Engineered T cells

Abstract

The present disclosure relates to T cells engineered to contain a heterologous nucleic acid sequence encoding double mutant transforming growth factor beta 1 (dmTGFB1) under the control of a promoter sequence. In certain embodiments, the cells further comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, and 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 XML format, which is incorporated herein by reference in its entirety. The XML copy was created on June 28, 2023, is titled 12793_0036-00304_SL_fhfgd, and is 359,125 bytes in size.

[0002] Related Applications This application incorporates by reference U.S. Provisional Application No. 63 / 367,275, filed June 29, 2022, the entire contents of which are expressly incorporated herein by reference. [Background technology]

[0003] Adaptive immunity is a defense mechanism by which the body can eliminate foreign pathogens. T cells are the immune cells that can mediate 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). While many autoreactive T cells are eliminated by clonal deletion in the thymus, other cells can escape clonal deletion and induce harmful immune responses. Ibid. Specialized T cells called regulatory T cells (Tregs) are 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., insufficient numbers of Tregs or improper functioning of Tregs) can contribute to autoimmune responses. Ibid.

[0005] Current therapies for treating autoimmune disorders aim to suppress adaptive immune processes or immune cell activation. While these therapies can suppress harmful immune responses (e.g., autoimmune responses), they can also suppress beneficial immune responses. Treg therapy has been used to suppress antigen-specific immune responses in various 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 therapy." Ibid. Therefore, there remains a need for effective T cell therapies (including Treg therapy) to suppress immune responses (including inflammation and autoimmunity). Summary of the Invention

[0006] The present disclosure provides double mutant transforming growth factor beta 1 (dmTGFB1) polypeptides, such as human dmTGFB1, and nucleic acids encoding same, as well as methods and uses thereof. The present disclosure provides T cells or populations of T cells engineered to contain a heterologous nucleic acid encoding dmTGFB under the control of a promoter sequence. The present disclosure also provides T cells or populations of T cells expressing dmTGFB under the control of a promoter sequence, as well as compositions and uses thereof, for example, to suppress immune response(s), including inflammation and autoimmunity.

[0007] In some embodiments, the cell further comprises a modification of an endogenous nucleic acid sequence encoding interferon-gamma (IFNG), wherein the modification knocks down expression of IFNG. In some embodiments, the cell further comprises a modification of an endogenous nucleic acid sequence encoding tumor necrosis factor alpha (TNFA), wherein the modification knocks down expression of TNFA. In some embodiments, the cell further comprises a modification of an endogenous nucleic acid sequence encoding interleukin-17a (IL17A), wherein the modification knocks down expression of IL17A. In some embodiments, the further modification comprises a modification of the endogenous nucleic acid sequence encoding TNFA and a modification of the endogenous nucleic acid sequence encoding IFNG. In some embodiments, the further modification comprises a modification of the endogenous nucleic acid sequence encoding TNFA and a modification of the endogenous nucleic acid sequence encoding IL17A. In some embodiments, the further modification comprises a modification of the endogenous nucleic acid sequence encoding TNFA, a modification of the endogenous nucleic acid sequence encoding IFNG, and a modification of the endogenous nucleic acid sequence encoding IL17A.

[0008] In some embodiments, the cells further comprise a heterologous nucleic acid encoding a regulatory T cell-promoting molecule.

[0009] The present disclosure provides a T cell or population of T cells engineered to contain a heterologous nucleic acid encoding a regulatory T cell-promoting molecule under the control of a promoter sequence, a modification in an endogenous nucleic acid sequence encoding tumor necrosis factor alpha (TNFA) that knocks down expression of TNFA, a modification in an endogenous nucleic acid sequence encoding tumor necrosis factor alpha (TNFA) that knocks down expression of TNFA, and a modification in an endogenous nucleic acid sequence encoding interleukin-17a (IL17A) that knocks down expression of IL17A. In certain embodiments, the T cell or population of T cells does not contain a modification in an endogenous nucleic acid sequence encoding interferon-gamma (IFNG) that knocks down expression of IFNG.

[0010] In some embodiments, the regulatory T cell-promoting molecule is interleukin-10 (IL10), cytotoxic T lymphocyte-associated protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), 5′-nucleotidase ecto (NT5E), interleukin-22 (IL-22), amphiregulin (AREG), interleukin-35 (IL 35), 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), BTB domain and CNC homolog 2 (BACH2), and interleukin 2 receptor subunit alpha (IL2RA, CD25). In some embodiments, the regulatory T cell-stimulating molecule is selected from IL10 or CTLA4. In some embodiments, the regulatory T cell-stimulating molecule is IL10 and CTLA4.

[0011] In some embodiments, a T cell or population of T cells is engineered to comprise heterologous nucleic acids encoding dmTGFB1 and IL10, each under the control of a promoter sequence, and a modification in an endogenous nucleic acid sequence encoding TNFA that knocks down expression of TNFA, a modification in an endogenous nucleic acid sequence encoding IFNG that knocks down expression of IFNG, or a modification in an endogenous nucleic acid sequence encoding IL17A that knocks down expression of IL17A. In some embodiments, the cells comprise modifications in endogenous nucleic acid sequences encoding each of IFNG and IL17A that knock down expression of each of IFNG and IL17A.

[0012] In some embodiments, a T cell or population of T cells is engineered to comprise heterologous nucleic acids encoding dmTGFB1 and CTLA4, each under the control of a promoter sequence, and a modification in an endogenous nucleic acid sequence encoding a TNFA, wherein the modification knocks down expression of the TNFA. In some embodiments, the T cell or population of T cells comprises an additional modification in an endogenous nucleic acid sequence encoding IFNG, wherein the modification knocks down expression of the TNFA. In some embodiments, the T cell or population of T cells comprises an additional modification in an endogenous nucleic acid sequence encoding IL17A, wherein the modification knocks down expression of the IL17A. In some embodiments, the T cell or population of T cells comprises a modification in an endogenous nucleic acid sequence encoding each of IFNG and TNFA, wherein the modification knocks down expression of the IFNG and TNFA, respectively. In some embodiments, the T cell or population of T cells comprises a modification in an endogenous nucleic acid sequence encoding each of IL17A and TNFA, wherein the modification knocks down expression of the IL17A and TNFA, respectively. In some embodiments, the T cell or population of T cells comprises a modification in an endogenous nucleic acid sequence encoding each of TNFA, IFNG, and IL17A, wherein the modification knocks down expression of each of TNFA, IFNG, and IL17A.

[0013] In some embodiments, a T cell or population of T cells is engineered to comprise heterologous nucleic acid sequences encoding dmTGFB1, IL10, and CTLA4, each under the control of a promoter sequence, and a modification in an endogenous nucleic acid sequence encoding a TNFA, wherein the modification knocks down expression of the TNFA. In some embodiments, the T cell or population of T cells comprises an additional modification in an endogenous nucleic acid sequence encoding IFNG, wherein the modification knocks down expression of IFNG. In some embodiments, the T cell or population of T cells comprises an additional modification in an endogenous nucleic acid sequence encoding IL17A, wherein the modification knocks down expression of IL17A. In some embodiments, the T cell or population of T cells comprises a modification in an endogenous nucleic acid sequence encoding each of IFNG and IL17A, wherein the modification knocks down expression of each of IFNG and IL17A.

[0014] 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 interleukin-2 (IL2), interleukin 6 (IL6), perforin 1 (PRFI), granzyme A (GZMA), granzyme B (GZMB), Fas ligand (FasL, NF superfamily, member 6), ryanodine receptor 2 (RYR2), and colony-stimulating factor 2 (CSF2). 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 RYR2.

[0015] 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).

[0016] 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 targeting 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). In some embodiments, the targeting receptor is targeted to mucosal vascular addressin cell adhesion molecule 1 (MADCAM1). In some embodiments, the targeting receptor is targeted to tumor necrosis factor alpha (TNFA).

[0017] 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 dmTGFB, e.g., as assessed by sequencing (e.g., NGS). In certain embodiments, additional modifications are present.

[0018] 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).

[0019] In some embodiments, at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, 80%, 85%, 90%, or 95% of a population of T cells comprise a modification (e.g., knockdown) in an 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 a population of T cells comprise a modification (e.g., knockdown) in a TNFA sequence, e.g., as assessed by sequencing (e.g., NGS).

[0020] 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 IL17A sequence, as assessed, for example, by sequencing (e.g., NGS).

[0021] 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 a 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 comprise an insertion of a sequence encoding a targeting receptor (e.g., a CAR), e.g., as assessed by sequencing (e.g., NGS).

[0022] The modifications described herein for knocking down gene expression 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 the 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, and the adeno-associated virus integration site 1 (AAVS1) locus.

[0023] 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).

[0024] In some embodiments, insertion of or modification (e.g., knockdown) of a sequence(s) described herein can be mediated by a guide RNA in combination with an RNA-guided DNA binding agent (e.g., a Cas nuclease). In some embodiments, insertion of or knockdown of a sequence(s) described herein 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 explanation of the drawings]

[0025] [Figure 1]Total TGF-β1 quantified in the culture supernatant is shown. [Figure 2] Active TGF-β1 quantified in the culture supernatant is shown. [Figure 3] Shown is the immediate suppression of autologous T cell proliferation by T cells overexpressing wild-type or mutant TGF-β1 as measured by CTV dilution. [Figure 4] 1 shows the survival probability over time after injection of engineered CD3+CD4+ cells into NOG mice. [Figure 5] Shown is the percent inhibition of cell proliferation by engineered T cells as measured by CTV dilution. [Figure 6] Total TGF-β1 production (pg / ml) by transduced cells is shown upon cell stimulation. [Figure 7] The production of active TGF-β1 (pg / ml) by the transduced cells upon cell stimulation is shown. [Figure 8] IFN-γ production (pg / ml) of transduced cells upon cell stimulation is shown. [Figure 9] TNF-α production (pg / ml) of transduced cells upon cell stimulation is shown. [Figure 10] IL-17a production (pg / ml) of transduced cells is shown upon cell stimulation. [Figure 11] IL-2 production (pg / ml) of transduced cells upon cell stimulation is shown. [Figure 12] IL-10 production (pg / ml) of transduced cells upon cell stimulation is shown. [Figure 13] IL-13 production (pg / ml) of transduced cells upon cell stimulation is shown. [Figure 14] The percentage of mice within each cohort surviving at each time point after injection of engineered suppressor T cells is shown. [Figure 15] The percentage of mice within each cohort surviving at each time point after injection of engineered suppressor T cells is shown. [Figure 16] The percentage of the initial body weight of each individual mouse in the treatment group is shown. [Figure 17] Colon length is given in cm. [Figure 18] The percentage of Tregs recovered from each tissue stained with Cell Trace Violet is shown. DETAILED DESCRIPTION OF THE INVENTION

[0026] 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. Rather, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the invention as defined by the accompanying embodiments.

[0027] The section headings used herein are for organizational purposes only and should not be construed as limiting the desired subject matter in any way. 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.

[0028] I. Definition Before describing the teachings of the present invention in detail, it should be understood that the present disclosure is not limited to particular compositions or process steps, as such may vary. It should be noted that, as used in this specification and the accompanying embodiments, 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, and so forth. 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 for or added to the listed items.

[0029] Numerical ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximations taking into account significant digits and inherent error in measurement. Also, 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.

[0030] Unless otherwise noted herein, embodiments described herein as "comprising" various components are also contemplated as "consisting of" or "consisting essentially of" the listed components, and embodiments described herein as "consisting of" various components are also contemplated as "comprising" or "consisting essentially of" the listed components. Also, embodiments described herein as "consisting essentially of" various components are also contemplated as "consisting of" or "including" the listed components (this interchangeability does not apply to the use of these terms in the claims).

[0031] The term "or" is used in its inclusive sense, ie, equivalent to "and / or," unless the context clearly dictates otherwise.

[0032] 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 the 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 the typical tolerances in the art. For example, "about" can be understood to mean about 2 standard deviations from the mean. In certain embodiments, about means + / - 10%. In certain embodiments, about means + / - 5%.

[0033] In some embodiments, a population of cells refers to a population of at least 10^3, 10^4, 10^5 or 10^6 cells, preferably 10^7, 2x10^7, 5x10^7, or 10^8 cells.

[0034] The term "at least" before a number or series of numbers is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that can 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 specified 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.

[0035] As used herein, "less than" or "less than" is understood to refer to the expression and the logical lower zero or neighboring 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" precedes a series of numbers or ranges, it is understood that each of the series or ranges is modified.

[0036] As used herein, ranges include both upper and lower limits.

[0037] As used herein, when the maximum amount of a value is represented by 100% (for example, 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.

[0038] In the event of a discrepancy between a sequence in this application and a designated accession number or position within an accession number, the sequence in this application will control.

[0039] Unless otherwise stated, the following terms and phrases, when used herein, are intended to have the following meanings:

[0040] 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 by at least 70%, 75%, 80%, 85%, 90%, 95%, or to below the level of detection in an assay, for example, compared to a suitable control in which the gene sequence is not modified. Protein knockdown can be measured by detecting the amount of protein in a tissue, cell population, or fluid of interest. Methods for measuring mRNA knockdown are known, including sequencing 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 fluids derived therefrom, such as serum or plasma. In some embodiments, "knockdown" can 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 mRNA sequences result in reduced expression of wild-type or full-length proteins. In some embodiments, "knockdown" can refer to some loss of expression of a particular gene product (e.g., an IFNG or TNFA gene product in a body fluid or tissue culture medium). Modification of the nucleic acid sequence encoding an endogenous nucleic acid sequence (e.g., an IFNG or TNFA) can result in knockdown.

[0041] As used herein, "T cell receptor" or "TCR" refers to a receptor in a T cell. Generally, a TCR is a heterodimeric receptor molecule comprising two TCR polypeptide chains, an α chain and a β chain. After antigen binding, the α chain and β chain TCR polypeptides complex with various CD3 molecules and can induce immune response(s), including inflammation and autoimmunity. As used herein, knockdown of a TCR refers to the partial or total knockdown of any TCR gene (e.g., deletion of a portion of the TRBC1 gene, alone or in combination with the partial or total knockdown of other TCR gene(s)).

[0042] "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.

[0043] "TRBC" refers to the T cell receptor β chain (e.g., TRBC1 and TRBC2). "TRBC1" and "TRBC2" refer to two homologous genes encoding the T cell receptor β chain, which is the gene product of the TRBC1 or TRBC2 gene.

[0044] 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.

[0045] 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 for TRBC2.

[0046] As used herein, "immune response" refers to one or more immune system reaction(s), e.g., an increase in the 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 (e.g., a microorganism, a virus, a prion, a fungus, etc.), an allergen (e.g., dust, pollen, a dust mite, etc.), a toxin (e.g., a chemical, a drug, etc.), or a physiological change (e.g., hypercholesterolemia, obesity, organ transplant, etc.)). An immune response can also include the 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 can target, attack, eliminate, or neutralize an antigen (e.g., foreign or self). An immune response can be desirable or undesirable. An immune response can be acute or chronic. An immune response can damage the cells, tissues, or organs against which the immune response is initiated.

[0047] As used herein, "autoimmune response" refers to one or more immune system reaction(s) against autoantigens (e.g., autoantigens presented by a subject's own cells, tissues, or organs). An autoimmune response can 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 can result in inflammation (e.g., persistent inflammation) or lead to autoimmune disease. An autoimmune response can target, attack, eliminate, or neutralize autoantigens produced by a subject's own cells, tissues, or organs, which can lead to autoimmune disease.

[0048] 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., immune system cell production or activity) compared to a suitable control (e.g., a control not treated with, 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 with, 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 count (e.g., lymphocyte count or spleen cell count), cell activity (e.g., T cell assay), or gene or protein expression (e.g., biomarker expression), and the 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).

[0049] 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 that are autologous or allogeneic.

[0050] As used herein, "immune disorder" refers to a disease or condition characterized by a pathological or unwanted 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, a subject with an immune disorder requires a suppressed immune response. In certain embodiments, a subject with an immune disorder requires increased immune tolerance.

[0051] "T cells" play a central role in immune responses following exposure to an antigen. T cells can be naturally occurring or non-naturally occurring, for example, when T cells are generated by engineering (e.g., from stem cells) or by transdifferentiation (e.g., somatic cell reprogramming). T cells can be distinguished from other lymphocytes by the presence of T cell receptors on their 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+. In some embodiments, T cells are CD8+.

[0052] "Regulatory T cells" or "Tregs" refer to specialized T cells that play a central role in suppressing excessive immune response(s), including inflammation and autoimmunity. Tregs can be naturally occurring or non-naturally occurring, e.g., when Tregs are generated by engineering, such as by inserting a sequence encoding a dmTGFB1 molecule. When generated by engineering, Tregs can further comprise modifications, e.g., by modifying (e.g., knocking down) endogenous nucleic acid sequences encoding IFNG, IL17A, and TNFA, and inserting at least one sequence(s) encoding a regulatory T cell-promoting molecule. Naturally occurring Tregs, or natural Tregs or nTregs (sometimes referred to as thymic Tregs or tTregs), are specialized T cells that typically develop in the thymus and function 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 regulatory T cell phenotypic characteristics and suppressive function by inserting a sequence encoding a dmTGFB1 molecule and, optionally, further modifying, e.g., 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, and these may be referred to as transduced or "engineered" T cells. In some embodiments, the engineered T cells comprise the insertion of a sequence encoding a dmTGFB1 molecule, modification of an endogenous nucleic acid sequence encoding IFNG, modification of an endogenous nucleic acid sequence encoding TNFA, and 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 comprise or consist of one or more indel or substitution mutations within the genomic sequence.

[0053] As used herein, dmTGFB1 refers to a mutant TGFB1 based on wild-type TGFB1 from any species, including, for example, human, mouse, rat, or cynomolgus monkey TGFB1. In certain embodiments, dmTGFB1 is human dmTGFB1, i.e., a mutant TGFB1 relative to wild-type human TGFB1. The coding sequence and amino acid sequence of wild-type TGFB1 are readily available in sequence databases such as NCBI, and exemplary sequences can be found under accession numbers NM_000660.7 and NP_000651.3 (human), NM_011577.2 and NP_035707.1 (mouse), NM_021578.2 and NP_067589.1 (rat), and XM_005589339.3 and XP_005589396.1 (cynomolgus monkey). Each accession number is incorporated by reference in the version available as of the filing date of this application. The ability to map mutations to the wild-type sequence is within the capabilities of one of ordinary skill in the art.

[0054] Transforming growth factor beta-1 (TGFB1) is synthesized as a large precursor molecule. The TGFB1 preprotein contains a 29-amino acid signal peptide that is proteolytically cleaved. TGFB1 is further cleaved after amino acid 278 to form the latency-associated peptide (LAP) and active TGFB1. LAP dimerizes through an interchain disulfide bond at C223 and C225. TGFB1 can be secreted as an inactive, small latent complex containing mature TGF-β1 homodimers noncovalently bound to LAP homodimers at LAP residues I53-L59. LAP protects the type II receptor binding site within mature TGFB1. Most cells secrete TGFB1 as a large latent complex (LLC) of TGF-β1 / LAP covalently linked between C33 of the LAP chain and the latent TGFB-binding protein (LTBP). LTBP facilitates TGFB1 folding, secretion, and potential targeting to the extracellular matrix. LLC activation occurs when the N-terminal domain of LTBP binds to the extracellular matrix.

[0055] Kamuracchi-Engelman disease (CED) is caused by domain-specific heterozygous mutations in the transforming growth factor-beta-1 gene (TGFB1, OMIM entry 190180, incorporated by reference in the version available as of the filing date of this application) on chromosome 19q13. Mutations reported in Kamuracchi-Engelman disease families include the following (from Janssen et al., 2006. J Med Genet. 43:1): [Table 1]

[0056] As shown in the table, the majority of pathogenic variants in CED patients result in single amino acid substitutions in the carboxy-terminus of the TGFB1 latency-associated peptide (LAP). The substitutions are close to the interchain disulfide bond site between LAP homodimers. These pathogenic variants inhibit LAP dimerization and binding to active TGFB1 (Walton et al., 2010. J Biol Chem. 285:17029-37), leading to increased release of active TGFB1 from cells. Walton et al. further demonstrated that the stability of the resulting large latent complex depends on the covalent dimerization of LAP, which is promoted by key residues at the dimer interface (F198, D199, V200, L208, F217, and L219). R218H mutant fibroblasts from patients with CED showed increased levels of active TGF-β1 in the cell culture medium compared with normal fibroblasts (Saito et al., 2001, J. Biol. Chem. 276:11469-72). In vitro analysis of R218C, H222D, and C225R mutant constructs also showed increased levels of active TGF-β1 in the culture medium of transfected cells. In contrast, the Leu11_Leu13dup and Tyr81His pathogenic variants caused decreased levels of secreted TGF-β1. However, in a luciferase reporter assay specific for TGF-β-induced transcriptional responses, the mutant cells showed increased luciferase activity, suggesting intracellular activation of the receptor (Janssens et al., 2003, J. Biol. Chem. 278:7718-24).

[0057] As used herein, in certain embodiments, dmTGFB1 refers to a TGFBl that contains mutations at two or more amino acid positions relative to wild-type TGFBl, which reduces the stability of the resulting large latent complex, which depends on covalent dimerization of LAP, compared to each TGFBl single mutant alone. The mutations at two or more amino acid positions may include mutations at positions selected from F198, D199, V200, L208, F217, L219, R218, H222, C223, and C225 relative to wild-type human TGFBl.

[0058] In certain embodiments, dmTGFB1 is TGFB1 that contains mutations at two or more amino acid positions compared to wild-type human TGFB1 at positions selected from F198, D199, V200, L208, F217, L219, R218, H222, C223, and C225, and is produced and secreted from cells in which TGFB1 is typically expressed. In some embodiments, dmTGFB1 contains a naturally occurring mutation, e.g., in the CED. In some embodiments, dmTGFB1 contains a non-naturally occurring mutation, e.g., a non-naturally occurring mutation in the CED.

[0059] In certain embodiments, the human dmTGFB1 comprises mutations at two or more amino acid positions selected from R218, H222, C223, and C225 compared to wild-type human TGFB1.

[0060] In certain embodiments, the human dmTGFB1 comprises two or more mutations selected from R218C / H, H222D, C223S / R / G, and C225R compared to wild-type human TGFB1.

[0061] In certain embodiments, the human dmTGFB1 comprises two or more mutations selected from R218C / H and C225R compared to wild-type human TGFB1.

[0062] The dmTGFB1 provided herein has more free active dmTGFB1 than any single mutant alone. In certain embodiments, the increase in active dmTGFB1 is at least additive compared to the activity of a single mutant alone. In certain embodiments, the increase in active dmTGFB1 is even more than additive compared to the activity of a single mutant alone. Methods for determining levels of total TGFB1 and active TGFB1 are known in the art, using commercially available kits (e.g., LEGEND MAX Total TGF-β1 ELISA kit (BioLegend, catalog number 436707) and LEGEND MAX Free Active TGF-β1 ELISA kit), as shown in the Examples below.

[0063] 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. 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. Regulatory T cell-promoting molecules can be used in combination with dmTGFB1 to promote the conversion of conventional T cells to regulatory T cells or confer regulatory activity to conventional T cells. Examples of immunosuppressive molecules that may be used in conjunction with dmTGFB1 include, but are not limited to, interleukin-10 (IL10), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), 5'-nucleotidase ecto (NT5E), interleukin-22 (IL22), amphiregulin (AREG), interleukin-35 (IL 35), 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), BTB domain and CNC homolog 2 (BACH2), and interleukin-2 receptor subunit alpha (IL2RA, CD25). 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 used in conjunction with dmTGFB1 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.

[0064] In some embodiments, conventional T cells can be engineered to modify, insert, or delete sequences within their genome, such that the "engineered" T cells exhibit one or more phenotypic characteristics and suppressive functions of natural regulatory T cells. For example, the "engineered" T cells exhibit suppressive activity in a mixed lymphocyte reaction assay, such as that provided in Example 2.4 below, or are capable of suppressing graft-versus-host disease, preferably statistically significantly, in a mouse model, such as that presented in Example 3.2 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 the modification (e.g., knockdown) of expression of pro-inflammatory cytokines (e.g., TNFA in combination with one or both of IFN-glucan and IL17A). 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.

[0065] As used herein, a "pro-inflammatory" molecule (e.g., a cytokine) increases the immune response as described herein and reduces the efficacy of Tregs in a dose-responsive manner, for example, in the mouse model of graft-versus-host disease presented in Example 3.2. Examples of pro-inflammatory molecules include, but are not limited to, IFNG, TNFA, IL17A, IL6, IL2, perforin 1 (PRF1), granzyme A (GZMA), granzyme B (GZMB), Fas ligand (FasL, NF superfamily, member 6), ryanodine receptor 2 (RYR2), and colony-stimulating factor 2 (CSF2).

[0066] 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 specific 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 that can activate a T cell upon binding of the 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, and myelin. These include 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).

[0067] 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 by reference in their entirety). Also contemplated are reverse universal CARs that facilitate immune cell-target cell binding via adapter molecules (see, e.g., WO2019238722, the contents of which are incorporated by reference in their entirety). CARs can be targeted to any antigen for which antibodies can be developed and are typically directed to molecules displayed on the surface of 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 containing 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, a CAR can target engineered T cells to inflamed tissues, e.g., a CAR targets TNFA to suppress immune responses in disorders such as rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, or Crohn's disease. In some embodiments, a CAR can target engineered T cells to inflamed tissues, e.g., a CAR targets TGF-b1 to suppress immune responses in disorders such as inflammatory bowel disease, ulcerative colitis, or Crohn's disease. In some embodiments, a CAR can target engineered T cells to neural tissues, e.g., a CAR targets MBP, MOG, or PLP1 to suppress immune responses in disorders such as multiple sclerosis. In some embodiments, a CAR can target engineered T cells to tissues containing mature B lymphocytes, e.g., a CAR targets TNFRSF17 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), desmoglein 3 (DSG3), or MHC class I HLA-A (HLA-A*02). Additional CAR targets (e.g., inflammatory antigens) are known in the art. See, e.g., WO2020092057A1, the contents of which are incorporated herein by reference in their entirety.

[0068] 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, preventing 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 characteristic of 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 treating autoimmune disorders (e.g., Remicade, Humira).

[0069] "Promoter" refers to a regulatory region that controls the expression of a gene to which it is linked.

[0070] "Polynucleotide" and "nucleic acid" are used herein to refer to polymeric compounds containing nucleosides or nucleoside analogs having nitrogen-containing heterocyclic bases or base analogs linked together along a backbone, and "polynucleotide" and "nucleic acid" 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 one or more of sugar-phosphodiester linkages, peptide-nucleic acid linkages ("peptide nucleic acids" or PNA, PCT Publication No. WO 95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar moiety of the nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions (e.g., 2'-methoxy, 2'-halide, or 2'-O-(2-methoxyethyl) (2'-O-moe) substitutions). Nitrogenous bases include conventional bases (A, G, C, T, U), their analogs (e.g., modified uridines, e.g., 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 5,378,825 and PCT Publication WO 93 / 13121). For a general discussion, see The Biochemistry of the Nucleic Acids, vol. 5-36, Adams et al., ed., 11 th ed., 1992). Nucleic acids can contain one or more "abasic" residues when the backbone does not contain a nitrogenous base at one or more positions in the polymer (U.S. Patent No. 5,585,481). Nucleic acids can contain only conventional RNA or DNA sugars, bases, and linkages, or can contain both conventional components 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 containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA-mimicking sugar conformation, enhancing hybridization affinity to complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). Nucleic acids include "unlocked nucleic acids" or UNAs. RNA and DNA can have different sugar moieties, which can be distinguished by the presence of uracil or its analogs in RNA and thymine or its analogs in DNA.

[0071] As used herein, "polypeptide" refers to a multimeric compound comprising amino acid residues capable of adopting a three-dimensional conformation. Polypeptides include, but are not limited to, enzymes, proenzyme proteins, regulatory proteins, structural proteins, receptors, nucleic acid binding proteins, antibodies, and the like. Polypeptides can, but need not, include post-translational modifications, unnatural amino acids, prosthetic groups, and the like.

[0072] As used herein, the "open reading frame" or "ORF" of a gene refers to a sequence of codons that specifies the amino acid sequence of the protein encoded by that gene. An ORF generally begins with a start codon (e.g., ATG in DNA or AUG in RNA) and ends with a stop codon (e.g., TAA, TAG, or TGA in DNA or UAA, UAG, or UGA in RNA).

[0073] "Guide RNA," "gRNA," and "guide" are used interchangeably herein to refer to either crRNA (also known as CRISPR RNA) or a combination of crRNA and trRNA (also known as tracrRNA). The crRNA and trRNA can associate as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). "Guide RNA" or "gRNA" refer to each type. The trRNA may be a naturally occurring sequence or a trRNA sequence that has modifications or variations compared to the naturally occurring sequence.

[0074] As used herein, "guide sequence" or "guide region" or "targeting sequence" or "spacer" or "spacer sequence" refers to a sequence within a gRNA that is complementary to a target sequence and functions to direct the gRNA to the target sequence for binding or modification (e.g., cleavage) by an RNA-guided nickase. A guide sequence can be 20 nucleotides in length, for example, in the case of Streptococcus pyogenes (i.e., Spy Cas9 (also referred to as SpCas9)) and related Cas9 homologs / orthologs. 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. A guide sequence can be 20-25 nucleotides in length, e.g., in the case of NmeCas9, it can be 20, 21, 22, 23, 24, or 25 nucleotides in length. For example, a 24 nucleotide long guide sequence can be used with Nme Cas9 (e.g., Nme2 Cas9).

[0075] In some embodiments, the target sequence, e.g., within a gene locus or on a chromosome, is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between the guide sequence and its corresponding target sequence can be about 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the guide sequence and target region can be 100% complementary or identical. In other embodiments, the guide sequence and target region can contain at least one mismatch. For example, the guide sequence and target sequence can contain one, two, three, or four mismatches, in which case 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 target region can contain one to four mismatches, in which case the guide sequence includes 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, in which case the guide sequence comprises 20 nucleotides. In some embodiments, the degree of complementarity or identity between the guide sequence and its corresponding target sequence may be at least 80%, 85%, preferably 90%, or 95%, for example, in which case the guide sequence comprises a sequence of 24 contiguous nucleotides. In some embodiments, the guide sequence and the target region may be 100% complementary or identical. In other embodiments, the guide sequence and the target region may contain at least one mismatch, i.e., one nucleotide that is not identical or complementary, relative to the reference sequence. For example, the guide sequence and the target sequence may contain one to two, preferably one or less, mismatches, in which case the total length of the target sequence is 19, 20, 21, 22, preferably 23 or 24 nucleotides or more. In some embodiments, the guide sequence and the target region may contain one to two mismatches, in which case the guide sequence comprises at least 24 or more nucleotides. In some embodiments, the guide sequence and the target region may contain 1-2 mismatches, in which case the guide sequence comprises 24 nucleotides.The location of the mismatch is known in the art, e.g., PAM-distal mismatches tend to be more tolerated than PAM-proximal matches, and mismatch tolerance at other positions is known in the art (see, e.g., Sternberg et al., 2015, Nature:527:110-113).

[0076] As used herein, "target sequence" or "genomic target sequence" refers to a sequence of nucleic acid in a target gene genome locus, either in the plus or minus strand, that has complementarity to a gRNA's guide sequence, i.e., is sufficiently complementary to the gRNA's guide sequence to enable specific binding of the guide to the target sequence. Interaction between the target sequence and the guide sequence induces an RNA-guided DNA-binding agent to bind to the target sequence and potentially nick or cleave within the target sequence (depending on the activity of the binding agent). The specific length of the target sequence and the number of possible mismatches between the target sequence and the guide sequence depend, for example, on the identity of the Cas9 nuclease guided by the gRNA. Because the nucleic acid substrate of a Cas protein is a double-stranded nucleic acid, the target sequence of the Cas protein 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 said to be "complementary to a target sequence," it is understood that the guide sequence can direct an RNA-guided DNA-binding agent (e.g., dCas9 or a disordered Cas9) to bind to the reverse complement of the target sequence. That is, in some embodiments, when a guide sequence binds to the reverse complement of a target sequence, the guide sequence is identical to a particular nucleotide of the target sequence (e.g., the target sequence without the PAM) except for the T to U substitution in the guide sequence.

[0077] Because the nucleic acid substrate of an RNA-guided DNA binding agent is a double-stranded nucleic acid, the target sequence of the RNA-guided DNA binding agent 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 specific nucleotide in the target sequence (e.g., the target sequence without a PAM), except for the substitution of U for T in the guide sequence.

[0078] As used herein, "RNA-guided DNA binding agent" refers to a polypeptide or polypeptide complex 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 cleavases / nickases. Exemplary RNA-guided DNA binding agents can include inactivated forms thereof ("dCas DNA binding agents") (e.g., when these agents are modified to enable DNA cleavage, e.g., via fusion with a FokI cleavase domain). "Cas nuclease," as used herein, encompasses Cas cleavases and Cas nickases. Cas cleavases and Cas nickases include the Csm or Cmr complex of a type III CRISPR system, Cas10, its Csm1 or Cmr2 subunit, the Cascade complex of a type I CRISPR system, its Cas3 subunit, and class 2 Cas nucleases. As used herein, a "Class 2 Cas nuclease" refers to 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 also have RNA-guided DNA cleavage 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 enable DNA cleavage or when they have C→T deaminase or A→G deaminase activity). Cas nickases contain a nuclease, and one of the RuvC or HNH domains of the Cas protein is cleaved by the nuclease so that only one strand is cleaved. In some embodiments, the RNA-guided DNA binder contains 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 Cpfl 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., a Cas nuclease, a Cas9 nuclease, or an S. pyogenes Cas9 nuclease or a Neisseria meningitidis Cas9 nuclease) includes delivery of a polypeptide or mRNA.

[0079] As used herein, the term "editor" or "base editor" refers to an agent comprising a polypeptide that can modify a base (e.g., A, T, C, G, or U) in a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, the editor is capable of deaminating a base in a nucleic acid. In some embodiments, the editor is capable of deaminating a base in a DNA molecule. In some embodiments, the editor is capable of deaminating a cytosine (C) in DNA. In some embodiments, the editor is a fusion protein comprising an RNA-guided nickase fused to a cytidine deaminase domain. In some embodiments, the editor is a fusion protein comprising an RNA-guided nickase fused to an APOBEC3A deaminase (A3A). In some embodiments, the editor comprises a Cas9 nickase fused to an APOBEC3A deaminase (A3A). In some embodiments, the editor is a fusion protein comprising an enzymatically inactive RNA-guided DNA-binding protein fused to a cytidine deaminase domain.

[0080] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to a guide RNA together with an RNA-guided DNA-binding agent, such as a Cas nuclease, e.g., 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, such as Cas9, to a target sequence, where the guide RNA hybridizes to the target sequence, the binding agent binds to the target sequence, and, if the binding agent is a cleavase or nickase, binding can be followed by cleavage or nicking.

[0081] As used herein, the term "uracil glycosylase inhibitor," "uracil-DNA glycosylase inhibitor," or "UGI" refers to a protein that can inhibit the uracil-DNA glycosylase (UDG) base excision repair enzyme (e.g., UniPROT ID: P14739, MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSKRTADGSEFESPKKKRKVE (SEQ ID NO: 226), or TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO: 227)).

[0082] 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 with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any of the Cas9 nucleic acid sequences or nucleic acid sequences encoding the amino acid sequences provided herein. In certain embodiments, the nucleotide sequence encoding the Cas9 amino acid sequence is not a naturally occurring Cas9 nucleotide sequence. Also contemplated are sequences with at least 95%, 96%, 97%, 98%, or 99% identity to any of the Cas9 amino acid sequences provided herein. In certain embodiments, the Cas9 amino acid sequence is not a naturally occurring Cas9 sequence. Exemplary open reading frames of Spy Cas9 Exemplary amino acid sequences of Spy Cas9 Exemplary open reading frames of Spy Cas9 Exemplary open reading frame of Spy Cas9 with Hibit tag Exemplary amino acid sequence of Spy Cas9 with Hibit tag Exemplary amino acid sequences of Spy Cas9 nickases Exemplary open reading frames of Nme2Cas9 Representative mRNA of Nme2Cas9 nickase Exemplary amino acid sequences of Nme2Cas9 Exemplary mRNAs Encoding UGIs (SEQ ID NO: 224) UGI open reading frame AUGGGACCGAAGAAGAAGAGAAAGGUCGGAGGAGGAAGCACAAACCUGUCGGACAUCAUCGAAAAGGAAACAGGAAAGCAGCUGGUCAUCCAGGAAUCGAUCCUGAUGCUGCCGGAAGAAGUCGAAGAAGUCAUCGGAAACAAGCCGGAA UCGGACAUCCUGGUCCACACAGCAUACGACGAAUCGACAGACGAAAACGUCAUGCUGCUGACAUCGGACGCACCGGAAUACAAGCCGUGGGCACUGGUCCAUGGACUCGAACGGAGAAAACAAGAUCAAGAUGCUGUGA (SEQ ID NO: 225) Exemplary Amino Acid Sequences of UGI MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSKRTADGSEFESPKKKRKVE (SEQ ID NO: 226) Exemplary Amino Acid Sequences of UGI TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO: 227)

[0083] 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, e.g., 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 binder binds to the target sequence. When the binder is a cleavase or nickase, binding can occur after double-stranded or single-stranded DNA cleavage.

[0084] As used herein, a first sequence is considered to "contain a sequence having at least X% identity to" a second sequence if alignment of the first sequence to the second sequence indicates that X% or more of the positions of the second sequence overall match the first sequence. For example, the sequence AAGA contains a sequence having 100% identity to the sequence AAG, because alignment results in 100% identity in that all three positions of the second sequence match. Differences between RNA and DNA (generally, the exchange of uridine with thymidine 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 nucleotide (e.g., thymidine, uridine, or modified uridine) has the same complement (e.g., adenosine for all 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 is 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 the appropriate selection of algorithm and parameter settings for aligning a given pair of sequences; for sequences of generally similar length and with predicted amino acid identity >50% or nucleotide identity >75%, the Needleman-Wunsch algorithm, using the default settings of the Needleman-Wunsch algorithm interface provided by the EBI on its web server at www.ebi.ac.uk, is generally appropriate.

[0085] As used herein, a first sequence is considered to be "X% complementary" to a second sequence if X% of the bases in the first sequence form base pairs with the second sequence. For example, a first sequence 5'AAGA3' is 100% complementary to a second sequence 3'TTCT5', and the second sequence is 100% complementary to the first sequence. In some embodiments, a first sequence 5'AAGA3' is 100% complementary to a second sequence 3'TTCTGTGA5', while the second sequence is 50% complementary to the first sequence.

[0086] 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). 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.

[0087] As used herein, "indel" refers to an insertion / deletion mutation that includes multiple nucleotides inserted or deleted at the site of a double-strand break (DSB) in a target nucleic acid. As used herein, in the case of indel formation that results in an insertion, the insertion is a random insertion at the site of the double-strand break and is not guided or based on a template sequence.

[0088] As used herein, "target sequence" refers to a nucleic acid sequence within 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.

[0089] As used herein, "polypeptide" refers to a wild-type or variant protein (e.g., a mutant, fragment, fusion, or a 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% of the functional activity of the wild-type polypeptide. In some embodiments, a 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, a variant polypeptide can be a highly active variant. In certain cases, a variant has about 80% to about 120%, about 140%, about 160%, about 180%, about 200%, about 300%, about 400%, about 500%, or more of the functional activity of a wild-type polypeptide. As used herein, "heterologous gene" refers to a gene introduced into a cell as an exogenous 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, and may be wild-type or variant. A heterologous gene may contain nucleotide sequences other than the sequence encoding the heterologous polypeptide (e.g., an internal ribosome entry site). A heterologous gene may be a gene naturally occurring in a genome, either wild-type or variant (e.g., mutant). For example, a cell may contain a gene of interest (wild-type or variant), but the same gene or a variant thereof may be introduced as an exogenous source for expression, e.g., at a highly expressed locus. A heterologous gene may also be a gene that does not naturally occur in the genome, or a gene that expresses a heterologous polypeptide that does not naturally occur in the genome. The terms "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 with respect to both its insertion site and its recipient cell.

[0090] A "safe harbor" locus is a locus in the genome into which a gene can be inserted without significant adverse effects on the cell. 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., a TRC gene, e.g., a TRAC gene) is advantageous for allogeneic cells. Other suitable safe harbor loci are known in the art.

[0091] II. Composition A. Engineered T cells Provided herein are T cells and populations of T cells that have been engineered to contain a modification comprising inserting into the cells a heterologous sequence encoding dmTGFB1 under the control of a promoter.

[0092] In some embodiments, T cells comprising a heterologous sequence encoding dmTGFB1 under the control of a promoter are further engineered to include further modification (e.g., knockdown) of the endogenous nucleic acid sequence encoding IFNG, modification (e.g., knockdown) of the endogenous nucleic acid sequence encoding TNFA, and insertion into the cells of 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, IDO1, ENTPD1, NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, BACH2, and IL2RA.

[0093] In some embodiments, T cells or populations of T cells comprising a heterologous sequence encoding a dmTGFB1 molecule under the control of a promoter are further 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 heterologous sequences encoding two or more regulatory T cell-promoting molecules, each under the control of a promoter sequence. For example, the engineered T cells comprise 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 and second promoters may be the same or different promoters. In certain embodiments, the heterologous sequence encoding the dmTGFB1 molecule is under the control of a promoter sequence controlling the expression of a regulatory T cell-promoting molecule. In certain embodiments, the heterologous sequence encoding the dmTGFB1 molecule is not under the control of a promoter sequence controlling the expression of a regulatory T cell-promoting molecule. In certain embodiments, the heterologous sequence encoding the dmTGFB1 molecule is under the control of a promoter sequence controlling the expression of a targeted receptor. In certain embodiments, the heterologous sequence encoding the dmTGFB1 molecule is not under the control of a promoter sequence controlling the expression of a targeted receptor.

[0094] In some embodiments, T cells or populations of T cells comprising a heterologous sequence encoding dmTGFB1 under the control of a promoter are further engineered to include a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, and insertion into the cells of heterologous sequence(s) encoding IL10 under the control of a promoter. In some embodiments, T cells comprising a heterologous sequence encoding dmTGFB1 under the control of a promoter are further engineered to include a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, and insertion into the cells of heterologous sequence(s) encoding CTLA4 under the control of a promoter. In some embodiments, the T cells comprising a heterologous sequence encoding dmTGFB1 under the control of a promoter are further engineered to comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A; insertion into the cell of heterologous sequence(s) encoding IL10 under the control of a promoter sequence; and insertion into the cell of heterologous sequence(s) encoding CTLA4 under the control of a promoter sequence.

[0095] In some embodiments, a T cell or population of T cells comprising a heterologous sequence encoding dmTGFB1 under the control of a promoter is further engineered to include a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, 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 naturally occurring regulatory T cells (nTregs) (e.g., suppression of an immune response or biomarker in an in vitro or in vivo assay (e.g., an animal model of GvHD)).

[0096] In some embodiments, the heterologous sequence(s) encoding dmTGFB1 or a regulatory T cell-promoting molecule are incorporated into an expression construct. In some embodiments, heterologous sequences encoding two or more molecules can be incorporated into two or more separate expression constructs. For example, a first heterologous sequence encoding dmTGFB1 is provided in a first expression construct, and a second heterologous sequence encoding a 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) are inserted into the genome, for example, as a targeted or non-targeted insertion.

[0097] In certain embodiments, the modification, eg, knockdown, of the endogenous nucleic acid sequence encoding IFNG or IL17A includes modification of IFNG and IL17A.

[0098] In some embodiments, the sequence(s) encoding dmTGFB1 or a regulatory T cell-promoting molecule can be inserted into a site selected from a TCR gene locus, e.g., a TRAC locus, a TNF gene locus, an IFNG gene locus, an IL17A locus, an IL6 locus, an IL2 locus, or an adeno-associated virus integration site 1 (AAVS1) locus.

[0099] In some embodiments, the engineered T cells or population of T cells are engineered, e.g., by gene editing, to contain a modification (e.g., an insertion) in a sequence encoding dmTGFB1, e.g., as assessed by sequencing (e.g., NGS), such that at least 30%, 35%, preferably at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells contain an insertion of a sequence encoding a dmTGFB1 molecule.

[0100] In some embodiments, the population of engineered T cells comprises a modified insertion of a sequence encoding dmTGFB1, e.g., generated by gene editing and as assessed by, e.g., 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 comprise an insertion of a sequence encoding a dmTGFB1 molecule, and further comprises 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 comprise an insertion, deletion, or replacement of an 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, compared to a suitable control (e.g., a control in which the TNFA gene is not modified, as quantified by ELISA or flow cytometry). For example, assays for TNFA protein and mRNA expression in populations of T cells are known in the art. In certain embodiments, knockdown of TNFA results in TNFA levels of 2500 pg / ml or less, as quantified, for example, using a custom U-PLEX Biomarker kit (Meso Scale Diagnostics, catalog number K15067L-2) according to the manufacturer's instructions.

[0101] In some embodiments, the population of engineered T cells comprises a modification (e.g., an insertion) of a sequence encoding dmTGFB1, e.g., by gene editing, and as assessed by, e.g., 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 comprise an insertion of a sequence encoding a dmTGFB1 molecule, and further comprises a modification (e.g., knockdown) of an IFNG sequence, e.g., 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 comprise 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 IFNG protein and mRNA expression in, for example, populations of T cells are known in the art. In certain embodiments, knockdown of IFNG results in IFNG levels of 300,000 pg / ml or less, as quantified, for example, using a custom U-PLEX Biomarker kit (Meso Scale Diagnostics, catalog number K15067L-2) according to the manufacturer's instructions.

[0102] In some embodiments, the population of engineered T cells comprises a modification (e.g., insertion) of a sequence encoding dmTGFB1, 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 comprise an insertion of a sequence encoding a dmTGFB1 molecule, and further comprises a modification (e.g., knockdown) of an IL17A sequence, e.g., 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 comprise an insertion, deletion, or substitution of the endogenous IL17A sequence. In some embodiments, expression of IL17A (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 IL17A gene is not modified, as quantified by ELISA or flow cytometry). Assays for IL17A protein and mRNA expression (e.g., in populations of T cells) are known in the art.

[0103] In some embodiments, the modification that knocks down expression of a gene (eg, TNFA, IFNG, or IL17A) is one or more of an insertion, deletion, or substitution.

[0104] In some embodiments, the engineered T cell or population of T cells comprises a modification (e.g., an insertion) of a sequence encoding dmTGFB1, e.g., by gene editing, e.g., as assessed by sequencing (e.g., NGS), wherein at least 30%, 35%, preferably at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells comprise an insertion of a sequence encoding a dmTGFB1 molecule, and further comprises an insertion of a sequence(s) encoding a regulatory T cell-promoting molecule, e.g., by gene editing, e.g., as assessed by sequencing (e.g., NGS), wherein at least 30%, 35%, preferably at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells comprise 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 without the regulatory T cell-promoting molecule gene insertion. In some embodiments, the engineered T cells include an insertion of sequence(s) encoding IL10 via gene editing, such that at least 30%, 35%, preferably at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells contain the insertion or sequence encoding IL10, e.g., as assessed by sequencing (e.g., NGS). In some embodiments, the inserted sequence(s) encoding IL10 results in a statistically significant increase in protein or mRNA expression compared to a suitable control, e.g., a regulatory T cell-promoting molecule. Assays for IL10 protein and mRNA expression, for example in populations of T cells, are known in the art (eg, ELISA and flow cytometry).In certain embodiments, the level of IL10 is at least 300 pg / ml, for example, as quantified using a custom U-PLEX Biomarker kit (Meso Scale Diagnostics, catalog number K15067L-2) according to the manufacturer's instructions.

[0105] In some embodiments, the engineered T cell or population of T cells comprises a modification (e.g., an insertion) of a sequence encoding dmTGFB1, 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 comprise an insertion of a sequence encoding a dmTGFB1 molecule, and further comprises an insertion of a 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 comprise the insertion or sequence encoding CTLA4. In some embodiments, the inserted sequence(s) encoding CTLA4 result in a statistically significant increase in protein or mRNA expression compared to a suitable control, e.g., a regulatory T cell-stimulating molecule. Assays for CTLA4 protein and mRNA expression, e.g., in populations of T cells, are described herein and known in the art (e.g., ELISA and flow cytometry).

[0106] In some embodiments, the population of T cells includes T cells that have been engineered, e.g., by gene editing, to include a modification (e.g., insertion) of a sequence encoding dmTGFB1, e.g., as assessed by sequencing (e.g., NGS), wherein 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 dmTGFB1 molecule, and are further engineered to include a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, 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% (e.g., within the detection limits of the assay used) of the T cells within a population of T cells are engineered to contain 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 within a population of T cells are engineered to contain a modification (e.g., knockdown) of a sequence(s) encoding a 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 within a population of T cells are engineered to contain 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 within a population of T cells are engineered to contain 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 within a population of T cells are engineered to contain an insertion of 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 within 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).

[0107] In some embodiments, the engineered T cell or population of T cells is engineered, e.g., by gene editing, to contain a modification (e.g., insertion) of a sequence encoding dmTGFB1, e.g., as assessed by sequencing (e.g., NGS), such that at least 30%, 35%, preferably at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells contain an insertion of a sequence encoding a dmTGFB1 molecule, and a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA and a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A. and insertion of a heterologous sequence(s) encoding a regulatory T cell-promoting molecule under the control of a promoter sequence, and further comprising modifications of endogenous nucleic acid sequences encoding interleukin 6 (IL6), interleukin 2 (IL2), perforin 1 (PRFI), granzyme A (GZMA), granzyme B (GZMB), Fas ligand (FasL, NF superfamily, member 6), ryanodine receptor 2 (RYR2), and colony-stimulating factor 2 (CSF2), which modifications knock down expression of IL17A, IL6, IL2, PRFI, GZMA, GZMB, FASL, RYR2, or CSF2, respectively. In certain embodiments, the further modification of the endogenous nucleic acid sequence comprises a modification of RYR2.

[0108] In certain embodiments, the modifications include modifications of IFNG and IL17A.

[0109] In some embodiments, the T cells or population of T cells are engineered using a gene editing system (e.g., using an RNA-guided DNA binder). In some embodiments, the T cells are engineered using a CRISPR / Cas gene editing system. In some embodiments, the T cells are engineered using a CRISPR / Cas type II gene editing system (e.g., using Cpfl). In some embodiments, the T cells are engineered using a CRISPR / Cas9 gene editing system (e.g., using SpyCas9). Exemplary Cas9 sequences are provided herein.

[0110] 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 for knockdown of IFNG and TNFA, respectively, are provided in Tables 1 and 2. Also provided are the genomic coordinates of the target for each listed guide sequence.

[0111] In some embodiments, the engineered T cells or population of T cells comprise IFNG and TNFA genes knocked down using guide RNAs disclosed herein in conjunction with an RNA-guided DNA-binding agent. In some embodiments, disclosed herein are engineered T cells by inducing cleavage (e.g., double-strand breaks (DSBs) or single-strand breaks (nicks)) in the IL-7A, IFNG, and TNFA genes of the T cells (e.g., using guide RNAs disclosed herein in conjunction with an RNA-guided DNA-binding agent (e.g., a CRISPR / Cas system)). The methods can be used in vitro or ex vivo, for example, in the production of cellular products for suppressing immune response(s) (including inflammation and autoimmunity). In some embodiments, the guide RNAs disclosed herein mediate target-specific cleavage by an RNA-guided DNA-binding agent (e.g., a Cas nuclease) at a site described herein in the IFNG gene. In some embodiments, the guide RNAs disclosed herein mediate target-specific cleavage by an RNA-guided DNA-binding agent (e.g., a Cas nuclease) at a site described herein within a TNFA gene. It will be understood that in some embodiments, the guide RNA comprises a guide sequence that binds to or is capable of binding to the aforementioned region.

[0112] Provided are engineered T cells or populations of T cells that comprise a genetic modification at a genomic coordinate selected from the genomic coordinates 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 the genomic coordinates 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.

[0113] In some embodiments, the guide RNAs disclosed herein comprise 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, the guide RNAs disclosed herein comprise 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.

[0114] In some embodiments, the guide RNAs disclosed herein comprise 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 RNAs disclosed herein comprise 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 RNAs disclosed herein comprise 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 RNAs disclosed herein comprise 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 RNAs disclosed herein comprise a guide sequence selected from the group of sequences in Table 1.

[0115] In some embodiments, the guide RNAs disclosed herein comprise 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 RNAs disclosed herein comprise 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 RNAs disclosed herein comprise 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 RNAs disclosed herein comprise 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 RNAs disclosed herein comprise a guide sequence selected from the group of sequences in Table 2.

[0116] Unless otherwise stated, all genomic coordinates are based on the human reference genome hg38.

[0117] 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 2] [Table 3-1] [Table 3-2] [Table 3-3]

[0118] Non-limiting modified guide sequences for TNFA knockdown are shown below (hg38 coordinates chr12:68158001-68158021, G019757): mC*mC*mA*GAGCAUCCAAAAGAGUGGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmUmGmCmU*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)

[0119] Non-limiting modified guide sequences for IFNG knockdown are shown below (hg38 coordinates chr6:31576805-31576825, G019753): mA*mG*mA*GCUCUUACCUACAACAUGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 120)

[0120] A non-limiting modified guide sequence for IL17A knockdown is shown below (hg38 coordinates chr6:52189069-52189089): mU*mC*mA*CAGAGGGAUAUCUCUCAGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 217)

[0121] An exemplary modified mock guide is shown below (hg38 coordinates chr1:0-20): mG*mA*mU*CACGUCGGCCGUUGGCGGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 121)

[0122] In some embodiments, disclosed herein are engineered T cells that have been engineered by using guide RNAs in conjunction with RNA-guided DNA binding agents to introduce or insert a heterologous dmTGFB1 nucleic acid into a genomic locus of a T cell or population of T cells, and constructs (e.g., donor constructs or templates) comprising a heterologous dmTGFB1 nucleic acid (e.g., for generating engineered T cells). In some embodiments, disclosed herein are engineered T cells that have been engineered by, for example, using guide RNAs in conjunction with RNA-guided DNA binding agents to express a heterologous dmTGFB1 from a genomic locus of a T cell or population of T cells, and constructs (e.g., donors) comprising a heterologous dmTGFB1 nucleic acid. In some embodiments, disclosed herein are engineered T cells that have been engineered by, for example, using guide RNAs in conjunction 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 dmTGFB1 gene. Also provided are cells and cell populations produced by the methods.

[0123] In some embodiments, disclosed herein are T cells engineered by introducing or inserting a heterologous dmTGFB1 nucleic acid into a genomic locus of a T cell, and T cells further engineered by using guide RNA with an RNA-guided DNA binder to introduce or insert a heterologous IL10 nucleic acid into a genomic locus of a T cell or population of T cells, as well as constructs (e.g., donor constructs or templates) comprising the heterologous IL10 nucleic acid (e.g., for generating engineered T cells). In some embodiments, disclosed herein are T cells engineered by, e.g., using guide RNA with an RNA-guided DNA binder to express heterologous IL10 from a genomic locus of a T cell or population of T cells, and constructs (e.g., donor) comprising the heterologous IL10 nucleic acid. In some embodiments, disclosed herein are T cells engineered by inducing 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, e.g., using a guide RNA in conjunction with an RNA-guided DNA binding agent (e.g., a CRISPR / Cas system) to insert an IL10 gene. Also provided are cells and cell populations produced by the method.

[0124] In some embodiments, disclosed herein are T cells engineered by introducing or inserting a heterologous dmTGFB1 nucleic acid into a genomic locus of a T cell, and T cells further engineered by using guide RNA with an RNA-guided DNA binder to introduce or insert a heterologous CTLA4 nucleic acid into a genomic locus of a T cell or population of T cells, as well as constructs (e.g., donor constructs or templates) comprising the heterologous CTLA4 nucleic acid (e.g., for generating engineered T cells). In some embodiments, disclosed herein are T cells engineered by, e.g., using guide RNA with an RNA-guided DNA binder to express a heterologous CTLA4 from a genomic locus of a T cell or population of T cells, and constructs (e.g., donor) comprising the heterologous CTLA4 nucleic acid. In some embodiments, disclosed herein are T cells engineered by inducing 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, e.g., using a guide RNA in conjunction with an RNA-guided DNA binding agent (e.g., a CRISPR / Cas system) to insert a CTLA4 gene. Also provided are cells and cell populations produced by the method.

[0125] In some embodiments, disclosed herein are T cells engineered by introducing or inserting a heterologous dmTGFB1 nucleic acid into a genomic locus of a T cell, and T cells further engineered by using guide RNAs 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) comprising the heterologous CTLA4 and IL10 nucleic acids (e.g., for generating engineered T cells). In some embodiments, disclosed herein are T cells engineered by, for example, using guide RNAs 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) comprising heterologous CTLA4 and IL10 nucleic acids. In some embodiments, disclosed herein are T cells engineered by, for example, using guide RNA in conjunction with an RNA-guided DNA-binding agent (e.g., a 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., a 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 methods.

[0126] Exemplary nucleotide and polypeptide sequences for 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 nucleic acid sequences encoding dmTGFB1, IL10, and CTLA4 are provided below. Other suitable dmTGFB1, IL10, and CTLA4 sequences are known in the art or can be designed based on the disclosure provided herein. See, e.g., Gorby et al., Engineered IL-10 variants elicit potent immunomodulatory activities at therapeutic low ligand doses, BioRxiv (2020); Saxton et al., Structure-based decoupling of the pro- and anti-inflammatory functions of interleukin-10, Science, (2021) 371:eabc8433 (doi:10.1126 / science.abc8433); WO2021243057; and Xu et al., Affinity and cross-reactivity engineering of CTLA4-Ig to modulate T cell costimulation, J Immunol (2012), the contents, variants (particularly IL-10 variants), and sequences of which are incorporated herein by reference. Methods for identifying alternative IL10 and CTLA4 sequences are also known in the art. See, e.g., above. For example, Gorby has described the anti-inflammatory and pro-cytotoxicity activities of IL-10. Recent studies have shown that, in addition to its anti-inflammatory activity, IL-10 can enhance the cytotoxic function of CD8 T cells, enhance their tumor targeting ability, and promote anti-cancer responses (Oft, 2019).This seems paradoxical, as IL-10 in the tumor microenvironment has been linked to tumor evasion of immune responses, likely due to its inhibitory effect on antigen presentation (Mannino et al., 2015; Yue et al., 1997). Despite this paradox, several studies have elegantly demonstrated that IL-10 can improve the production of the CD8 effector molecules granzyme B and interferon gamma both in vitro and in vivo (Emmerich et al., 2012; Mumm et al., 2011; Mumm and Oft, 2013). Currently, several clinical trials testing the antitumor properties of IL-10 are underway, and initial promising results have already been obtained (Naing et al., 2019). These studies used high doses of PEGylated IL-10 (pegylidecakin), which ensured prolonged retention of IL-10 in the circulation to ensure efficacy, reiterating the need for high and sustained IL-10 levels for effective in vivo IL-10 responses. Saxton et al. used yeast display-based directed evolution to engineer IL-10 variants. IL-10 is an immunomodulatory cytokine with both anti-inflammatory and immunostimulatory properties that is often dysregulated in disease. Mechanistically, IL-10 functions as a secreted homodimer that binds to two copies of a heterodimeric receptor complex containing the private receptor subunit IL-10Rα and the shared subunit IL-10Rβ. IL-10-dependent dimerization of IL-10Rα and IL-10Rβ then initiates activation of the transcription factor STAT3, which mediates the diverse biological effects of IL-10. Saxton et al. explain that IL-10 variants with varying IL-10Rβ binding strengths reveal significant differences in response thresholds between immune cell populations and provide a means to manipulate IL-10 cell type selectivity. Saxton et al. identified a "super 10" variant (D25A / E96A) that has enhanced affinity for IL-10Rβ, enabling assembly of the hexameric IL-10-IL-10Rα-IL-10Rβ complex.Other variants (e.g., D25K, D25A, N21A / R104A, and D25A / N21A / R104A based on the amino acid numbering of SEQ ID NO: 231) exhibit myeloid-polarizing activity by suppressing macrophage activation without stimulating inflammatory CD8+ T cells, thereby uncoupling the key opposing functions of IL-10. In certain embodiments, the engineered T cells provided herein utilize IL-10 variants with impaired immunostimulatory properties (referred to herein as "inhibitory IL-10 variants"), e.g., variants that retain myeloid-polarizing activity by suppressing macrophage activation and exhibit impaired stimulation of inflammatory CD8+ T cells. In certain embodiments, the inhibitory IL-10 variants used in the engineered T cells provided herein include substitutions selected from D25K, D25A, D25A / E96A, N21A / R104A, and D25A / N21A / R104A, e.g., D25A / E96A. The results by Saxton et al. provide a mechanistic blueprint for modulating the pleiotropic effects of IL-10. Additional inhibitory IL-10 variants are provided in WO2021243057, incorporated herein by reference, which provides methods for characterizing a number of IL-10 sequences and activities, including polypeptides with altered binding affinity to IL-10Rβ compared to the binding affinity of a reference IL-10 polypeptide lacking one or more amino acid substitutions. Additional variants are disclosed, including inhibitory IL-10 variants, which, like the D25K, D25A, D25A / E96A, N21A / R104A, and D25A / N21A / R104A variants, are predicted to exhibit myeloid-polarizing activity by suppressing macrophage activation without stimulating inflammatory CD8+ T cells. Embodiments provided herein include inhibitory IL-10 variants having one or more amino acid substitutions at positions corresponding to amino acid residues selected from D25, H14, N18, R24, D28, E74, H90, N92, E96, T100, and R104, and optionally further substitutions with one or more amino acids selected from N21, M22, R32, and S93.In certain embodiments, D25 is substituted with an amino acid selected from K, A, N, H, I, K, or V. In certain embodiments, E96 is substituted with an amino acid selected from A, N, D, Q, H, K, or S. Exemplary combinations of substitutions may include a) N18Y / N92Q / T100D / R104W, (b) N18Y / N21H / N92Q / E96D / T100V / R104W, (c) N18Y / N21H / E96H / T100V / R104W, (d) N18Y / D25A / N92Q / T100D / R104W, (e) N18Y / D25K / N92Q / T100D / R104W, and (f) N18Y / D25A / N92Q / E96A / T100D / R104W. Exemplary substitutions include (a) D25A, (b) D25K, (c) E96A, (d) E96K, (e) D25A / E96A, (f) N21A / R104A, (g) N21A / D25A, (h) N21A / D25A / E96A, and (i) N21A / M22A / D25A. Sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any of the nucleic acid sequences, amino acid sequences, or nucleic acid sequences encoding the amino acid sequences described herein, e.g., by mutation or truncation, are also contemplated. In some embodiments, nucleic acid sequences encoding any of the amino acid sequences provided herein are also provided.

[0127] 1 shows a non-limiting exemplary nucleic acid sequence encoding TGFB1.

[0128] Wild type TGFB1

[0129]

[0130] Double mutant (dm) TGFB1 (R218C, C225R)

[0131]

[0132] Non-limiting exemplary amino acid sequences of human TGFB1 Wild type MPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS (SEQ ID NO: 210) dmTGFB1 R218C, C225R MPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFHLSAHCSRDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS (SEQ ID NO: 214)

[0133] 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)

[0134] 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) Mature wild type IL10 SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 231) IL10(D25K) SPGQGTQSENSCTHFPGNLPNMLRKLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 232) IL10(D25E) SPGQGTQSENSCTHFPGNLPNMLRELRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 233) IL10(D25K / E96A) SPGQGTQSENSCTHFPGNLPNMLRKLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGANLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 234)

[0135] 1 shows a non-limiting exemplary nucleic acid sequence encoding TGFB1.

[0136] Wild type TGFB1

[0137] Double mutant TGFB1 (R218X, where X is H or C, C225R)

[0138] 1 shows a non-limiting exemplary nucleic acid sequence encoding CTLA4. Wild type CTLA4: ATGGCCTGCTTGGGCTTCCAAAGGCATAAAGCCCAGCTTAATCTTGCTACTCGCACGTGGCCCTGCACATTGCTCTTTTTCCTCCTGTTCATTCCCGTGTTTTGCAAGGCGATGCATGTGGCACAACCTGCCGTCGTTCTGGCATCATCAAGAGGTATTGCTAGCTTCGTTTGTGAGTACGCCTCCCCTGGAAAAGCGACGGAGGTGCGCGTCACTGTATTGCGGCAAGCCGACAGCCAAGTTACTGAAGTCTGCGCGGCAACGTATATGATGGGCAATGAGCTGACATTCCTTGACGATTCAATCTGCACGGGAACAAGTAGTGGTAACCAGGTGAATCTCACTATTCAAGGTCTGAGAGCCATGGACACCGGCCTCTACATTTGTAAGGTGGAGCTGATGTATCCTCCCCCATATTATCTGGGGATCGGAAATGGGACACAGATATATGTTATTGATCCCGAGCCATGTCCCGATAGTGACTTCCTCTTGTGGATACTTGCCGCTGTGAGCAGTGGTTTGTTTTTTTATTCATTCCTCCTTACGGCAGTATCACTTTCAAAAATGCTCAAGAAGCGAAGTCCTTTGACAACTGGCGTATATGTCAAAATGCCACCAACAGAGCCCGAATGTGAGAAACAGTTCCAGCCGTACTTTATTCCTATAAAC(SEQ ID NO: 126) High-affinity CTLA4 (belatacept; binding domain: A29Y, L104E): ATGGCCTGCTTGGGCTTCCAAAGGCATAAAGCCCAGCTTAATCTTGCTACTCGCACGTGGCCCTGCACATTGCTCTTTTTCCTCCTGTTCATTCCCGTGTTTTGCAAGGCGATGCATGTGGCACAACCTGCCGTCGTTCTGGCATCATCAAGAGGTATTGCTAGCTTCGTTTGTGAGTACGCCTCCCCTGGAAAATACACGGAGGTGCGCGTCACTGTATTGCGGCAAGCCGACAGCCAAGTTACTGAAGTCTGCGCGGCAACGTATATGATGGGCAATGAGCTGACATTCCTTGACGATTCAATCTGCACGGGAACAAGTAGTGGTAACCAGGTGAATCTCACTATTCAAGGTCTGAGAGCCATGGACACCGGCCTCTACATTTGTAAGGTGGAGCTGATGTATCCTCCCCCATATTATGAGGGGATCGGAAATGGGACACAGATATATGTTATTGATCCCGAGCCATGTCCCGATAGTGACTTCCTCTTGTGGATACTTGCCGCTGTGAGCAGTGGTTTGTTTTTTTATTCATTCCTCCTTACGGCAGTATCACTTTCAAAAATGCTCAAGAAGCGAAGTCCTTTGACAACTGGCGTATATGTCAAAATGCCACCAACAGAGCCCGAATGTGAGAAACAGTTCCAGCCGTACTTTATTCCTATAAAC(SEQ ID NO: 127) High affinity CTLA4 (binding domain: A29H): ATGGCCTGCTTGGGCTTCCAAAGGCATAAAGCCCAGCTTAATCTTGCTACTCGCACGTGGCCCTGCACATTGCTCTTTTTCCTCCTGTTCATTCCCGTGTTTTGCAAGGCGATGCATGTGGCACAACCTGCCGTCGTTCTGGCATCATCAAGAGGTATTGCTAGCTTCGTTTGTGAGTACGCCTCCCCTGGAAAACATACGGAGGTGCGCGTCACTGTATTGCGGCAAGCCGACAGCCAAGTTACTGAAGTCTGCGCGGCAACGTATATGATGGGCAATGAGCTGACATTCCTTGACGATTCAATCTGCACGGGAACAAGTAGTGGTAACCAGGTGAATCTCACTATTCAAGGTCTGAGAGCCATGGACACCGGCCTCTACATTTGTAAGGTGGAGCTGATGTATCCTCCCCCATATTATCTGGGGATCGGAAATGGGACACAGATATATGTTATTGATCCCGAGCCATGTCCCGATAGTGACTTCCTCTTGTGGATACTTGCCGCTGTGAGCAGTGGTTTGTTTTTTTATTCATTCCTCCTTACGGCAGTATCACTTTCAAAAATGCTCAAGAAGCGAAGTCCTTTGACAACTGGCGTATATGTCAAAATGCCACCAACAGAGCCCGAATGTGAGAAACAGTTCCAGCCGTACTTTATTCCTATAAAC(SEQ ID NO: 128) High-affinity CTLA4 (binding domain: K28H, A29H): (SEQ ID NO: 129)

[0139] 1 shows a non-limiting exemplary amino acid sequence of CTLA4. Wild type CTLA4: MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO: 130) High affinity CTLA4 (Belatacept; Binding domain: A29Y, L104E): MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO: 131) High affinity CTLA4 (binding domain: A29H): MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKHTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO: 132) High affinity CTLA4 (binding domain: K28H, A29H): MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGHHTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO: 133)

[0140] In some embodiments, engineered T cells or populations of T cells comprising a heterologous sequence encoding dmTGFB1 under the control of a promoter, further 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 cells heterologous sequence(s) encoding a regulatory T cell-promoting molecule under the control of a promoter sequence, exhibit at least one suppressive activity of natural regulatory T cells (nTregs) (e.g., suppression of immune response(s) or biomarkers in an in vitro or in vivo assay (e.g., an animal model of GvHD)). In some embodiments, engineered T cells or populations of T cells comprising a modified (e.g., knocked down) endogenous nucleic acid sequence encoding TNFA, a modified (e.g., knocked down) endogenous nucleic acid sequence encoding IFNG or IL17A, and insertion into the cells of heterologous sequence(s) encoding a regulatory T cell-promoting molecule under the control of a promoter sequence exhibit improved suppressive activity (e.g., increased suppression of immune responses or biomarkers in in vitro or in vivo assays (e.g., animal models of GvHD)) compared to nTregs. For example, in a mouse model of GvHD, mice administered engineered 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 insertion into the cells of 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). In certain embodiments, the engineered cells comprise modifications for knockdown of endogenous nucleic acid sequences encoding IFNG and IL17A, respectively. In certain embodiments, the regulatory T cell-promoting molecule comprises IL10 or CTLA4. In certain embodiments, the regulatory T cell-promoting molecule comprises IL10 and CTLA4.

[0141] In certain embodiments, an engineered T cell or population of T cells comprises two or more heterologous coding sequences under the control of a promoter, e.g., dmTGFB1 and a regulatory T cell-promoting molecule. In certain embodiments, each heterologous coding sequence is under the control of a separate promoter. In certain embodiments, two heterologous coding sequences are under the control of the same promoter. In certain embodiments, two or more heterologous coding sequences are under the control of the same promoter. In certain embodiments, when an engineered T cell or population of T cells comprises three or more heterologous coding sequences under the control of a promoter, e.g., dmTGFB1 and a regulatory T cell-promoting molecule, each heterologous sequence is independently under the control of a separate promoter or under the control of a promoter controlling the expression of multiple heterologous coding sequences.

[0142] B. Targeting Receptors In some embodiments, the engineered T cells comprising a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, and the insertion into the cells of heterologous sequence(s) encoding a dmTGFB1 molecule and a regulatory T cell-promoting molecule, each under the control of a promoter sequence, further comprise the insertion into the cells of heterologous sequence(s) encoding a targeting receptor. The sequence(s) encoding the targeting receptor are under the control of a promoter sequence (e.g., an endogenous promoter or a heterologous promoter). In certain embodiments, the engineered cells comprise modifications for knockdown of the endogenous nucleic acid sequences encoding IFNG and IL17A, respectively.

[0143] In some embodiments, the engineered T cells comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and insertion into the cells of heterologous sequence(s) encoding a regulatory T cell-promoting molecule under the control of a promoter sequence, further comprise insertion into the cells of heterologous sequence(s) encoding a targeting receptor. The sequence(s) encoding the targeting receptor are under the control of a promoter sequence (e.g., an endogenous promoter or a heterologous promoter). In certain embodiments, the engineered T cells do not comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0144] In some embodiments, the targeting receptor is a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a receptor for 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 the 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). In some embodiments, the targeting receptor targets MAdCAM-1. In some embodiments, the targeting receptor targets TNFA.

[0145] 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 a TNFA, an insertion of sequence(s) encoding a regulatory T cell-promoting molecule selected from IL10, CTLA4, IDO1, ENTPD1, NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, BACH2, and IL2RA, and an insertion of sequence(s) encoding a targeting receptor (e.g., a CAR (e.g., a CAR that targets MAdCAM-1)). Alternatively, the targeting receptor targets a TNFA.

[0146] In some embodiments, the engineered T cells comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, and insertion of sequences encoding dmTGFB1 and IL10, and a targeting receptor (e.g., a CAR (e.g., a CAR that targets MAdCAM-1)). Alternatively, the targeting receptor targets a TNFA.

[0147] In some embodiments, the engineered T cells comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, and insertion of sequences encoding dmTGFB1 and CTLA4, and a targeting receptor (e.g., a CAR (e.g., a CAR that targets MAdCAM-1)). Alternatively, the targeting receptor targets a TNFA.

[0148] In some embodiments, the engineered T cells comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, and insertion of a sequence encoding dmTGFB1, IL10, CTLA4, and a targeting receptor (e.g., a CAR (e.g., a CAR that targets MAdCAM-1)). Alternatively, the targeting receptor targets a TNFA.

[0149] In some embodiments, the engineered T cells comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and insertion of a sequence encoding IL10 and a targeting receptor (e.g., a CAR (e.g., a CAR that targets MAdCAM-1)). Alternatively, the targeting receptor targets TNFA. In certain embodiments, the engineered T cells do not comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0150] In some embodiments, the engineered T cells comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and insertion of a sequence encoding CTLA4 and a targeting receptor (e.g., a CAR (e.g., a CAR that targets MAdCAM-1)). Alternatively, the targeting receptor targets TNFA. In certain embodiments, the engineered T cells do not comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0151] In some embodiments, the engineered T cells comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and an insertion of a sequence encoding IL10, CTLA4, and a targeting receptor (e.g., a CAR (e.g., a CAR that targets MAdCAM-1)). Alternatively, the targeting receptor targets TNFA. In certain embodiments, the engineered T cells do not comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0152] In some embodiments, the sequence(s) encoding the targeting receptor are incorporated into an expression construct. In some embodiments, the expression construct comprising the sequence(s) encoding the targeting receptor further comprises sequence(s) encoding a regulatory T cell-promoting molecule, e.g., the sequence(s) encoding the targeting 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 targeting receptor does not further comprise 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 targeting receptor is an episomal expression construct. In some embodiments, the sequence(s) encoding the targeting receptor is inserted into the genome, e.g., as a targeted or non-targeted insertion.

[0153] In some embodiments, the sequence(s) encoding the targeted receptor can be inserted into a site selected from a TCR gene locus, e.g., a TRAC gene locus, a TNF gene locus, an IFNG gene locus, an IL17A gene locus, an IL6 gene locus, an IL2 gene locus, or an adeno-associated virus integration site 1 (AAVS1) locus.

[0154] In some embodiments, the engineered T cells comprise insertion of a sequence(s) encoding a targeted receptor by gene editing, e.g., as assessed by sequencing (e.g., NGS).

[0155] In some embodiments, the population of T cells comprises T cells that have been engineered to include a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, an insertion of a sequence encoding a regulatory T cell-promoting molecule, and an insertion of 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 within the population of T cells are engineered to include an insertion of sequence(s) encoding a targeting receptor, as assessed, for example, by sequencing (e.g., NGS). It is understood that T cell populations can be enriched for cell populations bearing target receptors using selection methods known in the art.

[0156] In some embodiments, disclosed herein are engineered T cells by using guide RNA in conjunction with an RNA-guided DNA binding agent 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) comprising 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, for example, using guide RNA in conjunction with an RNA-guided DNA binding agent 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) comprising the targeting receptor (e.g., CAR). In some embodiments, disclosed herein are engineered T cells by, for example, using 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 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.

[0157] In some embodiments, the targeting receptor (e.g., CAR) can confer targeting specificity to an engineered T cell that includes the targeting receptor (e.g., CAR), for example, to a particular cell, tissue, or organ.

[0158] In some embodiments, a targeting receptor (e.g., a CAR) can target the engineered T cells to the gastrointestinal system, for example, 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.

[0159] 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 the immune response in disorders such as inflammatory bowel disease, ulcerative colitis, or Crohn's disease.

[0160] 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.

[0161] In some embodiments, a targeting receptor (e.g., a CAR) can target the engineered T cells to tissues containing 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.

[0162] In some embodiments, the CAR is capable of targeting the 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.

[0163] 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.

[0164] 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.

[0165] 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 CD20 to suppress immune responses in disorders such as multiple sclerosis and systemic lupus erythematosus.

[0166] In some embodiments, a targeting receptor (e.g., a CAR) can target the engineered T cells to tissues containing 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.

[0167] 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).

[0168] Additional CAR targets (e.g., inflammatory antigens) are known in the art. See, e.g., WO2020092057A1, the contents of which are incorporated herein by reference 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 the engineered T cells. In some embodiments, insertion via 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.

[0169] In certain embodiments, the engineered T cell or population of T cells comprises two or more heterologous coding sequences under the control of a promoter, e.g., dmTGFB1, a regulatory T cell-promoting molecule, and a targeting receptor. In certain embodiments, each heterologous coding sequence is under the control of a separate promoter. In certain embodiments, two heterologous coding sequences are under the control of the same promoter. In certain embodiments, two or more heterologous coding sequences are under the control of the same promoter. In certain embodiments, when the engineered T cell or population of T cells comprises three or more heterologous coding sequences under the control of a promoter, e.g., dmTGFB1, a regulatory T cell-promoting molecule, and a targeting receptor, each heterologous sequence is independently under the control of a separate promoter or under the control of a promoter controlling the expression of multiple heterologous coding sequences.

[0170] In some embodiments, the engineered T cells or population of T cells do not comprise a heterologous target receptor.

[0171] CT cell receptor (TCR) In some embodiments, the engineered T cell or population of T cells comprising a modification (e.g., insertion into the cell) of a heterologous sequence(s) encoding a dmTGFB1 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).

[0172] In some embodiments, the engineered T cell or population of T cells comprising a modification comprising the insertion into the cell of heterologous sequence(s) encoding a dmTGFB1 molecule under the control of a promoter sequence further comprises a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, and the insertion into the cell of heterologous sequence(s) encoding a regulatory T cell-promoting molecule under the control of a promoter sequence, and further comprises a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TCR gene sequence(s).

[0173] 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 TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, and the insertion into the cells of heterologous sequences encoding a dmTGFB1 molecule and a regulatory T cell-promoting molecule, each 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).

[0174] In some embodiments, the engineered T cell or population of T cells comprising a modification comprising the insertion into the cell of heterologous sequence(s) encoding a dmTGFB1 molecule under the control of a promoter sequence further comprises modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, insertion into the cell of heterologous sequences encoding a dmTGFB1 molecule and a regulatory T cell-promoting molecule, each under the control of a promoter sequence, and insertion into the cell of heterologous sequence(s) encoding a targeted receptor, and further comprises modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TCR gene sequence(s).

[0175] In some embodiments, the engineered T cell or population of T cells comprising a modification comprising the insertion into the cell of 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 TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and the insertion into the cell of heterologous sequence(s) encoding a regulatory T cell-promoting molecule under the control of a promoter sequence, and further comprises a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TCR gene sequence(s). In some embodiments, the engineered T cell or population of T cells does not comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0176] In some embodiments, an engineered T cell or population of T cells comprising a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and the insertion into the cells of heterologous sequences encoding a first regulatory T cell-promoting molecule and a second regulatory T cell-promoting molecule, each 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). In certain embodiments, the engineered T cell or population of T cells does not comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0177] In some embodiments, the engineered T cell or population of T cells comprising a modification comprising insertion into the cell of heterologous sequence(s) encoding a regulatory T cell-promoting molecule under the control of a promoter sequence further comprises modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, insertion into the cell of heterologous sequences encoding a first regulatory T cell-promoting molecule and a second regulatory T cell-promoting molecule, each under the control of a promoter sequence, and insertion into the cell of heterologous sequence(s) encoding a targeting receptor, and further comprises modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TCR gene sequence(s). In certain embodiments, the engineered T cell or population of T cells does not comprise modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0178] Generally, TCRs are heterodimeric receptor molecules comprising 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 comprise 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.

[0179] In some embodiments, the engineered T cell or population of T cells comprises a modification comprising the insertion of a sequence encoding dmTGFB1.

[0180] In some embodiments, the engineered T cell or population of T cells comprising insertion into the cell of heterologous sequence(s) encoding a dmTGFB1 molecule under the control of a promoter sequence further comprises modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A; insertion of sequence(s) encoding a regulatory T cell-promoting molecule selected from IL10, CTLA4, IDO1, ENTPD1, NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, BACH2, and IL2RA; and modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TCR gene sequence(s).

[0181] In some embodiments, the engineered T cell or population of T cells comprising the insertion into the cell of heterologous sequence(s) encoding a dmTGFB1 molecule under the control of a promoter sequence further comprises a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TNFA in combination with a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, the insertion of sequence(s) encoding a regulatory T cell-promoting molecule selected from IL10, CTLA4, IDO1, ENTPD1, NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, BACH2, and a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TCR gene sequence(s).

[0182] In some embodiments, the engineered T cell or population of T cells comprising insertion into the cell of heterologous sequence(s) encoding a dmTGFB1 molecule under the control of a promoter sequence further comprises modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, insertion of sequence(s) encoding IL10 or CTLA4, and modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TCR gene sequence(s).

[0183] 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 TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, an insertion of sequence(s) encoding dmTGFB1, IL10, and CTLA4, and a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TCR gene sequence(s).

[0184] 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 TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, an insertion of a sequence(s) encoding a dmTGFB1 molecule and a regulatory T cell-promoting molecule, and a modification (e.g., knockdown) of an endogenous TCR gene sequence (e.g., a TRAC gene sequence).

[0185] 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 a TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, an insertion of sequence(s) encoding a dmTGFB1 molecule, an insertion of sequence(s) encoding a regulatory T cell-promoting molecule selected from IL10, CTLA4, IDO1, ENTPD1, NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, BACH2, and IL2RA, and a modification (e.g., knockdown) of an endogenous TCR gene (e.g., a TRAC gene sequence).

[0186] 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 TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, an insertion of a sequence encoding a dmTGFB1 molecule, an insertion of sequence(s) encoding IL10 or CTLA4, and a modification (e.g., knockdown) of a TCR gene (e.g., a TRAC gene sequence).

[0187] 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 TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, an insertion of a sequence encoding a dmTGFB1 molecule, an insertion of 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).

[0188] In some embodiments, engineered T cells or populations of T cells comprising insertion into cells of heterologous sequence(s) encoding a regulatory T cell-promoting molecule under the control of a promoter sequence further comprise modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, insertion of sequence(s) encoding IL10 or CTLA4, and modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TCR gene sequence(s). In certain embodiments, the engineered T cells or populations of cells do not comprise modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0189] 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 TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, an insertion of sequence(s) encoding IL10 and CTLA4, and a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TCR gene sequence(s). In certain embodiments, the engineered T cell or population of cells does not comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0190] 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 TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, an insertion of sequence(s) encoding a first regulatory T cell-promoting molecule and a second regulatory T cell-promoting molecule, and a modification (e.g., knockdown) of an endogenous TCR gene sequence (e.g., a TRAC gene sequence). In certain embodiments, the engineered T cell or population of cells does not comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0191] 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 TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, an insertion of sequence(s) encoding a regulatory T cell-promoting molecule selected from IL10, CTLA4, IDO1, ENTPD1, NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, BACH2, and IL2RA, and a modification (e.g., knockdown) of an endogenous TCR gene (e.g., a TRAC gene sequence). In certain embodiments, the engineered T cell or population of cells does not comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0192] 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 TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, an insertion of a sequence encoding a first regulatory T cell-promoting molecule, an insertion of sequence(s) encoding IL10 or CTLA4, and a modification (e.g., knockdown) of a TCR gene (e.g., a TRAC gene sequence). In certain embodiments, the engineered T cell or population of cells does not comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0193] 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 TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, an insertion of a sequence encoding a first regulatory T cell-promoting molecule, an insertion of a sequence encoding a second regulatory T cell-promoting molecule, and a modification (e.g., knockdown) of an endogenous TCR gene (e.g., a TRAC gene sequence). In certain embodiments, the engineered T cell or population of cells does not comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0194] In any of these embodiments, the engineered T cell or population of T cells can further comprise an insertion of a sequence(s) encoding a targeting receptor (e.g., a CAR (e.g., a CAR that targets MAdCAM-1)) described herein.

[0195] In any of these embodiments, the engineered T cell or population of T cells can further comprise an insertion of a sequence(s) encoding a targeting receptor (e.g., a CAR (e.g., a CAR that targets a TNFA)) described herein.

[0196] In some embodiments, the engineered T cells or population of T cells comprising the insertion into the cells of heterologous sequence(s) encoding a dmTGFB1 molecule under the control of a promoter sequence further comprise modification (e.g., knockdown) of the TRC gene sequence by gene editing, e.g., as assessed by sequencing (e.g., NGS), 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. 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, 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.

[0197] In some embodiments, the engineered T cell or population of T cells comprises insertion of a sequence(s) encoding a targeted receptor by gene editing, e.g., as assessed by sequencing (e.g., NGS).

[0198] In some embodiments, the population of T cells comprises T cells that are further engineered to include insertion into the cells of heterologous sequence(s) encoding dmTGFB1 molecules under the control of a promoter sequence, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding the insertion of sequence(s) 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 within the population of T cells are engineered to include a modification (e.g., knockdown) of at least one TCR gene sequence, as assessed, for example, by sequencing (e.g., NGS).

[0199] In some embodiments, the population of T cells comprises T cells that are further engineered to include insertion into the cells of heterologous sequence(s) encoding a dmTGFB1 molecule under the control of a promoter sequence, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, an insertion of sequence(s) 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 within the population of T cells are engineered to include a modification (e.g., knockdown) of at least one TCR gene sequence, as assessed, for example, by sequencing (e.g., NGS).

[0200] 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.

[0201] 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 production of cellular products for suppressing an immune response.

[0202] 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 understood that in some embodiments, the guide RNA comprises a guide sequence that binds to or is capable of binding to the aforementioned region.

[0203] D. Guide RNA In any embodiment herein, the guide RNA can further comprise a trRNA. In each composition and method embodiment 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 linkages between nucleotides that are not phosphodiester bonds.

[0204] 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.

[0205] 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 trRNA are covalently linked via a linker. In some embodiments, the sgRNA forms a stem-loop structure through base pairing between portions of the crRNA and the trRNA. In some embodiments, the crRNA and trRNA are covalently linked via one or more bonds that are not phosphodiester bonds.

[0206] In some embodiments, the trRNA can comprise all or a portion of a trRNA sequence derived from a naturally occurring CRISPR / Cas system. In some embodiments, the trRNA comprises a truncated or modified wild-type trRNA. The length of the trRNA depends on the CRISPR / Cas system used. In some embodiments, the trRNA comprises or consists of 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more than 100 nucleotides. In some embodiments, the trRNA can comprise a particular secondary structure, such as one or more hairpin or stem-loop structures, or one or more bulges.

[0207] In some embodiments, the target sequence or region can 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 can be 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the target sequence and the guide sequence of the gRNA can be 100% complementary or identical. In other embodiments, the target sequence and the guide sequence of the gRNA can contain one mismatch. For example, the target sequence and the guide sequence of the gRNA can contain one, two, three, four, or five mismatches, and the total length of the guide sequence is about 20 or 20 nucleotides. In some embodiments, the target sequence and the guide sequence of the gRNA can contain one to four mismatches, and the guide sequence is about 20 or 20 nucleotides. In some embodiments, the degree of complementarity or identity between the guide sequence and its corresponding target sequence can be at least 80%, 85%, preferably 90%, or 95%, for example, where the guide sequence comprises a sequence of 24 contiguous nucleotides. In some embodiments, the guide sequence and target region may be 100% complementary or identical. In other embodiments, the guide sequence and target region may contain at least one mismatch, i.e., one nucleotide that is not identical or complementary, relative to the reference sequence. For example, the guide sequence and target sequence may contain one to two, preferably one or fewer, mismatches, in which case the total length of the target sequence is 19, 20, 21, 22, preferably 23 or 24 nucleotides or more. In some embodiments, the guide sequence and target region may contain one to two mismatches, in which case the guide sequence comprises at least 24 or more nucleotides. In some embodiments, the guide sequence and target region may contain one to two mismatches, in which case the guide sequence comprises 24 nucleotides.

[0208] In any of the embodiments herein, each of the guide sequences herein can further comprise additional nucleotides to form a crRNA or guide RNA, for example, 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, for example, the following exemplary nucleotide sequence following the 3' end of the guide sequence: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 135), GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 136), GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGCACCGAGUCGGUGC (SEQ ID NO: 200), GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCACGAAAGGGCACCGAGUCGGUGC (SEQ ID NO: 201), GUUGUAGCUCCCUUUCUCAUUUCGGAAACGAAAUGAGAACCGUUGCUACAAUAAGGCCGUCUGAAAAGAUGUGCCGCAACGCUCUGCCCCUUAAAGCUUCUGCUUUAAGGGGCAUCGUUUA (SEQ ID NO: 202), GUUGUAGCUCCCUGAAACCGUUGCUACAAUAAGGCCGUCGAAAGAUGUGCCGCAACGCUCUGCCUUCUGGCAUCGUU (SEQ ID NO: 203).

[0209] In some embodiments, 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 strand opposite 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 NGG, NNGRRT, NNGRR(N), NNAGAAW, NNNNG(A / C)TT, and NNNNRYAC, for example, when the Cas system includes SpyCas9. In another embodiment, the PAM sequence includes NCC, N4GAYW, N4GYTT, N4GTCT, NNNNCC(a), NNNNCAAA (where N is defined as any nucleotide, W is defined as either A or T, R is defined as either A or G, and in (a), an A after the second C is preferred but not required), for example, when the Cas system includes NmeCas9.

[0210] In some embodiments, the guide RNA sequences provided herein are complementary to sequences adjacent to the PAM sequence.

[0211] In some embodiments, the guide RNA sequence comprises a sequence complementary to a sequence within a genomic region selected from the tables 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 tables 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 tables herein.

[0212] The guide RNAs disclosed herein mediate target-specific cleavage that results in a double-strand break (DSB). The guide RNAs disclosed herein mediate target-specific cleavage that results in a single-strand break (SSB or nick).

[0213] E. Chemically modified gRNA In some embodiments, the gRNA is chemically modified. A gRNA comprising 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 moieties used in place of, or in addition to, the canonical A, G, C, and U residues. In some embodiments, the 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 the following: (i) alteration (e.g., substitution) of one or both of the non-linking phosphate oxygens in the phosphodiester backbone linkage or one or both of the linking 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) modification or substitution of a naturally occurring nucleobase (including a non-canonical nucleobase) (exemplary base modifications); and (iv) modification of the 3' or 5' end of the oligonucleotide to provide exonuclease stability, for example, with a 2'O-me, 2' halide, or 2' deoxy substituted ribose, or an inverted abasic terminal nucleotide, or substitution of a phosphodiester with a phosphorothioate.

[0214] The chemical modifications listed above can be combined to yield modified gRNAs containing nucleosides and nucleotides (collectively "residues") that may have two, three, four, or more modifications. For example, modified residues can have modified sugars and modified nucleobases. In some embodiments, every base of a 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 a gRNA molecule are replaced with phosphorothioate groups. In some embodiments, a modified gRNA contains at least one modified residue at or near the 5' end of the RNA. In some embodiments, a 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.

[0215] In some embodiments, the guide RNAs disclosed herein comprise one of the modification patterns disclosed in WO2018107028 (the contents of which are incorporated herein by reference in relevant portions). In some embodiments, the guide RNAs disclosed herein comprise one of the structure / modification patterns disclosed in US20170114334 (which are incorporated herein by reference). In some embodiments, the guide RNAs disclosed herein comprise one of the structure / modification patterns disclosed in WO2017136794, WO2017004279, WO2019237069, US2018187186, US2019048338, WO2021119275, or WO2022125968 (which are incorporated herein by reference).

[0216] F. mRNA encoding an RNA-guided DNA binder In some embodiments, the cells or methods include an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA-binding agent (e.g., a Cas nuclease described herein). Cas9 ORFs are provided herein and are known in the art. As an example, the Cas9 ORF can be codon-optimized such that the coding sequence includes 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 for a given expression system (codon optimization). 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, WO2019 / 067910, and WO2022 / 125968 are incorporated herein by reference. Specifically, the ORF and Cas9 amino acid sequences 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 herein by reference.

[0217] 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.

[0218] In some embodiments, the mRNAs disclosed herein include 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 riboses of the first and second cap-proximal nucleotides of the mRNA both contain a 2'-hydroxyl. In Cap1, the riboses of the first and second transcribed nucleotides of the mRNA contain 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.

[0219] 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).

[0220] 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 from fresh leukopak samples, 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 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 their range 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 the blood or bone marrow can be screened with 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, e.g., Inaba, et al. (1992) J Exp Med. 176:1693-1702). Methods for promoting differentiation into CD4+ T cells are known in the art.

[0221] III. Method of Delivery The guide RNAs, RNA-guided DNA-binding agents (e.g., Cas nucleases), and nucleic acid sequences disclosed herein can be delivered in vitro or ex vivo to a cell or population of cells to produce engineered T cells, including the insertion of a sequence encoding dmTGFB1, optionally further comprising modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TNFA, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, and insertion of a sequence(s) encoding a regulatory T cell-promoting molecule (e.g., IL10, CTLA4), optionally further comprising insertion of a sequence(s) encoding a targeting receptor (e.g., a CAR), and optionally further comprising modification (e.g., knockdown) of a TCR sequence(s). The guide RNAs, RNA-guided DNA-binding agents, and nucleic acid constructs can be delivered individually or together in any combination, using the same or different delivery methods, as needed.

[0222] Conventional viral and non-viral gene delivery methods can be used to introduce guide RNAs 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.

[0223] 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.

[0224] Various delivery systems (e.g., vectors, liposomes, LNPs) containing guide RNAs, 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 performed by any route commonly used to introduce molecules into blood, body fluids, or cells for ultimate contact, including, but not limited to, injection, infusion, topical application, and electroporation. Suitable methods for administering such nucleic acids are available and well known to those skilled in the art.

[0225] In certain embodiments, the present 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., to modify (e.g., knockdown) IFNG and TNFA), or a donor construct comprising a sequence encoding a dmTGFB1 molecule, a regulatory T cell-promoting molecule (e.g., IL10), or a sequence(s) encoding 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-binding agent. In certain embodiments, the present invention includes DNA or RNA vectors encoding any one or more of the compositions described herein, or any combination. In some embodiments, the vector further comprises, for example, a promoter, an 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.

[0226] In certain embodiments, the present disclosure provides DNA or RNA vectors encoding a regulatory T cell-promoting molecule and a targeting receptor. Such vectors allow for the selection of cells based on the presence of the receptor for cells that also contain the coding sequence for the regulatory T cell-promoting molecule. Positive and negative selection based on the presence of cell surface molecules is known in the art.

[0227] 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 using multiple guide RNAs, the guide RNAs may be non-identical, such that they target different target sequences, or may be identical, such that they target the same target sequence. In some embodiments, the vector comprises multiple guide RNAs, each guide RNA may have different properties (e.g., activity or stability) within a complex with an RNA-guided DNA nuclease (e.g., a 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., a promoter, a 3' UTR, or a 5' UTR). In one embodiment, the promoter is a tRNA promoter (e.g., a 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 can 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 can be operably linked to a mouse or human U6 promoter. In other embodiments, the nucleotide sequence encoding the guide RNA can be operably linked to a mouse or human H1 promoter. In embodiments using multiple guide RNAs, the promoters used to drive expression can 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 can be provided on the same vector. In some embodiments, the nucleotides encoding the crRNA and the trRNA can be driven by the same promoter. In some embodiments, the crRNA and the trRNA can be transcribed into a single transcript. For example, crRNA and trRNA can be processed from a single transcript to form a dual-molecule guide RNA. Alternatively, crRNA and trRNA can be transcribed into a single-molecule guide RNA (sgRNA). In other embodiments, crRNA and trRNA can be driven by corresponding promoters on the same vector. In yet other embodiments, crRNA and trRNA can be encoded by different vectors.

[0228] In some embodiments, the nucleotide sequence encoding the guide RNA may be located on the same vector as the nucleotide sequence encoding the RNA-guided DNA-binding agent, such as a Cas protein. In some embodiments, expression of the guide RNA and the RNA-guided DNA-binding agent, such as a Cas protein, can be driven by their respective promoters. In some embodiments, expression of the guide RNA can be driven by the same promoter that drives 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, can be contained within a single transcript. For example, the guide RNA can 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 can be within the 5' UTR of the transcript. In other embodiments, the guide RNA can be within the 3' UTR of the transcript. In some embodiments, the intracellular half-life of the transcript can be reduced by incorporating the guide RNA within the 3' UTR of the transcript to shorten the length of the 3' UTR. In additional embodiments, the guide RNA can 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 from the transcript. In some embodiments, close temporal proximity of expression of the RNA guide DNA-binding agent (e.g., Cas protein) and guide RNA from the same vector can promote more efficient formation of CRISPR RNP complexes.

[0229] In some embodiments, the nucleotide sequence encoding the guide RNA or RNA-guided DNA binding agent can be located on the same vector containing a construct comprising a sequence encoding a dmTGFB1 molecule, a regulatory T cell-promoting molecule (e.g., IL10, CTLA4), or a targeting receptor (e.g., a CAR, e.g., MAdCAM-1 CAR). In some embodiments, the proximity of the construct comprising a sequence encoding a dmTGFB1 molecule, a regulatory T cell-promoting molecule (e.g., IL10, CTLA4), or a targeting receptor (e.g., a CAR, e.g., MAdCAM-1 CAR) and the guide RNA (or RNA-guided DNA binding agent) on the same vector can more efficiently promote insertion of the construct into the insertion site created by the guide RNA / RNA-guided DNA binding agent.

[0230] 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 genomic 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.

[0231] 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 Cas nuclease is a Cas9 nuclease from Neisseria meningitidis (i.e., Nme Cas9, e.g., Nme1, Nme2, or Nme3 Cas9). In some embodiments, the nucleotide sequence encoding the crRNA, trRNA, or crRNA and trRNA (which may be an sgRNA) comprises or consists of a guide sequence flanked by all or a portion of repeat sequences 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 conjunction with the crRNA, trRNA, or the crRNA and a trRNA in nature.

[0232] In some embodiments, the crRNA and trRNA are encoded by non-contiguous nucleic acids within a single 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 opposite strands of a single nucleic acid. In other embodiments, the crRNA and trRNA are encoded by the same strand of a single nucleic acid.

[0233] In some embodiments, the vector comprises a donor construct comprising a sequence encoding a dmTGFB1 molecule, 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 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, and 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 crRNA, trRNA, or crRNA and trRNA. In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding sgRNA and 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 crRNA, trRNA, and 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 Cas nuclease is a Cas9 nuclease from Neisseria meningitidis.In some embodiments, the nucleotide sequence encoding the crRNA, trRNA, or crRNA and trRNA (which may also be an sgRNA) comprises or consists of a guide sequence flanked by all or part of repeat sequences from a naturally occurring CRISPR / Cas system. A nucleic acid comprising or consisting of a crRNA, trRNA, or crRNA and trRNA can further comprise a vector sequence comprising or consisting of a nucleic acid not found with the crRNA, trRNA, or crRNA and trRNA in nature.

[0234] 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, minichromosomes, transposons, viral vectors, and expression vectors.

[0235] 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 characteristics of the vector. Such characteristics can include packaging capability, transduction efficiency, immunogenicity, genome integration, replication, transcription, and translation. In some embodiments, portions of the viral genome can be deleted to allow the virus to package larger foreign sequences. 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-deficient. 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 the viral components (e.g., viral proteins) required to amplify and package the vector into a viral particle. In such cases, one or more helper components (including one or more vectors encoding the 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 the viral components required for viral amplification and packaging.

[0236] 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.

[0237] 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. In certain embodiments, the AAV is acceptable for ex vivo use in human cells. In certain embodiments, the AAV is AAV6. 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 or public databases such as GenBank. As used herein, "AAV vector" refers to an AAV vector that contains heterologous sequences not derived from AAV (i.e., nucleic acid sequences heterologous to AAV), typically including sequences encoding a heterologous polypeptide 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. Generally, 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).

[0238] In some embodiments, the lentivirus can be integrating. In some embodiments, the lentivirus can be non-integrating. 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 for the 5' and 3' inverted terminal repeats (ITRs) and packaging signal ("I") have been deleted from the virus to increase packaging capacity. In yet other embodiments, the viral vector can be an HSV-1 vector. In some embodiments, HSV-1-based vectors are helper-dependent, while in other embodiments, they are helper-independent. For example, amplicon vectors that retain only the packaging sequence require a helper virus containing structural components for packaging, while a 30 kb deleted HSV-1 vector in which nonessential viral functions have been removed 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 viral head is emptied. In further embodiments, the viral vector may be a baculovirus vector. In still 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 necessary 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.

[0239] 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 to drive expression of one or more coding sequences when integrated into a cell (e.g., as exemplified herein, it uses the cell's endogenous promoter when inserted into a specific genomic locus of the cell). Suitable promoters 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 still other embodiments, the promoter may be shortened but retain its function. For example, the promoter may have a normal or reduced size suitable for proper packaging of the vector into a virus.

[0240] In some embodiments, the vector can include a nucleotide sequence encoding an RNA-guided DNA-binding agent, 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.

[0241] In some embodiments, the vector can comprise any one or more of the constructs described herein comprising a sequence encoding a dmTGFB1 molecule, 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 sequence of the dmTGFB1 molecule, a 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 dmTGFB1 molecule, a 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 dmTGFB1 molecule, regulatory T cell promoting molecule (e.g., IL10, CTLA4), or targeting receptor (e.g., a CAR, e.g., a MAdCAM-1 CAR) is not linked to a promoter driving expression of a heterologous gene.

[0242] In some embodiments, the promoter can be constitutive, inducible, or tissue-specific. In some embodiments, the promoter may be a constitutive promoter. Non-limiting exemplary constitutive promoters include the cytomegalovirus immediate-early promoter (CMV), the simian virus (SV40) promoter, the adenovirus major late (MLP) promoter, the Rous sarcoma virus (RSV) promoter, the mouse mammary tumor virus (MMTV) promoter, the phosphoglycerate kinase (PGK) promoter, the elongation factor alpha (EF1a) promoter, the ubiquitin promoter, the actin promoter, the tubulin promoter, the immunoglobulin promoter, functional fragments thereof, or a combination of any 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 the EF1a promoter. In some embodiments, the promoter may be an inducible promoter. Non-limiting exemplary 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).

[0243] In certain embodiments, the promoter is amenable for use in ex vivo applications in human cells.

[0244] In some embodiments, the promoter can be a tissue-specific promoter, for example, a promoter specific for expression in T cells.

[0245] 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.

[0246] In some embodiments, the vector system can include an inducible promoter to initiate expression only after delivery to a target cell. Non-limiting exemplary 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).

[0247] In additional embodiments, the vector system can include a tissue-specific promoter.

[0248] Non-limiting exemplary viral vector sequences are shown below. CTLA4 insert (nucleotide sequence) ATGGCCTGCTTGGGCTTCCAAAGGCATAAAGCCCAGCTTAATCTTGCTACTCGCACGTGGCCCTGCACATTGCTCTTTTTCCTCCTGTTCATTCCCGTGTTTTGCAAGGCGATGCATGTGGCACAACCTGCCGTCGTTCTGGCATCATCAAGAGGTATTGCTAGCTTCGTTTGTGAGTACGCCTCCCCTGGAAAAGCGACGGAGGTGCGCGTCACTGTATTGCGGCAAGCCGACAGCCAAGTTACTGAAGTCTGCGCGGCAACGTATATGATGGGCAATGAGCTGACATTCCTTGACGATTCAATCTGCACGGGAACAAGTAGTGGTAACCAGGTGAATCTCACTATTCAAGGTCTGAGAGCCATGGACACCGGCCTCTACATTTGTAAGGTGGAGCTGATGTATCCTCCCCCATATTATCTGGGGATCGGAAATGGGACACAGATATATGTTATTGATCCCGAGCCATGTCCCGATAGTGACTTCCTCTTGTGGATACTTGCCGCTGTGAGCAGTGGTTTGTTTTTTTATTCATTCCTCCTTACGGCAGTATCACTTTCAAAAATGCTCAAGAAGCGAAGTCCTTTGACAACTGGCGTATATGTCAAAATGCCACCAACAGAGCCCGAATGTGAGAAACAGTTCCAGCCGTACTTTATTCCTATAAACTGA(SEQ ID NO: 137) CTLA4 Insert (amino acid sequence) MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN(SEQ ID NO: 130) IL10 Insert (nucleotide sequence) ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAACTGA(SEQ ID NO: 138) IL10 Insert (amino acid sequence) MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN(SEQ ID NO: 124) Empty lentiviral vector Empty Lenticoverter Additional sequences are set forth throughout the specification and in the tables below, as well as in the Sequence Listing attached hereto, which forms a part of the specification.

[0249] Sequence Listing In the tables below, the terms "mA," "mC," "mU," or "mG" are used to indicate nucleotides that are 2'-O-Me modified.

[0250] In the table below, each "N" is used independently to represent any nucleotide (e.g., A, U, T, C, G). In certain embodiments, the nucleotide is an unmodified RNA nucleotide residue, i.e., a ribose sugar and a phosphodiester backbone.

[0251] In the table below, "*" is used to indicate a PS modification. In this application, the terms A*, C*, U*, or G* may be used to indicate a nucleotide that is linked to the next (e.g., 3') nucleotide by a PS bond.

[0252] When a DNA sequence (containing T) is referred to in relation to RNA, it is understood that T should be replaced with U (which may or may not be modified depending on the context), and vice versa.

[0253] In the table below, single letter amino acid abbreviations are used to indicate peptide sequences. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

Table 4-5

Table 4-6

Table 4-7

Table 4-8

Table 4-9

Table 4-10

Table 4-11

Table 4-12

Table 4-13

Table 4-14

Table 4-15

Table 4-16

Table 4-17

Table 4-18

[0254] Vectors containing donor constructs comprising sequences encoding guide RNAs, RNA-binding DNA binders, or dmTGFB1 molecules, regulatory T cell-promoting molecules (e.g., IL10, CTLA4), or targeting receptors (e.g., CARs) can be delivered individually or in any combination by liposomes, nanoparticles, exosomes, or microvesicles. Vectors can also be delivered by lipid nanoparticles (LNPs). Donor constructs comprising sequences encoding one or more guide RNAs, RNA-binding DNA binders (e.g., mRNA), or heterologous proteins can be delivered individually or in any combination by LNPs, liposomes, nanoparticles, exosomes, or microvesicles. Donor constructs comprising sequences encoding one or more guide RNAs, RNA-binding DNA binders (e.g., mRNA), or heterologous proteins can be delivered individually or in any combination by LNPs. In some embodiments, one or more guide RNAs and RNA-guided DNA binders (e.g., mRNA) are delivered by LNPs. Donor constructs can be delivered by viral vectors.

[0255] 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.

[0256] As used herein, lipid nanoparticles (LNPs) refer to particles comprising a plurality (i.e., two or more) of lipid molecules physically associated with one another by intermolecular forces. LNPs can be, for example, microspheres (including unilamellar and multilamellar vesicles, e.g., "liposomes," which are lamellar-phase lipid bilayers that are substantially spherical in some embodiments and, in certain embodiments, can include an aqueous core containing, for example, a substantial portion of RNA molecules), the dispersed phase of an emulsion, a micelle, or the internal phase of a suspension (see, e.g., International Publication No. WO2017173054, the entire contents of which are incorporated herein by reference). Any LNP known to those skilled in the art to be capable of delivering nucleotides to a subject can be utilized. Exemplary LNP formulations and methods for delivering agents to T cells for modification are described, for example, in WO2021222287.

[0257] In some embodiments, provided herein are methods for delivering any of the guide RNAs described herein or the donor constructs disclosed herein, alone or in combination, to a cell or cell population or a subject, wherein any one or more of the components are associated with LNPs. In some embodiments, the method further comprises an RNA-guided DNA-binding agent (e.g., Cas9 or a sequence encoding Cas9).

[0258] In some embodiments, provided herein are compositions comprising any of the guide RNAs described herein or donor constructs disclosed herein, alone or in combination, together with LNPs. In some embodiments, the compositions further comprise an RNA-guided DNA binding agent (e.g., Cas9 or a sequence encoding Cas9).

[0259] 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 WO2019067992, WO2017173054, WO2015095340, and WO2014136086, and the references cited therein. In some embodiments, the LNPs comprise a molar ratio of cationic lipid amine to 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 pH.

[0260] In some embodiments, LNPs related to 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.

[0261] 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., WO2019067992, WO2017173054, or WO2021222287 (the contents of which are incorporated herein by reference in their entirety).

[0262] In some embodiments, the LNP composition includes an RNA component and a lipid component, wherein 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 (distearoylphosphatidylcholine), and PEG-DMG (1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene glycol 2000 (PEG2k-DMG)). In certain embodiments, the lipid-nucleic acid assembly contained ionized 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 (distearoylphosphatidylserine), and PEG2k-DMG (1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene glycol 2000 (PEG2k-DMG)). In certain embodiments, the components are present in a molar ratio of 50:38:9:3 for lipid A, cholesterol, DSPC, and PEG-DMG, respectively. In certain embodiments, the components are present in a molar ratio of 35:47.5:15:2.5 for lipid A, cholesterol, DSPC, and PEG-DMG, respectively. The lipid nucleic acid assembly 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.

[0263] It will be apparent that the donor construct containing the guide RNA, RNA-guided DNA binder (e.g., Cas nuclease or a nucleic acid encoding a Cas nuclease), and a sequence encoding a dmTGFB1 molecule, a regulatory T cell-promoting molecule (e.g., IL10), or a targeting receptor (e.g., CAR) can be delivered using the same or different systems. For example, the guide RNA, Cas nuclease, and 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.

[0264] 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, e.g., in one vector, two vectors, individual vectors, one LNP, two LNPs, individual LNPs, or a combination 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, or more days). As a further example, the guide RNA and Cas nuclease can be delivered 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) as a vector or in association with an LNP.

[0265] IV. Methods for Manipulating T Cells The present disclosure provides methods for engineering T cells to include a modification by insertion into the cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence. The present disclosure provides methods for engineering T cells to include a modification by insertion into the cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence, further comprising a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, and a 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 methods for engineering T cells to include a modification by insertion into the cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence. The present disclosure provides methods for engineering T cells to include a modification by insertion into the cell of a heterologous sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, and a insertion into the cell of a heterologous sequence(s) encoding IL10 under the control of a promoter sequence. The present disclosure provides methods of engineering T cells to include a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, and insertion into the cell of heterologous sequence(s) encoding CTLA4 under the control of a promoter sequence. The present disclosure provides methods of engineering T cells to include a modification by insertion into the cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence. The present disclosure provides methods of engineering T cells to further include a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, and insertion into the cell of heterologous sequences encoding IL10 and CTLA4, each under the control of a promoter sequence. In certain embodiments, the engineered T cells include modifications in the endogenous nucleic acid sequences encoding IFNG and IL17A, respectively.

[0266] In some embodiments, the method comprises inserting into the cell a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence, modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding a TNFA, modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG or IL17A, and inserting into the cell heterologous sequence(s) encoding a regulatory T cell-stimulating molecule (e.g., IL10 or CTLA4), and further comprising modifying (e.g., knocking down) a TCR sequence(s). In certain embodiments, the engineered T cell comprises modifications in the endogenous nucleic acid sequences encoding IFNG and IL17A, respectively.

[0267] In some embodiments, the method comprises engineering a T cell to comprise the modification by insertion into the cell of an endogenous nucleic acid sequence encoding dmTGFB1 under the control of a promoter sequence, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TNFA, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, and insertion into the cell of heterologous sequence(s) encoding a regulatory T cell-promoting molecule (e.g., IL10 or CTLA4), and further comprising the insertion into the cell of heterologous sequence(s) encoding a targeting receptor (e.g., a CAR). In certain embodiments, the engineered T cell comprises modifications in the endogenous nucleic acid sequences encoding IFNG and IL17A, respectively.

[0268] In some embodiments, the method includes engineering a T cell to include modification by insertion into the cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A; insertion into the cell of heterologous sequence(s) encoding a regulatory T cell-stimulating 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., a CAR).

[0269] In some embodiments, the method comprises modifying by insertion into the cell a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence, modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding a TNFA, modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG or IL17A, inserting into the cell heterologous sequence(s) encoding a regulatory T cell-promoting molecule, and optionally knocking down a TCR gene and optionally inserting into the cell a targeting receptor (e.g., a CAR), engineered using the CRISPR / Cas system and guide RNA disclosed herein. In certain embodiments, the engineered T cells comprise modifications in the endogenous nucleic acid sequences encoding IFNG and IL17A, respectively.

[0270] The present disclosure provides methods of engineering T cells to include modification by insertion into the cell of a heterologous sequence encoding a regulatory T cell-promoting molecule under the control of a promoter sequence, and further including modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TNFA and modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A. The present disclosure provides methods of engineering T cells to include modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TNFA, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and insertion into the cell of heterologous sequence(s) encoding IL10 under the control of a promoter sequence. The present disclosure provides methods of engineering T cells to include modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TNFA, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and insertion into the cell of heterologous sequence(s) encoding CTLA4 under the control of a promoter sequence. The present disclosure provides methods of engineering T cells to further comprise modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IL17A, modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA, and inserting heterologous sequences encoding IL10 and CTLA4 into the cells, each under the control of a promoter sequence. In certain embodiments, the method does not comprise modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG.

[0271] In some embodiments, the method comprises modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA, modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IL17A, and inserting into the cell heterologous sequence(s) encoding a regulatory T cell-promoting molecule (e.g., IL10 or CTLA4), and further comprising modifying (e.g., knocking down) a TCR sequence(s). In certain embodiments, the method does not comprise modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG.

[0272] In some embodiments, the method comprises engineering a T cell to include modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA, modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IL17A, 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., a CAR). In certain embodiments, the method does not include modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG.

[0273] In some embodiments, the method involves engineering a T cell to include modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, 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., a CAR). In certain embodiments, the method does not include modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0274] In some embodiments, the method comprises modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA, modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IL17A, inserting into the cell heterologous sequence(s) encoding a regulatory T cell-promoting molecule, optionally knocking down a TCR gene, and optionally inserting into the cell a targeting receptor (e.g., a CAR), engineered using a CRISPR / Cas system and guide RNAs disclosed herein. In certain embodiments, the method does not comprise modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG.

[0275] In these embodiments, the inserted regulatory T cell-promoting molecule can be provided via a donor construct and can be selected from IL10, CTLA4, IDO1, ENTPD1, NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, BACH2, and IL2RA, and modifications (e.g., knockdown) of TCR gene sequence(s).

[0276] In these embodiments, the inserted targeting receptor can be provided via a donor construct. In some embodiments, the targeting receptor can be a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a receptor for 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 the 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 enables the cell to bind to a target site (e.g., a specific cell or tissue within an organism). In some of these embodiments, the targeting receptor is a CAR capable of targeting MAdCAM-1.

[0277] Suitable gene editing systems for engineering T cells to contain 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 through 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.

[0278] Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences in 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 desired DNA sequences and promote DNA cleavage at specific locations (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 engineered nuclease-mediated genome editing. Such methods and compositions for use therein are known in the art. See, e.g., WO2019147805, WO2014040370, and WO2018073393, the contents of which are incorporated herein by reference in their entireties.

[0279] Zinc finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain with a DNA cleavage domain. The zinc finger domain targets specific desired DNA sequences and can be engineered to allow zinc finger nucleases to target unique sequences within complex genomes. ZFNs typically use the nonspecific cleavage domain from the type II restriction endonuclease FokI as the cleavage domain. The cleavage is repaired by endogenous DNA repair machinery, allowing ZFNs to precisely alter the genomes of higher organisms. Such methods and compositions for use therein are known in the art. See, e.g., WO2011091324, the contents of which are incorporated herein by reference in their entirety.

[0280] 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 19–23 nucleotides long, forming a 19–21 nucleotide duplex) can reduce the activity of messenger RNA (mRNA) molecules by instructing the RNA-induced silencing complex (RISC) to degrade them, thereby preventing translation through post-transcriptional gene silencing. Short hairpin RNA (shRNA) is a type of siRNA in which the duplex-forming strand has a hairpin structure and is often generated by transcription from an expression vector. RNAi can also be achieved through long RNA duplex structures called Dicer substrate molecules, which are cleaved by the enzyme Dicer before loading into RISC and promoting cleavage of the target mRNA. Such methods and compositions 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 together with an expression vector.

[0281] 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., knocking 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).

[0282] In some embodiments, a donor construct containing the sequence for insertion (e.g., a sequence encoding dmTGFB1 or a regulatory T cell-promoting molecule (e.g., 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.

[0283] 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 sequences encoding dmTGFB1, a regulatory T cell-promoting molecule (e.g., IL10, CTLA4), or a targeting receptor (e.g., a CAR disclosed herein, e.g., a MAdCAM-1 CAR) can be inserted to express dmTGFB1, a regulatory T cell-promoting molecule (e.g., IL10, CTLA4), or a CAR (e.g., a MAdCAM-1 CAR). The targeting receptor (e.g., a CAR, e.g., a MAdCAM-1 CAR), or the inserted sequence, dmTGFB1, or regulatory T cell promoting molecule (e.g., IL10 or CTLA4), may be heterologous with respect to its insertion site or locus, e.g., a safe harbor locus or TCR locus where the dmTGFB1, regulatory T cell promoting molecule (e.g., IL10, CTLA4), or targeting receptor (e.g., a CAR, e.g., a MAdCAM-1 CAR) is not normally expressed, as described herein. In some embodiments, guide RNAs described herein can be used in conjunction with an RNA-guided DNA-binding agent (e.g., a Cas nuclease) according to the present methods to create an insertion site into which a donor construct comprising a sequence encoding dmTGFB1, a 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 inserted to express dmTGFB1, a regulatory T cell-promoting molecule (e.g., IL10, CTLA4), or a CAR (e.g., a MAdCAM-1 CAR). Guide RNAs for inserting dmTGFB1, a regulatory T cell-promoting molecule (e.g., IL10, CTLA4), or a targeting receptor (e.g., a CAR, e.g., a MAdCAM-1 CAR) into a specific genomic locus are exemplified and described herein. In an alternative embodiment, the targeting receptor is a TNFA-targeting receptor.

[0284] 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, IL17A, 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 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), and 2) transduced with a donor construct comprising nucleic acid sequence(s) encoding dmTGFB1, 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), or a TNFA-targeting receptor. In certain embodiments, the engineered cells are selected for expression of the targeting receptor.

[0285] In some embodiments, CD4+ T cells are engineered by transduction with gRNA (e.g., gRNA targeting IFNG, TNFA, IL17A, 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 nucleic acid sequence(s) encoding dmTGFB1, a regulatory T cell-promoting molecule (e.g., IL10 or CTLA4), and a targeting receptor (e.g., a CAR, e.g., MAdCAM-1 CAR) or a TNFA-targeting receptor, or 2) transduced with 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). In certain embodiments, the engineered cells are selected for expression of the targeting receptor.

[0286] In some embodiments, CD4+ T cells are engineered by transduction with gRNA (e.g., 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 nucleic acid sequence(s) encoding dmTGFB1, a regulatory T cell-promoting molecule (e.g., IL10 or CTLA4), and a targeting receptor (e.g., a CAR, e.g., MAdCAM-1 CAR) or a TNFA-targeting receptor, or 2) transduced with gRNA targeting a nucleic acid sequence encoding a pro-inflammatory cytokine (e.g., IFNG, IL17A, or TNFA), and an RNA-guided DNA binder (e.g., Cas nuclease).

[0287] As described herein, donor constructs comprising sequences encoding dmTGFB1, a regulatory T cell-promoting molecule (e.g., IL10, CTLA4), or a targeting receptor (e.g., a CAR), a guide RNA (e.g., a gRNA targeting IFNG, IL17A, TNFA, or a 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 sequences encoding dmTGFB1, a regulatory T cell-promoting molecule (e.g., IL10, CTLA4), or a targeting receptor (e.g., a CAR). In some embodiments, the guide RNA and Cas nuclease are associated with the LNP and delivered to a cell or population of cells after delivery of a donor construct comprising sequences encoding dmTGFB1, a regulatory T cell promoting molecule (e.g., IL10, CTLA4), or a targeting receptor (e.g., CAR).

[0288] 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 regulatory T cell properties (e.g., immune response suppression properties).

[0289] gRNAs, donor constructs, and RNA-guided DNA binders for modifying (e.g., knocking down) IFNG, TNFA, IL17A, or TCR gene expression, or for inserting sequences encoding dmTGFB1, regulatory T cell promoting molecules (e.g., IL10, CTLA4), or targeting receptors (e.g., CARs, e.g., MAdCAM-1 CARs) or TNFA targeting receptors, 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.

[0290] 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. While the use of the CRISPR / Cas system is exemplified herein, it will be understood that suitable variations on such systems 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 an 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.

[0291] 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 in the Type II CRISPR systems of S. pyogenes, S. aureus, Neisseria meningitidis, and other prokaryotes (see, e.g., the list in the next paragraph), as well as variant or mutant (e.g., engineered, non-naturally occurring, naturally occurring, or other variant) versions thereof. See, e.g., US20160312198, US20160312199.

[0292] 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。.

[0293] 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.

[0294] 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.

[0295] 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.

[0296] 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.

[0297] 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 the 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.

[0298] 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-strand 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 variation of a Cas nuclease (e.g., the Cas nucleases described above) in which the endonucleolytic activity site has been inactivated, for example, by one or more alterations (e.g., point mutations) in the catalytic domain. For a discussion of Cas nickases and exemplary catalytic domain alterations, see, e.g., U.S. Patent No. 8,889,356. In some embodiments, the Cas nickase (e.g., Cas9 nickase) has an inactivated RuvC or HNH domain.

[0299] 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 such that one of the nuclease domains is mutated or completely or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, a nickase with a RuvC domain that has 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 that has reduced activity is used. In some embodiments, a nickase with an inactive HNH domain is used.

[0300] In some embodiments, conserved amino acids within the Cas protein nuclease domain are substituted to reduce or alter nuclease activity. In some embodiments, the Cas nuclease may comprise 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 may comprise 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 sequence of Francisella novicida U112 Cpf1 (FnCpf1) (UniProtKB-A0Q7Q2(CPF1_FRATN))). Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain, or the RuvC or RuvC-like domain of N. meningitidis include Nme2Cas9D16A (HNH nickase) and Nme2Cas9H588A (RuvC nickase).

[0301] In some embodiments, a nickase is provided in combination with a pair of guide RNAs that are complementary to the sense and antisense strands of a target sequence, respectively. In this embodiment, the guide RNAs guide the nickase to the target sequence, where it introduces a DSB by generating nicks on opposite strands of the target sequence (i.e., double nicking). In some embodiments, a nickase is used with two separate guide RNAs that target opposite strands of DNA to generate a double nick within the target DNA. In some embodiments, a nickase is used with two separate guide RNAs that are selected to be in close proximity to generate a double nick within the target DNA.

[0302] In some embodiments, the RNA-guided DNA binding agent comprises (eg, is or comprises) one or more heterologous functional domains.

[0303] In some embodiments, the heterologous functional domain can facilitate the transport of the RNA-guided 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-guided DNA binding agent can be fused to one to five NLS(s). In some embodiments, the RNA-guided DNA binding agent can be fused to two, three, or four NLS(s). In some embodiments, the RNA-guided DNA binding agent can be fused to two NLS(s). In some embodiments, the RNA-guided DNA binding agent can be fused to one NLS. When one NLS is used, the NLS can be linked at the N-terminus or C-terminus of the RNA-guided DNA binding agent sequence. In some embodiments, the NLS is not linked to the C-terminus. It can also be inserted within the sequence of the RNA-guided DNA binding agent. In other embodiments, the RNA-guided DNA binding agent can be fused to multiple NLSs. In certain circumstances, at least two NLSs can be the same (e.g., two SV40 NLSs). In certain embodiments, at least two different NLSs are present in the RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is fused to two SV40 NLS sequences linked at the carboxy terminus. In some embodiments, the RNA-guided DNA binding agent can be fused to two NLSs, one linked at the N terminus and the other linked at the C terminus. In some embodiments, the RNA-guided DNA binding agent can be fused to three NLSs. In some embodiments, the RNA-guided DNA binding agent may not be fused to an NLS. In some embodiments, the NLS can be a monopartite sequence, such as 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 nucleoplasmin NLS, KRPAATKKAGQAKKKK (SEQ ID NO: 145).In certain embodiments, a single PKKKRKV (SEQ ID NO: 143) NLS can be attached at the C-terminus of the RNA-guided DNA binder. One or more linkers are optionally included in the fusion site.

[0304] V. Treatment method The present disclosure provides a method for suppressing an immune response in a subject, the method comprising administering engineered T cells comprising: insertion into the cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A; 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, the method comprising administering engineered T cells comprising modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A; and insertion into the cell of a heterologous sequence encoding IL10 under the control of a promoter sequence. The present disclosure provides a method for suppressing an immune response in a subject, the method comprising administering engineered T cells comprising: insertion into the cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A; and insertion into the cell of a heterologous sequence encoding CTLA4 under the control of a promoter sequence. The present disclosure provides a method for suppressing an immune response in a subject, the method comprising administering engineered T cells comprising: insertion into the cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A; and insertion into the cell of a heterologous sequence encoding IL10 or CTLA4, each under the control of a promoter sequence.

[0305] The present disclosure provides a method for treating an autoimmune disorder in a subject, the method comprising administering engineered T cells comprising: insertion into the cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; 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 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, the method comprising administering engineered T cells comprising: insertion into the cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; 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 a heterologous sequence encoding IL10 under the control of a promoter sequence. The present disclosure provides a method for treating an autoimmune disorder in a subject, the method comprising administering engineered T cells comprising: insertion into the cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; 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 CTLA4 under the control of a promoter sequence. The present disclosure provides a method for treating an autoimmune disorder in a subject, the method comprising administering engineered T cells comprising: insertion into the cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; 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 sequences encoding IL10 and CTLA4, each under the control of a promoter sequence.

[0306] The present disclosure provides a method for treating GvHD in a subject, comprising administering engineered T cells comprising: insertion into the cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFN-γ or IL17A; 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 engineered T cells comprising: insertion into the cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFN-γ or IL17A; 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, the method comprising administering engineered T cells comprising: insertion into the cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFN-γ or IL17A; and insertion into the cell of heterologous sequence(s) encoding CTLA4 under the control of a promoter sequence. The present disclosure provides a method for treating GvHD in a subject, the method comprising administering engineered T cells comprising: insertion into the cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFN-γ or IL17A; and insertion into the cell of heterologous sequences encoding IL10 and CTLA4, each under the control of a promoter sequence.

[0307] The present disclosure provides a method for suppressing an immune response in a subject, the method comprising administering engineered T cells that comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and insertion into the cells 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, the method comprising administering engineered T cells that comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and insertion into the cells of a heterologous sequence encoding IL10 under the control of a promoter sequence. The present disclosure provides methods for suppressing an immune response in a subject, the method comprising administering engineered T cells that comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and insertion into the cells of a heterologous sequence encoding CTLA4 under the control of a promoter sequence. The present disclosure provides methods for suppressing an immune response in a subject, the method comprising administering engineered T cells that comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and insertion into the cells of heterologous sequences encoding IL10 and CTLA4, each under the control of a promoter sequence. In certain embodiments, the method does not include administering engineered T cells that comprise a modification, including knockdown, of an endogenous nucleic acid sequence encoding IFNG.

[0308] The present disclosure provides a method for treating GvHD in a subject, the method comprising administering engineered T cells that comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and insertion into the cells of 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, the method comprising administering engineered T cells that comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and insertion into the cells of heterologous sequence(s) encoding IL10 under the control of a promoter sequence. The present disclosure provides methods for treating GvHD in a subject, the methods comprising administering engineered T cells comprising a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and insertion into the cell of heterologous sequence(s) encoding CTLA4 under the control of a promoter sequence. The present disclosure provides methods for treating GvHD in a subject, the methods comprising administering engineered T cells comprising a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and insertion into the cell of heterologous sequences encoding IL10 and CTLA4, each under the control of a promoter sequence. In certain embodiments, the methods do not include administering engineered T cells comprising a modification, including knockdown, of an endogenous nucleic acid sequence encoding IFNG.

[0309] In some embodiments, the method comprises administering engineered T cells that comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding INFG or IL17A, and insertion of a sequence(s) encoding a regulatory T cell-promoting molecule, and further comprise a modification (e.g., knockdown) of a TCR sequence(s).

[0310] In some embodiments, the method comprises administering engineered T cells that comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and insertion of sequence(s) encoding a regulatory T cell-promoting molecule, and further comprise a modification (e.g., knockdown) of a TCR sequence(s). In certain embodiments, the method does not comprise administering engineered T cells that comprise a modification that comprises knockdown of an endogenous nucleic acid sequence encoding IFNG.

[0311] In some embodiments, the method comprises administering engineered T cells that comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, and an insertion of sequence(s) encoding a regulatory T cell-promoting molecule, and further comprise an insertion of sequence(s) encoding a targeting receptor (e.g., a CAR).

[0312] In some embodiments, the method comprises administering engineered T cells that comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and an insertion of sequence(s) encoding a regulatory T cell-promoting molecule, and further comprise an insertion of sequence(s) encoding a targeting receptor (e.g., a CAR). In certain embodiments, the method does not comprise administering engineered T cells that comprise a modification that comprises knockdown of an endogenous nucleic acid sequence encoding IFNG.

[0313] In some embodiments, the 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, 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., a CAR) can target the engineered T cells to tissues containing 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., a 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., a CAR) can target the engineered T cells to B cells, e.g., the targeting receptor is a CAR that targets CD19, for example, to suppress immune responses in disorders such as multiple sclerosis and systemic lupus erythematosus. In some embodiments, the 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, for example, to suppress immune responses in disorders such as multiple sclerosis and systemic lupus erythematosus. In some embodiments, the targeting receptor (e.g., a CAR) can target the engineered T cells to inflamed tissue, for example, the targeting receptor is a CAR that targets TNFA, for example, 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 containing mature B lymphocytes, e.g., the 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., the targeting receptor is a CAR that targets citrullinated vimentin, e.g., to suppress immune responses in disorders such as rheumatoid arthritis.

[0314] 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).

[0315] In some embodiments, the method comprises administering engineered T cells that comprise modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, insertion of sequence(s) encoding a regulatory T cell-promoting molecule, and insertion of sequence(s) encoding a targeting receptor (e.g., CAR), and further comprise modification (e.g., knockdown) of TCR sequence(s).

[0316] In some embodiments, the method comprises administering engineered T cells that comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, an insertion of sequence(s) encoding a regulatory T cell-promoting molecule, and an insertion of sequence(s) encoding a targeting receptor (e.g., a CAR), and further comprise a modification (e.g., knockdown) of a TCR sequence(s). In certain embodiments, the method does not comprise administering engineered T cells that comprise a modification that comprises knockdown of an endogenous nucleic acid sequence encoding IFNG.

[0317] In some embodiments, the inserted sequence(s) 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.

[0318] In some embodiments, the method comprises administering a population of T cells comprising engineered T cells 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).

[0319] 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.

[0320] 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 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). [Example]

[0321] 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.

[0322] 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 an RNA cargo concentration of approximately 0.45 mg / mL.

[0323] 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 molar ratios of 35:47.5:15:2.5 for lipid, cholesterol, DSPC, and PEG-DMG, 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.

[0324] LNPs were prepared using a cross-flow technique that utilizes impingement jet mixing of lipids in ethanol with two volumes of RNA solution and one volume of water. The lipids in ethanol were mixed with two volumes of RNA solution through a mixing cross. A fourth water stream was mixed with the cross's outlet stream through an in-line tee (see Figure 2 in WO2016010840). The LNPs were held at room temperature for 1 hour and further diluted with water (approximately 1:1 v / v). The 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 a PD-10 desalting column (GE). Alternatively, the LNPs were optionally concentrated using a 100 kDa Amicon spin filter and buffer-exchanged into TSS using a PD-10 desalting column (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.

[0325] 1.2. In vitro transcription of mRNA (IVT) Capped and polyadenylated mRNA containing N1-methylpseudo-U was generated by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase. Plasmid DNA containing the T7 promoter, transcription sequence, and polyadenylation region was linearized by incubation with XbaI at 37°C for 2 hours 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. The linearized plasmid was purified from enzymes and buffer salts. IVT reactions to generate modified mRNA were performed at 37°C for 1.5–4 h with 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 1x reaction buffer. 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 the RNeasy Maxi Kit (Qiagen) according to the manufacturer's instructions. Alternatively, mRNA was purified using a precipitation protocol (sometimes followed by HPLC-based purification). Briefly, after DNase digestion, mRNA was purified using LiCl, ammonium acetate, and sodium acetate precipitations. In the case of HPLC-purified mRNA, the mRNA was purified by RP-IP HPLC after LiCl precipitation and reconstitution (see, e.g., Kariko, et al. Nucleic Acids Research, 2011, Vol. 39, No. 21 e142). Fractions selected for pooling were combined and desalted by sodium acetate / ethanol precipitation as described above.In a further alternative method, mRNA was purified by LiCl precipitation and then further purified by tangential flow filtration. RNA concentration was quantified by measuring light absorbance at 260 nm (Nanodrop), and transcripts were analyzed by capillary electrophoresis on a Bioanlayzer (Agilent).

[0326] Streptococcus pyogenes ("Spy") Cas9 mRNA encoding the open reading frame (SEQ ID NO: 17) was generated from plasmid DNA 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-methylpseudouridine 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. Guide RNAs are chemically synthesized by methods known in the art.

[0327] 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 instructions.

[0328] To quantitatively determine the editing efficiency at the target position in the genome, deep sequencing is used to identify the presence of insertions and deletions introduced by gene editing.Based on the target site in the target gene (e.g., TRAC), PCR primers are designed to amplify the target genomic region.Primer sequences are designed according to standard methods in the art.

[0329] Additional PCR was performed according to the manufacturer's operating instructions (Illumina), and sequencing chemistry was applied. Amplicons were sequenced on an Illumina MiSeq or NextSeq instrument. After removing reads with low quality scores, reads were aligned to the human reference genome (e.g., hg38). The resulting file containing the reads was mapped to the reference genome (BAM file), 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. The 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).

[0330] Example 2. Suppressive activity of engineered T cells expressing TGFB1 alleles The suppressive phenotype of T cells was assessed by lentiviral overexpression of a constitutively active TGFB1 mutant that uncouples TGFB1 from LAP. TGFB1 is a well-known suppressive cytokine that is produced in a proform consisting of TGFB1 and latency-associated peptide (LAP). TGFB1 activation requires cleavage of the LAP accessory protein.

[0331] Example 2.1. Preparation of T Cells Human CD3+CD4+ T cells were prepared from fresh leukopaks (AllCells, donor no. 32015). CD3+CD4+ T cells were isolated by negative selection using a human CD4+ T cell isolation kit (Miltenyi, catalog no. 130-096-533) according to the manufacturer's instructions. CD3+CD4+ T cells were cultured at 1x10^ in a total of 5ml of OpTmizer basal medium containing CTS OpTmizer T Cell Expansion SFM (Gibco, Cat. No. A3705001), 1% penicillin-streptomycin, 1X Glutamax, 10mM HEPES, 2.5% human AB serum (Gemini, Cat. No. 100-512), supplemented with 200U / mL recombinant human interleukin-2 (Miltenyi Biotec, Cat. No. 130-097-746), 5ng / mL recombinant human interleukin-7 (StemCell technologies, Cat. No. 78053.1), and 5ng / mL recombinant human interleukin-15 (StemCell technologies, Cat. No. 78031.1). 6 Cells were plated at a density of 1000 cells / mL. Cells were activated by adding 25 μL / mL of ImmunoCult Human CD3 / CD28 T cell Activator (Stemcell Technologies, Cat. No. 10991) and cultured at 37°C for 48 hours before lentiviral transduction.

[0332] Example 2.2. T Cell Transduction and Cell Sorting Activated CD3+CD4+ T cells were transduced with lentiviral constructs to overexpress wild-type or mutant TGFB1. 48 hours after activation, CD3+CD4+ T cells were harvested, washed, and cultured at 1x10^ in OpTmizer basal medium containing CTS OpTmizer T Cell Expansion SFM (Gibco, Cat. No. A3705001), 1% penicillin-streptomycin, 1X Glutamax, 10mM HEPES, and 2.5% human AB serum (Gemini, Cat. No. 100-512).6 The cells were resuspended at a density of 100 cells / 100 μL. The concentrated viral supernatant was added to the CD3+CD4+ T cells and centrifuged at 1000 × g 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 20 mL of OpTmizer basal medium supplemented with 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), 500 U / mL IL-2, 5 ng / mL IL-7, and 5 ng / mL IL-17. Transduced CD3+CD4+ T cells were cultured for 3–4 days and then 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 30 mL of OpTmizer basal medium containing CTS OpTmizer T Cell Expansion SFM (Gibco, Cat. No. A3705001), 1% penicillin-streptomycin, 1X Glutamax, 10 mM HEPES, and 2.5% human AB serum (Gemini, Cat. No. 100-512), supplemented with 500 U / mL IL-2, 5 ng / mL IL-7, and 5 ng / mL IL-17.

[0333] Natural regulatory T cells (nTregs) were prepared using methods known in the art. Briefly, CD25+ cells were separated from autologous CD4+ T cells in PBMCs and isolated in Example 2.1 using CD25 microbeads (Miltenyi, catalog number 130-092-983) according to the manufacturer's instructions. Isolated CD25+CD4+ T cells were plated at 5 million cells / well in a 6-well Grex plate containing 30 ml of 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), and supplemented with 1000 U / mL recombinant human interleukin-2 (Miltenyi Biotec, Catalog No. 130-097-746). Cells were activated by adding 25 μL / mL ImmunoCult Human CD3 / CD28 T cell Activator (Stemcell Technologies, Catalog No. 10991) and cultured at 37°C for 5 days. Activated CD3+CD4+CD25+ T cells were harvested and labeled with 50 μg / mL biotinylated anti-LAP antibody (Miltenyi, Cat. No. 130-095-213) for 15 minutes at 4°C. The labeled cells were washed twice and further labeled with anti-biotin microbeads (Miltenyi, Cat. No. 130-090-485) according to the manufacturer's instructions. Cells were separated using an LS column (Miltenyi, Cat. No. 130-042-401). Isolated natural Tregs were cultured in 6-well Grex plates in 30 mL OpTmizer basal medium containing CTS OpTmizer T Cell Expansion SFM (Gibco, catalog no. A3705001), 1% penicillin-streptomycin, 1X Glutamax, 10 mM HEPES, and 2.5% human AB serum (Gemini, catalog no. 100-512) supplemented with 1000 U / mL IL-2.

[0334] Example 2.3 Verification of TGFB1 protein expression TGFB1 protein expression from various lentiviral constructs was verified by ELISA. One hundred thousand sorted, transduced T cells were plated in U-bottom plates containing OpTmizer basal media containing CTS OpTmizer T Cell Expansion SFM (Gibco, Catalog No. A3705001), 1% penicillin-streptomycin, 1X Glutamax, 10 mM HEPES, and 2.5% human AB serum (Gemini, Catalog No. 100-512). Some wells also received 25 μL / mL of ImmunoCult Human CD3 / CD28 / 2 T cell Activator (Stemcell Technologies, Catalog No. 10990). Wells that did not receive anti-CD3 / CD28 stimulation were used to assess background cytokine release. Cells were cultured at 37°C for 48 hours. After 48 hours of culture, the culture plates were centrifuged, and the supernatants were collected and cryopreserved for subsequent cytokine quantification using the LEGEND MAX Total TGF-β1 ELISA kit (BioLegend, catalog no. 436707) or the LEGEND MAX Free Active TGF-β1 ELISA kit according to the manufacturer's instructions. Total TGFB1 in the culture supernatants is shown in Table 1 and Figure 1. Active TGFB1 protein in the culture supernatants is shown in Table 2 and Figure 2. CD3+CD4+ T cells transduced with a lentiviral expression vector carrying the TGFB1 R218H C225R coding sequence secreted elevated levels of active TGF-β1. [Table 5] [Table 6]

[0335] Example 2.4 Suppressive capacity of engineered T cells expressing mutant TGF-β1 The suppressive function of selected transduced CD3+CD4+ T cells was assayed using a mixed lymphocyte reaction (MLR) assay. The MLR is an inflammatory response triggered by T cells recognizing other leukocytes (allogeneic leukocytes) as foreign. Tregs suppress the MLR inflammatory response by suppressing the proliferation and production of inflammatory cytokines by inflammatory T cells. Cell proliferation was measured by labeling the initial population with an intracellular fluorescent dye, which becomes diluted with each generation as part of the cellular contents.

[0336] MLR assays were performed in 96-well U-bottom plates using OpTmizer basal medium containing CTS OpTmizer T Cell Expansion SFM (Gibco, catalog no. A3705001), 1% penicillin-streptomycin, 1X Glutamax, 10 mM HEPES, and 2.5% human AB serum (Gemini, catalog no. 100-512). Responder cells were non-transduced CD3+CD4+ T cells from the same donor as the transduced T cells (autologous T cells). These non-transduced cells were labeled with CellTrace Violet (CTV) (Thermofisher Scientific, catalog no. C34557) according to the manufacturer's instructions. CD3-depleted PBMCs from an allogeneic donor were used to stimulate non-transduced responder T cells. Cultures were prepared by combining 50,000 CTV-labeled autologous T cells per well with 50,000 CD3-depleted allogeneic PBMCs and approximately 50,000 (1:1), 16,666 (3:1), or 5,555 (9:1) sorted transduced CD3+CD4+ T cells. After 5 days of incubation at 37°C, the culture plates were centrifuged, and the culture supernatants were collected for cytokine quantification. The cell pellets were resuspended in FACS buffer containing APC / Fire 750 anti-CD4 and incubated at 4°C for 30 minutes. Cells were then washed, 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 signal, and finally by the undiluted CTV population. The inhibition of CTV dilution was calculated using the following formula:

number

[0337] 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 Figure 3 and Table 3. Table 3 - Percentage inhibition of autologous T cell proliferation by T cells overexpressing wild-type or mutant TGF-β1 as measured by CTV dilution [Table 7]

[0338] Example 3. Suppressive activity of engineered T cells expressing active TGFB1 mutants with disrupted cytokine genes Example 3.1. T Cell Engineering We evaluated the in vivo suppressive function of T cells expressing the TGFβ1 R218H C225R mutant in a mouse model of graft-versus-host disease (GvHD). Transduced T cells expressing mutant TGFB1 were further disrupted in one or more of the cytokine genes tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), or interleukin-17a (IL17α).

[0339] T cells were isolated and lentivirally transduced as described in Example 2 and further engineered for lipid nanoparticle (LNP) delivery of Cas9 mRNA and guide RNA. LNPs were generated as described in Example 1 using a 35:47.5:15:2.5 molar ratio of lipid A, cholesterol, DSPC, and PEG2k-DMG. LNPs were formulated to achieve a lipid amine:RNA phosphate (N:P) molar ratio of approximately 6. LNPs were prepared with a 1:1 weight ratio of gRNA:Cas9 mRNA cargo. LNPs were prepared using G019753 (IFNg), G019757 (TNFa), or G027259 (IL17a) to disrupt target genes.

[0340] Each LNP preparation was incubated for 15 minutes at 37°C in OpTmizer basal medium containing CTS OpTmizer T Cell Expansion SFM (Gibco, Cat. No. A3705001), 1% penicillin-streptomycin, 1X Glutamax, 10 mM HEPES, 5% human AB serum (Gemini, Cat. No. 100-512), 200 U / mL recombinant human interleukin-2, 5 ng / ml recombinant human interleukin-7, and 5 ng / ml recombinant human interleukin-15, and supplemented with 5 μg / ml recombinant human ApoE3 (Peprotech, Cat. No. 350-02). On day 3 after activation and transduction, the transduced T cells were washed and suspended in serum-free OpTmizer medium containing 200 U / mL recombinant human interleukin-2, 5 ng / mL recombinant human interleukin-7, and 5 ng / mL recombinant human interleukin-15 at a concentration of 1 million T cells per mL of medium and plated at 5 million cells per group in T-25 flasks.

[0341] The pre-incubated LNP mixture was added to each T-25 flask to obtain a final total RNA concentration of 2.5 μg / ml. For groups with multiple knockouts, 2.5 μg / ml of LNP was used. Twenty-four hours after LNP treatment, T cells were harvested, washed, and expanded in Optimizer medium containing 5% human serum and cytokines as described in Example 2 until the day of injection (15 days after activation). Cells were sorted approximately 5–6 days after activation as described in Example 2.

[0342] Example 3.2 In vivo evaluation of immunosuppression in a GvHD model CD3+CD4+ T cells selected for in vivo injection were harvested 15 days post-activation. PBMCs autologous to the engineered T cells were thawed as described above in the Examples. PBMCs were added to each assay population at a 1:1 ratio, and cells were resuspended in RPMI supplemented with 2% FBS and 10 mM HERES to a concentration of 8 x 10 6 / 100μL. The PBMC-only group was 4×10^ 6Resuspended in 100 μL.

[0343] Female NOG mice (NOD.Cg-Prkdc scid Il2rg tm1Sug Cells were conditioned for transplantation by sublethally irradiating (200 rads) cells with X-rays (RS-2000 irradiator; Rad Source Technologies) one day prior to injection. Cohorts of irradiated NOG mice were intravenously injected with 100 μL of each test cell population described in the previous paragraph. Eight irradiated mice injected with 100 μL of RPMI supplemented with 2% FBS and 10 mM HEPES served as irradiation-only controls. Body weight was monitored daily. Mice were sacrificed upon a 20% weight loss. The results are shown in Figure 4 and Table 4. [Table 8]

[0344] Example 3.3. Mixed Lymphocyte Reaction Assay of Suppressor Function Engineered T cells from the injected populations were assessed for suppressive function in vitro using a mixed lymphocyte reaction (MLR) as described in Example 2 at the ratios of CTV-labeled T cells:engineered T cells listed in Figure 5 and Table 5. [Table 9]

[0345] Example 3.4. TGFB1 Expression in Transduced CD3+CD4+ T Cells Engineered T cells from the injected population were assessed for total and activated TGFB1 levels by ELISA. Sorted transduced CD3+CD4+ T cells were stimulated to assess cytokine profiles. Sorted transduced CD3+CD4+ T cells were plated at 1x10^5 T cells / well in U-bottom culture plates in a total of 200μL of OpTmizer basal medium containing CTS OpTmizer T Cell Expansion SFM (Gibco, Catalog No. A3705001), 1% penicillin-streptomycin, 1X Glutamax, 10mM HEPES, 2.5% human AB serum (Gemini, Catalog No. 100-512), and 25μL / mL ImmunoCult Human CD3 / CD28 T cell activator (Stemcell Technologies, Catalog No. 10991) and cultured at 37°C for 48 hours. After 48 hours of culture, the culture plates were centrifuged, and the supernatants were collected and cryopreserved. Cytokine quantification was then performed using the LEGEND MAX Total TGF-β1 ELISA kit (BioLegend, catalog no. 436707) and the LEGEND MAX Free Active TGF-β1 ELISA kit according to the manufacturer's instructions. Total TGF-β1 in the culture supernatants is shown in Table 6 and Figure 6. Active TGF-β1 in the culture supernatants is shown in Table 7 and Figure 7. [Table 10] [Table 11]

[0346] Example 3.5 Cytokine Profile Analysis of Transduced CD3+CD4+ T Cells Engineered T cells from the population used for injection were assessed for levels of INFg, TNFα, IL17a, IL10, IL2, and IL13 by ELISA. Sorted transduced CD3+CD4+ T cells were stimulated to assess cytokine profiles. Sorted transduced CD3+CD4+ T cells were plated at 1x10^5 T cells / well in U-bottom culture plates in 200μL of OpTmizer basal medium containing CTS OpTmizer T Cell Expansion SFM (Gibco, Cat. No. A3705001), 1% penicillin-streptomycin, 1X Glutamax, 10mM HEPES, 2.5% human AB serum (Gemini, Cat. No. 100-512), and 25uL / mL ImmunoCult Human CD3 / CD28 T cell activator (Stemcell Technologies, Cat. No. 10991) and cultured at 37°C for 48 hours. After 48 hours of incubation, the culture plates were centrifuged, and the supernatants were collected and cryopreserved. Cytokine quantification was then performed using a custom U-PLEX Biomarker kit (Meso Scale Diagnostics, catalog number K15067L-2) according to the manufacturer's instructions. Specifically, the U-PLEX Biomarker kit was used to quantify the following human cytokines: IFN-γ, TNFA, IL17A, IL2, IL13, and IL10. The U-PLEX Biomarker plate was read using a Meso Quickplex SQ120 instrument (Meso Scale Discovery), and the data were analyzed using the Discovery Workbench 4.0 software package (Meso Scale Discovery). The results are shown in Table 8 and Figures 8–13. [Table 12]

[0347] Example 4: In vivo evaluation of mutant IL-10, CD25 expression and IL-2 knockout in suppressor T cells Example 4.1. T Cell Engineering To generate engineered suppressor T cells (supT cells), CD4+ T cells were treated according to Table 9.

[0348] Isolated CD4+ T cells were activated in 5 mL of OpTmizer containing 200 U / mL IL-2, 5 ng / mL IL-7, 5 ng / mL IL-15, and 25 μL / mL Immunocult (Stemcell Technologies, 10991) by plating at 5x10^6 cells / well in 6-well plates. Two days after activation (Day 2), cells were transduced with lentivirus strains encoding CTLA4 (SEQ ID NO: 130), IL-10 (SEQ ID NO: 124), and mScarlett according to Table 9, and with lentiviruses encoding CTLA4 (SEQ ID NO: 130), IL-10DE (SEQ ID NO: 229), and mScarlett. The concentrated viral supernatant was added to T cells and centrifuged at 1000 x g for 60 minutes at 37°C. 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) plate containing T cell basal medium supplemented with 500 U / mL IL-2, 5 ng / mL IL-7, and 5 ng / mL IL-15. Cells were split periodically, and medium or cytokines were replenished. On day 3, T cells transduced with CTLA4, IL-10, and mScarlet were further transduced with a lentivirus encoding CD25 (NCBI Reference Sequence: NP_000408.1) using the same method as for day 2 transduction. [Table 13]

[0349] On day 4, T cells were edited according to Table 9 using LNPs delivering Cas9 mRNA and guide RNA targeting INF-gamma (G019753), LNPs delivering Cas9 mRNA and guide RNA targeting TNF-alpha (G019757), and in some groups, LNPs delivering Cas9 mRNA and guide RNA targeting IL-2 (G021925). LNPs were produced as described in Example 1 with lipid A, cholesterol, DSPC, and PEG2k-DMG in a molar ratio of 35:47.5:15:2.5, respectively. LNPs were formulated to achieve a lipid amine:RNA phosphate (N:P) molar ratio of approximately 6. LNPs were prepared with a gRNA:Cas9 mRNA cargo weight ratio of 1:1.

[0350] Each LNP preparation was incubated in T cell basal medium supplemented with 5 μg / ml recombinant human ApoE3 (Peprotech, Catalog No. 350-02) for 15 minutes at 37°C. One day after transduction, T cells were washed and suspended in serum-free T cell basal medium containing 1000 U / ml recombinant human interleukin-2, 10 ng / ml recombinant human interleukin-7, and 10 ng / ml recombinant human interleukin-15 at a concentration of 1 million T cells per ml of medium and plated at 5 million cells per group into T-25 flasks.

[0351] The pre-incubated LNP mixture was added to each T-25 flask to obtain a final total RNA concentration of 1.25 μg / ml, as described in Table 9. For multiple knockout groups, 1.25 μg / ml of LNP was used. Twenty-four hours after LNP treatment, T cells were harvested, washed, and expanded by culture in OpTmizer medium containing 5% human serum with cytokines, as described in Example 2. On day 5, transduced samples were sorted by flow cytometry to obtain an enriched mScarlet+ population.

[0352] CD4+CD25+ T cell populations are known to be enriched for natural Tregs (nTregs). CD4+CD25+ cells were isolated by methods known in the art. CD25+ cells were activated on day 0 by plating 5x10^6 cells / well in a 6-well Grex containing 30 mL T cell basal medium supplemented with 1000 U / mL IL-2 and 25 μL / mL ImmunoCult (Stemcell Technologies, 10991). On day 5, CD4+CD25+ samples were stained with antibodies and sorted by flow cytometry to obtain a purified LAP+ population for use as a non-transduced "nTreg" population in in vivo mouse studies. Latency-associated peptide (LAP) is a marker for activated Treg cells.

[0353] Example 4.2. In vivo functional characterization of suppressor T cells T cells for in vivo injection were harvested 14 days after activation. PBMCs autologous to the engineered T cells were thawed as described above in the Examples. PBMCs were added to each assay population at a 1:1 ratio, and cells were resuspended at 1.5 x 10^7 / mL in RPMI supplemented with 2% FBS and 10 mM HERES. PBMC-only groups were resuspended at 3 x 10^6 / mL.

[0354] Female NOG mice (NOD.Cg-Prkdcscid Il2rgtm1Sug / JicTac; Taconic, catalog number NOG-F) were conditioned for cell transplantation by sublethally irradiating (150 rads) using X-rays (RS-2000 irradiator; Rad Source Technologies) one day prior to injection. Cohorts of irradiated NOG mice were intravenously injected with 100 μL of each test cell population described in the previous paragraph. Ten irradiated mice injected with 100 μL of RPMI supplemented with 2% FBS and 10 mM HEPES served as irradiation-only controls. Body weight was monitored daily. Mice were sacrificed when they lost 20% of their body weight. The results are shown in Figure 14 and Table 10. [Table 14]

[0355] Example 5. In vivo evaluation of mutant IL-10 expression in suppressor T cells Example 5.1. T Cell Engineering To generate engineered suppressor T cells (supT cells), CD4+ T cells were engineered according to Table 11.

[0356] Sorted CD4+ T cells were activated in 5 mL of OpTmizer containing 200 U / mL IL-2, 5 ng / mL IL-7, 5 ng / mL IL-15, and 25 μL / mL Immunocult (Stemcell Technologies, 10991) by plating at 5x10^6 cells / well in 6-well plates. Two days after activation (day 2), cells were transduced with lentivirus strains encoding CTLA4 (SEQ ID NO: 130), IL-10 (SEQ ID NO: 124), and mScarlet; lentivirus encoding CTLA4 (SEQ ID NO: 130), IL-10DE (SEQ ID NO: 229), and mScarlet; or lentivirus encoding GFP, according to Table 11. Concentrated viral supernatant was added to T cells and centrifuged at 1000xg for 60 minutes at 37°C. 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 OpTmizer supplemented with 500 U / mL IL-2, 5 ng / mL IL-7, and 5 ng / mL IL-15. Cells were split periodically and medium or cytokines were replenished. [Table 15]

[0357] On day 3, T cells were edited using LNPs delivering Cas9 mRNA and a guide RNA targeting INF-gamma (G019753) and LNPs delivering Cas9 mRNA and a guide RNA targeting TNF-alpha (G019757) according to Table 11. LNPs were produced as described in Example 1 with lipid A, cholesterol, DSPC, and PEG2k-DMG in a molar ratio of 35:47.5:15:2.5, respectively. LNPs were formulated to achieve a lipid amine:RNA phosphate (N:P) molar ratio of approximately 6. LNPs were prepared with a gRNA:Cas9 mRNA cargo weight ratio of 1:1.

[0358] Each LNP preparation was incubated in T cell basal medium supplemented with 5 μg / ml recombinant human ApoE3 (Peprotech, Cat. No. 350-02) for 15 minutes at 37°C. One day after transduction, T cells were washed and suspended in serum-free OpTmizer medium containing 1000 U / ml recombinant human interleukin-2, 10 ng / ml recombinant human interleukin-7, and 10 ng / ml recombinant human interleukin-15 at a concentration of 1 million T cells per ml of medium, and 5 million cells per group were plated into T-25 flasks.

[0359] The pre-incubated LNP mixture was added to each T-25 flask to obtain a final concentration of 1.25 μg / ml of total RNA, as described in Table 11. For multiple knockout groups, 1.25 μg / ml of LNP was used. Twenty-four hours after LNP treatment, T cells were harvested, washed, and expanded by culture in OpTmizer medium containing 5% human serum with cytokines, as described in Example 2. On day 5, transduced samples were sorted by flow cytometry to obtain an enriched mScarlet+ population.

[0360] CD4+CD25+ T cell populations are known to be enriched for natural Tregs (nTregs). CD4+CD25+ cells and CD4+CD25+CD127-CD45RA+ were isolated by methods known in the art. Isolated cells were activated on day 0 by plating 5x10^6 cells / well into a 6-well Grex containing 30 mL of OpTmizer supplemented with 1000 U / mL IL-2 and 25 μL / mL ImmunoCult (Stemcell Technologies, 10991). On day 5, samples were stained with antibodies and sorted by flow cytometry to obtain a purified LAP+ population for use as "LAP nTregs" and an enriched CD45+ population for use as "CD45RA nTregs" for use in in vivo mouse studies. Latency-associated peptide (LAP) is a marker for activated Treg cells.

[0361] Example 5.2. In vivo functional characterization of suppressor T cells T cells for in vivo injection were harvested 13 days after activation. PBMCs autologous to the engineered T cells were thawed as described above in the Examples. PBMCs were added to each assay population at a 1:1 ratio, and cells were resuspended at 5 x 10^7 / mL in RPMI supplemented with 2% FBS and 10 mM HERES. The PBMC-only group was resuspended at 2.5 x 10^7 / mL.

[0362] Female NOG mice (NOD.Cg-Prkdcscid Il2rgtm1Sug / JicTac; Taconic, catalog number NOG-F) were conditioned for cell transplantation by sublethally irradiating (150 rads) using X-rays (RS-2000 irradiator; Rad Source Technologies) one day prior to injection. Cohorts of irradiated NOG mice were intravenously injected with 100 μL of each test cell population described in the previous paragraph. Ten irradiated mice injected with 100 μL of RPMI supplemented with 2% FBS and 10 mM HEPES served as irradiation-only controls. Body weight was monitored daily. Mice were sacrificed when they lost 20% of their body weight. The results are shown in Figure 15 and Table 12. [Table 16]

[0363] Example 6: Suppressive capacity of engineered T cells in an inflammatory bowel disease model Example 6.1 Evaluation of nTreg efficacy in a humanized mouse colitis model Tregs are known to suppress the induction of colitis in preclinical models (see, e.g., Goettel et al., Low-Dose Interleukin-2 Ameliorates Colitis in a Preclinical Humanized Mouse Model. Cell Mol Gastroenterol Hepatol. 2019;8(2):193-195) or in CD45RBhi transmigration models of IBD (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).

[0364] Natural Tregs were isolated using methods known in the art and administered to a humanized mouse model of IBD. Colitis was induced in TNBS-sensitized mice by application of trinitrobenzene sulfonic acid (TNBS) to the colon. Briefly, NSG mice were reconstituted with human CD34+ cells obtained from a commercial source (Jackson Laboratory). After a 1-week acclimation period, mice were injected subcutaneously (sc) with TNBS or vehicle and then challenged with either vehicle or natural Tregs (nTregs) [1x10^]. 6

[00136] was injected intraperitoneally (ip). Three days later, mice received an enema of vehicle (50% ethanol) or TNBS in 50% ethanol, as described in Table 13. Body weights were recorded daily, and the results are shown in Figure 16 (+ / - SEM) and Table 14. [Table 17] [Table 18]

[0365] Mice were sacrificed three days after enema. Colon length was measured to assess the preventive effect against colitis. The spleen, mesenteric lamina propria (mLN), and colonic lamina propria (cLP) were collected and stained with CellTrace Violet to determine the persistence of Tregs in various tissues throughout the model. The results are shown in Figure 17 (+ / - SEM) and Figure 18 (+ / - SEM) and Tables 15 and 16, respectively. [Table 19] [Table 20]

[0366] These data indicate that nTregs are protective against colitis in this humanized mouse model, as evidenced by rebound weight loss and longer colon lengths in TNBS-treated mice receiving nTregs compared with TNBS-treated mice receiving vehicle control. CellTrace Violet staining indicates that nTregs persisted in all compartments evaluated throughout the experimental period.

[0367] Example 6.2: Comparison of the efficacy of nTregs and engineered T cells in a humanized mouse model of colitis Based on this demonstration of the efficacy of nTregs in protecting against colitis in a humanized mouse model, engineered T cells were evaluated for their protective effects against colitis in the same mouse model. Engineered T cells were prepared generally as described in Example 3.1 and engineered to disrupt TNFA and IFNG and to express TGFBl R218H C225R, CTLA4, and IL-10 D25A / E96A. Additionally, this study included a time course to assess outcomes. As described above, NSG mice reconstituted with human CD34+ cells were obtained from a commercial source (Jackson Laboratory). After a one-week acclimation period, mice were injected subcutaneously with TNBS or vehicle, followed by intraperitoneal injections of vehicle, nTregs, or engineered Tregs, as shown in Table 17. One week later, mice were given an enema of vehicle (50% ethanol) or TNBS in 50% ethanol. Body weights were recorded daily. On days 3, 5, and 7 after the enema, 6 mice from group 1 and 8 mice from groups 2, 3, and 4 are sacrificed. [Table 21]

[0368] Body weight will be monitored throughout the study. At the time of sacrifice, colons will be harvested and measured for length. Colon sections will be analyzed using five-parameter blinded histology after H&E staining. This study confirms the results of a single-time point study that demonstrated the protective effect offered by nTregs in a colitis model and demonstrates the efficacy of engineered T cells in exerting a protective effect against colitis in the same model. This study will be used to select the optimal time point after enema to evaluate study results in the model.

[0369] Example 6.3: Evaluating the efficacy of targeted engineered T cells in a humanized mouse model of colitis Further studies will be conducted to determine the effectiveness of targeting engineered T cells using a chimeric antigen receptor (CAR) that binds to mouse MACAM, a surface protein present in various tissues of the gastrointestinal tract. As in the previous experiment, T cells will be engineered generally as described in Example 4. The T cells will be further edited using SpyCas9 and known methods to insert a construct for expression of the MACAM CAR into the TRAC locus. To enable the delivered engineered cells to differentiate from endogenous mouse T cells, the engineered T cells will also be transduced with a lentivirus expressing either green fluorescent protein (GFP) or mScarlet. Time points for the study will be selected based on the time course study described above. Experimental groups are described in Table 18. [Table 22]

[0370] Body weight will be monitored throughout the study. At the time of sacrifice, colons will be harvested and measured for length. Colon sections will be analyzed using five-parameter blinded histology after H&E staining. The spleen, mesenteric lamina propria, and colonic lamina propria will be harvested and analyzed by bulk RNA-seq using standard methods for the transplanted engineered T cells and endogenous T effector cells. Flow cytometry will also be performed on T cells from the spleen, mesenteric lamina propria, and colonic lamina propria, staining for CD3, CD4, CD8, hCD45, mCD45, and live / dead cells identified by GFP or mScarlet for the delivered engineered T cells.

[0371] This study confirms the results of previous studies demonstrating the protective effects offered by nTregs and engineered T cells in a colitis model. This study demonstrates that targeted engineered T cells also exert a protective effect in a colitis model.

Claims

1. 1. An engineered T cell, comprising: The engineered T cell comprises a heterologous nucleic acid sequence encoding double mutant transforming growth factor beta 1 (dmTGFB1) under the control of a promoter sequence.

2. i) a modification of an endogenous nucleic acid sequence encoding tumor necrosis factor alpha (TNFA), wherein the modification knocks down expression of said TNFA; ii) a modification of an endogenous nucleic acid sequence encoding interferon-gamma (IFNG), wherein the modification knocks down expression of said IFNG, or a modification of an endogenous nucleic acid sequence encoding interleukin 17A (IL17A), wherein the modification knocks down expression of said IL17A; 2. The engineered T cell of claim 1, further comprising:

3. 3. The engineered T cell of claim 1 or 2, comprising a modification of an endogenous nucleic acid sequence encoding IFNG, wherein the modification knocks down expression of the IFNG, and a modification of an endogenous nucleic acid sequence encoding IL17A, wherein the modification knocks down expression of the IL17A.

4. 1. An engineered T cell, comprising: i) a heterologous nucleic acid sequence encoding a regulatory T cell-promoting molecule under the control of a promoter sequence; ii) a modification of an endogenous nucleic acid sequence encoding interleukin 17A (IL17A), wherein the modification knocks down expression of said IL17A; and iii) a modification of an endogenous nucleic acid sequence encoding tumor necrosis factor alpha (TNFA), wherein the modification knocks down expression of said TNFA; and The engineered T cell comprising:

5. The regulatory T cell-promoting molecule is selected from the group consisting of interleukin-10 (IL10), cytotoxic T lymphocyte-associated protein 4 (CTLA4), 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, and CD2 74 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), BTB domain and CNC homolog 2 (BACH2), and interleukin 2 receptor subunit alpha (IL2RA).

6. 6. The engineered T cell of claim 5, wherein the regulatory T cell promoting molecule is IL10.

7. The engineered T cell of claim 5, wherein the regulatory T cell-promoting molecule is CTLA4.

8. 8. The engineered T cell of any of claims 5-7, wherein the regulatory T cell-promoting molecule is a first regulatory T cell-promoting molecule and further comprises a heterologous nucleic acid sequence encoding a second regulatory T cell-promoting molecule under the control of a promoter sequence.

9. 9. The engineered T cell of claim 8, wherein the first and second regulatory T cell promoting molecules are IL10 and CTLA4.

10. 10. The engineered T cell of any one of claims 1-9, 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), granzyme B (GZMB), Fas ligand (FASL, NF superfamily, member 6), ryanodine receptor 2 (RYR2), and colony-stimulating factor 2 (CSF2), wherein the modification knocks down expression of the IL17A, IL2, IL6, PRF1, GZMA, GZMB, FASL, RYR2, or CSF2, respectively.

11. 11. The engineered T cell of any one of claims 1-10, further comprising a modification of an endogenous nucleic acid sequence encoding an endogenous T cell receptor (TCR), wherein said modification knocks down expression of said endogenous TCR.

12. 12. The engineered T cell of any one of claims 1 to 11, further comprising a heterologous coding sequence for a targeting receptor under the control of a promoter sequence.

13. 13. The engineered T cell of claim 12, wherein the targeting 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).

14. 13. The engineered T cell of claim 12, wherein the targeting receptor is targeted to MADCAM1.

15. 15. The engineered T cell of any of claims 12-14, wherein the targeting receptor comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

16. 16. The engineered T cell of any one of claims 12 to 15, wherein the heterologous nucleic acid sequence encoding the targeted receptor is incorporated into an expression construct.

17. 17. The engineered T cell of any one of claims 12 to 16, wherein the heterologous nucleic acid sequence encoding a targeting receptor is in an expression construct that does not include a nucleic acid sequence encoding a regulatory T cell-stimulating molecule.

18. 18. The engineered T cell of any one of claims 8-17, wherein the heterologous nucleic acid sequence encoding the first regulatory T cell-promoting molecule is incorporated into an expression construct and the heterologous nucleic acid sequence encoding the second regulatory T cell-promoting molecule is incorporated into an expression construct.

19. 19. The engineered T cell of any one of claims 8 to 18, wherein the heterologous nucleic acid sequence encoding the first regulatory T cell-promoting molecule and the heterologous nucleic acid sequence encoding the second regulatory T cell-promoting molecule are incorporated into separate expression constructs.

20. 19. The engineered T cell of claim 17 or 18, wherein the heterologous nucleic acid sequence encoding the first regulatory T cell-promoting molecule and the heterologous nucleic acid sequence encoding the second regulatory T cell-promoting molecule are combined into a single expression construct.

21. 21. The engineered T cell of claims 16 or 18-20, wherein the expression construct further comprises a nucleic acid sequence encoding the targeting receptor.

22. 22. The engineered T cell of any one of claims 15 to 21, wherein at least one heterologous coding sequence is in an episomal expression construct.

23. 21. The engineered T cell of any one of claims 1 to 20, wherein at least one heterologous nucleic acid sequence is inserted into the genome.

24. 24. The engineered T cell of claim 23, wherein the insertion into the genome is a non-targeted insertion.

25. 24. The engineered T cell of claim 23, wherein the insertion is a targeted insertion.

26. 26. The engineered T cell of claim 25, 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.

27. 27. The engineered T cell of claim 26, wherein the TCR gene locus is the T cell receptor alpha constant (TRAC) locus.

28. 28. The engineered T cell of any one of claims 2 to 27, wherein the modification that knocks down expression of a gene comprises one or more of an insertion, deletion, or substitution.

29. A population of cells comprising the engineered cells of any one of claims 1 to 28.

30. 29. A population of engineered T cells comprising the engineered T cells of any one of claims 1 to 28, wherein at least 30%, preferably at least 40% of the cells of said population comprise a heterologous nucleic acid sequence encoding dmTGFB1 under the control of a promoter sequence.

31. at least 50%, preferably at least 70%, of the cells of said population comprise a modification of the endogenous nucleic acid sequence encoding TNFA; at least 50%, preferably at least 70% of the cells of said population contain a modification of the endogenous nucleic acid sequence encoding IFNG, or 31. The population of engineered T cells of claim 30, wherein at least 50%, preferably at least 70% of the cells of said population comprise a modification of the endogenous nucleic acid sequence encoding IL17A.

32. at least 50%, preferably at least 70% of the cells of said population comprise a modification of the endogenous nucleic acid sequence encoding IFNG, 32. The population of engineered T cells of claim 30 or 31, wherein at least 50%, preferably at least 70% of the cells of said population comprise a modification in an endogenous nucleic acid sequence encoding interleukin (IL17A), said modification knocking down expression of said IL17A.

33. at least 50%, preferably at least 70%, of the cells of said population comprise a modification of the endogenous nucleic acid sequence encoding TNFA; 33. The population of engineered T cells of claim 31 or 32, wherein at least 50%, preferably at least 70% of the cells of said population comprise a modification in an endogenous nucleic acid sequence encoding interleukin (IL17A), said modification knocking down expression of said IL17A.

34. 34. The population of engineered T cells of any of claims 31 to 33, wherein at least 30%, preferably at least 40% of the cells of the population comprise a heterologous nucleic acid sequence encoding a regulatory T cell-stimulating molecule under the control of a promoter sequence.

35. 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%, preferably at least 70%, of the cells of said population comprise a modification of the endogenous nucleic acid sequence encoding TNFA; 30. The population of engineered T cells of claim 29, wherein at least 50%, preferably at least 70% of the cells of said population comprise a modification of the endogenous nucleic acid sequence encoding IL17A.

36. 36. The population of engineered T cells of claim 35, wherein at least 50%, preferably at least 70% of the cells of said population comprise a modification of the endogenous nucleic acid sequence encoding IFNG.

37. 37. The population of engineered T cells of any of claims 29-36, wherein the percentage of cells containing the insertion or modification is determined by the percentage of reads by next generation sequencing (NGS).

38. 35. The population of engineered T cells of claim 34, wherein the regulatory T cell-promoting molecule is selected from interleukin-10 (IL10), cytotoxic T lymphocyte-associated protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), 5'-nucleotidase ecto (NT5E), interleukin-22 (IL22), amphiregulin (AREG), forkhead box P3 (FOXP3), IKAROS family zinc finger 2 (IKZF2), familial eosinophilia (EOS), interferon regulatory factor 4 (IRF4), lymphoid enhancer-binding factor 1 (LEF1), BTB domain and CNC homolog 2 (BACH2), and interleukin-2 receptor subunit alpha (IL2RA).

39. 39. The population of engineered T cells of any of claims 34 to 38, wherein the regulatory T cell promoting molecule is IL10.

40. 40. The population of engineered T cells of any of claims 34 to 39, wherein the regulatory T cell-promoting molecule is CTLA4.

41. 41. The population of engineered T cells of any of claims 34-40, wherein the regulatory T cell-promoting molecule is a first regulatory T cell-promoting molecule and further comprises a heterologous nucleic acid sequence encoding a second regulatory T cell-promoting molecule under the control of a promoter sequence.

42. 42. The engineered T cell of claim 41, wherein the first and second regulatory T cell-promoting molecules are IL10 and CTLA4.

43. and wherein said population of cells further comprises a modification of at least one endogenous nucleic acid sequence encoding interleukin 17A (IL17A), interleukin 6 (IL6), interleukin 2 (IL2), perforin 1 (PRF1), granzyme A (GZMA), granzyme B (GZMB), Fas ligand (FASL, NF superfamily, member 6), ryanodine receptor 2 (RYR2), and colony stimulating factor 2 (CSF2), wherein said population of cells is at least 70% of said population of cells, preferably at least 10% of said population of cells.

43. The population of engineered T cells of any one of claims 29-42, wherein at least 80% of the engineered T cells comprise a modification of at least one of the IL17A, IL6, IL2, PRF1, GZMA, GZMB, FASL, RYR2, or CSF2, respectively, wherein the modification knocks down expression of the at least one of the IL17A, IL6, IL2, PRF1, GZMA, GZMB, FASL, RYR2, or CSF2, respectively.

44. 44. The population of engineered T cells of claim 43, wherein the modification of at least one endogenous nucleic acid sequence encoding interleukin 2 (IL2), wherein the population of cells comprises the modification of IL2 in at least 70% of the population of cells, preferably at least 80% of the population of cells, and wherein the modification knocks down expression of the IL2.

45. 45. The population of engineered T cells of any one of claims 29 to 44, wherein at least 50%, preferably at least 70% of the cells comprise a knockdown of the TCR.

46. 46. The population of engineered T cells of any one of claims 29 to 45, wherein at least 30%, preferably at least 40% of the cells comprise an insertion of a nucleic acid coding sequence for a targeting receptor.

47. 47. The population of engineered T cells of claim 46, wherein the targeting receptor specifically binds 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).

48. 48. The population of engineered T cells of claim 46 or 47, wherein the targeting receptor comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

49. 49. The population of engineered T cells of any one of claims 46 to 48, wherein the heterologous nucleic acid sequence encoding the target receptor is incorporated into an expression construct.

50. 50. The population of engineered T cells of claim 49, wherein the heterologous nucleic acid sequence encoding a targeted receptor is in an expression construct that does not include a nucleic acid sequence encoding a regulatory T cell-promoting molecule.

51. 51. The population of engineered T cells of any one of claims 29-50, wherein the heterologous nucleic acid sequence encoding a first of the at least one regulatory T cell-promoting molecule is incorporated into an expression construct, and the heterologous nucleic acid sequence encoding a second of the at least one regulatory T cell-promoting molecule is incorporated into an expression construct.

52. 52. The population of engineered T cells of claim 51 , wherein the heterologous nucleic acid sequence encoding the first regulatory T cell-promoting molecule and the heterologous nucleic acid sequence encoding the second regulatory T cell-promoting molecule are incorporated into separate expression constructs.

53. 52. The population of engineered T cells of claim 51 , wherein the heterologous nucleic acid sequence encoding the first regulatory T cell-promoting molecule and the heterologous nucleic acid sequence encoding the second regulatory T cell-promoting molecule are combined into a single expression construct.

54. 54. The population of engineered T cells of claims 49-53, wherein the expression construct further comprises a nucleic acid sequence encoding a targeting receptor.

55. 55. The population of engineered T cells of any one of claims 29 to 54, wherein at least one heterologous coding sequence is in an episomal expression construct.

56. 56. The population of engineered T cells of any one of claims 29 to 55, wherein at least one heterologous coding sequence is inserted into the genome.

57. 57. The population of engineered T cells of claim 56, wherein said insertion into the genome is a non-targeted insertion.

58. 57. The population of engineered T cells of claim 56, wherein the insertion is a targeted insertion.

59. 59. The population of engineered T cells of claim 58, wherein the targeted insertion is into a site selected from a TCR gene locus, a TNF gene locus, an IL2 gene locus, an IL6 gene locus, an IL17A gene locus, an IFNG gene locus, an adeno-associated virus integration site 1 (AAVS1) locus.

60. 60. The population of engineered T cells of claim 59, wherein the TCR gene locus is the T cell receptor alpha constant (TRAC) locus.

61. 61. The population of engineered T cells of claims 29-60, wherein the modification that knocks down expression of a gene comprises one or more of an insertion, deletion, or substitution.

62. A pharmaceutical composition comprising any of the engineered T cells of claims 1 to 28 or the population of engineered T cells of claims 29 to 61.

63. A method or use comprising administering to a subject a cell according to any one of claims 1 to 28, or a population of cells according to any one of claims 29 to 61, or a pharmaceutical composition according to claim 62.

64. 64. The method or use of claim 63, wherein the subject is in need of immunosuppression.

65. 65. The method or use of claim 63 or 64 for the treatment of an immune disorder.

66. 66. The method or use according to any one of claims 63 to 65 for the treatment of an autoimmune disease.

67. 67. The method or use of claim 66, wherein the autoimmune disease is selected from ulcerative colitis, Crohn's disease, rheumatoid arthritis, psoriasis, multiple sclerosis, systemic lupus erythematosus, and type 1 diabetes.

68. 66. The method or use of any one of claims 63 to 65 for the treatment of graft-versus-host disease (GvHD).

69. A polypeptide comprising dmTGFB1.

70. 70. The polypeptide of claim 69, wherein the dmTGFB1 is human dmTGFB1.

71. 71. The polypeptide of claim 70, wherein the dmTGFB1 comprises mutations at two or more amino acids selected from F198, D199, V200, L208, F217, L219, R218, H222, C223, and C225 compared to wild-type human TGFB1.

72. 72. The polypeptide of claim 70 or 71, wherein the dmTGFB1 comprises mutations at two or more amino acids selected from R218, H222, C223, and C225 compared to wild-type human TGFB1.

73. The polypeptide of any one of claims 70 to 72, wherein the dmTGFB1 comprises two or more amino acid mutations selected from R218C / H, H222D, C223S / R / G, and C225R compared to wild-type human TGFB1.

74. 73. The polypeptide of any one of claims 70 to 72, wherein the dmTGFB1 comprises the amino acid mutations R218C / H and C225R.

75. A nucleic acid sequence encoding a polypeptide according to any one of claims 69 to 74.

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