are modified to allow DNA cleavage or when they have C→T deaminase or A→G deaminase activity). In some embodiments, the RNA-guided DNA binder comprises a deaminase domain and an RNA-guided DNA nickase (e.g., Cas9 nickase).Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins and modifications thereof. The Cpf1 protein (Zetsche et al., Cell, 163:1-13 (2015)) also contains a RuvC-like nuclease domain. The Cpf1 sequence of Zetsche is incorporated by reference in its entirety. See, e.g., Tables S1 and S3 of Zetsche. See, e.g., Makarova et al., Nat Rev Microbial, 13(11)722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015). As used herein, delivery of an RNA-guided DNA-binding agent (e.g., Cas nuclease, Cas9 nuclease, or S. pyogenes Cas9 nuclease) includes delivery of a polypeptide or mRNA.

[0055] Exemplary nucleotide and polypeptide sequences of Cas9 molecules are provided below. Methods for identifying alternative nucleotide sequences encoding Cas9 polypeptide sequences (including alternative naturally occurring variants) are known in the art. Also contemplated are sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any of the Cas9 nucleic acid sequences, amino acid sequences, or nucleic acid sequences encoding amino acid sequences provided herein. Exemplary open reading frames of Cas9 Exemplary amino acid sequences of Cas9 Exemplary open reading frames of Cas9 Exemplary open reading frames for Cas9 with Hibit tags Exemplary amino acid sequences of Cas9 with Hibit tags

[0056] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to a guide RNA together with an RNA-guided DNA binding agent (e.g., a Cas nuclease, such as a Cas cleavase, a Cas nickase, or a dCas DNA binding agent (e.g., Cas9)). In some embodiments, the guide RNA guides the RNA-guided DNA binding agent (e.g., Cas9) to a target sequence, the guide RNA hybridizes to the target sequence, and the binding agent binds to the target sequence. When the binding agent is a cleavase or nickase, binding can occur after double-stranded or single-stranded DNA cleavage.

[0057] As used herein, a first sequence is considered to "contain a sequence having at least X% identity to" a second sequence if an alignment of the first sequence to the second sequence shows that, overall, X% or more of the positions of the second sequence match the first sequence. For example, the sequence AAGA contains a sequence having 100% identity to the sequence AAG, since the sequence AAGA has a match to all three positions in the second sequence, resulting in 100% identity in the alignment. Differences between RNA and DNA (generally uridine to thymidine exchange or vice versa) and the presence of nucleoside analogs such as modified uridines do not contribute to identity or complementarity between polynucleotides, as long as the related nucleotides (e.g., thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as their complement). Thus, for example, in the sequence 5'-AXG, where X is any modified uridine, such as pseudouridine, N1-methylpseudouridine, or 5-methoxyuridine, it would be considered 100% identical to AUG, since all are perfectly complementary to the same sequence (5'-CAU). Exemplary alignment algorithms include the Smith-Waterman and Needleman-Wunsch algorithms, which are well known in the art. Those skilled in the art will understand which choice of algorithm and parameter settings is appropriate for aligning a given pair of sequences; generally for sequences of similar length and with predicted amino acid identity >50% or nucleotide identity >75%, the Needleman-Wunsch algorithm using default settings of the Needleman-Wunsch algorithm interface provided by the EBI on its web server at www.ebi.ac.uk is generally appropriate.

[0058] As used herein, a first sequence is considered to be "X% complementary" to a second sequence if X% of the bases of the first sequence base pair with the second sequence. For example, the first sequence 5'AAGA3' is 100% complementary to the second sequence 3'TTCT5', and the second sequence is 100% complementary to the first sequence. In some embodiments, the first sequence 5'AAGA3' is 100% complementary to the second sequence 3'TTCTGTGA5', while the second sequence is 50% complementary to the first sequence.

[0059] As used herein, "mRNA" is used herein to refer to a polynucleotide that is entirely or predominantly RNA or modified RNA and contains an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by ribosomes and aminoacylated tRNAs). An mRNA can include a phosphate-sugar backbone that includes ribose residues or analogs thereof (e.g., 2'-methoxyribose residues). In some embodiments, the sugars of the mRNA phosphate-sugar backbone consist essentially of ribose residues, 2'-methoxyribose residues, or a combination thereof.

[0060] As used herein, "indel" refers to an insertion / deletion mutation consisting of multiple nucleotides that are inserted or deleted at the site of a double-stranded break (DSB) in a target nucleic acid.

[0061] As used herein, "target sequence" refers to a nucleic acid sequence in a target gene that has complementarity to a guide sequence of a gRNA. The interaction of the target sequence with the guide sequence causes the RNA-guided DNA binding agent to bind, and potentially (depending on the activity of the agent) nick or cleave, within the target sequence.

[0062] As used herein, "polypeptide" refers to a wild-type or variant protein (e.g., mutant, fragment, fusion, or combination thereof). A variant polypeptide can have at least or about 5%, 10%, 15%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% functional activity of the wild-type polypeptide. In some embodiments, the variant is at least 70%, 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of the wild-type polypeptide. In some embodiments, the variant polypeptide can be a highly active variant. In certain cases, the variant has about 80% to about 120%, about 140%, about 160%, about 180%, about 200%, about 300%, about 400%, about 500%, or more functional activity of the wild-type polypeptide.

[0063] As used herein, a "heterologous gene" refers to a gene that has been introduced into a cell as a foreign source (e.g., a gene inserted into a genomic locus, such as a safe harbor locus, including a TCR gene locus). That is, the introduced gene is heterologous with respect to its insertion site. A polypeptide expressed from such a heterologous gene is referred to as a "heterologous polypeptide." A heterologous gene may be naturally occurring or engineered, wild type or variant. A heterologous gene may include nucleotide sequences other than the sequence encoding the heterologous polypeptide (e.g., an internal ribosome entry site). A heterologous gene may be a gene that is naturally occurring in the genome, either as a wild type or variant (e.g., mutant). For example, a cell contains a gene of interest (as a wild type or variant), but the same gene or a variant thereof can be introduced as a foreign source for expression, for example, at a highly expressed locus. A heterologous gene may also be a gene that is not naturally occurring in the genome, or a gene that expresses a heterologous polypeptide that is not naturally occurring in the genome. "Heterologous gene," "foreign gene," and "transgene" are used interchangeably. In some embodiments, a heterologous gene or transgene comprises a foreign nucleic acid sequence (e.g., a nucleic acid sequence that is not endogenous to a recipient cell). In some embodiments, a heterologous gene or transgene comprises a foreign nucleic acid sequence (e.g., a nucleic acid sequence that does not naturally occur in a recipient cell). For example, a heterologous gene can be heterologous both with respect to its insertion site and with respect to its recipient cell.

[0064] A "safe harbor" locus is a locus in a genome where a gene can be inserted without significant adverse effects on cells. Non-limiting examples of safe harbor loci targeted by nuclease(s) for use herein include AAVS1 (PPP1 R12C), TCR, B2M, and any locus targeted for knockdown as described herein (e.g., TNFA, IFNG, IL17A, and IL6 genomic loci). In some embodiments, insertion into a locus(s) targeted for knockdown (e.g., TRC gene, e.g., TRAC gene) is favorable for allogeneic cells. Other suitable safe harbor loci are known in the art.

[0065] II. Composition A. Engineered T Cells Provided herein are T cells and populations of T cells engineered to include a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and insertion into the cells of a heterologous sequence(s) encoding a regulatory T cell-promoting molecule under the control of a promoter sequence, and compositions and uses thereof. In some embodiments, the regulatory T cell-promoting molecule is selected from IL10, CTLA4, TGFB1, IDO1, ENTPD1, ​​NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, and BACH2.

[0066] In some embodiments, a T cell or population of T cells is engineered to include a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and the insertion into the cells of heterologous sequences encoding two or more regulatory T cell-promoting molecules, each under the control of a promoter sequence. For example, the engineered T cell includes a first heterologous sequence encoding a first regulatory T cell-promoting molecule under the control of a first promoter, and a second heterologous sequence encoding a second regulatory T cell-promoting molecule under the control of a second promoter. The first promoter and the second promoter may be the same promoter or different promoters.

[0067] In some embodiments, a T cell or population of T cells is engineered to include a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and insertion into the cell of a heterologous sequence(s) encoding IL10 under the control of a promoter. In some embodiments, a T cell is engineered to include a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and insertion into the cell of a heterologous sequence(s) encoding CTLA4 under the control of a promoter. In some embodiments, a T cell is engineered to include a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and insertion into the cell of a heterologous sequence encoding IL10 under the control of a promoter, and insertion into the cell of a heterologous sequence(s) encoding CTLA4 under the control of a promoter.

[0068] In some embodiments, a T cell or population of T cells is engineered to contain a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and insertion into the cells of a heterologous sequence(s) encoding a regulatory T cell-promoting molecule under the control of a promoter, and exhibits at least one suppressive activity of a natural regulatory T cell (nTreg) (e.g., suppression of an immune response or biomarker in an in vitro or in vivo assay (e.g., an animal model of GvHD)).

[0069] In some embodiments, the heterologous sequence(s) encoding the regulatory T cell-promoting molecule are incorporated into an expression construct. In some embodiments, the heterologous sequences encoding two or more regulatory T cell-promoting molecules can be incorporated into two or more separate expression constructs. For example, a first heterologous sequence encoding a first regulatory T cell-promoting molecule is provided in a first expression construct, and a second heterologous sequence encoding a second regulatory T cell-promoting molecule is provided in a second separate expression construct. In some embodiments, the expression construct is an episomal expression construct. In some embodiments, the heterologous sequence(s) encoding the regulatory T cell-promoting molecule are inserted into the genome, for example, as a targeted or non-targeted insertion.

[0070] In some embodiments, the sequence(s) encoding the regulatory T cell promoting molecule can be inserted into a site selected from the TCR gene locus, e.g., the TRAC gene locus, the TNF gene locus, the IFNG gene locus, the IL17A gene locus, the IL6 gene locus, the IL2 gene locus, or the adeno-associated virus integration site 1 (AAVS1) locus.

[0071] In some embodiments, a population of engineered T cells includes a modification (e.g., knockdown) of a TNFA sequence by gene editing, e.g., as assessed by sequencing (e.g., NGS), where at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, 80%, 85%, 90%, or 95% of the cells include an insertion, deletion, or substitution of the endogenous TNFA sequence. In some embodiments, expression of TNFA (full-length wild-type protein or mRNA) is reduced by at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, 80%, 85%, 90%, 95%, or below the detection limit of the assay, as compared to a suitable control (e.g., a control in which the TNFA gene is not modified, as quantified by ELISA or flow cytometry). Assays for expression of TNFA protein and mRNA (e.g., in a population of T cells) are known in the art and are provided herein (see Examples 2 and 3). In certain embodiments, knockdown of TNFA results in TNFA levels below 2500 pg / ml by the methods set forth in Examples 2 and 3.

[0072] In some embodiments, a population of engineered T cells includes a modification (e.g., knockdown) of the IFNG sequence by gene editing, e.g., as assessed by sequencing (e.g., NGS), where at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, 80%, 85%, 90%, or 95% of the cells include an insertion, deletion, or substitution of the endogenous IFNG sequence. In some embodiments, expression of IFNG (full-length wild-type protein or mRNA) is reduced by at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, 80%, 85%, 90%, 95%, or below the detection limit of the assay, compared to a suitable control (e.g., a control in which the IFNG gene is not modified, as quantified by ELISA or flow cytometry). Assays for expression of IFNG protein and mRNA (e.g., in a population of T cells) are known in the art and are provided herein (see Examples 2 and 3). In certain embodiments, knockdown of IFNG results in IFNG levels below 300,000 pg / ml by the methods set forth in Examples 2 and 3.

[0073] In some embodiments, the modification that knocks down expression of a gene (eg, TNFA or IFNG) is one or more of an insertion, deletion, or substitution.

[0074] In some embodiments, the engineered T cell or population of T cells includes an insertion, e.g., by gene editing, of a sequence(s) encoding a regulatory T cell promoting molecule, e.g., as assessed by sequencing (e.g., NGS), where at least 30%, 35%, preferably at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells include an insertion of a sequence encoding a regulatory T cell promoting molecule. In some embodiments, the inserted regulatory T cell promoting molecule (e.g., IL10) results in a statistically significant increase in protein or mRNA expression, e.g., as quantified by ELISA or flow cytometry, compared to a suitable control in which the regulatory T cell promoting molecule gene is not inserted. In some embodiments, the engineered T cells include an insertion of sequence(s) encoding IL10 by gene editing, e.g., as assessed by sequencing (e.g., NGS), where at least 30%, 35%, preferably at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells include an insertion or sequence encoding IL10. In some embodiments, the inserted sequence(s) encoding IL10 results in a statistically significant increase in protein or mRNA expression, e.g., in a regulatory T cell promoting molecule, compared to a suitable control. Assays for IL10 protein and mRNA expression, e.g., in a population of T cells, are described herein and known in the art (e.g., ELISA and flow cytometry). In certain embodiments, the level of IL10 is at least 300 pg / ml, as quantified by the methods of Examples 2 and 3.

[0075] In some embodiments, the engineered T cells or population of T cells includes an insertion of sequence(s) encoding CTLA4, e.g., by gene editing, e.g., as assessed by sequencing (e.g., NGS), where at least 30%, 35%, preferably at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells include an insertion or sequence encoding CTLA4. In some embodiments, the inserted sequence(s) encoding CTLA4 results in a statistically significant increase in protein or mRNA expression, e.g., in a regulatory T cell promoting molecule, compared to a suitable control. Assays for CTLA4 protein and mRNA expression, e.g., in a population of T cells, are described herein and known in the art (e.g., ELISA and flow cytometry).

[0076] In some embodiments, the population of T cells includes T cells engineered to include a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and an insertion of a sequence encoding a regulatory T cell-promoting molecule. In some embodiments, at least 40%, 45%, preferably at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the T cells in the population of T cells (e.g., within the detection limits of the assay used) are engineered to include a heterologous regulatory T cell-promoting molecule, e.g., as assessed by sequencing (e.g., NGS). In some embodiments, at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, preferably at least 80%, 85%, 90%, 95%, or 100% of the T cells in a population of T cells are engineered to include a modification (e.g., knockdown) of a sequence(s) encoding TNFA, e.g., as assessed by sequencing (e.g., NGS). In some embodiments, at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the T cells in a population of T cells are engineered to include a modification (e.g., knockdown) of a sequence(s) encoding IFNG, e.g., as assessed by sequencing (e.g., NGS). In some embodiments, at least 40%, 45%, preferably at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the T cells in a population of T cells are engineered to include an insertion of a sequence encoding a regulatory T cell-promoting molecule, e.g., as assessed by sequencing (e.g., NGS). In some embodiments, at least 30%, 35%, preferably at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the T cells in a population of T cells are engineered to include an insertion of a sequence(s) encoding IL10, e.g., as assessed by sequencing (e.g., NGS).In some embodiments, at least 30%, 35%, preferably at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the T cells in a population of T cells are engineered to contain an insertion of sequence(s) encoding CTLA4, e.g., as assessed by sequencing (e.g., NGS).

[0077] In some embodiments, the engineered T cell or population of T cells comprising a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and the insertion of a heterologous sequence(s) encoding a regulatory T cell-promoting molecule under the control of a promoter sequence further comprises a modification of an endogenous nucleic acid sequence encoding interleukin 17A (IL17A), interleukin 6 (IL6), interleukin 2 (IL2), perforin 1 (PRF1), granzyme A (GZMA), or granzyme B (GZMB), which knocks down expression of IL17A, IL6, IL2, PRF1, GZMA, or GZMB, respectively.

[0078] In some embodiments, the T cells or population of T cells are engineered with a gene editing system (e.g., with an RNA-guided DNA binder). In some embodiments, the T cells are engineered with a CRISPR / Cas gene editing system. In some embodiments, the T cells are engineered with a CRISPR / Cas type II gene editing system (e.g., with Cpf1). In some embodiments, the T cells are engineered with a CRISPR / Cas9 gene editing system (e.g., with SpyCas9). Exemplary Cas9 sequences are provided herein.

[0079] In some embodiments, T cells or populations of T cells are engineered with guide RNAs that specifically target sites within the IFNG and TNFA genes to provide knockdown of the IFNG and TNFA genes. Exemplary sequences are shown in Tables 1 and 2. Also shown are the genomic coordinates of the targets of each listed guide sequence.

[0080] In some embodiments, the engineered T cell or population of T cells includes IFNG and TNFA genes knocked down using guide RNAs disclosed herein with RNA-guided DNA binding agents. In some embodiments, disclosed herein are engineered T cells by inducing breaks (e.g., double-strand breaks (DSBs) or single-strand breaks (nicks)) in the IFNG and TNFA genes of the T cell (e.g., using guide RNAs disclosed herein with RNA-guided DNA binding agents (e.g., CRISPR / Cas systems)). The methods can be used in vitro or ex vivo, for example, in the manufacture of cellular products for suppressing immune response(s) (including inflammation and autoimmunity). In some embodiments, the guide RNAs disclosed herein mediate targeted cleavage by an RNA-guided DNA binding agent (e.g., Cas nuclease) at a site described herein in the IFNG gene. In some embodiments, the guide RNAs disclosed herein mediate targeted cleavage by an RNA-guided DNA binding agent (e.g., Cas nuclease) at a site described herein in the TNFA gene. It will be appreciated that in some embodiments, the guide RNA comprises a guide sequence that binds or is capable of binding to the aforementioned regions.

[0081] Provided are engineered T cells or populations of T cells that comprise a genetic modification at a genomic coordinate selected from those listed in Table 1, e.g., cells that comprise an indel or substitution mutation within any listed genomic range in IFNG. Also provided are engineered T cells that comprise a genetic modification at a genomic coordinate selected from those listed in Table 2, e.g., cells that comprise an indel or substitution mutation within any listed genomic range in TNFA. In some embodiments, the engineered T cells comprise a modification in a genomic coordinate region selected from Table 1 and a modification in a genomic coordinate region selected from Table 2.

[0082] In some embodiments, a guide RNA disclosed herein comprises a guide sequence that is 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group of sequences in Table 1 or Table 2. In some embodiments, a guide RNA disclosed herein comprises a guide sequence that is 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group of sequences in Table 1 or Table 2.

[0083] In some embodiments, the guide RNA disclosed herein comprises a guide sequence having at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of sequences that are 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group of sequences in Table 1. In some embodiments, the guide RNA disclosed herein comprises a guide sequence of at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group of sequences in Table 1. In some embodiments, the guide RNA disclosed herein comprises a guide sequence that is 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group of sequences in Table 1. In some embodiments, the guide RNA disclosed herein comprises a guide sequence that is 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group of sequences in Table 1. In some embodiments, the guide RNA disclosed herein comprises a guide sequence that is selected from the group of sequences in Table 1.

[0084] In some embodiments, the guide RNA disclosed herein comprises a guide sequence having at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of sequences that are 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group of sequences in Table 2. In some embodiments, the guide RNA disclosed herein comprises a guide sequence of at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group of sequences in Table 2. In some embodiments, the guide RNA disclosed herein comprises a guide sequence that is 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group of sequences in Table 2. In some embodiments, the guide RNA disclosed herein comprises a guide sequence that is 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group of sequences in Table 2. In some embodiments, the guide RNA disclosed herein comprises a guide sequence that is selected from the group of sequences in Table 2.

[0085] Unless otherwise stated, all genomic coordinates are based on the human reference genome hg38.

[0086] In certain embodiments, a guide RNA comprising a guide sequence targeting IFNG and a guide RNA comprising a guide sequence targeting TNFA are included. [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

[0087] Non-limiting modified guide sequences for TNFA knockdown are shown below (hg38 coordinates chr12:68158001-68158021): mC*mC*mA*GAGCAUCCAAAAGAGUGGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmGmUmGmCmAmCmCmGmAmGmUmCmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 119) (wherein m is a 2'-OMe modified nucleotide / nucleoside residue, * indicates a phosphorothioate bond between residues, and capital letters indicate residues, preferably containing a ribose sugar)

[0088] Non-limiting modified guide sequences for IFNG knockdown are shown below (hg38 coordinates chr6:31576805-31576825): mA*mG*mA*GCUCUUACCUACAACAUGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 120)

[0089] An exemplary modified mock guide is shown below (hg38 coordinates chr1:0-20): mG*mA*mU*CACGUCGGCCGUUGGCGGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 121)

[0090] In some embodiments, disclosed herein are engineered T cells by using guide RNA with an RNA-guided DNA binding agent to introduce or insert a heterologous IL10 nucleic acid into a genomic locus of a T cell or population of T cells, and constructs (e.g., donor constructs or templates) that include a heterologous IL10 nucleic acid (e.g., for generating engineered T cells). In some embodiments, disclosed herein are engineered T cells by using, for example, guide RNA with an RNA-guided DNA binding agent to express a heterologous IL10 into a genomic locus of a T cell or population of T cells, and constructs (e.g., donors) that include a heterologous IL10 nucleic acid. In some embodiments, disclosed herein are engineered T cells by using, for example, guide RNA with an RNA-guided DNA binding agent (e.g., CRISPR / Cas system) to induce a break (e.g., a double-strand break (DSB) or a single-strand break (nick)) in the genome of a T cell or population of T cells to insert an IL10 gene. Also provided are cells and cell populations generated by the methods.

[0091] In some embodiments, disclosed herein are engineered T cells by using guide RNA with an RNA-guided DNA binding agent to introduce or insert a heterologous CTLA4 nucleic acid into a genomic locus of a T cell or population of T cells, and constructs (e.g., donor constructs or templates) that include a heterologous CTLA4 nucleic acid (e.g., for generating engineered T cells). In some embodiments, disclosed herein are engineered T cells by using, for example, guide RNA with an RNA-guided DNA binding agent to express a heterologous CTLA4 into a genomic locus of a T cell or population of T cells, and constructs (e.g., donors) that include a heterologous CTLA4 nucleic acid. In some embodiments, disclosed herein are engineered T cells by using, for example, guide RNA with an RNA-guided DNA binding agent (e.g., CRISPR / Cas system) to induce a break (e.g., a double-strand break (DSB) or a single-strand break (nick)) in the genome of a T cell or population of T cells to insert a CTLA4 gene. Also provided are cells and cell populations generated by the methods.

[0092] In some embodiments, disclosed herein are engineered T cells by using guide RNA with RNA-guided DNA binders to introduce or insert heterologous CTLA4 and IL10 nucleic acids into a genomic locus of a T cell or population of T cells, as well as one or more constructs (e.g., donor constructs or templates) that include heterologous CTLA4 and IL10 nucleic acids (e.g., for generating engineered T cells). In some embodiments, disclosed herein are engineered T cells by using guide RNA with RNA-guided DNA binders to express heterologous CTLA4 and IL10 from a genomic locus of a T cell or population of T cells, as well as one or more constructs (e.g., donor constructs or templates) that include heterologous CTLA4 and IL10 nucleic acids. In some embodiments, disclosed herein are engineered T cells, for example, by using guide RNA in conjunction with an RNA-guided DNA binding agent (e.g., CRISPR / Cas system) to induce breaks (e.g., double-strand breaks (DSBs) or single-strand breaks (nicks)) in the genome of a T cell or population of T cells to insert CTLA4 and IL10 genes. In some embodiments, the guide RNA mediates target-specific cleavage by an RNA-guided DNA binding agent (e.g., Cas nuclease) at a site described herein to insert sequences encoding two or more regulatory T cell-promoting molecules, e.g., IL10 and CTLA4. It will be appreciated that in some embodiments, the guide RNA comprises a guide sequence that binds or is capable of binding to the aforementioned regions. Also provided are cells and cell populations produced by the method.

[0093] Exemplary nucleotide and polypeptide sequences of regulatory T cell promoting molecules are provided below. Methods for identifying alternative nucleotide sequences encoding polypeptide sequences (including alternative naturally occurring variants and non-human homologs) are known in the art. Exemplary IL10 and CTLA4 encoding nucleic acid sequences are provided below. Other suitable IL10 and CTLA4 sequences are also known in the art. See, for example, Gorby et al., Engineered IL-10 variants elicit potent immuno-modulatory activities at therapeutic low ligand doses, BioRxiv (2020) and Xu et al., Affinity and cross-reactivity engineering of CTLA4-Ig to modulate T cell costimulation, J Immunol (2012), the contents and sequences of which are incorporated herein by reference. Methods for identifying alternative IL10 and CTLA4 sequences are also known in the art. See, for example, above. Also contemplated are sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, e.g., by mutation or truncation, to any of the nucleic acid sequences, amino acid sequences, or nucleic acid sequences encoding amino acid sequences described herein. In some embodiments, nucleic acid sequences encoding any of the amino acid sequences provided herein are also provided.

[0094] 1 shows a non-limiting exemplary nucleic acid sequence encoding IL10. Wild type IL10: ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAAC(SEQ ID NO: 122) High affinity IL10 (N36I, N110I, K117N, F129L): (SEQ ID NO:123)

[0095] 1 shows a non-limiting exemplary amino acid sequence of IL10. Wild type IL10: MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 124) High affinity IL10 (N36I, N110I, K117N, F129L): MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGILPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVISLGENLNTLRLRLRRCHRLLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 125)

[0096] 1 shows a non-limiting exemplary nucleic acid sequence encoding CTLA4. Wild type CTLA4: (SEQ ID NO:126) High affinity CTLA4 (Belatacept; Binding domains: A29Y, L104E): ATGGCCTGCTTGGGCTTCCAAAGGCATAAAGCCCAGCTTAATCTTGCTACTCGCACGTGGCCCTGCACATTGCTCTTTTTCCTCCTGTTCATTCCCGTGTTTTGCAAGGCGATGCATGTGGCACAACCTGCCGTCGTTCTGGCATCATCAAGAGGTATTGCTAGCTTCGTTTGTGAGTACGCCTCCCCTGGAAAATACACGGAGGTGCGCGTCACTGTATTGCGGCAAGCCGACAGCCAAGTTACTGAAGTCTGCGCGGCAACGTATATGATGGGCAATGAGCTGACATTCCTTGACGATTCAATCTGCACGGGAACAAGTAGTGGTAACCAGGTGAATCTCACTATTCAAGGTCTGAGAGCCATGGACACCGGCCTCTACATTTGTAAGGTGGAGCTGATGTATCCTCCCCCATATTATGAGGGGATCGGAAATGGGACACAGATATATGTTATTGATCCCGAGCCATGTCCCGATAGTGACTTCCTCTTGTGGATACTTGCCGCTGTGAGCAGTGGTTTGTTTTTTTATTCATTCCTCCTTACGGCAGTATCACTTTCAAAAATGCTCAAGAAGCGAAGTCCTTTGACAACTGGCGTATATGTCAAAATGCCACCAACAGAGCCCGAATGTGAGAAACAGTTCCAGCCGTACTTTATTCCTATAAAC(SEQ ID NO: 127) High affinity CTLA4 (binding domain: A29H): ATGGCCTGCTTGGGCTTCCAAAGGCATAAAGCCCAGCTTAATCTTGCTACTCGCACGTGGCCCTGCACATTGCTCTTTTTCCTCCTGTTCATTCCCGTGTTTTGCAAGGCGATGCATGTGGCACAACCTGCCGTCGTTCTGGCATCATCAAGAGGTATTGCTAGCTTCGTTTGTGAGTACGCCTCCCCTGGAAAACATACGGAGGTGCGCGTCACTGTATTGCGGCAAGCCGACAGCCAAGTTACTGAAGTCTGCGCGGCAACGTATATGATGGGCAATGAGCTGACATTCCTTGACGATTCAATCTGCACGGGAACAAGTAGTGGTAACCAGGTGAATCTCACTATTCAAGGTCTGAGAGCCATGGACACCGGCCTCTACATTTGTAAGGTGGAGCTGATGTATCCTCCCCCATATTATCTGGGGATCGGAAATGGGACACAGATATATGTTATTGATCCCGAGCCATGTCCCGATAGTGACTTCCTCTTGTGGATACTTGCCGCTGTGAGCAGTGGTTTGTTTTTTTATTCATTCCTCCTTACGGCAGTATCACTTTCAAAAATGCTCAAGAAGCGAAGTCCTTTGACAACTGGCGTATATGTCAAAATGCCACCAACAGAGCCCGAATGTGAGAAACAGTTCCAGCCGTACTTTATTCCTATAAAC(SEQ ID NO: 128) High affinity CTLA4 (binding domains: K28H, A29H): (SEQ ID NO:129)

[0097] 1 shows a non-limiting exemplary amino acid sequence of CTLA4. Wild type CTLA4: MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO: 130) High affinity CTLA4 (Belatacept; Binding domains: A29Y, L104E): MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO: 131) High affinity CTLA4 (binding domain: A29H): MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKHTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO: 132) High affinity CTLA4 (binding domains: K28H, A29H): MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGHHTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO: 133)

[0098] In some embodiments, an engineered T cell or population of T cells comprising a modified (e.g., knocked down) endogenous nucleic acid sequence encoding IFNG, a modified (e.g., knocked down) endogenous nucleic acid sequence encoding TNFA, and an insertion into the cells of a heterologous sequence(s) encoding a regulatory T cell-promoting molecule under the control of a promoter sequence exhibits at least one suppressive activity of a naturally occurring regulatory T cell (nTreg) (e.g., suppression of an immune response(s) or biomarker in an in vitro or in vivo assay (e.g., an animal model of GvHD)). In some embodiments, an engineered T cell or population of T cells comprising a modified (e.g., knocked down) endogenous nucleic acid sequence encoding IFNG, a modified (e.g., knocked down) endogenous nucleic acid sequence encoding TNFA, and an insertion into the cells of a heterologous sequence(s) encoding a regulatory T cell-promoting molecule under the control of a promoter sequence exhibits improved suppressive activity (e.g., increased suppression of an immune response or biomarker in an in vitro or in vivo assay (e.g., an animal model of GvHD)) compared to nTreg. For example, in a mouse model of GvHD, mice administered engineered T cells comprising a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and insertion into the cells of a heterologous sequence(s) encoding a regulatory T cell-promoting molecule under the control of a promoter sequence exhibit improved survival compared to controls (e.g., mice administered PBMCs).

[0099] B. Targeting Receptors In some embodiments, the engineered T cell comprising a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and the insertion into the cell of a heterologous sequence(s) encoding a regulatory T cell-promoting molecule under the control of a promoter sequence, further comprises the insertion into the cell of a heterologous sequence(s) encoding a targeting receptor, the sequence(s) encoding the targeting receptor being under the control of a promoter sequence (e.g., an endogenous promoter or a heterologous promoter).

[0100] In some embodiments, the targeting receptor is a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a receptor of a cell surface molecule operably linked through at least a transmembrane domain within an internal signaling domain, which can activate a T cell upon binding of an extracellular receptor portion. In some embodiments, the targeting receptor can be a receptor present on the surface of a cell (e.g., a T cell) that allows the cell to bind to a target site (e.g., a specific cell or tissue within an organism). The targeting receptor does not have to be an antigen receptor, for example, the targeting receptor can be an RGD peptide that can target an integrin. In some embodiments, the targeting receptor targets a molecule selected from the group consisting of MAdCAM-1, TNFA, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), desmoglein 3 (DSG3), and MHC class I HLA-A (HLA-A*02).

[0101] In some embodiments, the engineered T cell comprises a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and the insertion of sequence(s) encoding a regulatory T cell-promoting molecule selected from IL10, CTLA4, TGFB1, IDO1, ENTPD1, ​​NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, and BACH2, and the insertion of sequence(s) encoding a targeting receptor (e.g., a CAR (e.g., a CAR that targets MAdCAM-1)).

[0102] In some embodiments, the engineered T cells comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, an insertion of sequence(s) encoding IL10, and an insertion of sequence(s) encoding a targeting receptor (e.g., a CAR (e.g., a CAR that targets MAdCAM-1)).

[0103] In some embodiments, the engineered T cells comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, an insertion of sequence(s) encoding CTLA4, and an insertion of sequence(s) encoding a targeting receptor (e.g., a CAR (e.g., a CAR that targets MAdCAM-1)).

[0104] In some embodiments, the engineered T cells comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, an insertion of sequence(s) encoding IL10, an insertion of sequence(s) encoding CTLA4, and an insertion of sequence(s) encoding a targeting receptor (e.g., a CAR (e.g., a CAR that targets MAdCAM-1)).

[0105] In some embodiments, the sequence(s) encoding the targeted receptor are incorporated into an expression construct. In some embodiments, the expression construct comprising the sequence(s) encoding the targeted receptor further comprises a sequence(s) encoding a regulatory T cell promoting molecule, e.g., the sequence(s) encoding the targeted receptor and the sequence(s) encoding the regulatory T cell promoting molecule are incorporated into the same expression construct. In some embodiments, the expression construct comprising the sequence(s) encoding the targeted receptor does not further comprise a sequence(s) encoding a regulatory T cell promoting molecule, e.g., the sequence(s) encoding the regulatory T cell promoting molecule are incorporated into a separate expression construct. In some embodiments, the expression construct comprising the sequence(s) encoding the targeted receptor is an episomal expression construct. In some embodiments, the sequence(s) encoding the targeted receptor is inserted into the genome, e.g., as a targeted or non-targeted insertion.

[0106] In some embodiments, the sequence(s) encoding the targeted receptor can be inserted into a site selected from the TCR gene locus, e.g., the TRAC gene locus, the TNF gene locus, the IFNG gene locus, the IL17A gene locus, the IL6 gene locus, the IL2 gene locus, or the adeno-associated virus integration site 1 (AAVS1) locus.

[0107] In some embodiments, the engineered T cells include insertion of a sequence(s) encoding a targeting receptor by gene editing, e.g., as assessed by sequencing (e.g., NGS). In some embodiments, the population of T cells includes T cells that have been engineered to include a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, an insertion of a sequence encoding a regulatory T cell promoting molecule, and an insertion of a sequence(s) encoding a targeting receptor (e.g., CAR). In some embodiments, at least 40%, 45%, preferably at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the T cells in the population of T cells are engineered to include insertion of a sequence(s) encoding a targeting receptor, e.g., as assessed by sequencing (e.g., NGS). It is understood that the T cell population can be enriched for the cell population bearing the target receptor using selection methods known in the art.

[0108] In some embodiments, disclosed herein are engineered T cells by using guide RNA with RNA-guided DNA binding agents to introduce or insert a targeting receptor (e.g., CAR) nucleic acid into a genomic locus of a T cell or population of T cells, and constructs (e.g., donor constructs or templates) that include the targeting receptor (e.g., CAR) nucleic acid (e.g., for generating engineered T cells). In some embodiments, disclosed herein are engineered T cells by using, for example, guide RNA with RNA-guided DNA binding agents to express a targeting receptor (e.g., CAR) into a genomic locus of a T cell or population of T cells, and constructs (e.g., donors) that include a targeting receptor (e.g., CAR). In some embodiments, disclosed herein are engineered T cells by using, for example, guide RNA with RNA-guided DNA binding agents (e.g., CRISPR / Cas systems) to induce a break (e.g., a double-strand break (DSB) or a single-strand break (nick)) in the genome of a T cell or population of T cells to insert a targeting receptor (e.g., CAR). Also provided are cells and cell populations produced by the methods.

[0109] In some embodiments, a targeting receptor (e.g., a CAR) can confer targeting specificity to an engineered T cell that contains the targeting receptor (e.g., a CAR), for example, to a particular cell, tissue, or organ.

[0110] In some embodiments, a targeting receptor (e.g., a CAR) can target the engineered T cells to the gastrointestinal system, e.g., the targeting receptor is a CAR that targets MAdCAM-1 to suppress immune responses in disorders such as inflammatory bowel disease, ulcerative colitis, or Crohn's disease.

[0111] In some embodiments, a targeting receptor (e.g., a CAR) can target the engineered T cells to inflamed tissue, e.g., the targeting receptor is a CAR that targets TNFA to suppress immune responses in disorders such as inflammatory bowel disease, ulcerative colitis, or Crohn's disease.

[0112] In some embodiments, a targeting receptor (e.g., a CAR) can target the engineered T cells to endothelial cells, e.g., the targeting receptor is a CAR that targets CEACAM6 to suppress immune response(s) (including inflammation) in disorders such as Crohn's disease.

[0113] In some embodiments, a targeting receptor (e.g., a CAR) can target the engineered T cells to tissues that contain endothelial cells, for example, the targeting receptor is a CAR that targets VCAM-1 to suppress immune responses in disorders such as Crohn's disease and multiple sclerosis.

[0114] In some embodiments, the CAR is capable of targeting engineered T cells to synovial tissue, e.g., the targeting receptor is a CAR that targets citrullinated vimentin to suppress immune responses in disorders such as rheumatoid arthritis.

[0115] In some embodiments, a targeting receptor (e.g., a CAR) can target the engineered T cells to neural tissue, e.g., the targeting receptor is a CAR that targets MBP, MOG, or PLP1, e.g., to suppress immune responses in disorders such as multiple sclerosis.

[0116] In some embodiments, a targeting receptor (e.g., a CAR) can target the engineered T cells to B cells, for example, the targeting receptor is a CAR that targets CD19 to suppress immune responses in disorders such as multiple sclerosis and systemic lupus erythematosus.

[0117] In some embodiments, a targeting receptor (e.g., a CAR) can target the engineered T cells to B cells, e.g., the targeting receptor is a CAR that targets CD20 to suppress immune responses in disorders such as multiple sclerosis and systemic lupus erythematosus.

[0118] In some embodiments, a targeting receptor (e.g., a CAR) can target the engineered T cells to tissues that contain mature B lymphocytes, e.g., the targeting receptor is a CAR that targets TNFRSF17 to suppress immune responses in disorders such as systemic lupus erythematosus.

[0119] In some embodiments, the targeting receptor (e.g., CAR) targets SCL2A2. In some embodiments, the targeting receptor (e.g., CAR) targets DPP6. In some embodiments, the targeting receptor (e.g., CAR) targets GAD2. In some embodiments, the targeting receptor (e.g., CAR) targets DSG3. In some embodiments, the targeting receptor (e.g., CAR) targets MHC class I HLA-A (HLA-A*02).

[0120] Additional CAR targets (e.g., inflammatory antigens) are known in the art. See, for example, WO2020092057A1, the contents of which are incorporated herein in their entirety. In some embodiments, insertion can be assessed by detecting the amount of protein or mRNA in engineered T cells, a population of engineered T cells, a tissue, a body fluid of interest, or tissue culture medium containing engineered T cells. In some embodiments, insertion by gene editing can be assessed by sequencing, e.g., next generation sequencing (NGS). Assays for protein and mRNA expression of targeted receptors (e.g., CARs) are described herein and known in the art.

[0121] In some embodiments, the engineered T cells or population of T cells do not contain a xenogeneic target receptor.

[0122] CT cell receptor (TCR) In some embodiments, the engineered T cell or population of T cells comprising a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and the insertion into the cell of a heterologous sequence(s) encoding a regulatory T cell-promoting molecule under the control of a promoter sequence, further comprises a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TCR gene sequence(s).

[0123] In some embodiments, the engineered T cell or population of T cells comprising modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, insertion into the cell of heterologous sequence(s) encoding a regulatory T cell-promoting molecule under the control of a promoter sequence, and insertion into the cell of heterologous sequence(s) encoding a targeted receptor further comprises modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TCR gene sequence(s).

[0124] Generally, TCRs are heterodimeric receptor molecules that contain two TCR polypeptide chains, α and β. Suitable α and β genomic sequences or loci to target for knockdown are known in the art. In some embodiments, the engineered T cells contain a modification (e.g., knockdown) of a TCR α chain gene sequence (e.g., TRAC). See, e.g., NCBI Gene ID: 28755; Ensembl: ENSG00000277734 (T cell receptor alpha constant), US2018 / 0362975, and WO2020081613.

[0125] In some embodiments, the engineered T cell or population of T cells comprises a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, an insertion of sequence(s) encoding a regulatory T cell-promoting molecule selected from IL10, CTLA4, TGFB1, IDO1, ENTPD1, ​​NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, and BACH2, and a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TCR gene sequence(s).

[0126] In some embodiments, the engineered T cell or population of T cells comprises a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, an insertion of a sequence(s) encoding IL10 or CTLA4, and a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TCR gene sequence(s).

[0127] In some embodiments, the engineered T cell or population of T cells comprises a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, an insertion of a sequence(s) encoding a regulatory T cell-promoting molecule, and a modification (e.g., knockdown) of an endogenous TCR gene sequence (e.g., a TRAC gene sequence).

[0128] In some embodiments, the engineered T cell or population of T cells comprises a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and

[0129] A sequence(s) encoding a regulatory T cell promoting molecule selected from: These include insertions of IL10, CTLA4, TGFB1, IDO1, ENTPD1, ​​NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, and BACH2, as well as modifications (e.g., knockdown) of endogenous TCR genes (e.g., TRAC gene sequences).

[0130] In some embodiments, the engineered T cell or population of T cells comprises a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, an insertion of a sequence(s) encoding IL10 or CTLA4, and a modification (e.g., knockdown) of a TCR gene (e.g., a TRAC gene sequence).

[0131] In some embodiments, the engineered T cell or population of T cells comprises a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, an insertion of a sequence(s) encoding a regulatory T cell-promoting molecule, and a modification (e.g., knockdown) of an endogenous TCR gene (e.g., a TRAC gene sequence).

[0132] In any of these embodiments, the engineered T cell or population of T cells can further include an insertion of a sequence(s) encoding a targeting receptor (e.g., a CAR, e.g., a CAR that targets MAdCAM-1) as described herein.

[0133] In some embodiments, the engineered T cells or population of T cells comprises a modification (e.g., knockdown) of the TRC gene sequence by gene editing, such that at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, 80%, 85%, 90%, or 95% of the cells comprise an insertion, deletion, or replacement of the endogenous TRC gene sequence, as assessed, for example, by sequencing (e.g., NGS). In some embodiments, TRC is reduced by at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, 80%, 85%, 90%, 95%, or below the detection limit of the assay, as compared to a suitable control (e.g., a control in which the TRC gene is not modified). Assays for TRC protein and mRNA expression are known in the art.

[0134] In some embodiments, the engineered T cell or population of T cells includes the insertion of a sequence(s) encoding a targeted receptor by gene editing, e.g., as assessed by sequencing (e.g., NGS).

[0135] In some embodiments, the population of T cells includes T cells that have been engineered to include a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, an insertion of a sequence encoding a regulatory T cell promoting molecule, and a modification (e.g., knockdown) of at least one TCR gene sequence. In some embodiments, at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the T cells in the population of T cells are engineered to include a modification (e.g., knockdown) of at least one TCR gene sequence, e.g., as assessed by sequencing (e.g., NGS).

[0136] In some embodiments, a guide RNA that specifically targets a site within a TCR gene (e.g., the TRAC gene) is used to provide modification (e.g., knockdown) of the TCR gene.

[0137] In some embodiments, the TCR gene is modified (e.g., knocked down) in a T cell using a guide RNA in conjunction with an RNA-guided DNA binding agent. In some embodiments, disclosed herein are T cells engineered by, for example, using a guide RNA in conjunction with an RNA-guided DNA binding agent (e.g., a CRISPR / Cas system) to induce a break (e.g., a double-strand break (DSB) or a single-strand break (nick)) in the TCR gene of the T cell. The methods can be used in vitro or ex vivo, for example, in the manufacture of a cellular product for suppressing an immune response.

[0138] In some embodiments, the guide RNA mediates target-specific cleavage by an RNA-guided DNA-binding agent (e.g., a Cas nuclease) at a site described herein within the TCR gene. It will be appreciated that in some embodiments, the guide RNA comprises a guide sequence that binds or is capable of binding to the aforementioned region.

[0139] D. Guide RNA In any embodiment herein, the guide RNA can further comprise a trRNA. In each of the composition and method embodiments described herein, the crRNA and trRNA can be associated on a single RNA (sgRNA) or on separate RNAs (dgRNA). In the context of an sgRNA, the components of the crRNA and trRNA can be covalently linked, for example, via a phosphodiester bond or other covalent bond. In some embodiments, the sgRNA includes one or more bonds between nucleotides that are not phosphodiester bonds.

[0140] In each of the composition, use, and method embodiments described herein, the guide RNA can comprise two RNA molecules as a "dual guide RNA" or "dgRNA." The dgRNA comprises a first RNA molecule comprising a crRNA, e.g., comprising a guide sequence as provided herein, and a second RNA molecule comprising a trRNA. The first and second RNA molecules may not be covalently linked, but can form an RNA duplex via base pairing between a portion of the crRNA and a portion of the trRNA.

[0141] In each of the composition, use, and method embodiments described herein, the guide RNA can comprise a single RNA molecule, referred to as a "single guide RNA" or "sgRNA." The sgRNA can comprise a crRNA (or a portion thereof) comprising a guide sequence as set forth herein covalently linked to a trRNA. The sgRNA can comprise 15, 16, 17, 18, 19, or 20 consecutive nucleotides of a guide sequence as set forth herein. In some embodiments, the crRNA and the trRNA are covalently linked via a linker. In some embodiments, the sgRNA forms a stem-loop structure via base pairing between a portion of the crRNA and a portion of the trRNA. In some embodiments, the crRNA and the trRNA are covalently linked via one or more bonds that are not phosphodiester bonds.

[0142] In some embodiments, the trRNA can include all or part of the trRNA sequence from a naturally occurring CRISPR / Cas system. In some embodiments, the trRNA includes a truncated or modified wild-type trRNA. The length of the trRNA depends on the CRISPR / Cas system used. In some embodiments, the trRNA includes or consists of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 nucleotides. In some embodiments, the trRNA can include a certain secondary structure, such as one or more hairpin or stem-loop structures, or one or more bulge structures.

[0143] In some embodiments, the target sequence or region may be complementary to the guide sequence of the guide RNA. In some embodiments, the degree of complementarity or identity between the guide sequence of the guide RNA and its corresponding target sequence may be 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the target sequence and the guide sequence of the gRNA may be 100% complementary or identical. In other embodiments, the target sequence and the guide sequence of the gRNA may include one mismatch. For example, the target sequence and the guide sequence of the gRNA may include 1, 2, 3, 4, or 5 mismatches, and the total length of the guide sequence is about 20 or 20. In some embodiments, the target sequence and the guide sequence of the gRNA may include 1 to 4 mismatches, when the guide sequence is about 20 or 20 nucleotides.

[0144] In any embodiment herein, each of the guide sequences herein can further comprise additional nucleotides to form a crRNA or guide RNA, such as the following exemplary nucleotide sequence (5' to 3' direction) following the 3' end of the guide sequence: GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 134). In the case of an sgRNA, the above guide sequences can further comprise additional nucleotides to form an sgRNA, such as the following exemplary nucleotide sequence (5' to 3' direction) following the 3' end of the guide sequence: GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 134). It may further include GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 135) or GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 136).

[0145] In any embodiment, the guide RNA disclosed herein binds to a region upstream of a protospacer adjacent motif (PAM). As will be understood by those skilled in the art, the PAM sequence occurs on the opposite strand to the strand containing the target sequence and varies depending on the CRISPR / Cas system. That is, the PAM sequence is on the complementary strand of the target strand (the strand containing the target sequence to which the guide RNA binds). In some embodiments, the PAM is selected from the group consisting of NGG, NNGRRT, NNGRR(N), NNAGAAW, NNNNG(A / C)TT, and NNNNRYAC.

[0146] In some embodiments, the guide RNA sequences provided herein are complementary to sequences adjacent to the PAM sequence.

[0147] In some embodiments, the guide RNA sequence comprises a sequence complementary to a sequence within a genomic region selected from the table herein according to the coordinates of the human reference genome hg38. In some embodiments, the guide RNA sequence comprises a sequence complementary to a sequence comprising 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides from within a genomic region selected from the table herein. In some embodiments, the guide RNA sequence comprises a sequence complementary to a sequence comprising 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides spanning a genomic region selected from the table herein.

[0148] The guide RNAs disclosed herein mediate target-specific cleavage resulting in a double-stranded break (DSB). The guide RNAs disclosed herein mediate target-specific cleavage resulting in a single-stranded break (SSB or nick).

[0149] E. Chemically modified gRNA In any embodiment herein, the gRNA can be chemically modified. A gRNA that includes one or more modified nucleosides or nucleotides is referred to as a "modified" gRNA or a "chemically modified" gRNA to account for the presence of one or more non-natural or naturally occurring components or structures used in place of or in addition to the canonical A, G, C, and U residues. In some embodiments, a modified gRNA is synthesized with non-canonical nucleosides or nucleotides, referred to herein as "modified." Modified nucleosides and nucleotides can include one or more of: (i) alteration (e.g., substitution) of one or both of the non-linked phosphate oxygens in the phosphodiester backbone linkages or one or both of the linked phosphate oxygens (exemplary backbone modifications); (ii) alteration (e.g., substitution) of a component of the ribose sugar, for example, of the 2' hydroxyl on the ribose sugar (exemplary sugar modifications); (iii) wholesale replacement of the phosphate moiety with a "dephosphorylated" linker (exemplary backbone modifications); (iv) modification or substitution of a naturally occurring nucleobase (including non-canonical nucleobases) (exemplary base modifications); (v) substitution or modification of the ribose-phosphate backbone (exemplary backbone modifications); (vi) modification of the 3' or 5' end of the oligonucleotide, for example, removal, modification, or substitution of a terminal phosphate group, or conjugation of a moiety, cap, or linker (such 3' or 5' cap modifications can include sugar or backbone modifications); and (vii) modification or replacement of the sugar (exemplary sugar modifications).

[0150] The chemical modifications listed above can be combined to obtain modified gRNAs that contain nucleosides and nucleotides (collectively "residues") that can have two, three, four, or more modifications. For example, modified residues can have modified sugars and modified nucleobases. In some embodiments, every base of the gRNA is modified, e.g., all bases have modified phosphate groups, such as phosphorothioate groups. In certain embodiments, all, or substantially all, of the phosphate groups of the gRNA molecule are replaced with phosphorothioate groups. In some embodiments, the modified gRNA contains at least one modified residue at or near the 5' end of the RNA. In some embodiments, the modified gRNA contains at least one modified residue at or near the 3' end of the RNA. Certain gRNAs contain at least one modified residue at or near the 5' and 3' ends of the RNA.

[0151] In some embodiments, the guide RNA disclosed herein comprises one of the modification patterns disclosed in WO2018 / 107028 (the contents of which are incorporated herein by reference in relevant parts). In some embodiments, the guide RNA disclosed herein comprises one of the structure / modification patterns disclosed in US20170114334 (which are incorporated herein by reference). In some embodiments, the guide RNA disclosed herein comprises one of the structure / modification patterns disclosed in WO2017 / 136794, WO2017004279, US2018187186, US2019048338 (which are incorporated herein by reference).

[0152] F. mRNA encoding an RNA-guided DNA binder In some embodiments, the cell or method comprises an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA-binding agent (e.g., a Cas nuclease as described herein). The Cas9 ORF is provided herein and known in the art. As an example, the Cas9 ORF can be codon-optimized such that the coding sequence comprises one or more alternative codons for one or more amino acids. As used herein, "alternative codons" refers to variations in codon usage for a given amino acid, which may or may not be preferred or optimized codons (codon-optimized) for a given expression system. Preferred codon usage, or codons that are well tolerated in a given expression system, are known in the art. The Cas9 coding sequences, Cas9 mRNA, and Cas9 protein sequences of WO2013 / 176772, WO2014 / 065596, WO2016 / 106121, and WO2019 / 067910 are incorporated herein by reference. In particular, the ORF and Cas9 amino acid sequence in the table in paragraph

[0449] of WO2019 / 067910, and the Cas9 mRNA and ORF in paragraphs

[0214] to

[0234] of WO2019 / 067910 are incorporated by reference in this specification.

[0153] In some embodiments, the modified ORF can include modified uridines at at least one, more than one, or all uridine positions. In some embodiments, the modified uridine is a uridine modified at the 5 position, e.g., with a halogen, methyl, or ethyl. In some embodiments, the modified uridine is a pseudouridine modified at the 1 position, e.g., with a halogen, methyl, or ethyl. The modified uridine can be, for example, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine, or a combination thereof. In some embodiments, the modified uridine is 5-methoxyuridine. In some embodiments, the modified uridine is 5-iodouridine. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of N1-methylpseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5-methoxyuridine.

[0154] In some embodiments, the mRNA disclosed herein comprises a 5' cap (e.g., Cap0, Cap1, or Cap2). The 5' cap is generally a 7-methylguanine ribonucleotide (which may be further modified, e.g., ARCA (anti-reverse cap analog; Thermo Fisher Scientific, catalog number AM8045) is a cap analog containing 7-methylguanine 3'-methoxy-5'-triphosphate attached to the 5' position of a guanine ribonucleotide) attached via a 5'-triphosphate to the 5' position of the first nucleotide of the 5'→3' strand of the mRNA (i.e., the first cap-proximal nucleotide). In Cap0, the ribose of the first and second cap-proximal nucleotides of the mRNA both comprise a 2'-hydroxyl. In Cap1, the ribose of the first and second transcribed nucleotides of the mRNA comprise a 2'-methoxy and a 2'-hydroxyl, respectively. See, e.g., CleanCap™ AG (m7G(5')ppp(5')(2'OMeA)pG; TriLink Biotechnologies, Catalog No. N-7113) or CleanCap™ GG (m7G(5')ppp(5')(2'OMeG)pG; TriLink Biotechnologies, Catalog No. N-7133). In Cap2, the riboses of the first and second cap-proximal nucleotides of the mRNA both contain 2'-methoxy. See, e.g., Katibah et al. (2014) Proc Natl Acad Sci USA 111(33):12025-30; Abbas et al. (2017) Proc Natl Acad Sci USA 114(11):E2106-E2115.

[0155] In some embodiments, the mRNA further comprises a polyadenylation (polyA) tail. In some embodiments, the polyA tail comprises 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines (SEQ ID NO: 147), optionally up to 300 adenines (SEQ ID NO: 148). In some embodiments, the polyA tail comprises 95, 96, 97, 98, 99, or 100 adenine nucleotides (SEQ ID NO: 149).

[0156] G. Manipulation of T Cells The engineered cells provided herein are prepared from a cell population enriched for CD4+ T cells. Such cells can be readily obtained, for example, from fresh leukopak samples, which are commercially available from a variety of sources, including StemCell Technologies. CD4+ T cells can be routinely isolated using commercially available kits, for example, by negative selection using a human CD4+ T cell isolation kit. However, methods for preparing CD4+ T cells from other sources are also known in the art. For example, pluripotent cells, such as hematopoietic stem cells (HSCs, e.g., isolated from bone marrow or umbilical cord blood), hematopoietic progenitor cells (e.g., lymphoid progenitor cells), or mesenchymal stem cells (MSCs), can be used to obtain CD4+ T cells. Pluripotent cells can develop into multiple cell types, but the breadth of differentiation is more limited than that of pluripotent cells. Pluripotent cells can be derived from established cell lines or isolated from human bone marrow or umbilical cord. For example, HSCs can be isolated from patients or healthy donors following G-CSF-induced mobilization, plerixafor-induced mobilization, or a combination thereof. To separate HSCs from blood or bone marrow, cells in blood or bone marrow can be screened by antibodies that bind to unwanted cells (e.g., antibodies against CD4 and CD8 (T cells), CD45 (B cells), GR-I (granulocytes), and Iad (differentiated antigen-presenting cells)) (see, for example, Inaba, et al. (1992) J Exp Med. 176:1693-1702). Methods for promoting differentiation into CD4+ T cells are known in the art.

[0157] III. Method of Delivery The guide RNA, RNA-guided DNA binding agent (e.g., Cas nuclease), and nucleic acid sequences disclosed herein can be delivered in vitro or ex vivo using various known suitable methods available in the art to a cell or population of cells that includes a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and an insertion of a sequence(s) encoding a regulatory T cell promoting molecule (e.g., IL10, CTLA4), and optionally further includes an insertion of a sequence(s) encoding a targeting receptor (e.g., CAR), and optionally further includes a modification (e.g., knockdown) of a TCR sequence(s). The guide RNA, RNA-guided DNA binding agent, and nucleic acid construct can be delivered individually or together in any combination, using the same or different delivery methods as needed.

[0158] Conventional viral and non-viral based gene delivery methods can be used to introduce guide RNA and RNA-guided DNA binders and donor constructs into cells (e.g., mammalian cells) and target tissues. As further provided herein, non-viral vector delivery systems include nucleic acids, such as non-viral vectors, plasmid vectors, and, for example, naked nucleic acids, as well as nucleic acids complexed with delivery vehicles (e.g., liposomes, lipid nanoparticles (LNPs), or poloxamers). Viral vector delivery systems include DNA and RNA viruses.

[0159] Methods and compositions for non-viral delivery of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, LNPs, polycation or lipid:nucleic acid conjugates, naked nucleic acids (e.g., naked DNA / RNA), artificial virions, and drug-enhanced uptake of DNA. Sonoporation, for example, using the Sonitron 2000 system (Rich-Mar), can also be used to deliver nucleic acids.

[0160] Various delivery systems (e.g., vectors, liposomes, LNPs) including guide RNA, RNA-guided DNA binding agents, and donor constructs, alone or in combination, can also be administered to cells or cell cultures ex vivo. Administration can be by any route that is normally used to introduce molecules into blood, body fluids, or ultimately into contact with cells, including, but not limited to, injection, infusion, topical application, and electroporation. Suitable methods for administering such nucleic acids are available and are well known to those skilled in the art.

[0161] In certain embodiments, the disclosure provides DNA or RNA vectors encoding any of the compositions disclosed herein, e.g., a guide RNA comprising any one or more of the guide sequences described herein (e.g., for modifying (e.g., knocking down) IFNG and TNFA), or a donor construct comprising a sequence(s) encoding a regulatory T cell promoting molecule (e.g., IL10), or a targeting receptor (e.g., a CAR, e.g., a MAdCAM-1 CAR). In some embodiments, the vector also comprises a sequence encoding an RNA-guided DNA binder. In certain embodiments, the invention includes DNA or RNA vectors encoding any one or more of the compositions described herein, or in any combination. In some embodiments, the vector further comprises, e.g., a promoter, enhancer, and regulatory sequences. In some embodiments, a vector comprising a guide RNA comprising any one or more of the guide sequences described herein also comprises one or more nucleotide sequence(s) encoding a crRNA, a trRNA, or a crRNA and a trRNA, as disclosed herein.

[0162] In certain embodiments, the present disclosure provides DNA or RNA vectors encoding regulatory T cell promoting molecules and targeting receptors. Such vectors allow for the selection of cells based on the presence of receptors for cells that also contain coding sequences for regulatory T cell promoting molecules. Positive and negative selection based on the presence of cell surface molecules are known in the art.

[0163] In some embodiments, the vector comprises a nucleotide sequence encoding a guide RNA as described herein. In some embodiments, the vector comprises one copy of the guide RNA. In other embodiments, the vector comprises multiple copies of the guide RNA. In embodiments with multiple guide RNAs, the guide RNAs may be non-identical in that they target different target sequences, or may be identical in that they target the same target sequence. In some embodiments where the vector comprises multiple guide RNAs, each guide RNA may have other distinct properties (e.g., activity or stability) in a complex with an RNA-guided DNA nuclease (e.g., Cas RNP complex). In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to at least one transcriptional or translational control sequence (e.g., promoter, 3'UTR, or 5'UTR). In one embodiment, the promoter is a tRNA promoter (e.g., tRNA Lys3) or tRNA chimeras. See Mefferd et al., RNA. 2015 21:1683-9; Scherer et al., Nucleic Acids Res. 2007 35:2620-2628. In some embodiments, the promoter may be recognized by RNA polymerase III (Pol III). Non-limiting examples of Pol III promoters include the U6 promoter and the H1 promoter. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human U6 promoter. In other embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human H1 promoter. In embodiments using multiple guide RNAs, the promoters used to drive expression may be the same or different. In some embodiments, the nucleotides encoding the crRNA of the guide RNA and the nucleotides encoding the trRNA of the guide RNA may be provided on the same vector. In some embodiments, the nucleotides encoding the crRNA and the trRNA may be driven by the same promoter. In some embodiments, the crRNA and the trRNA may be transcribed into a single transcript. For example, the crRNA and trRNA can be processed from a single transcript to form a dual molecule guide RNA. Alternatively, the crRNA and trRNA can be transcribed into a single molecule guide RNA (sgRNA). In other embodiments, the crRNA and trRNA can be driven by corresponding promoters on the same vector. In yet other embodiments, the crRNA and trRNA can be encoded by different vectors.

[0164] In some embodiments, the nucleotide sequence encoding the guide RNA may be located on the same vector that contains the nucleotide sequence encoding the RNA-guided DNA-binding agent, such as a Cas protein. In some embodiments, the expression of the guide RNA and the expression of the RNA-guided DNA-binding agent, such as a Cas protein, may be driven by their respective promoters. In some embodiments, the expression of the guide RNA may be driven by the same promoter that drives the expression of the RNA-guided DNA-binding agent, such as a Cas protein. In some embodiments, the guide RNA and the RNA-guided DNA-binding agent, such as a Cas protein transcript, may be contained within a single transcript. For example, the guide RNA may be present within the untranslated region (UTR) of the RNA-guided DNA-binding agent, such as a Cas protein transcript. In some embodiments, the guide RNA may be within the 5'UTR of the transcript. In other embodiments, the guide RNA may be within the 3'UTR of the transcript. In some embodiments, the intracellular half-life of the transcript may be reduced by accommodating the guide RNA within the 3'UTR of the transcript to shorten the length of the 3'UTR. In additional embodiments, the guide RNA may be within an intron of the transcript. In some embodiments, suitable splice sites can be added to the intron within which the guide RNA is located to ensure that the guide RNA is properly spliced ​​out of the transcript. In some embodiments, close temporal proximity expression of the RNA-guided DNA-binding agent (e.g., Cas protein) and the guide RNA from the same vector can promote more efficient formation of the CRISPR RNP complex.

[0165] In some embodiments, the nucleotide sequence encoding the guide RNA or RNA-guided DNA binding agent can be located on the same vector that includes a construct comprising a sequence encoding a regulatory T cell promoting molecule (e.g., IL10, CTLA4) or a targeting receptor (e.g., a CAR, e.g., a MAdCAM-1 CAR). In some embodiments, the proximity of a construct comprising a sequence encoding a regulatory T cell promoting molecule (e.g., IL10, CTLA4) or a targeting receptor (e.g., a CAR, e.g., a MAdCAM-1 CAR) and a guide RNA (or RNA-guided DNA binding agent) on the same vector can more efficiently facilitate insertion of the construct into the insertion site created by the guide RNA / RNA-guided DNA binding agent.

[0166] In certain embodiments, DNA and RNA vectors can contain multiple open reading frames for expression under a single promoter present either within the vector or at the genome insertion site.In such embodiments, a coding sequence for a self-cleaving peptide can be included between the open reading frames.The self-cleaving peptide can be, for example, a 2A peptide, such as a P2A peptide, an E2A peptide, an F2A peptide, or a T2A peptide.

[0167] In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding the sgRNA and an mRNA encoding the RNA-guided DNA binder, which may be a Cas protein, e.g., Cas9 or Cpf1. In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding the crRNA, trRNA and an mRNA encoding the RNA-guided DNA binder, which may be a Cas protein, e.g., Cas9 or Cpf1. In one embodiment, the Cas9 is from Streptococcus pyogenes (i.e., Spy Cas9). In some embodiments, the nucleotide sequence encoding the crRNA, trRNA, or the crRNA and trRNA (which may be an sgRNA) comprises or consists of a guide sequence flanked by all or a portion of a repeat sequence from a naturally occurring CRISPR / Cas system. The nucleic acid comprising or consisting of the crRNA, trRNA, or the crRNA and trRNA can further comprise a vector sequence comprising or consisting of a nucleic acid not found in nature with the crRNA, trRNA, or the crRNA and trRNA.

[0168] In some embodiments, the crRNA and trRNA are encoded by non-contiguous nucleic acids within one vector. In other embodiments, the crRNA and trRNA may be encoded by contiguous nucleic acids. In some embodiments, the crRNA and trRNA are encoded by opposing strands of a single nucleic acid. In other embodiments, the crRNA and trRNA are encoded by the same strand of a single nucleic acid.

[0169] In some embodiments, the vector comprises a donor construct comprising a sequence encoding a regulatory T cell promoting molecule (e.g., IL10) or a targeting receptor (e.g., a CAR, e.g., MAdCAM-1) as disclosed herein. In some embodiments, in addition to the donor construct disclosed herein, the vector can further comprise a nucleic acid encoding a guide RNA as described herein, or a nucleic acid encoding an RNA-guided DNA binder (e.g., a Cas nuclease such as Cas9). In some embodiments, the nucleic acid encoding the RNA-guided DNA binder is each or both present on a vector separate from the vector comprising the donor construct disclosed herein. In any embodiment, the vector can comprise other sequences, including but not limited to promoters, enhancers, regulatory sequences, as described herein. In some embodiments, the promoter does not drive expression of the regulatory T cell promoting molecule (e.g., IL10) or targeting receptor (e.g., a CAR, e.g., MAdCAM-1) of the donor construct. In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding a crRNA, a trRNA, or a crRNA and a trRNA. In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding the sgRNA and an mRNA encoding an RNA-guided DNA nuclease, which may be a Cas nuclease (e.g., Cas9). In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding the crRNA, trRNA and an mRNA encoding an RNA-guided DNA nuclease, which may be a Cas nuclease (e.g., Cas9). In some embodiments, the Cas9 is from Streptococcus pyogenes (i.e., Spy Cas9). In some embodiments, the nucleotide sequence encoding the crRNA, trRNA, or the crRNA and trRNA (which may be an sgRNA) comprises or consists of a guide sequence flanked by all or a portion of a repeat sequence from a naturally occurring CRISPR / Cas system.A nucleic acid comprising or consisting of a crRNA, a trRNA, or a crRNA and a trRNA can further comprise a vector sequence comprising or consisting of a nucleic acid not found in nature with the crRNA, the trRNA, or the crRNA and a trRNA.

[0170] In some embodiments, the vector can be circular. In other embodiments, the vector can be linear. In some embodiments, the vector can be encapsulated in a lipid nanoparticle, a liposome, a non-lipid nanoparticle, or a viral capsid. Non-limiting examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes, mini-chromosomes, transposons, viral vectors, and expression vectors.

[0171] In some embodiments, the vector can be a viral vector. In some embodiments, the viral vector can be genetically modified from its wild-type counterpart. For example, the viral vector can include one or more nucleotide insertions, deletions, or substitutions to facilitate cloning or to alter one or more properties of the vector. Such properties can include packaging ability, transduction efficiency, immunogenicity, genomic integration, replication, transcription, and translation. In some embodiments, portions of the viral genome can be deleted to allow the virus to package foreign sequences of large size. In some embodiments, the viral vector can have enhanced transduction efficiency. In some embodiments, the immune response elicited by the virus in the host can be reduced. In some embodiments, a viral gene that facilitates integration of viral sequences into the genome (e.g., integrase) can be mutated to render the virus non-integrating. In some embodiments, the viral vector can be replication-defective. In some embodiments, the viral vector can include foreign transcriptional or translational control sequences to drive expression of coding sequences on the vector. In some embodiments, the virus can be helper-dependent. For example, a virus may require one or more helper viruses to provide viral components (e.g., viral proteins) required for amplifying and packaging a vector into a viral particle. In such cases, one or more helper components (including one or more vectors encoding viral components) can be introduced into a cell or population of cells along with the vector systems described herein. In other embodiments, the virus can be helper-free. For example, the virus may be capable of amplifying and packaging a vector without a helper virus. In some embodiments, the vector systems described herein can also encode viral components required for amplifying and packaging the virus.

[0172] Non-limiting examples of viral vectors include adeno-associated viral (AAV) vectors, lentiviral vectors, adenoviral vectors, helper-dependent adenoviral vectors (HDAd), herpes simplex viral (HSV-1) vectors, bacteriophage T4, baculoviral vectors, and retroviral vectors. In some embodiments, the viral vector can be an AAV vector. In other embodiments, the viral vector can be a lentiviral vector.

[0173] In some embodiments, "AAV" refers to all serotypes, subtypes, and naturally occurring AAVs as well as recombinant AAVs. "AAV" can be used to refer to the virus itself or its derivatives. The term "AAV" includes AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAVrh.64R1, AAVhu.37, AAVrh.8, AAVrh.32.33, AAV8, AAV9, AAV-DJ, AAV2 / 8, AAVrh10, AAVLK03, AV10, AAV11, AAV12, rh10, and hybrids thereof, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. The genomic sequences of various serotypes of AAV, as well as the sequences of native terminal repeats (TRs), Rep proteins, and capsid subunits, are known in the art. Such sequences can be found in the literature and in public databases such as GenBank. As used herein, "AAV vector" refers to an AAV vector that contains heterologous sequences that are not derived from AAV (i.e., nucleic acid sequences that are heterologous to AAV), and typically contains sequences that code for heterologous polypeptides of interest. The construct can include AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAVrh.64R1, AAVhu.37, AAVrh.8, AAVrh.32.33, AAV8, AAV9, AAV-DJ, AAV2 / 8, AAVrh10, AAVLK03, AV10, AAV11, AAV12, rh10, and hybrids thereof, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV capsid sequences. In general, the heterologous nucleic acid sequence (transgene) is flanked by at least one, and generally two, AAV inverted terminal repeats (ITRs). AAV vectors can be either single-stranded (ssAAV) or self-complementary (scAAV).

[0174] In some embodiments, the lentivirus can be integrative. In some embodiments, the lentivirus can be non-integrative. In some embodiments, the viral vector can be an adenovirus vector. In some embodiments, the adenovirus can be a high cloning capacity or "gutless" adenovirus, in which all viral coding regions except the 5' and 3' inverted terminal repeats (ITRs) and the packaging signal ("I") are deleted from the virus to increase packaging capacity. In yet other embodiments, the viral vector can be an HSV-1 vector. In some embodiments, the HSV-1 based vector is helper dependent, while in other embodiments, the vector is helper independent. For example, an amplicon vector that retains only the packaging sequence requires a helper virus that has structural components for packaging, while a 30 kb deleted HSV-1 vector that removes non-essential viral functions does not require a helper virus. In additional embodiments, the viral vector can be bacteriophage T4. In some embodiments, bacteriophage T4 may be capable of packaging any linear or circular DNA or RNA molecule when the head of the virus is emptied. In further embodiments, the viral vector may be a baculovirus vector. In yet further embodiments, the viral vector may be a retrovirus vector. In embodiments using AAV or other vectors with relatively small cloning capacity, the use of two or more vectors may be required to deliver all components of the vector system as disclosed herein. For example, one AAV vector may contain a sequence encoding an RNA-guided DNA-binding agent such as a Cas protein (e.g., Cas9), while a second AAV vector may contain one or more guide sequences.

[0175] In some embodiments, the vector may be capable of driving expression of one or more nuclease components in a cell. In some embodiments, the vector does not contain a promoter that drives expression of one or more coding sequences when integrated into a cell (e.g., uses the cell's endogenous promoter when inserted into a specific genomic locus of the cell, as exemplified herein). Promoters suitable for driving expression in different types of cells (e.g., CD4+ T cells) are known in the art. In some embodiments, the promoter may be wild type. In other embodiments, the promoter may be modified for more efficient or effective expression. In yet other embodiments, the promoter may be truncated and still retain its function. For example, the promoter may have a normal size or a reduced size suitable for proper packaging of the vector into a virus.

[0176] In some embodiments, the vector can include a nucleotide sequence encoding an RNA-guided DNA binder, such as a Cas protein (e.g., Cas9) described herein. In some embodiments, the nuclease encoded by the vector can be a Cas protein. In some embodiments, the vector system can include one copy of the nucleotide sequence encoding the nuclease. In other embodiments, the vector system can include two or more copies of the nucleotide sequence encoding the nuclease. In some embodiments, the nucleotide sequence encoding the nuclease can be operably linked to at least one transcriptional or translational control sequence. In some embodiments, the nucleotide sequence encoding the nuclease can be operably linked to at least one promoter.

[0177] In some embodiments, the vector can include any one or more of the constructs comprising sequences encoding a regulatory T cell promoting molecule (e.g., IL10, CTLA4) or a targeting receptor (e.g., a CAR, e.g., a MAdCAM-1 CAR) as described herein. In some embodiments, the sequence of the regulatory T cell promoting molecule (e.g., IL10, CTLA4) or a targeting receptor (e.g., a CAR, e.g., a MAdCAM-1 CAR) can be operably linked to at least one transcriptional or translational control sequence. In some embodiments, the sequence of the regulatory T cell promoting molecule (e.g., IL10, CTLA4) or a targeting receptor (e.g., a CAR, e.g., a MAdCAM-1 CAR) can be operably linked to at least one promoter. In some embodiments, the sequence of the regulatory T cell promoting molecule (e.g., IL10, CTLA4) or a targeting receptor (e.g., a CAR, e.g., a MAdCAM-1 CAR) is not linked to a promoter driving expression of a heterologous gene.

[0178] In some embodiments, the promoter may be constitutive, inducible, or tissue-specific. In some embodiments, the promoter may be a constitutive promoter. Non-limiting examples of constitutive promoters include cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late promoter (MLP), Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor alpha (EF1a) promoter, ubiquitin promoter, actin promoter, tubulin promoter, immunoglobulin promoter, functional fragments thereof, or any combination of the foregoing. In some embodiments, the promoter may be a CMV promoter. In some embodiments, the promoter may be a truncated CMV promoter. In other embodiments, the promoter may be an EF1a promoter. In some embodiments, the promoter may be an inducible promoter. Non-limiting examples of inducible promoters include promoters inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter can be a promoter with a low basal (uninduced) expression level, such as the Tet-On® promoter (Clontech).

[0179] In some embodiments, the promoter can be a tissue-specific promoter, for example, a promoter specific for expression in T cells.

[0180] In some embodiments, the composition comprises a vector system. In some embodiments, the vector system can comprise one single vector. In other embodiments, the vector system can comprise two vectors. In additional embodiments, the vector system can comprise three vectors. When different guide RNAs are used for multiplexing, or when multiple copies of a guide RNA are used, the vector system can comprise four or more vectors.

[0181] In some embodiments, the vector system can include an inducible promoter to initiate expression only after delivery to a target cell. Non-limiting examples of inducible promoters include promoters that can be induced by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter can be a promoter with a low basal (uninduced) expression level, such as the Tet-On® promoter (Clontech).

[0182] In additional embodiments, the vector system can include a tissue-specific promoter.

[0183] Non-limiting exemplary viral vector sequences are shown below. CTLA4 lentiviral insert (nucleotide sequence) ATGGCCTGCTTGGGCTTCCAAAGGCATAAAGCCCAGCTTAATCTTGCTACTCGCACGTGGCCCTGCACATTGCTCTTTTTCCTCCTGTTCATTCCCGTGTTTTGCAAGGCGATGCATGTGGCACAACCTGCCGTCGTTCTGGCATCATCAAGAGGTATTGCTAGCTTCGTTTGTGAGTACGCCTCCCCTGGAAAAGCGACGGAGGTGCGCGTCACTGTATTGCGGCAAGCCGACAGCCAAGTTACTGAAGTCTGCGCGGCAACGTATATGATGGGCAATGAGCTGACATTCCTTGACGATTCAATCTGCACGGGAACAAGTAGTGGTAACCAGGTGAATCTCACTATTCAAGGTCTGAGAGCCATGGACACCGGCCTCTACATTTGTAAGGTGGAGCTGATGTATCCTCCCCCATATTATCTGGGGATCGGAAATGGGACACAGATATATGTTATTGATCCCGAGCCATGTCCCGATAGTGACTTCCTCTTGTGGATACTTGCCGCTGTGAGCAGTGGTTTGTTTTTTTATTCATTCCTCCTTACGGCAGTATCACTTTCAAAAATGCTCAAGAAGCGAAGTCCTTTGACAACTGGCGTATATGTCAAAATGCCACCAACAGAGCCCGAATGTGAGAAACAGTTCCAGCCGTACTTTATTCCTATAAACTGA(SEQ ID NO: 137) CTLA4 lentiviral insert (amino acid sequence) MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN*(SEQ ID NO: 130) IL10 lentiviral insert (nucleotide sequence) ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAACTGA(SEQ ID NO: 138) IL10 lentiviral insert (amino acid sequence) MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN*(SEQ ID NO: 124) FOXP3 lentiviral insert (nucleotide sequence) FOXP3 lentiviral insert (amino acid sequence) MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQAD HLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP** (SEQ ID NO: 140) Empty Wrench Vector Empty Wrench Vector

[0184] A vector comprising a donor construct comprising a sequence encoding a guide RNA, an RNA-binding DNA binder, or a regulatory T cell promoting molecule (e.g., IL10, CTLA4), or a targeting receptor (e.g., CAR) can be delivered by a liposome, nanoparticle, exosome, or microvesicle, either individually or in any combination. The vector can also be delivered by a lipid nanoparticle (LNP). A donor construct comprising one or more guide RNAs, an RNA-binding DNA binder (e.g., mRNA), or a sequence encoding a heterologous protein can be delivered by a LNP, liposome, nanoparticle, exosome, or microvesicle, either individually or in any combination. A donor construct comprising one or more guide RNAs, an RNA-binding DNA binder (e.g., mRNA), or a sequence encoding a heterologous protein can be delivered by a LNP, either individually or in any combination. In some embodiments, one or more guide RNAs and an RNA-leading DNA binder (e.g., mRNA) are delivered by a LNP. The donor construct can be delivered by a viral vector.

[0185] Lipid nanoparticles (LNPs) are a well-known means for delivering nucleotide and protein cargo and can be used to deliver any of the guide RNAs, RNA-guided DNA binders, or donor constructs disclosed herein.

[0186] As used herein, lipid nanoparticles (LNPs) refer to particles that contain a plurality (i.e., two or more) of lipid molecules physically associated with each other by intermolecular forces. LNPs can be, for example, microspheres (unilamellar and multilamellar vesicles, e.g., "liposomes": in some embodiments, substantially spherical, and in more particular embodiments, contain a lamellar phase lipid bilayer that can contain an aqueous core (e.g., containing a substantial portion of RNA molecules), the dispersed phase in an emulsion, a micelle, or the internal phase in a suspension (see, for example, WO2017173054, the contents of which are incorporated herein by reference in their entirety). Any LNP known to those skilled in the art to be capable of delivering nucleotides to a subject can be utilized.

[0187] In some embodiments, provided herein is a method for delivering any of the guide RNAs described herein or donor constructs disclosed herein, alone or in combination, to a cell or cell population or subject, wherein any one or more of the components are associated with LNP. In some embodiments, the method further comprises an RNA-guided DNA binding agent (e.g., Cas9 or a sequence encoding Cas9).

[0188] In some embodiments, compositions are provided herein that include any of the guide RNAs described herein or donor constructs disclosed herein, alone or in combination, with LNPs. In some embodiments, the compositions further include an RNA-guided DNA binding agent (e.g., Cas9 or a sequence encoding Cas9).

[0189] In some embodiments, the LNPs comprise a cationic lipid. In some embodiments, the LNPs comprise (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate (also known as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate) or another ionizable lipid. See, e.g., the lipids of WO2019 / 067992, WO2017 / 173054, WO2015 / 095340, and WO2014 / 136086, and references therein. In some embodiments, the LNPs comprise a molar ratio of cationic lipid amine:RNA phosphate (N:P) of about 4.5, 5.0, 5.5, 6.0, or 6.5. In some embodiments, the terms cationic and ionizable in the context of LNP lipids are interchangeable, e.g., ionizable lipids are cationic depending on the pH.

[0190] In some embodiments, the LNPs associated with the constructs disclosed herein are used in the preparation of cell-based therapeutics for suppressing immune responses. Methods for the preparation of cell-based therapeutics and reagents for use in cell-based therapeutics are known in the art.

[0191] In some embodiments, any of the guide RNAs described herein, RNA-guided DNA binding agents, or donor constructs disclosed herein, alone or in combination, whether naked or part of a vector, are formulated in or administered via lipid nanoparticles.See, e.g., WO2019 / 067992 or WO2017 / 173054, the contents of which are incorporated herein by reference in their entirety.

[0192] In some embodiments, LNP compositions are included that include an RNA component and a lipid component, where the lipid component includes an amine lipid (e.g., a biodegradable, ionizable lipid). In some cases, the lipid component includes a biodegradable, ionizable lipid, cholesterol, DSPC, and PEG-DMG. In certain embodiments, the lipid-nucleic acid assembly included the ionizable lipid A((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate (also known as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate), cholesterol, DSPC, and PEG2k-DMG. In certain embodiments, the components are present in a molar ratio of 50:38:9:3, respectively. The lipid nucleic acid assemblies can be formulated with a lipid amine:RNA phosphate (N:P) molar ratio of about 6 and a gRNA:mRNA weight ratio of 2:1, 1:1, or 1:2.

[0193] It will be apparent that the donor constructs including the guide RNA, the RNA-guided DNA binder (e.g., Cas nuclease or a nucleic acid encoding a Cas nuclease), and the sequence encoding the regulatory T cell promoting molecule (e.g., IL10) or the targeting receptor (e.g., CAR) can be delivered using the same or different systems. For example, the guide RNA, Cas nuclease, and the construct can be delivered by the same vector (e.g., AAV). Alternatively, the Cas nuclease (as a protein or mRNA) or gRNA can be delivered by a plasmid or LNP, while the donor construct can be delivered by a vector such as AAV.

[0194] The different delivery systems can be delivered simultaneously or in any order. In some embodiments, the donor construct, guide RNA, and Cas nuclease can be delivered simultaneously, for example, in one vector, two vectors, individual vectors, one LNP, two LNPs, individual LNPs, or combinations thereof. In some embodiments, the donor construct can be delivered as a vector or in association with LNPs, alone or together, or as a ribonucleoprotein (RNP), prior to delivery of the guide RNA or Cas nuclease (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more). As a further example, the guide RNA and Cas nuclease can be delivered either alone or together, or as a ribonucleoprotein (RNP), prior to delivery of the construct (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days), either as a vector or in association with a LNP.

[0195] IV. Methods for Engineering T Cells The present disclosure provides a method of engineering a T cell to include modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG, modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA, and inserting into the cell a heterologous sequence(s) encoding a regulatory T cell promoting molecule under the control of a promoter sequence. The present disclosure provides a method of engineering a T cell to include modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG, modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA, and inserting into the cell a heterologous sequence(s) encoding IL10 under the control of a promoter sequence. The present disclosure provides a method of engineering a T cell to include modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG, modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA, and inserting into the cell a heterologous sequence(s) encoding CTLA4 under the control of a promoter sequence. The present disclosure provides methods of engineering T cells to include modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG, modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA, and inserting into the cell heterologous sequences encoding IL10 and CTLA4, each under the control of a promoter sequence.

[0196] In some embodiments, the method includes engineering the T cell to include modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and insertion into the cell of heterologous sequence(s) encoding a regulatory T cell-promoting molecule (e.g., IL10 or CTLA4), and further including modification (e.g., knockdown) of a TCR sequence(s).

[0197] In some embodiments, the method includes engineering a T cell to include modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG, modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA, and inserting into the cell heterologous sequence(s) encoding a regulatory T cell promoting molecule (e.g., IL10 or CTLA4), and further including inserting into the cell heterologous sequence(s) encoding a targeting receptor (e.g., CAR).

[0198] In some embodiments, the method includes engineering a T cell to include modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, insertion into the cell of heterologous sequence(s) encoding a regulatory T cell promoting molecule (e.g., IL10 or CTLA4), modification (e.g., knockdown) of a TCR sequence(s), and insertion into the cell of heterologous sequence(s) encoding a targeting receptor (e.g., CAR).

[0199] In some embodiments, the modification (e.g., knockdown) of the endogenous nucleic acid sequence encoding IFNG, the modification (e.g., knockdown) of the endogenous nucleic acid sequence encoding TNFA, the insertion into the cell of heterologous sequence(s) encoding a regulatory T cell-promoting molecule, the optional knockdown of a TCR gene, and the optional insertion into the cell of a targeted receptor (e.g., a CAR) are engineered using the CRISPR / Cas system and guide RNAs disclosed herein.

[0200] In these embodiments, the inserted regulatory T cell promoting molecule can be provided via a donor construct. The regulatory T cell promoting molecule provided via the donor construct can be selected from IL10, CTLA4, TGFB1, IDO1, ENTPD1, ​​NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, and BACH2, and modification (e.g., knockdown) of the TCR gene sequence(s).

[0201] In these embodiments, the targeting receptor to be inserted can be provided via the donor construct. In some embodiments, the targeting receptor can be a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a receptor of a cell surface molecule operably linked via at least a transmembrane domain within an internal signaling domain that can activate a T cell upon binding of an extracellular receptor portion. In some embodiments, the targeting receptor can be a receptor present on the surface of a cell (e.g., a T cell) that allows the cell to bind to a target site (e.g., a specific cell or tissue within an organism). In these embodiments, the targeting receptor is a CAR that can target MAdCAM-1.

[0202] Suitable gene editing systems for engineering T cells to include insertions and modifications (e.g., knockdown) are disclosed herein and known in the art. In some embodiments, gene editing systems include, but are not limited to, CRISPR / Cas systems, zinc finger nuclease (ZFN) systems, and transcription activator-like effector nuclease (TALEN) systems. Generally, gene editing systems involve the use of engineered cleavage systems to induce double-strand breaks (DSBs) or nicks (e.g., single-strand breaks, or SSBs) within a target DNA sequence. Cleavage or nicking can occur by the use of specific nucleases (e.g., engineered ZFNs, TALENs) or by using CRISPR / Cas systems with engineered guide RNAs to guide specific cleavage or nicking of the target DNA sequence (e.g., CRISPR / Cas9 systems). Additionally, targeted nucleases based on the Argonaute system have been developed (e.g., from T. thermophilus known as "TtAgo"; see Swarts et al (2014) Nature 507(7491):258-261), which may also have the potential to be used in genome editing and gene therapy.

[0203] Transcription Activator-Like Effector Nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences of DNA. They are created by fusing a TAL effector DNA binding domain to a DNA cleavage domain (a nuclease that cleaves DNA strands). Transcription Activator-Like Effectors (TALEs) can be engineered to bind to a desired DNA sequence and promote DNA cleavage at a specific location (see, e.g., Boch, TALEs of genome targeting Nature Biotech. 29:135-136 (2011)). Restriction enzymes can be introduced into cells for use in gene editing or for in situ genome editing, a technique known as genome editing with engineered nucleases. Such methods and compositions for use therein are known in the art. See, e.g., WO2019147805, WO2014040370, WO2018073393, the contents of which are incorporated herein by reference in their entirety.

[0204] Zinc finger nucleases (ZFNs) are artificial restriction enzymes that are generated by fusing a zinc finger DNA binding domain with a DNA cleavage domain. The zinc finger domain can be engineered to target specific desired DNA sequences, enabling the zinc finger nuclease to target unique sequences in complex genomes. The non-specific cleavage domain of the type IIs restriction endonuclease FokI is typically used as the cleavage domain in ZFNs. The cleavage is repaired by endogenous DNA repair machinery, allowing ZFNs to precisely modify the genome of higher organisms. Such methods and compositions for use therein are known in the art. See, for example, WO2011091324, the contents of which are incorporated herein in their entirety.

[0205] RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression or translation by neutralizing target mRNA molecules. Small interfering RNA (siRNA) is central to RNA interference. RNA is the direct product of genes, and these small RNAs (typically each strand is 19-23 nucleotides long and forms a duplex of 19-21 nucleotides) can reduce the activity of messenger RNA (mRNA) molecules by instructing the RNA-induced silencing (RISC) complex to degrade the messenger RNA (mRNA) molecule, thereby preventing translation via post-transcriptional gene silencing. Short hairpin RNA (shRNA) is an siRNA in which the strand that forms the duplex region has a hairpin structure, and is often generated by transcription from an expression vector. RNAi can also be achieved by long RNA duplex structures, termed dicer substrate molecules, which are cleaved by the enzyme dicer before being loaded into RISC, facilitating cleavage of the target mRNA. Such methods and compositions of use are known in the art. In the compositions and methods provided herein, the RNA molecule for promoting RNA interference is preferably provided as an expression vector for durability.See, for example, WO2018208837, the contents of which are incorporated herein by reference in their entirety.In some embodiments, RNAi is used with an expression vector.

[0206] It will be understood that the present disclosure contemplates insertion methods performed with or without the guide RNAs disclosed herein (e.g., using a ZFN system to cause cleavage in a target DNA sequence to create an insertion site for a construct). In methods using guide RNAs disclosed herein, the methods include the use of a CRISPR / Cas system to modify (e.g., knock down) a nucleic acid sequence encoding a TNFA, IFNG, or TCR. It will also be understood that the present disclosure contemplates methods of modifying (e.g., knock down) a TNFA, IFNG, or TCR, which can be performed without the guide RNAs disclosed herein (e.g., using a ZFN system to cause cleavage in a target DNA sequence to create an insertion site for a construct).

[0207] In some embodiments, a donor construct containing the sequence for insertion (e.g., a sequence encoding IL10 or CTLA4) is inserted into the genomic locus of the sequence to be targeted for modification (e.g., knockdown), e.g., a TCR gene.

[0208] In some embodiments, a CRISPR / Cas system (e.g., guide RNA and RNA-guided DNA binder) can be used to create an insertion site at a desired locus in the genome, into which a donor construct comprising a sequence encoding IL10, CTLA4, or a targeting receptor (e.g., a CAR disclosed herein, e.g., a MAdCAM-1 CAR) can be inserted to express IL10, CTLA4, or a CAR (e.g., a MAdCAM-1 CAR). The targeting receptor (e.g., a CAR, e.g., a MAdCAM-1 CAR), IL10, or CTLA4 can be heterologous with respect to its insertion site or locus, e.g., a safe harbor locus or a TCR locus where IL10, CTLA4, or a targeting receptor (e.g., a CAR, e.g., a MAdCAM-1 CAR) is not normally expressed, as described herein. In some embodiments, guide RNAs as described herein can be used in accordance with the methods with an RNA-guided DNA binding agent (e.g., Cas nuclease) to create an insertion site into which a donor construct comprising a sequence encoding IL10, CTLA4, or a targeting receptor (e.g., a CAR, e.g., a MAdCAM-1 CAR) can be inserted to express IL10, CTLA4, or a CAR (e.g., a MAdCAM-1 CAR). Guide RNAs for inserting IL10, CTLA4, or a targeting receptor (e.g., a CAR, e.g., a MAdCAM-1 CAR) into a particular genomic locus are exemplified and described herein.

[0209] In some embodiments, CD4+ T cells are engineered by transduction (e.g., using viral or non-viral delivery) with gRNA (e.g., gRNA targeting IFNG, TNFA, or TCR for knockdown), RNA-guided DNA binder (e.g., Cas nuclease), and donor construct. In some embodiments, engineered T cells are 1) transduced with gRNA targeting a nucleic acid sequence encoding a pro-inflammatory cytokine (e.g., IFNG or TNFA), RNA-guided DNA binder (e.g., Cas nuclease), and 2) transduced with a donor construct comprising a nucleic acid sequence(s) encoding a regulatory T cell promoting molecule (e.g., IL10 or CTLA4) and a targeting receptor (e.g., a CAR, e.g., a MAdCAM-1 CAR). In certain embodiments, engineered cells are selected for expression of a targeting receptor.

[0210] In some embodiments, CD4+ T cells are engineered by transduction with a gRNA (e.g., a gRNA targeting IFNG, TNFA, or TCR for knockdown), an RNA-guided DNA binder (e.g., a Cas nuclease), and a donor construct. In some embodiments, engineered T cells are 1) transduced with a donor construct comprising a nucleic acid sequence(s) encoding a regulatory T cell promoting molecule (e.g., IL10 or CTLA4) and a targeted receptor (e.g., a CAR, e.g., a MAdCAM-1 CAR), and 2) transduced with a gRNA targeting a nucleic acid sequence encoding a pro-inflammatory cytokine (e.g., IFNG or TNFA), an RNA-guided DNA binder (e.g., a Cas nuclease). In certain embodiments, engineered cells are selected for expression of a targeted receptor.

[0211] In some embodiments, CD4+ T cells are engineered by transduction with a gRNA (e.g., a gRNA targeting IFNG, TNFA, or TCR for knockdown), an RNA-guided DNA binder (e.g., Cas nuclease), and a donor construct. In some embodiments, the engineered T cells are 1) transduced with a donor construct comprising a nucleic acid sequence(s) encoding a regulatory T cell promoting molecule (e.g., IL10 or CTLA4) and a targeting receptor (e.g., a CAR, e.g., a MAdCAM-1 CAR), and 2) transduced with a gRNA targeting a nucleic acid sequence encoding a pro-inflammatory cytokine (e.g., IFNG or TNFA), an RNA-guided DNA binder (e.g., Cas nuclease).

[0212] As described herein, the donor construct comprising a sequence encoding IL10, CTLA4, or a targeted receptor (e.g., CAR), a guide RNA (e.g., gRNA targeting IFNG, TNFA, or TCR for knockdown), and an RNA-guided DNA binder can be delivered using any suitable delivery system and method known in the art. In some embodiments, the guide RNA and Cas nuclease are associated with LNPs and delivered to a cell or population of cells prior to delivery of the donor construct comprising a sequence encoding IL10, CTLA4, or a targeted receptor (e.g., CAR). In some embodiments, the guide RNA and Cas nuclease are associated with LNPs and delivered to a cell or population of cells after delivery of the donor construct comprising a sequence encoding IL10, CTLA4, or a targeted receptor (e.g., CAR).

[0213] In some embodiments, administration of the gRNAs, donor constructs, and RNA-guided DNA binding agents described herein to naturally occurring T cells can convert naturally occurring T cells (e.g., CD4+ T cells) into cells that exhibit properties of regulatory T cells (e.g., immune response suppression properties).

[0214] gRNAs, donor constructs, and RNA-guided DNA binders for modifying (e.g., knocking down) IFNG, TNFA, or TCR gene expression, or for inserting sequences encoding IL10, CTLA4, or a targeted receptor (e.g., a CAR, e.g., a MAdCAM-1 CAR), can be introduced into conventional T cells or populations of conventional T cells to generate the engineered T cells or populations of T cells described herein.

[0215] Methods using various RNA-guided DNA-binding agents (e.g., nucleases, e.g., Cas nucleases, e.g., Cas9) are also well known in the art. Although the use of the CRISPR / Cas system is exemplified herein, it will be understood that suitable variations on the system can also be used. Depending on the context, it will be understood that the RNA-guided DNA-binding agent can be provided as a nucleic acid (e.g., DNA or mRNA), e.g., as an mRNA encoding the RNA-guided DNA-binding agent provided above, or as a protein. In some embodiments, the method can be performed in a cell that already contains or expresses the RNA-guided DNA-binding agent.

[0216] In some embodiments, the RNA-guided DNA binding agent (e.g., Cas9 nuclease) has cleavage activity, which may also be referred to as double-stranded endonuclease activity. In some embodiments, the RNA-guided DNA binding agent (e.g., Cas9 nuclease) has nickase activity, which may also be referred to as single-stranded endonuclease activity. In some embodiments, the RNA-guided DNA binding agent comprises a Cas nuclease. Examples of Cas nucleases include those of S. pyogenes, S. aureus, and other prokaryotic type II CRISPR systems (see, e.g., the list in the next paragraph), as well as variants or mutants (e.g., engineered, non-naturally occurring, naturally occurring, or other variants) versions thereof. See, e.g., US2016 / 0312198 A1; US2016 / 0312199 A1.

[0217] Non-limiting examples of species from which Cas nucleases may be derived include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp.、Crocosphaera watsonii、Cyanothece sp.、Microcystis aeruginosa、Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohlobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, Acidaminococcus sp.、Lachnospiraceae bacterium ND2006、、、Acaryochloris marina is included。.

[0218] In some embodiments, the Cas nuclease is a Cas9 nuclease from Streptococcus pyogenes. In some embodiments, the Cas nuclease is a Cas9 nuclease from Streptococcus thermophilus. In some embodiments, the Cas nuclease is a Cas9 nuclease from Neisseria meningitidis. In some embodiments, the Cas nuclease is a Cas9 nuclease from Staphylococcus aureus. In some embodiments, the Cas nuclease is a Cpf1 nuclease from Francisella novicida. In some embodiments, the Cas nuclease is a Cpf1 nuclease from Acidaminococcus sp. In some embodiments, the Cas nuclease is a Cpf1 nuclease from Lachnospiraceae bacterium ND2006. In further embodiments, the Cas nuclease is a Cpf1 nuclease from Francisella tularensis, Lachnospiraceae bacterium, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium, Parcubacteria bacterium, Smithella, Acidaminococcus, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi, Leptospira inadai, Porphyromonas crevioricanis, Prevotella disiens, or Porphyromonas macacae. In certain embodiments, the Cas nuclease is a Cpf1 nuclease from Acidaminococcus or Lachnospiraceae.

[0219] In some embodiments, the gRNA together with the RNA-guided DNA-binding agent is referred to as a ribonucleoprotein complex (RNP). In some embodiments, the RNA-guided DNA-binding agent is a Cas nuclease. In some embodiments, the gRNA together with the Cas protein is referred to as a Cas RNP. In some embodiments, the RNP comprises type I, type II, or type III components. In some embodiments, the Cas nuclease is a Cas9 protein from a type II CRISPR / Cas system. In some embodiments, the gRNA together with Cas9 is referred to as a Cas9 RNP.

[0220] Wild-type Cas9 has two nuclease domains, RuvC and HNH. The RuvC domain cleaves the non-target DNA strand, and the HNH domain cleaves the target strand of DNA. In some embodiments, the Cas9 protein comprises multiple RuvC domains or multiple HNH domains. In some embodiments, the Cas9 protein is wild-type Cas9. In each of the composition, use, and method embodiments, Cas induces a double-strand break in the target DNA.

[0221] In some embodiments, chimeric Cas nucleases are used in which one domain or region is replaced with a portion of a different protein. In some embodiments, the Cas nuclease domain may be replaced with a domain from a different nuclease, such as Fok1. In some embodiments, the Cas nuclease may be a modified nuclease.

[0222] In other embodiments, the Cas protein may be from a type I CRISPR / Cas system. In some embodiments, the Cas nuclease may be a component of a cascade complex of a type I CRISPR / Cas system. In some embodiments, the Cas nuclease may be a Cas3 protein. In some embodiments, the Cas protein may be from a type III CRISPR / Cas system. In some embodiments, the Cas nuclease may have RNA cleavage activity.

[0223] In some embodiments, the RNA-guided DNA-binding agent has single-stranded nickase activity, i.e., it can cleave one DNA strand, resulting in a single-stranded break, also known as a "nick." In some embodiments, the RNA-guided DNA-binding agent comprises a Cas nickase. A nickase is an enzyme that creates a nick in dsDNA, i.e., it cleaves one strand of the DNA double helix, but not the other. In some embodiments, the Cas nickase is a version of a Cas nuclease (e.g., a Cas nuclease described above) in which the endonuclease active site has been inactivated, e.g., by one or more modifications (e.g., point mutations) in the catalytic domain. See, e.g., U.S. Patent No. 8,889,356 for a discussion of Cas nickases and exemplary catalytic domain modifications. In some embodiments, the Cas nickase (e.g., Cas9 nickase) has an inactivated RuvC or HNH domain.

[0224] In some embodiments, the RNA-guided DNA binder is modified to contain only one functional nuclease domain. For example, the drug protein can be modified to reduce its nucleic acid cleavage activity by mutating or completely or partially deleting one of the nuclease domains. In some embodiments, a nickase with a RuvC domain with reduced activity is used. In some embodiments, a nickase with an inactive RuvC domain is used. In some embodiments, a nickase with an HNH domain with reduced activity is used. In some embodiments, a nickase with an inactive HNH domain is used.

[0225] In some embodiments, conserved amino acids in the Cas protein nuclease domain are substituted to reduce or modify nuclease activity. In some embodiments, the Cas nuclease can include an amino acid substitution in the RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015) Cell Oct 22:163(3):759-771. In some embodiments, the Cas nuclease can include an amino acid substitution in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al (2015). Further exemplary amino acid substitutions include D917A, E1006A, and D1255A (based on the Francisella novicida U112 Cpf1 (FnCpf1) sequence (UniProtKB: A0Q7Q2(CPF1_FRATN))).

[0226] In some embodiments, the nickase is provided in combination with a pair of guide RNAs that are complementary to the sense and antisense strands of the target sequence, respectively. In this embodiment, the guide RNA guides the nickase to the target sequence and introduces a DSB by generating a nick on the opposite strand of the target sequence (i.e., double nicking). In some embodiments, the nickase is used with two separate guide RNAs that target opposite strands of DNA to generate a double nick in the target DNA. In some embodiments, the nickase is used with two separate guide RNAs that are selected to be in close proximity to generate a double nick in the target DNA.

[0227] In some embodiments, the RNA-guided DNA binding agent comprises (eg, is or comprises a fusion polypeptide) one or more heterologous functional domains.

[0228] In some embodiments, the heterologous functional domain can facilitate the transport of the RNA-leading DNA binding agent into the cell nucleus. For example, the heterologous functional domain can be a nuclear localization signal (NLS). In some embodiments, the RNA-leading DNA binding agent can be fused with 1-10 NLS(s). In some embodiments, the RNA-leading DNA binding agent can be fused with 1-5 NLS(s). In some embodiments, the RNA-leading DNA binding agent can be fused with one NLS. When one NLS is used, the NLS can be attached at the N-terminus or C-terminus of the RNA-leading DNA binding agent sequence. It can also be inserted within the sequence of the RNA-leading DNA binding agent. In other embodiments, the RNA-leading DNA binding agent can be fused with multiple NLSs. In some embodiments, the RNA-leading DNA binding agent can be fused with 2, 3, 4, or 5 NLSs. In some embodiments, the RNA-leading DNA binding agent can be fused with two NLSs. In certain circumstances, the two NLSs can be the same (e.g., two SV40 NLSs) or different. In some embodiments, the RNA-guided DNA-binding agent is fused with two SV40 NLS sequences bound at the carboxy terminus. In some embodiments, the RNA-guided DNA-binding agent can be fused with two NLSs, one bound at the N terminus and the other bound at the C terminus. In some embodiments, the RNA-guided DNA-binding agent can be fused with three NLSs. In some embodiments, the RNA-guided DNA-binding agent can be unfused to an NLS. In some embodiments, the NLS can be a monopartite sequence, such as, for example, the SV40 NLS, PKKKRKV (SEQ ID NO: 143) or PKKKRRV (SEQ ID NO: 144). In some embodiments, the NLS can be a bipartite sequence, such as the NLS of nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO: 145). In certain embodiments, a single PKKKRKV (SEQ ID NO: 143) NLS can be bound at the C terminus of the RNA-guided DNA-binding agent.One or more linkers are optionally included at the fusion site.

[0229] V. Treatment method The present disclosure provides a method for suppressing an immune response in a subject, comprising administering an engineered T cell comprising a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and insertion into the cell of a heterologous sequence(s) encoding a regulatory T cell promoting molecule under the control of a promoter sequence.The present disclosure provides a method for suppressing an immune response in a subject, comprising administering an engineered T cell comprising a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and insertion into the cell of a heterologous sequence(s) encoding IL10 under the control of a promoter sequence. The present disclosure provides a method for suppressing an immune response in a subject, comprising administering an engineered T cell that includes a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and insertion into the cell of a heterologous sequence(s) encoding CTLA4 under the control of a promoter sequence. The present disclosure provides a method for suppressing an immune response in a subject, comprising administering an engineered T cell that includes a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and insertion into the cell of a heterologous sequence encoding IL10 and CTLA4.

[0230] The present disclosure provides a method for treating an autoimmune disorder in a subject, comprising administering an engineered T cell comprising a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and insertion into the cell of a heterologous sequence(s) encoding a regulatory T cell promoting molecule under the control of a promoter sequence.The present disclosure provides a method for treating an autoimmune disorder in a subject, comprising administering an engineered T cell comprising a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and insertion into the cell of a heterologous sequence(s) encoding IL10 under the control of a promoter sequence. The present disclosure provides a method for treating an autoimmune disorder in a subject, comprising administering an engineered T cell that comprises a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and insertion into the cell of a heterologous sequence(s) encoding CTLA4 under the control of a promoter sequence.The present disclosure provides a method for treating an autoimmune disorder in a subject, comprising administering an engineered T cell that comprises a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and insertion into the cell of a heterologous sequence encoding IL10 and CTLA4, each under the control of a promoter sequence.

[0231] The present disclosure provides a method for treating GvHD in a subject, comprising administering an engineered T cell comprising a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and insertion into the cell of a heterologous sequence(s) encoding a regulatory T cell-promoting molecule under the control of a promoter sequence.The present disclosure provides a method for treating GvHD in a subject, comprising administering an engineered T cell comprising a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and insertion into the cell of a heterologous sequence(s) encoding IL10 under the control of a promoter sequence. The present disclosure provides a method for treating GvHD in a subject, comprising administering an engineered T cell that comprises a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and insertion into the cell of a heterologous sequence(s) encoding CTLA4 under the control of a promoter sequence.The present disclosure provides a method for treating GvHD in a subject, comprising administering an engineered T cell that comprises a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and insertion into the cell of a heterologous sequence encoding IL10 and CTLA4, each under the control of a promoter sequence.

[0232] In some embodiments, the method includes administering an engineered T cell that includes modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and insertion of a sequence(s) encoding a regulatory T cell-promoting molecule, and further includes modification (e.g., knockdown) of a TCR sequence(s).

[0233] In some embodiments, the method includes administering engineered T cells that include modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and insertion of sequence(s) encoding a regulatory T cell-promoting molecule, and further include insertion of sequence(s) encoding a targeting receptor (e.g., CAR).

[0234] In some embodiments, the targeting receptor (e.g., CAR) can target the engineered T cells to the gastrointestinal system, e.g., the targeting receptor is a CAR that targets MAdCAM-1, e.g., to suppress immune responses (including inflammation) in disorders such as inflammatory bowel disease, ulcerative colitis, or Crohn's disease. In some embodiments, the targeting receptor (e.g., CAR) can target the engineered T cells to tissues that include endothelial cells, e.g., the targeting receptor is a CAR that targets VCAM-1, e.g., to suppress immune responses (including inflammation) in disorders such as Crohn's disease and multiple sclerosis. In some embodiments, the targeting receptor (e.g., CAR) can target the engineered T cells to endothelial cells, e.g., the targeting receptor is a CAR that targets CEACAM6, e.g., to suppress immune responses in disorders such as Crohn's disease. In some embodiments, the targeting receptor (e.g., CAR) can target the engineered T cells to B cells, e.g., the targeting receptor is a CAR that targets CD19, e.g., to suppress immune responses in disorders such as multiple sclerosis and systemic lupus erythematosus. In some embodiments, the targeting receptor (e.g., CAR) can target the engineered T cells to B cells, e.g., the targeting receptor is a CAR that targets CD20, e.g., to suppress immune responses in disorders such as multiple sclerosis and systemic lupus erythematosus. In some embodiments, the targeting receptor (e.g., CAR) can target the engineered T cells to inflamed tissues, e.g., the targeting receptor is a CAR that targets TNFA, e.g., to suppress immune responses in disorders such as inflammatory bowel disease, ulcerative colitis, or Crohn's disease. In some embodiments, a targeting receptor (e.g., a CAR) can target the engineered T cells to neural tissue, e.g., the targeting receptor is a CAR that targets MBP, MOG, or PLP, e.g., to suppress immune responses in disorders such as multiple sclerosis.In some embodiments, the targeting receptor (e.g., a CAR) can target the engineered T cells to tissues that contain mature B lymphocytes, e.g., a targeting receptor is a CAR that targets TNFRSF17, e.g., to suppress immune responses in disorders such as systemic lupus erythematosus. In some embodiments, the targeting receptor (e.g., a CAR) can target the engineered T cells to synovial tissue, e.g., a targeting receptor is a CAR that targets citrullinated vimentin, e.g., to suppress immune responses in disorders such as rheumatoid arthritis.

[0235] In some embodiments, the targeting receptor is a CAR that targets DPP6, SCL2A2, glutamic acid decarboxylase (GAD2), desmoglein 3 (DSG3), and MHC class I HLA-A (HLA-A*02).

[0236] In some embodiments, the method includes administering an engineered T cell that includes modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, insertion of a sequence(s) encoding a regulatory T cell-promoting molecule, and insertion of a sequence(s) encoding a targeting receptor (e.g., a CAR), and further includes modification (e.g., knockdown) of a TCR sequence(s).

[0237] In some embodiments, the sequence(s) to be inserted are inserted into the sequence(s) to be modified (e.g., knocked down). For example, a CAR sequence is inserted into a TNFA genomic sequence, thereby modifying (e.g., knocking down) the TNFA sequence.

[0238] In some embodiments, the method comprises administering a population of T cells comprising T cells that have been engineered as described above, in some embodiments, at least 40%, 45%, preferably at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the T cells in the population of T cells are engineered, e.g., as assessed by sequencing (e.g., NGS).

[0239] In some embodiments, the autoimmune disorder is selected from ulcerative colitis, Crohn's disease, rheumatoid arthritis, psoriasis, multiple sclerosis, systemic lupus erythematosus, type 1 diabetes, and graft-versus-host disease (GvHD). In some embodiments, the engineered T cells are used autologous or allogeneic.

[0240] In some embodiments, the efficacy of treatment with the engineered T cells described above can be assessed in an animal model (e.g., a mouse model) of graft-versus-host disease by measuring the body weight or survival rate of the animals after administration of the engineered T cells (animals are sacrificed after losing a significant portion of their body weight, e.g., 20% of their starting body weight). In some embodiments, an effective treatment results in a statistically significant increase in survival rate compared to a suitable control (e.g., animals treated with PBMCs). EXAMPLES

[0241] The following examples are provided to illustrate certain disclosed embodiments and should not be construed as limiting the scope of the disclosure in any way.

[0242] Example 1. General Method 1.1. Preparation of lipid nanoparticles Generally, lipid components were dissolved in 100% ethanol at various molar ratios. RNA cargo (e.g., Cas9 mRNA and sgRNA) was dissolved in 25 mM citrate buffer, 100 mM NaCl, pH 5.0, resulting in a concentration of RNA cargo of approximately 0.45 mg / mL.

[0243] Unless otherwise specified, the lipid-nucleic acid assemblies contained ionizable lipid A ((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate (also known as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate)), cholesterol, DSPC, and PEG2k-DMG in a molar ratio of 50:38.5:10:1.5, respectively. Lipid nucleic acid assemblies were formulated at a lipid amine:RNA phosphate (N:P) molar ratio of approximately 6 and a gRNA:mRNA weight ratio of 1:1 unless otherwise specified.

[0244] LNPs were prepared using a cross-flow technique that utilizes impinging jet mixing of lipids in ethanol with two volumes of RNA solution and one volume of water. Lipids in ethanol were mixed with two volumes of RNA solution through a mixing cross. A fourth water stream was mixed with the outlet stream of the cross through an in-line tee (see FIG. 2 of WO2016010840). LNPs were held at room temperature for 1 hour and further diluted with water (approximately 1:1 v / v). LNPs were concentrated using tangential flow filtration on a flat sheet cartridge (Sartorius, 100 kD MWCO) and buffer exchanged into 50 mM Tris, 45 mM NaCl, 5% (w / v) sucrose, pH 7.5 (TSS) using PD-10 desalting columns (GE). Alternatively, LNPs were optionally concentrated using 100 kDa Amicon spin filters and buffer exchanged into TSS using PD-10 desalting columns (GE). The resulting mixture was then filtered using a 0.2 μM sterile filter, and the final LNPs were stored at 4° C. or −80° C. until further use.

[0245] 1.2. In vitro transcription of mRNA (IVT) Capped and polyadenylated mRNA containing N1-methylpseudo-U was generated by in vitro transcription using linearized plasmid DNA template and T7 RNA polymerase. Plasmid DNA containing the T7 promoter, sequence for transcription, and polyadenylation region was linearized by incubation with XbaI for 2 hours at 37°C using the following conditions: 200 ng / μL plasmid, 2 U / μL XbaI (NEB), and 1× reaction buffer. XbaI was inactivated by heating the reaction at 65°C for 20 minutes. Linearized plasmid was purified from enzymes and buffer salts. IVT reactions to generate modified mRNA were performed in the following conditions: 50 ng / μL linearized plasmid; 2–5 mM each of GTP, ATP, CTP, and N1-methylpseudo-UTP (Trilink); 10–25 mM ARCA (Trilink); 5 U / μL T7 RNA polymerase (NEB); 1 U / μL mouse RNase inhibitor (NEB); 0.004 U / μL inorganic E. coli pyrophosphatase (NEB); and 1× reaction buffer by incubating at 37°C for 1.5–4 h. TURBO DNase (ThermoFisher) was added to a final concentration of 0.01 U / μL, and the reaction was incubated for an additional 30 min to remove the DNA template. mRNA was purified using the MegaClear Transcription Clean-up kit (ThermoFisher) or RNeasy Maxi kit (Qiagen) according to the manufacturer's protocol. Alternatively, mRNA was purified by precipitation protocols, optionally followed by HPLC-based purification. Briefly, after DNase digestion, mRNA was purified using LiCl precipitation, ammonium acetate precipitation, and sodium acetate precipitation. For HPLC-purified mRNA, after LiCl precipitation and reconstitution, mRNA was purified by RP-IP HPLC (see, e.g., Kariko, et al. Nucleic Acids Research, 2011, Vol. 39, No. 21 e142). The fractions selected for pooling were combined and desalted by sodium acetate / ethanol precipitation as described above.Alternatively, mRNA was purified by LiCl precipitation followed by further purification by tangential flow filtration. RNA concentration was quantified by measuring the light absorption at 260 nm (Nanodrop) and transcripts were analyzed by capillary electrophoresis with a Bioanlayzer (Agilent).

[0246] Streptococcus pyogenes ("Spy") Cas9 mRNA was generated from plasmid DNA encoding an open reading frame according to the nucleic acid sequence described herein. For the mRNA nucleic acid sequence below, it is understood that T should be replaced with U (which was N1-methyl pseudouridine as described above). The messenger RNA used in the examples includes a 5' cap and a 3' polyA tail, e.g., up to 100 nt (SEQ ID NO: 146).

[0247] 1.3. Next Generation Sequencing ("NGS") and Analysis of On-Target Editing Efficiency Genomic DNA was extracted using QuickExtract™ DNA Extraction Solution (Lucigen, Cat. No. QE09050) according to the manufacturer's protocol.

[0248] To quantitatively determine the editing efficiency at the target position in the genome, deep sequencing was used to identify the presence of insertions and deletions introduced by gene editing. PCR primers were designed based on the target site in the gene of interest (e.g., TRAC) to amplify the genomic segment of interest. Primer sequence design was performed as standard in the field.

[0249] An additional PCR was performed according to the manufacturer's protocol (Illumina) to add sequencing chemistry. Amplicons were sequenced on an Illumina MiSeq instrument. After removing reads with low quality scores, reads were aligned to the human reference genome (e.g., hg38). The resulting files containing the reads were mapped to the reference genome (BAM files), reads overlapping the target region of interest were selected, and the number of reads containing indels relative to the number of wild-type reads was calculated. Editing percentage (e.g., "editing efficiency" or "percent editing" or "percent indels") is defined as the total number of sequence reads with indels divided by the total number of sequence reads (including wild-type).

[0250] Example 2. Suppressive ability of T cells overexpressing Treg-associated factors CD3+CD4+ T cells were transduced to overexpress the Treg-associated transcription factors Foxp3, Foxp3 and IL10, IL10, CTLA4, and IL10 and CTLA4, and assayed for their ability to suppress inflammatory immune responses.

[0251] 2.1 Preparation of T cells Human CD3+CD4+ T cells were prepared in-house from fresh leukopak (StemCell Technologies, donor no. RG1953). For in-house preparation, CD3+CD4+ T cells were isolated by negative selection using human CD4+T cell isolation kit (Miltenyi, catalog no. 130-096-533) according to the manufacturer's protocol. Isolated CD3+CD4+ T cells were frozen in Cryostor CS10 freezing media (catalog no. 07930) until further use. On the day of activation, frozen CD3+CD4+ T cells were thawed using a 37°C water bath. Thawed CD3+CD4+ T cells were added at 1×10 6At a density of cells / mL, the cells were cultured in 10% (v / v) fetal bovine serum (Gibco, Cat. No. A31605-01), 1x Glutamax (Gibco, Cat. No. 35050-061), 50 μM 2-mercaptoethanol (Gibco, Cat. No. 31350-010), 1x non-essential amino acids (Corning, Cat. No. 25-0250-CI), 1 mM sodium pyruvate (Corning, Cat. No. 25-000-CI), 10 mM HEPES buffer (Gibco, Cat. No. 15630-080), 1x penicillin-streptomycin (Gibco, Cat. No. 15140-122), and supplemented with the following cytokines: 100 U / mL recombinant human interleukin-2 (StemCell Technologies, Cat. No. 78036.1), 5 ng / mL recombinant human interleukin-7 (StemCell Technologies, Cat. No. 78036.1), 10 ng / mL recombinant human interleukin-8 (StemCell Technologies, Cat. No. 78036.1), 10 ng / mL recombinant human interleukin-9 (StemCell Technologies, Cat. No. 78036.1), 10 ng / mL recombinant human interleukin-10 (StemCell Technologies, Cat. No. 78036.1), 10 ng / mL recombinant human interleukin-11 (StemCell Technologies, Cat. No. 78036.1), 10 ng / mL recombinant human interleukin-12 (StemCell Technologies, Cat. No. 78036.1), 10 ng / mL recombinant human interleukin-13 (StemCell Technologies, Cat. No. 7803 Cells were plated in a total of 5ml of T cell RPMI media composed of RPMI1640 (Corning, Catalog No. 10-040-CV) containing 5ng / ml recombinant human interleukin-15 (StemCell technologies, Catalog No. 78053.1), and 5ng / ml recombinant human interleukin-15 (StemCell technologies, Catalog No. 78031.1). Cells were activated by adding 25uL / mL ImmunoCult Human CD3 / CD28 / CD2 T cell Activator (Stemcell Technologies, Catalog No. 10990) and cultured at 37°C for 48 hours prior to lentiviral transduction.

[0252] 2.2 T cell transduction and cell sorting Activated CD3+CD4+ T cells were transduced with lentiviral constructs individually or together to overexpress IL10, CTLA4, and FOX3P. Forty-eight hours after activation, CD3+CD4+ T cells were harvested, washed, and cultured at 1 × 10 6The cells were resuspended in T cell RPMI medium at a density of cells / 100uL. 100uL of concentrated viral supernatant was added to the CD3+CD4+ T cells and centrifuged at 1000xg for 60 minutes at 37°C. After transduction, the CD3+CD4+ T cells were resuspended in the cell / viral supernatant mixture and transferred to a single well of a 6-well G-rex (Wilson Wolf; Catalog No. 80240M) containing 20mL of T cell RPMI medium supplemented with 500U / mL IL-2, 5ng / mL IL-7, and 5ng / mL IL-17. The transduced CD3+CD4+ T cells were cultured for 4-6 days and sorted using a BD FACSAria™ Fusion Cell Sorter (BD Biosciences) to isolate cells expressing the target of interest. After sorting, CD3+CD4+ T cells were cultured in 6-well Grex plates in 20 mL of T cell RPMI medium containing the components mentioned in 2.1 and further supplemented with 500 U / mL IL-2, 5 ng / mL IL7, and 5 ng / mL IL17 until day 25.

[0253] Natural regulatory T cells (nTreg) were prepared using methods known in the art. Briefly, autologous PBMCs were thawed and processed using a CD4+ T cell negative selection kit (Miltenyi, Cat. No. 130-096-533) according to the manufacturer's instructions to isolate and process CD4+ T cells. CD3+ CD4+ T cells were isolated at 1×10 7Cells were resuspended in FACS buffer at 100 cells / mL and stained with BV421 anti-CD4 (Biolegend, Cat. No. 300532), APC anti-CD25 (Biolegend, Cat. No. 302610), and PE-Cy7 anti-CD127 (Biolegend, Cat. No. 351320) for 30 min at 4° C. The top 3–5% of high expressing CD25+ cells were sorted by FACS from the CD4+CD127- population and placed into T cell RPMI medium cultures containing 50% fetal bovine serum. Sorted CD3+CD4+CD25+CD127-nTregs were plated in 6-well Grex in 20 mL of T cell RPMI medium supplemented with the components described in Example 2.1, additionally supplemented with 500 U / mL IL-2 (Stemcell Technologies, Cat. No. 78036.1), 100 nM rapamycin (Millipore Sigma, Cat. No. 553211), and 25 μl / mL anti-CD3 / 28 / 2 Immunocult T cell activator (Stemcell Technologies, Cat. No. 10990). IL-2 (Stemcell Technologies, Cat. No. 78036.1) and rapamycin (Millipore Sigma, Cat. No. 553211) were added every other day for 7 days, at which point only 500 U / ml IL-2 (Stemcell Technologies, Cat. No. 78036.1) was added every other day. On day 12, nTregs were harvested, washed, and plated in 6-well Grex plates containing T cell RPMI medium supplemented with the components mentioned in A.1, 500 U / mL IL-2 (Stemcell Technologies, Cat. No. 78036.1), until the day of injection.

[0254] 2.3 Validation of target expression Target expression was verified by flow cytometry. 500,000 transduced CD3+CD4+ T cells were permeabilized using FoxP3 / Transcription Factor Staining Buffer Set (eBioscience, Cat. No. 00-5523-00) according to the manufacturer's instructions. After permeabilization, transduced CD3+CD4+ T cells were incubated with an antibody panel consisting of either (1) PerCP / Cy 5.5 anti-FoxP3 (BD ​​Biosciences, Cat. No. 561493), eFluor660 anti-Eos (Invitrogen, Cat. No. 50-5758-80), Pacific blue anti-Helios (Biolegend, Cat. No. 137220), or (2) BV421 anti-IL-10 (Biolegend, Cat. No. 501422) and APC anti-CTLA4 (Biolegend, Cat. No. 369612). Stained transduced CD3+CD4+ T cells were run on a Cytoflex flow cytometer (Beckman Coulter) and analyzed using the FlowJo software package. Transduced CD3+CD4+ T cells were gated based on size and shape before quantifying targets in the antibody panel. Target overexpression was characterized relative to transduced controls. The desired expression level for a given target was equal to or greater than nTreg (Table 4 and Figure 1A-E). nTreg samples expressed high levels of Foxp3, Helios, and Eos, which correlates with a highly pure and suppressive phenotype. [Table 3]

[0255] 2.4 In vivo evaluation of immunosuppression in GvHD models A mouse model of graft-versus-host disease was used to assess the in vivo suppressive function of selected transformed CD3+CD4+ T cells.

[0256] Sorted CD3+CD4+ T cells for in vivo injection were harvested and processed using a dead cell removal kit (Miltenyi, Cat. No. 130-090-101) according to the manufacturer's instructions. Autologous PBMCs were thawed as described in the above examples. PBMCs were added to each assay population at a 1:1 ratio, and cells were resuspended in PBS to a concentration of 6×10 6 / 150 μL. PBMC only group was 3 × 10 6 Resuspended in 150 μL.

[0257] Female NOG mice (NOD.Cg-Prkdc scid Il2rg tm1Sug / JicTac; Taconic, Cat. No. NOG-F) were conditioned for cell transplantation by sublethally irradiating (200 rads) with X-rays (RS-2000 irradiator; Rad Source Technologies) one day prior to injection. Cohorts of irradiated NOG mice were intravenously injected with 150 μL of each test cell population. Five irradiated mice were not injected and served as irradiation only controls. Body weight was monitored daily. Mice with a 20% weight loss were sacrificed and the cellular composition of the spleen was evaluated. Survival times were plotted to understand the survival rate of mice in each test group. As shown in Table 5 and Figure 2A, only T cells transduced with both IL-10 and CTLA4 extended survival to levels similar to nTregs. [Table 4]

[0258] To confirm human leukocyte engraftment, spleen composition was assessed. At the time of euthanasia, the spleen of each animal was collected and placed into a PBS-containing gentleMACS C tube (Miltenyi, catalog number 130-096-334). Spleens were dissociated using a gentleMACS Octo Dissociator machine (Miltenyi, 130-095-937, program mSpleen01_01). The cell suspension was filtered through a 70 micron cell strainer (Corning, catalog number 08-771-2) and cells were counted using a Vi-CELL XR Cell Viability Analyzer (Beckman Coulter). Approximately 1 million viable splenocytes were resuspended in FACS buffer and stained with a panel of antibodies consisting of anti-human CD3 (Alexa Fluor 488 (Biolegend, Cat. No. 317310) or PerCP / Cyanine 5.5 (Biolegend, Cat. No. 300327)), BV650 anti-human CD19 (Biolegend, Cat. No. 302238), APC anti-human CD45 (BD Pharmigen, Cat. No. 561864), APC-Fire 750 anti-human CD4 (Biolegend, Cat. No. 300560), and BV421 anti-mouse Ter119 (Biolegend, Cat. No. 116234) for 30 minutes at 4°C. Splenocytes were washed with FACS buffer, processed on a Cytoflex flow cytometer (Beckman Coulter), and analyzed using the FlowJo software package. CD4 T cells were defined as Ter119-CD45+CD19-CD3+CD4+. CD8 T cells were defined as Ter119-CD45+CD19-CD3+CD4-. B cells were defined as Ter119-CD45+CD19+CD3-. To quantify the cell numbers of the individual populations, the percentage of each population relative to the total number of splenocytes harvested was applied. Mice treated with lentiviral vector-transduced T cells to overexpress both IL10 and CTLA4, as well as nTregs, had lower percentages and numbers of B cells than untreated PBMC mice. Data are shown in Table 6 and Figure 2B. [Table 5]

[0259] 2.5 Cytokine profile analysis of transduced CD3+CD4+ T cells The selected transduced CD3+CD4+ T cells were stimulated to assess the cytokine profile. The selected transduced CD3+CD4+ T cells were plated at 1×10^5 T cells / well in a total of 200μL T cell RPMI medium with or without 25uL / mL ImmunoCult Human CD3 / CD28 / CD2 T cell Activator (Stemcell Technologies, Cat. No. 10990) in U-bottom culture plates and cultured at 37℃ for 48 hours. After 48 hours of culture, the culture plates were centrifuged, the supernatants were collected, frozen and stored, and then cytokine quantification was performed using a custom U-PLEX Biomarker kit (Meso Scale Diagnostics, Cat. No. K15067L-2) according to the manufacturer's instructions. Specifically, the U-PLEX Biomarker kit was used to quantify the following human cytokines: IFNG, TNFA, IL6, IL2, IL13, and IL10. The U-PLEX Biomarker plates were read using a Meso Quickplex SQ120 instrument (Meso Scale Discovery) and data were analyzed using the Discovery Workbench 4.0 software package (Meso Scale Discovery). The results are shown in Table 7 and Figures 3A-3F. CD3+CD4+ T cells transduced with a lentiviral expression vector carrying sequences encoding IL10 secreted large amounts of IL10 upon TCR stimulation. Overexpression of IL10 also increased secretion of IL6, IFNG, and TNFA. In contrast, T cells transduced with a lentiviral expression vector carrying sequences encoding FoxP3 showed reduced expression of all quantified cytokines. Natural Tregs also showed reduced secretion of quantified cytokines, characteristic of highly pure and suppressive nTregs. [Table 6]

[0260] 2.6 Mixed lymphocyte reaction assay of suppressor function The mixed lymphocyte reaction (MLR) was used to assay the suppressive function of the selected transduced CD3+CD4+ T cells. The MLR is an inflammatory response triggered by T cells recognizing allogeneic leukocytes from another donor as foreign. Tregs can suppress this inflammatory response. Therefore, the MLR is a standard assay to evaluate the suppressive capacity of Tregs, including engineered Tregs. If Tregs are suppressive, there will be less proliferation and production of inflammatory cytokines by the responding inflammatory T cells.

[0261] MLR was performed in 96-well U-bottom plates using T cell RPMI medium. Untransduced CD3+CD4+ T cells were labeled with CellTrace Violet (CTV) (Thermofisher Scientific, Cat. No. C34557) according to the manufacturer's instructions and used as responder cells. CD3-depleted PBMCs from a different allogeneic donor than those used for transduced T cells were processed using Dead cell removal kit (Miltenyi, Cat. No. 130-090-101) according to the manufacturer's instructions. Cultures were prepared by combining 50,000 CTV-labeled T cells per well with 50,000 CD3-depleted PBMCs and approximately 50,000 (1:1), 16,666 (4:1), 5,555 (16:1), 1,851 (64:1), or 617 (256:1) sorted transduced CD3+CD4+ T cells. After 5 days of incubation at 37°C, culture plates were centrifuged and culture supernatants were harvested for cytokine quantification. Cell pellets were resuspended in FACS buffer containing APC / Fire 750 anti-CD4 and incubated at 4°C for 30 min. Cells were then washed and processed on a CytoFlex flow cytometer (Beckman Coulter) and analyzed using the FlowJo software package. Cells were first gated by positive CD4 expression, then by CTV expression, and finally by the undiluted CTV population. The inhibition of CTV dilution was calculated using the following formula:

number

[0262] where y = mean fluorescence intensity of the entire CTV-labeled population / mean fluorescence intensity of the undiluted portion of the CTV-labeled population. The data are shown in Table 8 and Figure 4. [Table 7]

[0263] Example 3. Suppressive capacity of engineered T cells Because CD3+CD4+ T cells transduced with lentiviral vectors to promote overexpression of IL-10 and CTLA-4 showed increased production of IFNG and TNFA, these cells were further engineered to disrupt the genes encoding IFNG and TNFA, and the suppressive capacity of these cells was assessed in vitro and in vivo.

[0264] 3.1 T cell manipulation Human CD3+CD4+ T cells were isolated from leukopaks, activated, and transduced with lentiviral constructs to promote overexpression of IL10 and CTLA4 as described in Example 2.2. One day after transduction, transduced cells were engineered with Cas9 to disrupt the TNFA and IFNG genes. LNPs containing Cas9 mRNA and sgRNA targeting IFNG (G019753; IFNG guide sequence CCAGAGCAUCCAAAAGAGUG (SEQ ID NO: 14)) or TNFA (G019757; TNFA guide sequence AGAGCUCUUACCUACAACAU (SEQ ID NO: 58)) were formulated as described in Example 1. G019753: mC*mC*mA*GAGCAUCCAAAAGAGUGGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmGmUmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 119) G019757: mA*mG*mA*GCUCUUACCUACAACAUGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 120)

[0265] Each LNP preparation was incubated at 37°C for 15 minutes in OpTmizer basal medium containing CTS OpTmizer T Cell Expansion SFM (Gibco, Catalog No. A3705001), 1% penicillin-streptomycin, 1X Glutamax, 10 mM HEPES, 2.5% human AB serum (Gemini, Catalog No. 100-512), 200U / mL recombinant human interleukin-2, 5ng / ml recombinant human interleukin-7, and 5ng / ml recombinant human interleukin-15, and 10ug / ml recombinant human ApoE3 (Peprotech, Catalog No. 350-02). 48 hours after activation, transduced T cells were washed and suspended in OpTmizer basal medium containing 200U / mL recombinant human interleukin-2, 5ng / ml recombinant human interleukin-7, and 5ng / ml recombinant human interleukin-15 with 2.5% human AB serum (Gemini, Cat. No. 100-512). The pre-incubated LNP mixture was added to each 15mL tube to give a final concentration of 5ug / ml total RNA. In the double knockout group, the final concentration was 10ug / ml total RNA. Media supplemented with ApoE3 was used as a vehicle control. After 24 hours, T cells were collected, washed, and cultured in T cell RPMI medium with cytokines as described in Example 2 to expand until the day of injection (15 days after activation).

[0266] 3.2 Flow cytometry analysis of protein expression in engineered CD3+CD4+ T cells Target expression was verified by flow cytometry as in Example 2. Data are shown in Table 9 and Figures 5A-5E. [Table 8]

[0267] 3.3 In vivo evaluation of inflammatory responses in GvHD models The in vivo suppressive function of engineered CD3+CD4+ T cells overexpressing IL10 and CTLA4 alone (mock KO) or in combination with editing to knockdown expression of IFNG, TNFA, or double knockdown of IFNG and TNFA was assessed using a graft-versus-host disease mouse model as in Example 2. Survival data are shown in Table 10 and Figure 6A. Human leukocyte engraftment was assessed by spleen composition as in Example 2. Data are shown in Table 11 and Figure 6B. [Table 9] [Table 10]

[0268] 3.4 Cytokine profile of CD3+CD4+ engineered cells Selected transformed CD3+CD4+ T cells were stimulated and cytokine profiles assessed in triplicates as in Example 2. The results are shown in Table 12 and Figures 7A-7F. [Table 11]

[0269] 3.5 Mixed lymphocyte reaction assay of suppressor function A mixed lymphocyte reaction was used to assay the suppressive function of the transduced cells as in Example 2, using the ratios of CTV-labeled T cells:engineered T cells listed in Table 13. The data are shown in Table 13 and Figure 8. [Table 12]

[0270] Example 4. In vivo evaluation of inflammatory responses in GvHD models The in vivo suppressive function of engineered CD3+CD4+ T cells edited to overexpress wild-type or high affinity versions of IL10 and CTLA4 and knockdown expression of IFNG, TNFA was assessed in a mouse model of graft-versus-host disease as in Example 2, except that 5x10^6 PBMCs and Tregs were injected. Survival data are shown in Table 14 and Figure 9A. Human leukocyte engraftment was assessed by spleen composition as in Example 2. Data are shown in Table 15 and Figure 9B. [Table 13] [Table 14]

[0271] Example 5. Suppressive capacity of engineered T cells after exposure to inflammatory cytokines Because the stability of the Treg suppressive phenotype in an inflammatory environment is an important consideration for Treg therapy, we therefore exposed sTregs to the proinflammatory cytokines TNF-α, IL-6, and IL-1β and assessed their suppressive function in vitro.

[0272] 5.1 T cell manipulation Human CD3+CD4+ T cells were isolated from leukopaks, activated, and transduced with lentiviral constructs to promote overexpression of IL10 and CTLA4 as described in Example 2.2. One day after transduction, transduced cells were engineered with Cas9 to disrupt the TNFA and IFNG genes using gRNAs G019754 (INFG) and G019760 (TNFA) as described in Example 3.1.

[0273] A portion of the sorted engineered CD3+CD4+ T cells were cultured in the presence of a cocktail of proinflammatory cytokines (100 ng / mL each of TNF-α (Miltenyi; 130-094-014), IL-1β (Miltenyi; 130-093-898), and IL-6 (Miltenyi; 130-095-365)). TNF-α, IL-1β, and IL-6 were replenished every 2 days for 8 days, at which point their function was assessed using an MLR suppression assay.

[0274] Target expression was verified by flow cytometry as in Example 2. Data are shown in Table 16. [Table 15] Sorted transduced CD3+CD4+ T cells were stimulated in triplicate and cytokine profiles assessed as in Example 2. Data are shown in Tables 17 and 18. [Table 16] [Table 17]

[0275] 5.2 Mixed lymphocyte reaction assay of suppressor function As in Example 2, a mixed lymphocyte reaction was used to assay the suppressive function of transduced cells at a 1:1 ratio of CTV-labeled T cells to engineered T cells with or without the addition of 100 ng / mL each of TNF-α, IL-1β, and IL-6. The data are shown in Tables 19 and 20, and Figures 10A-B. [Table 18] [Table 19]

[0276] Example 6. Suppressive capacity of engineered T cells in an inflammatory bowel disease model Tregs are known to suppress the induction of colitis in preclinical models. Furthermore, mutations in IL-10 are known to be a risk factor for the development of colitis in humans. Importantly, inflamed colons are known to express high levels of MAdCAM-1, an adhesion molecule for lymphocytes. T cells that overexpress IL10 and CTLA4, edited to disrupt genes encoding IFNG and TNFA, and engineered to express an anti-MAdCAM CAR, have been shown to suppress the induction of colitis in humanized mouse models of inflammatory bowel disease (IBD) (see, e.g., Gottel et al., Low-Dose Interleukin-2 Ameliorates Colitis in a Preclinical Humanized Mouse Model. Cell Mol Gastroenterol Hepatol. 2019;8(2):193-195), or CD45RB in IBD. hi It has been used for treatment in migration models (see, e.g., Asseman et al., An Essential Role for Interleukin 10 in the Function of Regulatory T Cells That Inhibit Intestinal Inflammation. J Expt Med. 190(7):995-1003).

[0277] Briefly, on day 0, NOG mice were injected with 20 × 10 6Humanized mouse IBD models are induced by injecting PBMCs and administering an enema containing 50 μL of 2,4-dinitrobenzenesulfonic acid (DNBS) (Sigma-Aldrich, Cat. No. 556971) suspended in 50% aqueous ethanol (X w / v). To evaluate engineered T cells in a preventative setting, 20×10^6 engineered T cells provided herein are co-transplanted with PBMCs from the same donor on day 0. In a therapeutic setting of the humanized model, PBMCs are injected on the day of the DNBS enema, and 20×10^6 engineered T cells from the same donor as the PBMCs are transplanted on various days thereafter. Mice are euthanized and colons are collected after a 20% weight loss or at a predefined time in the absence of at least a 20% weight loss. Mice are analyzed for the extent of colitis using known methods. For example, the extent of colitis is determined by the total length of the colon and the ratio of colon length:body weight. Colons are fixed in formalin and histologically analyzed for colonic epithelial thickening. Prophylactic and therapeutic treatment of mice with engineered T cells with a combination of CTLA4 and IL10 insertion and knockout of both IFNG and TNFA significantly reduces epithelial thickening. In some embodiments, prophylactic and therapeutic treatments include administering engineered T cells with CTLA4 insertion and knockout of both IFNG and TNFA significantly reduces epithelial thickening. In some embodiments, prophylactic and therapeutic treatments include administering engineered T cells with IL10 insertion and knockout of both IFNG and TNFA significantly reduces epithelial thickening.

[0278] T helper cell type 1-mediated / CD45RB in IBD hi The transplantation model was to transfer CD45RB IgG from BALB / c (Taconic; BALB) mice to immunodeficient SCID mice (Taconic; CB17SC). hi Colitis is induced by the transfer of CD3+CD4+CD25- T cells. Colitis is due to the development of a Th1 response because polarized Th1 cells are present in the intestinal lesions. 低 CD4+ T cell subsets and normal and pathological CD45RB 高Co-transplantation of CD45RB cells from normal mice prevents the development of colitis. 低 It has been shown that CD4+ subsets contain a population of regulatory T cells that can control inflammatory responses in the intestine (Powrid et al., Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in CB-17 scid mice. Int. Immunol. 5:1461-1471). Cell populations are isolated using known methods and prepared for intraperitoneal injection. Engineered Tregs and nTregs are prepared as in Example 2 above. Mice are euthanized after 20% weight loss or at a predefined time in the absence of at least 20% weight loss, and their colons are collected. The extent of colitis in mice is analyzed using known methods as provided in humanized mouse models of IBD. Treatment of mice with engineered T cells with the combination of CTLA4 and IL10 insertion and knockout of both IFNG and TNFA significantly reduces epithelial thickening by more than 50%. In some embodiments, the prophylactic and therapeutic treatments include administering engineered T cells with an insertion of CTLA4 and knockout of both IFNG and TNFA, which significantly reduces epithelial thickening by more than 50%. In some embodiments, the prophylactic and therapeutic treatments include administering engineered T cells with an insertion of IL10 and knockout of both IFNG and TNFA, which significantly reduces epithelial thickening by more than 50%. The treatments also reduce weight loss and extend survival.

Claims

1. 1. An engineered T cell, comprising: i) a heterologous nucleic acid encoding a regulatory T cell-promoting molecule under the control of a promoter sequence; ii) a modification of an endogenous nucleic acid sequence encoding interferon-gamma (IFNG), said modification knocking down expression of said IFNG; iii) a modification of an endogenous nucleic acid sequence encoding tumor necrosis factor alpha (TNFA), wherein the modification knocks down expression of the TNFA.

2. The regulatory T cell promoting molecule is selected from the group consisting of interleukin-10 (IL10), cytotoxic T lymphocyte-associated protein 4 (CTLA4), transforming growth factor beta 1 (TGFB1), indoleamine 2,3-dioxygenase 1 (IDO1), ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), 5'-nucleotidase ecto (NT5E), interleukin-22 (IL-22), amphiregulin (AREG), indole-2, ... cell promoting molecule (TCT1), indole-2, cytotoxic T cell promoting molecule (TCT1), indole-2, cytotoxic T cell promoting molecule (TCT1), indole-2, cytotoxic T cell promoting molecule (TCT1), indole-2, cytotoxic T cell promoting molecule (TCT1), indole-2, cytotoxic T cell promoting molecule (TCT1), indole-2, cytotoxic T cell promoting molecule (TCT1), indole-2, cytotoxic T cell promoting molecule (TCT1), indole-2, cytotoxic T cell promoting molecule (TCT1), indole-2, cytotoxic T cell promoting molecule (TCT1), indole-2, cytotoxic T cell promoting molecule (TCT1), indole-2, cytotoxic T cell promoting molecule (TCT1), indole-2, cytotoxic T cell promoting molecule (TCT1), indole-2, cytotoxic T cell promoting 2. The engineered T cell of claim 1, wherein the T cell is selected from interleukin-35 (IL35), GARP, CD274 molecule (CD274), forkhead box P3 (FOXP3), IKAROS family zinc finger 2 (IKZF2), familial eosinophilia (EOS), interferon regulatory factor 4 (IRF4), lymphoid enhancer binding factor 1 (LEF1), and BTB domain and CNC homolog 2 (BACH2).

3. The engineered T cell of claim 1 or 2, wherein the regulatory T cell promoting molecule is IL10 or CTLA4.

4. 4. The engineered T cell of any of claims 1 to 3, wherein the regulatory T cell-promoting molecule is a first regulatory T cell-promoting molecule and further comprises a heterologous nucleic acid encoding a second regulatory T cell-promoting molecule under the control of a promoter sequence, optionally wherein the first and second regulatory T cell-promoting molecules are IL10 and CTLA4. (i) further comprising a modification of an endogenous nucleic acid sequence encoding interleukin 17A (IL17A), interleukin 2 (IL2), interleukin 6 (IL6), perforin 1 (PRF1), granzyme A (GZMA), or granzyme B (GZMB), wherein the modification knocks down expression of the IL17A, IL2, IL6, PRF1, GZMA, or GZMB, respectively; and / or (ii) further comprising a modification of an endogenous nucleic acid sequence encoding an endogenous T cell receptor (TCR), wherein the modification knocks down expression of the endogenous TCR. The engineered T cell of any one of claims 1 to 4.

6. 6. The engineered T cell of any one of claims 1 to 5, further comprising a heterologous coding sequence for a targeting receptor under the control of a promoter sequence.

7. (i) the targeted receptor is targeted to a ligand selected from mucosal vascular addressin cell adhesion molecule 1 (MADCAM1), tumor necrosis factor alpha (TNFA), CEA cell adhesion molecule 6 (CEACAM6), vascular cell adhesion molecule 1 (VCAM1), citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNF receptor superfamily member 17 (TNFRSF17), dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), desmoglein 3 (DSG3), and MHC class I HLA-A (HLA-A*02); and / or (ii) the targeting receptor comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR); The engineered T cell of claim 6.

8. 8. The engineered T cell of claim 7, wherein the heterologous nucleic acid encoding the targeting receptor is incorporated into an expression construct.

9. 9. The engineered T cell of any one of claims 6 to 8, wherein the heterologous nucleic acid encoding a targeting receptor is in an expression construct that does not include a nucleic acid encoding a regulatory T cell promoting molecule.

10. 10. The engineered T cell of any one of claims 4 to 9, wherein the heterologous nucleic acid encoding the first regulatory T cell-promoting molecule is incorporated into an expression construct and the heterologous nucleic acid encoding the second regulatory T cell-promoting molecule is incorporated into an expression construct.

11. 11. The engineered T cell of any one of claims 4 to 10, wherein the heterologous nucleic acid encoding the first regulatory T cell-promoting molecule and the heterologous nucleic acid encoding the second regulatory T cell-promoting molecule are incorporated into separate expression constructs.

12. 11. The engineered T cell of claim 9 or 10, wherein the heterologous nucleic acid encoding the first regulatory T cell-promoting molecule and the heterologous nucleic acid encoding the second regulatory T cell-promoting molecule are integrated into a single expression construct.

13. 13. The engineered T cell of claims 8 or 10-12, wherein the expression construct further comprises a nucleic acid encoding a targeting receptor.

14. The engineered T cell of any one of claims 8 to 13, wherein at least one heterologous coding sequence is in an episomal expression construct.

15. The engineered T cell of any one of claims 1 to 13, wherein at least one heterologous coding sequence is inserted into the genome.

16. 16. The engineered T cell of claim 15, wherein the insertion into the genome is a non-targeted insertion.

17. The engineered T cell of claim 15, wherein the insertion is a targeted insertion.

18. 18. The engineered T cell of claim 17, wherein the targeted insertion is into a site selected from a TCR gene locus, a TNF gene locus, an IFNG gene locus, an IL17A gene locus, an IL6 gene locus, an IL2 gene locus, an adeno-associated virus integration site 1 (AAVS1) locus, and optionally, the TCR gene locus is the T cell receptor alpha constant (TRAC) locus.

19. 19. The engineered T cell of any one of claims 1-18, wherein the modification that knocks down expression of a gene comprises one or more of an insertion, deletion, or substitution.

20. A population of cells comprising an engineered T cell according to any one of claims 1 to 19.

21. A population of cells comprising an engineered T cell according to any one of claims 1 to 20, wherein at least 30%, preferably at least 40% of the cells of said population comprise a heterologous nucleic acid sequence encoding a regulatory T cell promoting molecule under the control of a promoter sequence, at least 50% of the cells of said population, preferably at least 70% of the cells contain a modification of the endogenous nucleic acid sequence encoding IFNG, A population of cells, wherein at least 50%, preferably at least 70%, of the cells in said population comprise a modification of an endogenous nucleic acid sequence encoding TNFA.

22. 22. The population of engineered T cells of claim 21, wherein the percentage of cells containing the insertion or modification is determined by the percentage of reads by next generation sequencing (NGS).

23. 23. The population of engineered T cells of claim 21 or 22, further comprising a modification of at least one endogenous nucleic acid sequence encoding interleukin 17A (IL17A), interleukin 6 (IL6), interleukin 2 (IL2), perforin 1 (PRFl), granzyme A (GZMA), or granzyme B (GZMB), wherein the population of cells comprises at least one modification of the IL17A, IL6, IL2, PRFl, GZMA, or GZMB, respectively, in at least 70% of the population of cells, preferably at least 80% of the population of cells, wherein the modification knocks down expression of the at least one of the IL17A, IL6, IL2, PRFl, GZMA, or GZMB, respectively.

24. 24. The population of engineered T cells of any one of claims 21 to 23, wherein at least 50%, preferably at least 70% of the cells comprise a knockdown of the TCR.

25. 25. The population of engineered T cells of any one of claims 21 to 24, wherein at least 30%, preferably at least 40% of the cells comprise an insertion of a nucleic acid coding sequence for a targeting receptor.

26. A pharmaceutical composition comprising an engineered T cell according to claims 1 to 19 or a population of engineered T cells according to claims 20 to 25.

27. A composition comprising a cell according to any one of claims 1 to 19 or a population of cells according to any one of claims 20 to 25, or a pharmaceutical composition according to claim 26, for use in the treatment of an immune disorder.

28. 27. A composition comprising a cell according to any one of claims 1 to 19 or a population of cells according to any one of claims 20 to 25, or a pharmaceutical composition according to claim 26, for use in the treatment of an autoimmune disease, optionally wherein the autoimmune disease is selected from ulcerative colitis, Crohn's disease, rheumatoid arthritis, psoriasis, multiple sclerosis, systemic lupus erythematosus, and type 1 diabetes.

29. A composition comprising a population of cells according to any one of claims 1 to 19 or cells according to any one of claims 20 to 25, or a pharmaceutical composition according to claim 26, for use in the treatment of graft-versus-host disease (GvHD).

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