Engineered T cells

Abstract

The present disclosure relates to T cells engineered to comprise a heterologous nucleic acid sequence encoding a double mutant transforming growth factor [beta] 1 (dmTGFB1) under the control of a promoter sequence. In certain embodiments, the cells further comprise modifications of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modifications, e.g., knock-down, of an endogenous nucleic acid sequence encoding IFNG or IL17A; and insertion of sequences encoding regulatory T cell promoting molecules, and compositions and uses thereof.

Description

[0001] Sequence Listing

[0002] This application contains a sequence listing submitted electronically in XML format and incorporated herein in its entirety by reference. The XML copy was created on June 28, 2023, named 12793_0036-00304_SL_fhfgd, and is 359,125 bytes in size.

[0003] Related Applications

[0004] This application hereby incorporates by reference U.S. Provisional Application No. 63 / 367,275, filed on June 29, 2022, the entire contents of which are expressly incorporated herein by reference. Background Art

[0005] Adaptive immunity is a defense mechanism by which the body is able to eliminate foreign pathogens. T cells are 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 arises from the rearrangement of the TCR α and β loci.

[0006] A feature of adaptive immunity is the ability to distinguish "self" from "non-self" antigens. Autoimmune and autoinflammatory disorders are characterized by pathogenic immune responses to "self" antigens. Some rearrangements of the TCR α and β loci produce autoreactive T cells. Owen et al., Regulatory T Cell Development in the Thymus, J Immunol 203 (8) (2019). Many autoreactive T cells are eliminated by clonal deletion in the thymus, but other cells can escape clonal deletion and trigger harmful immune responses. Ibid. Specialized T cells called regulatory T cells (Treg) are very important for "self" tolerance. Ibid. For example, in graft-versus-host disease, Tregs can suppress excessive immune responses, autoimmune responses, and undesirable immune responses. Ibid. Dysregulation of Tregs (e.g., if the number of Tregs is insufficient or if Tregs are not functioning properly) may lead to autoimmune responses. Ibid.

[0007] Therapies currently used to treat autoimmune disorders are intended to inhibit the activation of adaptive immune processes or immune cells. Although these therapies can inhibit harmful immune responses, such as autoimmune responses, they can also inhibit beneficial immune responses. Treg therapy has been used to inhibit antigen-specific immune responses in different diseases, including graft-versus-host disease (GvHD), in which donor cells mediate immune attacks on host tissues after hematopoietic stem cell transplantation. Pierini et al., TCells Expressing Chimeric Antigen Receptor Promoter Immun e Tolerance, JCI Insight 2 (20) (2017). However, "the clinical implementation of Treg-based therapies still faces major challenges". Ibid. Therefore, there is still a need for effective T cell therapies, including Treg therapies, for suppressing immune responses, including inflammation and autoimmunity. Summary of the invention

[0008] The present disclosure provides double mutant transforming growth factor β1 (dmTGFB1) polypeptides, such as human dmTGFB1, and nucleic acids encoding the same; and methods and uses thereof. The present disclosure provides T cells or T cell populations engineered to contain a heterologous nucleic acid encoding dmTGFB under the control of a promoter sequence. The present disclosure also provides T cells or T cell populations expressing dmTGFB under the control of a promoter sequence, and compositions and uses thereof, for example, for inhibiting immune responses, including inflammation and autoimmunity.

[0009] In some embodiments, the cell further comprises a modification of an endogenous nucleic acid sequence encoding interferon-γ (IFNG), wherein the modification knocks down the expression of the IFNG. In some embodiments, the cell comprises a modification of an endogenous nucleic acid sequence encoding tumor necrosis factor alpha (TNFA), wherein the modification knocks down the 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 the expression of the IL17A. In some embodiments, further modifications include modifications of an endogenous nucleic acid sequence encoding TNFA and modifications of an endogenous nucleic acid sequence encoding IFNG. In some embodiments, further modifications include modifications of an endogenous nucleic acid sequence encoding TNFA and modifications of an endogenous nucleic acid sequence encoding IL17A. In some embodiments, further modifications include modifications of an endogenous nucleic acid sequence encoding TNFA, modifications of an endogenous nucleic acid sequence encoding IFNG, and modifications of an endogenous nucleic acid sequence encoding IL17A.

[0010] In some embodiments, the cells further comprise a heterologous nucleic acid encoding a regulatory T cell promoting molecule.

[0011] The present disclosure provides T cells or T cell populations, which are engineered to contain heterologous nucleic acids encoding regulatory T cell promoting molecules under the control of a promoter sequence; modifications of endogenous nucleic acid sequences encoding tumor necrosis factor alpha (TNFA), wherein the modifications knock down the expression of TNF A, and modifications of endogenous nucleic acid sequences encoding interleukin-17a (IL17A), wherein the modifications knock down the expression of the IL17A. In certain embodiments, T cells or T cell populations do not contain modifications of endogenous nucleic acid sequences encoding interferon-gamma (IFNG), wherein the modifications knock down the expression of the IFNG.

[0012] In some embodiments, 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), ecto-5'-nucleotidase (NT5E), interleukin-22 (IL-22), amphiregulin (AREG), interleukin-35 (IL35), GARP, CD274 molecule (CD274), forkhead box P3 (FOXP 3), IKAROS family zinc finger 2 (IKZF2), familial eosinophilia (EO S), 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 promoting molecule is IL10 or CTLA4. In some embodiments, the regulatory T cell promoting molecules are IL10 and CTLA4.

[0013] In some embodiments, T cells or T cell populations are engineered to include heterologous nucleic acids encoding dmTGFB1 and IL10, each of which is under the control of a promoter sequence; a modification of an endogenous nucleic acid sequence encoding TNFA, wherein the modification knocks down the expression of TNFA; and a modification of an endogenous nucleic acid sequence encoding IFNG, wherein the modification knocks down the expression of IFNG, or a modification of an endogenous nucleic acid sequence encoding IL17A, wherein the modification knocks down the expression of IL17A. In some embodiments, cells comprise modifications of endogenous nucleic acid sequences encoding IFNG and IL17A, respectively, wherein the modification knocks down the expression of each of the IFNG and IL17A.

[0014] In some embodiments, T cells or T cell populations are engineered to include heterologous nucleic acids encoding dmTGFB1 and CTLA4, each of which is under the control of a promoter sequence; and modifications of endogenous nucleic acid sequences encoding TNFA, wherein the modifications knock down the expression of TNFA. In some embodiments, T cells or T cell populations include further modifications of endogenous nucleic acid sequences encoding IFNG, wherein the modifications knock down the expression of IFNG. In some embodiments, T cells or T cell populations include further modifications of endogenous nucleic acid sequences encoding IL17A, wherein the modifications knock down the expression of IL17A. In some embodiments, T cells or T cell populations include modifications of endogenous nucleic acid sequences encoding IFNG and TNFA, respectively, wherein the modifications knock down the expression of IFNG and TNFA, respectively. In some embodiments, T cells or T cell populations include modifications of endogenous nucleic acid sequences encoding IL17A and TNFA, respectively, wherein the modifications knock down the expression of IL17A and TNFA, respectively. In some embodiments, the T cell or population of T cells comprises modifications of endogenous nucleic acid sequences encoding TNFA, IFNG, and IL17A, respectively, wherein the modifications knock down the expression of each of TNFA, IFNG, and IL17A.

[0015] In some embodiments, T cells or T cell populations are engineered to include heterologous nucleic acid sequences encoding dmTGFB1, IL10, and CTLA4, each of which is under the control of a promoter sequence; and modification of an endogenous nucleic acid sequence encoding TNFA, wherein the modification knocks down the expression of TNFA. In some embodiments, T cells or T cell populations include further modification of an endogenous nucleic acid sequence encoding IFNG, wherein the modification knocks down the expression of IFN G. In some embodiments, T cells or T cell populations include further modification of an endogenous nucleic acid sequence encoding IL17A, wherein the modification knocks down the expression of IL17A. In some embodiments, T cells or T cell populations include modification of endogenous nucleic acid sequences encoding IFN G and IL17A, respectively, wherein the modification knocks down the expression of IFNG and IL17A, respectively.

[0016] In some embodiments, T cells or T cell populations are further engineered to include modifications of endogenous nucleic acid sequences encoding interleukin-2 (IL2), interleukin 6 (IL6), perforin 1 (PRF1), granzyme A (GZMA), granzyme B (GZMB), Fas ligand (FasL, NF superfamily, member 6), ranolidine receptor 2 (RYR2), and colony stimulating factor 2 (CSF2), e.g., knockdown. In some embodiments, T cells or T cell populations are further engineered to include modifications of endogenous nucleic acid sequences encoding RYR2, e.g., knockdown.

[0017] In some embodiments, the T cell or T cell population is further engineered to comprise a modification, e.g., a knockdown, of an endogenous nucleic acid sequence encoding an endogenous T cell receptor (TCR).

[0018] In some embodiments, T cell or T cell colony is further engineered to include the heterologous coding sequence of the targeting receptor under the control of the 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 targets a ligand selected from the following: 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), glutamate decarboxylase (GAD2), desmoglein 3 (DS G3) and MHC class I HLA-A (HLA-A*02). In some embodiments, the targeted receptor targets mucosal vascular addressin cell adhesion molecule 1 (MADCAM1). In some embodiments, the targeted receptor targets tumor necrosis factor alpha (TNFA).

[0019] 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 T cell population comprises an insertion of a sequence encoding dmTGFB, e.g., as assessed by sequencing, e.g., NGS. In certain embodiments, there are further modifications.

[0020] 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 T cell population comprises an insertion of a sequence encoding a regulatory T cell promoting molecule, e.g., as assessed 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 T cell population comprises a modification in the IFNG sequence, such as a knockdown, for example, as assessed by sequencing, such as NGS. In some embodiments, at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, 80%, 85%, 90% or 95% of the T cell population comprises a modification in the TNFA sequence, such as a knockdown, for example, as assessed by sequencing, such as NGS.

[0022] In some embodiments, at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, 80%, 85%, 90% or 95% of the T cell population comprises a modification in the IL17A sequence, e.g., knockdown, e.g., as assessed by sequencing, e.g., NGS.

[0023] In some embodiments, at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, 80%, 85%, 90% or 95% of the T cell populations include modifications in the TCR sequence, such as knocking down, for example, as assessed by sequencing, such as NGS. In some embodiments, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the T cell populations include insertions of sequences encoding targeting receptors (such as CAR), for example, as assessed by sequencing, such as NGS.

[0024] Modifications for knocking down gene expression as described herein may include one or more of insertion, deletion or substitution. Heterologous sequences as described herein may be incorporated into expression constructs. Multiple heterologous sequences may be incorporated into single expression constructs or separate expression constructs. Heterologous sequences as described herein may be incorporated into free expression constructs. Heterologous sequences as described herein may be inserted into genomes, such as non-targeted insertions or targeted insertions. In some embodiments, the targeted insertion is in a site selected from TCR loci, TNF loci, IFNG loci, IL17A loci, IL6 loci, IL2 loci, adeno-associated virus integration site 1 (AAVS1) loci.

[0025] Also provided herein are pharmaceutical compositions and uses of engineered T cells. In some embodiments, engineered T cells and pharmaceutical compositions thereof can be administered to subjects requiring immunosuppression. In some embodiments, engineered T cells and pharmaceutical compositions thereof can be used to treat 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).

[0026] In some embodiments, insertion of one or more sequences described herein or modification (e.g., knockdown) of one or more sequences can be mediated by a combination of a guide RNA and an RNA-guided DNA binder (e.g., a Cas nuclease). In some embodiments, insertion of one or more sequences described herein or knockdown of one or more sequences can be mediated by another suitable gene editing system (e.g., a zinc finger nuclease (ZFN) system or a transcription activator-like effector nuclease (TALEN) system). BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Total TGF-β1 quantified in culture supernatants is shown.

[0028] Figure 2 Active TGF-β1 quantified in the culture supernatant is shown.

[0029] Figure 3 Shown are the percentage inhibition of autologous T cell proliferation by T cells overexpressing wild-type or mutant TGF-β1 as measured by CTV dilution.

[0030] Figure 4 Shown is the probability of survival over time after injection of NOG mice with engineered CD3+CD4+ cells.

[0031] Figure 5 The percent inhibition of cell proliferation by engineered T cells as measured by CTV dilution is shown.

[0032] Figure 6 Shown are total TGF-β1 production (pg / ml) by transduced cells following cell stimulation.

[0033] Figure 7 Active TGF-β1 production (pg / ml) of transduced cells after cell stimulation is shown.

[0034] Figure 8 IFN-γ production (pg / ml) by transduced cells after cell stimulation is shown.

[0035] Fig. 9 TNF-α production (pg / ml) by transduced cells after cell stimulation is shown.

[0036] Fig.10 IL-17a production (pg / ml) by transduced cells after cell stimulation is shown.

[0037] Fig.11 IL-2 production (pg / ml) by transduced cells after cell stimulation is shown.

[0038] Fig.12 IL-10 production (pg / ml) by transduced cells after cell stimulation is shown.

[0039] Fig.13 IL-13 production (pg / ml) by transduced cells after cell stimulation is shown.

[0040] Fig.14 Shown are the percentages of mice in each cohort that survived at each time point after injection with engineered suppressor T cells.

[0041] Fig.15 Shown are the percentages of mice in each cohort that survived at each time point after injection with engineered suppressor T cells.

[0042] Fig.16 The percentage of initial body weight of individual mice in the treatment groups is shown.

[0043] Fig.17 Colon length (in cm) is shown.

[0044] Fig.18 The percentage of Tregs recovered from each tissue stained with Cell Trace Violet is shown. DETAILED DESCRIPTION

[0045] Reference will now be made in detail to certain embodiments of the present invention, examples of which are shown in the accompanying drawings. Although the present invention is described in conjunction with the embodiments shown, it should be understood that it is not intended to limit the present invention to those embodiments. On the contrary, the present invention is intended to cover all replacements, modifications and equivalents, which may be included in the present invention limited by the attached embodiments.

[0046] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter in any way. If any material incorporated by reference conflicts with any term defined in this specification or any other express content of this specification, the present specification controls.

[0047] I. Definitions

[0048] Before describing the present teachings in detail, it should be understood that the present disclosure is not limited to specific compositions or process steps, as they may vary. It should be noted that, unless the context clearly indicates otherwise, as used in this specification and the attached embodiments, the singular forms "a / an" and "the" include plural referents. Thus, for example, reference to "a conjugate" includes a plurality of conjugates, reference to "a cell" includes a plurality of cells or cell populations, etc. As used herein, the term "includes" and its grammatical variants are intended to be non-restrictive, such that the recitation of an item in a list does not exclude other similar items that may be substituted or added to the listed items.

[0049] Numerical ranges include the values ​​defining the ranges. Taking into account significant figures and errors associated with measurement, measured values ​​and measurable values ​​should be understood as approximate values. In addition, the use of "comprise / comprises / comprising", "contain / contains / containing", "include / includes / including" is not intended to be limiting. It should be understood that the foregoing general description and detailed description are merely exemplary and illustrative, and not limiting of the teachings.

[0050] Unless otherwise specified in the specification, embodiments described in the specification as "comprising" various components are also contemplated as "consisting of" or "consisting essentially of" the recited components; embodiments described in the specification as "consisting of" various components are also contemplated as "comprising" or "consisting essentially of" the recited components; and embodiments described in the specification as "consisting essentially of" various components are also contemplated as "consisting of" or "comprising" the recited components (this interchangeability does not apply to the use of these terms in the claims).

[0051] Unless the context clearly dictates otherwise, the term "or" is used in an inclusive sense, ie, equivalent to "and / or".

[0052] When used before a list or range, the term "about" modifies each member of the list or each endpoint of the range. The term "about" or "approximately" refers to the acceptable error of a particular value determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. The term "about" is used herein to refer to within the typical tolerance range in this field. For example, "about" can be understood as about 2 standard deviations of the mean. In certain embodiments, about refers to + / -10%. In certain embodiments, about refers to + / -5%.

[0053] In some embodiments, a cell population refers to a population of at least 10^3, 10^4, 10^5, or 10^6 cells, preferably 10^7, 2×10^7, 5×10^7, or 10^8 cells.

[0054] The term "at least" before a number or a series of numbers is understood to include the number adjacent to the term "at least", and all subsequent numbers or integers that can be logically included, as clearly seen 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 nucleic acid molecule of 20 nucleotides" means that 17, 18, 19 or 20 nucleotides have the indicated characteristics. When at least appears before a series of numbers or ranges, it should be understood that "at least" can modify each number in the series or range.

[0055] As used herein, "not greater than" or "less than" is understood to be a value adjacent to the phrase and a logical low value or integer, which is logically zero from the context. For example, a duplex region of "no more than 2 nucleotide base pairs" has 2, 1, or 0 nucleotide base pairs. When "not greater than" or "less than" appears before a series of values ​​or ranges, it should be understood to modify each value in the series or range.

[0056] As used herein, ranges include upper and lower limits.

[0057] As used herein, it is understood that when the maximum amount of a value is represented by 100% (e.g., 100% inhibition or 100% encapsulation), 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 unable to detect the material for encapsulation outside the vesicle.

[0058] If there is a conflict between a sequence in this application and a designated accession number or a position in an accession number, the sequence in this application controls.

[0059] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings.

[0060] As used herein, "knock down" refers to the expression of a specific gene product (e.g., full-length or wild-type mRNA, protein, or both) reduced by gene editing, such as in cells, cell colonies, tissues, or organs. In some embodiments, gene editing can be assessed by sequence, such as next generation sequencing (NGS). Compared with a suitable control (e.g., wherein the gene sequence is not modified), expression can be reduced by at least 70%, 75%, 80%, 85%, 90%, 95%, or reduced to a detection level lower than that determined. The knock down of protein can be measured by detecting the amount of protein from tissues, cell colonies, or fluids of interest. The method for measuring mRNA knockdown is known, and includes sequencing of mRNA separated from tissues or cell colonies of interest. Flow cytometry analysis is a known method for measuring protein expression knockdown. For secreted proteins, knockdown can be assessed in fluids (e.g., tissue culture medium or blood, or serum or plasma derived therefrom). In some embodiments, "knockdown" may refer to some loss of expression of a particular gene product, such as 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 cell population. It is well understood what changes in the mRNA sequence will lead to reduced expression of a wild-type or full-length protein. In some embodiments, "knockdown" may refer to some loss of expression of a particular gene product (e.g., IFNG or TNFA gene product in body fluids or tissue culture medium). Modification of an endogenous nucleic acid sequence (e.g., encoding IFNG or TNFA) may result in knockdown.

[0061] As used herein, "T cell receptor" or "TCR" refers to a receptor in a T cell. Typically, a TCR is a heterodimeric receptor molecule that contains two TCR polypeptide chains, α and β. The α and β chain TCR polypeptides can complex with various CD3 molecules and induce immune responses, including inflammation and autoimmunity, upon antigen binding. As used herein, knockdown of a TCR refers to partial or complete knockdown of any TCR gene, such as partial deletion of the TRBC1 gene, alone or in combination with partial or complete knockdown of other TCR genes.

[0062] "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 for TRAC.

[0063] "TRBC" is used to refer to T cell receptor beta chain, for example, TRBC1 and TRBC2. "TRBC1" and "TRBC2" refer to two homologous genes encoding T cell receptor beta chain, which are the gene products of TRBC1 or TRBC2 gene.

[0064] The human wild-type TRBC1 sequence is available at NCBI gene ID: 28639; Ense mbl: ENSG00000211751. T cell receptor beta constant, V segment translation product, BV 05S1J2.2, TCRBC1 and TCRB are gene synonyms for TRBC1.

[0065] The human wild-type TRBC2 sequence is available at NCBI gene ID: 28638; Ense mbl: ENSG00000211772. T cell receptor beta constant, V segment translation product and TCRBC2 are gene synonyms for TRBC2.

[0066] As used herein, "immune response" refers to one or more immune system responses, for example, an increase in the production or activity of immune system cells compared to an unstimulated control immune system, such as, 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. The immune system may be exposed to antigens that may cause an immune response, such as foreign or self-antigens, such as, but not limited to, pathogens (microorganisms, viruses, prions, fungi, etc.), allergens (dust, pollen, dust mites, etc.), toxins (chemical substances, drugs, etc.) or physiological changes (hypercholesterolemia, obesity, organ transplantation, etc.). The immune response may also include the response of donor cells mediating immune attacks on host tissues after hematopoietic stem cell transplantation in GvHD. The immune response may cause inflammation. The immune response may target, attack, remove or neutralize antigens, such as foreign or self-antigens. The immune response may be required or may not be required. The immune response may be acute or chronic. An immune response can damage the cells, tissues, or organs in which the immune response was initiated.

[0067] As used herein, "autoimmune reaction" refers to one or more immune system reactions to autoantigens, such as reactions produced by cells, tissues or organs of the subject's own. Compared with a suitable control (e.g., a healthy control), an autoimmune reaction can result in an increase in the production or activity of immune system cells, such as, 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. An autoimmune reaction can result in inflammation, such as long-term inflammation, or result in an autoimmune disease. An autoimmune reaction can target, attack, remove or neutralize autoantigens produced by cells, tissues or organs of the subject's own body, which can result in an autoimmune disease.

[0068] As used herein, "suppressing" an immune response refers to reducing or inhibiting the level of one or more immune system responses, such as the yield or activity of immune system cells compared to a suitable control (e.g., not treated with engineered T cells as described herein or before being treated with the engineered T cells). "Suppressing" an immune response may refer to a reduction in the yield or activity of immune system cells compared to a suitable control (e.g., not treated with engineered T cells as described herein or before being treated with the engineered T cells). "Suppressing" an immune response may refer to increasing immune tolerance. For example, the yield or activity of immune system cells can be measured by: cell counts, such as lymphocyte counts or spleen cell counts; cell activity, such as T cell assays; or gene or protein expression, such as biomarker expression; wherein the yield or activity is reduced by 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or below the detection level of the assay compared to a suitable control (e.g., not treated with engineered T cells as described herein or before being treated with the engineered T cells).

[0069] As used herein, "autoimmune disease" or "autoimmune disorder" refers to a condition characterized by a pathological immune response to 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, the engineered T cells have autologous or allogeneic use.

[0070] As used herein, an "immune disorder" is understood to be a disease or condition characterized by a pathological or undesirable 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 suppression of the immune response. In certain embodiments, a subject with an immune disorder requires an increase in immune tolerance.

[0071] "T cells" play a central role in the immune response after exposure to an antigen. T cells can be naturally occurring, or, for example, when T cells are formed by engineering (e.g., from stem cells) or by transdifferentiation (e.g., reprogramming somatic cells), they are non-natural. T cells can be distinguished from other lymphocytes by the presence of T cell receptors on the cell surface. This definition includes conventional 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 γδT cells. In some embodiments, T cells are CD4+. In some embodiments, T cells are CD3+ / CD4+. In some embodiments, T cells are CD8+.

[0072] "Regulatory T cells" or "Tregs" refer to specialized T cells that play a central role in suppressing excessive immune responses, including inflammation and autoimmunity. Tregs can be naturally occurring or non-natural, for example, when Tregs are formed by engineering, including by inserting sequences encoding dmTGFB1 molecules. When formed by engineering, for example, by modification (e.g., knockdown) of endogenous nucleic acid sequences encoding IFNG, IL17A, and TNFA and insertion of at least one sequence encoding regulatory T cell-promoting molecules, Tregs may include further modifications. Naturally occurring Tregs or natural Tregs or nTregs, sometimes also referred to as thymic Tregs or tTregs, are specialized T cells that typically develop in the thymus and function by suppressing excessive immune responses. In some embodiments, cells such as conventional T cells or conventional T cell populations, such as T cell populations that are not enriched for the presence of nTreg cells, can be engineered by inserting sequences encoding dmTGFB1 molecules, and optionally further modifying endogenous nucleic acid sequences encoding, for example, TNFA and IFNG, such as knocking down nucleic acid sequences encoding TNFA and IFNG, and inserting sequences encoding regulatory T cell-promoting molecules into cells to express phenotypic characteristics and inhibitory functions of regulatory T cells, and these can be referred to as transduced or "engineered" T cells. In some embodiments, engineered T cells include insertion of sequences encoding dmTGFB1 molecules; modification of endogenous nucleic acid sequences encoding IFNG and modification of endogenous nucleic acid sequences encoding TNFA, and insertion of heterologous regulatory T cell-promoting molecules (such as IL10 or CTLA4). Modifications of endogenous nucleic acid sequences (e.g., modifications that knock down endogenous gene expression) can include or consist of one or more insertion, deletion or substitution mutations in the genomic sequence.

[0073] As used herein, dmTGFB1 is 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 TG FB1. The coding and amino acid sequences of wild-type TGFB1 can be conveniently obtained in sequence databases (including NCBI), and exemplary sequences can be found in 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 form obtained as of the filing date of this application. The ability to map mutations onto the wild-type sequence is well within the capabilities of those skilled in the art.

[0074] Transforming growth factor β-1 (TGFB1) is synthesized as a large precursor molecule. The TGFB1 proprotein contains a 29 amino acid signal peptide that is cleaved by proteolysis. TGFB1 is further cleaved after amino acid 278 to form the latency associated peptide (LAP) and active TGFB1. LAP forms a dimer with an interchain disulfide bond at C223 and C225. TGFB1 can be secreted as an inactive small latent complex consisting of a mature TGF-β1 homodimer non-covalently associated with a LAP homodimer at LAP residues I53-L59. LAP shields the type II receptor binding site in mature TGFB1. Most cells secrete TGFB1 as a large latent complex (LLC) of TGF-β1 / LAP that is covalently bound between C33 in the LAP chain and the latent TGFB binding protein (LTBP). LTBP promotes TGFB 1 folding, secretion, and possibly targeting to the extracellular matrix. LLC activation occurs through binding of the N-terminal domain of LTBP to the extracellular matrix.

[0075] Progressive diaphyseal dysplasia (CED) is caused by domain-specific heterozygous mutations in the transforming growth factor-β-1 gene (TGFB1; OMIM entry 190180, incorporated by reference in the form available on the filing date of this application) on chromosome 19q13. Mutations reported in families with progressive diaphyseal dysplasia include (from Janssen et al., 2006. J Med Genet. 43:1.):

[0076] Exon DNA mutations Protein mutation 1 28_36 Repeat L10-L12 duplication 1 241T→C Y81H 2 463C→T R156C 4 652C→T R218C 4 653G→A R218H 4 664C→G H222D 4 ？ C223S 4 667T→C C223R 4 667T→G C223G 4 673T→C C225R

[0077] As shown in the table, most pathogenic variants in individuals with CED result in a single amino acid substitution in the carboxyl terminus of TGF B1 latent associated peptide (LAP). The substitution occurs near the site of the interchain disulfide bond between LAP homodimers. These pathogenic variants disrupt the dimerization of LAP and the binding to active TGFB1 (Walton et al., 2010. J Biol Chem. 285: 17029–37), resulting in an increase in active TGFB1 released 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 (F198, D199, V200, L208, F217 and L219) at the dimer interface. Compared with normal fibroblasts, R218H mutant fibroblasts of individuals with CE D showed an increase in active TGF B1 in the cell culture medium (Saito et al., 2001. J Biol Chem. 276: 11469–72). In vitro analysis of R218C, H222D and C225R mutant constructs also showed an increase in active TGF-β1 in the culture medium of transfected cells. In contrast, Leu11_Leu13dup and Tyr81His pathogenic variants lead to a decrease in TGFB1 secretion. However, in a luciferase reporter gene assay for TGFB1-induced transcriptional responses, mutant cells showed an increase in luciferase activity, indicating that the receptor is activated in the cell (Janssens et al., 2003. J Biol Chem. 278: 7718–24).

[0078] As used herein, in certain embodiments, dmTGFB1 is a TGFB1 comprising mutations at two or more amino acid positions relative to wild-type TGFB1, which mutants reduce the stability of the resulting large latent complex that relies on LAP covalent dimerization relative to each TGFB1 single mutant alone. The mutations at two or more amino acid positions can include mutations at positions selected from F198, D199, V200, L208, F217, L219, R218, H222, C223 and C225 relative to wild-type human TGFB1.

[0079] In certain embodiments, dmTGFB1 is a TGFB1 comprising a mutation at two or more amino acid positions relative to wild-type TGFB1, the mutation being at a position selected from F198, D199, V200, L208, F217, L219, R218, H222, C223, and C225 relative to wild-type human TGFB1, wherein when dmTGFB1 is produced, it is secreted from cells that normally express TGFB1. In some embodiments, dmTGFB1 comprises a naturally occurring mutation, e.g., in a CED. In some embodiments, dmTGFB1 comprises a non-naturally occurring mutation, e.g., a non-naturally occurring mutation in a CED.

[0080] In certain embodiments, human dmTGFB1 comprises mutations at two or more amino acid positions selected from R218, H222, C223, and C225 relative to wild-type human TGFB1.

[0081] In certain embodiments, human dmTGFB1 comprises two or more mutations selected from R218C / H, H222D, C223S / R / G, and C225R relative to wild-type human TGFB1.

[0082] In certain embodiments, human dmTGFB1 comprises two or more mutations selected from R218C / H and C225R relative to wild-type human TGFB1.

[0083] The dmTGFB1 provided herein has more free active dmTGFB1 relative to any of the individual single mutants. In certain embodiments, the increase in active dmTGFB1 is at least additive compared to the activity of the individual single mutants. In certain embodiments, the increase in active dmTGFB1 is significantly more than additive compared to the activity of the individual single mutants. Methods for determining the 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 No. 436707) and LEGEND MAX Free Active TGF-β1 ELISA Kit) and as provided in the following examples.

[0084] As used herein, "regulatory T cell promoting molecules" refer to molecules that promote the conversion of conventional T cells into regulatory T cells, including immunosuppressive molecules and Treg transcription factors. In addition, regulatory T cell promoting molecules also refer to molecules that confer regulatory activity to conventional T cells, including Treg-related immunosuppressive molecules and transcription factors. Regulatory T cell promoting molecules can be used in combination with dmTGFB1, which promote the conversion of conventional T cells into regulatory T cells, or confer regulatory activity to conventional T cells. Examples of immunosuppressive molecules for use in combination 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), ecto-5'-nucleotidase (NT5E), interleukin-22 (IL22), amphiregulin (AREG), interleukin-35 (IL35), leucine-rich repeat-containing protein 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, such as IL10 and CTLA4, ENTPD1 and NT5E, and IL 22 and AREG. Specifically, provided herein is a combination of IL10 and CTLA4 used in combination with dmTGFB1. In some embodiments, the expression of immunosuppressive molecules can be promoted by the expression of transcription factors (e.g., FoxP3, Helios, Eos, IRF4, Lef1, or BACH2).

[0085] In some embodiments, conventional T cells can be engineered to modify, insert or delete sequences in the genome, and the "engineered" T cells exhibit phenotypic characteristics and inhibitory functions of one or more natural regulatory T cells. For example, the "engineered" T cells provided in Example 2.4 below exhibit inhibitory activity in a mixed lymphocyte reaction assay, or are preferably able to inhibit the graft-versus-host disease in the mouse model presented in Example 3.2 below in a statistically significant manner (see also, e.g., Parmar et al., Ex vivo fucosylation of third-party human regulatory T cells enhances anti-graft-ve rsus-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 inserting the coding sequence of regulatory T cell-promoting molecules and by modifying (e.g., knocking down) the expression of proinflammatory cytokines (e.g., TNFA with one or both of IFNG and IL17A). In some embodiments, the starting T cell population used for engineering is not enriched for the presence of natural Tregs, for example, the starting T cell population has less than 20% natural Tregs.

[0086] As used herein, "pro-inflammatory" molecules (e.g., cytokines) increase the immune response described herein, for example, in a dose-response manner, reduce the efficacy of Tregs 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), ranolidine receptor 2 (RYR2), and colony stimulating factor 2 (CSF2).

[0087] As used herein, "targeting receptor" refers to a receptor present on the surface of a cell (eg, a T cell) to allow the cell to bind to a target site (eg, a specific cell or tissue in an organism). Targeting receptors include, but are not limited to, chimeric antigen receptors (CARs), T cell receptors (TCRs), and receptors for cell surface molecules that are operably linked by at least one transmembrane domain in an internal signaling domain that is capable of activating T cells upon binding to an extracellular receptor portion of a protein, such as mucosal addressin cell adhesion molecule-1 (MADCAM-1), TNFA, CEA cell adhesion molecule 6 (CEACAM6), vascular cell adhesion molecule 1 (VCAM1), citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNF receptor superfamily member 17 (TNFRSF17), dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), desmoglein 3 (DSG3), and MHC HLA-A class I (HLA-A*02).

[0088] As used herein, "chimeric antigen receptor" refers to an extracellular antigen recognition domain, for example, scFv, VHH, nanobody; it is operably connected to an intracellular signaling domain, and when the antigen binds, it activates T cells. CAR consists of four regions: antigen recognition domain, extracellular hinge region, transmembrane domain, and intracellular T cell signaling domain. Such receptors are well known in the art (see, for example, WO2020092057, WO2019191114, WO2019147805, WO2018208837, the corresponding contents of these documents are incorporated herein by reference). It is also considered that a reverse universal CAR (see, for example, WO2019238722, the contents of which are incorporated herein as a whole) that promotes immune cells to bind to target cells by an adapter molecule. CAR can be targeted to any antigen that can produce antibodies, and is generally directed to molecules displayed on the surface of cells or tissues to be targeted. In some embodiments, CAR can target engineered T cells to the gastrointestinal tract, such as CAR targeting MAdCAM-1. In some embodiments, CAR can target engineered T cells to tissues containing endothelial cells, such as CAR targeting VCAM-1, for example, for suppressing immune responses in conditions such as Crohn's disease and multiple sclerosis. In some embodiments, CAR can target engineered T cells to endothelial cells, such as CAR targeting CEACAM6, for example, for suppressing immune responses in conditions such as Crohn's disease. In some embodiments, CAR can target engineered T cells to pre-B cells, such as CAR targeting CD19, for example, for suppressing immune responses in conditions such as multiple sclerosis and systemic lupus erythematosus. In some embodiments, CAR can target engineered T cells to B lymphocytes, such as CAR targeting CD20, for example, for suppressing immune responses in conditions such as multiple sclerosis and systemic lupus erythematosus. In some embodiments, CAR can target engineered T cells to inflammatory tissues, such as CAR targeting TNFA, for example, for suppressing immune responses in conditions such as rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis or Crohn's disease. In some embodiments, CAR can target engineered T cells to inflammatory tissues, such as CAR targeting TGF-b1, for example, for suppressing immune responses in conditions such as inflammatory bowel disease, ulcerative colitis, or Crohn's disease. In some embodiments, CAR can target engineered T cells to neural tissues, such as CAR targeting MBP, MOG, or PLP1, for example, for suppressing immune responses in conditions such as multiple sclerosis. In some embodiments, CAR can target engineered T cells to tissues containing mature B lymphocytes, such as CAR targeting TNFRSF17, for example, for suppressing immune responses in conditions such as systemic lupus erythematosus.In some embodiments, CAR can target engineered T cells to synovial tissue, such as CAR targeting citrullinated vimentin, for example, for suppressing immune responses in conditions such as rheumatoid arthritis. In some embodiments, CAR targets dipeptidyl peptidase-like 6 (DPP 6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), desmoglein 3 (DSG3) or MHC class I HLA-A (HLA-A*02). Additional CAR targets (such as inflammatory antigens) are known in the art. See, for example, WO2020092057A1, the contents of which are incorporated herein in their entirety.

[0089] As used herein, "treatment" refers to any application or application of a disease or condition of a subject for treatment, and includes suppressing the disease, preventing its development, 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 autoimmune or inflammatory reactions or conditions can include alleviating inflammation associated with a particular condition, thereby alleviating disease-specific symptoms. Treatment with engineered T cells as described herein can be used before, after, or in combination with other therapeutic agents (e.g., anti-inflammatory agents, immunosuppressants, or biological agents for treating autoimmune conditions, such as Remicade, Humira).

[0090] "Promoter" refers to a regulatory region that controls the expression of a gene to which the gene is associated.

[0091] "Polynucleotide" and "nucleic acid" are used herein to refer to polymeric compounds comprising nucleosides or nucleoside analogs having nitrogen-containing heterocyclic bases or base analogs linked together along a backbone, including polymers of conventional RNA, DNA, mixed RNA-DNA, and analogs thereof. The nucleic acid "backbone" can be composed of a variety of bonds, including one or more sugar-phosphodiester bonds, peptide-nucleic acid bonds ("peptide nucleic acid" or PNA; PCT No. WO 95 / 32305), phosphorothioate bonds, methylphosphonate bonds, or combinations thereof. The sugar portion of the nucleic acid can be ribose, deoxyribose, or a similar compound having a substitution, for example, a 2'methoxy, 2'halide, or 2'-O-(2-methoxyethyl) (2'-O-moe) substitution. The nitrogenous base can be a conventional base (A, G, C, T, U), an analog thereof (e.g., a modified uridine, such as 5-methoxyuridine, pseudouridine or N1-methylpseudouridine or others); inosine; a derivative of a purine or pyrimidine (e.g., N 4 -methyl deoxyguanosine, deaza-purine or aza-purine, deaza-pyrimidine or aza-pyrimidine, a pyrimidine base having a substituent at the 5-position or 6-position (e.g., 5-methylcytosine), a purine base having a substituent at the 2-position, 6-position or 8-position, 2-amino-6-methylaminopurine, O6 -methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine and O 4 -alkyl-pyrimidine; U.S. Pat. No. 5,378,825 and PCT No. WO 93 / 13121). For a general discussion, see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th edition, 1992). Nucleic acids can include one or more "absic" residues, in which the backbone does not include a nitrogenous base at the polymer position (U.S. Pat. No. 5,585,481). Nucleic acids can contain only conventional RNA or DNA sugars, bases and bonds, or can contain conventional components and substitutes (e.g., conventional bases with 2' methoxy bonds, or polymers containing conventional bases and one or more base analogs). Nucleic acids include "locked nucleic acids" (LNA) (analogs of LNA nucleotide monomers containing one or more bicyclic furanose units locked in RNA that mimic sugar conformations), which enhance hybridization affinity for complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43 (42): 13233-41). Nucleic acids include "unlocked nucleic acids" or UNA. RNA and DNA may have different sugar moieties, and may differ in the presence of uracil or its analogs in RNA and thymine or its analogs in DNA.

[0092] As used herein, "polypeptide" refers to a polymeric compound comprising amino acid residues that can adopt a three-dimensional conformation. Polypeptides include, but are not limited to, enzymes, enzyme precursor proteins, regulatory proteins, structural proteins, receptors, nucleic acid binding proteins, antibodies, etc. Polypeptides may, but need not, contain post-translational modifications, non-natural amino acids, prosthetic groups, etc.

[0093] As used herein, the "open reading frame" or "ORF" of a gene refers to a sequence consisting of a series of codons that determine the amino acid sequence of the protein encoded by the gene. An ORF usually 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).

[0094] "Guide RNA", "gRNA" and "guide" are used interchangeably herein to refer to crRNA (also known as CRISPR RNA) or a combination of crRNA and trRNA (also known as tracr RNA). CrRNA and trRNA can be combined as a single RNA molecule (single guide RNA, sgRNA) or as two separate RNA molecules (dual guide RNA, dgRNA). "Guide RNA" or "gRNA" refers to each type. TrRNA can be a naturally occurring sequence, or a trRNA sequence with modifications or variations compared to a naturally occurring sequence.

[0095] As used herein, "guide sequence" or "guide region" or "targeting sequence" or "spacer" or "spacer sequence" and the like refer to a sequence within a gRNA that is complementary to a target sequence and has a function of guiding gRNA to a target sequence to allow an RNA-guided nickase to bind or modify (e.g., cleave). The guide sequence can be 20 nucleotides in length, for example, in Streptococcus pyogenes (i.e., Spy Cas9 (also referred to as SpCas9)) and related Cas9 homologs / orthologs. Shorter or longer sequences can also be used as guides, for example, with a length of 15, 16, 17, 18, 19, 21, 22, 23, 24, or 25 nucleotides. The guide sequence can be 20-25 nucleotides in length, for example, in the case of Nme Cas9, for example, the length can be 20, 21, 22, 23, 24, or 25 nucleotides. For example, a guide sequence of 24 nucleotides in length can be used with an Nme Cas9, e.g., Nme2 Cas9.

[0096] In some embodiments, for example, the target sequence is in a genomic locus or on a chromosome and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between the guide sequence and its corresponding target sequence may be about 75%, 80%, 85%, 90%, 95% or 100%. 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. For example, the guide sequence and the target sequence may contain 1, 2, 3 or 4 mismatches, wherein the total length of the target sequence is at least 17, 18, 19, 20 or more base pairs. In some embodiments, the guide sequence and the target region may contain 1-4 mismatches, wherein the guide sequence comprises at least 17, 18, 19, 20 or more nucleotides. In some embodiments, the guide sequence and the target region may contain 1, 2, 3 or 4 mismatches, wherein the guide sequence comprises 20 nucleotides. In some embodiments, the degree of complementarity or identity between the guide sequence and its corresponding target sequence is at least 80%, 85%, preferably 90%, or 95%, for example, when the guide sequence comprises a sequence of 24 consecutive nucleotides. In some embodiments, the guide sequence and the target region can be 100% complementary or identical. In other embodiments, the guide sequence and the target region can contain at least one mismatch, that is, one non-identical or non-complementary nucleotide, depending on the reference sequence. For example, the guide sequence and the target sequence can contain 1-2, preferably no more than 1 mismatch, wherein the total length of the target sequence is 19, 20, 21, 22, preferably 23 or 24 nucleotides, or more nucleotides. In some embodiments, the guide sequence and the target region can contain 1 to 2 mismatches, wherein the guide sequence comprises at least 24 or more nucleotides. In some embodiments, the guide sequence and the target region can contain 1 to 2 mismatches, wherein the guide sequence comprises 24 nucleotides. The mismatch positions are known in the art, for example, PAM distal mismatches are often more acceptable than PAM proximal matches. Mismatch tolerance at other positions is known in the art (see, eg, Sternberg et al., 2015, Nature: 527: 110-113).

[0097] As used herein, "target sequence" or "genomic target sequence" refers to a sequence of a nucleic acid in the genomic locus of a target gene of a positive or negative strand, which is complementary to the guide sequence of a gRNA, that is, sufficiently complementary to the guide sequence of a gRNA to allow guidance to specifically bind to the target sequence. The interaction between the target sequence and the guide sequence guides the RNA-guided DNA binder to bind within the target sequence, and may cut or cleave therein (depending on the activity of the agent). For example, the specific length of the target sequence and the number of possible mismatches between the target sequence and the guide sequence depend on the identity of the Cas9 nuclease guided by the gRNA. The target sequence of the Cas protein includes the positive and negative strands of the genomic DNA (i.e., a given sequence and the reverse complementary sequence of the sequence), because the nucleic acid substrate of the Cas protein is a double-stranded nucleic acid. Therefore, when the guide sequence is referred to as "complementary to the target sequence", it should be understood that the guide sequence can guide the RNA-guided DNA binder (e.g., dCas9 or damaged Cas9) to bind to the reverse complementary sequence of the target sequence. Thus, in some embodiments, where the guide sequence binds to the reverse complement of a target sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., the target sequence does not include a PAM), but T in the guide sequence is replaced by U.

[0098] The target sequence of the RNA-guided DNA binder includes the positive and negative strands of the genomic DNA (i.e., a given sequence and the reverse complement of the sequence), because the nucleic acid substrate of the RNA-guided DNA binder is a double-stranded nucleic acid. Therefore, when the guide sequence is referred to as "complementary to the 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. Therefore, in some embodiments, when the guide sequence binds to the reverse complement of the target sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., the target sequence does not include a PAM), except that T in the guide sequence is replaced by U.

[0099] As used herein, "RNA-guided DNA binders" refer to polypeptides or polypeptide complexes with RNA and DNA binding activity, or DNA binding subunits of such complexes, wherein the DNA binding activity is sequence-specific and depends on the sequence of the RNA. The term RNA-guided DNA binders also includes nucleic acids encoding such polypeptides. Exemplary RNA-guided DNA binders include Cas lyases / nickases. Exemplary RNA-guided DNA binders may include their inactivated forms ("dCas DNA binders"), for example, if these agents are modified to allow DNA cleavage, for example, by fusion with the FokI lyase domain. As used herein, "Cas nucleases" include Cas lyases and Cas nickases. Cas lyases and Cas nickases include Csm or Cmr complexes of type III CRISPR systems, their Cas10, Csm1 or Cmr2 subunits, Cascade complexes of type I CRISPR systems, their Cas3 subunits and class 2 Cas nucleases. As used herein, "class 2 Cas nucleases" are single-chain polypeptides with RNA-guided DNA binding activity. Class 2 Cas nucleases include Class 2 Cas lyases / nickases (e.g., H840A, D10A, or N863A variants), which further have RNA-guided DNA lyase or nickase activity, and Class 2 dCas DNA binders, in which the lyase / nickase activity is inactivated), for example, if those agents are modified to allow DNA cleavage, or have C to T deaminase or A to G deaminase activity. Cas nickases include one of the RuvC or HNH domains of the Cas protein so that only a single strand is cleaved by the nuclease. In some embodiments, the RNA-guided DNA binder comprises a deaminase region and an RNA-guided DNA nickase, such as a 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. Cpf1 protein (Zetsche et al., Cell, 163: 1-13 (2015)) also contains a RuvC-like nuclease domain. Zetsche's Cpf1 sequence is incorporated herein by reference in its entirety. See, e.g., Zetsche, Table S1 and Table S3.See, e.g., Makarova et al., Nat Rev Microbiol, 13(11):722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015). As used herein, delivery of an RNA-guided DNA binder (e.g., Cas nuclease, Cas9 nuclease, or Streptococcus pyogenes Cas9 nuclease or Neisseria meningitidis Cas9 nuclease) includes delivery of a polypeptide or mRNA.

[0100] As used herein, the term "editor" or "base editor" refers to an agent comprising a polypeptide capable of modifying a base (e.g., A, T, C, G, or U) within a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, the editor is capable of deaminating a base within a nucleic acid. In some embodiments, the editor is capable of deaminating a base within a DNA molecule. 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 AP OBEC3A 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 a DNA-binding protein guided by an enzymatically inactive RNA fused to a cytidine deaminase domain.

[0101] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to a guide RNA and an RNA-guided DNA binder, such as a Cas nuclease, e.g., a Cas lyase, a Cas nickase, or a dCas DNA binder (e.g., Cas9). In some embodiments, the guide RNA guides the RNA-guided DNA binder (e.g., Cas9) to a target sequence, and the guide RNA hybridizes to the target sequence and the agent binds to the target sequence; where the agent is a lyase or nickase, binding may be followed by cleavage or cutting.

[0102] As used herein, the term "uracil glycosylase inhibitor", "uracil-DNA glycosylase inhibitor" or "UGI" refers to a protein capable of inhibiting the uracil-DNA glycosylase (UDG) base excision repair enzyme (e.g., UniPROTID: P14739; MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVML LTSDAPEYKPWALVIQDSNGENKIKMLSGGSKRTADGSEFESPKK KRKVE (SEQ ID NO: 226); or TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO: 227)).

[0103] Exemplary nucleotide and polypeptide sequences of Cas9 molecules are provided below. Methods for identifying alternative nucleotide sequences (including alternative naturally occurring variants) encoding Cas9 polypeptide sequences are known in the art. It is also contemplated that there are sequences with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with any Cas9 nucleic acid sequence or nucleic acid sequence encoding an amino acid sequence provided herein. In certain embodiments, the nucleotide sequence encoding the Cas9 amino acid sequence is not a naturally occurring Cas9 nucleotide sequence. It is also contemplated that there are sequences with at least 95%, 96%, 97%, 98% or 99% identity with any one of the Cas9 amino acid sequences provided herein. In certain embodiments, the Cas9 amino acid sequence is not a naturally occurring Cas9 sequence.

[0104] Example open reading frame of Spy Cas9

[0105]

[0106] Exemplary amino acid sequence of Spy Cas9

[0107]

[0108] Example open reading frame of Spy Cas9

[0109]

[0110] Exemplary open reading frame of Spy Cas9 with Hibit tag

[0111]

[0112] Exemplary amino acid sequence of Spy Cas9 with Hibit tag

[0113]

[0114] Exemplary amino acid sequence of Spy Cas9 nickase

[0115]

[0116] Exemplary open reading frame of Nme2Cas9

[0117]

[0118] Exemplary mRNA for Nme2Cas9 nickase

[0119]

[0120] Exemplary amino acid sequence of Nme2Cas9

[0121]

[0122] Exemplary mRNA encoding UGI

[0123] GGGAGACCCAAGCUGGCUAGCUCCCGCAGUCGGCGUCCAGCGGCUCUGCUUGUUCGUGUGUGUGUCGUUGCAGGCCUUAUUCGGAUCCGCCACCAUGGGACCGAAGAAGAAGAGAAAGGUCGGAGGAGGAAGCACAAACCUGUCGGACAUCAUCGAAAAGGAAACAGGAAAGCAGCUGGUCAUCCAGGAAUCGAUCCUGAUGCUGCCGGAAGAAGUCGAAGAAGUCAUCGGAAACAAGCCGGAAUCGGACAUCCUGGUCCACACAGCAUACGACGAAUCGACAGACGAAAACGUCAUGCUGCUGACAUCGGACGCACCGGAAUACAAGCCGUGGGCACUGGUCAUCCAGGACUCGAACGGAGAAAACAAGAUCAAGAUGCUGUGAUAGUCUAGACAUCACAUUUAAAAGCAUCUCAGCCUACCAUGAGAAUAAGAGAAAGAAAAUGAAGAUCAAUAGCUUAUUCAUCUCUUUUUCUUUUUCGUUGGUGUAAAGCCAACACCCUGUCUAAAAAACAUAAAUUUCUUUAAUCAUUUUGCCUCUUUUCUCUGUGCUUCAAUUAAUAAAAAAUGGAAAGAACCUCGAGUCUAG(SEQ ID NO:224)

[0124] Open reading frame of UGI

[0125] AUGGGACCGAAGAAGAAGAGAAAGGUCGGAGGAGGAAGCACAAACCUGUCGGACAUCAUCGAAAAGGAAACAGGAAAGCAGCUGGUCAUCCAGGAAUCGAUCCUGAUGCUGCCGGAAGAAGUCGAAGAAGUCAUCGGAAACAAGCCGG AAUCGGACAUCCUGGUCCACACAGCAUACGACGAAUCGACAGACGAAAACGUCAUGCUGCUGACAUCGGACGCACCGGAAUACAAGCCGUGGGCACUGGUCAUCCAGGACUCGAACGGAGAAAACAAGAUCAAGAUGCUGUGA(SEQID NO:225)

[0126] Exemplary amino acid sequences of UGI

[0127] MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSKRTADGSEFESPKKKRKVE(SEQ ID NO:226)

[0128] Exemplary amino acid sequences of UGI

[0129] TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML(SEQ ID NO:227)

[0130] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to a guide RNA and an RNA-guided DNA binder, such as a Cas nuclease, such as a Cas lyase, a Cas nickase, or a dCas DNA binder (e.g., Cas9). In some embodiments, the guide RNA guides the RNA-guided DNA binder (e.g., Cas9) to a target sequence, and the guide RNA hybridizes with the target sequence and the agent binds to the target sequence; in the case where the agent is a lyase or nickase, the binding may be followed by double-stranded DNA cleavage or single-stranded DNA cleavage.

[0131] As used herein, a first sequence is said to "comprise a sequence having at least X% identity with a second sequence" if an alignment of the first sequence with a second sequence shows that X% or more of the positions of the second sequence match the first sequence as a whole. For example, the sequence AAGA comprises a sequence having 100% identity with the sequence AAG, since the alignment would give 100% identity since all three positions with the second sequence match. Differences between RNA and DNA (typically uridine exchanged for thymidine or vice versa) and the presence of nucleoside analogs (such as modified uridine) do not result in differences in identity or complementarity between polynucleotides as long as the relevant nucleotides (e.g., thymidine, uridine, or modified uridine) have the same complementary sequence (e.g., adenosine of all thymidine, uridine, or modified uridines, another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as complementary sequences). Thus, for example, the sequence 5'-AXG (wherein X is any modified uridine, such as pseudouridine, N1-methyl pseudouridine or 5-methoxyuridine) is considered to be 100% identical to AUG, since both are fully complementary to the same sequence (5'-CAU). Exemplary alignment algorithms are the Smith-Waterman and Needle man-Wunsch algorithms, which are well known in the art. One skilled in the art will understand what algorithm and parameter settings are appropriate for a given pair of sequences to be aligned; for sequences of roughly similar length and expected identity of >50% for amino acids or >75% for nucleotides, the Needleman-Wunsch algorithm with default settings of the Needleman-Wunsch algorithm interface provided by EBI on the www.ebi.ac.uk web server is generally appropriate.

[0132] As used herein, a first sequence is said to be "X% complementary" to a second sequence if X% of its bases are paired with the second sequence. For example, the first sequence 5'AAGA3' is 100% complementary to the second sequence 3'TTCT5', and the second sequence is 100% complementary to the first sequence. In some embodiments, the first sequence 5'AAGA3' is 100% complementary to the second sequence 3'TTCTGTGA5', and the second sequence is 50% complementary to the first sequence.

[0133] As used herein, "mRNA" refers herein to a polynucleotide that is entirely or predominantly RNA or modified RNA and comprises 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). The mRNA can comprise a phosphate-sugar backbone, including ribose residues or analogs thereof, such as 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.

[0134] As used herein, "indel" refers to an insertion / deletion mutation consisting of a number of nucleotides inserted or deleted at a double-strand break (DSB) site of a target nucleic acid. As used herein, when an indel is formed to produce an insertion, the insertion is a random insertion at the double-strand break site and is not guided or based on a template sequence.

[0135] As used herein, "target sequence" refers to a nucleic acid sequence in a target gene that is complementary to the guide sequence of a gRNA. The interaction of the target sequence and the guide sequence guides the RNA-guided DNA binding agent to bind within the target sequence and may cut or cleave therein (depending on the activity of the agent).

[0136] As used herein, "polypeptide" refers to wild-type or variant proteins (e.g., mutants, fragments, fusions, or combinations thereof). Variant polypeptides may have at least or about 5%, 10%, 15%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% functional activity of a wild-type polypeptide. In some embodiments, the variant has at least 70%, 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of the wild-type polypeptide. In some embodiments, the variant polypeptide may be a hyperactive variant. In some cases, variants have about 80% to about 120%, 140%, 160%, 180%, 200%, 300%, 400%, 500% or more functional activity of wild-type polypeptides. As used herein, "heterologous gene" refers to a gene that has been introduced into a cell as an exogenous source (e.g., inserted at a genomic locus, such as a safe harbor locus including a TCR locus). That is, the introduced gene is heterologous with respect to its insertion site. The polypeptide expressed by this heterologous gene is referred to as a "heterologous polypeptide". A heterologous gene can be naturally occurring or engineered, and can be a wild type or a variant. A heterologous gene can include a nucleotide sequence (e.g., an internal ribosome entry site) that is different from a sequence encoding a heterologous polypeptide. A heterologous gene can be a naturally occurring gene in a genome, a wild type or variant form (e.g., a mutant). For example, although a cell contains a gene of interest (wild type or variant form), the same gene or its variant can be introduced as an exogenous source for, for example, expression at a highly expressed locus. A heterologous gene can also be a gene that occurs naturally in a non-genome, or a gene that expresses a heterologous polypeptide that occurs naturally in a non-genome. "Heterologous gene," "foreign gene," and "transgene" are used interchangeably. In some embodiments, a heterologous gene or transgene includes an exogenous 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 includes an exogenous nucleic acid sequence, e.g., a nucleic acid sequence that does not occur naturally in a recipient cell. For example, a heterologous gene can be heterologous with respect to its insertion site and its recipient cell.

[0137] "Safe harbor" locus is a locus within the genome, wherein a gene can be inserted without producing a significant deleterious effect on the cell. Non-limiting examples of safe harbor loci targeted by nucleases used herein include AAVS1 (PPP1R12C), TCR, B2M and any locus of the targeting knockdown described herein, such as TNFA, IFNG, IL17A and IL6 genomic loci. In some embodiments, insertion at one or more loci (such as TRC genes, such as TRAC genes) knocked down by targeting is advantageous for allogeneic cells. Other suitable safe harbor loci are known in the art.

[0138] II. Composition

[0139] A. Engineered T cells

[0140] Provided herein are T cells and T cell populations engineered to contain a modification comprising insertion into the cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter.

[0141] In some embodiments, T cells comprising a heterologous sequence encoding dmTGFB1 under the control of a promoter are further engineered to comprise additional modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and insertion of a heterologous sequence encoding a regulatory T cell promoting molecule under the control of a promoter sequence into the cell, as well as 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.

[0142] In some embodiments, a T cell or T cell population comprising a heterologous sequence encoding a dmTGFB1 molecule under the control of a promoter is further engineered to include a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IF NG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and insertion of heterologous sequences encoding two or more regulatory T cell promoting molecules, each under the control of a promoter sequence, into the cell. For example, an engineered T cell comprises a first heterologous sequence encoding a first regulatory T cell promoting molecule under the control of a first promoter and a second heterologous sequence encoding a second regulatory T cell promoting molecule under the control of a second promoter. The first promoter and the second promoter may be the same promoter or different promoters. In certain embodiments, the heterologous sequence encoding the dmTGFB1 molecule is under the control of a promoter sequence controlling the expression of the 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 the 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 the targeting receptor. In certain embodiments, the heterologous sequence encoding the dmTGFB1 molecule is not under the control of a promoter sequence that controls expression of the targeted receptor.

[0143] In some embodiments, a T cell or T cell population comprising a heterologous sequence encoding dmTGFB1 under the control of a promoter is 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; and insertion of a heterologous sequence encoding IL10 under the control of a promoter into the cell. In some embodiments, a T cell comprising a heterologous sequence encoding dmTGFB1 under the control of a promoter is 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; and insertion of a heterologous sequence encoding CTL A4 under the control of a promoter into the cell. In some embodiments, T cells comprising a heterologous sequence encoding dmTGFB1 under the control of a promoter are further engineered to 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 into the cell of a heterologous sequence encoding IL10 under the control of a promoter, and insertion into the cell of a heterologous sequence encoding CTLA4 under the control of a promoter.

[0144] In some embodiments, a T cell or T cell population comprising a heterologous sequence encoding dmTGFB1 under the control of a promoter is further engineered to comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA or IL17A; and insertion into the cell of a heterologous sequence encoding a regulatory T cell promoting molecule under the control of a promoter, and exhibits at least one suppressive activity of a naturally occurring regulatory T cell (nTreg), e.g., suppression of an immune response or biomarker in an in vitro or in vivo assay (e.g., an animal model of GvHD).

[0145] In some embodiments, a heterologous sequence encoding dmTGFB1 or a regulatory T cell promoting molecule is 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 is inserted into the genome, for example, a targeted or non-targeted insertion.

[0146] In certain embodiments, modification (eg, knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A includes modification of both IFNG and IL17A.

[0147] In some embodiments, the sequence encoding dmTGFB1 or a regulatory T cell promoting molecule can be inserted into a site selected from the following: a TCR locus, such as a TRAC locus; a TNF locus, an IFNG locus, an IL17A locus, an IL6 locus, an IL2 locus, or an adeno-associated virus integration site 1 (AAVS1) locus.

[0148] In some embodiments, the engineered T cell or T cell population is engineered to contain a modification (e.g., 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 contain an insertion of a sequence encoding a dmTGFB1 molecule.

[0149] In some embodiments, the engineered T cell population comprises an insertion modification of the sequence encoding dmTGFB1, e.g., generated by gene editing, and, 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 the sequence encoding the dmTGFB1 molecule, and further comprises a modification (e.g., knockdown) in the TNFA sequence by gene editing, e.g., as assessed by sequencing (e.g., NGS), wherein 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 in the endogenous TNFA sequence. In some embodiments, the 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 to below the detection limit of the assay, for example, where the TNFA gene is not modified, as determined by, for example, ELISA or flow cytometry. For example, in T cell populations, assays for TNFA protein and mRNA expression are known in the art. In certain embodiments, the knockdown of TNFA produces TNFA levels of 2500 pg / ml or less, as determined, for example, using a custom U-PLEX biomarker kit (Meso Scale Diagnostics, catalog number K15067L-2) according to the manufacturer's instructions.

[0150] In some embodiments, the engineered T cell population comprises a modification (e.g., insertion) of the 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 the sequence encoding the dmTGFB1 molecule, and further comprises a modification (e.g., knockdown) in the IFNG sequence by gene editing, e.g., as assessed by sequencing (e.g., NGS), wherein 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 in the endogenous IFNG sequence. In some embodiments, compared with a suitable control, the 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 reduced to below the detection limit of the assay, for example, wherein the IFNG gene is not modified, as determined by, for example, ELISA or flow cytometry. Determination of IFNG protein and mRNA expression in, for example, T cell populations is known in the art. In certain embodiments, the knockdown of IFNG produces 300,000 pg / ml or lower IFNG levels, as determined, for example, using a custom U-PLEX biomarker kit (MesoScale Diagnostics, catalog number K15067L-2) according to the manufacturer's instructions.

[0151] In some embodiments, the engineered T cell population comprises a modification (e.g., insertion) of the 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 the sequence encoding the dmTGFB1 molecule, and further comprises a modification (e.g., knockdown) in the IL17A sequence by gene editing, e.g., as assessed by sequencing (e.g., NGS), wherein 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 in the endogenous IL17A sequence. In some embodiments, the 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 to below the detection limit of the assay, compared to a suitable control, e.g., wherein the IL17A gene is not modified, as determined by, e.g., ELISA or flow cytometry. Assays for IL17A protein and mRNA expression, e.g., in T cell populations, are known in the art.

[0152] In some embodiments, the modification that knocks down the expression of a gene (eg, TNFA, IFNG, or IL17A) is one or more of an insertion, a deletion, or a substitution.

[0153] In some embodiments, the engineered T cell or T cell population 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), 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 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, compared with a suitable control, the inserted regulatory T cell promoting molecule (eg, IL10) causes the expression of protein or mRNA to be statistically significantly increased, for example, wherein the regulatory T cell promoting molecule gene is not inserted, as determined by, for example, ELISA or flow cytometry. In some embodiments, the engineered T cell comprises the insertion of a sequence encoding IL10 by gene editing, for example, as assessed by sequencing (eg, 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 or sequence encoding IL10. In some embodiments, compared with a suitable control, the inserted sequence encoding IL10 causes the expression of protein or mRNA to be statistically significantly increased, for example, wherein regulatory T cells promote molecules. The determination of IL10 protein and mRNA expression in, for example, a T cell population is known in the art, such as ELISA and flow cytometry. In certain embodiments, the level of IL10 is at least 300 pg / ml as determined, for example, using a custom U-PLEX biomarker kit (Meso Scale Diagnostics, catalog number K15067L-2) according to the manufacturer's instructions.

[0154] In some embodiments, the engineered T cells or T cell populations comprise a modification (e.g., 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 comprise an insertion of a sequence encoding CTLA4, 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 or sequence encoding CTLA4. In some embodiments, the inserted sequence encoding CTLA4 results in a statistically significant increase in expression of protein or mRNA, e.g., wherein a regulatory T cell promoting molecule, compared to an appropriate control. Assays for CTLA4 protein and mRNA expression in, e.g., T cell populations are described herein and are known in the art, e.g., ELISA and flow cytometry.

[0155] In some embodiments, the T cell population comprises T cells engineered to comprise a modification (e.g., 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 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; 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 limit of the assay used) of the T cells in a T cell population are engineered to contain a heterologous regulatory T cell promoting molecule, for example, as assessed by sequencing (e.g., NGS). In some embodiments, at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, preferably at least 80%, 85%, 90%, 95% or 100% of the T cells in a T cell population are engineered to contain a modification (e.g., knockdown) of a sequence encoding TNFA, for example, as assessed by sequencing (e.g., NGS). In some embodiments, at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, 80%, 85%, 90%, 95% or 100% of the T cells in the T cell population are engineered to include a modification (e.g., knockdown) of a sequence encoding IFNG, for example, as assessed by sequencing (e.g., NGS). In some embodiments, at least 40%, 45%, preferably at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the T cells in the T cell population are engineered to include an insertion of a sequence encoding a regulatory T cell promoting molecule, for example, as assessed by sequencing (e.g., NGS). In some embodiments, at least 30%, 35%, preferably at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the T cells in the T cell population are engineered to include the insertion of the sequence encoding IL10, for example, as assessed by sequencing (e.g., NGS). In some embodiments, at least 30%, 35%, preferably at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the T cells in the T cell population are engineered to include the insertion of the sequence encoding CTLA4, for example, as assessed by sequencing (e.g., NGS).

[0156] In some embodiments, the engineered T cell or T cell population is engineered to comprise a modification (e.g., 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 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 a heterologous sequence encoding a regulatory T cell promoting molecule under the control of a promoter sequence, and further comprise a sequence encoding interleukin 6 (IL6), interleukin 2 (IL2), perforin 1 (PRF1), granzyme A (GZM A), granzyme B (GZMB), Fas ligand (FasL, NF superfamily, member 6), ranolidine receptor 2 (RYR2) and colony stimulating factor 2 (CSF2) modification of the endogenous nucleic acid sequence, wherein the modification knocks down the expression of IL17A, IL6, IL2, PRF1, GZMA, GZMB, FASL, RYR2 or CSF2, respectively. In certain embodiments, the further modification of the endogenous nucleic acid sequence includes modification of RYR2.

[0157] In certain embodiments, the modifications include modification of IFNG and IL17A.

[0158] In some embodiments, a gene editing system is used, such as an engineered T cell or T cell population using a DNA binder guided by RNA. In some embodiments, a CRISPR / Cas gene editing system is used to engineer T cells. In some embodiments, a CRISPR / Cas II type gene editing system is used, such as an engineered T cell using Cpf1. In some embodiments, a CRISPR / Cas9 gene editing system is used, such as an engineered T cell using SpyCas9. Exemplary Cas9 sequences are provided herein.

[0159] In some embodiments, a guide RNA specifically targeting a site within the IFNG and TNFA genes is used to engineer a T cell or T cell population to provide knockdown of the IFNG and TNFA genes. Exemplary sequences for knockdown of IFNG and TNFA are provided in Tables 1 and 2, respectively, along with the genomic coordinates of the target of each listed guide sequence.

[0160] In some embodiments, engineered T cells or T cell populations include IFNG and TNFA genes knocked down using guide RNA and RNA-guided DNA binders disclosed herein. In some embodiments, disclosed herein are T cells engineered by, for example, inducing breaks (e.g., double-strand breaks (DSBs) or single-strand breaks (nicks)) in IL-7A, IFNG, and TNFA genes of T cells using guide RNA and RNA-guided DNA binders (e.g., CRISPR / Cas systems) disclosed herein. The method can be used in vitro or ex vivo, for example, to manufacture cell products that inhibit immune responses (including inflammation and autoimmunity). In some embodiments, the guide RNA disclosed herein mediates target-specific cleavage at sites described herein in the IFNG gene by RNA-guided DNA binders (e.g., Cas nucleases). In some embodiments, the guide RNA disclosed herein mediates target-specific cleavage at sites described herein in the TNFA gene by RNA-guided DNA binders (e.g., Cas nucleases). It should be understood that in some embodiments, the guide RNA comprises a guide sequence that binds or can bind to the region.

[0161] Provided are engineered T cells or T cell populations comprising genetic modifications at genomic coordinates selected from those listed in Table 1, e.g., cells comprising indels or substitution mutations within any of the listed genomic ranges within IFNG. Also provided are engineered T cells comprising genetic modifications at genomic coordinates selected from those listed in Table 2, e.g., cells comprising indels or substitution mutations within any of the listed genomic ranges within TNFA. In some embodiments, the engineered T cells will comprise modifications within a genomic coordinate region selected from Table 1 and modifications within a genomic coordinate region selected from Table 2.

[0162] In some embodiments, the guide RNA disclosed herein comprises a guide sequence having 95%, 90%, 85%, 80% or 75% identity to a sequence selected from the group of sequences in Table 1 or Table 2. In some embodiments, the guide RNA disclosed herein comprises a guide sequence having 95%, 90%, 85%, 80% or 75% identity to a sequence selected from the group of sequences in Table 1 or Table 2.

[0163] In some embodiments, the guide RNA disclosed herein comprises a guide sequence having at least 15, 16, 17, 18, 19, or 20 consecutive nucleotides of a sequence selected from the group consisting of: a sequence having 95%, 90%, 85%, 80%, or 75% identity to a sequence selected from the group of sequences in Table 1. In some embodiments, the guide RNA disclosed herein comprises a guide sequence having at least 17, 18, 19, or 20 consecutive nucleotides of a sequence selected from the group of sequences in Table 1. In some embodiments, the guide RNA disclosed herein comprises a guide sequence having 95%, 90%, 85%, 80%, or 75% identity to a sequence selected from the group of sequences in Table 1. In some embodiments, the guide RNA disclosed herein comprises a guide sequence having 17, 18, 19, or 20 consecutive nucleotides of a sequence selected from the group of sequences in Table 1. In some embodiments, the guide RNA disclosed herein comprises a guide sequence selected from the group of sequences in Table 1.

[0164] In some embodiments, the guide RNA disclosed herein comprises a guide sequence having at least 15, 16, 17, 18, 19, or 20 consecutive nucleotides of a sequence selected from the group consisting of: a sequence having 95%, 90%, 85%, 80%, or 75% identity to a sequence selected from the group of sequences in Table 2. In some embodiments, the guide RNA disclosed herein comprises a guide sequence having at least 17, 18, 19, or 20 consecutive nucleotides of a sequence selected from the group of sequences in Table 2. In some embodiments, the guide RNA disclosed herein comprises a guide sequence having 95%, 90%, 85%, 80%, or 75% identity to a sequence selected from the group of sequences in Table 2. In some embodiments, the guide RNA disclosed herein comprises a guide sequence having 17, 18, 19, or 20 consecutive nucleotides of a sequence selected from the group of sequences in Table 2. In some embodiments, the guide RNA disclosed herein comprises a guide sequence selected from the group of sequences in Table 2.

[0165] Unless otherwise stated, genomic coordinates are always based on the human reference genome hg38.

[0166] In certain embodiments, a guide RNA comprising a guide sequence targeting IFNG and a guide RNA comprising a guide sequence targeting TNFA are included.

[0167] Table 1: Human guide sequences and chromosome coordinates for knockdown of IFNG

[0168]

[0169]

[0170] Table 2: Human guide sequences and chromosome coordinates for knockdown of TNFA

[0171]

[0172]

[0173]

[0174]

[0175]

[0176] A non-limiting modified guide sequence for knocking down TNFA is shown below (hg38 coordinates chr12:68158001-68158021, G019757):

[0177] mC*mC*mA*GAGCAUCCAAAAGAGUGGUUUUAGAmGmCm UmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCC GUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*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 comprising ribose.

[0178] A non-limiting modified guide sequence for knocking down IFNG is shown below (hg38 coordinates chr6:31576805-31576825, G019753):

[0179] mA*mG*mA*GCUCUUACCUACAACAUGUUUUAGAmGmCm UmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCC GUUAUCAmAmCmUmUmGmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU(SEQ ID NO:120).

[0180] A non-limiting modified guide sequence for knocking down IL17A is shown below (hg38 coordinates chr6:52189069-52189089):

[0181] mU*mC*mA*CAGAGGGAUAUCUCUCAGUUUUAGAmGmCm UmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCC GUUAUCAmAmCmUmUmGmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU(SEQ ID NO:217).

[0182] An exemplary modified simulation guide is shown below (hg38 coordinates chr1:0-20):

[0183] mG*mA*mU*CACGUCGGCCGUUGGCGGUUUUAGAmGmCm UmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCC GUUAUCAmAmCmUmUmGmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU(SEQ ID NO:121).

[0184] In some embodiments, disclosed herein are T cells engineered by introducing or inserting heterologous dmTGFB1 nucleic acids into or into the genomic loci of T cells or T cell populations, for example, to make engineered T cells, using guide RNAs with RNA-guided DNA binders and constructs (e.g., donor constructs or templates) comprising heterologous dmTGFB1 nucleic acids. In some embodiments, disclosed herein are T cells engineered by expressing heterologous dmTGFB1 from the genomic loci of T cells or T cell populations, for example, using guide RNAs with RNA-guided DNA binders and constructs (e.g., donors) comprising heterologous dmTGFB1 nucleic acids. In some embodiments, disclosed herein are T cells engineered by inducing breaks (e.g., double-strand breaks (DSBs) or single-strand breaks (nicks)) in the genome of T cells or T cell populations, for example, using guide RNAs with RNA-guided DNA binders (e.g., CRISPR / Cas systems) to insert dm TGFB1 genes. Cells and cell populations prepared by the method are also provided.

[0185] In some embodiments, disclosed herein is a T cell engineered by introducing or inserting a heterologous dmTGFB1 nucleic acid into a genomic locus of a T cell, which is further engineered by introducing or inserting a heterologous IL10 nucleic acid into a genomic locus of a T cell or a population of T cells using a guide RNA with an RNA-guided DNA binder and a construct (e.g., a donor construct or template) comprising a heterologous IL10 nucleic acid, for example, to prepare an engineered T cell. In some embodiments, disclosed herein is a T cell engineered by, for example, expressing heterologous IL10 from a genomic locus of a T cell or a population of T cells using a guide RNA with an RNA-guided DNA binder and a construct (e.g., a donor) comprising a heterologous IL10 nucleic acid. In some embodiments, disclosed herein is a T cell engineered by, for example, inducing a break (e.g., a double-strand break (DSB) or a single-strand break (nick)) in the genome of a T cell or a population of T cells using a guide RNA with an RNA-guided DNA binder (e.g., a CRISPR / Cas system) to insert an IL10 gene. Cells and cell populations produced by the methods are also provided.

[0186] In some embodiments, disclosed herein is a T cell engineered by introducing or inserting a heterologous dmTGFB1 nucleic acid into a genomic locus of a T cell, which is further engineered by introducing or inserting a heterologous CTLA4 nucleic acid into a T cell or a T cell population using a guide RNA and a DNA binder guided by RNA and a construct (e.g., a donor construct or template) containing a heterologous CTLA4 nucleic acid, For example, to prepare an engineered T cell. In some embodiments, disclosed herein is a T cell engineered by the following means: For example, using a guide RNA and a DNA binder guided by RNA and a construct (e.g., a donor) containing a heterologous CTLA4 nucleic acid, heterologous CTLA4 is expressed from a genomic locus of a T cell or a T cell population. In some embodiments, disclosed herein is a T cell engineered by the following means: For example, using a guide RNA and a DNA binder guided by RNA (e.g., a CRISPR / Cas system), a break (e.g., a double-strand break (DSB) or a single-strand break (nick)) is induced in the genome of a T cell or a T cell population to insert a CTLA4 gene. Cells and cell populations produced by the methods are also provided.

[0187] In some embodiments, disclosed herein is a T cell engineered by introducing or inserting a heterologous dmTGFB1 nucleic acid into a genomic locus of a T cell, the T cell being further engineered by introducing or inserting a heterologous CTLA4 nucleic acid and a heterologous IL10 nucleic acid into a genomic locus of a T cell or a population of T cells, for example, to prepare an engineered T cell, using a guide RNA with an RNA-guided DNA binder and one or more constructs (e.g., a donor construct or template) comprising a heterologous CTLA4 nucleic acid and a heterologous IL10 nucleic acid. In some embodiments, disclosed herein is a T cell engineered by expressing heterologous CTLA4 and heterologous IL10 from a genomic locus of a T cell or a population of T cells, for example, using a guide RNA with an RNA-guided DNA binder and one or more constructs (e.g., a donor construct or template) comprising a heterologous CTLA4 nucleic acid and a heterologous IL10 nucleic acid. In some embodiments, disclosed herein are T cells engineered in the following manner: for example, using guide RNA and RNA-guided DNA binders (e.g., CRISPR / Cas systems) to induce breaks (e.g., double-strand breaks (DSBs) or single-strand breaks (nicks)) in the genome of a T cell or T cell population to insert CTLA4 genes and IL10 genes. In some embodiments, the guide RNA mediates target-specific cleavage at sites described herein by RNA-guided DNA binders (e.g., Cas nucleases) for inserting sequences encoding two or more regulatory T cell-promoting molecules (e.g., IL10 and CTLA4). It should be understood that in some embodiments, the guide RNA comprises a guide sequence that binds or is capable of binding to the region. Cells and cell populations prepared by the method are also provided.

[0188] Provided below are exemplary nucleotide and polypeptide sequences of regulatory T cell promoting molecules. Methods for identifying alternative nucleotide sequences encoding polypeptide sequences (including alternative naturally occurring variants and non-human homologs) are known in the art. Provided below are exemplary nucleic acid sequences encoding dmTGFB1, IL10 and CTLA4. Other suitable dmTGFB1, IL10 and CTLA4 sequences are known in the art or can be designed according to the disclosure provided herein. See, for example, Gorby et al., Engineered IL-10 variants elimi t potent immuno-modulatory 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 (specifically IL-10 variants) and sequences of these documents are hereby incorporated by reference. Methods for identifying alternative IL10 and CTLA4 sequences are also known in the art. See, for example, ibid. For example, Gorby disclosed the anti-inflammatory and pro-cytotoxic activities of IL-10. Recent studies have shown that in addition to its anti-inflammatory activity, IL-10 can also enhance the cytotoxic function of CD8 T cells, enhancing their ability to target tumors and enhance anticancer responses (Oft, 2019). This seems somewhat contradictory, as IL-10 in the tumor microenvironment has been associated with tumor escape from immune responses, most likely due to IL-10's inhibitory effects on antigen presentation (Mannino et al., 2015; Yue et al., 1997). Despite this contradiction, several studies have clearly demonstrated that IL-10 can improve the production of CD8 effector molecules granzyme B and interferon gamma in vivo and in vitro (Emmerich et al., 2012; Mumm et al., 2011; Mumm and Oft, 2013). Several clinical trials are currently testing the anti-tumor properties of IL-10, and preliminary good results have been achieved (Naing et al., 2019).In these trials, high doses of PEGylated IL-10 (Pegilodekakin) were used, which resulted in long-term retention of IL-10 in the circulation to ensure efficacy, again highlighting that effective IL-10 in vivo responses require high concentrations and sustained levels of IL-10. Saxton et al. used yeast display-based directed evolution to engineer IL-10 variants. IL-10 is an immunomodulatory cytokine with anti-inflammatory and immunostimulatory properties that is often dysregulated in disease. Mechanistically, IL-10 functions as a secreted homodimer that engages with two copies of a heterodimeric receptor complex containing a private receptor subunit IL-10Rα and a shared subunit IL-10Rβ. IL-10-dependent dimerization of IL-10Rα and IL-10Rβ then initiates activation of the transcription factor STAT3, thereby mediating the multiple biological effects of IL-10. Saxton et al. teach IL-10 variants with a range of IL-10Rβ binding strengths, revealing significant differences in response thresholds for different immune cell populations, providing 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 and is able to assemble a hexameric IL-10–IL-10Rα–IL-10Rβ complex. Additional variants (e.g., D25K, D25A, N21A / R104A, and D25A / N21A / R104A based on amino acid numbering of SEQ ID NO:231) display myeloid-biased activity by inhibiting macrophage activation but not stimulating inflammatory CD8+ T cells, thereby unraveling a major opposing function of IL-10. In certain embodiments, IL-10 variants with impaired immunostimulatory properties (referred to herein as "inhibitory IL-10 variants") are used in the engineered T cells provided herein, for example, variants that retain myeloid biased activity and exhibit impaired inflammatory CD8+T cell stimulation by inhibiting macrophage activation. 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 of Saxton et al. provide a mechanistic blueprint for adjusting the pleiotropic effects of IL-10. Additional inhibitory IL-10 variants are provided in WO2021243057, which is incorporated herein by reference, providing a number of IL-10 sequences, including polypeptides having altered binding affinity to IL-10Rβ compared to the binding affinity of a reference IL-10 polypeptide lacking one or more amino acid substitutions, and methods of characterizing activity.Disclosed are additional variants, including inhibitory IL-10 variants, and predicted to exhibit myeloid-biased activity by inhibiting macrophage activation but not stimulating inflammatory CD8+T cells, similar to D25K, D25A, D25A / E96A, N21A / R104A, and D25A / N21A / R104A variants. 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, optionally including further substitutions at 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 with any nucleic acid sequence, amino acid sequence, or nucleic acid sequence encoding an amino acid sequence described herein, for example due to mutation or truncation, are also contemplated. In some embodiments, nucleic acid sequences encoding any amino acid sequence provided herein are also provided.

[0189] Non-limiting exemplary nucleic acid sequences encoding TGFB1 are provided:

[0190] Wild-type TGFB1

[0191]

[0192] Double mutant (dm)TGFB1(R218C, C225R)

[0193]

[0194] Non-limiting exemplary amino acid sequence of human TGFB1

[0195] Wild type

[0196] MPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS(SEQ ID NO:210)

[0197] dmTGFB1 R218C, C225R

[0198] MPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLS FDVTGVVRQWLSRGGEIEGFHLSAHCSRDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS(SEQ ID NO: 214) provides a non-limiting exemplary nucleic acid sequence encoding IL10:

[0199] Wild-type IL10:

[0200] (SEQ ID NO:122)

[0201] IL10 (N36I, N110I, K117N, F129L):

[0202] ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCATCCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGatcTCCCTGGGGGAGAACCTGAATACCCTCAGGCTGAGGCTACGGCGCTGTCATCGActcCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAAC(SEQ ID NO:123)

[0203] An exemplary non-limiting amino acid sequence of IL10 is provided:

[0204] Wild-type IL10:

[0205] MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN(SEQ ID NO:124)

[0206] High-affinity IL10 (N36I, N110I, K117N, F129L):

[0207] MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGILPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVISLGENLNTLRLRLRRCHRLLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN(SEQ ID NO:125)

[0208] Mature wild-type IL10

[0209] SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN(SEQ ID NO:231)

[0210] IL10(D25K)

[0211] SPGQGTQSENSCTHFPGNLPNMLRKLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN(SEQ ID NO:232)

[0212] IL10(D25E)

[0213] SPGQGTQSENSCTHFPGNLPNMLRELRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN(SEQ ID NO:233)

[0214] IL10(D25K / E96A)

[0215] A non-limiting exemplary nucleic acid sequence encoding TGFB1 is provided by SPGQGTQSENSCTHFPGNLPNMLRKLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGANLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 234):

[0216] Wild-type TGFB1

[0217] Double mutant TGFB1 (R218X, where X is H or C, C225R)

[0218] Non-limiting exemplary nucleic acid sequences encoding CTLA4 are provided:

[0219] Wild-type CTLA4:

[0220] ATGGCCTGCTTGGGCTTCCAAAGGCATAAAGCCCAGCTTAATCTTGCTACTCGCACGTGGCCCTGCACATTGCTCTTTTTCCTCCTGTTCATTCCCGTGTTTTGCAAGGCGATGCATGTGGCACAACCTGCCGTCGTTCTGGCATCATCAAGAGGTATTGCTAGCTTCGTTTGTGAGTACGCCTCCCCTGGAAAAGCGACGGAGGTGCGCGTCACTGTATTGCGGCAAGCCGACAGCCAAGTTACTGAAGTCTGCGCGGCAACGTATATGATGGGCAATGAGCTGACATTCCTTGACGATTCAATCTGCACGGGAACAAGTAGTGGTAACCAGGTGAATCTCACTATTCAAGGTCTGAGAGCCATGGACACCGGCCTCTACATTTGTAAGGTGGAGCTGATGTATCCTCCCCCATATTATCTGGGGATCGGAAATGGGACACAGATATATGTTATTGATCCCGAGCCATGTCCCGATAGTGACTTCCTCTTGTGGATACTTGCCGCTGTGAGCAGTGGTTTGTTTTTTTATTCATTCCTCCTTACGGCAGTATCACTTTCAAAAATGCTCAAGAAGCGAAGTCCTTTGACAACTGGCGTATATGTCAAAATGCCACCAACAGAGCCCGAATGTGAGAAACAGTTCCAGCCGTACTTTATTCCTATAAAC(SEQ IDNO:126)

[0221] High affinity CTLA4 (belatacept; binding domains: A29Y, L104E):

[0222] ATGGCCTGCTTGGGCTTCCAAAGGCATAAAGCCCAGCTTAATCTTGCTACTCGCACGTGGCCCTGCACATTGCTCTTTTTCCTCCTGTTCATTCCCGTGTTTTGCAAGGCGATGCATGTGGCACAACCTGCCGTCGTTCTGGCATCATCAAGAGGTATTGCTAGCTTCGTTTGTGAGTACGCCTCCCCTGGAAAATACACGGAGGTGCGCGTCACTGTATTGCGGCAAGCCGACAGCCAAGTTACTGAAGTCTGCGCGGCAACGTATATGATGGGCAATGAGCTGACATTCCTTGACGATTCAATCTGCACGGGAACAAGTAGTGGTAACCAGGTGAATCTCACTATTCAAGGTCTGAGAGCCATGGACACCGGCCTCTACATTTGTAAGGTGGAGCTGATGTATCCTCCCCCATATTATGAGGGGATCGGAAATGGGACACAGATATATGTTATTGATCCCGAGCCATGTCCCGATAGTGACTTCCTCTTGTGGATACTTGCCGCTGTGAGCAGTGGTTTGTTTTTTTATTCATTCCTCCTTACGGCAGTATCACTTTCAAAAATGCTCAAGAAGCGAAGTCCTTTGACAACTGGCGTATATGTCAAAATGCCACCAACAGAGCCCGAATGTGAGAAACAGTTCCAGCCGTACTTTATTCCTATAAAC(SEQ IDNO:127)

[0223] High affinity CTLA4 (binding domain: A29H):

[0224] ATGGCCTGCTTGGGCTTCCAAAGGCATAAAGCCCAGCTTAATCTTGCTACTCGCACGTGGCCCTGCACATTGCTCTTTTTCCTCCTGTTCATTCCCGTGTTTTGCAAGGCGATGCATGTGGCACAACCTGCCGTCGTTCTGGCATCATCAAGAGGTATTGCTAGCTTCGTTTGTGAGTACGCCTCCCCTGGAAAACATACGGAGGTGCGCGTCACTGTATTGCGGCAAGCCGACAGCCAAGTTACTGAAGTCTGCGCGGCAACGTATATGATGGGCAATGAGCTGACATTCCTTGACGATTCAATCTGCACGGGAACAAGTAGTGGTAACCAGGTGAATCTCACTATTCAAGGTCTGAGAGCCATGGACACCGGCCTCTACATTTGTAAGGTGGAGCTGATGTATCCTCCCCCATATTATCTGGGGATCGGAAATGGGACACAGATATATGTTATTGATCCCGAGCCATGTCCCGATAGTGACTTCCTCTTGTGGATACTTGCCGCTGTGAGCAGTGGTTTGTTTTTTTATTCATTCCTCCTTACGGCAGTATCACTTTCAAAAATGCTCAAGAAGCGAAGTCCTTTGACAACTGGCGTATATGTCAAAATGCCACCAACAGAGCCCGAATGTGAGAAACAGTTCCAGCCGTACTTTATTCCTATAAAC(SEQ IDNO:128)

[0225] High affinity CTLA4 (Binding domain: K28H, A29H):

[0226] ATGGCCTGCTTGGGCTTCCAAAGGCATAAAGCCCAGCTTAATCTTGCTACTCGCACGTGGCCCTGCACATTGCTCTTTTTCCTCCTGTTCATTCCCGTGTTTTGCAAGGCGATGCATGTGGCACAACCTGCCGTCGTTCTGGCATCATCAAGAGGTATTGCTAGCTTCGTTTGTGAGTACGCCTCCCCTGGACATCACACGGAGGTGCGCGTCACTGTATTGCGGCAAGCCGACAGCCAAGTTACTGAAGTCTGCGCGGCAACGTATATGATGGGCAATGAGCTGACATTCCTTGACGATTCAATCTGCACGGGAACAAGTAGTGGTAACCAGGTGAATCTCACTATTCAAGGTCTGAGAGCCATGGACACCGGCCTCTACATTTGTAAGGTGGAGCTGATGTATCCTCCCCCATATTATCTGGGGATCGGAAATGGGACACAGATATATGTTATTGATCCCGAGCCATGTCCCGATAGTGACTTCCTCTTGTGGATACTTGCCGCTGTGAGCAGTGGTTTGTTTTTTTATTCATTCCTCCTTACGGCAGTATCACTTTCAAAAATGCTCAAGAAGCGAAGTCCTTTGACAACTGGCGTATATGTCAAAATGCCACCAACAGAGCCCGAATGTGAGAAACAGTTCCAGCCGTACTTTATTCCTATAAAC(SEQ IDNO:129)

[0227] An exemplary non-limiting amino acid sequence of CTLA4 is provided:

[0228] Wild-type CTLA4:

[0229] MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN(SEQ ID NO:130)

[0230] High-affinity CTLA4 (Belatacept; binding domain: A29Y, L104E):

[0231] MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN(SEQ ID NO:131)

[0232] High-affinity CTLA4 (binding domain: A29H):

[0233] MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKHTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN(SEQ ID NO:132)

[0234] High-affinity CTLA4 (binding domain: K28H, A29H):

[0235] MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGHHTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVN LTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN(SEQ ID NO:133)

[0236] In some embodiments, an engineered T cell or T cell population comprising a heterologous sequence encoding dmTGFB1 under the control of a promoter, a further modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and the insertion of a heterologous sequence encoding a regulatory T cell promoting molecule into a cell under the control of a promoter sequence exhibits at least one suppressive activity of a naturally occurring regulatory T cell (nTreg), for example, suppression of an immune response or biomarker in an in vitro or in vivo assay (e.g., an animal model of GvHD). In some embodiments, an engineered T cell or T cell population 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 of a heterologous sequence encoding a regulatory T cell promoting molecule into a cell under the control of a promoter sequence exhibits improved suppressive activity, for example, increased suppression of an immune response or biomarker in an in vitro or in vivo assay (e.g., an animal model of GvHD) compared to nTreg. For example, in a mouse model of GvHD, compared to a control (e.g., a mouse receiving PBMC), a mouse receiving an engineered T cell comprising a modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding IFNG, a modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding TNFA, and an insertion of a heterologous sequence encoding a regulatory T cell promoting molecule into a cell under the control of a promoter sequence showed an improved survival rate. In certain embodiments, the engineered cell comprises a modification of an endogenous nucleic acid sequence encoding IFNG and IL17A respectively. In certain embodiments, the regulatory T cell promoting molecule includes IL10 or CTLA4. In certain embodiments, the regulatory T cell promoting molecule includes IL10 and CTLA4.

[0237] In certain embodiments, engineered T cells or T cell colonies include two or more heterologous coding sequences under the control of a promoter, for example, dmTGFB1 and regulatory T cell promoting molecules. 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 engineered T cells or T cell colonies include three or more heterologous coding sequences (e.g., dmTG FB1 and regulatory T cell promoting molecules) under the control of a promoter, each heterologous sequence is independently under the control of a separate promoter or under the control of a promoter controlling the expression of more than one heterologous coding sequence.

[0238] B. Targeting receptors

[0239] In some embodiments, the engineered T cells comprising modifications (e.g., knockdown) of endogenous nucleic acid sequences encoding TNFA; modifications (e.g., knockdown) of endogenous nucleic acid sequences encoding IFNG or IL17A; and insertion of heterologous sequences encoding dmTGFB1 molecules and regulatory T cell promoting molecules, each under the control of a promoter sequence, into cells also comprise insertion of heterologous sequences encoding targeting receptors into cells. The sequences encoding targeting receptors are under the control of promoter sequences (e.g., endogenous promoters or heterologous promoters). In certain embodiments, the engineered cells comprise modifications of endogenous nucleic acid sequences encoding IFNG and IL17A, respectively, for knockdown.

[0240] In some embodiments, the engineered T cells comprising modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and insertion of a heterologous sequence encoding a regulatory T cell promoting molecule into the cell under the control of a promoter sequence further comprise insertion of a heterologous sequence encoding a targeting receptor into the cell. The sequence encoding the targeting receptor is 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 modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0241] In some embodiments, the targeting receptor is a receptor for a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a cell surface molecule, which is operably connected by at least one transmembrane domain in an internal signaling domain, which can activate T cells when binding to an extracellular receptor portion. In some embodiments, the targeting receptor can be a receptor present on the surface of a cell (e.g., a T cell) to allow the cell to bind to a target site (e.g., a specific cell or tissue in an organism). The targeting receptor does not need to be an antigen receptor, for example, the targeting receptor can be an RGD peptide capable of targeting 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), MO G (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), glutamate 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 TNF A.

[0242] In some embodiments, the engineered T cells include modifications (e.g., knockdown) of endogenous nucleic acid sequences encoding IFNG; modifications (e.g., knockdown) of endogenous nucleic acid sequences encoding TNFA; insertion of sequences encoding regulatory T cell-promoting molecules selected from IL10, CTLA4, IDO1, ENTPD1, ​​NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, BACH 2, and IL2RA; and insertion of sequences encoding targeting receptors (e.g., CARs, e.g., CARs targeting MAdCAM-1). Alternatively, the targeting receptor targets TNFA.

[0243] In some embodiments, the engineered T cells 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 sequences encoding dmTGFB1 and IL10, and a targeting receptor, e.g., CAR, e.g., a CAR targeting MAdCAM-1. Alternatively, the targeting receptor targets TNFA.

[0244] In some embodiments, the engineered T cells 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 sequences encoding dmTGFB1 and CTLA4, and a targeting receptor, e.g., CAR, e.g., a CAR targeting MAdCAM-1. Alternatively, the targeting receptor targets TNFA.

[0245] In some embodiments, the engineered T cells comprise modifications (e.g., knockdown) of endogenous nucleic acid sequences encoding TNFA; modifications (e.g., knockdown) of endogenous nucleic acid sequences encoding IFNG or IL17A; insertion of sequences encoding dmTGFB1, IL10, CTLA4, and targeting receptors, e.g., CARs, e.g., CARs targeting MAdCAM-1. Alternatively, the targeting receptor targets TNFA.

[0246] In some embodiments, the engineered T cells include modifications (e.g., knockdown) of endogenous nucleic acid sequences encoding TNFA; modifications (e.g., knockdown) of endogenous nucleic acid sequences encoding IL17A, insertion of sequences encoding IL10, and targeting receptors, e.g., CARs, e.g., CARs targeting MAdCAM-1. Alternatively, the targeting receptor targets TNFA. In certain embodiments, the engineered T cells do not include modifications (e.g., knockdown) of endogenous nucleic acid sequences encoding IFNG.

[0247] In some embodiments, the engineered T cells include modifications (e.g., knockdown) of endogenous nucleic acid sequences encoding TNFA; modifications (e.g., knockdown) of endogenous nucleic acid sequences encoding IL17A; insertion of sequences encoding CTLA4, and targeting receptors, e.g., CARs, e.g., CARs targeting MAdCAM-1. Alternatively, the targeting receptor targets TNFA. In certain embodiments, the engineered T cells do not include modifications (e.g., knockdown) of endogenous nucleic acid sequences encoding IFNG.

[0248] In some embodiments, engineered T cells 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 of sequences encoding IL10, CTLA4, and targeting receptors, e.g., CARs, e.g., CARs targeting MAdCAM-1. Alternatively, the targeting receptor targets TNFA. In certain embodiments, engineered T cells do not include modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0249] In some embodiments, the sequence encoding the targeting receptor is incorporated into the expression construct. In some embodiments, the expression construct comprising the sequence encoding the targeting receptor also comprises the sequence encoding the regulatory T cell promoting molecule, for example, the sequence encoding the targeting receptor and the sequence encoding the regulatory T cell promoting molecule are incorporated into the same expression construct. In some embodiments, the expression construct comprising the sequence encoding the targeting receptor does not further comprise the sequence encoding the regulatory T cell promoting molecule, for example, the sequence encoding the regulatory T cell promoting molecule is incorporated into a separate expression construct. In some embodiments, the expression construct comprising the sequence encoding the targeting receptor is a free expression construct. In some embodiments, the sequence encoding the targeting receptor is inserted into the genome, for example, a targeted or non-targeted insertion.

[0250] In some embodiments, the sequence encoding the targeting receptor can be inserted into a site selected from the TCR locus, such as the TRAC locus, TNF locus, IFNG locus, IL17A locus, IL6 locus, IL2 locus, or adeno-associated virus integration site 1 (AAVS1) locus.

[0251] In some embodiments, the engineered T cells comprise the insertion of a sequence encoding a targeting receptor by gene editing, e.g., as assessed by sequencing (e.g., NGS).

[0252] In some embodiments, the T cell population includes T cells, which are engineered to include modifications (e.g., knockdown) of endogenous nucleic acid sequences encoding TNFA, modifications (e.g., knockdown) of endogenous nucleic acid sequences encoding IFNG or IL17A, insertion of sequences encoding regulatory T cell-promoting molecules, and insertion of sequences encoding targeting receptors (e.g., CAR). In some embodiments, at least 40%, 45%, preferably at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the T cells in the T cell population are engineered to include insertion of sequences encoding targeting receptors, for example, as assessed by sequencing (e.g., NGS). It should be understood that selection methods known in the art can be used to enrich T cell populations from cell populations with targeting receptors.

[0253] In some embodiments, disclosed herein is a T cell engineered in the following manner: using a DNA binder guided by guide RNA and RNA and a construct (e.g., a donor construct or template) comprising a targeting receptor (e.g., CAR nucleic acid), introducing or inserting a targeting receptor (e.g., CAR nucleic acid) in a T cell (e.g., in a genomic locus of a T cell or T cell colony), for example, to manufacture engineered T cells. In some embodiments, disclosed herein is a T cell engineered in the following manner: for example, using a DNA binder guided by guide RNA and RNA and a construct (e.g., donor) comprising a targeting receptor (e.g., CAR nucleic acid), expressing a targeting receptor (e.g., CAR) from a genomic locus of a T cell or T cell colony. In some embodiments, disclosed herein is a T cell engineered in the following manner: for example, using a DNA binder guided by guide RNA and RNA (e.g., CRISPR / Cas system), inducing a break (e.g., double-strand break (DSB) or single-strand break (nick)) in the genome of a T cell or T cell colony to insert a targeting receptor (e.g., CAR). Cells and cell colonies prepared by the method are also provided.

[0254] In some embodiments, the targeting receptor (e.g., CAR) is capable of conferring target specificity to an engineered T cell comprising the targeting receptor (e.g., CAR), such as a specific cell, tissue, or organ.

[0255] In some embodiments, the targeting receptor (e.g., CAR) is capable of targeting the engineered T cells to the gastrointestinal system, for example, the targeting receptor is a CAR targeting MAdCAM-1, for example, for inhibiting immune responses in disorders such as inflammatory bowel disease, ulcerative colitis, or Crohn's disease.

[0256] In some embodiments, the targeting receptor (e.g., CAR) is capable of targeting the engineered T cells to inflammatory tissues, for example, the targeting receptor is a CAR targeting TNFA, for example, for suppressing immune responses in conditions such as inflammatory bowel disease, ulcerative colitis, or Crohn's disease.

[0257] In some embodiments, the targeting receptor (e.g., CAR) is capable of targeting the engineered T cells to endothelial cells, for example, the targeting receptor is a CAR targeting CEACAM6, for example, for suppressing immune responses, including inflammation, in disorders such as Crohn's disease.

[0258] In some embodiments, the targeting receptor (e.g., CAR) is capable of targeting the engineered T cells to tissues containing endothelial cells, for example, the targeting receptor is a CAR targeting VCAM-1, for example, for suppressing immune responses in disorders such as Crohn's disease and multiple sclerosis.

[0259] In some embodiments, the CAR is capable of targeting engineered T cells to synovial tissue, e.g., the targeting receptor is a CAR that targets citrullinated vimentin, e.g., for suppressing immune responses in disorders such as rheumatoid arthritis.

[0260] In some embodiments, the targeting receptor (e.g., CAR) is capable of targeting the engineered T cells to neural tissue, for example, the targeting receptor is a CAR targeting MBP, MOG, or PLP1, for example, for suppressing immune responses in disorders such as multiple sclerosis.

[0261] In some embodiments, the targeting receptor (e.g., CAR) is capable of targeting the engineered T cells to B cells, for example, the targeting receptor is a CAR targeting CD19, for example, for suppressing immune responses in disorders such as multiple sclerosis and systemic lupus erythematosus.

[0262] In some embodiments, the targeting receptor (e.g., CAR) is capable of targeting the engineered T cells to B cells, for example, the targeting receptor is a CAR targeting CD20, for example, for suppressing immune responses in disorders such as multiple sclerosis and systemic lupus erythematosus.

[0263] In some embodiments, the targeting receptor (e.g., CAR) is capable of targeting the engineered T cells to tissues containing mature B lymphocytes, for example, the targeting receptor is a CAR targeting TNFRSF17, for example, for suppressing immune responses in disorders such as systemic lupus erythematosus.

[0264] 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).

[0265] Other CAR targets (e.g., inflammatory antigens) are known in the art. See, for example, WO2020092057A1, the contents of which are incorporated herein in their entirety. In some embodiments, insertion can be assessed by detecting the amount of protein or mRNA in engineered T cells, engineered T cell colonies, tissues, body fluids of interest, or tissue culture media containing engineered T cells. In some embodiments, insertion by gene editing can be assessed by sequence, such as next generation sequencing (NGS). The determination of protein and mRNA expression of targeting receptors (e.g., CAR) is described herein and is known in the art.

[0266] In certain embodiments, the engineered T cell or T cell population comprises two or more heterologous coding sequences, for example, dmTGFB1, regulatory T cell promoting molecules and targeting receptors; they are under the control of a promoter. 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 T cell population comprises three or more heterologous coding sequences, for example, dmTGFB1, regulatory T cell promoting molecules and targeting receptors; they are under the control of a promoter, each heterologous sequence is independently under the control of a separate promoter or under the control of a promoter that controls the expression of more than one heterologous coding sequence.

[0267] In some embodiments, the engineered T cell or T cell population does not include a heterologous targeting receptor.

[0268] T cell receptor (TCR)

[0269] In some embodiments, the engineered T cell or T cell population comprising a modification (e.g., insertion into the cell) of a heterologous sequence encoding a dmTGF Bl molecule under the control of a promoter sequence also comprises a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TCR gene sequence.

[0270] In some embodiments, the engineered T cell or T cell population comprising modifications (including insertion into the cell of a heterologous sequence encoding a dmTGFB1 molecule under the control of a promoter sequence) also 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; and insertion into the cell of a heterologous sequence encoding a regulatory T cell promoting molecule under the control of a promoter sequence; and also comprises modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TCR gene sequence.

[0271] In some embodiments, the engineered T cell or T cell population 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 cell 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.

[0272] In some embodiments, the engineered T cell or T cell population comprising modifications (including insertion into the cell of a heterologous sequence encoding a dmTGFB1 molecule under the control of a promoter sequence) also 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 modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TCR gene sequence.

[0273] In some embodiments, the engineered T cell or T cell population comprising a modification (including insertion of a heterologous sequence encoding a regulatory T cell promoting molecule under the control of a promoter sequence into a cell) 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; and insertion of a heterologous sequence encoding a regulatory T cell promoting molecule under the control of a promoter sequence into a cell; and also comprising modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TCR gene sequence. In certain embodiments, the engineered T cell or cell population does not comprise modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0274] In some embodiments, the engineered T cell or T cell population comprising modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A; and the insertion of a heterologous sequence encoding a first regulatory T cell promoting molecule and a second regulatory T cell promoting molecule into a cell, each under the control of a promoter sequence, further comprises modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TCR gene sequence. In certain embodiments, the engineered T cell or cell population does not comprise modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0275] In some embodiments, the engineered T cell or T cell population comprising a modification (including insertion of a heterologous sequence encoding a regulatory T cell promoting molecule under the control of a promoter sequence into a cell) 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 of a heterologous sequence encoding a first regulatory T cell promoting molecule and a second regulatory T cell promoting molecule into a cell, each under the control of a promoter sequence; and modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TCR gene sequence. In certain embodiments, the engineered T cell or cell population does not comprise modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0276] Typically, TCR is a heterodimeric receptor molecule containing two TCR polypeptide chains α and β. Suitable α and β genomic sequences or loci for targeted knockdown are known in the art. In some embodiments, engineered T cells comprise modifications (e.g., knockdowns) of TCR α chain gene sequences (e.g., TRAC). See, e.g., NCBI gene ID: 28755; Ensembl: ENSG00000277734 (T cell receptor α constant), US2018 / 0362975 and WO2020081613.

[0277] In some embodiments, the engineered T cell or population of T cells comprises a modification comprising the insertion of a sequence encoding dmTGFB1.

[0278] In some embodiments, the engineered T cell or T cell population comprising the insertion of a heterologous sequence encoding a dmTGF B1 molecule under the control of a promoter sequence into the cell 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 IFNG or IL17A; an insertion of a sequence encoding a regulatory T cell promoting molecule selected from the group consisting of IL10, CTLA4, IDO1, ENTPD1, ​​NT5E, IL22, AREG, IL35, GARP, CD274, FO XP3, IKZF2, EOS, IRF4, LEF1, BACH2, and IL2RA; and a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TC R gene sequence.

[0279] In some embodiments, the engineered T cell or T cell population comprising the insertion of a heterologous sequence encoding a dmTGF B1 molecule under the control of a promoter sequence into the cell further comprises 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 IFN G or IL17A; insertion of a sequence encoding a regulatory T cell promoting molecule selected from IL10, CT LA4, IDO1, ENTPD1, ​​NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, BACH2 and; and a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TC R gene sequence.

[0280] In some embodiments, the engineered T cell or T cell population comprising the insertion of a heterologous sequence encoding a dmTGF B1 molecule under the control of a promoter sequence into the cell also 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 IL10 or CTLA4, and a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TCR gene sequence.

[0281] In some embodiments, the engineered T cell or T cell population comprises a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNF A, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, an insertion of a sequence encoding dmTGFB1, IL10, and CTLA4, and a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TCR gene sequence.

[0282] In some embodiments, the engineered T cells or T cell populations comprise modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNF A, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A, insertion of sequences encoding a dmTGFB1 molecule and a regulatory T cell promoting molecule, and modification (e.g., knockdown) of an endogenous TCR gene sequence (e.g., a TRAC gene sequence).

[0283] In some embodiments, the engineered T cell or T cell population comprises a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNF A; a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A;

[0284] Insertion of a sequence encoding a dmTGFB1 molecule; Insertion of a sequence 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 TCR gene (e.g., a TRAC gene sequence).

[0285] In some embodiments, the engineered T cells or T cell populations comprise modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNF A, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A; insertion of a sequence encoding a dmTGFB1 molecule, insertion of a sequence encoding IL10 or CTLA4; and modification (e.g., knockdown) of a TCR gene (e.g., a TRAC gene sequence).

[0286] In some embodiments, the engineered T cell or T cell population comprises a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNF A; 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 a sequence encoding a regulatory T cell promoting molecule; and a modification (e.g., knockdown) of an endogenous TCR gene (e.g., a TRAC gene sequence).

[0287] In some embodiments, the engineered T cell or T cell population comprising the insertion of a heterologous sequence encoding a regulatory T cell promoting molecule under the control of a promoter sequence into the cell 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 IL 17A; insertion of a sequence encoding IL10 or CTLA4, and modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding a TCR gene sequence. In certain embodiments, the engineered T cell or cell population does not comprise modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0288] In some embodiments, the engineered T cell or T cell population comprises modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding TNF A, modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding IL17A, insertion of sequences encoding IL10 and CTLA4, and modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding a TCR gene sequence. In certain embodiments, the engineered T cell or cell population does not comprise modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding IFNG.

[0289] In some embodiments, the engineered T cell or T cell population comprises a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNF A; a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A; insertion of a sequence encoding a first regulatory T cell promoting molecule and a second regulatory T cell promoting molecule, and modification (e.g., knockdown) of an endogenous TCR gene sequence (e.g., TRAC gene sequence). In certain embodiments, the engineered T cell or cell population does not comprise a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0290] In some embodiments, engineered T cells or T cell colonies include modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding TNF A; modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding IL17A; insertion of a sequence encoding a first regulatory T cell promoting molecule; insertion of a sequence encoding a regulatory T cell promoting molecule, the regulatory T cell promoting molecule being selected from IL10, CTLA4, IDO1, ENTPD1, ​​NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, BACH 2, and IL2RA; and modification (e.g., knocking down) of an endogenous TCR gene (e.g., TRAC gene sequence). In certain embodiments, engineered T cells or cell colonies do not include modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding IFNG.

[0291] In some embodiments, the engineered T cell or T cell population comprises modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNF A, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A; insertion of a sequence encoding a first regulatory T cell promoting molecule, insertion of a sequence encoding IL10 or CTLA4; and modification (e.g., knockdown) of a TCR gene (e.g., a TRAC gene sequence). In certain embodiments, the engineered T cell or cell population does not comprise modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0292] In some embodiments, the engineered T cell or T cell population comprises modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNF A; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A; insertion of a sequence encoding a first regulatory T cell promoting molecule, insertion of a sequence encoding a second regulatory T cell promoting molecule, and modification (e.g., knockdown) of an endogenous TCR gene (e.g., a TRAC gene sequence). In certain embodiments, the engineered T cell or cell population does not comprise modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0293] In any of these embodiments, the engineered T cell or population of T cells may further comprise the insertion of a sequence encoding a targeting receptor (e.g., a CAR, e.g., a CAR targeting MAdCAM-1) as described herein.

[0294] In any of these embodiments, the engineered T cell or T cell population may further comprise the insertion of a sequence encoding a targeting receptor (e.g., a CAR, e.g., a CAR targeting TNFA) as described herein.

[0295] In some embodiments, the engineered T cell or T cell population comprising the insertion of a heterologous sequence encoding a dmTGF B1 molecule under the control of a promoter sequence into a cell comprises a modification (e.g., knockdown) of a TRC gene sequence by gene editing, for example, as assessed by sequencing (e.g., NGS), wherein 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 in an endogenous TRC gene sequence. In some embodiments, compared to a suitable control, TRC is reduced by at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, 80%, 85%, 90%, 95%, or is reduced to a detection limit below the assay, for example, wherein the TRC gene is not modified. Determination of TRC protein and mRNA expression is known in the art.

[0296] In some embodiments, the engineered T cell or T cell population comprises the insertion of a sequence encoding a targeting receptor by gene editing, e.g., as assessed by sequencing (e.g., NGS).

[0297] In some embodiments, the T cell population comprises T cells comprising an insertion of a heterologous sequence encoding a dmTGFB1 molecule under the control of a promoter sequence into the cell, 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 a regulatory T cell promoting molecule, and a modification (e.g., knockdown) of at least one TCR gene sequence. In some embodiments, at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, 80%, 85%, 90%, 95% or 100% of the T cells in the T cell population are engineered to comprise a modification (e.g., knockdown) of at least one TCR gene sequence, e.g., as assessed by sequencing (e.g., NGS).

[0298] In some embodiments, the T cell population comprises T cells comprising an insertion of a heterologous sequence encoding a dmTGFB1 molecule under the control of a promoter sequence into the cell, 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, an insertion of a sequence encoding a regulatory T cell promoting molecule, and a modification (e.g., knockdown) of at least one TCR gene sequence. In some embodiments, at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, 80%, 85%, 90%, 95% or 100% of the T cells in the T cell population are engineered to comprise a modification (e.g., knockdown) of at least one TCR gene sequence, e.g., as assessed by sequencing (e.g., NGS).

[0299] In some embodiments, a guide RNA that specifically targets a site within a TCR gene (e.g., a TRAC gene) is used to provide modification (e.g., knockdown) of the TCR gene.

[0300] In some embodiments, guide RNA and RNA-guided DNA binders are used in T cells to modify (e.g., knock down) TCR genes. In some embodiments, disclosed herein are T cells engineered in the following manner: for example, using guide RNA and RNA-guided DNA binders (e.g., CRISPR / Cas systems), inducing breaks (e.g., double-strand breaks (DSBs) or single-strand breaks (nicks)) in the TCR genes of T cells. The method can be used in vitro or in vitro for, for example, manufacturing cell products that suppress immune responses.

[0301] In some embodiments, the guide RNA mediates target-specific cleavage at a site described herein within a TCR gene by a DNA binder (e.g., Cas nuclease) guided by RNA. It should be understood that in some embodiments, the guide RNA comprises a guide sequence that binds or is capable of binding to the region.

[0302] D. Guide RNA

[0303] In any embodiment herein, the guide RNA may further include trRNA. In each composition and method embodiment described herein, crRNA and trRNA may be associated as a single RNA (sgRNA), or may be on a separate RNA (dgRNA). In the case of sgRNA, the crRNA and trRNA components may be covalently linked, for example, by a phosphodiester bond or other covalent bond. In some embodiments, sgRNA includes one or more bonds between nucleotides that are not phosphodiester bonds.

[0304] In each of the compositions, uses, and method embodiments described herein, the guide RNA may comprise two RNA molecules as a "dual guide RNA" or "dgRNA". The dgRNA comprises a first RNA molecule and a second RNA molecule, the first RNA molecule comprising a crRNA, the crRNA comprising a guide sequence such as shown herein, and the second RNA molecule comprising a trRNA. The first and second RNA molecules may not be covalently linked, but may form an RNA duplex by base pairing between the crRNA and trRNA portions.

[0305] In each of the compositions, uses, and method embodiments described herein, the guide RNA may comprise a single RNA molecule as a "single guide RNA" or "sgRNA". The sgRNA may comprise a crRNA (or a portion thereof), the crRNA comprising a guide sequence covalently linked to the trRNA as shown herein. The sgRNA may comprise 15, 16, 17, 18, 19, or 20 consecutive nucleotides of the guide sequence as shown herein. In some embodiments, the crRNA and trRNA are covalently linked via a linker. In some embodiments, the sgRNA forms a stem-loop structure via base pairing between the crRNA and trRNA portions. In some embodiments, the crRNA and trRNA are covalently linked via one or more bonds that are not phosphodiester bonds.

[0306] In some embodiments, trRNA may include all or part of a trRNA sequence from a naturally occurring CRISPR / Cas system. In some embodiments, trRNA includes truncated or modified wild-type trRNA. The length of trRNA depends on the CRISPR / Cas system used. In some embodiments, trRNA includes 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more than 100 nucleotides or is composed of the nucleotides. In some embodiments, trRNA may include certain secondary structures, such as one or more hairpins or stem-loop structures, or one or more protruding structures.

[0307] In some embodiments, the target sequence or region may be complementary to the guide sequence of the guide RNA. In some embodiments, the complementarity or degree of identity between the guide sequence of the guide RNA and its corresponding target sequence may be 75%, 80%, 85%, 90%, 95% or 100%. In some embodiments, the target sequence and guide sequence of the gRNA may be 100% complementary or identical. In other embodiments, the target sequence and guide sequence of the gRNA may contain one mismatch. For example, the target sequence and guide sequence of the gRNA may contain 1, 2, 3, 4 or 5 mismatches, wherein the total length of the specified sequence is about 20 or 20. In some embodiments, the target sequence and guide sequence of the gRNA may contain 1-4 mismatches, wherein 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 is at least 80%, 85%, preferably 90%, or 95%, for example, when the guide sequence comprises a sequence of 24 consecutive 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 non-identical or non-complementary nucleotide, depending on the reference sequence. For example, the guide sequence and the target sequence may contain 1-2, preferably no more than 1 mismatch, wherein 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 1 to 2 mismatches, wherein the guide sequence comprises at least 24 or more nucleotides. In some embodiments, the guide sequence and the target region may contain 1 to 2 mismatches, wherein the guide sequence comprises 24 nucleotides.

[0308] In any of the embodiments herein, each guide sequence herein may further comprise additional nucleotides to form a crRNA or guide RNA, for example, the following exemplary nucleotide sequence following the guide sequence at its 3' end: GUUUUAGAGC UAUGCUGUUUUG (SEQ ID NO: 134) in 5' to 3' orientation. In the case of sgRNA, the above guide sequence may further comprise additional nucleotides to form the sgRNA, for example, the following exemplary nucleotide sequences after the guide sequence at its 3' end: GUUUUAGAGCUAG AAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 135); GU UUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 136); GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGGCACCGAGUCGGUGC (SEQ ID NO: 200); GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCACGAAAGGGCACCGAGUCGGUGC (SEQ ID NO: 2 NO:201);GUUGUAGCUCCCUUUCUCAUUUCGGAAACGAAAUGAGA ACCGUUGCUACAAUAAGGCCGUCUGAAAAGAUGUGCCGCAA CGCUCUGCCCCUUAAAGCUUCUGCUUUAAGGGGCAUCGUUUA (SEQ ID NO:202);GUUGUAGCUCCCUGAAACCGUUGCUACA AUAAGGCCGUCGAAAGAUGUGCCGCAACGCUCUGCCUUCUGGCAUCGUU (SEQ ID NO: 203).

[0309] In any embodiment, the guide RNA disclosed herein is bound to the upstream region of the protospacer adjacent motif (PAM). As will be appreciated by those skilled in the art, the PAM sequence appears on the chain opposite to the chain containing the target sequence, and varies with the CRISPR / Cas system. That is, the PAM sequence is on the complementary strand of the target strand (the strand containing the target sequence bound by the guide RNA). In some embodiments, PAM is selected from NGG, NNGRRT, NNGRR (N), NNAGAAW, NNNNG (A / C) TT and NNNNRYAC, for example, when the Cas system includes SpyCas9. In other embodiments, the PAM sequence includes NCC, N4GAYW, N4GYTT, N4GTCT, NNNNCC (a), NNNNCAAA (wherein N is defined as any nucleotide, W is defined as A or T, and R is defined as A or G; and (a) is preferably but not necessarily, and the second C is followed by A), for example, when the Cas system includes NmeCas9.

[0310] In some embodiments, the guide RNA sequence provided herein is complementary to a sequence adjacent to a PAM sequence.

[0311] In some embodiments, the guide RNA sequence comprises a sequence complementary to a sequence within a genomic region selected from the table herein according to coordinates in 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 within the genomic region from the table herein. In some embodiments, the guide RNA sequence comprises a sequence complementary to a sequence comprising 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides spanning a genomic region selected from the table herein.

[0312] The guide RNAs disclosed herein mediate target-specific cleavage, resulting in double-strand breaks (DSBs). The guide RNAs disclosed herein mediate target-specific cleavage, resulting in single-strand breaks (SSBs or nicks).

[0313] E. Chemically modified gRNA

[0314] 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 describe the presence of one or more non-natural or naturally occurring components or configurations that are used to replace or supplement canonical A, G, C, and U residues. In some embodiments, a modified gRNA is synthesized with non-canonical nucleosides or nucleotides, referred to herein as "modified". Modified nucleosides and nucleotides may include one or more of the following: (i) changes (e.g., substitutions) in one or both of the non-linked phosphate oxygens or one or more of the linked phosphate oxygens in the phosphodiester backbone linkage (exemplary backbone modifications); (ii) changes (e.g., substitutions) in the composition of the ribose sugar, e.g., changes (e.g., substitutions) in the 2' hydroxyl group on the ribose sugar (exemplary sugar modifications); (iii) modifications or substitutions of naturally occurring nucleobases, including non-canonical nucleobases (exemplary base modifications); and (iv) modifications of the 3' or 5' end of the oligonucleotide that provide exonuclease stability, e.g., modifications of the ribose sugar to 2'O-me, 2' halide, or 2' deoxy substituted ribose; or inversion of an abasic terminal nucleotide, or substitution of a phosphodiester by a phosphorothioate.

[0315] Chemical modifications (such as those listed above) can be combined to provide a modified gRNA comprising nucleosides and nucleotides (collectively referred to as "residues"), which can have two, three, four or more modifications. For example, the modified residue can have a modified sugar and a modified nucleobase. In some embodiments, each base of the gRNA is modified, for example, all bases have a modified phosphate group, such as a thiophosphate group. In certain embodiments, all or substantially all phosphate groups of the gRNA molecule are replaced by thiophosphate groups. In some embodiments, the modified gRNA includes at least one modified residue at or near the 5' end of the RNA. In some embodiments, the modified gRNA includes at least one modified residue at or near the 3' end of the RNA. Some gRNAs include at least one modified residue at or near the 5' end and 3' end of the RNA.

[0316] In some embodiments, the guide RNA disclosed herein includes one of the modification patterns disclosed in WO2018107028, the content of which is hereby incorporated by reference into the relevant parts. In some embodiments, the guide RNA disclosed herein includes one of the structures / modification patterns disclosed in US20170114334, which is incorporated herein by reference. In some embodiments, the guide RNA disclosed herein includes one of the structures / modification patterns disclosed in WO2017136794, WO2017004279, WO2019237069, US2018187186, US2019048338, WO2021119275 or WO2022125968, which are hereby incorporated by reference.

[0317] F. mRNA encoding RNA-guided DNA binders

[0318] In some embodiments, the cell or method comprises mRNA, and the mRNA comprises an open reading frame (ORF) of a DNA binder guided by encoding RNA, such as a Cas nuclease as described herein. Cas9 ORF is provided herein, and is known in the art. As an example, Cas9 ORF can be codon-optimized so that the coding sequence includes one or more alternative codons for one or more amino acids. As used herein, "alternative codons" refer to changes in the use of codons for a given amino acid, and for a given expression system, may or may not be preferred or optimized codons (codon-optimized). Preferred codon usage or well-tolerated codons in a given expression system are known in the art. WO2013 / 176772, WO2014 / 065596, WO2016 / 106121, WO2019 / 067910, and WO 2022 / 125968 Cas9 coding sequences, Cas9 mRNA, and Cas9 protein sequences are hereby incorporated by reference. In particular, 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] -

[0234] of WO2019 / 067910 are incorporated herein by reference.

[0319] In some embodiments, the modified ORF may include modified uridines at at least one, multiple or all uridine positions. In some embodiments, the modified uridine is a uridine modified at the 5 position, for example, with a halogen, a methyl or an ethyl. In some embodiments, the modified uridine is a pseudouridine modified at the 1 position, for example, with a halogen, a methyl or an ethyl. The modified uridine may be, for example, a pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine or a combination thereof. In some embodiments, the modified uridine is a 5-methoxyuridine. In some embodiments, the modified uridine is a 5-iodouridine. In some embodiments, the modified uridine is a pseudouridine. In some embodiments, the modified uridine is an 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-methyl pseudouridine 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.

[0320] In some embodiments, the mRNA disclosed herein comprises a 5' cap, such as Cap0, Cap1, or Cap2. The 5' cap is typically a 7-methylguanine ribonucleotide (which can be further modified, for example, ARCA (anti-reverse cap analog; Therm oFisher Scientific catalog number AM8045) is a cap analog comprising 7-methylguanine 3'-methoxy-5'-triphosphate linked to the 5' position of the first nucleotide of the 5'-to-3' strand of the mRNA) via a 5'-triphosphate. In Cap0, the ribose of the first and second proximal cap nucleotides of the mRNA both comprise a 2'-hydroxyl group. In Cap1, the ribose of the first and second transcribed nucleotides of the mRNA comprise a 2'-methoxy group and a 2'-hydroxyl group, respectively. See, for example, CleanCap TM AG (m7G (5') ppp (5') (2'OM eA) pG; TriLink Biotechnologies Cat. No. N-7113) or CleanCap TMGG (m7G (5') ppp (5') (2'OMeG) pG; TriLink Biotechnologies catalog number N-7133). In Cap2, the ribose of the first and second cap-proximal nucleotides of the mRNA both contain a 2'-methoxy group. 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.

[0321] In some embodiments, the mRNA further comprises a polyadenylation (poly-A) tail. In some embodiments, the poly-A 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 poly-A tail comprises 95, 96, 97, 98, 99 or 100 adenine nucleotides (SEQ ID NO: 149).

[0322] G. T cells for engineering

[0323] The engineered cells provided herein are prepared by a cell colony rich in CD4+T cells. Such cells can be easily obtained from fresh leukopak samples, which can be obtained commercially from various sources (including, for example, StemCell Technologies). CD4+T cells can be separated using conventional methods, such as by using the negative selection of a human CD4+T cell separation kit, using a commercially available kit. However, the method for preparing CD4+T cells from other sources is also known in the art. For example, multipotent cells such as hematopoietic stem cells (HSC, such as those separated from bone marrow or umbilical cord blood), hematopoietic progenitor cells (such as lymphoid progenitor cells) or mesenchymal stem cells (MSC) can be used to obtain CD4+T cells. Multipotent cells can develop into more than one cell type, but are more limited than multipotent cells in the breadth of differentiation. Multipotent cells can be derived from established cell lines or separated from human bone marrow or umbilical cord. For example, HSC can be separated from a patient or a healthy donor after mobilization induced by G-CSF, mobilization induced by plerixafor, or a combination thereof. To isolate HSC from blood or bone marrow, cells in the blood or bone marrow can be selected with antibodies that bind to unwanted cells, such as 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.

[0324] III. Delivery Methods

[0325] Guide RNA, RNA-guided DNA binders (e.g., Cas nucleases) and nucleic acid sequences disclosed herein can be delivered to cells or cell populations in vitro or ex vivo for the production of engineered T cells, comprising insertion of sequences encoding dmTGFB1; optionally, modification of endogenous nucleic acid sequences encoding TNFA (e.g., knocking down), modification of endogenous nucleic acid sequences encoding IFNG or IL17A (e.g., knocking down), insertion of sequences encoding regulatory T cell-promoting molecules (e.g., IL10, CTLA4); and optionally, insertion of sequences encoding targeting receptors (e.g., CAR), and optionally, modification of TCR sequences using various known and suitable methods available in the art (e.g., knocking down). Guide RNA, RNA-guided DNA binders and nucleic acid constructs can be delivered alone or in any combination, using the same or different delivery methods as appropriate.

[0326] Conventional viral and non-viral gene delivery methods can be used to introduce guide RNA and RNA-guided DNA binders and donor constructs in cells (e.g., mammalian cells) and target tissues. As further provided herein, non-viral vector delivery system nucleic acids, such as non-viral vectors, plasmid vectors and, for example, naked nucleic acids, and nucleic acids compounded with delivery vehicles, such as liposomes, lipid nanoparticles (LNPs) or poloxamer (poloxamer). Viral vector delivery systems include DNA and RNA viruses.

[0327] Methods and compositions for non-viral delivery of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, LNPs, polycations or lipids: nucleic acid conjugates, naked nucleic acids (e.g., naked DNA / RNA), artificial virosomes, and agent-enhanced DNA uptake. Acoustic perforation using, for example, the Sonitron 2000 system (Rich-Mar) can also be used to deliver nucleic acids.

[0328] Various delivery systems (e.g., carriers, liposomes, LNPs) containing guide RNA, RNA-guided DNA binders, and donor constructs can also be administered to cells or cell cultures ex vivo, either alone or in combination. Administration is by any route commonly used to introduce molecules into final contact with blood, fluids, or cells, including but not limited to injection, infusion, topical application, and electroporation. Suitable methods for administering such nucleic acids are available and are well known to those skilled in the art.

[0329] In certain embodiments, the disclosure provides a DNA or RNA vector encoding any one of the compositions disclosed herein, for example, a guide RNA comprising any one or more of the guide sequences described herein, for example, for modifying (e.g., knocking down) IFNG and TNFA, or comprising a sequence encoding a dmTGFB1 molecule, a donor construct encoding a sequence of a regulatory T cell promoting molecule (e.g., IL10) or a targeting receptor (e.g., CAR, e.g., MAdCAM-1CAR). In some embodiments, the vector also includes a sequence encoding a DNA binder guided by RNA. In certain embodiments, the present invention includes a DNA or RNA vector encoding any one or more or any combination thereof in the compositions described herein. In some embodiments, the vector also includes, for example, a promoter, an enhancer, and a regulatory sequence. In some embodiments, the vector comprising a guide RNA containing any one or more guide sequences described herein also includes one or more nucleotide sequences encoding crRNA, trRNA, or crRNA and trRNA, as disclosed herein.

[0330] In certain embodiments, the invention provides DNA or RNA vectors encoding regulatory T cell promoting molecules and targeting receptors. Such vectors allow selection of cells based on the presence of cell receptors that also contain the coding sequence of regulatory T cell promoting molecules. Positive and negative selection methods based on the presence of cell surface molecules are known in the art.

[0331] In some embodiments, the vector comprises a nucleotide sequence encoding a guide RNA as described herein. In some embodiments, the vector comprises a copy of the guide RNA. In other embodiments, the vector comprises more than one copy of the guide RNA. In embodiments with more than one guide RNA, the guide RNAs may be different so that they target different target sequences, or may be the same because they target the same target sequence. In embodiments where the vector comprises more than one guide RNA, each guide RNA may have other different properties, such as activity or stability in a complex with a DNA nuclease guided by RNA (e.g., a Cas RNP complex). In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to at least one transcription or translation control sequence, such as a promoter, a 3'UTR, or a 5'UTR. In one embodiment, the promoter may be a tRNA promoter, such as a tRNA. Lys3, or tRNA chimera. See Meffer d 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 U6 and H1 promoters. 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 with more than one guide RNA, 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 nucleotides encoding 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 bimolecular guide RNA. Alternatively, crRNA and trRNA can be transcribed into single molecule guide RNA (sgRNA). In other embodiments, crRNA and trRNA can be driven by their corresponding promoters on the same vector. In other embodiments, crRNA and trRNA can be encoded by different vectors.

[0332] In some embodiments, the nucleotide sequence encoding the guide RNA may be located on the same vector containing the nucleotide sequence encoding the DNA binder (e.g., Cas protein) guided by RNA. In some embodiments, the expression of the guide RNA and the DNA binder (e.g., Cas protein) guided by RNA may be driven by their own corresponding promoters. In some embodiments, the expression of the guide RNA may be driven by the same promoter that drives the expression of the DNA binder (e.g., Cas protein) guided by RNA. In some embodiments, the guide RNA and the DNA binder (e.g., Cas protein) transcripts guided by RNA may be contained in a single transcript. For example, the guide RNA may be in the untranslated region (UTR) of the DNA binder (e.g., Cas protein) transcript guided by RNA. In some embodiments, the guide RNA may be in the 5'UTR of the transcript. In other embodiments, the guide RNA may be in the 3'UTR of the transcript. In some embodiments, the intracellular half-life of the transcript may be shortened by containing the guide RNA in its 3'UTR, thereby shortening the length of its 3'UTR. In other embodiments, the guide RNA may be in the intron of the transcript. In some embodiments, a suitable splice site can be added at the intron, and the guide RNA is located within the intron so that the guide RNA is correctly spliced ​​out of the transcript. In some embodiments, the expression of the RNA-guided DNA binder (e.g., Cas protein) and the guide RNA from the same vector very close in time can promote more efficient formation of the CRISPR RNP complex.

[0333] In some embodiments, the nucleotide sequence encoding the DNA binder guided by guide RNA or RNA can be located on the same vector comprising a construct, 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., CAR, such as MAdCAM-1CAR). 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., CAR, such as MAdCAM-1CAR); and the guide RNA (or RNA-guided DNA binder) on the same vector can promote the construct to be more effectively inserted into the insertion site generated by the guide RNA / RNA-guided DNA binder.

[0334] In certain embodiments, DNA and RNA vectors may include one or more open reading frames expressed under a single promoter present in the vector or at the genomic insertion site. In such embodiments, the coding sequence of the self-cleaving peptide may be included between the open reading frames. The self-cleaving peptide may be, for example, a 2A peptide, such as a P2A peptide, an E2A peptide, a F2A peptide, or a T2A peptide.

[0335] In some embodiments, the vector comprises one or more nucleotide sequences encoding sgRNA and mRNA encoding an RNA-guided DNA binder, which may be a Cas protein, such as Cas9 or Cpf1. In some embodiments, the vector comprises one or more nucleotide sequences encoding crRNA, trRNA, and mRNA, which encode an RNA-guided DNA binder, which may be a Cas protein, such as Cas9 or Cpf1. In one embodiment, Cas9 is from Streptococcus pyogenes (i.e., Spy Cas9). In some embodiments, the Cas nuclease is a Cas9 nuclease from Neisseria meningitidis (i.e., NmeCas9, e.g., Nme1, Nme2, or Nme3 Cas9). In some embodiments, the nucleotide sequence encoding crRNA, trRNA, or crRNA and trRNA (which may be sgRNA) comprises or consists of a guide sequence flanked by all or part of a repeat sequence from a naturally occurring CRISPR / Cas system. The nucleic acid comprising or consisting of crRNA, trRNA, or crRNA and trRNA may further comprise a vector sequence, wherein the vector sequence comprises or consists of a nucleic acid that is not naturally found with the crRNA, trRNA, or crRNA and trRNA.

[0336] In some embodiments, crRNA and trRNA are encoded by discontinuous nucleic acids in a vector. In other embodiments, crRNA and trRNA can be encoded by continuous nucleic acids. In some embodiments, crRNA and trRNA are encoded by opposite strands of a single nucleic acid. In other embodiments, crRNA and trRNA are encoded by the same strand of a single nucleic acid.

[0337] In some embodiments, the vector comprises a donor construct comprising a sequence encoding a dmTGFB 1 molecule, a regulatory T cell promoting molecule (e.g., IL10) or a targeting receptor (e.g., CAR, e.g., MAdCAM-1), as disclosed herein. In some embodiments, in addition to the donor construct disclosed herein, the vector may also include a nucleic acid encoding a guide RNA described herein or a nucleic acid encoding a DNA binder guided by RNA (e.g., Cas nuclease, e.g., Cas9). In some embodiments, each or both of the nucleic acids encoding the DNA binder guided by RNA are located on a different vector from the vector comprising the donor construct disclosed herein. In any embodiment, the vector may include other sequences, including but not limited to promoters, enhancers, and regulatory sequences, as described herein. In some embodiments, the promoter does not drive the expression of regulatory T cells (e.g., IL10) or targeting receptors (e.g., CAR, e.g., MAdCAM-1) of the donor construct. In some embodiments, the vector comprises one or more nucleotide sequences encoding crRNA, trRNA, or crRNA and trRNA. In some embodiments, the vector comprises one or more nucleotide sequences encoding sgRNA and mRNA encoding RNA-guided DNA nucleases, which may be Cas nucleases (e.g., Cas9). In some embodiments, the vector comprises one or more nucleotide sequences encoding crRNA, trRNA, and mRNA, which mRNA encodes RNA-guided DNA nucleases, which may be Cas nucleases, such as Cas9. In some embodiments, 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 crRNA, trRNA, or crRNA and trRNA (which may be sgRNA) comprises a guide sequence flanking all or part of a repetitive sequence from a naturally occurring CRISPR / Cas system or is composed of the guide sequence. A nucleic acid comprising crRNA, trRNA, or crRNA and trRNA or consisting thereof may also comprise a vector sequence, wherein the vector sequence comprises a nucleic acid not naturally found with crRNA, trRNA, or crRNA and trRNA or is composed of the nucleic acid.

[0338] In some embodiments, the carrier can be circular. In other embodiments, the carrier can be linear. In some embodiments, the carrier can be encapsulated in lipid nanoparticles, liposomes, non-lipid nanoparticles or viral capsids. Non-limiting exemplary carriers include plasmids, phagemids, cosmids, artificial chromosomes, minichromosomes, transposons, viral vectors and expression vectors.

[0339] In some embodiments, the vector may be a viral vector. In some embodiments, the viral vector may be genetically modified from its wild-type counterpart. For example, the viral vector may include the insertion, deletion or substitution of one or more nucleotides to promote cloning or change one or more characteristics of the vector. These characteristics may include packaging capacity, transduction efficiency, immunogenicity, genome integration, replication, transcription and translation. In some embodiments, a part of the viral genome may be deleted so that the virus can package an exogenous sequence with a larger size. In some embodiments, the viral vector may have an enhanced transduction efficiency. In some embodiments, the immune response induced by the virus in a may be reduced. In some embodiments, the viral gene (such as, for example, integrase) that promotes the integration of viral sequences into the genome may be mutated so that the virus becomes non-integrated. In some embodiments, the viral vector may be replication-deficient. In some embodiments, the viral vector may include exogenous transcription or translation control sequences to drive the expression of the coding sequence on the vector. In some embodiments, the virus may be helper virus-dependent. For example, the virus may need one or more helper viruses to provide the viral components (such as, for example, viral proteins) required for amplification and packaging of the vector into the viral particles. In this case, one or more auxiliary components (including one or more vectors encoding viral components) can be introduced into a cell or cell population together with the vector system described herein. In other embodiments, the virus can be helper-free. For example, the virus may be able to amplify and package the vector without a helper virus. In some embodiments, the vector system described herein can also encode the viral components required for viral amplification and packaging.

[0340] Non-limiting exemplary viral vectors include adeno-associated virus (AAV) vectors, lentiviral vectors, adenoviral vectors, helper virus-dependent adenoviral vectors (HDAd), herpes simplex virus (HSV-1) vectors, bacteriophage T4, baculovirus 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.

[0341] In some embodiments, "AAV" refers to all serotypes, subtypes and naturally occurring AAVs and 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, AAV10, AAV11, AAV12, rh10 and hybrids thereof, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV and sheep AAV. In certain embodiments, AAV can be used for ex vivo applications of human cells. In certain embodiments, AAV is AAV6. The genomic sequences of various serotypes of AAV, as well as the sequences of natural terminal repeats (TR), Rep proteins and capsid subunits are known in the art. These sequences can be found in the literature or in public databases (such as GenBank). "AAV vector" as used herein refers to an AAV vector comprising a heterologous sequence of non-AAV origin (i.e., a nucleic acid sequence heterologous to AAV), typically comprising a sequence encoding a heterologous polypeptide of interest. The construct may comprise 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. Typically, the heterologous nucleic acid sequence (transgene) is flanked by at least one, typically two, AAV inverted terminal repeats (ITRs). AAV vectors may be single-stranded (ssAAV) or self-complementary (scAAV).

[0342] In some embodiments, the lentivirus may be integrative. In some embodiments, the lentivirus may be non-integrative. In some embodiments, the viral vector may be an adenovirus vector. In some embodiments, the adenovirus may be a highly cloning or "gutless" adenovirus, in which all coding viral regions except the 5' and 3' inverted terminal repeats (ITRs) and packaging signals ('I') are deleted from the virus to increase its packaging capacity. In other embodiments, the viral vector may be an HSV-1 vector. In some embodiments, the HSV-1-based vector is helper virus-dependent, while in other embodiments, it is helper virus-independent. For example, an amplicon vector that retains only the packaging sequence requires a helper virus with a packaging structural component, while a 30kb deleted HSV-1 vector that removes non-essential viral functions does not require a helper virus. In other embodiments, the viral vector may be bacteriophage T4. In some embodiments, when the head of the virus is emptied, bacteriophage T4 may be able to package any linear or circular DNA or RNA molecule. In other embodiments, the viral vector may be a baculovirus vector. In other embodiments, the viral vector can be a retroviral vector. In embodiments using AAV or other vectors with less cloning ability, it may be necessary to use more than one vector to deliver all components of the vector system disclosed herein. For example, an AAV vector can include a sequence encoding a DNA binder (e.g., Cas protein (e.g., Cas9)) guided by RNA, and a second AAV vector can include one or more guide sequences.

[0343] In some embodiments, the vector system can drive the expression of one or more nuclease components in the cell. In some embodiments, the vector does not include a promoter (e.g., using the endogenous promoter of the cell, such as when inserted into the specific genomic locus of the cell, as illustrated herein) that drives the expression of one or more encoding sequences once integrated into the cell. Suitable promoters for driving the expression in different types of cells (e.g., CD4+T cells) are known in the art. In some embodiments, the promoter can be wild-type. In other embodiments, the promoter can be modified to obtain more efficient or more effective expression. In other embodiments, the promoter can be truncated, but still retains its function. For example, the promoter can have a normal size or a reduced size suitable for correctly packaging the vector into the virus.

[0344] In some embodiments, the vector may include a nucleotide sequence encoding an RNA-guided DNA binder (e.g., a Cas protein described herein (e.g., Cas9)). In some embodiments, the nuclease encoded by the vector may be a Cas protein. In some embodiments, the vector system may include a copy of the nucleotide sequence encoding the nuclease. In other embodiments, the vector system may include more than one copy of the nucleotide sequence encoding the nuclease. In some embodiments, the nucleotide sequence encoding the nuclease may be operably linked to at least one transcription or translation control sequence. In some embodiments, the nucleotide sequence encoding the nuclease may be operably linked to at least one promoter.

[0345] In some embodiments, the vector may include any one or more constructs, the constructs including sequences encoding dmTGFB1 molecules, regulatory T cells promoting molecules (eg, IL10, CTLA4) or targeting receptors (eg, CAR, such as MAdCAM-1CAR), as described herein. In some embodiments, the sequence of dmTGFB1 molecules, regulatory T cells promoting molecules (eg, IL10, CTLA4) or targeting receptors (eg, CAR, such as MAdCAM-1CAR) may be operably connected to at least one transcription or translation control sequence. In some embodiments, the sequence of dmTGFB1 molecules, regulatory T cells promoting molecules (eg, IL10, CTLA4) or targeting receptors (eg, CAR, such as MAdCAM-1CAR) may be operably connected to at least one promoter. In some embodiments, the sequence of dmTGFB1 molecules, regulatory T cells promoting molecules (eg, IL10, CTLA4) or targeting receptors (eg, CAR, such as MAdCAM-1CAR) is not connected to a promoter driving the expression of a heterologous gene.

[0346] In some embodiments, the promoter can be constitutive, inducible or tissue-specific. In some embodiments, the promoter can be a constitutive promoter. Non-limiting exemplary constitutive promoters include cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (Rous sarcoma virus; RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor-α (EF1a) promoter, ubiquitin promoter, actin promoter, tubulin promoter, immunoglobulin promoter, its functional fragment or any combination of the foregoing. In some embodiments, the promoter can be a CMV promoter. In some embodiments, the promoter can be a truncated CMV promoter. In other embodiments, the promoter can be an EF1a promoter. In some embodiments, the promoter can be an inducible promoter. Non-limiting exemplary inducible promoters include promoters that can be induced by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohols. In some embodiments, an inducible promoter can be a promoter with a low basal (non-induced) expression level, such as, for example, Promoter (Clontech).

[0347] In certain embodiments, the promoters are useful for ex vivo applications in human cells.

[0348] In some embodiments, the promoter can be a tissue-specific promoter, such as a promoter specific for expression in T cells.

[0349] In some embodiments, the composition comprises a vector system. In some embodiments, the vector system can include a single vector. In other embodiments, the vector system can include two vectors. In other embodiments, the vector system can include three vectors. When different guide RNAs are used for multiplexing, or when multiple copies of guide RNA are used, the vector system can include more than three vectors.

[0350] In some embodiments, the vector system may include an inducible promoter to initiate expression only after it is delivered to the target cell. Non-limiting exemplary inducible promoters include promoters that can be induced by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, an inducible promoter can be a promoter with a low basal (non-induced) expression level, such as, for example Promoter (Clontech).

[0351] In additional embodiments, the vector system may comprise a tissue-specific promoter.

[0352] The following provides non-limiting exemplary viral vector sequences:

[0353] CTLA4 insert (nucleotide sequence)

[0354] ATGGCCTGCTTGGGCTTCCAAAGGCATAAAGCCCAGCTTAATCTTGCTACTCGCACGTGGCCCTGCACATTGCTCTTTTTCCTCCTGTTCATTCCCGTGTTTTGCAAGGCGATGCATGTGGCACAACCTGCCGTCGTTCTGGCATCATCAAGAGGTATTGCTAGCTTCGTTTGTGAGTACGCCTCCCCTGGAAAAGCGACGGAGGTGCGCGTCACTGTATTGCGGCAAGCCGACAGCCAAGTTACTGAAGTCTGCGCGGCAACGTATATGATGGGCAATGAGCTGACATTCCTTGACGATTCAATCTGCACGGGAACAAGTAGTGGTAACCAGGTGAATCTCACTATTCAAGGTCTGAGAGCCATGGACACCGGCCTCTACATTTGTAAGGTGGAGCTGATGTATCCTCCCCCATATTATCTGGGGATCGGAAATGGGACACAGATATATGTTATTGATCCCGAGCCATGTCCCGATAGTGACTTCCTCTTGTGGATACTTGCCGCTGTGAGCAGTGGTTTGTTTTTTTATTCATTCCTCCTTACGGCAGTATCACTTTCAAAAATGCTCAAGAAGCGAAGTCCTTTGACAACTGGCGTATATGTCAAAATGCCACCAACAGAGCCCGAATGTGAGAAACAGTTCCAGCCGTACTTTATTCCTATAAACTGA(SEQ ID NO:137)

[0355] CTLA4 insert (amino acid sequence)

[0356] MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN(SEQ ID NO:130)

[0357] IL10 insert (nucleotide sequence)

[0358] ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAACTGA(SEQ ID NO:138)

[0359] IL10 insert (amino acid sequence)

[0360] MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN(SEQ ID NO:124)

[0361] Empty lentiviral vector

[0362]

[0363] Empty lentiviral vector

[0364]

[0365] Additional sequences are provided throughout the specification and in the tables below; and the Sequence Listing filed herewith constitutes a part of the specification.

[0366] Sequence Listing

[0367] In the following tables, the terms "mA", "mC", "mU" or "mG" are used to indicate a nucleotide that has been modified with 2'-O-Me.

[0368] In the following table, each "N" is used to independently represent any nucleotide (e.g., A, U, T, C, G). In certain embodiments, the nucleotides are unmodified RNA nucleotide residues, i.e., ribose and a phosphodiester backbone.

[0369] In the following table, "*" is used to indicate a PS modification. In the present application, the terms A*, C*, U* or G* may be used to indicate a nucleotide that is linked to the next (eg, 3') nucleotide via a PS bond.

[0370] It will be understood that if a DNA sequence (comprising T) is referred to with respect to RNA, T should be replaced with U (modified or unmodified depending on the context), and vice versa.

[0371] In the table below, peptide sequences are provided using single amino acid letter codes.

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383] The carrier of the donor construct comprising a guide RNA, RNA binding DNA binding agent or a sequence comprising coding dmTGFB1 molecules, regulatory T cells promoting molecules (such as IL10, CTLA4) or targeting receptors (such as CAR) can be delivered by liposomes, nanoparticles, exosomes or microvesicles alone or in any combination. The carrier can also be delivered by lipid nanoparticles (LNP). One or more guide RNAs, RNA binding DNA binding agents (such as mRNA) or donor constructs comprising sequences encoding heterologous proteins can be delivered by LNP, liposomes, nanoparticles, exosomes or microvesicles alone or in any combination. One or more guide RNAs, RNA binding DNA binding agents (such as mRNA) or donor constructs comprising sequences encoding heterologous proteins can be delivered by LNP alone or in any combination. In some embodiments, one or more guide RNAs and RNA-guided DNA binding agents (such as, mRNA) are delivered by LNP. The donor construct can be delivered by viral vectors.

[0384] Lipid nanoparticles (LNPs) are a well-known means for delivering nucleotide and protein cargoes, and can be used to deliver any guide RNA, RNA-guided DNA binder, or donor construct disclosed herein.

[0385] As used herein, lipid nanoparticle (LNP) refers to a particle comprising multiple (i.e., more than one) lipid molecules, which are physically associated with each other by intermolecular forces. LNP can be, for example, a microsphere (including unilamellar and multilamellar vesicles, such as "liposomes"-lamellar phase lipid bilayers, which are substantially spherical in some embodiments, and in more specific embodiments, an aqueous core can be included, such as the main part of an RNA molecule), an internal phase in a dispersed phase, micelle or suspension in an emulsion (see, for example, WO2017173054, the contents of which are incorporated herein by reference in their entirety). Any LNP capable of delivering nucleotides to a subject known to those skilled in the art can be used. For example, exemplary LNP preparations and methods for delivering medicaments to T cells for modification are provided in WO2021222287.

[0386] In some embodiments, provided herein is a method for delivering any guiding RNA described herein or a donor construct disclosed herein to a cell or a cell colony or a subject alone or in combination, wherein any one or more components are associated with LNP. In some embodiments, the method further comprises a DNA binder (e.g., Cas9 or a sequence encoding Cas9) guided by RNA.

[0387] In some embodiments, a composition is provided herein, comprising any guide RNA described herein or a donor construct disclosed herein, alone or in combination with LNP. In some embodiments, the composition further comprises a DNA binder (e.g., Cas9 or a sequence encoding Cas9) guided by RNA.

[0388] In some embodiments, LNP comprises cationic lipid.In some embodiments, LNP comprises octadecane-9,12-dienoic acid (9Z, 12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester, also known as (9Z, 12Z)-octadecane-9,12-dienoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester or another ionizable lipid.See, e.g., lipids of WO2019067992, WO2017173054, WO2015095340 and WO2014136086, and references provided therein. In some embodiments, the LNP comprises 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 are interchangeable in the context of LNP lipids, e.g., where the ionizable lipid is cationic, depending on the pH.

[0389] In some embodiments, LNPs associated with constructs disclosed herein are used to prepare cell-based drugs for suppressing immune responses.Methods for preparing cell-based therapeutics and agents for cell-based therapeutics are known in the art.

[0390] In some embodiments, any guide RNA described herein, RNA-guided DNA binder, or donor construct disclosed herein (alone or in combination), whether naked or as part of a vector, is formulated in or administered via a lipid nanoparticle; see, e.g., WO2019067992, WO2017173054, or WO2021222287, the contents of which are hereby incorporated by reference in their entireties.

[0391] In some embodiments, LNP compositions, it comprises: RNA component and lipid component, wherein lipid component comprises amine lipid, for example biodegradable, ionizable lipid.In some cases, lipid component comprises 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 contains an ionizable lipid A (octadec-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester, also known as (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester), cholesterol, DSPC (distearoylphosphatidylcholine) and PEG2k-DMG (1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene glycol 2000 (PEG2k-DMG)). In certain embodiments, each component is present in a molar ratio of 50 lipid A:38 cholesterol:9DSPC:3PEG-DMG, respectively. In certain embodiments, the components are present in a molar ratio of 35 lipid A: 47.5 cholesterol: 15 DSPC: 2.5 PEG-DMG. The lipid nucleic acid assembly can be formulated to have a molar ratio of lipid amine to RNA phosphate (N: P) of about 6, and a weight ratio of gRNA to mRNA of 2:1, 1:1 or 1:2.

[0392] It is obvious that the DNA binding agent (for example, Cas nuclease or nucleic acid encoding Cas nuclease) guided by guide RNA, RNA and the donor construct comprising the sequence encoding dmTGFB1 molecule, regulatory T cell promoting molecule (such as IL10) or targeting receptor (such as CAR) can be delivered using the same or different systems. For example, guide RNA, Cas nuclease and construct can be carried by the same carrier (for example, AAV). Alternatively, Cas nuclease (as protein or mRNA) or gRNA can be carried by plasmid or LNP, and donor construct can be carried by carrier (such as AAV).

[0393] Different delivery systems can deliver simultaneously or in any order.In some embodiments, donor construct, guide RNA and Cas nuclease can be delivered simultaneously, for example, in a carrier, two carriers, a single carrier, an LNP, two LNPs, a single LNP or its combination.In some embodiments, donor construct can be used as a carrier or associated with LNP, before delivering guide RNA or Cas nuclease (for example, about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or more days) as a carrier or with LNP alone or together associated or as ribonucleoprotein (RNP) delivery. As another example, the guide RNA and Cas nuclease (as a vector or associated with LNPs alone or together or as a ribonucleoprotein (RNP)) can be delivered as a vector or associated with LNPs prior to delivery of the construct (e.g., about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or more days).

[0394] IV. Methods of Engineering T Cells

[0395] The present disclosure provides a method for engineering T cells to include a modification, the modification being performed by inserting a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence into a cell. The present disclosure provides a method for engineering T cells, which 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, and insertion of a heterologous sequence encoding a regulatory T cell promoting molecule into a cell under the control of a promoter sequence. The present disclosure provides a method for engineering T cells to include a modification, the modification being performed by inserting a heterologous sequence encoding dmTG FB1 under the control of a promoter sequence into a cell. The present disclosure provides a method for engineering T cells, which 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, and insertion of a heterologous sequence encoding IL10 under the control of a promoter sequence into a cell. The present disclosure provides a method for engineering T cells 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 IFNG or IL17A, and insertion of a heterologous sequence encoding CTLA4 under the control of a promoter sequence into a cell. The present disclosure provides a method for engineering T cells to include modification by insertion of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence into a cell. The present disclosure provides a method for engineering T cells, which further includes 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, and insertion of heterologous sequences encoding IL10 and CT LA4, each under the control of a promoter sequence, into a cell. In certain embodiments, the engineered T cells include modifications of endogenous nucleic acid sequences encoding IFNG and IL17A, respectively.

[0396] In some embodiments, the method includes engineering T cells to include modifications (by insertion of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence into the cell), modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A; insertion of a heterologous sequence encoding a regulatory T cell promoting molecule (e.g., IL10 or CTLA4) into the cell, and further comprising modifications (e.g., knockdown) of a TCR sequence. In certain embodiments, the engineered T cells include modifications of endogenous nucleic acid sequences encoding IFNG and IL17A, respectively.

[0397] In some embodiments, the method includes engineering T cells to include modification (by inserting a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence into a cell), modification of an endogenous nucleic acid sequence encoding TNFA (e.g., knocking down); modification of an endogenous nucleic acid sequence encoding IFNG or IL17A (e.g., knocking down); insertion of a heterologous sequence encoding a regulatory T cell promoting molecule (e.g., IL10 or CTLA4) into a cell, and also including insertion of a heterologous sequence encoding a targeting receptor (e.g., CAR) into a cell. In certain embodiments, engineered T cells include modifications of endogenous nucleic acid sequences encoding IFNG and IL17A, respectively.

[0398] In some embodiments, the method includes engineering T cells to include modifications (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 TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG or IL17A; insertion into the cell of a heterologous sequence encoding a regulatory T cell promoting molecule (e.g., IL10 or CTLA4), modification (e.g., knockdown) of a TCR sequence, and insertion into the cell of a heterologous sequence encoding a targeting receptor (e.g., CAR).

[0399] In some embodiments, the method includes engineering using a CRISPR / Cas system and a guide RNA disclosed herein, i.e., modification by insertion of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence into a cell, modification of an endogenous nucleic acid sequence encoding TNFA (e.g., knocking down); modification of an endogenous nucleic acid sequence encoding IFNG or IL17A (e.g., knocking down); insertion of a heterologous sequence encoding a regulatory T cell promoting molecule into a cell, optional knocking down of a TCR gene, and optional insertion of a targeting receptor (e.g., CAR) into a cell. In certain embodiments, engineered T cells include modifications of endogenous nucleic acid sequences encoding IFNG and IL17A, respectively.

[0400] The present disclosure provides a method for engineering T cells to include a modification, the modification is carried out by inserting a heterologous sequence encoding a regulatory T cell promoting molecule into a cell under the control of a promoter sequence, and also includes a modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding TNFA; and a modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding IL17A. The present disclosure provides a method for engineering T cells to include a modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding IL17A, and an insertion of a heterologous sequence encoding IL10 under the control of a promoter sequence into a cell. The present disclosure provides a method for engineering T cells to include a modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding IL17A, and an insertion of a heterologous sequence encoding CTLA4 under the control of a promoter sequence into a cell. The present disclosure provides a method for engineering T cells, which further comprises modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding TNFA, modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding IL17A, and insertion of heterologous sequences encoding IL10 and CTLA4, each under the control of a promoter sequence, into a cell. In certain embodiments, the method does not include modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding IFNG.

[0401] In some embodiments, the method comprises engineering T cells to include modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNF A, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, insertion of a heterologous sequence encoding a regulatory T cell promoting molecule (e.g., IL10 or CTLA 4) into the cell, and further comprising modification (e.g., knockdown) of a TCR sequence. In certain embodiments, the method does not include modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0402] In some embodiments, the method includes engineering T cells to include modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding TN FA, modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding IL17A, insertion of a heterologous sequence encoding a regulatory T cell promoting molecule (e.g., IL10 or CTLA 4) into the cell, and also includes insertion of a heterologous sequence encoding a targeting receptor (e.g., CAR) into the cell. In certain embodiments, the method does not include modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding IFNG.

[0403] In some embodiments, the method includes engineering T cells to include modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding TNF A, modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding IL17A, insertion of a heterologous sequence encoding a regulatory T cell promoting molecule (e.g., IL10 or CTLA 4) into the cell, modification (e.g., knocking down) of a TCR sequence, and insertion of a heterologous sequence encoding a targeting receptor (e.g., CAR) into the cell. In certain embodiments, the method does not include modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding IFNG.

[0404] In some embodiments, the method includes modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding TNFA using a CRISPR / Cas system and a guide RNA disclosed herein, modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding IL17A, insertion of a heterologous sequence encoding a regulatory T cell promoting molecule into a cell, optional knocking down of a TCR gene, and optional insertion of a targeting receptor (e.g., CAR) into a cell. In certain embodiments, the method does not include modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding IFNG.

[0405] In these embodiments, the regulatory T cell promoting molecules to be inserted can be provided by a donor construct. The regulatory T cell promoting molecules provided by the donor construct can be selected from IL10, CTLA4, IDO1, ENTPD1, ​​NT5E, IL22, AREG, IL35, GAR P, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, BACH2 and IL2RA, and modifications (e.g., knockdown) of TCR gene sequences.

[0406] In these embodiments, the targeting receptor to be inserted can be provided by a donor construct. In some embodiments, the targeting receptor can be a receptor of a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a cell surface molecule, the receptor being operably connected by at least one transmembrane domain in an internal signaling domain, and the internal signaling domain can activate T cells after binding to an extracellular receptor portion. In some embodiments, the targeting receptor can be a receptor present on the surface of a cell (eg, a T cell) to allow the cell to bind to a target site (eg, a specific cell or tissue in an organism). In some embodiments of these embodiments, the targeting receptor is a CAR capable of targeting MAdCAM-1.

[0407] Disclosed herein and known in the art for engineering T cells to include insertion and modification (e.g., knockdown) of suitable gene editing systems. In some embodiments, gene editing systems include but are not limited to CRISPR / Cas systems; zinc finger nuclease (ZFN) systems; transcription activator-like effector nuclease (TALEN) systems. Typically, gene editing systems involve inducing double-strand breaks (DSBs) or cuts (e.g., single-strand breaks or SSBs) in target DNA sequences using engineered cleavage systems. Cleavage or cuts can be directed by using specific nucleases (e.g., engineered ZFNs, TALENs) or using CRISPR / Cas systems and engineered guide RNAs to guide specific cleavage or cuts of target DNA sequences, such as CRISPR / Cas9 systems. In addition, targeted nucleases (e.g., from Streptococcus thermophilus (T.th ermophilus), referred to as "TtAgo", see Swarts et al. (2014) Nature 507 (7491): 258-261) are being developed based on the Argonaute system, which may also have the potential for genome editing and gene therapy.

[0408] Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cut specific DNA sequences. They are made by fusing the TAL effector DNA binding domain with a DNA cleavage domain (a nuclease that cuts the DNA chain). Transcription activator-like effectors (TALEs) can be engineered to bind to the desired DNA sequence, thereby promoting DNA cleavage at a specific position (see, for example, Boch, TALEs of genome targeting Nature Biotech. 29: 135–136 (2011)). Restriction enzymes can be introduced into cells for gene editing or in situ genome editing, a technique referred to as genome editing with engineered nucleases. Such methods and compositions used therein are known in the art. See, for example, WO2019147805, WO2014040370, WO2018073393, the contents of which are incorporated herein in their entirety.

[0409] Zinc finger nucleases (ZFNs) are artificial restriction enzymes produced by fusing zinc finger DNA binding domains with DNA cleavage domains. Zinc finger domains can be engineered to target specific desired DNA sequences, enabling zinc finger nucleases to target unique sequences in complex genomes. The non-specific cleavage domain of type II restriction endonuclease FokI is commonly used as the cleavage domain in ZFNs. Cleavage is repaired by endogenous DNA repair mechanisms, allowing ZFNs to precisely alter the genomes of higher organisms. Such methods and compositions used therein are known in the art. See, for example, WO2011091324, the contents of which are incorporated herein in their entirety.

[0410] 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 the core of RNA interference. RNA is the direct product of genes, and these small RNAs (usually 19-23 nucleotides per chain, forming a duplex of 19-21 nucleotides) can guide RNA-induced silencing (RISC) complexes to degrade messenger RNA (mRNA) molecules, and thus prevent translation by post-transcriptional gene silencing to reduce its activity. Short hairpin RNA (shRNA) is siRNA as single-stranded RNA, in which the chain forming the duplex region has a hairpin structure, which is usually produced by transcription from an expression vector. RNAi can also be completed by a longer RNA duplex structure called a Dicer substrate molecule, which is cleaved by the enzyme Dicer before being loaded into RISC to promote the cleavage of target mRNA. This method and composition for use are known in the art. In the compositions and methods provided herein, it is preferred to provide RNA molecules that promote RNA interference as persistent expression vectors, see, for example, WO2018208837, the contents of which are introduced in full herein. In some embodiments, RNAi is used with an expression vector.

[0411] It should be understood that the present disclosure contemplates methods of insertion with or without the guide RNA disclosed herein (e.g., using a ZFN system to cause a break in the target DNA sequence, thereby creating a site for inserting a construct). For methods using the guide RNA disclosed herein, the method includes using a CRISPR / Cas system to modify (e.g., knock down) a nucleic acid sequence encoding TNFA, IFNG, or TCR. It should also be understood that the present disclosure contemplates methods of modifying (e.g., knocking down) TNFA, IFNG, or TCR, which can be performed without the guide RNA disclosed herein (e.g., using a ZFN system to cause a break in the target DNA sequence, thereby creating a site for inserting a construct).

[0412] In some embodiments, a donor construct comprising a sequence for insertion (e.g., a sequence encoding dmTGF B1 or a regulatory T cell promoting molecule (e.g., IL10 or CTLA4)) is inserted into the genomic locus of a sequence targeted for modification (e.g., knockdown) (e.g., a TCR gene).

[0413] In some embodiments, the CRISPR / Cas system (e.g., guide RNA and RNA-guided DNA binders) can be used to generate an insertion site at a desired locus within the genome, where a donor construct comprising a sequence encoding dmTGFB1 disclosed herein can be inserted; regulatory T cell promoting molecules (e.g., IL10, CTLA4); or a targeting receptor (e.g., CAR, e.g., MAdCAM-1CAR) to express dmTGFB1, regulatory T cell promoting molecules (e.g., IL10, CTLA4) or CAR (e.g., MAdCAM-1CAR). Targeting receptors (e.g., CAR, e.g., MAdCAM-1CAR); inserting sequences, dmTGFB1 or regulatory T cell promoting molecules (e.g., IL10 or CTLA4) can be heterologous in terms of their insertion sites or loci, such as dmTGFB1; regulatory T cell promoting molecules (e.g., IL10, CTLA4); or targeting receptors (e.g., CAR, e.g., MAdCAM-1CAR) are not normally expressed in a safe harbor locus or TCR locus, as described herein. In some embodiments, the guide RNA described herein can be used with an RNA-guided DNA binder (e.g., Cas nuclease) according to the methods of the present invention to produce an insertion site at which a donor construct comprising a sequence encoding dmTGFB1; regulatory T cell promoting molecules (e.g., IL10, CTLA4); or a targeting receptor (e.g., CAR, e.g., MAdCAM-1CAR) can be inserted to express dmTGFB1; regulatory T cell promoting molecules (e.g., IL10, CTLA4) or CAR (e.g., MAdCAM-1CAR). This article illustrates and describes guide RNAs for inserting dmTGFB1; regulatory T cell promoting molecules (e.g., IL10, CTLA4); or targeting receptors (e.g., CAR, e.g., MAdCAM-1CAR) into specific genomic loci. In alternative embodiments, the targeting receptor is a TNFA targeting receptor.

[0414] 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), RNA-guided DNA binders (e.g., Cas nucleases), and donor constructs. In some embodiments, engineered T cells are: 1) transduced with gRNA targeting nucleic acid sequences encoding proinflammatory cytokines (e.g., IFNG or TNFA), RNA-guided DNA binders (e.g., Cas nucleases), and 2) transduced with donor constructs comprising nucleic acid sequences encoding dmTGFB1, regulatory T cell-promoting molecules (e.g., IL10 or CTLA4), and targeting receptors (e.g., CARs, e.g., MAdCAM-1CARs), or TNFA targeting receptors. In certain embodiments, engineered cells are selected for expression of targeted receptors.

[0415] In some embodiments, CD4+T cells are engineered by transduction with gRNA (e.g., gRNA targeting IFNG, TNFA, IL17A, or TCR for knockdown), RNA-guided DNA binders (e.g., Cas nucleases) and donor constructs. In some embodiments, engineered T cells: 1) transduced with a donor construct comprising a nucleic acid sequence encoding dmTGFB1, regulatory T cell-promoting molecules (e.g., IL10 or CTLA4) and a targeting receptor (e.g., CAR, e.g., MAd CAM-1CAR) or TNFA targeting receptor, and 2) transduced with a gRNA targeting a nucleic acid sequence encoding a proinflammatory cytokine (e.g., IFNG or TNFA), an RNA-guided DNA binder (e.g., Cas nuclease). In certain embodiments, engineered cells are selected for expression of a targeting receptor.

[0416] In some embodiments, CD4+T cells are engineered by transduction with gRNA (e.g., gRNA targeting IFNG, TNFA, or TCR for knockdown), RNA-guided DNA binders (e.g., Cas nucleases), and donor constructs. In some embodiments, engineered T cells are: 1) transduced with a donor construct comprising a nucleic acid sequence encoding dmTGFB1, a regulatory T cell promoting molecule (e.g., IL10 or CTLA4), and a targeting receptor (e.g., CAR, e.g., MAdCAM-1CAR) or a TNFA targeting receptor, and 2) transduced with a gRNA targeting a nucleic acid sequence encoding a proinflammatory cytokine (e.g., IFNG, IL17A, or TNFA) and an RNA-guided DNA binder (e.g., Cas nuclease).

[0417] As described herein, any suitable delivery system and method known in the art can be used to deliver a donor construct comprising coding dmTGFB1; regulatory T cells promote molecules (e.g., IL10, CTLA4); or targeting receptors (e.g., CAR) sequences, guide RNA (e.g., targeting IFNG, IL17A, TNFA gRNA, or TCR for knocking down) and RNA-guided DNA binders. In some embodiments, guide RNA and Cas nucleases are associated with LNPs and delivered to cells or cell colonies before delivering donor constructs comprising coding dmTGFB1; regulatory T cells promote molecules (e.g., IL10, CTLA4); or targeting receptors (e.g., CAR) sequences. In some embodiments, guide RNA and Cas nucleases are associated with LNPs and delivered to cells or cell colonies after delivering donor constructs comprising coding dmTGFB1; regulatory T cells promote molecules (e.g., IL10, CTLA4) or targeting receptors (e.g., CAR) sequences.

[0418] In some embodiments, administration of the gRNAs, donor constructs, and RNA-guided DNA binders described herein to naturally occurring T cells can convert naturally occurring T cells (e.g., CD4+ T cells) into cells that exhibit characteristics of regulatory T cells (e.g., immune response suppression characteristics).

[0419] gRNA, donor constructs, and RNA-guided DNA binders for modifying (e.g., knocking down) IFNG, TNFA, IL17A or TCR gene expression or inserting sequences encoding dmTGFB1; regulatory T cell promoting molecules (e.g., IL10, CTLA4) or targeting receptors (e.g., CAR, e.g., MAdCAM-1CAR) or TNFA targeting receptors can be introduced into conventional T cells or conventional T cell populations to produce engineered T cells or T cell populations described herein.

[0420] Methods of using various RNA-guided DNA binders (e.g., nucleases, such as Cas nucleases, such as Cas9) are also well known in the art. Although the use of the CRISPR / Cas system is illustrated herein, it will be understood that appropriate variations of the system may also be used. It will be understood that, depending on the context, the RNA-guided DNA binder may be provided as a nucleic acid (e.g., DNA or mRNA), such as the mRNA encoding the RNA-guided DNA binder provided above, or provided as a protein. In some embodiments, the methods of the present invention may be implemented in cells that already contain or express the RNA-guided DNA binder.

[0421] In some embodiments, the DNA binder (e.g., Cas9 nuclease) guided by RNA has lyase activity, which may also be referred to as double-stranded endonuclease activity. In some embodiments, the DNA binder (e.g., Cas9 nuclease) guided by RNA has nickase activity, which may also be referred to as single-stranded endonuclease activity. In some embodiments, the DNA binder guided by RNA includes Cas nuclease. Examples of Cas nucleases include type II CRISPR systems of Streptococcus pyogenes, Staphylococcus aureus (S.aureus), Neisseria meningitidis, and other prokaryotes (see, e.g., the list in the next paragraph), and variants or mutants thereof (e.g., engineered, non-naturally occurring, naturally occurring, or other variants) of those types. See, e.g., US20160312198; US20160312199.

[0422] Non-limiting exemplary species from which Cas nucleases can be derived include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Ga mmaproteobacterium, Neisseria meningitides, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dasyphylla, dassonvillei), Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporang ium roseum, Streptosporang ium roseum, Alicyclobacillusacido caldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactoba cillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium), Polaromonas naph thalenivorans, Polaromonas sp.), Croco osphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptorbecscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius th ermophiles, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans ferrooxidans), Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoaltero monas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp.), Microcoleuschthonoplastes, Oscillatoria sp.), Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculu m lavamentivorans, Corynebacterium diphtheria, Acidaminococcus sp., Lachnospiraceae bacterium ND2006 and Acaryochloris marina.

[0423] 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 an aminoacidococcus species. In some embodiments, the Cas nuclease is a Cpf1 nuclease from Lachnospiraceae ND2006. In other embodiments, the Cas nuclease is from Francisella tularensis, Lachnospiraceae, Butyrivibrioproteoclasticus, Peregrinibacteria bacterium, Parcubacteria bacterium, Smithella, Acidaminococcus, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi, Leptospira in adai, Porphyromonas crevioricanis, Prevotella disiens, or Porphyromonas cynomolgus. In certain embodiments, the Cas nuclease is a Cpf1 nuclease from Acidaminococcus or Lachnospiraceae.

[0424] In some embodiments, the gRNA and the DNA binder guided by RNA are referred to as ribonucleoprotein complexes (RNPs). In some embodiments, the DNA binder guided by RNA is a Cas nuclease. In some embodiments, the gRNA and the Cas nuclease are referred to as Cas RNPs together. In some embodiments, RNPs include 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 and Cas9 are referred to as Cas9 RNPs together.

[0425] 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 DNA strand. In some embodiments, the Cas9 protein comprises more than one RuvC domain or more than one HNH domain. 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.

[0426] In some embodiments, a chimeric Cas nuclease is used in which a domain or region of a protein is replaced by a portion of a different protein. In some embodiments, the Cas nuclease domain can be replaced by a domain from a different nuclease (such as Fok1). In some embodiments, the Cas nuclease can be a modified nuclease.

[0427] In other embodiments, the Cas nuclease may be from a type I CRISPR / Cas system. In some embodiments, the Cas nuclease may be a component of a Cascade complex of a type I CRISPR / Cas system. In some embodiments, the Cas nuclease may be a Cas3 protein. In some embodiments, the Cas nuclease may be from a type III CRISPR / Cas system. In some embodiments, the Cas nuclease may have RNA cleavage activity.

[0428] In some embodiments, the RNA-guided DNA binder has single-stranded nickase activity, i.e., it can cut one DNA strand to produce a single-strand break, also referred to as a "nick". In some embodiments, the RNA-guided DNA binder includes a Cas nickase. A nickase is an enzyme that produces a nick in dsDNA, i.e., it cuts one strand of the DNA double helix without cutting the other strand. In some embodiments, the Cas nickase is a form of a Cas nuclease (e.g., the Cas nuclease described above), wherein the endonuclease active site is inactivated, for example, by one or more changes (e.g., point mutations) in the catalytic domain. For a discussion of Cas nickases and exemplary catalytic domain changes, see, e.g., U.S. Pat. No. 8,889,356. In some embodiments, a Cas nickase (such as a Cas9 nickase) has an inactivated RuvC or HNH domain.

[0429] In some embodiments, the RNA-guided DNA binder is modified to contain only one functional nuclease domain. For example, the agent protein can be modified so 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 with reduced activity is used. In some embodiments, a nickase with an inactive RuvC domain is used. In some embodiments, a nickase with a HNH domain with reduced activity is used. In some embodiments, a nickase with an inactive HNH domain is used.

[0430] In some embodiments, the conservative amino acids in the Cas protein nuclease domain are replaced to reduce or change the nuclease activity. In some embodiments, the Cas nuclease may be included in the amino acid replacement in the RuvC or RuvC-like nuclease domain. Exemplary amino acid replacements in the RuvC or RuvC-like nuclease domain include D10A (based on Streptococcus pyogenes Cas9 protein). See, for example, Zetsche et al. (2015) Cell October 22: 163 (3): 759-771. In some embodiments, the Cas nuclease may be included in the amino acid replacement in the HNH or HNH-like nuclease domain. Exemplary amino acid replacements in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A and D986A (based on Streptococcus pyogenes Cas9 protein). See, for example, Zetsche et al. (2015). Other exemplary amino acid substitutions include D917A, E1006A, and D1255A (based on the F. novicida U112 Cpf1 (FnCpf1) sequence (UniProtKB-A0Q7Q2(CPF1_FRATN)). Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain or RuvC or RuvC-like domain of N. meningitidis include Nme2Cas9D16A (HNH nickase) and Nme2Cas9H588A (RuvC nickase).

[0431] In some embodiments, a nickase is provided that is combined with a pair of guide RNAs complementary to the sense strand and antisense strand of a target sequence, respectively. In this embodiment, the guide RNA directs the nickase to the target sequence, and introduces DSB by producing a nick (i.e., double nicking) on ​​the relative strand of the target sequence. In some embodiments, the nickase is used together with two separate guide RNAs of the relative strand of the target DNA to produce double nicking in the target DNA. In some embodiments, the nickase is used together with two separate guide RNAs selected to be very close to produce double nicking in the target DNA.

[0432] In some embodiments, the RNA-guided DNA-binding agent comprises one or more heterologous functional domains (e.g., is or comprises a fusion polypeptide).

[0433] In some embodiments, the heterologous functional domain can promote the RNA-guided DNA binder to be transported to the nucleus. For example, the heterologous functional domain can be a nuclear localization signal (NLS). In some embodiments, the RNA-guided DNA binder can be fused with 1-5 NLS. In some embodiments, the RNA-guided DNA binder can be fused with 2, 3 or 4 NLS. In some embodiments, the RNA-guided DNA binder can be fused with two NLS. In some embodiments, the RNA-guided DNA binder can be fused with one NLS. When using one NLS, the NLS can be connected to the N-terminal or C-terminal end of the DNA binder sequence guided by the RNA. In some embodiments, the NLS is not connected to the C-terminal. It can also be inserted into the DNA binder sequence guided by the RNA. In other embodiments, the RNA-guided DNA binder can be fused with more than one NLS. In some cases, at least two NLS are identical (for example, two SV40 NLS). In certain embodiments, there are at least two different NLSs in the RNA-guided DNA binder. In some embodiments, the RNA-guided DNA binder is fused to two SV40 NLS sequences connected at the carboxyl terminus. In some embodiments, the RNA-guided DNA binder can be fused to two NLSs, one connected at the N-terminus and the other connected at the C-terminus. In some embodiments, the RNA-guided DNA binder can be fused to 3 NLSs. In some embodiments, the RNA-guided DNA binder may not be fused to the NLS. In some embodiments, the NLS can be a single-part sequence, such as, for example, SV40 NLS, PKKKRKV (SEQ ID NO: 143) or PKKKRRV (SEQ ID NO: 144). In some embodiments, the NLS can be a two-part sequence, such as the NLS of nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO: 145). In a specific embodiment, a single PKKKRKV (SEQ ID NO: 143) NLS can be connected to the C-terminus of the RNA-guided DNA binder. The fusion site optionally includes one or more linkers.

[0434] V. Treatment Methods

[0435] The present disclosure provides a method for suppressing an immune response in a subject, comprising administering an engineered T cell comprising an insertion of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence into a cell; 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; and insertion of a heterologous sequence encoding a regulatory T cell promoting molecule under the control of a promoter sequence into a cell. The present disclosure provides a method for suppressing an immune response in a subject, comprising administering an engineered T cell comprising an insertion of a heterologous sequence encoding IL10 under the control of a promoter sequence into a cell, 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. The present disclosure provides a method for suppressing an immune response in a subject, comprising administering an engineered T cell comprising an insertion of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence into a cell; 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; and insertion of a heterologous sequence encoding CTLA4 under the control of a promoter sequence into a cell. The present disclosure provides a method for suppressing an immune response in a subject, comprising administering an engineered T cell comprising an insertion of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence into a cell; 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; and insertion of heterologous sequences encoding IL10 and CTLA4, each under the control of a promoter sequence, into a cell.

[0436] The present disclosure provides a method for treating an autoimmune disorder in a subject, comprising administering an engineered T cell comprising an insertion into a 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 a cell of a heterologous sequence encoding a regulatory T cell promoting molecule under the control of a promoter sequence. The present disclosure provides a method for treating an autoimmune disorder in a subject, comprising administering an engineered T cell comprising an insertion into a 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 a 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, comprising administering an engineered T cell comprising an insertion of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence into a cell; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; and insertion of a heterologous sequence encoding CTLA4 under the control of a promoter sequence into a cell. The present disclosure provides a method for treating an autoimmune disorder in a subject, comprising administering an engineered T cell comprising an insertion of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence into a cell; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; and insertion of heterologous sequences encoding IL10 and CTLA4, each under the control of a promoter sequence, into a cell.

[0437] The present disclosure provides a method for treating GvHD in a subject, comprising administering an engineered T cell comprising an insertion of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence into a cell; 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; and insertion of a heterologous sequence encoding a regulatory T cell promoting molecule under the control of a promoter sequence into a cell. The present disclosure provides a method for treating GvHD in a subject, comprising administering an engineered T cell comprising an insertion of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence into a cell; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding INFG or IL17A, and insertion of a heterologous sequence encoding IL10 under the control of a promoter sequence into a cell. The present disclosure provides a method for treating GvHD in a subject, comprising administering an engineered T cell comprising an insertion of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence into a cell; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IF NG or IL17A; and insertion of a heterologous sequence encoding CTLA4 under the control of a promoter sequence into a cell. The present disclosure provides a method for treating GvHD in a subject, comprising administering an engineered T cell comprising an insertion of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence into a cell; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding INFG or IL17A; and insertion of heterologous sequences encoding IL10 and CTLA4, each under the control of a promoter sequence, into a cell.

[0438] The present disclosure provides a method for suppressing an immune response in a subject, comprising administering an engineered T cell comprising a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and insertion of a heterologous sequence encoding a regulatory T cell promoting molecule into a cell under the control of a promoter sequence. The present disclosure provides a method for suppressing an immune response in a subject, comprising administering an engineered T cell comprising a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and insertion of a heterologous sequence encoding IL10 into a cell under the control of a promoter sequence. The present disclosure provides a method for suppressing an immune response in a subject, comprising administering an engineered T cell comprising a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and insertion of a heterologous sequence encoding CTLA4 into a cell under the control of a promoter sequence. The present disclosure provides a method for suppressing an immune response in a subject, comprising administering engineered T cells, wherein the engineered T cells comprise modifications (e.g., knocking down) of an endogenous nucleic acid sequence encoding TNFA, modifications (e.g., knocking down) of an endogenous nucleic acid sequence encoding IL17A; and insertion of heterologous sequences encoding IL10 and CTLA4, each under the control of a promoter sequence, into cells. In certain embodiments, the method does not include administering engineered T cells comprising modifications (including, knocking down) of an endogenous nucleic acid sequence encoding IFNG.

[0439] The present invention provides a method for treating GvHD in a subject, comprising administering an engineered T cell comprising a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and insertion of a heterologous sequence encoding a regulatory T cell promoting molecule into the cell under the control of a promoter sequence. The present invention provides a method for treating GvHD in a subject, comprising administering an engineered T cell comprising a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and insertion of a heterologous sequence encoding IL10 into the cell under the control of a promoter sequence. The present invention provides a method for treating GvHD in a subject, comprising administering an engineered T cell comprising a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A, and insertion of a heterologous sequence encoding CTLA4 into the cell under the control of a promoter sequence. The present disclosure provides a method for treating GvHD in a subject, comprising administering an engineered T cell comprising an insertion of a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA into the cell; a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IL17A; and an insertion of a heterologous sequence encoding IL10 and CTLA 4, each under the control of a promoter sequence, into the cell. In certain embodiments, the method does not include administering an engineered T cell comprising a modification (including, knockdown) of an endogenous nucleic acid sequence encoding IFNG.

[0440] In some embodiments, the method comprises 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 INFG or IL17A, an insertion of a sequence encoding a regulatory T cell promoting molecule, and further comprising a modification (e.g., knockdown) of a TCR sequence.

[0441] In some embodiments, the method includes administering engineered T cells comprising modifications (e.g., knockdown) of endogenous nucleic acid sequences encoding TNFA, modifications (e.g., knockdown) of endogenous nucleic acid sequences encoding IL17A, insertion of sequences encoding regulatory T cell promoting molecules, and further comprising modifications (e.g., knockdown) of TCR sequences. In certain embodiments, the method does not include administering engineered T cells comprising modifications (including, knockdown) of endogenous nucleic acid sequences encoding IFNG.

[0442] In some embodiments, the method comprises 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 IFNG or IL17A, an insertion of a sequence encoding a regulatory T cell promoting molecule, and further comprising an insertion of a sequence encoding a targeting receptor (e.g., CAR).

[0443] In some embodiments, the method includes administering engineered T cells, the engineered T cells comprising modifications (e.g., knocking down) of endogenous nucleic acid sequences encoding TNFA, modifications (e.g., knocking down) of endogenous nucleic acid sequences encoding IL17A, insertion of sequences encoding regulatory T cell-promoting molecules, and further comprising insertion of sequences encoding targeting receptors (e.g., CAR). In certain embodiments, the method does not include administering engineered T cells comprising modifications (including, knocking down) of endogenous nucleic acid sequences encoding IFNG.

[0444] In some embodiments, a targeting receptor (e.g., CAR) can target engineered T cells to the gastrointestinal system, for example, a targeting receptor is a CAR targeting MAdCAM-1, for example, for suppressing immune responses in conditions such as inflammatory bowel disease, ulcerative colitis, or Crohn's disease, including inflammation. In some embodiments, a targeting receptor (e.g., CAR) can target engineered T cells to tissues comprising endothelial cells, for example, a targeting receptor is a CAR targeting VCAM-1, for example, for suppressing immune responses in conditions such as Crohn's disease and multiple sclerosis, including inflammation. In some embodiments, a targeting receptor (e.g., CAR) can target engineered T cells to endothelial cells, for example, a targeting receptor is a CAR targeting CEACAM6, for example, for suppressing immune responses in conditions such as Crohn's disease. In some embodiments, a targeting receptor (e.g., CAR) can target engineered T cells to B cells, for example, a targeting receptor is a CAR targeting CD19, for example, for suppressing immune responses in conditions such as multiple sclerosis and systemic lupus erythematosus. In some embodiments, targeting receptors (e.g., CAR) can target engineered T cells to B cells, for example, targeting receptors are CARs targeting CD20, for example, for suppressing immune responses in diseases such as multiple sclerosis and systemic lupus erythematosus. In some embodiments, targeting receptors (e.g., CAR) can target engineered T cells to inflammatory tissues, for example, targeting receptors are CARs targeting TNFA, for example, for suppressing immune responses in diseases such as inflammatory bowel disease, ulcerative colitis or Crohn's disease. In some embodiments, targeting receptors (e.g., CAR) can target engineered T cells to neural tissues, for example, targeting receptors are CARs targeting MBP, MOG or PLP, for example, for suppressing immune responses in diseases such as multiple sclerosis. In some embodiments, targeting receptors (e.g., CAR) can target engineered T cells to tissues comprising mature B lymphocytes, for example, targeting receptors are CARs targeting TNFRSF17, for example, for suppressing immune responses in diseases such as systemic lupus erythematosus. In some embodiments, the targeting receptor (e.g., CAR) is capable of targeting the engineered T cells to synovial tissue, for example, the targeting receptor is a CAR that targets citrullinated vimentin, for example, for suppressing immune responses in disorders such as rheumatoid arthritis.

[0445] In some embodiments, the targeted receptor is a CAR targeting DPP6, SCL2A2, glutamate decarboxylase (GAD2), desmoglein 3 (DSG3), and MHC class I HLA-A (HLA-A*02).

[0446] In some embodiments, the method comprises 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 IFNG or IL17A, an insertion of a sequence encoding a regulatory T cell promoting molecule, an insertion of a sequence encoding a targeting receptor (e.g., CAR), and further comprising a modification (e.g., knockdown) of a TCR sequence.

[0447] In some embodiments, the method includes administering engineered T cells, the engineered T cells comprising modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding TNFA, modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding IL17A, insertion of a sequence encoding a regulatory T cell promoting molecule, insertion of a sequence encoding a targeting receptor (e.g., CAR), and further comprising modification (e.g., knocking down) of a TCR sequence. In certain embodiments, the method does not include administering engineered T cells comprising modification (including, knocking down) of an endogenous nucleic acid sequence encoding IFNG.

[0448] In some embodiments, the sequence to be inserted is inserted into the sequence to be modified (e.g., knocked down), for example, the CAR sequence is inserted into the TNFA genomic sequence, thereby modifying (e.g., knocking down) the TNFA sequence.

[0449] In some embodiments, the method includes administering a T cell colony 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 T cell colony are engineered, for example, as assessed by sequencing (e.g., NGS).

[0450] 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 have autologous or allogeneic use.

[0451] In some embodiments, the effectiveness of treatment using the above-described engineered T cells can be assessed in an animal model of graft-versus-host disease (e.g., a mouse model) by measuring the body weight or survival rate of the animal after administration of the engineered T cells (wherein the animal is sacrificed after a substantial portion of the body weight (e.g., 20% of the initial body weight) is lost). In some embodiments, effective treatment results in a statistically significant increase in survival rate compared to an appropriate control (e.g., an animal treated with PBMCs).

[0452] Example

[0453] 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.

[0454] Example 1. General method

[0455] 1.1. Preparation of lipid nanoparticles

[0456] Typically, lipid components were dissolved in 100% ethanol at various molar ratios. RNA cargo (eg, Cas9 mRNA and sgRNA) was dissolved in 25 mM citrate buffer, 100 mM NaCl (pH 5.0), resulting in a concentration of RNA cargo of approximately 0.45 mg / mL.

[0457] Unless otherwise stated, lipid nucleic acid assemblies contained ionizable lipid A octadec-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester, also known as ((9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester), cholesterol, DSPC, and PEG2k-DMG, respectively, at a molar ratio of 35 lipid A:47.5 cholesterol:15 DSPC:2.5 PEG-DMG. Unless otherwise stated, lipid nucleic acid assemblies were formulated at a lipid amine to RNA phosphate (N:P) molar ratio of about 6 and a gRNA to mRNA weight ratio of 1:1.

[0458] LNPs were prepared using a cross-flow technique that utilizes an impingement jet mixing of lipid-containing ethanol with two volumes of RNA solution and one volume of water. The lipid-containing ethanol was mixed with the two volumes of RNA solution through a cross mixer. A fourth stream of water was mixed with the outlet water stream of the cross tube through an inline tee (see WO2016010840). Figure 2 ). The LNPs were kept at room temperature for 1 hour and further diluted with water (about 1: 1 v / v). The LNPs were concentrated using tangential flow filtration on a flat filter cartridge (Sartorius, 100 kDM WCO), and the buffer was 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 the buffer was exchanged into TSS using a PD-10 desalting column (GE). The resulting mixture was then filtered using a 0.2 μm sterile filter. The final LNPs were stored at 4 ° C or -80 ° C until further use.

[0459] 1.2. In vitro transcription of mRNA (“IVT”)

[0460] Using the linearized plasmid DNA template and T7 RNA polymerase, capped and polyadenylated mRNA containing N1-methyl pseudo-U was generated by in vitro transcription. Plasmid DNA containing the T7 promoter, transcription sequence and polyadenylation region was linearized by incubation with XbaI for 2 hours at 37°C using the following conditions: 200 ng / μL plasmid, 2U / μL XbaI (NEB) and 1x reaction buffer. XbaI was inactivated by heating the reaction at 65°C for 20 minutes. The linearized plasmid was purified from the enzyme and buffer salts. IVT reactions to produce modified mRNA were performed by incubation at 37°C for 1.5-4 hours under the following conditions: 50 ng / μL linearized plasmid; 2-5 mM each of GTP, ATP, CTP, and N1-methyl pseudoUTP (Trilink); 10-25 mM ARCA (Trilink); 5 U / μL T7 RNA polymerase (NEB); 1 U / μL murine RNase inhibitor (NEB); 0.004 U / μL inorganic E. coli pyrophosphatase (NEB); and 1× reaction buffer. TURBO DNase (ThermoFisher) was added to a final concentration of 0.01 U / μL, and the reactions were incubated for an additional 30 minutes to remove the DNA template. mRNA was purified using the MegaClear Transcription Cleanup Kit (ThermoFisher) or the RNeasy Maxi Kit (Qiagen) according to the manufacturer's protocol. Alternatively, mRNA was purified by a precipitation protocol, followed in some cases by HPLC-based purification. In brief, after DNA enzyme digestion, LiCl precipitation, ammonium acetate precipitation and sodium acetate precipitation are used to purify mRNA. For the mRNA of HPLC purification, after LiCl precipitation and reconstruction, by RP-IP HPLC purification mRNA (see, for example, Kariko et al. Nucleic Acids Research, 2011, volume 39, 21e142 phase). The fraction combination selected for merging, and desalination by sodium acetate / ethanol precipitation as described above. In other alternative methods, mRNA is purified by LiCl precipitation method, then further purified by tangential flow filtration. RNA concentration is determined by measuring the absorbance (Nanodrop) at 260nm, and transcripts are analyzed by capillary electrophoresis by Bioanlayzer (Agilent).

[0461] Streptococcus pyogenes ("Spy") Cas9 mRNA encoding an open reading frame (SEQ ID NO: 17) was generated from plasmid DNA according to the nucleic acid sequences described herein. For the following mRNA nucleic acid sequences, it should be understood that T should be replaced by U (N1-methyl pseudouridine as described above). The messenger RNA used in the examples includes a 5' cap and a 3' poly-A tail, for example, up to 100 nt. The guide RNA was chemically synthesized by methods known in the art.

[0462] 1.3. Next Generation Sequencing (“NGS”) and Analysis of On-Target Editing Efficiency

[0463] According to the manufacturer's protocol, use QuickExtract TM Genomic DNA was extracted using DNA extraction solution (Lucigen, catalog number QE09050).

[0464] In order to quantitatively determine the editing efficiency of the target position in the genome, deep sequencing is used to identify the presence of insertions and deletions introduced by gene editing. PCR primers are designed around the target site in the gene of interest (e.g., TRAC), and the genomic region of interest is amplified. Primer sequence design is performed according to the standards of the art.

[0465] Additional PCR is performed according to the manufacturer's protocol (Illumina) to add chemicals for sequencing. The amplicons are sequenced on Illumina MiSeq or NextSeq instruments. After eliminating those reads with low quality scores, the reads are aligned with a human reference genome (e.g., hg38). The resulting file containing reads is plotted onto a reference genome (BAM file), where reads overlapping the target region of interest are selected, and the number of wild-type reads and the number of reads containing indels are calculated. The editing percentage (e.g., "editing efficiency" or "editing percentage" or "indel percentage") is defined as the ratio of the total number of sequence reads with indels to the total number of sequence reads, including wild type.

[0466] Example 2. Suppressive activity of engineered T cells expressing TGFB1 alleles

[0467] The suppressive phenotype of T cells was assessed by lentiviral overexpression of a constitutively active TGFB1 mutant (TGFB1 separated from LAP). TGFB1 is a well-known suppressive cytokine that is produced in a precursor form consisting of TGFB1 and latency-associated peptide (LAP). Activation of TGFB1 requires cleavage of the LAP accessory protein.

[0468] Example 2.1. T cell preparation

[0469] Human CD3+CD4+T cells were prepared from fresh leukocytes (AllCells, donor #32015). CD3+CD4+T cells were isolated by negative selection using a human CD4+T cell isolation kit (Milten yi; catalog number 130-096-533) according to the manufacturer's protocol. CD3+CD4+T cells were isolated at 1×10^ 6 Cells were plated at a density of 10 cells / ml in a total of 5 ml of OpTmizer basal medium, which contained CTS OpTmizer T cell expansion SFM (Gibco, product catalog number A3705001), 1% penicillin-streptomycin, 1×Glutamax, 10 mM HEPES, 2.5% human AB serum (Gemini, product catalog number 100-512), and supplemented with 200 U / ml recombinant human interleukin-2 (Miltenyi Biotec; product catalog number 130-097-746), 5 ng / ml recombinant human interleukin 7 (StemCell technologies, product catalog number 78053.1) and 5 ng / ml recombinant human interleukin 15 (StemCell technologies, product catalog number 78031.1). Cells were activated by adding 25 μL / mL ImmunoCult Human CD3 / CD28 T Cell Activator (Stemcell Technologies, Cat. No. 10991) and incubated at 37°C for 48 hours prior to lentiviral transduction.

[0470] Example 2.2. T cell transduction and cell sorting

[0471] To overexpress wild-type or mutant TGFB1, activated CD3+CD4+ T cells were transduced with lentiviral constructs. Forty-eight hours after activation, CD3+CD4+ T cells were harvested, washed, and cultured at 1×10^ 6The cells were resuspended at a density of 10 cells / 100 μL OpTmizer basal medium, which contains CTS OpTmizer T cell expansion SFM (Gibco, catalog number A3705001), 1% penicillin-streptomycin, 1×Glutamax, 10 mM HEPES, 2.5% human AB serum (Gemini, catalog number 100-512). 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, CD3+CD4+T cells were resuspended in the cell / virus supernatant mixture and transferred to a single well of a 6-well G-rex (Wilson Wolf; catalog number 80240M) containing 20 mL of OpTmizer basal medium, which contained CTS OpTmizer T cell expansion SFM (Gibco, catalog number A3705001), 1% penicillin-streptomycin, 1×Glutamax, 10 mM HEPES, 2.5% human AB serum (Gemin i, catalog number 100-512), supplemented with 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 quantified using a BD FACSAria TM Fusion cell sorter (BD Biosciences) was used to isolate cells expressing the target of interest. After sorting, CD3+CD4+T cells were cultured in 6-well Grex plates with 30 mL OpTmizer basal medium, which contained CTS OpTmizer T cell expansion SFM (Gibco, catalog number A3705001), 1% penicillin-streptomycin, 1×Glutamax, 10 mM HEPES, 2.5% human AB serum (Gemini, catalog number 100-512), and supplemented with 500 U / mL IL-2, 5 ng / mL IL7, and 5 ng / mL IL17.

[0472] Natural regulatory T cells (nTreg) were prepared using methods known in the art. In brief, CD25 microbeads (Miltenyi, catalog number 130-092-983) were used to separate CD25+ cells from the PBMC autologous CD4+T cells separated from Example 2.1 according to the manufacturer's instructions. The separated CD25+CD4+T cells were plated in 6-well Grex with 5 million per well, and the wells contained 30ml OpTmizer basal medium, which contained CTS OpTmizer T cell expansion SFM (Gibco, catalog number A3705001), 1% penicillin-streptomycin, 1×Glutamax, 10mM HEPES, 2.5% human AB serum (Gemini, catalog number 100-512), and supplemented with 1000U / mL recombinant human interleukin-2 (Miltenyi Biotec; catalog number 130-097-746). Cells were activated by adding 25 μL / mL ImmunoCult human CD3 / CD28 T cell activator (Stemcell Technologies, catalog number 10991) and culturing 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; catalog number 130-095-213) for 15 minutes at 4°C. The labeled cells were washed twice and further labeled with anti-biotin microbeads (Miltenyi; catalog number 130-090-485) according to the manufacturer's instructions. Cells were separated using LS columns (Miltenyi; catalog number 130-042-401). The isolated natural Tregs were cultured in 6-well Grex plates, each well containing 30 ml of OpTmizer basal medium, which contained CTS OpTmizer T cell expansion SFM (Gibco, product catalog number A3705001), 1% penicillin-streptomycin, 1×Glutamax, 10 mM HEPES, 2.5% human AB serum (Gemini, product catalog number 100-512), and supplemented with 1000 U / mL IL-2.

[0473] Example 2.3 Verification of TGFB1 protein expression

[0474] TGFB1 protein expression of various lentiviral constructs was verified by ELISA. One hundred thousand sorted, transduced T cells were placed in a U-bottom plate containing OpTmi zer basal medium, which contained CTS OpTmizer T cell expansion SFM (Gibco, catalog number A3705001), 1% penicillin-streptomycin, 1×Glutamax, 10mM HEPES, 2.5% human AB serum (Gemini, catalog number 100-512). Some wells also received 25 μL / mL ImmunoCult human CD3 / CD28 / 2T cell activator (Stemc ell Technologies, catalog number 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 frozen for subsequent cytokine quantification using the LE GEND MAX Total TGF-β1 ELISA Kit (BioLegend, catalog number 436707) or the LEGEND MAX Free Active TGF-β1 ELISA Kit according to the manufacturer's instructions. Total TGFB1 in the culture supernatant is shown in Tables 1 and Figure 1 The active TGFB1 protein in the culture supernatant is shown in Table 2 and Figure 2 CD3+CD4+ T cells transduced with a lentiviral expression vector encoding the sequence of TGFB1 R218H C225R secreted high levels of active TGF-β1.

[0475] Table 1 - Total TGF-β1 quantified in culture supernatants (in pg / ml)

[0476]

[0477] Table 2 - Active TGF-β1 quantified in culture supernatant (in pg / ml)

[0478]

[0479] Example 2.4 Suppressive ability of engineered T cells expressing mutant TGF-β1

[0480] The mixed lymphocyte reaction (MLR) assay is used to measure the suppressive function of sorted, transduced CD3+CD4+ T cells. The MLR is an inflammatory response caused by T cells recognizing another type of leukocyte (allogeneic leukocyte) as foreign. Tregs suppress the MLR inflammatory response by inhibiting the proliferation and production of inflammatory cytokines caused by inflammatory T cells. Cell proliferation is measured by labeling the initial population with an intracellular fluorescent dye that is diluted as it is passed to each generation as part of the cell contents.

[0481] MLR assays were performed in 96-well U-bottom plates using OpTmizer basal medium containing CTS OpTmizer T cell expansion SFM (Gibco, catalog number A3705001), 1% penicillin-streptomycin, 1×Glutamax, 10 mM HEPES, 2.5% human AB serum (Gemini, catalog number 100-512). Non-transduced CD3+CD4+ T cells from the same donor as the transduced T cells (autologous T cells) were used as responder cells. These non-transduced cells were labeled with CellTrace Violet (CTV) (Thermofisher Scientific; catalog number C34557) according to the manufacturer's instructions. CD3-depleted PBMCs from allogeneic donors can be used to stimulate reactive non-transduced T cells relative to transduced T cells. Cultures were prepared by combining 50,000 CTV-labeled autologous T cells, 50,000 CD3-deleted allogeneic PBMCs, and approximately 50,000 (1 to 1), 16,666 (3 to 1), or 5,555 (9 to 1) sorted transduced CD3+CD4+T cells per well. After culturing for 5 days at 37°C, the culture plates were centrifuged and the culture supernatant was harvested for cytokine quantification. The cell pellet was resuspended in FACS buffer containing APC / Fire 750 anti-CD4 and placed at 4°C for 30 minutes. The 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 CTV signal, and finally undiluted CTV population. The inhibition of CTV dilution was calculated using the following formula:

[0482] (log2(y) of CTV T cells - log2(y) of CTV T cells with Tregs)) / log2(y) of CTV T cells*100

[0483] Where y = average fluorescence intensity of the entire CTV-marked population / average fluorescence intensity of the undiluted portion of the CTV-marked population. Data are shown in Figure 3and Table 3.

[0484] Table 3 - Percent inhibition of autologous T cell proliferation by T cells overexpressing wild-type or mutant TGF-β1 as measured by CTV dilution

[0485]

[0486]

[0487] Example 3. Suppressive activity of engineered T cells expressing active TGFB1 mutants with cytokine gene disruption

[0488] Example 3.1. T cell engineering

[0489] The in vivo suppressive function of T cells expressing the TGFβ1 R218H C225R mutant was evaluated using a graft-versus-host disease (GvHD) mouse model. Transduced T cells expressing mutant TGFB1 were evaluated by further disruption of one or more of the following cytokine genes: tumor necrosis factor-α (TNFa), interferon gamma (IFNg), or interleukin 17a (IL17a).

[0490] T cells were isolated and transduced with lentivirus as described in Example 2, and further engineered T cells were delivered with lipid nanoparticles (LNPs) of Cas9 mRNA and guide RNA. LNPs were produced using a molar ratio of 35 lipid A / 47.5 cholesterol / 15 DSPC / 2.5 PEG2k-DMG as described in Example 1. LNPs were prepared with a lipid amine to RNA phosphate (N:P) molar ratio of about 6. LNPs were prepared with a gRNA to Cas9 mRNA carrier ratio of 1:1 by weight. In order to break the target gene, LNPs were prepared with G019753 (IFNg), G019757 (TNFa) or G027259 (IL17a).

[0491] Each LNP formulation was incubated at 37°C for 15 minutes in OpTmizer basal medium containing CTS OpTmizer T cell expansion SFM (Gibco, catalog number A3705001), 1% penicillin-streptomycin, 1X glutamine, 10 mM HEPES, 5% human AB serum (Gemini, catalog number 100-512), 200 U / mL recombinant human interleukin-2, 5 ng / ml recombinant human interleukin 7 and 5 ng / ml recombinant human interleukin-15 supplemented with 5 ug / ml recombinant human ApoE3 (Peprotech, catalog number 350-02). On day 3 after activation and transduction, 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 / mL medium and plated in T-25 flasks at 5 million cells per group.

[0492] The LNP mixture of pre-incubation is added in each T-25 flask, to produce a final concentration of 2.5 μ g / mL total RNA. Multiple knockout groups use 2.5 μ g / mL of LNP respectively. Twenty-four hours after LNP treatment, collect, wash T cells, and cultivate in the Optimizer culture medium with 5% human serum and cytokine as described in Example 2, to expand until injection day (the 15th day after activation). Sorting cells about five to six days after activation as described in Example 2.

[0493] Example 3.2 In vivo evaluation of immunosuppression in the GvHD model

[0494] Sorted CD3+CD4+T cells were harvested 15 days after activation for in vivo injection. Autologous PBMCs engineered with T cells were thawed as described in the above examples. PBMCs were added to each assay population at a 1:1 ratio and the cells were resuspended in RPMI (supplemented with 2% FBS and 10 mM HEPES) until 8×10^ 6 / 100μL. Only PBMC group with 4×10^ 6 Resuspend in 100 μL.

[0495] Female NOG mice (NOD.Cg-Prkdc scid Iq tm1Sug / JicTac; Taconic, catalog number NOG-F) was treated with sublethal radiation (200 rads) using X-rays (RS-2000 irradiator; RadSource Technologies) one day before injection for cell transplantation. 100 μL of each test cell population was injected intravenously into the irradiated NOG mouse cohort as described in the above paragraph. Eight irradiated mice were injected with 100 μL RPMI (supplemented with 2% FBS and 10 mM HEPES) as a control that received only radiation. Body weight was monitored daily. Mice were sacrificed when they lost 20% of their body weight. The results are shown in Figure 4 and Table 4.

[0496] Table 4 - Percentage of survival days after injection of engineered CD3+CD4+ cells

[0497]

[0498] Example 3.3. Mixed lymphocyte reaction assay for suppressive function

[0499] The engineered T cells from the population for injection were evaluated for suppressive function in vitro using a mixed lymphocyte reaction (MLR) as described in Example 2, where the ratio of CTV-labeled T cells to engineered T cells was as follows: Figure 5 and as described in Table 5.

[0500] Table 5 - Percent inhibition of autologous T cell proliferation by engineered T cells measured by CTV dilution

[0501]

[0502] Example 3.4. TGFB1 expression in transduced CD3+CD4+ T cells

[0503] The total TGFb1 and active TGFB1 levels of engineered T cells from the colony for injection were assessed by ELISA. The transduced CD3+CD4+T cells of the sorting were stimulated to assess their cytokine profile. The transduced CD3+CD4+T cells after sorting were seeded in U-bottom culture plates at a density of 1x10^5T cells / well, and the culture fluid totaled 200 μL OpT mizer basal medium including CTS OpTmizer T cell expansion SFM (Gibco, catalog number A3705001), 1% penicillin-streptomycin, 1XGlutamax, 10mM HEPES, 2.5% human AB serum (Gemini, catalog number 100-512) and 25uL / mL Immu noCult human CD3 / CD28 T cell activator (Stemcell Technologies, catalog number 10991), and cultured for 48 hours at 37°C. After 48 hours of culture, the culture plates were centrifuged and the supernatants were collected and frozen for subsequent cytokine quantification using the LEGEND MAX Total TGF-β1 ELISA Kit (BioLegend, catalog number 436707) and the LEGEND MAX Free Active TGF-β1 ELISA Kit according to the manufacturer's instructions. Total TGFB1 in the culture supernatant is shown in Tables 6 and Figure 6 The active TGFB1 in the culture supernatant is shown in Table 7 and Figure 7 shown.

[0504] Table 6 - Total TGF-β1 production by transduced cells after cell stimulation (pg / ml)

[0505]

[0506] Table 7 - Active TGF-β1 production by transduced cells after cell stimulation (pg / ml)

[0507]

[0508]

[0509] Example 3.5 Cytokine profile analysis of transduced CD3+CD4+ T cells

[0510] The INF g, TNFa, IL17a, IL10, IL2 and IL13 levels of the engineered T cells from the colony for injection were assessed by ELISA. The CD3+CD4+T cells of the transduction of the sorting were stimulated to assess its cytokine profile. The transduction CD3+CD4+T cells after sorting were inoculated in a U-bottom culture plate at a density of 1x10^5T cells / well, and the culture fluid totaled 200 μL OpTmizer basal medium including CTS OpTmizer T cell expansion SF M (Gibco, catalog number A3705001), 1% penicillin-streptomycin, 1X Glutamax, 10mM HEPES, 2.5% human AB serum (Gemini, catalog number 100-512) and 25uL / mL ImmunoCult human CD3 / CD28 T cell activator (Stemcell Technologies, catalog number 10991), and cultured for 48 hours at 37°C. After 48 hours of culture, the plates were centrifuged and the supernatants were collected and frozen for subsequent cytokine quantification 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: IFNG, 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 was analyzed using the Discovery Workbench 4.0 software package (Meso Scale Discovery). The results are shown in Table 8 and Figure 8-13 middle.

[0511] Table 8 - In vitro cytokine production by transduced cells following cell stimulation (pg / ml)

[0512]

[0513] Example 4: In vivo evaluation of the effects of mutant IL-10, CD25 expression and IL-2 knockout on suppressor T cells

[0514] Example 4.1. T cell engineering

[0515] To generate engineered suppressor T cells (supT cells), CD4+ T cells were treated according to Table 9.

[0516] The isolated CD4+T cells were activated in 5 mL OpTmizer (containing 200 U / mL IL-2, 5 ng / mL IL-7, 5 ng / mL IL-15 and 25 uL / mL Immunocult (Stemcell Technologies, 10991)) in a 6-well plate at 5 x 10^6 cells / well. Two days after activation (day 2), the cells were transduced with lentivirus encoding CTLA4 (SEQ ID NO: 130), IL-10 (SEQ ID NO: 124) and mScarlett and lentivirus encoding CTLA4 (SEQ ID NO: 130), IL-10DE (SEQ ID NO: 229) and mScarlett according to Table 9. The concentrated viral supernatant was added to the T cells and centrifuged at 1000 × g for 60 minutes at 37°C. T cells were resuspended in the cell / virus supernatant mixture and transferred to a single well of a 6-well G-rex (Wilson Wolf; Catalog No. 80240M) containing T cell basal medium supplemented with 500U / mL IL-2, 5ng / mL IL-7, and 5ng / mL IL-15. The cells were split and supplemented with medium or cytokines regularly. On day 3, T cells transduced with CTLA4, IL-10, and mScarlet were further transduced with a lentivirus encoding CD25 (NCBI Reference Sequence Number: NP_000408.1) in the same manner as the transduction on day 2.

[0517] Table 9. T cell editing treatment groups. "LV" stands for lentivirus.

[0518]

[0519] At the 4th day, T cells were edited according to Table 9, using LNP to deliver Cas9 mRNA and the guide RNA targeting INF-gamma (G019753), using LNP to deliver Cas9mRNA and the guide RNA targeting TNF-alpha (G019757), and in some groups, LNP was also used to deliver Cas9 mRNA and the guide RNA targeting IL-2 (G021925). LNPs were produced using a molar ratio of 35 lipid A / 47.5 cholesterol / 15DSPC / 2.5PEG2k-DM G as described in Example 1. LNPs were prepared with a lipid amine to RNA phosphate (N:P) molar ratio of about 6. LNPs were prepared with a gRNA to Cas9 mRNA carrier ratio of 1:1 by weight.

[0520] Each LNP formulation was incubated in T cell basal medium supplemented with 5ug / ml recombinant human ApoE3 (Peprotech, Cat.350-02) at 37°C for 15 minutes. One day after transduction, T cells were washed and suspended in serum-free T cell basal medium containing 1000U / mL recombinant human interleukin-2, 10ng / ml recombinant human interleukin-7 and 10ng / ml recombinant human interleukin-15 at a concentration of 1 million T cells / mL medium and plated in T-25 flasks at 5 million cells per group.

[0521] The pre-incubated LNP mixture was added to each T-25 flask as described in Table 9 to produce a final concentration of 1.25 μg / mL total RNA. Multiple knockout groups were each treated with 1.25 μg / mL LNP. Twenty-four hours after LNP treatment, T cells were collected, washed, and cultured in OpTmizer medium with 5% human serum and cytokines as described in Example 2 to expand. On the 5th day, the transduced samples were sorted by flow cytometry to obtain enriched mScarlet+ populations.

[0522] It is known that CD4+CD25+T cell colonies are rich in natural Treg (nTreg). CD4+CD25+ cells are separated by methods known in the art. On the 0th day, CD25+ cells are activated by placing 5×10^6 cells / well in 6-well Grex, and the wells contain 30mL T cell basal medium supplemented with 1000U / mL IL-2 and 25μL / mL ImmunoCult (Stemcell Technologies, 10991). On the 5th day, CD4+CD25+ samples were stained with antibodies and sorted by flow cytometry to obtain purified LAP+ populations, which were used as untransduced "nTreg" groups in mouse in vivo studies. Latency-associated peptide (LAP) is a marker for activated Treg cells.

[0523] Example 4.2. In vivo functional characterization of suppressor T cells

[0524] T cells were harvested 14 days after activation for in vivo injection. The engineered T cell autologous PBMCs were thawed as described in the above examples. PBMCs were added to each assay population at a 1:1 ratio and the cells were resuspended in RPMI (supplemented with 2% FBS and 10mM HEPES) until 1.5×10^7 / mL. The PBMC group alone was resuspended at 3×10^6 / mL.

[0525] Female NOG mice (NOD.Cg-Prkdcscid Il2rgtm1Sug / JicTac; Taconic, catalog number NOG-F) were treated with sublethal radiation (150 rads) using X-rays (RS-2000 irradiator; Rad Source Technologies) one day before injection for cell transplantation. 100 μL of each test cell population was injected intravenously into the irradiated NOG mouse cohort as described in the above paragraph. Ten irradiated mice were injected with 100 μL RPMI (supplemented with 2% FBS and 10 mM HEPES) as a control that received only radiation. Body weight was monitored daily. Mice were sacrificed when they lost 20% of their body weight. The results are shown in Fig.14 and Table 10.

[0526] Table 10. Survival percentage of mice in each cohort that survived at each time point after injection.

[0527]

[0528] Example 5. In vivo evaluation of mutant IL-10 expression in suppressor T cells

[0529] Example 5.1. T cell engineering

[0530] To generate engineered suppressor T cells (supT cells), CD4+ T cells were engineered according to Table 11.

[0531] The isolated CD4+T cells were activated in 5 mL OpTmizer (containing 200 U / mL IL-2, 5 ng / mL IL-7, 5 ng / mL IL-15 and 25 uL / mL Immunocult (Stemcell Technologies, 10991)) in a 6-well plate at 5 x 10^6 cells / well. Two days after activation (day 2), cells were transduced with lentivirus encoding CTLA4 (SEQ ID NO: 130), IL-10 (SEQ ID NO: 124) and mScarlet and lentivirus encoding CTLA4 (SEQ ID NO: 130), IL-10DE (SEQ ID NO: 229) and mScarlet or lentivirus encoding GFP according to Table 11. The concentrated viral supernatant was added to the T cells and centrifuged at 1000 × g for 60 minutes at 37°C. T cells were resuspended in the cell / virus supernatant mixture and transferred to a single well of a 6-well G-rex (Wilson Wolf; Catalog No. 80240M) containing an OpTmizer supplemented with 500 U / mL IL-2, 5 ng / mL IL-7, and 5 ng / mL IL- 15. Cells were split and supplemented with medium or cytokines regularly.

[0532] Table 11. T cell editing treatment groups. "LV" stands for lentivirus.

[0533]

[0534] On the 3rd day, T cells were edited according to Table 11, using LNP to deliver Cas9 mRNA and guide RNA targeting INF-gamma (G019753), and using LNP to deliver Cas9 mRNA and guide RNA targeting TNF-alpha (G019757). LNPs were produced using a molar ratio of 35 lipid A / 47.5 cholesterol / 15 DSPC / 2.5 PEG2k-DMG as described in Example 1. LNPs were prepared with a lipid amine to RNA phosphate (N: P) molar ratio of about 6. LNPs were prepared with a gRNA to Cas9 mRNA carrier ratio of 1: 1 by weight.

[0535] Each LNP formulation was incubated in T cell basal medium supplemented with 5ug / ml recombinant human ApoE3 (Peprotech, Cat.350-02) at 37°C for 15 minutes. One day after transduction, T cells were washed and suspended in serum-free OpTmizer medium containing 1000U / mL recombinant human interleukin-2, 10ng / ml recombinant human interleukin-7 and 10ng / ml recombinant human interleukin-15 at a concentration of 1 million T cells / mL medium and plated in T-25 flasks at 5 million cells per group.

[0536] The pre-incubated LNP mixture was added to each T-25 flask as described in Table 11 to produce a final concentration of 1.25 μg / mL total RNA. Multiple knockout groups were each treated with 1.25 μg / mL LNP. Twenty-four hours after LNP treatment, T cells were collected, washed, and cultured in OpTmizer medium with 5% human serum and cytokines as described in Example 2 to expand. On the 5th day, the transduced samples were sorted by flow cytometry to obtain an enriched mScarlet+ population.

[0537] It is known that CD4+CD25+T cell populations are rich in natural Treg (nTreg). CD4+CD25+ cells and CD4+CD25+CD127-CD45RA+ are separated by methods known in the art. On day 0, the separated cells were activated by placing 5×10^6 cells / well in a 6-well Grex, with 30mL OpTmizer supplemented with 1000U / mL IL-2 and 25μL / mL Imm unoCult (Stemcell Technologies, 10991). On day 5, the samples were stained with antibodies and sorted by flow cytometry to obtain purified LAP+ populations, which were used as "LAPnTreg" groups in in vivo studies in mice, and enriched CD45+ populations were obtained, which were used as "CD45RA nTre g" groups in in vivo studies in mice. Latency-associated peptide (LAP) is a marker for activated Treg cells.

[0538] Example 5.2. In vivo functional characterization of suppressor T cells

[0539] T cells were harvested 13 days after activation for in vivo injection. The engineered T cell autologous PBMCs were thawed as described in the above examples. PBMCs were added to each assay population at a 1:1 ratio and the cells were resuspended in RPMI (supplemented with 2% FBS and 10mM HEPES) until 5×10^7 / mL. The PBMC group alone was resuspended at 2.5×10^7 / mL.

[0540] Female NOG mice (NOD.Cg-Prkdcscid Il2rgtm1Sug / JicTac; Taconic, catalog number NOG-F) were treated with sublethal radiation (150 rads) using X-rays (RS-2000 irradiator; Rad Source Technologies) one day before injection for cell transplantation. 100 μL of each test cell population was injected intravenously into the irradiated NOG mouse cohort as described in the above paragraph. Ten irradiated mice were injected with 100 μL RPMI (supplemented with 2% FBS and 10 mM HEPES) as a control that received only radiation. Body weight was monitored daily. Mice were sacrificed when they lost 20% of their body weight. The results are shown in Fig.15 and Table 12.

[0541] Table 12. Survival percentage of mice in each cohort that survived at each time point post injection.

[0542]

[0543] Example 6: Suppressive capacity of engineered T cells in an inflammatory bowel disease model

[0544] Example 6.1 Evaluation of the efficacy of nTregs in a humanized mouse model of colitis

[0545] Tregs are known to inhibit the induction of colitis in preclinical models (see, e.g., Goet tel 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 transfer 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).

[0546] Natural Tregs were isolated using methods known in the art and applied to a humanized mouse model of IBD, in which trinitrobenzene sulfonic acid (TNBS) was applied to the colon of TNBS-sensitized mice to induce colitis. Briefly, NSG mice were reconstituted with human CD34+ cells obtained from a commercial source (Jackson Laboratory). After a 1-week adaptation period, mice were injected subcutaneously (sc) with TNBS or vehicle, and mice were injected intraperitoneally (ip) with vehicle or natural Tregs (nTreg) [1×10^ 6 After three days, mice received enema of vehicle (50% ethanol) or TNBS in 50% ethanol as described in Table 13. Body weight was recorded daily and the results are shown in Fig.16 (+ / - SEM) and Table 14.

[0547] Table 13. Experimental design

[0548]

[0549] Table 14. Percentage of initial body weight of individual mice in treatment groups.

[0550]

[0551]

[0552] On day 3 after enema, mice were euthanized. Colon length was determined to assess protection against colitis. Spleen, mesenteric lamina propria (mLN), and colonic lamina propria (cLP) were harvested and stained with CellTrace Violet to determine the persistence of Tregs in various tissues for the duration of the model. Results are shown in Fig.17 (+ / -SEM) and 18 (+ / -SEM) and in Tables 15 and 16.

[0553] Table 15. Colon length (in cm).

[0554]

[0555]

[0556] Table 16. Percentage of Tregs stained with Cell Trace Violet recovered from each tissue of individual mice.

[0557]

[0558] These data demonstrate that nTregs can provide protection against colitis in a humanized mouse model, as nTreg-treated TNBS-treated mice experienced rebound weight loss and increased colon length compared to vehicle control-treated TNBS-treated mice. CellTrace Violet staining demonstrated persistence of nTregs in all evaluated compartments throughout the experimental period.

[0559] Example 6.2: Comparison of the efficacy of nTregs and engineered T cells in a humanized mouse model of colitis

[0560] Based on this proof of the efficacy of nTreg providing protection against colitis in a humanized mouse model, the protective effect of engineered T cells against colitis was evaluated in the same mouse model. Engineered T cells are generally prepared as described in Example 3.1, and are engineered to break TNFA and IFNG, and provide the expression of TGFB1 R218H C225R, CTLA4 and IL-10D25A / E96A. The study also includes the time course of the evaluation results. As described above, NSG mice were reconstructed with human CD34+ cells obtained from a commercial source (Jackson Laboratory). As described in Table 17, after a 1-week adaptation period, mice were subcutaneously injected with TNBS or vehicle, and mice were intraperitoneally injected with vehicle, nTreg or engineered Treg. After one week, mice received enema of vehicle (50% ethanol) or TNBS in 50% ethanol. Body weight was recorded every day. On days 3, 5, and 7 after enema, 6 mice in group 1 and 8 mice in groups 2, 3, and 4 were euthanized.

[0561] Table 17. Experimental treatment groups.

[0562]

[0563] Body weights were monitored throughout the study. At euthanasia, colons were harvested and measured to determine length. Colon sections were analyzed using 5-parameter blinded histology after H&E staining. This study confirmed the results of single time point studies, demonstrating that nTregs provide protection in a colitis model, and demonstrated that engineered T cells provide protection against colitis in the same model. This study was used to select the optimal time after enema to evaluate the findings in the model.

[0564] Example 6.3: Efficacy evaluation of targeted engineered T cells in a humanized mouse model of colitis

[0565] Further studies were conducted to determine the effect of targeting engineered T cells using a chimeric antigen receptor (CAR) that binds mouse MadCAM (a surface protein present in various tissues in the gastrointestinal tract). As in previous experiments, T cells are generally engineered as described in Example 4. SpyCas9 and known methods are used to further edit T cells to insert constructs for expressing MadCAM CAR into the TRAC locus. In order to distinguish the delivered engineered cells from endogenous mouse T cells, engineered T cells were also transduced with a lentivirus expressing green fluorescent protein (GFP) or mScarlet. The time points used in the study were selected according to the above-mentioned time course studies. The experimental groups are described in Table 18.

[0566] Table 18. Experimental design

[0567]

[0568] Body weight was monitored throughout the study. At euthanasia, colons were harvested and measured to determine length. Colon sections were analyzed using 5-parameter blinded histology after H&E staining. Spleen, mesenteric lamina propria, and colonic lamina propria were harvested for analysis by Bulk RNA-seq using conventional methods for transferred engineered T cells and endogenous T effector cells. Flow cytometric analysis of T cells from spleen, mesenteric lamina propria, and colonic lamina propria was also performed by staining for CD3, CD4, CD8, hCD45, mCD45, and live / dead for delivered engineered T cells that could be identified by GFP or mScarlet.

[0569] This study confirms the results of previous studies demonstrating the protection provided by nTregs and engineered T cells in a colitis model. This study shows that targeted engineered T cells can also provide protection in a colitis model.

Claims

1. An engineered T cell comprising: A heterologous nucleic acid sequence encoding a double mutant transforming growth factor β1 (dmTGFB1) under the control of a promoter sequence.

2. The engineered T cell of claim 0, further comprising: i) modification of an endogenous nucleic acid sequence encoding tumor necrosis factor alpha (TNFA), wherein the modification knocks down the expression of TNFA; and ii) modification of an endogenous nucleic acid sequence encoding interferon-17 (IFNG), wherein the modification knocks down the expression of the IFNG, or modification of an endogenous nucleic acid sequence encoding interleukin 17A (IL17A), wherein the modification knocks down the expression of IL17A.

3. An engineered T cell as described in claim 1 or claim 2, which comprises a modification of an endogenous nucleic acid sequence encoding IFNG, wherein the modification knocks down the expression of IFNG, and a modification of an endogenous nucleic acid sequence encoding IL07A, wherein the modification knocks down the expression of IL17A.

4. 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) modification of an endogenous nucleic acid sequence encoding interleukin 17A (IL17A), wherein the modification knocks down the expression of the IL17A; and iii) modification of an endogenous nucleic acid sequence encoding tumor necrosis factor alpha (TNFA), wherein said modification knocks down the expression of TNFA.

5. The engineered T cell of claim 4, 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), ecto-5'-nucleotidase (NT5E), interleukin-22 (IL-22), amphiregulin (AREG), interleukin-35 (IL35), GARP, CD274 molecule (CD274), forkhead box P3 (FOXP3), IKAROS family zinc finger 2 (IKZF2), familial eosinophilia (EOS), interferon regulatory factor 4 (IRF4), lymphoid enhancer binding factor 1 (LEF1), BTB domain and CNC homolog 2 (BACH2) and interleukin 2 receptor subunit alpha (IL2RA).

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

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

10. The engineered T cell of any one of claims 1 to 9, further comprising modifications of endogenous nucleic acid sequences 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), ranolidine receptor 2 (RYR2) and colony stimulating factor 2 (CSF2), wherein the modifications knock down the expression of the IL17A, the IL2, the IL6, the PRF1, the GZMA, the GZMB, the FASL, the RYR2 or the CSF2, respectively.

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

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. An engineered T cell as described in claim 12, wherein the targeting receptor targets a ligand selected from the following: 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), glutamate decarboxylase (GAD2), desmoglein 3 (DSG3) and MHC class I HLA-A (HLA-A*02).

14. The engineered T cell of claim 12, wherein the targeting receptor targets MADCAM1.

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

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

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 comprise a nucleic acid sequence encoding a regulatory T cell promoting molecule.

18. An engineered T cell as described in any one of claims 8 to 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. 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. The engineered T cell of claim 17 or claim 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 a single expression construct.

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

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

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

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

26. An engineered T cell as described in claim 25, wherein the targeted insertion is in a site selected from the group consisting of a TCR locus, a TNF locus, an IFNG locus, an IL17A locus, an IL6 locus, an IL2 locus, and an adeno-associated virus integration site 1 (AAVS1) locus.

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

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

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

30. An engineered T cell population 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 the population comprise a heterologous nucleic acid sequence encoding dmTGFB1 under the control of a promoter sequence.

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

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

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

34. The engineered T cell population of any one 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 promoting molecule under the control of a promoter sequence.

35. The engineered T cell population of claim 29, 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 promoting molecule under the control of a promoter sequence; At least 50%, preferably at least 70%, of the cells of the population comprise a modification of an endogenous nucleic acid sequence encoding TNFA; and At least 50%, preferably at least 70% of the cells of the population comprise a modification of the endogenous nucleic acid sequence encoding IL17A.

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

37. The engineered T cell population of any one of claims 29 to 36, wherein the percentage of cells comprising an insertion or modification is determined by the percentage of reads measured by next generation sequencing (NGS).

38. An engineered T cell population as described in 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), ecto-5'-nucleotidase (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. The engineered T cell population of any one of claims 34 to 38, wherein the regulatory T cell promoting molecule is IL10.

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

41. An engineered T cell population as described in any one of claims 34 to 40, wherein the regulatory T cell promoting molecule is a first regulatory T cell promoting molecule, and the engineered T cell population further comprises a heterologous nucleic acid sequence encoding a second regulatory T cell promoting molecule under the control of a promoter sequence.

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

43. The engineered T cell population of any one of claims 29 to 42, further comprising 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), ranolidine receptor 2 (RYR2), and colony stimulating factor 2 (CSF2), wherein at least 70% of the cells In a cell population, preferably at least 80% of the cell population, the cell population comprises a modification in at least one of the IL17A, the IL6, the IL2, the PRF1, the GZMA, the GZMB, the FASL, the RYR2 or the CSF2, respectively, wherein the modification knocks down the expression of at least one of the IL17A, the IL6, the IL2, the PRF1, the GZMA, the GZMB, the FASL, the RYR2 or the CSF2, respectively.

44. An engineered T cell population as described in claim 43, wherein the modification of at least one endogenous nucleic acid sequence encoding interleukin 2 (IL2), wherein in at least 70% of the cell population, preferably at least 80% of the cell population, the cell population contains the modification of the IL2, wherein the modification knocks down the expression of the IL2.

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

46. ​​The engineered T cell population 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 encoding sequence for a targeting receptor.

47. An engineered T cell population as described in claim 46, wherein the targeting receptor specifically binds to a ligand selected from the following: 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), glutamate decarboxylase (GAD2), desmoglein 3 (DSG3) and MHC class I HLA-A (HLA-A*02).

48. An engineered T cell population as described in claim 46 or claim 47, wherein the targeting receptor comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

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

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

51. An engineered T cell population as described in any one of claims 29 to 50, wherein the heterologous nucleic acid sequence encoding a first of 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. The engineered T cell population 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. The engineered T cell population 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 a single expression construct.

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

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

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

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

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

59. An engineered T cell population as described in claim 58, wherein the targeted insertion is in a site selected from the group consisting of a TCR locus, a TNF locus, an IL2 locus, an IL6 locus, an IL17A locus, an IFNG locus, and an adeno-associated virus integration site 1 (AAVS1) locus.

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

61. The engineered T cell population of any one of claims 29 to 60, wherein the modification to knock down gene expression comprises one or more of an insertion, a deletion, or a substitution.

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

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

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

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

66. The method of use as claimed in any one of claims 63 to 65 for the treatment of an autoimmune disease.

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. The method or use of any one of claims 63 to 65, for treating graft versus host disease (GvHD).

69. A polypeptide comprising dmTGFB1.

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

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 relative to wild-type human TGFB1.

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

74. 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 the polypeptide of any one of claims 69 to 74.

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