engineered T cells

Abstract

The present disclosure relates to T cells engineered to include a heterologous nucleic acid sequence encoding a double mutant transforming growth factor beta 1 (dmTGFB1) and a modification, e.g., knockdown, of an endogenous nucleic acid sequence encoding TGFBR2 under the control of a promoter sequence. In certain embodiments, 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; and an insertion of sequence(s) encoding a regulatory T cell promoter molecule, and provides compositions and uses thereof.

Description

Technology Field

[0001] Related applications

[0002] This application claims the benefit and priority of U.S. provisional application serial number 63 / 612,847 filed December 20, 2023 and U.S. provisional application serial number 63 / 727,648 filed December 3, 2024, the entire contents of which are incorporated herein by reference.

[0003] Sequence list

[0004] This application is filed together with a sequence list in electronic format. A sequence list file named 12793_0041-00304_SL was created on December 18, 2024, and has a size of 800,521 bytes. The entirety of the sequence list in electronic format is incorporated herein by reference. Background Technology

[0005] Adaptive immunity is a defense mechanism that enables the body to eliminate foreign pathogens. T cells are immune cells capable of mediating this immune response. The T cell receptor (TCR) is a protein complex on the surface of T cells that recognizes antigens. T cell diversity is induced by the rearrangement of the TCR alpha and beta loci.

[0006] One characteristic of adaptive immunity is the ability to distinguish between "self" antigens and "non-self" antigens. Autoimmune and autoinflammatory disorders are characterized by pathogenic immune responses to "self" antigens. Some rearrangements of the TCR alpha and beta loci generate self-reactive T cells. Owen et al., Regulatory T Cell Development in the Thymus, J Immunol 203(8)(2019). Many self-reactive T cells are eliminated by clonal deletion in the thymus, but others evade clonal deletion and can trigger harmful immune responses. Id.Specialized T cells called regulatory T cells (Tregs) are used for "self" tolerance. Id Treg can suppress excessive immune responses, autoimmune responses, and undesirable immune responses, for example, in graft-versus-host disease. Id Dysregulation of Treg, for example, if the number of Tregs is insufficient or Tregs do not function properly, can contribute to an autoimmune response. Id .

[0007] Current therapies for treating autoimmune disorders aim to suppress adaptive immune processes or the activation of immune cells. While these therapies can suppress harmful immune responses, such as autoimmune responses, they can also suppress beneficial immune responses. Treg therapies have been used to suppress antigen-specific immune responses in various diseases, including graft-versus-host disease (GvHD), in which donor cells mediate an immune attack on host tissues following hematopoietic stem cell transplantation. Pierini et al., T Cells Expressing Chimeric Antigen Receptor Promoter Immune Tolerance, JCI Insight 2(20)(2017). However, "major challenges regarding the clinical implementation of Treg-based therapies" still exist. Id Therefore, there is still a need for effective T cell therapies, including Treg therapies, to suppress immune response(s), including inflammation and autoimmunity.

[0008] One aspect of the present disclosure relates to a engineered T cell comprising: i) a heterogeneous nucleic acid sequence encoding a double mutant transforming growth factor beta 1 (dmTGFB1) under the control of a promoter sequence; and ii) a modification of an endogenous nucleic acid sequence encoding transforming growth factor beta receptor 2 (TGFBR2), wherein the modification knocks down the expression of TGFBR2.

[0009] In some embodiments, dmTGFB1 comprises mutations in two or more amino acids selected from F198, D199, V200, L208, F217, L219, R218, H222, C223, and C225 compared to wild-type human TGFB1. In some embodiments, dmTGFB1 comprises mutations in two or more amino acids selected from R218, H222, C223, and C225 compared to wild-type human TGFB1. In some embodiments, dmTGFB1 comprises mutations in two or more amino acids selected from R218C / H, H222D, C223S / R / G, and C225R compared to wild-type human TGFB1. In some embodiments, dmTGFB1 comprises amino acid mutations R218C / H and C225R compared to wild-type human TGFB1.

[0010] In some embodiments, the engineered T cells are CD4+ T cells.

[0011] In some embodiments, the engineered T cell further comprises i) a modification of an endogenous nucleic acid sequence encoding tumor necrosis factor alpha (TNFA) that knocks down the expression of TNFA; ii) a modification of an endogenous nucleic acid sequence encoding interferon-gamma (IFNG) that knocks down the expression of IFNG; or iii) a heterogeneous nucleic acid sequence encoding a regulatory T cell promoter molecule under the control of a promoter sequence.

[0012] In some embodiments, the engineered T cell comprises i) a modified endogenous nucleic acid sequence encoding tumor necrosis factor alpha (TNFA), wherein the modification knocks down the expression of TNFA; ii) a modified endogenous nucleic acid sequence encoding interferon-gamma (IFNG), wherein the modification knocks down the expression of IFNG; and iii) a heterogeneous nucleic acid sequence encoding a regulatory T cell promoter molecule under the control of a promoter sequence.

[0013] In some embodiments, the regulatory T cell promoter is interleukin-10 (IL10), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), T cell immune receptor having Ig and ITIM domains (TIGIT), indoleamine 2,3-deoxygenase 1 (IDO1), ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), 5'-nucleotidase ecto (NT5E), interleukin-22 (IL-22), ampyregulin (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), lymphatic enhancer binding factor 1 (LEF1), BTB domain and CNC homolog 2 (BACH2), and It is selected from interleukin 2 receptor subunit alpha (IL2RA).

[0014] In some embodiments, the regulatory T cell promoter is IL10 or CTLA4.

[0015] In some embodiments, the regulatory T cell promoter is a first regulatory T cell promoter and further comprises a heterogeneous nucleic acid sequence encoding a second regulatory T cell promoter under the control of a promoter sequence. In some embodiments, the first and second regulatory T cell promoter molecules are IL10 and CTLA4.

[0016] In some embodiments, IL10 comprises a mutation at one or more amino acids selected from D25, E96, N21, and R104 compared to mature wild-type human IL10. In some embodiments, IL10 comprises one or more amino acid mutations selected from D25A / K, E96A, N21A, and R104A compared to mature wild-type human IL10. In some embodiments, IL10 comprises a combination of amino acid mutations selected from D25K, D25A, D25A / E96A, D25K / E96A, N21A / R104A, D25A / N21A / R104A, and D25K / N21A / R104A compared to mature wild-type human IL10. In some embodiments, IL10 comprises mature wild-type human IL10.

[0017] In some embodiments, the engineered T cell comprises a modification of an endogenous nucleic acid sequence encoding at least one of a T-cell receptor, an MHC class I molecule, or an MHC class II molecule. In some embodiments, the engineered T cell comprises a modification of an endogenous nucleic acid sequence encoding a protein selected from TRAC, HLA-A, HLA-B, and CIITA, wherein the modification knocks down the expression of the protein. In some embodiments, the protein is TRAC, and the modification knocks down the expression of TRAC. In some embodiments, the protein is HLA-A, and the modification knocks down the expression of HLA-A. In some embodiments, the protein is HLA-B, and the modification knocks down the expression of HLA-B. In some embodiments, the protein is CIITA, and the modification knocks down the expression of CIITA.

[0018] In some embodiments, the engineered T cell further comprises a) a modification of the endogenous nucleic acid sequence encoding TRAC, wherein the modification knocks down the expression of TRAC; b) a modification of the endogenous nucleic acid sequence encoding HLA-A, wherein the modification knocks down the expression of HLA-A; c) a modification of the endogenous nucleic acid sequence encoding HLA-B, wherein the modification knocks down the expression of HLA-B; and d) a modification of the endogenous nucleic acid sequence encoding CIITA, wherein the modification knocks down the expression of CIITA.

[0019] Another aspect of the present disclosure relates to an engineered T cell comprising: i) a modification of a first endogenous nucleic acid sequence encoding tumor necrosis factor alpha (TNFA), wherein the modification of the first endogenous nucleic acid knocks down the expression of TNFA; ii) a modification of a second endogenous nucleic acid sequence encoding interferon-gamma (IFNG), wherein the modification of the second endogenous nucleic acid knocks down the expression of IFNG; iii) a modification of a third endogenous nucleic acid sequence encoding a protein selected from TRAC, HLA-A, HLA-B, and CIITA, wherein the modification of the third endogenous nucleic acid knocks down the expression of the protein; and iv) a heterologous nucleic acid sequence encoding a regulatory T cell promoter molecule under the control of a promoter sequence.

[0020] In some embodiments, the protein is TRAC, and the third modification knocks down the expression of TRAC. In some embodiments, the protein is HLA-A, and the third modification knocks down the expression of HLA-A. In some embodiments, the protein is HLA-B, and the third modification knocks down the expression of HLA-B. In some embodiments, the protein is CIITA, and the third modification knocks down the expression of CIITA.

[0021] Another aspect of the present disclosure is i) a modification of a first endogenous nucleic acid sequence encoding tumor necrosis factor alpha (TNFA), wherein the modification knocks down the expression of TNFA; ii) a modification of a second endogenous nucleic acid sequence encoding interferon-gamma (IFNG), wherein the modification knocks down the expression of IFNG; iii) a modification of a third endogenous nucleic acid sequence encoding TRAC, wherein the modification of the third endogenous nucleic acid sequence knocks down the expression of TRAC; iv) a modification of a fourth endogenous nucleic acid sequence encoding HLA-A, wherein the modification of the fourth endogenous nucleic acid sequence knocks down the expression of HLA-A; v) a modification of a fifth endogenous nucleic acid sequence encoding HLA-B, wherein the modification of the fifth endogenous nucleic acid sequence knocks down the expression of HLA-B; vi) a modification of a sixth endogenous nucleic acid sequence encoding CIITA, wherein the modification of the sixth endogenous nucleic acid sequence knocks down the expression of CIITA; and vii) the invention relates to an engineered T cell comprising a heterologous nucleic acid sequence encoding a regulatory T cell promoter molecule under the control of a promoter sequence.

[0022] In some embodiments, the regulatory T cell promoter is interleukin-10 (IL10), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), T cell immune receptor having Ig and ITIM domains (TIGIT), indoleamine 2,3-deoxygenase 1 (IDO1), ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), 5'-nucleotidase ecto (NT5E), interleukin-22 (IL-22), ampyregulin (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), lymphatic enhancer binding factor 1 (LEF1), BTB domain and CNC homolog 2 (BACH2), and It is selected from interleukin 2 receptor subunit alpha (IL2RA).

[0023] In some embodiments, the regulatory T cell promoter is IL10 or CTLA4.

[0024] In some embodiments, the regulatory T cell promoter is a first regulatory T cell promoter and further comprises a heterogeneous nucleic acid sequence encoding a second regulatory T cell promoter under the control of a promoter sequence.

[0025] In some embodiments, the first and second regulatory T cell promoter molecules are IL10 and CTLA4. In some embodiments, IL10 comprises a mutation at one or more amino acids selected from D25, E96, N21, and R104 compared to mature wild-type human IL10. In some embodiments, IL10 comprises one or more amino acid mutations selected from D25A / K, E96A, N21A, and R104A compared to mature wild-type human IL10. In some embodiments, IL10 comprises a combination of amino acid mutations selected from D25K, D25A, D25A / E96A, D25K / E96A, N21A / R104A, D25A / N21A / R104A, and D25K / N21A / R104A compared to mature wild-type human IL10. In some embodiments, IL10 comprises mature wild-type human IL10.

[0026] In some embodiments, the engineered T cells further comprise modifications to the endogenous nucleic acid sequences encoding interleukin 17A (IL17A), interleukin-2 (IL2), interleukin 6 (IL6), perforin 1 (PRF1), granzyme A (GZMA), granzyme B (GZMB), natural killer cell granule protein 7 (NKG7), Fas ligand (FASL, NF superfamily, member 6), lyanodine receptor 2 (RYR2), and colony-stimulating factor 2 (CSF2), wherein the modifications knock down the expression of IL17A, IL2, IL6, PRF1, GZMA, GZMB, FASL, RYR2, or CSF2, respectively.

[0027] In some embodiments, the engineered T cells further include a modification of the endogenous nucleic acid sequence encoding the endogenous T cell receptor (TCR), wherein the modification knocks down the expression of the endogenous TCR.

[0028] In some embodiments, the engineered T cell further comprises a heterologous coding sequence for a targeting receptor under the control of a promoter sequence. In some embodiments, the targeting receptor is mucosal vascular addressin cell adhesion molecule 1 (MADCAM1), tumor necrosis factor alpha (TNFA), CD70 molecule (CD70), 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 (ligand) superfamily member 7 (TNFSF7), 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 It is targeted to a ligand selected from HLA-A (HLA-A*02).

[0029] In some embodiments, the targeting receptor includes a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0030] In some embodiments, a heterogeneous nucleic acid sequence encoding a targeting receptor is incorporated into an expression construct. In some embodiments, the heterogeneous nucleic acid sequence encoding a targeting receptor is in an expression construct that does not contain a nucleic acid sequence encoding a regulatory T cell promoter molecule. In some embodiments, the heterogeneous nucleic acid sequence encoding a targeting receptor is in an expression construct that contains a nucleic acid sequence encoding a regulatory T cell promoter molecule.

[0031] In some embodiments, a heterogeneous nucleic acid sequence encoding a first regulatory T cell promoter molecule is incorporated into an expression construct, and a heterogeneous nucleic acid sequence encoding a second regulatory T cell promoter molecule is incorporated into an expression construct. In some embodiments, the heterogeneous nucleic acid sequence encoding the first regulatory T cell promoter molecule and the heterogeneous nucleic acid sequence encoding the second regulatory T cell promoter molecule are incorporated into separate expression constructs. In some embodiments, the heterogeneous nucleic acid sequence encoding the first regulatory T cell promoter molecule and the heterogeneous nucleic acid sequence encoding the second regulatory T cell promoter molecule are incorporated into a single expression construct.

[0032] In some embodiments, at least one heterologous coding sequence is present in the episome expression construct. In some embodiments, at least one heterologous nucleic acid sequence is inserted into the genome. In some embodiments, the insertion into the genome is a non-targeted insertion. In some embodiments, the insertion is a targeted insertion. In some embodiments, the targeted insertion is inserted into a site selected from the TCR locus, TNF locus, IFNG locus, IL17A locus, IL6 locus, IL2 locus, and the adeno-associated virus integration site 1 (AAVS1) locus. In some embodiments, the TCR locus is the T cell receptor alpha-constant (TRAC) locus.

[0033] In some embodiments, modifications that knock down gene expression include one or more of insertions, deletions, or substitutions. In some embodiments, the engineered T cells are CD4+ T cells.

[0034] Further aspects of the present disclosure relate to a population of engineered T cells comprising engineered T cells as described herein. In some embodiments, the population of engineered T cells comprises engineered T cells as described herein, wherein at least 30% or at least 40% of the cells in the population comprise a heterologous nucleic acid sequence encoding dmTGFB1 under the control of a promoter sequence; and at least 50% or at least 70% of the cells in the population comprise a modification of an endogenous nucleic acid sequence encoding TGFBR2. In some embodiments, the population of engineered T cells comprises engineered T cells as described herein, wherein at least 50% or at least 70% of the cells in the population comprise a modification of an endogenous nucleic acid sequence encoding TNFA; at least 50% or at least 70% of the cells in the population comprise a modification of an endogenous nucleic acid sequence encoding IFNG; or at least 30% or at least 40% of the cells in the population comprise a heterologous nucleic acid sequence encoding a regulatory T cell promoter molecule under the control of a promoter sequence. In some embodiments, the population of engineered T cells comprises engineered T cells as described herein, wherein the percentage of cells including insertions or modifications is determined by the percentage of readouts by next-generation sequencing (NGS).

[0035] One aspect of the present disclosure relates to a pharmaceutical composition comprising engineered T cells or an engineered T cell population as described herein.

[0036] Further aspects of the present disclosure relate to methods or uses of administering engineered T cells, engineered T cell populations, or pharmaceutical compositions as described herein to a subject. In some embodiments, the subject requires immunosuppression. In some embodiments, the methods or uses described herein are for the treatment of immune disorders. In some embodiments, the methods or uses described herein are for the treatment of autoimmune diseases.

[0037] In some embodiments, the autoimmune disease is selected from ulcerative colitis, Crohn's disease, rheumatoid arthritis, psoriasis, multiple sclerosis, systemic lupus erythematosus, and type 1 diabetes. In some embodiments, the method or use described herein is for the treatment of graft-versus-host disease (GvHD).

[0038] In some embodiments, the subject receives a transplant or a blood transfusion. In some embodiments, the subject will receive a transplant or a blood transfusion. In some embodiments, the subject is in the process of receiving a transplant or a blood transfusion. In some embodiments, the subject has received a transplant or an infusion. In some embodiments, the transplant or blood transfusion comprises a solid organ or a population of cells. In some embodiments, the transplant or blood transfusion comprises cells from a donor other than the subject.

[0039] In some embodiments, the subject is administered engineered T cells, an population of engineered T cells, or a pharmaceutical composition as described herein before receiving a transplant or blood transfusion. In some embodiments, the subject is administered engineered T cells, an population of engineered T cells, or a pharmaceutical composition as described herein simultaneously with receiving a transplant or blood transfusion. In some embodiments, the subject is administered engineered T cells, an population of engineered T cells, or a pharmaceutical composition as described herein after receiving a transplant or blood transfusion. Brief explanation of the drawing

[0040] Figure 1 shows the percentage of inhibition of cell proliferation by engineered T cells as measured by CTV dilution. Figure 2 shows the survival percentage over time after injecting engineered T cells into NOG mice. Figure 3 shows the persistence profile of engineered T cells. Figure 4 shows in vivo IL-10 production (pg / ml). Figure 5 shows the results of an in vivo study on CAR targeting by synTreg in a xenoGvHD mouse model, plotting the survival percentage of mice in each cohort surviving at each time point after injection. Figure 6 shows the results of an in vivo study on CAR targeting by synTreg in a xenoGvHD mouse model, illustrating the average percentage of synTreg among all human CD45+ cells recovered from the mouse spleen at sacrifice. Figure 7 shows the results of an in vivo study on CAR targeting by synTreg in a xenoGvHD mouse model and plots the average GvHD scores for each experimental condition. Figure 8 shows the results of an in vivo study on CAR targeting by synTreg in a xenoGvHD mouse model and plots the average IL-10 serum levels of mice in each treatment group. Figure 9 shows the inhibitory activity of synTreg isolated from the spleen, measured as the inhibitory activity against the proliferation of Tresp. Figures 10a-10d show the secretion of pro-inflammatory cytokines (IFN-gamma, Fig. 10a; TNF-alpha, Fig. 10b; IL-2, Fig. 10c; IL-10, Fig. 10d) by Tconv compared to synTreg. Figures 11a-11c show the activation profiles of Tconv (Figure 11a), synTreg (Figure 11b), and targeted CAR synTreg (Figure 11c) as measured by CD69 levels upon exposure to murine MAdCAM-1. Figure 12 shows the mean cytokine secretion in pg / ml upon restimulation with INFg, TNFa, IL-10, and TFG-b. Cells were stimulated with murine MAdCAM-1 (MC-1) or Immunocult (IC). Figure 13 shows the mean percentage of inhibition of cell proliferation in a mixed lymphocyte response using synTreg and T cells derived from patients with ulcerative colitis. HD, healthy donor. UC, ulcerative colitis. Figure 14 shows the mean cytokine secretion of IFNg (Figure 14a) and TNFa (Figure 14b) in a mixed lymphocyte response using synTreg and T cells derived from patients with ulcerative colitis. HD, healthy donor. UC, ulcerative colitis. Figure 15 shows the colon weight:length ratio at the time of sacrifice. Figure 16 shows the average histological score for colon samples. Figure 17 shows the colon weight:length ratio at the time of sacrifice. Figure 18 shows the average percentage of synTregs among CD4+ cell populations in mouse spleen. Figure 19 shows the average percentage of synTregs among CD4+ cell populations in mouse mesenteric lymph nodes (MLN). Figure 20 shows the colon weight:length ratio after treatment in a winter colitis model. Figure 21 shows the histological scores for colon sections after treatment in a winter colitis model. Figures 22a-22d show cytokine expression in CD4+ cells from mesenteric lymph nodes after treatment with TNFa (Figure 22a), IFNg (Figure 22b), IL-22 (Figure 22c), and IL-17 (Figure 22d), respectively, in a synchocolitis model. Figures 23a and 23b show cytokine levels in the medium harvested after colon explant culture for IFNg (Figure 23a) and TNFa (Figure 23b), respectively. Figure 24 shows the lipocalin levels in the stool after treatment of a winter colitis model. Specific details for implementing the invention

[0041] Specific embodiments of aspects of the present disclosure will now be described in detail, examples of which are illustrated in the accompanying drawings. While the present disclosure is described together with the illustrative embodiments, it will be understood that the present disclosure is not intended to be limited to these embodiments. On the contrary, the present disclosure is intended to encompass all alternatives, modifications, and equivalents that may be included within the present disclosure as defined by the attached embodiments.

[0042] The section titles used herein are for organizational purposes only and should not be construed as limiting the subject matter in any way. In the event that any material incorporated by reference conflicts with any term defined herein or any other express content of this specification, this specification shall prevail.

[0043] I. Definition

[0044] Before describing the contents of this teaching in detail, it should be understood that the present disclosure is not limited to specific compositions or process steps and that the contents themselves may vary. It should be noted that, as used in this specification and in the appended embodiments, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Thus, for example, references to “conjugates” include plural conjugates, and references to “cells” include plural cells or cell populations, etc. As used herein, the term “includes” and its grammatical variations are intended to be non-limiting and do not exclude other similar items that may replace or add to the items listed in the list.

[0045] Numerical ranges include the number defining the range. Measured values ​​and measurable values ​​are understood to be approximations, taking into account significant figures and errors associated with the measurement. Additionally, the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" is not intended to be limiting. Both the general description and the detailed description above are to be understood as merely illustrative and descriptive and not limiting to the content of this teaching.

[0046] Unless specifically stated in the specification, embodiments of the specification that enumerate various components as “comprising” are also deemed to be “consisting” or “essentially composed” of the enumerated components; embodiments of the specification that enumerate various components as “comprising” are also deemed to be “comprising” or “essentially composed” of the enumerated components; embodiments of the specification that enumerate various components as “essentially composed” are also deemed to be “consisting” or “comprising” of the enumerated components (this compatibility does not apply to the use of these terms in the claims).

[0047] The term "or" is used in an inclusive sense, i.e., equivalent to "and / or," unless the context clearly indicates otherwise.

[0048] The term “about” modifies each member of the list or each endpoint of the range when used prior to a list or range. The terms “about” or “approximate” mean an acceptable margin of error for a specific value determined by a person of ordinary skill in the art, depending in part on the method by which the value is measured or determined. The term “about” is used herein to mean within the typical tolerance range in the art. For example, “about” may be understood as about 2 standard deviations from the mean. In certain embodiments, about means + / -10%. In certain embodiments, about means + / -5%.

[0049] In some embodiments, the cell population refers to a population of at least 10^3, 10^4, 10^5, or 10^6 cells, for example, at least 10^7, 2 x 10^7, 5 x 10^7, or 10^8 cells.

[0050] The term "at least" preceding a number or a series of numbers is understood to include any number adjacent to the term "at least" and any subsequent number or integer that may be logically and clearly included in the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 17 nucleotides of a 20-nucleotide nucleic acid molecule" means that 17, 18, 19, or 20 nucleotides have the specified properties. If "at least" precedes a series of numbers or a range, it is understood that "at least" may modify each number in the series or range.

[0051] As used herein, “less than” or “less than” is understood as a value adjacent to the phrase and logically lower than a value or integer, or logically up to 0 in the context. For example, a bilayer region of “2 or fewer nucleotide base pairs” has 2, 1, or 0 nucleotide base pairs. When “less than” or “less than” precedes a series of numbers or ranges, each number in the series or range is understood to be modified.

[0052] As used herein, the range includes both an upper limit and a lower limit.

[0053] As used herein, when the maximum value is expressed as 100% (e.g., 100% inhibition or 100% encapsulation), it is understood that the value is limited by the detection method. For example, 100% inhibition is understood as inhibition to a level below the detection level of the assay, and 100% encapsulation is understood as the material to be encapsulated being undetectable outside the vesicle.

[0054] In the event of a conflict between the sequence of the present application and the indicated accession number or a position within the accession number, the sequence of the present application prevails.

[0055] Unless otherwise noted, the following terms and phrases as used herein are intended to have the following meanings.

[0056] As used herein, “knockdown” refers to a reduction in the expression of a specific gene product (e.g., full-length or wild-type mRNA, protein, or both) by gene editing in, for example, a cell, cell population, tissue, or organ. In some embodiments, gene editing may be evaluated by sequencing, for example, by next-generation sequencing (NGS). Expression may be reduced, for example, to at least 70%, 75%, 80%, 85%, 90%, 95%, or below the detection level of a assay compared to a suitable control in which the gene sequence is not modified. Knockdown of a protein may be measured by detecting the amount of protein in a tissue, cell population, or fluid of interest. Methods for measuring knockdown of mRNA are known and include sequencing of mRNA isolated from the tissue or cell population of interest. Flow cytometry is a known method for measuring knockdown of protein expression. In the case of secreted proteins, knockdown may be evaluated in tissue culture media or fluids such as blood, or serum or plasma derived therefrom. In some embodiments, “knockdown” may refer to a partial loss of expression of a specific gene product, for example, a reduction in the amount of full-length wild-type mRNA that is transcribed or translated into a full-length protein, or a reduction in the amount of protein expressed by a cell population. It is well known that changes in the mRNA sequence will result in reduced expression of the wild-type or full-length protein. In some embodiments, “knockdown” may refer to a partial loss of expression of a specific gene product, for example, the TGFBR2, IFNG, or TNFA gene product in body fluid or tissue culture medium. Modification of an endogenous nucleic acid sequence, for example, a sequence encoding TGFBR2, IFNG, or TNFA, may result in knockdown.

[0057] As used herein, “T cell receptor” or “TCR” refers to a receptor within a T cell. Generally, a TCR is a heterodimeric receptor molecule containing two TCR polypeptide chains, α and β. The α and β chain TCR polypeptides can form complexes with various CD3 molecules and can induce immune response(s), including inflammation and autoimmunity, after antigen binding. As used herein, TCR knockdown refers to the knockdown of any TCR gene, either partially or wholly, e.g., deletion of part of the TRBC1 gene alone, or in combination with the knockdown of other TCR genes, either partially or wholly.

[0058] "TRAC" is used to refer to the T cell receptor α 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.

[0059] "TRBC" is used to refer to T-cell receptor β-chains, e.g., TRBC1 and TRBC2. "TRBC1" and "TRBC2" refer to two homologous genes encoding the T-cell receptor β-chain, which is the gene product of the TRBC1 or TRBC2 gene.

[0060] The human wild-type TRBC1 sequence is available at NCBI Gene ID: 28639; Ensemble: ENSG00000211751. T-cell receptor beta constant, V_segment Translation Product, BV05S1J2.2, TCRBC1, and TCRB are gene synonyms for TRBC1.

[0061] The human wild-type TRBC2 sequence is available at NCBI Gene ID: 28638; Ensemble: ENSG00000211772. T-cell receptor beta constant, V_segment translation product, and TCRBC2 are gene synonyms for TRBC2.

[0062] As used herein, “immune response” refers to one or more immune system response(s), e.g., increased production or activity of immune system cells, e.g., non-limitingly T cells, B cells, natural killer cells, monocytes, neutrophils, eosinophils, basophils, mast cells, erythrocytes, dendritic cells, antigen-presenting cells, macrophages, or phagocytes, compared to an unstimulated control immune system. Exposure of the immune system to antigens, e.g., foreign or self-antigens, e.g., non-limitingly pathogens (microorganisms, viruses, prions, fungi, etc.), allergens (dust, pollen, house dust mites, etc.), toxins (chemicals, drugs, etc.), or physiological changes (hypercholesterolemia, obesity, organ transplantation, etc.) may trigger an immune response. The immune response may also include a response mediated by donor cells of an immune attack on host tissues following hematopoietic stem cell transplantation in GvHD. Inflammation may occur as a result of the immune response. Alternatively or additionally, the immune response may also include a response in which host cells mediate an immune attack on donor tissue following solid organ transplantation or the introduction of other non-host cells via transplantation or transfusion. The immune response may target, attack, eliminate, or neutralize antigens, e.g., foreign or self-antigens. The immune response may be desirable or undesirable. The immune response may be acute or chronic. The immune response may damage the cells, tissues, or organs where the immune response occurs.

[0063] As used herein, “autoimmune response” refers to one or more immune system response(s) to self-antigens generated by, for example, the subject’s own cells, tissues, or organs. An autoimmune response may result in increased production or activity of immune system cells, for example, to a non-limiting extent, T cells, B cells, natural killer cells, monocytes, neutrophils, eosinophils, basophils, mast cells, erythrocytes, dendritic cells, antigen-presenting cells, macrophages, or phagocytes, compared to a suitable control, for example, a healthy control. An autoimmune response may result in inflammation, for example, persistent inflammation, or lead to an autoimmune disease. An autoimmune response may target, attack, eliminate, or neutralize self-antigens generated by the subject’s own cells, tissues, or organs that may lead to an autoimmune disease.

[0064] As used herein, “suppressing” immune response(s) refers to reducing or inhibiting the level of one or more immune system response(s), e.g., the production or activity of immune system cells, compared to a suitable control, e.g., a control not treated with the engineered T cells described herein or prior to treatment. “Suppressing” immune response(s) may refer to reduced production or activity of immune system cells compared to a suitable control, e.g., a control not treated with the engineered T cells described herein or prior to treatment. “Suppressing” immune response may refer to increasing immune tolerance. For example, the production or activity of immune system cells may be measured by cell counting, e.g., lymphocyte counting or splenocyte counting; cell activity, e.g., T cell assay; or gene or protein expression, e.g., biomarker expression; Here, production or activity is reduced to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or less than the detection level of the assay compared to a suitable control, for example, a control not treated with or prior to treatment with the engineered T cells described herein.

[0065] As used herein, “autoimmune disease” or “autoimmune disorder” refers to a pathological condition characterized by a pathological immune response to the 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 are autologous or allogeneic.

[0066] As used herein, "immune disorder" is understood as a disease or condition characterized by a pathological or undesirable immune response of a subject. In certain embodiments, the immune disorder is an autoimmune disease. In certain embodiments, the immune disorder is a GvHD. In certain embodiments, the subject with the immune disorder requires suppression of the immune response. In certain embodiments, the subject with the immune disorder requires an increase in immune tolerance.

[0067] "T cells" play a central role in the immune response following exposure to an antigen. T cells can be naturally occurring, or non-natural when they are formed by manipulation, for example, from stem cells, or by transdifferentiation, for example, the reprogramming of somatic cells. T cells can be distinguished from other lymphocytes by the presence of T cell receptors on the cell surface. This definition includes ordinary adaptive T cells, including helper CD4+ T cells, cytotoxic CD8+ T cells, memory T cells, and regulatory CD4+ T cells, and innate-like T cells, including natural killer T cells, mucosal-associated constant T cells, and gamma delta T cells. In some embodiments, the T cells are CD4+. In some embodiments, the T cells are CD3+ / CD4+. In some embodiments, the T cells are CD8+. In some embodiments, T cells may be further characterized as stem-cell-like memory T cells (Tscm) (CD62L+CD45RO-), central memory T cells (Tcm;) (CD62L+CD45RO+), effector memory T cells (Tem;) (CD62L-CD45RO+), and terminal effector T cells (Tte;) (CD62L-CD45RO-); and the population of T cells may include any one or more of these T cell subtypes.

[0068] "Regulatory T cells" or "Treg" refer to specialized T cells that play a central role in suppressing excessive immune response(s), including inflammation and autoimmunity. Tregs may be naturally occurring (also referred to interchangeably herein as natural Tregs ("nTreg") and thymic Tregs ("tTreg"), or unnatural when Tregs are formed by manipulation, for example, including the insertion of a sequence encoding the dmTGFB1 molecule. When formed by manipulation, Tregs may include, for example, modifications to endogenous nucleic acid sequences encoding TGFBR2, IFNG, and TNFA, or additional modifications by the insertion of at least one sequence(s) encoding knockdown and regulatory T cell promoter molecules. Naturally occurring Tregs, natural Tregs, or nTregs, sometimes referred to as thymic Tregs or tTregs, are specialized T cells that typically develop in the thymus and function by suppressing excessive immune response(s). In some embodiments, cells similar to normal T cells or a normal T cell population, for example, a T cell population not enriched for the presence of nTreg cells, may include the insertion of a sequence encoding the dmTGFB1 molecule, optionally additionally, for example For example, endogenous nucleic acid sequences encoding TGFBR2, TNFA, and IFNG may be further modified, e.g., knockdown of nucleic acid sequences encoding TGFBR2, TNFA, and IFNG, and sequence(s) encoding regulatory T cell promoter molecules may be inserted into cells to be engineered to exhibit regulatory T cell phenotypic characteristics and repressive functions, and these may be referred to as transduced or “engineered” T cells. In some embodiments, engineered T cells include the insertion of a sequence encoding a dmTGFB1 molecule; modification of an endogenous nucleic acid sequence encoding TGFBR2, modification of an endogenous nucleic acid sequence encoding IFNG, and modification of an endogenous nucleic acid sequence encoding TNFA; and the insertion of a heterologous regulatory T cell promoter molecule such as IL10 or CTLA4.Modifications to endogenous nucleic acid sequences, for example, modifications that knock down the expression of endogenous genes, may include or consist of one or more indels or substitution mutations in the genome sequence.

[0069] As used herein, dmTGFB1 is a mutant TGFB1 based on wild-type TGFB1 from any species, including, for example, human, mouse, rat, or synomolgus TGFB1. In certain embodiments, dmTGFB1 is human dmTGFB1, i.e., a mutant TGFB1 relative to wild-type human TGFB1. Code sequences and amino acid sequences for wild-type TGFB1 are readily available 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 (synomolgus). Each accession number is incorporated by reference to the version available as of the filing date of this application. The ability to map mutations to wild-type sequences is within the capability of those skilled in the art.

[0070] Transforming growth factor beta-1 (TGFB1) is synthesized as a large precursor molecule. The TGFB1 free protein contains a 29-amino acid signal peptide that is proteolytically cleaved. TGFB1 is further cleaved after amino acid 278 to form the latent-associated peptide (LAP) and active TGFB1. LAP is dimerized by interchain disulfide linkages at C223 and C225. TGFB1 can be secreted as an inactive small latent complex consisting of a mature TGF-β1 homomer non-covalently linked to the LAP homomer at LAP residues 153–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 covalently linked between C33 within the LAP chain and the latent TGFB-binding protein (LTBP). LTBP facilitates TGFB1 folding, secretion, and possibly targeting to the extracellular matrix. Activation of LLC occurs through the N-terminal domain of LTBP, which binds to the extracellular matrix.

[0071] Camurati-Engelmann disease (CED) results from a domain-specific heterozygous mutation in the transforming growth factor-beta-1 gene (TGFB1; OMIM entry 190180, incorporated by reference as a version available on the filing date of this application) on chromosome 19q13. Mutations reported in the Camurati-Engelmann disease family include the following (derived from Janssen et al., 2006. J Med Genet. 43:1):

[0072]

[0073] As shown in the table, most pathogenic variants in individuals with CED result in a single amino acid substitution at the carboxyl terminus of the TGFB1 latent-associated peptide (LAP). The substitution is located near the interchain disulfide bond site between LAP homomers. These pathogenic variants interfere with the dimerization of LAP and its binding to active TGFB1 (Walton et al., 2010. J Biol Chem. 285:17029-37), thereby increasing the release of active TGFB1 from cells. Walton et al. further demonstrated that the stability of the resulting large latent complex depends on the covalent dimerization of LAP facilitated by key residues (F198, D199, V200, L208, F217, and L219) at the dimer interface. R218H mutant fibroblasts from individuals with CED showed increased active TGFB1 in cell medium compared to normal fibroblasts (Saito et al., 2001. J Biol Chem. 276:11469-72). In vitro analysis of R218C, H222D, and C225R mutant constructs also showed increased active TGF-β1 in the medium of transfected cells. In contrast, the Leu11_Leu13dup and Tyr81His pathogenic variants reduced the amount of secreted TGFB1. However, in a luciferase reporter assay specific to TGFB1-induced transcriptional responses, the mutant cells showed increased luciferase activity, suggesting intracellular activation of the receptor (Janssens et al., 2003. J Biol Chem. 278:7718-24).

[0074] As used herein, in certain embodiments, dmTGFB1 is a TGFB1 containing mutations at two or more amino acid positions relative to wild-type TGFB1, which reduces the stability of the resulting large latent complex dependent on the covalent dimerization of LAP compared to each TGFB1 single mutant alone. Mutations at two or more amino acid positions may include mutations at positions selected from F198, D199, V200, L208, F217, L219, R218, H222, C223, and C225 relative to wild-type human TGFB1.

[0075] In certain embodiments, dmTGFB1 is TGFB1 comprising mutations at two or more amino acid positions relative to wild-type TGFB1 at positions 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 a cell in which TGFB1 is typically expressed. In some embodiments, dmTGFB1 comprises mutations that occur naturally, for example, in CED. In some embodiments, dmTGFB1 comprises non-natural mutations that do not occur naturally, for example, in CED.

[0076] In certain embodiments, human dmTGFB1 comprises mutations at two or more amino acid positions selected from R218, H222, C223, and C225 compared to wild-type human TGFB1.

[0077] In certain embodiments, human dmTGFB1 comprises two or more mutations selected from R218C / H, H222D, C223S / R / G, and C225R compared to wild-type human TGFB1.

[0078] In certain embodiments, human dmTGFB1 comprises two or more mutations selected from R218C / H and C225R compared to wild-type human TGFB1, optionally R218H and C225R.

[0079] The dmTGFB1 provided herein has more free, active dmTGFB1 than any single mutant alone. In certain embodiments, the increase in active dmTGFB1 is at least additive compared to the activity of a single mutant alone. In certain embodiments, the increase in active dmTGFB1 is much greater than additive compared to the activity of a single mutant alone. Methods for determining the levels of total TGFB1 and active TGFB1 are known in the art, as provided in the following examples, using commercially available kits (e.g., LEGEND MAX Total TGF-β1 ELISA Kit (BioLegend, Catalog 436707), and LEGEND MAX Free Active TGF-β1 ELISA Kit).

[0080] As used herein, "regulatory T cell promoter" refers to a molecule that promotes the conversion of normal T cells into regulatory T cells, comprising an immunosuppressive molecule and a Treg transcription factor. Additionally, the regulatory T cell promoter also refers to a molecule that confers regulatory activity to normal T cells, comprising a Treg-associated immunosuppressive molecule and a transcription factor. The regulatory T cell promoter may be used in combination with dmTGFB1 to promote the conversion of normal T cells into regulatory T cells or to confer regulatory activity to normal T cells. Examples of immunosuppressive molecules used in combination with dmTGFB1 include Interleukin-10 (IL10), Cytotoxic T-Lymphocyte-Associated Protein 4 (CTLA4), T-cell Immunoreceptor with Ig and ITIM Domains (TIGIT), Indoleamine 2,3-Deoxygenase 1 (IDO1), Ectonucleoside Triphosphate Diphosphohydrolase 1 (ENTPD1), 5'-Nucleotidase Ecto (NT5E), Interleukin-22 (IL22), Ampyregulin (AREG), Interleukin-35 (IL35), Leucine-Rich Repeat-Containing 32 (GARP), CD274 molecule (CD274), Forkhead Box P3 (FOXP3), IKAROS Family Zinc Finger 2 (IKZF2), Familial Eosinophilia (EOS), Interferon Regulatory Factor 4 (IRF4), and Lymphatic Enhancer Binding Factor. It includes, but is not limited to, 1 (LEF1), BTB domain and CNC homolog 2 (BACH2), and interleukin 2 receptor subunit alpha (IL2RA, CD25). In some embodiments, the regulatory T cell promoter molecules may be used in specific combinations, for example, IL10 and CTLA4, ENTPD1 and NT5E, and IL22 and AREG. In particular, a combination of IL10 and CTLA4 for use with dmTGFB1 is provided herein. In some embodiments, the expression of the immunosuppressive molecule may be promoted by the expression of transcription factors such as FoxP3, Helios, Eos, IRF4, Lef1, or BACH2.

[0081] In some embodiments, normal T cells may be engineered to modify, insert, or delete sequences within the genome, and “engineered” T cells exhibit one or more phenotypic features and repressive functions of natural regulatory T cells. For example, “engineered” T cells may exhibit repressive activity in a mixed lymphocyte response assay as provided in Example 2.4 below, or inhibit graft-versus-host disease in a statistically significant manner, for example, in the mouse model presented in Example 3.2 below (see also, e.g., Parmar et al., Ex vivo fucosylation of third-party human regulatory T cells enhances anti-graft-versus-host disease potency in vivo, Blood 125(9)(2015)). In some embodiments, “engineered” T cells are normal T cells modified by inserting the coding sequence of a regulatory T cell promoter molecule and having modified expression of pro-inflammatory cytokines such as TNFA, e.g., knockdown, in combination with one or both of IFNG and IL17A. In some embodiments, the starting T cell population for the operation is not enriched with the presence of natural Tregs, for example, the starting T cell population has less than 20% natural Tregs.

[0082] As used herein, "pro-inflammatory" molecules, e.g., cytokines, increase the immune response as described herein and, e.g., reduce the efficacy of Treg in a dose-response manner in a 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), natural killer cell granule protein 7 (NKG7), Fas ligand (FasL, NF superfamily, member 6), lyanodine receptor 2 (RYR2), and colony-stimulating factor 2 (CSF2).

[0083] As used herein, "targeting receptor" refers to a receptor present on the surface of a cell, e.g., a T cell, that causes the cell to bind to a target site, e.g., a specific cell or tissue within an organism. Targeting receptors are receptors for cell surface molecules operably linked via at least a transmembrane domain to an internal signaling domain capable of activating T cells upon binding to chimeric antigen receptors (CARs), T-cell receptors (TCRs), and the extracellular receptor portion of a protein, e.g., mucosal addressin cell adhesion molecule-1 (MADCAM-1), TNFA, CD70, 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 (ligand) superfamily member 7 (TNFSF7), TNF receptor superfamily member 17 (TNFRSF17), dipeptidyl peptidase-like 6 (DPP6), and solute carrier family 2 member. Includes, but not limited to, 2 (SCL2A2), glutamate decarboxylase (GAD2), demoglain 3 (DSG3) and MHC class I HLA-A (HLA-A*02).

[0084] As used herein, "chimeric antigen receptor" refers to an extracellular antigen recognition domain, e.g., scFv, VHH, nanobodies; which is operably connected to an intracellular signaling domain that activates T cells when an antigen binds. The CAR consists of four regions: an antigen recognition domain, an extracellular hinge region, a transmembrane domain, and an intracellular T-cell signaling domain. Such receptors are well known in the art (e.g., see WO2020092057, WO2019191114, WO2019147805, WO2018208837, the relevant parts of which are incorporated herein by reference). An inverted universal CAR that facilitates the binding of immune cells to target cells via an adapter molecule (e.g. see WO2019238722, the entire contents of which are incorporated herein by reference) is also considered. CAR can be targeted against any antigen to which antibodies may develop and is typically induced by a molecule appearing on the surface of the targeted cell or tissue. In some embodiments, CAR can target engineered T cells to the gastrointestinal tract, for example, CAR targets MAdCAM-1. In some embodiments, CAR can target engineered T cells to activated immune cells (e.g., T cells or B cells expressing upregulated targets), for example, CAR targets CD70. In some embodiments, CAR can target engineered T cells to tissues containing endothelial cells, for example, CAR targets VCAM-1 to suppress the immune response in disorders such as Crohn's disease and multiple sclerosis. In some embodiments, CAR can target engineered T cells to endothelial cells, for example, CAR targets CEACAM6 to suppress the immune response in disorders such as Crohn's disease.In some embodiments, the CAR may target engineered T cells to pre-B cells, for example, the CAR may target CD19 to suppress the immune response in disorders such as multiple sclerosis and systemic lupus erythematosus. In some embodiments, the CAR may target engineered T cells to B lymphocytes, for example, the CAR may target CD20 to suppress the immune response in disorders such as multiple sclerosis and systemic lupus erythematosus. In some embodiments, the CAR may target engineered T cells to inflammatory tissue, for example, the CAR may target TNFA to suppress the immune response in disorders such as rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, or Crohn's disease. In some embodiments, the CAR may target engineered T cells to inflammatory tissue, for example, the CAR may target TGF-b1 to suppress the immune response in disorders such as inflammatory bowel disease, ulcerative colitis, or Crohn's disease. In some embodiments, the CAR may target engineered T cells to neural tissue, for example, the CAR may target MBP, MOG, or PLP1 to suppress the immune response in disorders such as multiple sclerosis. In some embodiments, the CAR may target engineered T cells to immune cells expressing TNFSF7, including but not limited to activated T lymphocytes, B lymphocytes, dendritic cells, and / or NK cells, for example, the CAR may target TNFSF7 to suppress the immune response in disorders such as Crohn's disease, ulcerative colitis, systemic lupus erythematosus, or multiple sclerosis. In some embodiments, the CAR can target engineered T cells to tissues containing mature B lymphocytes, for example, the CAR targets TNFRSF17 to suppress the immune response in disorders such as systemic lupus erythematosus.In some embodiments, the CAR may target engineered T cells to synovial tissue, for example, the CAR targets citrullinated vimentin to suppress the immune response in disorders such as rheumatoid arthritis. In some embodiments, the CAR targets dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), demoglain 3 (DSG3), or MHC class I HLA-A (HLA-A*02). Additional CAR targets, for example, inflammatory antigens, are known in the art. For example. Refer to WO2020092057A1, the contents of which are incorporated herein in their entirety.

[0085] As used herein, “treatment” refers to any administration or application of a therapeutic agent for a disease or disorder in a subject, including inhibition of the disease, inhibition of its progression, alleviation of one or more symptoms of the disease, cure of the disease, prevention of one or more symptoms of the disease, or prevention of the recurrence of one or more symptoms of the disease. Treating an autoimmune or inflammatory response or disorder results in the alleviation of disease-specific symptoms by alleviating inflammation associated with the specific disorder. Treatment with engineered T cells described herein may be used before, after, or in combination with additional therapeutic agents, e.g., anti-inflammatory agents, immunosuppressants, or biological agents for the treatment of autoimmune disorders, e.g., Remicade, Humira.

[0086] A "promoter" refers to a regulatory region that controls the expression of a gene to which the regulatory region is connected.

[0087] "Polynucleotide" and "nucleic acid" are used herein to refer to a multimeric compound comprising a nucleoside or a nucleoside analog having nitrogenous heterocyclic bases or base analogs linked together along a backbone, including conventional RNA, DNA, mixed RNA-DNA, and polymers that are analogs thereof. The nucleic acid "backbone" may consist of various linkages including one or more of sugar-phosphodiester linkages, peptide-nucleic acid linkages ("peptide nucleic acid" or PNA; PCT No. WO 95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar moiety of the nucleic acid may be ribose, deoxyribose, or a similar compound having a substitution, e.g., 2' methoxy, 2' halide, or 2'-O-(2-methoxyethyl)(2'-O-moe) substitution. Nitrogenous bases are ordinary bases (A, G, C, T, U), analogs thereof (e.g., modified uridins such as 5-methoxyuridine, pseudouridine, or N1-methylpseudouridine); inosine; derivatives of purines or pyrimidines (e.g., N 4 -methyl deoxyguanosine, deaza- or aza-purine, deaza- or aza-pyrimidine, pyrimidine base having a substituent at the 5 or 6 position (e.g., 5-methylcytosine), purine base having a substituent at the 2, 6 or 8 position, 2-amino-6-methylaminopurine, O 6 -methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine and O 4 -alkyl-pyrimidine; may be U.S. Patent No. 5,378,825 and PCT No. WO 93 / 13121). For general discussion, The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11 thRefer to ed., 1992). The nucleic acid may comprise one or more “basic” residues in which the backbone does not contain a nitrogenous base for the polymer position(s) (U.S. Patent No. 5,585,481). The nucleic acid may comprise only a conventional RNA or DNA sugar, base, and linkage, or may comprise both a conventional component and a substitution (e.g., a conventional base having a 2' methoxy linkage, or a polymer containing both a conventional base and one or more base analogs). The nucleic acid comprises “locked nucleic acid” (LNA), which is an analog containing one or more LNA nucleotide monomers having a bicyclic furanose unit locked in RNA that mimics a sugar form that enhances 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 due to the presence of uracil or its analogues in RNA and thymine or its analogues in DNA.

[0088] As used herein, the term "polypeptide" refers to a multimeric compound containing amino acid residues capable of adopting a three-dimensional structure. Polypeptides include, but are not limited to, enzymes, enzyme precursor proteins, regulatory proteins, structural proteins, receptors, nucleoprotein-binding proteins, antibodies, etc. Polypeptides may include, but are not limited to, post-translational modifications, non-natural amino acids, prosthetic groups, etc.

[0089] As used herein, a “open read frame” or “ORF” of a gene refers to a sequence consisting of a series of codons that specify the amino acid sequence of the protein encoded by the gene. An ORF generally begins with a start codon (e.g., ATG in DNA or AUG in RNA) and ends with a stop codon, e.g., TAA, TAG, or TGA in DNA or UAA, UAG, or UGA in RNA.

[0090] "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 tracrRNA). crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or as two separate RNA molecules (double guide RNA, dgRNA). "Guide RNA" or "gRNA" refers to each type. trRNA may be a naturally occurring sequence or a trRNA sequence having a modification or variation relative to the naturally occurring sequence.

[0091] As used herein, “guide sequence,” “guide region,” “targeting sequence,” “spacer,” “spacer sequence,” etc. refer to a sequence within gRNA that functions to guide the gRNA to a target sequence for binding or modification (e.g., cleavage) by an RNA-guided nikcasase that is complementary to the target sequence. The guide sequence may be a nucleotide of length 20, for example, in the case of Streptococcus pyogenes (i.e., Spy Cas9 (also referred to as SpCas9)) and related Cas9 homologs / centrosomes. Additionally, shorter or longer sequences of lengths of, for example, 15, 16, 17, 18, 19, 21, 22, 23, 24, or 25 nucleotides may be used as a guide. The guide sequence may be 20 to 25 nucleotides long, for example, for Nme Cas9, 20, 21, 22, 23, 24, or 25 nucleotides long. For example, a guide sequence of 24 nucleotides long may be used with Nme Cas9, for example, Nme2 Cas9.

[0092] In some embodiments, the target sequence is located, for example, on a genomic locus or 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 one, two, three, or four mismatches, wherein the total length of the target sequence is at least 17, 18, 19, or 20 base pairs. In some embodiments, the guide sequence and the target region may contain 1 to 4 mismatches, wherein the guide sequence comprises at least 17, 18, 19, or 20 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, for example, at least 80%, 85%, 90%, or 95% 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, that is, one nucleotide that is not identical or complementary according to the reference sequence. For example, the guide sequence and the target sequence may contain 1-2 mismatches, preferably 1 or fewer mismatches, wherein the total length of the target sequence is 19, 20, 21, 22, 23, or 24 or more nucleotides.In some embodiments, the guide sequence and the target region may contain one or two mismatches, wherein the guide sequence comprises at least 24 or more nucleotides. In some embodiments, the guide sequence and the target region may contain one or two mismatches, wherein the guide sequence comprises 24 nucleotides. Mismatch locations are known to the art as provided herein, for example, PAM distal mismatches tend to be more tolerant than PAM proximal matches. Tolerance for mismatches at other locations is known to the art (see, for example, Sternberg et al., 2015, Nature:527:110-113).

[0093] As used herein, "target sequence" or "genome target sequence" refers to a nucleic acid sequence located at a target gene genomic locus on the positive or negative strand, which is complementary to the gRNA guide sequence, that is, sufficiently complementary to the gRNA guide sequence to allow specific binding of the guide to the target sequence. The interaction between the target sequence and the guide sequence induces the RNA-guided DNA binder to the target sequence and potentially nicking or cleavage (depending on the activity of the agent). The specific length of the target sequence and the number of possible mismatches between the target sequence and the guide sequence depend, for example, on the identity of the Cas9 nuclease induced by the gRNA. The target sequence for the Cas protein includes both the positive and negative strands of the genomic DNA (i.e., the given sequence and its reverse complement) because the nucleic acid substrate for the Cas protein is a double-stranded nucleic acid. Therefore, when a guide sequence is described as "complementary to a target sequence," it should be understood that the guide sequence can induce an RNA-guided DNA binder (e.g., dCas9 or damaged Cas9) to bind to the reverse complement of the target sequence. Thus, in some embodiments, when the guide sequence binds to the reverse complement of the target sequence, the guide sequence is identical to a specific nucleotide of the target sequence (e.g., a target sequence not containing PAM), except for the substitution of U instead of T in the guide sequence.

[0094] The target sequence for the RNA-guided DNA binder includes both the positive and negative strands of genomic DNA (i.e., the given sequence and the reverse complement of this sequence) because the nucleic acid substrate for the RNA-guided DNA binder is a double-stranded nucleic acid. Therefore, when a guide sequence is said to be "complementary to the target sequence," it should be understood that the guide sequence is capable of inducing the guide RNA to bind to the reverse complement of the target sequence. Thus, in some embodiments, when the guide sequence binds to the reverse complement of the target sequence, the guide sequence is identical to a specific nucleotide of the target sequence (e.g., a target sequence not containing PAM), except for a substitution of T to U within the guide sequence.

[0095] As used herein, “RNA-guided DNA-binding agent” means a polypeptide or a complex of polypeptides having RNA and DNA binding activity, or a DNA-binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the sequence of the RNA. The term RNA-guided DNA-binding agent also includes a nucleic acid encoding such a polypeptide. An exemplary RNA-guided DNA-binding agent includes a Cas cleavage / nikcasase. An exemplary RNA-guided DNA-binding agent may include an inactivated form thereof (“dCas DNA-binding agent”), for example, if the corresponding agent is modified to allow DNA cleavage, for example, through fusion with a FokI cleavage domain. “Cas nuclease” as used herein includes a Cas cleavage and a Cas nikcasase. Cas cleavage enzymes and Cas nickases include the Csm or Cmr complex of a type III CRISPR system, its Cas10, Csm1, or Cmr2 subunits, the cascade complex of a type I CRISPR system, its Cas3 subunit, and class 2 Cas nucleases. As used herein, "class 2 Cas nuclease" is a single-strand polypeptide having RNA-guided DNA binding activity. Class 2 Cas nucleases further include class 2 Cas cleavage enzyme / nickase having RNA-guided DNA cleavage or nickase activity (e.g., H840A, D10A, or N863A variants), and class 2 dCas DNA-binding agents in which the cleavage enzyme / nickase activity is inactivated, for example, if the agent is modified to allow DNA cleavage or has C-to-T deaminase or A-to-G deaminase activity. Cas nickases include nucleases in which one of the RuvC or HNH domains of the Cas protein is cleaved so that only a single strand is cleaved by the nuclease.In some embodiments, the RNA-guided DNA-binding agent comprises a deaminase domain and an RNA-guided DNA nikase, e.g., Cas9 nikase. Class 2 Cas nucleases include, e.g., 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 included in its entirety for reference. For example, Zetsche, Tables S1 and S3 reference For example, Makarova et al. Nat Rev Microbiol , 13(11): 722-36(2015); Shmakov et al., Molecular Cell, See 60:385-397 (2015). As used herein, RNA-guided DNA-binding agents (e.g., Cas nuclease, Cas9 nuclease, or S. phyogenes ( S. pyogenes ) Cas9 nuclease or Neisseria meningitidis( Neisseria meningitidis The delivery of ) Cas9 nuclease includes the delivery of polypeptides or mRNA.

[0096] As used herein, the terms “editor” or “base editor” refer to an agent comprising a polypeptide capable of modifying bases (e.g., A, T, C, G, or U) within a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, the editor may deaminate bases within the nucleic acid. In some embodiments, the editor may deaminate bases within a DNA molecule. In some embodiments, the editor may deaminate cytosine (C) in DNA. In some embodiments, the editor is a fusion protein comprising an RNA-guided nikcase fused to a cytidine deaminase domain. In some embodiments, the editor is a fusion protein comprising an RNA-guided nikcase fused to an APOBEC3A deaminase (A3A). In some embodiments, the editor comprises a Cas9 nikcase fused to an APOBEC3A deaminase (A3A). In some embodiments, the editor is a fusion protein comprising an enzymatically inactive RNA-guided DNA-binding protein fused to a cytidine deaminase domain.

[0097] As used herein, “ribonucleoprotein (RNP)” or “RNP complex” refers to a guide RNA together with an RNA-guided DNA binder such as a Cas nuclease, e.g., a Cas cleavage enzyme, a Cas nickase, or a dCas DNA binder (e.g., Cas9). In some embodiments, the guide RNA guides an RNA-guided DNA binder, such as Cas9, to a target sequence, the guide RNA hybridizes with the target sequence and the agent binds to the target sequence; if the agent is a cleavage enzyme or a nickase, it may cleave or nick after binding.

[0098] As used herein, the terms “uracil glycosylase inhibitor,” “uracil-DNA glycosylase inhibitor” or “UGI” refer to proteins capable of inhibiting uracil-DNA glycosylase (UDG) base-excision repair enzymes (e.g., UniPROT ID: P14739; MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSKRTADGSEFESPKKKRKVE (SEQ No. 226); or TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML (SEQ No. 227)).

[0099] Exemplary nucleotide and polypeptide sequences of a Cas9 molecule are provided below. Methods for identifying alternative nucleotide sequences encoding a Cas9 polypeptide sequence, including alternative naturally occurring variants, are known in the art. Sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any Cas9 nucleic acid sequence or a nucleic acid sequence encoding an amino acid sequence provided herein are also considered. In certain embodiments, a nucleotide sequence encoding a Cas9 amino acid sequence is not a naturally occurring Cas9 nucleotide sequence. Sequences having at least 95%, 96%, 97%, 98%, or 99% identity with any Cas9 amino acid sequence provided herein are also considered. In certain embodiments, a Cas9 amino acid sequence is not a naturally occurring Cas9 sequence.

[0100] Exemplary open read frame for Spy Cas9

[0101]

[0102]

[0103]

[0104] Exemplary amino acid sequence for Spy Cas9

[0105]

[0106] Exemplary open read frame for Spy Cas9

[0107]

[0108]

[0109]

[0110] Exemplary open read frame for Spy Cas9 with Hibit tag

[0111]

[0112]

[0113]

[0114] Exemplary amino acid sequence for Spy Cas9 with Hibit tag

[0115]

[0116] Exemplary amino acid sequence for Spy Cas9 nikcase

[0117]

[0118]

[0119] Exemplary open read frame for Nme2Cas9

[0120]

[0121]

[0122] Exemplary mRNA for Nme2Cas9 nickase

[0123]

[0124]

[0125] Exemplary amino acid sequence for Nme2Cas9

[0126]

[0127] Exemplary mRNA encoding UGI

[0128]

[0129] Open read frame for UGI

[0130]

[0131] Exemplary amino acid sequence for UGI

[0132]

[0133] Exemplary amino acid sequence for UGI

[0134]

[0135] As used herein, “ribonucleoprotein (RNP)” or “RNP complex” refers to a guide RNA together with an RNA-guided DNA binder such as a Cas nuclease, e.g., a Cas cleavage enzyme, a Cas nikcaase, or a dCas DNA binder (e.g., Cas9). In some embodiments, the guide RNA guides an RNA-guided DNA binder, such as Cas9, to a target sequence, the guide RNA hybridizes with the target sequence, and the agent binds to the target sequence; where the agent is a cleavage enzyme or a nikcaase, a double-stranded DNA cleavage or a single-stranded DNA cleavage may follow after binding.

[0136] As used herein, a first sequence is considered to "contain a sequence having at least X% identity" with respect to a second sequence if the alignment of the first sequence with respect to the second sequence shows that at least X% of the positions of the second sequence match the first sequence. For example, the sequence AAGA contains a sequence having 100% identity with the sequence AAG, because the alignment would provide 100% identity in that it matches all three positions of the second sequence. Differences between RNA and DNA (generally the exchange of uridine to thymidine or vice versa) and the presence of nucleoside analogs such as modified uridine do not contribute to differences in identity or complementarity among polynucleotides, as long as the related nucleotides (e.g., thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as complement). Thus, for example, the sequence 5'-AXG, where X is any modified uridine such as pseudouridine, N1-methylpseudouridine, or 5-methoxyuridine, is considered 100% identical to AUG in that both are perfectly complementary to the same sequence (5'-CAU). An exemplary alignment algorithm is the Smith-Waterman and Needleman-Wunsch algorithm, which is well known in the art. A person of ordinary skill in the art will understand which choice of algorithm and parameter settings is appropriate for a given pair of sequences to be aligned; generally, for sequences that are similar in length and have an expected identity of >50% for amino acids or >75% for nucleotides, the Needleman-Wunsch algorithm provided by EBI on the www.ebi.ac.uk web server is generally appropriate.

[0137] As used herein, if X% of the bases of the first sequence are paired with the second sequence, the first sequence is considered to be “X% complementary” to the second sequence. For example, the first sequence 5’AAGA3’ is 100% complementary to the second sequence 3’TTCT5’, and the second sequence is 100% complementary to the first sequence. In some embodiments, the first sequence 5’AAGA3’ is 100% complementary to the second sequence 3’TTCTGTGA5’, while the second sequence is 50% complementary to the first sequence.

[0138] As used herein, “mRNA” is used to refer to a polynucleotide comprising an open read frame that is wholly or primarily RNA, or modified RNA, and can be translated into a polypeptide (i.e., can serve as a substrate for translation by ribosomes and amino-acylated tRNA). mRNA may comprise a phosphate-sugar backbone comprising ribose residues or analogs thereof, for example, 2'-methoxyribose residues. In some embodiments, the sugar of the mRNA phosphate-sugar backbone essentially consists of ribose residues, 2'-methoxyribose residues, or a combination thereof.

[0139] As used herein, “insertion / deletion” refers to an insertion / deletion mutation consisting of multiple nucleotides that are inserted or deleted at a double-strand break (DSB) site in the target nucleic acid. As used herein, when the insertion / deletion type results in an insertion, the insertion is a random insertion at the double-strand break site and is not induced by or based on a template sequence.

[0140] As used herein, “target sequence” refers to a nucleic acid sequence within a target gene that is complementary to the guide sequence of the gRNA. The interaction between the target sequence and the guide sequence induces an RNA-guided DNA binder to the target sequence and potentially nick or cleave it (depending on the activity of the agent).

[0141] As used herein, “polypeptide” refers to a wild-type or variant protein (e.g., a mutant, a fragment, a fusion, or a combination thereof). The variant polypeptide 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% of the functional activity of the wild-type polypeptide. In some embodiments, the variant is at least 70%, 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of the wild-type polypeptide. In some embodiments, the variant polypeptide may be an overactive variant. In certain cases, the variant retains about 80% to about 120%, 140%, 160%, 180%, 200%, 300%, 400%, 500% or more of the functional activity of the wild-type polypeptide. As used herein, “heterogeneous gene” refers to a gene introduced into a cell as an exogenous source (e.g., inserted into a genomic locus such as a safe harbor locus containing a TCR locus). That is, the introduced gene is heterogeneous with respect to the insertion site. A polypeptide expressed from such a heterogeneous gene is referred to as a “heterogeneous polypeptide.” The heterogeneous gene may be naturally occurring or engineered and may be wild-type or variant. The heterogeneous gene may include a nucleotide sequence other than the sequence encoding the heterogeneous polypeptide (e.g., an internal ribosome entry site). Heterogeneous genes may be genes or variants (e.g., mutants) that occur naturally in the genome as wild types. For example, even if a cell contains the gene of interest (as a wild type or variant), the same gene or its variant may be introduced as an exogenous source for expression, for example, at a highly expressed locus.A heterogene may also be a gene that does not naturally occur in the genome or expresses a heterogeneous polypeptide that does not naturally occur in the genome. "Heterogeneous gene," "exogenous gene," and "transplant gene" are used interchangeably. In some embodiments, the heterogeneous gene or transplant gene comprises an exogenous nucleic acid sequence, for example, the nucleic acid sequence is not endogenous to the recipient cell. In some embodiments, the heterogeneous gene or transplant gene comprises an exogenous nucleic acid sequence, for example, a nucleic acid sequence that does not naturally occur in the recipient cell. For example, the heterogeneous gene may be heterogeneous with respect to the insertion site of the heterogeneous gene and its recipient cell.

[0142] A "safe harbor" locus is a locus within the genome where a gene can be inserted without significant adverse effects on the cell. Non-limiting examples of safe harbor locus targeted by the nuclease(s) used herein include AAVS1 (PPP1 R12C), TCR, B2M, and any locus targeted for knockdown described herein, e.g., TNFA, IFNG, IL17A, and IL6 genomic locus. In some embodiments, insertion into locus or locus targeted for knockdown, such as the TRC gene, e.g., the TRAC gene, is advantageous for allogeneic cells. Other suitable safe harbor locus is known in the art.

[0143] II. Composition

[0144] A. Targeting receptors

[0145] In some embodiments, engineered T cells comprising heterogeneous sequence(s) encoding a dmTGFB1 molecule and modifications of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown, further comprise intracellular insertion of heterogeneous sequence(s) encoding a targeting receptor, optionally further comprising modifications of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown, modifications of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown, and / or intracellular insertion of heterogeneous sequence(s) encoding a regulatory T cell promoter molecule, each under the control of a promoter sequence. The sequence(s) encoding the targeting receptor are under the control of a promoter sequence, such as an endogenous promoter or a heterogeneous promoter. In certain embodiments, the regulatory T cell promoter molecule comprises IL10 or CTLA4. In certain embodiments, the regulatory T cell promoter molecule comprises IL10 and CTLA4.

[0146] In some embodiments, the targeting receptor is a receptor for a cell surface molecule operably linked through at least a transmembrane domain within an internal signaling domain capable of activating a T cell upon binding to a chimeric antigen receptor (CAR), a T-cell receptor (TCR), or an extracellular receptor portion. In some embodiments, the targeting receptor may 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 within an organism. The targeting receptor does not need to be an antigen receptor, and, for example, the targeting receptor may 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, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), demoglain 3 (DSG3), and MHC class I HLA-A (HLA-A*02).

[0147] In some embodiments, the engineered T cells are modified endogenous nucleic acid sequences encoding TGFBR2, e.g., knockdown, modified endogenous nucleic acid sequences encoding TNFA, e.g., knockdown; Modification of an endogenous nucleic acid sequence encoding an IFNG, e.g., knockdown, dmTGFB1, IL10, CTLA4, and insertion of a sequence encoding a CAR targeting an e.g., MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), demoglain 3 (DSG3), or MHC class I HLA-A (HLA-A*02).

[0148] In some embodiments, sequence(s) encoding the targeting receptor are incorporated into an expression construct. In some embodiments, an expression construct containing sequence(s) encoding the targeting receptor further contains sequence(s) encoding a regulatory T cell promoter molecule, for example, the sequence(s) encoding the targeting receptor and the sequence(s) encoding the regulatory T cell promoter molecule are incorporated into the same expression construct. In some embodiments, an expression construct containing sequence(s) encoding the targeting receptor does not further contain sequence(s) encoding the regulatory T cell promoter molecule, for example, the sequence(s) encoding the regulatory T cell promoter molecule are incorporated into a separate expression construct. In some embodiments, an expression construct containing sequence(s) encoding the targeting receptor is an episomal expression construct. In some embodiments, the sequence(s) encoding the targeting receptor are inserted into the genome, for example, as a targeted or non-targeted insert.

[0149] In some embodiments, the sequence(s) encoding the targeting receptor may be inserted into a site selected from a TCR locus, e.g., TRAC locus, TNF locus, IFNG locus, IL17A locus, IL6 locus, IL2 locus, or adeno-associated virus integration site 1 (AAVS1) locus.

[0150] In some embodiments, the engineered T cells include the insertion of sequence(s) encoding a targeting receptor by gene editing, as evaluated, for example, by sequencing, for example, NGS.

[0151] In some embodiments, the T cell population comprises T cells engineered to include the insertion of a sequence encoding dmTGFB1, a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown, a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown, a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; the insertion of a sequence encoding a regulatory T cell promoter molecule, and the insertion of sequence(s) encoding a targeting receptor, e.g., CAR. In some embodiments, 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 sequence(s) encoding a targeting receptor, as evaluated by, e.g., sequencing, e.g., NGS. It is understood that T cell populations can be enriched for cell populations having targeting receptors using selection methods known in the art.

[0152] In some embodiments, the present invention discloses engineered T cells by introducing or inserting a targeting receptor, e.g., CAR nucleic acid, into a T cell, e.g., into a genomic locus of a T cell or T cell population, using a construct (e.g., a donor construct or template) comprising a guide RNA and a targeting receptor, e.g., CAR nucleic acid, together with an RNA-guided DNA binder. In some embodiments, the present invention discloses engineered T cells by expressing a targeting receptor, such as CAR, from a genomic locus of a T cell or T cell population, e.g., using a construct (e.g., a donor) comprising a guide RNA and a targeting receptor nucleic acid, such as CAR, together with an RNA-guided DNA binder. In some embodiments, the present invention relates to a break (e.g., double-strand break (DSB) or single-strand break(nick)) within the genome of a T cell or T cell population for inserting a targeting receptor, e.g., CAR, using guide RNA together with an RNA-guided DNA-binding agent (e.g., CRISPR / Cas system). T cells engineered by induction are initiated. Cells and cell populations produced by the method are also provided.

[0153] In some embodiments, a targeting receptor, e.g., CAR, can confer target specificity to engineered T cells containing the targeting receptor, e.g., CAR, e.g., to specific cells, tissues, or organs.

[0154] In some embodiments, a targeting receptor, e.g., a CAR, may target engineered T cells to the gastrointestinal tract, e.g., the targeting receptor is a CAR that targets MAdCAM-1 to suppress an immune response in disorders such as inflammatory bowel disease, ulcerative colitis, or Crohn's disease.

[0155] In some embodiments, a targeting receptor, e.g., a CAR, can target engineered T cells against inflammatory tissue, e.g., the targeting receptor is a CAR that targets TNFA to suppress an immune response in disorders such as inflammatory bowel disease, ulcerative colitis, or Crohn's disease.

[0156] In some embodiments, a targeting receptor, e.g., a CAR, may target engineered T cells against inflammatory tissue and / or activated immune cells, e.g., the targeting receptor is a CAR that targets CD70 to suppress the immune response in disorders such as inflammatory bowel disease, ulcerative colitis, or Crohn's disease.

[0157] In some embodiments, a targeting receptor, e.g., a CAR, can target engineered T cells against endothelial cells, e.g., the targeting receptor is a CAR that targets CEACAM6 to suppress immune response(s) including inflammation in disorders such as, e.g., Crohn's disease.

[0158] In some embodiments, a targeting receptor, e.g., a CAR, can target engineered T cells to tissues containing endothelial cells, e.g., the targeting receptor is a CAR that targets VCAM-1 to suppress the immune response in disorders such as Crohn's disease and multiple sclerosis.

[0159] In some embodiments, the CAR can target engineered T cells to synovial tissue, for example, the targeting receptor is a CAR that targets citrullinated vimentin to suppress the immune response in disorders such as rheumatoid arthritis.

[0160] In some embodiments, a targeting receptor, e.g., a CAR, can target engineered T cells to neural tissue, e.g., the targeting receptor is a CAR that targets MBP, MOG, or PLP1 to suppress an immune response in disorders such as multiple sclerosis.

[0161] In some embodiments, a targeting receptor, e.g., a CAR, can target engineered T cells against B cells, e.g., a targeting receptor is a CAR that targets CD19 to suppress the immune response in disorders such as multiple sclerosis and systemic lupus erythematosus.

[0162] In some embodiments, a targeting receptor, e.g., a CAR, can target engineered T cells against B cells, e.g., the targeting receptor is a CAR that targets CD20 to suppress the immune response in disorders such as multiple sclerosis and systemic lupus erythematosus.

[0163] In some embodiments, a targeting receptor, e.g., CAR, may target engineered T cells to immune cells expressing TNFSF7, including but not limited to activated T lymphocytes, B lymphocytes, dendritic cells and / or NK cells, e.g., the CAR targets TNFSF7 to suppress the immune response in disorders such as, e.g., Crohn's disease, ulcerative colitis, systemic lupus erythematosus, or multiple sclerosis.

[0164] In some embodiments, a targeting receptor, e.g., a CAR, may target engineered T cells to tissues containing mature B lymphocytes, e.g., the targeting receptor is a CAR that targets TNFRSF17 to suppress an immune response in disorders such as systemic lupus erythematosus.

[0165] 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).

[0166] Additional CAR targets, for example, inflammatory antigens, are known in the art. For example, Refer to WO2020092057A1, the contents of which are incorporated herein in their entirety. In some embodiments, insertions may be evaluated by detecting the amount of protein or mRNA in engineered T cells, engineered T cell populations, tissues, body fluids of interest, or tissue culture media containing engineered T cells. In some embodiments, insertions by gene editing may be evaluated by sequencing, e.g., next-generation sequencing (NGS). Testing for protein and mRNA expression of targeting receptors, such as CARs, is described herein and is known in the art.

[0167] In embodiments disclosed herein, where the target of a targeting receptor, e.g., CAR, is also present on the surface of a cell, e.g., immune cell or T-cell, the engineered T-cell or population of T-cells may further include a modification of the endogenous nucleic acid sequence encoding the target, e.g., knockdown.

[0168] In certain embodiments, the engineered T cell or T cell population comprises two or more heterogeneous coding sequences, e.g., dmTGFB1, a regulatory T cell promoter, and a targeting receptor; under the control of a promoter. In certain embodiments, each heterogeneous coding sequence is under the control of a separate promoter. In certain embodiments, two heterogeneous coding sequences are under the control of the same promoter. In certain embodiments, two or more heterogeneous 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 heterogeneous coding sequences, e.g., dmTGFB1, a regulatory T cell promoter(s), and a targeting receptor; under the control of the promoter, each heterogeneous sequence is independently under the control of a separate promoter or under the control of a promoter that controls the expression of one or more heterogeneous coding sequences.

[0169] In some embodiments, the engineered T cells or T cell populations do not contain heterologous targeting receptors.

[0170] B. Engineered T cells

[0171] The present invention provides T cells and T cell populations engineered to include modifications comprising intracellular insertion of a heterologous sequence encoding dmTGFB1 under the control of a promoter and modifications of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown.

[0172] In some embodiments, T cells comprising a heterologous sequence encoding dmTGFB1 and a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., a knockdown, under the control of a promoter are further manipulated to include a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., a knockdown, a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., a knockdown, and / or intracellular insertion of heterologous sequence(s) encoding a regulatory T cell promoter molecule under the control of a promoter sequence, as well as compositions and uses thereof. In some embodiments, the regulatory T cell promoter molecule is selected from IL10, CTLA4, TIGIT, IDO1, ENTPD1, ​​NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, BACH2, and IL2RA.

[0173] In some embodiments, a T cell or population of T cells comprising a heterogeneous sequence encoding a dmTGFB1 molecule under the control of a promoter is further engineered to include a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., a knockdown; a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., a knockdown; a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., a knockdown; and the intracellular insertion of a heterogeneous sequence encoding two or more regulatory T cell promoter molecules, each under the control of a promoter sequence. For example, the engineered T cell comprises a first heterogeneous sequence encoding a first regulatory T cell promoter molecule under the control of a first promoter and a second heterogeneous sequence encoding a second regulatory T cell promoter 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 heterogeneous sequence encoding the dmTGFB1 molecule is under the control of a promoter sequence that controls the expression of a regulatory T cell promoter molecule. In certain embodiments, the heterogeneous sequence encoding the dmTGFB1 molecule is not under the control of a promoter sequence that controls the expression of a regulatory T cell promoter molecule. In certain embodiments, the heterogeneous sequence encoding the dmTGFB1 molecule is under the control of a promoter sequence that controls the expression of a targeting receptor. In certain embodiments, the heterogeneous sequence encoding the dmTGFB1 molecule is not under the control of a promoter sequence that controls the expression of a targeting receptor.

[0174] In some embodiments, a T cell or T cell population comprising a heterologous sequence encoding dmTGFB1 and a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown, under the control of a promoter is further manipulated to include a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; intracellular insertion of heterologous sequence(s) encoding IL10 under the control of a promoter; and intracellular insertion of heterologous sequence(s) encoding CTLA4 under the control of a promoter.

[0175] In some embodiments, a T cell or T cell population comprising a heterologous sequence encoding dmTGFB1 and a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown, under the control of a promoter is further engineered to include a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; intracellular insertion of heterologous sequence(s) encoding IL10 under the control of a promoter; and intracellular insertion of heterologous sequence(s) encoding CTLA4 under the control of a promoter, and exhibits at least one inhibitory activity of a naturally occurring regulatory T cell (nTreg), such as inhibition of an immune response or biomarker in an in vitro or in vivo assay, e.g., an animal model of GvHD.

[0176] In some embodiments, heterogeneous sequence(s) encoding dmTGFB1 or one or more regulatory T cell promoter molecules are incorporated into an expression construct. In some embodiments, heterogeneous sequences encoding two or more molecules may be incorporated into two or more separate expression constructs. For example, a first heterogeneous sequence encoding dmTGFB1 is provided in a first expression construct, a second heterogeneous sequence encoding a regulatory T cell promoter molecule is provided in a second separate expression construct, and a third heterogeneous sequence encoding a regulatory T cell promoter molecule is provided in a third separate expression construct. In an alternative example, the first heterogeneous sequence encoding dmTGFB1 is provided in the same expression construct as the second heterogeneous sequence encoding a regulatory T cell promoter molecule and the third heterogeneous sequence encoding a regulatory T cell promoter molecule. In some embodiments, one or more expression constructs are episomal expression constructs. In some embodiments, one or more heterogeneous sequences are inserted into the genome, for example, targeted or untargeted inserts.

[0177] In certain embodiments, a modification of the endogenous nucleic acid sequence encoding TGFBR2, e.g., the knockdown comprises a modification of TGFBR2. In certain embodiments, a modification of the endogenous nucleic acid sequence encoding IFNG, e.g., the knockdown comprises a modification of IFNG. In certain embodiments, a modification of the endogenous nucleic acid sequence encoding TNFA, e.g., the knockdown comprises a modification of TNFA.

[0178] In some embodiments, the sequence(s) encoding dmTGFB1 or a regulatory T cell promoter molecule may be inserted into a site selected from a TCR locus, e.g., TRAC locus; TNF locus, IFNG locus, IL17A locus, IL6 locus, IL2 locus, or adeno-associated virus integration site 1 (AAVS1) locus.

[0179] In some embodiments, the engineered T cells or T cell population are engineered to include modifications such as the insertion of a sequence encoding dmTGFB1 by gene editing, for example, as evaluated by sequencing such as NGS, for example, wherein at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells include the insertion of a sequence encoding the dmTGFB1 molecule.

[0180] In some embodiments, the engineered T cell population comprises an insertion variant of the sequence encoding dmTGFB1 generated, for example, by gene editing and as evaluated by sequencing such as NGS, for example, wherein at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells comprise an insertion variant of the sequence encoding the dmTGFB1 molecule, and further comprises a variant within the TGFBR2 sequence, for example, knockdown, by gene editing as evaluated by sequencing such as NGS, for example, wherein at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells comprise a variant within the endogenous TGFBR2 sequence Includes insertions, deletions, or substitutions. In some embodiments, the expression of TGFBR2 (full length, wild-type protein, or mRNA) is reduced by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or below the detection limit of the assay compared to a suitable control in which the TGFBR2 gene is not modified, for example, as determined by ELISA or flow cytometry. For example, assays for TGFBR2 protein and mRNA expression in T cell populations are known in the art.

[0181] In some embodiments, the engineered T cell population comprises an insertion modification of a sequence encoding dmTGFB1 generated, for example, by gene editing and as evaluated, for example, by sequencing, e.g., NGS, wherein at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells comprise an insertion of a sequence encoding a dmTGFB1 molecule, and further comprises a modification within a TNFA sequence by gene editing, for example, as evaluated, by sequencing, e.g., NGS, e.g., knockdown, wherein at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells comprise a modification within an endogenous TNFA sequence Includes insertions, deletions, or substitutions. In some embodiments, the expression of TNFA (full length, wild-type protein, or mRNA) is reduced by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or below the detection limit of the assay compared to a suitable control, for example, in which the TNFA gene is not modified, as determined by, for example, ELISA or flow cytometry. For example, assays for TNFA protein and mRNA expression in T cell populations are known in the art. In certain embodiments, TNFA knockdown results in TNFA levels of 2500 pg / ml or less, as determined, for example, using a custom U-PLEX biomarker kit (Meso Scale Diagnostics, catalog K15067L-2) according to the manufacturer's instructions.

[0182] In some embodiments, the engineered T cell population comprises modifications such as the insertion of a sequence encoding dmTGFB1 by gene editing, for example, as evaluated by sequencing, for example, NGS, wherein at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells comprise the insertion of a sequence encoding the dmTGFB1 molecule, and further comprises modifications within an IFNG sequence by gene editing, for example, as evaluated by sequencing, for example, NGS, for example, knockdown, for example, wherein at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells comprise modifications within an endogenous IFNG sequence Includes insertions, deletions, or substitutions. In some embodiments, the expression of IFNG (full length, wild-type protein, or mRNA) is reduced to at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or below the detection limit of the assay compared to a suitable control in which the IFNG gene is not modified, for example, as determined by ELISA or flow cytometry. For example, assays for IFNG protein and mRNA expression in T cell populations are known in the art. In certain embodiments, IFNG knockdown results in an IFNG level of 300,000 pg / ml or less, as determined, for example, using a custom U-PLEX biomarker kit (Meso Scale Diagnostics, catalog K15067L-2) according to the manufacturer's instructions.

[0183] In some embodiments, modifications that knock down the expression of a gene, e.g., TGFBR2, TNFA, or IFNG, are one or more of insertions, deletions, or substitutions.

[0184] In some embodiments, the engineered T cells or T cell population comprises, for example, a modification, e.g., an insertion of a sequence encoding dmTGFB1 by gene editing, e.g., as evaluated by sequencing, e.g., NGS, wherein at least 30%, 35%, 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, for example, an insertion of sequence(s) encoding a regulatory T cell promoter molecule by gene editing, e.g., as evaluated by sequencing, e.g., NGS, e.g., wherein at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, of the cells 80%, 85%, 90%, or 95% comprises the insertion of a sequence encoding a regulatory T cell promoter molecule. In some embodiments, the inserted regulatory T cell promoter molecule, e.g., IL10, results in statistically significantly increased expression of protein or mRNA compared to a suitable control, e.g., without the insertion of the regulatory T cell promoter molecule gene, as determined by ELISA or flow cytometry. In some embodiments, the engineered T cells comprise the insertion of sequence(s) encoding IL10 by gene editing, e.g., as evaluated by sequencing, e.g., NGS, wherein at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells comprise the insertion of the sequence encoding IL10. In some embodiments, the inserted sequence(s) encoding IL10 result in statistically significantly increased expression of protein or mRNA compared to a suitable control, e.g., the regulatory T cell promoter here.For example, assays for IL10 protein and mRNA expression in T cell populations are known in the art, such as ELISA and flow cytometry. In a specific embodiment, the level of IL10 is at least 300 pg / ml, as determined according to the manufacturer's instructions using, for example, a custom U-PLEX biomarker kit (Meso Scale Diagnostics, catalog K15067L-2).

[0185] In some embodiments, the engineered T cells or T cell population comprises modifications such as the insertion of a sequence encoding dmTGFB1 by gene editing, for example, as evaluated by sequencing, for example, NGS, wherein at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells comprise the insertion of a sequence encoding the dmTGFB1 molecule, and further comprise the insertion of sequence(s) encoding CTLA4 by gene editing, for example, as evaluated by sequencing, for example, NGS, wherein at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, of the cells 80%, 85%, 90%, or 95% include the insertion of a sequence encoding CTLA4. In some embodiments, the inserted sequence(s) encoding CTLA4 result in statistically significantly increased expression of protein or mRNA compared to a suitable control, e.g., the regulatory T cell promoter molecule here. For example, assays for CTLA4 protein and mRNA expression in T cell populations are described herein and are known in the art, e.g., ELISA and flow cytometry.

[0186] In some embodiments, the T cell population comprises modifications such as the insertion of a sequence encoding dmTGFB1 by gene editing, for example, as evaluated by sequencing, for example, NGS, wherein at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells are engineered to include the insertion of a sequence encoding the dmTGFB1 molecule, and further comprises T cells engineered to include modifications of an endogenous nucleic acid sequence encoding TGFBR2, for example, knockdown; modifications of an endogenous nucleic acid sequence encoding TNFA, for example, knockdown; modifications of an endogenous nucleic acid sequence encoding IFNG, for example, knockdown; and the insertion of a sequence encoding a regulatory T cell promoter molecule. In some embodiments, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of T cells in a T cell population (e.g., within the detection limit of the assay used) are engineered to contain a heterologous regulatory T cell promoting molecule as evaluated, for example, by sequencing, e.g., NGS. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of T cells in a T cell population are engineered to contain a modification, e.g., a knockdown, of the sequence(s) encoding TGFBR2 as evaluated, for example, by sequencing, e.g., NGS. In some embodiments, at least 50%, 55%, 60%, 65%, at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the T cells in the T cell population are manipulated to include a modification, e.g., knockdown of the sequence(s) encoding TNFA, as evaluated, e.g., by sequencing, e.g., NGS.In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the T cells in the T cell population are manipulated to include a modification, e.g., knockdown of the sequence(s) encoding IFNG, as evaluated, e.g., by sequencing, e.g., NGS. In some embodiments, 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 manipulated to include an insertion of a sequence encoding a regulatory T cell promoter, as evaluated, e.g., by sequencing, e.g., NGS. In some embodiments, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the T cells in the T cell population are manipulated to include the insertion of sequence(s) encoding IL10, as evaluated, for example, by sequencing, for example, NGS. In some embodiments, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the T cells in the T cell population are manipulated to include the insertion of sequence(s) encoding CTLA4, as evaluated, for example, by sequencing, for example, NGS.

[0187] In some embodiments, the engineered T cells or T cell population are engineered to include modifications, such as the insertion of a sequence encoding dmTGFB1 by gene editing, for example, as evaluated by sequencing, for example, NGS, wherein at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells include the insertion of a sequence encoding the dmTGFB1 molecule, and modifications to an endogenous nucleic acid sequence encoding TGFBR2, for example, knockdown; modifications to an endogenous nucleic acid sequence encoding TNFA, for example, knockdown; modifications to an endogenous nucleic acid sequence encoding IFNG, for example, knockdown; and further comprises insertion of heterogeneous sequence(s) encoding regulatory T cell promoter molecules under the control of the promoter sequence, and further comprises modification of endogenous nucleic acid sequences encoding interleukin 17A (IL17A), interleukin 6 (IL6), interleukin 2 (IL2), perforin 1 (PRF1), granzyme A (GZMA), granzyme B (GZMB), natural killer cell granule protein 7 (NKG7), Fas ligand (FasL, NF superfamily, member 6), lyanodine receptor 2 (RYR2) and colony stimulating factor 2 (CSF2), wherein each modification knocks down the expression of IL17A, IL6, IL2, PRF1, GZMA, GZMB, NKG7, FASL, RYR2 or CSF2, and each additional modification of the endogenous nucleic acid sequence comprises a modification of IL17A.

[0188] In some embodiments, T cells or T cell populations are manipulated using a gene editing system, for example, using an RNA-guided DNA binder. In some embodiments, T cells are manipulated using a CRISPR / Cas gene editing system. In some embodiments, T cells are manipulated using a CRISPR / Cas type II gene editing system, for example, using Cpf1. In some embodiments, T cells are manipulated using a CRISPR / Cas9 gene editing system, for example, using SpyCas9. An exemplary Cas9 sequence is provided herein.

[0189] In some embodiments, T cells or T cell populations are engineered using guide RNA that specifically targets target sites within the TGFBR2, IFNG, and TNFA genes to provide knockdown of the TGFBR2, IFNG, and TNFA genes. Exemplary sequences for the knockdown of IFNG and TNFA are provided in Tables 1 and 2, respectively, as are the genomic coordinates for the targets of each listed guide sequence. An exemplary sequence for the knockdown of TGFBR2 is Table 3 It is provided in, and the genomic coordinates of the target for each listed guide sequence are also likewise.

[0190] In some embodiments, the engineered T cells or T cell population comprises the TGFBR2, IFNG, and TNFA genes, which are knocked down using the guide RNA disclosed herein in combination with an RNA-guided DNA binder. In some embodiments, the present invention discloses engineered T cells by inducing breakage (e.g., double-strand break (DSB) or single-strand break (nick)) within the T cells using the guide RNA disclosed herein in combination with an RNA-guided DNA binder (e.g., a CRISPR / Cas system), for example. The method may be used in vitro or in vitro in the preparation of cell products for suppressing immune response(s), including inflammation and autoimmunity, for example. In some embodiments, the guide RNA disclosed herein is used with an RNA-guided DNA binder (e.g., at the site described herein within the TGFBR2 gene) It mediates target-specific cleavage by Cas nucleases. In some embodiments, the guide RNA disclosed herein is an RNA-guided DNA binder (e.g., at the site described herein within the IFNG gene) It mediates target-specific cleavage by Cas nucleases. In some embodiments, the guide RNA disclosed herein is an RNA-guided DNA binder (e.g., at the site described herein within the TNFA gene) It mediates target-specific cleavage by Cas nucleases. In some embodiments, it will be understood that the guide RNA includes a guide sequence that binds to or can bind to the region.

[0191] Engineered T cells or T cell populations containing genetic modifications at genome coordinates selected from the genome coordinates listed in Table 1 are provided, for example, cells containing insertion / deletion or substitution mutations within any listed genome range in IFNG are provided. Engineered T cells containing genetic modifications at genome coordinates selected from the coordinates listed in Table 2 are also provided, for example, cells containing insertion / deletion or substitution mutations within any listed genome range in TNFA are provided. In some embodiments, the engineered T cells will include modifications within the genome coordinate region selected from Table 1 and modifications within the genome coordinate region selected from Table 2. In Table 3 Manipulated T cells or T cell populations containing genetic modifications at selected genomic coordinates from the listed genomic coordinates are provided, for example, cells containing insertion / deletion or substitution mutations within any listed genomic range within TGFBR2.

[0192] In some embodiments, the guide RNA disclosed herein is Table 1, Table 2 or Table 3 It includes a guide sequence that is 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the sequence group of. In some embodiments, the guide RNA disclosed herein is Table 1, Table 2, or Table 3 It includes a guide sequence that is 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the sequence group.

[0193] 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 a group of sequences that are 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group of sequences in Table 1. In some embodiments, the guide RNA disclosed herein comprises a guide sequence 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% identical to a sequence selected from the group of sequences in Table 1. In some embodiments, the guide RNA disclosed herein comprises a guide sequence 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 sequence group of Table 1.

[0194] 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 a group of sequences that are 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group of sequences in Table 2. In some embodiments, the guide RNA disclosed herein comprises a guide sequence 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 that is 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group of sequences in Table 2. In some embodiments, the guide RNA disclosed herein comprises a guide sequence that is 17, 18, 19, or 20 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 sequence group of Table 2.

[0195] In some embodiments, the guide RNA disclosed herein is Table 3 It comprises a guide sequence having at least 15, 16, 17, 18, 19, or 20 consecutive nucleotides of a sequence selected from a group of sequences that are 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from a group of sequences. In some embodiments, the guide RNA disclosed herein is Table 3 It comprises a guide sequence having at least 17, 18, 19, or 20 consecutive nucleotides of a sequence selected from the sequence group of. In some embodiments, the guide RNA disclosed herein comprises a guide sequence that is 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the sequence group of Table 2. In some embodiments, the guide RNA disclosed herein is Table 3It includes a guide sequence which is 17, 18, 19, or 20 consecutive nucleotides of a sequence selected from the sequence group. In some embodiments, the guide RNA disclosed herein is Table 3 It includes a guide sequence selected from the sequence group.

[0196] Overall, genome coordinates follow the human reference genome hg38 unless otherwise noted.

[0197] In certain embodiments, a guide RNA comprising a guide sequence targeting IFNG and a guide RNA comprising a guide sequence targeting TNFA are included.

[0198] In some embodiments, the engineered T cells may be engineered autologous T cells or engineered allogeneic T cells. In autologous T cell therapy, the T cells may be engineered from T cells derived from the same patient as the patient to be treated with the engineered T cells. In some embodiments, the engineered autologous T cells may be generated by extracting T cells from the patient and modifying the T cells according to the modifications described herein, and the engineered autologous T cells are reintroduced into the same patient. In some embodiments, the engineered autologous T cells may be generated for each individual patient and administered on a patient-by-patient basis.

[0199] In some embodiments, the engineered T cells may be engineered allogeneic T cells. In allogeneic T cell therapy, T cells may be engineered from donor cells and then administered to one or more patients using an "off-the-shelf" approach.

[0200] engineered allogeneic T cells

[0201] In some embodiments, the engineered T cells described herein are engineered allogeneic T cells. Accordingly, the present invention provides compositions and methods for producing “ready-made” T cells by altering, for example, inhibiting or reducing the expression and / or function (e.g., the expression level of a functional version) of a gene target or a protein encoded by a gene target, for example, by modifying, for example, inhibiting or reducing the efficacy (e.g., reducing or eliminating undesirable immunogenicity (e.g., host-to-graft response or graft-to-host response)), function, proliferation, stimulation, or survival of transplanted cells, for example, engineered T cells as described herein, for example, allogeneic engineered T cells for immunotherapy.

[0202] "Off-the-shelf" therapies using donor (allogeneic) cells eliminate the need to retrieve and modify the patient's own lymphocytes. However, allogeneic T cells can trigger undesirable immune responses or have a short lifespan. Often, immune rejection of allogeneic cells results from a mismatch of major histocompatibility complex (MHC) molecules between the donor and the recipient. For example, slight differences in MHC alleles between individuals can cause the recipient's T cells to become activated. During T cell development, while an individual's T cell repertoire is tolerant to its own MHC molecules, T cells that recognize MHC molecules from another individual may persist in the circulation and are referred to as allogeneic reactive T-cells. Allogeneic reactive T-cells are activated, for example, by the presence of cells from another individual expressing MHC molecules within the body, and can cause, for instance, graft-versus-host disease and transplant rejection. Additionally, refer to WO / 2023 / 245108, the entire contents of which are incorporated herein by reference, for example.

[0203] In some embodiments, the gene target is an allogeneic T-cell protein such as HLA-A, HLA-B, TRAC, or CIITA. Without being bound by theory, inhibition or elimination (e.g., through genetic modification) of the level of the allogeneic T-cell target or the expression level of the allogeneic T-cell target gene target is believed to improve the function of cells, e.g., transplanted, e.g., engineered allogeneic T-cells, by reducing or eliminating graft-versus-host response or host-versus-graft response, or to make said transplanted cells resistant to immunosuppressive therapy.

[0204] In some embodiments, the compositions and methods described herein may be used to improve the function (e.g., by reducing or eliminating undesirable immunogenicity (e.g., host-to-graft response or graft-to-host response), survival, proliferation, and / or efficacy of cells, e.g., allogeneic engineered T-cells described herein) by modifying the genes of major histocompatibility complex components, e.g., HLA proteins, e.g., HLA-A and / or HLA-B. Without being bound by theory, the reduction or absence of expression of mismatched (e.g., not matching the type of subject receiving cell therapy) HLA proteins (or components) is considered to reduce or eliminate host-to-graft disease by eliminating host T-cell receptor recognition and response to the mismatched (e.g., allogeneic) graft tissue. Thus, such an approach could be used to generate "off-the-shelf" T-cells (Torikai et al., 2012 Blood 119, 5697-5705).

[0205] In some embodiments, the compositions and methods described herein may be used to improve the function (e.g., by reducing or eliminating undesirable immunogenicity (e.g., host-to-graft response or graft-to-host response)), survival, proliferation, and / or efficacy of cells, e.g., the allogeneic engineered T cells described herein, by modifying the gene of a T-cell receptor (TCR) component, e.g., TRAC. Without being bound by theory, the reduction or absence of expression of a functional T-cell receptor component is considered to reduce or eliminate the presence of the TCR on the surface of said cells, thereby eliminating T-cell receptor recognition and response to it in host tissues, and thus reduce or prevent graft-to-host disease. Thus, this approach may be used to generate "off-the-shelf" T-cells.

[0206] In some embodiments, the compositions and methods described herein may be used to improve the function (e.g., by reducing or eliminating undesirable immunogenicity (e.g., host-to-graft response or graft-to-host response), survival, proliferation, and / or efficacy of cells, e.g., allogeneic engineered T-cells described herein, by modifying a gene encoding a protein that regulates the expression of one or more components of the major histocompatibility complex, e.g., CIITA. While not intending to be bound by theory, it is believed that reducing or eliminating the expression of a modulator of MHC class II expression, e.g., CIITA, reduces or eliminates host-graft disease by reducing or eliminating the expression of MHC class II molecules on allogeneic cells, thereby reducing or eliminating the expression of mismatched (e.g., not matched with the type of subject receiving cell therapy) MHC class II proteins (or components), e.g., by eliminating host T-cell receptor recognition and response to mismatched (e.g., allogeneic) graft tissues, e.g., allogeneic engineered T-cells, as described herein. Thus, this approach could be used to generate "off-the-shelf" T-cells.

[0207] In some embodiments, to further reduce or eliminate host versus graft disease response upon administration of cells, it may be beneficial to reduce or eliminate the expression of one or more MHC class I molecules and one or more MHC II molecules in, for example, T-cells, e.g., allogeneic engineered T-cells as described herein. Accordingly, in embodiments of the cells and methods of the present disclosure, cells may be contacted with a composition of the present disclosure comprising a gRNA molecule for HLA-A and / or HLA-B, e.g., as described herein (e.g., a composition comprising gRNA and a Cas9 molecule) (e.g., so that the expression of one or more MHC class I molecules is reduced or eliminated in said cells), and a composition of the present disclosure comprising a gRNA molecule for CIITA, e.g., as described herein (e.g., a composition comprising gRNA and a Cas9 molecule) (e.g., so that the expression of one or more MHC class II molecules is reduced or eliminated). In an embodiment of the cell and method of the present disclosure, the cell may also be in contact with a composition of the present disclosure (e.g., a composition comprising gRNA and a Cas9 molecule) comprising a component of the TCR, e.g., a gRNA molecule for TRAC, e.g., a gRNA molecule as described herein (e.g., a composition comprising gRNA and a Cas9 molecule) (e.g., so as to reduce or eliminate the expression of one or more components of the T cell receptor, e.g., the TCR). In some embodiments, the cell of the present disclosure has reduced or eliminated the expression of the TCR (e.g., as detected by flow cytometry), reduced or eliminated the expression of one or more MHC class I molecules (e.g., as detected by flow cytometry), and reduced or eliminated the expression of one or more MHC class II molecules (e.g., as detected by flow cytometry).

[0208] In some embodiments, “off-the-shelf” allogeneic T cells and allogeneic T cell populations are engineered to include modifications comprising: intracellular insertion of heterogeneous sequence(s) encoding IL10 under the control of a promoter; intracellular insertion of heterogeneous sequence(s) encoding CTLA4 under the control of a promoter; modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding HLA-A, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding HLA-B, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding CIITA, e.g., knockdown; and modification of an endogenous nucleic acid sequence encoding TRAC, e.g., knockdown. In some embodiments, “off-the-shelf” allogeneic T cells and allogeneic T cell populations further comprise a targeting receptor as described herein.

[0209] In some embodiments, “off-the-shelf” allogeneic T cells and allogeneic T cell populations are engineered to include: intracellular insertion of heterogeneous sequence(s) encoding dmTGFB1 under the control of a promoter; intracellular insertion of heterogeneous sequence(s) encoding IL10 under the control of a promoter; intracellular insertion of heterogeneous sequence(s) encoding CTLA4 under the control of a promoter; modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding HLA-A, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding HLA-B, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding CIITA, e.g., knockdown; and modification of an endogenous nucleic acid sequence encoding TRAC, e.g., knockdown. In some embodiments, "off-the-shelf" allogeneic T cells and allogeneic T cell populations further comprise targeting receptors as described herein.

[0210] In some embodiments, "off-the-shelf" allogeneic T cells and allogeneic T cell populations include: intracellular insertion of heterogeneous sequence(s) encoding dmTGFB1 under the control of a promoter; intracellular insertion of heterogeneous sequence(s) encoding IL10 under the control of a promoter; intracellular insertion of heterogeneous sequence(s) encoding CTLA4 under the control of a promoter; modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding HLA-A, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding HLA-B, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding CIITA, e.g., knockdown; and is engineered to include modifications to the endogenous nucleic acid sequence encoding TRAC, for example, modifications including knockdown. In some embodiments, "off-the-shelf" allogeneic T cells and allogeneic T cell populations further include targeting receptors as described herein.

[0211] In some embodiments, the “off-the-shelf” allogeneic T cells and allogeneic T cell population are CD3+ cells. In some embodiments, the “off-the-shelf” allogeneic CD3+ T cells and allogeneic CD3+ T cell population may first be engineered to include a modification comprising one or more of: a modification of an endogenous nucleic acid sequence encoding HLA-A, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding HLA-B, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding CIITA, e.g., knockdown; and a modification of an endogenous nucleic acid sequence encoding TRAC, e.g., knockdown, and then selected for CD4+ T cells or CD8+ T cells, and then further engineered to produce the engineered T cells described herein. The order in which the different modifications are engineered is, for example, that the “off-the-shelf” allogeneic CD3+ T cells and allogeneic CD3+ T cell population are engineered to include the following modifications:

[0212] (a) Intracellular insertion of heterologous sequence(s) encoding IL10 under the control of a promoter; intracellular insertion of heterologous sequence(s) encoding CTLA4 under the control of a promoter; and modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown;

[0213] (b) intracellular insertion of heterologous sequence(s) encoding dmTGFB1 under the control of a promoter; intracellular insertion of heterologous sequence(s) encoding IL10 under the control of a promoter; intracellular insertion of heterologous sequence(s) encoding CTLA4 under the control of a promoter; and modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; or

[0214] (c) Intracellular insertion of heterologous sequence(s) encoding dmTGFB1 under the control of a promoter; intracellular insertion of heterologous sequence(s) encoding IL10 under the control of a promoter; intracellular insertion of heterologous sequence(s) encoding CTLA4 under the control of a promoter; modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; and modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown,

[0215] and subsequently engineered to include a targeting receptor.

[0216] In another embodiment, "off-the-shelf" allogeneic CD3+ T cells and allogeneic CD3+ T cell populations are engineered to include a targeting receptor and then engineered to include the modifications (a), (b), or (c) described above.

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228] A non-restrictive modified guide sequence for TNFA knockdown is presented below (hg38 coordinates chr12:68158001-68158021, G019757):

[0229]

[0230] Here, m is a 2'-OMe modified nucleotide / nucleoside residue, * indicates a phosphorothioate link between residues, and uppercase letters indicate residues, e.g., residues containing ribose sugars.

[0231] A non-restrictive modified guide sequence for IFNG knockdown is presented below (hg38 coordinates chr6:31576805-31576825, G019753):

[0232]

[0233] A non-restrictive modified guide sequence for IL17A knockdown is presented below (hg38 coordinates chr6:52189069-52189089):

[0234]

[0235] An exemplary modified simulation guide is presented below (hg38 coordinates chr1:0-20):

[0236]

[0237] A non-restrictive modified guide sequence for TGFBR2 knockdown is presented below (hg38 coordinates chr3:30671941-30671961):

[0238]

[0239] A non-restrictive modified guide sequence for HLA-A knockdown is presented below (hg38 coordinates chr6:29942891-29942915, G034202):

[0240]

[0241] Here, m is a 2'-OMe modified nucleotide / nucleoside residue, * indicates a phosphorothioate link between residues, and uppercase letters indicate residues, e.g., residues containing ribose sugars.

[0242] A non-restrictive modified guide sequence for HLA-B knockdown is presented below (hg38 coordinates chr6:31355222-31355246, G034208):

[0243]

[0244] Here, m is a 2'-OMe modified nucleotide / nucleoside residue, * indicates a phosphorothioate link between residues, and uppercase letters indicate residues, e.g., residues containing ribose sugars.

[0245] A non-restrictive modified guide sequence for CIITA knockdown is presented below (hg38 coordinates chr16:10906643-10906667, G034619):

[0246]

[0247] Here, m is a 2'-OMe modified nucleotide / nucleoside residue, * indicates a phosphorothioate link between residues, and uppercase letters indicate residues, e.g., residues containing ribose sugars.

[0248] A non-restrictive modified guide sequence for TRAC knockdown is presented below (hg38 coordinates Chr14: 22547524- 22547544, G025420):

[0249]

[0250] Here, m is a 2'-OMe modified nucleotide / nucleoside residue, * indicates a phosphorothioate link between residues, and uppercase letters indicate residues, e.g., residues containing ribose sugars; or

[0251]

[0252] Here, m is a 2'-OMe modified nucleotide / nucleoside residue, * indicates a phosphorothioate link between residues, and uppercase letters indicate residues, e.g., residues containing ribose sugars.

[0253] In some embodiments, the present invention discloses a T cell engineered by introducing or inserting a heterologous dmTGFB1 nucleic acid into a genomic locus of a T cell or T cell population using a construct (e.g., a donor construct or template) comprising a guide RNA and a heterologous dmTGFB1 nucleic acid together with an RNA-guided DNA binder to produce the engineered T cell, for example. In some embodiments, the present invention discloses a T cell engineered by expressing heterologous dmTGFB1 from a genomic locus of a T cell or T cell population using a construct (e.g., a donor) comprising a guide RNA and a heterologous dmTGFB1 nucleic acid together with an RNA-guided DNA binder, for example. In some embodiments, the present invention discloses, for example, an RNA-guided DNA binder (e.g., Breaks (e.g., double-strand breaks (DSB) or single-strand breaks (nicks)) within the genome of T cells or a T cell population for inserting the dmTGFB1 gene using guide RNA with the CRISPR / Cas system T cells engineered by induction are initiated. Cells and cell populations produced by the method are also provided.

[0254] In some embodiments, the present invention discloses a T cell that is further engineered, for example, by introducing or inserting a heterologous dmTGFB1 nucleic acid into a genomic locus of a T cell to produce engineered T cells, and engineered by introducing or inserting a heterologous IL10 nucleic acid into a genomic locus of a T cell or T cell population using a construct (e.g., a donor construct or template) comprising a guide RNA and a heterologous IL10 nucleic acid together with an RNA-guided DNA binder. In some embodiments, the present invention discloses a T cell engineered by expressing heterologous IL10 from a genomic locus of a T cell or T cell population using, for example, a construct (e.g., a donor) comprising a guide RNA and a heterologous IL10 nucleic acid together with an RNA-guided DNA binder. In some embodiments, the present invention discloses, for example, an RNA-guided DNA binder (e.g., Using guide RNA in conjunction with the CRISPR / Cas system, to insert the IL10 gene, breakage (e.g., double-stranded) within the genome of T cells or a T cell population Break (DSB) or single-strand break (nick) T cells engineered by induction are initiated. Cells and cell populations produced by the method are also provided.

[0255] In some embodiments, the present invention discloses a T cell that is further engineered, for example, by introducing or inserting a heterologous dmTGFB1 nucleic acid into a genomic locus of a T cell to produce engineered T cells, and engineered by introducing or inserting a heterologous CTLA4 nucleic acid into a genomic locus of a T cell or T cell population using a construct (e.g., a donor construct or template) comprising a guide RNA and a heterologous CTLA4 nucleic acid together with an RNA-guided DNA binder. In some embodiments, the present invention discloses a T cell engineered by expressing heterologous CTLA4 from a genomic locus of a T cell or T cell population using, for example, a construct (e.g., a donor) comprising a guide RNA and a heterologous CTLA4 nucleic acid together with an RNA-guided DNA binder. In some embodiments, the present invention discloses, for example, an RNA-guided DNA binder (e.g., Breaks (e.g., double-strand breaks (DSB) or single-strand breaks (nicks)) within the genome of T cells or T cell populations for inserting the CTLA4 gene using guide RNA with the CRISPR / Cas system T cells engineered by induction are initiated. Cells and cell populations produced by the method are also provided.

[0256] In some embodiments, the present invention discloses a T cell engineered by introducing or inserting a heterologous dmTGFB1 nucleic acid into a genomic locus of a T cell, for example, to produce engineered T cells, and further engineered by introducing or inserting heterologous CTLA4 nucleic acid and heterologous IL10 nucleic acid into a genomic locus of a T cell or T cell population using one or more constructs (e.g., a donor construct or a template) comprising a guide RNA and heterologous CTLA4 nucleic acid and heterologous IL10 nucleic acid together with an RNA-guided DNA binder. In some embodiments, the present invention discloses a T cell engineered by expressing heterologous CTLA4 and heterologous IL10 from a genomic locus of a T cell or T cell population using one or more constructs (e.g., a donor construct or a template) comprising a guide RNA and heterologous CTLA4 nucleic acid and heterologous IL10 nucleic acid together with an RNA-guided DNA binder. In some embodiments, the present invention discloses, for example, an RNA-guided DNA binder (e.g., Breaks (e.g., double-strand breaks (DSB) or single-strand breaks (nicks)) within the genome of T cells or T cell populations for inserting the CTLA4 and IL10 genes using guide RNA in conjunction with the CRISPR / Cas system T cells engineered by induction are initiated. In some embodiments, the guide RNA mediates target-specific cleavage by an RNA-guided DNA-binding agent (e.g., Cas nuclease) at the site described herein for the insertion of sequence(s) encoding two or more regulatory T cell promoter molecules, e.g., IL10 and CTLA4. In some embodiments, it will be understood that the guide RNA comprises a guide sequence that binds to or can bind to said site. Cells and cell populations produced by the method are also provided.

[0257] Exemplary nucleotide and polypeptide sequences of regulatory T cell promoter molecules are provided below. Methods for identifying alternative nucleotide sequences encoding polypeptide sequences, including alternative naturally occurring variants and non-human homologs, are known in the art. Exemplary nucleic acid sequences encoding dmTGFB1, IL10, and CTLA4 are provided below. Other suitable dmTGFB1, IL10, and CTLA4 sequences may be known in the art or designed based on the disclosures provided herein. For example, Refer to Gorby et al., Engineered IL-10 variants elicit 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 (particularly IL-10 variants), and sequences of which are incorporated herein by reference. Methods for identifying alternative IL10 and CTLA4 sequences are also known in the art. For example, the same literature ( id.) See. For example, Gorby initiates the anti-inflammatory and cytotoxic activities of IL-10. In addition to anti-inflammatory activity, recent studies have shown that IL-10 can enhance the cytotoxic function of CD8 T cells, thereby increasing their ability to target tumors and promoting anticancer responses (Oft, 2019). This appears paradoxical, as IL-10 is associated with tumor-evasion immune responses in the tumor microenvironment, most likely due to its inhibitory effect on antigen presentation (Mannino et al., 2015; Yue et al., 1997). Despite this paradox, several studies have subtly demonstrated that IL-10 can improve the production of CD8 effector molecules granzyme B and interferon gamma both in vitro and in vivo (Emmerich et al., 2012; Mumm et al., 2011; Mumm and Oft, 2013). Currently, there are several clinical trials testing the antitumor properties of IL-10, and they have already yielded promising results in the early stages (Naing et al., 2019). In these trials, high doses of pegylated IL-10 (pegilodecakin) were used, which resulted in prolonged IL-10 retention in the circulatory system to ensure efficacy, and this, in turn, highlighted that an effective IL-10 response in vivo requires high concentrations and sustained levels of IL-10. Saxton et al. engineered IL-10 variants using yeast display-based induced evolution. IL-10 is an immunomodulatory cytokine possessing both anti-inflammatory and immunostimulatory properties, and it is often unregulated in diseases. Mechanistically, IL-10 functions as a secreted homodimer that binds two copies of a heterodimeric receptor complex containing the quasi-receptor subunit IL-10Rα and the quasi-receptor subunit IL-10Rβ. IL-10-dependent dimerization of IL-10Rα and IL-10Rβ sequentially initiates the activation of the transcription factor STAT3, which mediates the various biological effects of IL-10.Saxton et al. teach that IL-10 variants with varying IL-10Rβ binding strengths reveal substantial differences in response thresholds across immune cell populations, thereby providing a method to manipulate IL-10 cell type selectivity. Saxton et al. identified a "Super-10" variant (D25A / E96A based on the amino acid numbering of SEQ ID NO. 231) having enhanced affinity for IL-10Rβ that enables the assembly of a hexamer IL-10-IL-10Rα-IL-10Rβ complex. Additional variants (e.g., D25K, D25A, N21A / R104A, and D25A / N21A / R104A based on the amino acid numbering of SEQ ID NO. 231) exhibited myeloid-biased activity by inhibiting macrophage activation without stimulating inflammatory CD8+ T cells, thereby isolating the major opposing functions of IL-10. In certain embodiments, an IL-10 variant referred herein as a "suppressive IL-10 variant" having impaired immunostimulatory properties, for example, a variant that possesses myeloid-biased activity by inhibiting macrophage activation and exhibits impaired stimulation of inflammatory CD8+ T cells, is used in the engineered T cells provided herein. In certain embodiments, the suppressive IL-10 variant used in the engineered T cells provided herein comprises substitutions selected from D25K, D25A, D25A / E96A, N21A / R104A, and D25A / N21A / R104A, for example, D25A / E96A. The results of Saxton et al. provide a mechanical blueprint for tuning the pleiotropic action of IL-10. Additional inhibitory IL-10 variants are provided in WO2021243057 incorporated herein by reference, and provide a plurality of IL-10 sequences comprising polypeptides having altered binding affinities for IL-10Rβ compared to the binding affinity of a reference IL-10 polypeptide lacking one or more amino acid substitutions, and a method for characterizing activity.Additional variants including inhibitory IL-10 variants are disclosed and are predicted to exhibit myeloid-biased activity by inhibiting macrophage activation without stimulating inflammatory CD8+ T cells, similar to D25K, D25A, D25A / E96A, N21A / R104A, and D25A / N21A / R104A variants. The embodiments provided herein include inhibitory IL-10 variants having one or more amino acid substitutions at positions corresponding to amino acid residues selected from D25, H14, N18, R24, D28, E74, H90, N92, E96, T100, and R104, and optionally include additional 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 substitution 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 are included. For example, any nucleic acid sequence, amino acid sequence, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any nucleic acid sequence, amino acid sequence, or a nucleic acid sequence encoding an amino acid sequence described herein due to mutation or truncation is also considered.In some embodiments, a nucleic acid sequence encoding any amino acid sequence provided herein is also provided.

[0258] An exemplary non-restrictive nucleic acid sequence encoding TGFB1 is provided:

[0259] Wild-type TGFB1

[0260]

[0261] Double mutant (dm) TGFB1(R218H, C225R)

[0262]

[0263] Non-restrictive exemplary amino acid sequence of human TGFB1

[0264] Wild type

[0265]

[0266] dmTGFB1 R218C, C225R

[0267]

[0268] An exemplary non-restrictive nucleic acid sequence encoding IL10 is provided:

[0269] Wild type IL10:

[0270]

[0271] High-affinity IL10 (N36I, N110I, K117N, F129L):

[0272]

[0273] An exemplary non-restrictive amino acid sequence of IL10 is provided:

[0274] Wild type IL10:

[0275]

[0276] High-affinity IL10 (N36I, N110I, K117N, F129L):

[0277]

[0278] Mature wild-type IL10

[0279]

[0280] IL10(D25K)

[0281]

[0282] IL10(D25E)

[0283]

[0284] IL10(D25K / E96A)

[0285]

[0286] An exemplary non-restrictive nucleic acid sequence encoding TGFB1 is provided:

[0287] Wild-type TGFB1 (see paragraph [000145] and sequence number 204 above)

[0288] Double mutant TGFB1 (R218C, C225R)

[0289]

[0290] Double mutant TGFB1 (R218H, C225R) (see paragraph [000347] and sequence number

[0214] above).

[0291] An exemplary non-restrictive nucleic acid sequence encoding CTLA4 is provided:

[0292] Wild-type CTLA4:

[0293]

[0294] High-affinity CTLA4 (belatacept; binding domain: A29Y, L104E):

[0295]

[0296] High-affinity CTLA4 (binding domain: A29H):

[0297]

[0298] High-affinity CTLA4 (binding domains: K28H, A29H):

[0299]

[0300] An exemplary non-restrictive amino acid sequence of CTLA4 is provided:

[0301] Wild-type CTLA4:

[0302]

[0303] High-affinity CTLA4 (velatacept; binding domain: A29Y, L104E):

[0304]

[0305] High-affinity CTLA4 (binding domain: A29H):

[0306]

[0307] High-affinity CTLA4 (binding domains: K28H, A29H):

[0308]

[0309] In some embodiments, engineered T cells or a T cell population comprising a heterogeneous sequence encoding dmTGFB1 under the control of a promoter; a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., a knockdown; a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., a knockdown; a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., a knockdown; and the insertion into cells of heterogeneous sequence(s) encoding regulatory T cell promoter molecules under the control of a promoter sequence exhibit at least one inhibitory activity of naturally occurring regulatory T cells (nTreg), such as the inhibition of immune response(s) or biomarkers in in vitro or in vivo assays, e.g., in an animal model of GvHD. For example, in a mouse model of GvHD, a heterogeneous sequence encoding dmTGFB1 under the control of a promoter; a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., a knockdown; a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., a knockdown; Mice receiving engineered T cells comprising modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; and insertion into cells of heterologous sequence(s) encoding a regulatory T cell promoter molecule under the control of a promoter sequence exhibit improved survival compared to a control, e.g., mice receiving PBMC. In certain embodiments, the regulatory T cell promoter molecule comprises IL10 or CTLA4. In certain embodiments, the regulatory T cell promoter molecule comprises IL10 and CTLA4.

[0310] In a specific embodiment, the engineered T cell or T cell population comprises two or more heterogeneous coding sequences, e.g., dmTGFB1 and a regulatory T cell promoter, under the control of a promoter. In a specific embodiment, each heterogeneous coding sequence is under the control of a separate promoter. In a specific embodiment, two heterogeneous coding sequences are under the control of the same promoter. In a specific embodiment, two or more heterogeneous coding sequences are under the control of the same promoter. In a specific embodiment, when the engineered T cell or T cell population comprises three or more heterogeneous coding sequences, e.g., dmTGFB1 and a regulatory T cell promoter, under the control of a promoter, each heterogeneous sequence is independently under the control of a separate promoter or under the control of a promoter that controls the expression of one or more heterogeneous coding sequences.

[0311] C. T cell receptor (TCR)

[0312] In some embodiments, the engineered T cell or T cell population comprises modifications such as intracellular insertion of heterogeneous sequence(s) encoding a dmTGFB1 molecule under the control of a promoter sequence, and further comprises modifications of an endogenous nucleic acid sequence encoding TCR gene sequence(s), e.g., knockdown, and modifications of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown, further comprising modifications of an endogenous nucleic acid sequence encoding TCR gene sequence(s), e.g., knockdown.

[0313] In some embodiments, the engineered T cell or T cell population comprising the intracellular insertion of heterologous sequence(s) encoding a dmTGFB1 molecule under the control of a promoter sequence and the modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown, further comprising the modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; the modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; and / or the intracellular insertion of heterologous sequence(s) encoding a regulatory T cell promoter molecule under the control of a promoter sequence further comprises the modification of an endogenous nucleic acid sequence encoding TCR gene sequence(s), e.g., knockdown.

[0314] In some embodiments, the engineered T cell or T cell population comprises a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; and intracellular insertion of a heterogeneous sequence encoding a dmTGFB1 molecule and a regulatory T cell promoter molecule, each under the control of a promoter sequence, and further comprises a modification of an endogenous nucleic acid sequence encoding TCR gene sequence(s), e.g., knockdown.

[0315] In some embodiments, the engineered T cell or T cell population comprising a modification including the insertion into the cell of a heterogeneous sequence(s) encoding a dmTGFB1 molecule under the control of a promoter sequence comprises: a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; further comprising the insertion into the cell of a heterogeneous sequence encoding a dmTGFB1 molecule and a regulatory T cell promoter molecule, each under the control of a promoter sequence, and the insertion into the cell of a heterogeneous sequence(s) encoding a targeting receptor; and further comprising a modification of an endogenous nucleic acid sequence encoding TCR gene sequence(s), e.g., knockdown.

[0316] Generally, a TCR is a heterodimeric receptor molecule containing two TCR polypeptide chains, α and β. Genomic sequences or loci suitable for targets for knockdown are known in the art. In some embodiments, engineered T cells comprise a TCR α-chain gene sequence, e.g., a modification of TRAC, e.g., knockdown. e.g. See NCBI gene ID: 28755; ensemble: ENSG00000277734 (T-cell receptor alpha constant), US 2018 / 0362975, and WO2020081613.

[0317] In some embodiments, the engineered T cell or T cell population includes a modification comprising the insertion of a sequence encoding dmTGFB1.

[0318] In some embodiments, the engineered T cell or T cell population comprising intracellular insertion of heterologous sequence(s) encoding a dmTGFB1 molecule under the control of a promoter sequence and modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown, further comprises modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; insertion of sequence(s) encoding a regulatory T cell promoter molecule selected from IL10, CTLA4, TIGIT, IDO1, ENTPD1, ​​NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, BACH2, and IL2RA; and modification of an endogenous nucleic acid sequence encoding TCR gene sequence(s), e.g., knockdown.

[0319] In some embodiments, the engineered T cell or T cell population comprising the intracellular insertion of heterogeneous sequence(s) encoding the dmTGFB1 molecule under the control of the promoter sequence and the modification of the endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown, further comprises the modification of the endogenous nucleic acid sequence encoding TNFA, e.g., knockdown, the modification of the endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; the insertion of sequence(s) encoding a regulatory T cell promoter molecule selected from IL10, CTLA4, TIGIT, IDO1, ENTPD1, ​​NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, BACH2; and the modification of the endogenous nucleic acid sequence encoding the TCR gene sequence(s), e.g., knockdown.

[0320] In some embodiments, the engineered T cell or T cell population comprising intracellular insertion of heterologous sequence(s) encoding the dmTGFB1 molecule under the control of the promoter sequence and modification of the endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown, further comprises modification of the endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modification of the endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; insertion of sequence(s) encoding IL10 or CTLA4, and modification of the endogenous nucleic acid sequence encoding TCR gene sequence(s), e.g., knockdown.

[0321] In some embodiments, the engineered T cell or T cell population comprises a modification of the endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; a modification of the endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; a modification of the endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; insertion of sequence(s) encoding dmTGFB1, IL10, and CTLA4; and a modification of the endogenous nucleic acid sequence encoding TCR gene sequence(s), e.g., knockdown.

[0322] In some embodiments, the engineered T cell or T cell population comprises a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; an insertion of a sequence encoding a dmTGFB1 molecule, an insertion of sequence(s) encoding a regulatory T cell promoter molecule, and a modification of an endogenous TCR gene, e.g., TRAC gene sequence, e.g., knockdown.

[0323] In some embodiments, the engineered T cell or T cell population comprises a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; an insertion of sequence(s) encoding the dmTGFB1 molecule; an insertion of sequence(s) encoding a regulatory T cell promoter selected from IL10, CTLA4, TIGIT, IDO1, ENTPD1, ​​NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, BACH2, and IL2RA; and a modification of an endogenous TCR gene, e.g., TRAC gene sequence, e.g., knockdown.

[0324] In some embodiments, the engineered T cell or T cell population comprises a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; an insertion of a sequence encoding the dmTGFB1 molecule, an insertion of sequence(s) encoding IL10 or CTLA4, and a modification of a TCR gene, e.g., TRAC gene sequence, e.g., knockdown.

[0325] In any of these embodiments, the engineered T cell or T cell population may further comprise the insertion of sequence(s) encoding a CAR that targets a targeting receptor as described herein, e.g., MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), demoglain 3 (DSG3), and MHC class I HLA-A (HLA-A*02).

[0326] In some embodiments, engineered T cells or a T cell population comprising intracellular insertion of heterologous sequence(s) encoding a dmTGFB1 molecule under the control of a promoter sequence further comprises modification of the TRC gene sequence by gene editing, e.g., knockdown, as evaluated by sequencing, e.g., NGS, wherein at least 50%, 55%, 60%, 65%, at least 70%, 75%, 80%, 85%, 90%, or 95% of the cells comprise insertions, deletions, or substitutions in the endogenous TRC gene sequence. In some embodiments, TRC is reduced, e.g., by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or below the detection limit of the assay compared to a suitable control in which the TRC gene is not modified. Testing for TRC protein and mRNA expression is known in this technical field.

[0327] In some embodiments, the engineered T cells or T cell populations include the insertion of sequence(s) encoding a targeting receptor by gene editing, as evaluated, for example, by sequencing, for example, NGS.

[0328] In some embodiments, the T cell population comprises T cells further engineered to include insertion into the cell of heterologous sequence(s) encoding the dmTGFB1 molecule under the control of the promoter sequence, and modification of the endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown. In some embodiments, the T cell population comprises T cells further comprising modification of the endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modification of the endogenous nucleic acid sequence encoding the insertion of sequence(s) encoding the regulatory T cell promoter molecule, e.g., knockdown; and / or modification of at least one TCR gene sequence, e.g., knockdown. In some embodiments, 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 at least one modification of a TCR gene sequence, e.g., knockdown, as evaluated by sequencing, e.g., NGS.

[0329] In some embodiments, the T cell population comprises T cells further engineered to include insertion into the cell of heterologous sequence(s) encoding a dmTGFB1 molecule under the control of a promoter sequence, and modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; insertion of sequence(s) encoding a regulatory T cell promoter molecule, and modification of at least one TCR gene sequence, e.g., knockdown. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the T cells in the T cell population are manipulated to include at least one modification of a TCR gene sequence, e.g., knockdown, as evaluated by sequencing, e.g., NGS.

[0330] In some embodiments, a guide RNA that specifically targets a region within a TCR gene, e.g., a TRAC gene, is used to provide a modification of the TCR gene, e.g., a knockdown.

[0331] In some embodiments, the TCR gene is modified, for example, knocked down in T cells using guide RNA in combination with an RNA-guided DNA binder. In some embodiments, the invention discloses a T cell engineered by inducing a break (e.g., double-strand break (DSB) or single-strand break (nick)) within the T cell's TCR gene using guide RNA in combination with, for example, an RNA-guided DNA binder (e.g., a CRISPR / Cas system). The method may be used in vitro or in vitro, for example, in the production of cell products to suppress an immune response.

[0332] In some embodiments, the guide RNA mediates target-specific cleavage by an RNA-guided DNA binder (e.g., Cas nuclease) at the region described herein within the TCR gene. In some embodiments, it will be understood that the guide RNA comprises a guide sequence that binds to or can bind to said region.

[0333] D. Guide RNA

[0334] In any embodiment of the present invention, the guide RNA may further comprise trRNA. In each composition and method embodiment described herein, the crRNA and trRNA may be associated as a single RNA (sgRNA) or may exist on separate RNAs (dgRNA). In the context of sgRNA, the crRNA and trRNA components may be covalently linked, for example, through phosphodiester bonds or other covalent bonds. In some embodiments, the sgRNA comprises one or more links between nucleotides that are not phosphodiester links.

[0335] In each of the composition, use, and method embodiments described herein, the guide RNA may comprise two RNA molecules as "double guide RNA" or "dgRNA". The dgRNA comprises, for example, a first RNA molecule comprising a crRNA comprising the guide sequence presented herein and a second RNA molecule comprising a trRNA. The first and second RNA molecules may not be covalently linked, but may form an RNA double strand through base pairing between portions of the crRNA and trRNA.

[0336] In each of the composition, use, 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 part thereof) comprising the guide sequence presented herein that is covalently linked to a trRNA. The sgRNA may comprise 15, 16, 17, 18, 19, or 20 consecutive nucleotides of the guide sequence presented herein. In some embodiments, the crRNA and trRNA are covalently linked through a linker. In some embodiments, the sgRNA forms a stem-loop structure through base pairing between the crRNA portion and the trRNA portion. In some embodiments, the crRNA and trRNA are covalently linked through one or more bonds other than phosphodiester bonds.

[0337] In some embodiments, trRNA may comprise all or part of a trRNA sequence derived from a naturally occurring CRISPR / Cas system. In some embodiments, trRNA comprises truncated or modified wild-type trRNA. The length of trRNA depends on the CRISPR / Cas system used. In some embodiments, trRNA comprises or consists of 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more than 100 nucleotides. In some embodiments, trRNA may comprise specific secondary structures, such as, for example, one or more hairpin or stem-loop structures or one or more ridge structures.

[0338] In some embodiments, the target sequence or region may be complementary to the guide sequence of the guide RNA. In some embodiments, the degree of complementarity or identity between the guide sequence of the guide RNA and its corresponding target sequence may be 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the target sequence and the guide sequence of the gRNA may be 100% complementary or identical. In other embodiments, the target sequence and the guide sequence of the gRNA may contain one mismatch. For example, the target sequence and the guide sequence of the gRNA may contain 1, 2, 3, 4, or 5 mismatches, where the total length of the guide sequence is about 20 or 20. In some embodiments, the target sequence and the guide sequence of the gRNA may contain 1 to 4 mismatches, where 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, for example, at least 80%, 85%, 90%, or 95% 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, that is, one nucleotide that is not identical or complementary according to the reference sequence. For example, the guide sequence and the target sequence may contain one or two, for example, one or fewer mismatches, wherein the total length of the target sequence is 19, 20, 21, 22, 23, or 24 nucleotides or more. In some embodiments, the guide sequence and the target region may contain one or two mismatches, wherein the guide sequence comprises at least 24 nucleotides. In some embodiments, the guide sequence and the target region may contain 1-2 mismatches, wherein the guide sequence contains 24 nucleotides.

[0339] In any embodiment of the present invention, each guide sequence of the present invention may further comprise additional nucleotides to form crRNA or guide RNA, for example, having the following exemplary nucleotide sequence following the guide sequence at its 3' end: GUUUUAGAGCUAUGCUGUUUUG in a 5' to 3' orientation (SEQ ID NO. 134).

[0340] In the case of sgRNA, the guide sequence may additionally include additional nucleotides to form sgRNA, for example, having the following exemplary nucleotide sequence following the 3' end of the guide sequence:

[0341]

[0342] In any embodiment, the guide RNA disclosed herein binds to an upstream region of a prophosphate adjacent motif (PAM). As understood by those skilled in the art, the PAM sequence occurs on the opposite strand of the strand containing the target sequence and varies depending on the CRISPR / Cas system. That is, the PAM sequence is on the complement strand of the target strand (the strand containing the target sequence to which the guide RNA binds). In some embodiments, the PAM is selected from NGG, NNGRRT, NNGRR(N), NNAGAAW, NNNNG(A / C)TT, and NNNNRYAC, for example, when the Cas system includes SpyCas9. In another embodiment, the PAM sequence comprises, for example, NCC, N4GAYW, N4GYTT, N4GTCT, NNNNCC(a), NNNNCAAA (wherein N is defined as any nucleotide, W is defined as either A or T, and R is defined as either A or G; and in (a), A is preferred but not required after the second C).

[0343] In some embodiments, the guide RNA sequence provided herein is complementary to the sequence adjacent to the PAM sequence.

[0344] In some embodiments, the guide RNA sequence comprises a sequence complementary to a sequence within a genomic region selected in 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 from within a genomic region selected in 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 in the table of the present invention.

[0345] The guide RNA disclosed herein mediates target-specific cleavage to cause double-strand break (DSB). The guide RNA disclosed herein mediates target-specific cleavage to cause single-strand break (SSB or nick).

[0346] E. Chemically modified gRNA

[0347] In some embodiments, gRNA is chemically modified. gRNA comprising one or more modified nucleosides or nucleotides is referred to as “modified” gRNA or “chemically modified” gRNA to describe the presence of one or more non-natural or naturally occurring components or compositions used in place of or in addition to the regular A, G, C, and U residues. In some embodiments, modified gRNA having non-regular nucleosides or nucleotides is synthesized and is referred to herein as “modified.” Modified nucleosides and nucleotides may comprise one or more of the following: (i) a change, e.g., substitution, of one or both of unlinked phosphate oxygens or one or more linked phosphate oxygens in a phosphodiester backbone linkage of the nucleoside (exemplary backbone modification); (ii) a change, e.g., substitution, of a ribose sugar component, e.g., a change, e.g., of the 2’ hydroxyl of the ribose sugar (exemplary sugar modification); (iii) a modification or substitution of a naturally occurring nucleobase containing a non-regular nucleobase (exemplary base modification); and (iv) modification of the 3' or 5' end of the oligonucleotide to provide exonuclease stability, e.g., 2' O-me, 2' halide, or 2' deoxy-substituted ribose; or use of an inversely baseless terminal nucleotide, or replace the phosphodiester with a phosphothioate.

[0348] Chemical modifications such as those listed above may be combined to provide modified gRNA comprising nucleosides and nucleotides (collectively “residues”) having two, three, four or more modifications. For example, modified residues may have modified sugars and modified nucleobases. In some embodiments, all bases of the gRNA are modified, for example, all bases have modified phosphate groups such as phosphorothioate groups. In certain embodiments, all or substantially all of the phosphate groups of the gRNA molecule are replaced with phosphorothioate groups. In some embodiments, the modified gRNA comprises at least one modified residue at or near the 5’ end of the RNA. In some embodiments, the modified gRNA comprises at least one modified residue at or near the 3’ end of the RNA. Certain gRNA comprises at least one modified residue at or near the 5’ end and 3’ end of the RNA.

[0349] In some embodiments, the guide RNA disclosed herein comprises one of the modification patterns disclosed in WO2018107028, the contents of which are incorporated herein by reference in the relevant parts. In some embodiments, the guide RNA disclosed herein comprises one of the structure / modification patterns disclosed in US20170114334, incorporated herein by reference. In some embodiments, the guide RNA disclosed herein comprises one of the structure / modification patterns disclosed in WO2017136794, WO2017004279, WO2019237069, US2018187186, US2019048338, WO2021119275, or WO2022125968, incorporated herein by reference.

[0350] F. mRNA encoding an RNA-guided DNA binder

[0351] In some embodiments, the cell or method comprises mRNA containing an open read frame (ORF) encoding an RNA-guided DNA-binding agent, such as the Cas nuclease described herein. Cas9 ORFs are provided herein and are known in the art. As an example, a Cas9 ORF may be codon optimized such that the coding sequence includes one or more alternative codons for one or more amino acids. As used herein, "alternative codons" refer to variations in the codon usage for a given amino acid and may or may not be codons that are desirable or optimized for a given expression system (codon optimization). Desired codon usages, or codons that are well tolerated for a given expression system, are known in the art. The Cas9 coding sequences, Cas9 mRNA, and Cas9 protein sequences of WO2013 / 176772, WO2014 / 065596, WO2016 / 106121, WO2019 / 067910, and WO2022 / 125968 are incorporated herein by reference. In particular, the ORF and Cas9 amino acid sequences in the table of paragraph

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

[0214] –

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

[0352] In some embodiments, the modified ORF may comprise a modified uridine at at least one, multiple, or all uridine positions. In some embodiments, the modified uridine is a uridine modified with, for example, a halogen, methyl, or ethyl at the 5 position. In some embodiments, the modified uridine is a pseudouridine modified with, for example, a halogen, methyl, or ethyl at the 1 position. The modified uridine may be, for example, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-ioduridine, or a combination thereof. In some embodiments, the modified uridine is 5-methoxyuridine. In some embodiments, the modified uridine is 5-ioduridine. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of N1-methyl pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-ioduridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-ioduridine. In some embodiments, the modified uridine is a combination of 5-ioduridine and 5-methoxyuridine.

[0353] In some embodiments, the mRNA disclosed herein comprises a 5' cap such as Cap0, Cap1, or Cap2. The 5' cap is generally a 7-methylguanine ribonucleotide linked via a 5'-triphosphate to the 5' position of the first nucleotide of the 5' to 3' chain of the mRNA, i.e., the first cap-proximal nucleotide (this may be further modified, for example, ARCA (anti-reverse cap analog; Thermo Fisher Scientific catalog number AM8045) is a cap analog comprising a 7-methylguanine 3'-methoxy-5'-triphosphate linked to the 5' position of the guanine ribonucleotide). In Cap0, the riboses of the first and second cap-proximal nucleotides of the mRNA both contain a 2'-hydroxyl. In Cap1, the riboses of the first and second transcribed nucleotides of the mRNA each contain a 2'-methoxyl and a 2'-hydroxyl. For example, CleanCap ™ AG(m7G(5')ppp(5')(2'OMeA)pG; TriLink Biotechnologies Catalog No. N-7113) or CleanCap ™ Refer to GG(m7G(5')ppp(5')(2'OMeG)pG; TriLink Biotechnologies Catalog No. N-7133). In Cap2, the riboses of the first and second cap-proximal nucleotides of the mRNA both contain a 2'-methoxy. For example, Katibah et al. (2014) Proc Natl Acad Sci USA 111(33):12025-30; Abbas et al. (2017) Proc Natl Acad Sci USA See 114(11):E2106-E2115.

[0354] In some embodiments, the mRNA further comprises a poly-adenylated (poly-A) tail. In some embodiments, the poly-A tail comprises 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines (SEQ No. 147), optionally up to 300 adenines (SEQ No. 148). In some embodiments, the poly-A tail comprises 95, 96, 97, 98, 99, or 100 adenine nucleotides (SEQ No. 149).

[0355] G. T cells for manipulation

[0356] The engineered cells provided herein are produced from a cell population enriched with T cells. In some embodiments, the T cells are CD4+ T cells. CD4+ T cells may be autologous or allogeneic cells. These cells can be readily obtained from commercially available fresh Leucopec samples from various sources, for example, StemCell Technologies. CD4+ T cells can be isolated using commercially available kits by routine methods, for example, by negative selection using a human CD4+ T cell isolation kit. However, methods for producing CD4+ T cells from other sources are also known in the art. For example, CD4+ T cells can be obtained using pluripotent cells such as hematopoietic stem cells (HSCs isolated from bone marrow or umbilical cord blood), hematopoietic progenitor cells (e.g., lymphoid progenitor cells), or mesenchymal stem cells (MSCs). Pluripotent cells can develop into more than one cell type, but their range of differentiation is more limited than that of pluripotent cells. Pluripotent cells can be derived from established cell lines or isolated from human bone marrow or umbilical cord. For example, HSCs can be isolated from a patient or a healthy donor following G-CSF-induced mobilization, plerixaphor-induced mobilization, or a combination thereof. To isolate HSCs from blood or bone marrow, cells within the blood or bone marrow can be panned by 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.T cells can generate more cells than, for example, more than 500 times, more than 600 times, more than 700 times, more than 800 times, more than 900 times, or more than 1000 times, compared to the expansion of tTreg according to methods known in the art, as measured on days 8, 9, 10, 11, 12, 13, 14, and 15 after the first day of editing.

[0357] In some embodiments, the engineered product is a cell population enriched with CD8+ T cells. The CD8+ T cells may be autologous or allogeneic cells. Like CD4+ T cells, CD8+ T cells may be isolated from a sample, e.g., a commercially available sample or a patient sample, or may be produced from other sources according to methods known in the art. For example, sources may include pluripotent cells such as hematopoietic stem cells (HSCs isolated from bone marrow or umbilical cord blood), hematopoietic progenitor cells (e.g., lymphoid progenitor cells), or mesenchymal stem cells (MSCs). Methods for promoting differentiation into CD8+ T cells are known in the art. T cells engineered from CD8+ T cells as described herein can generate more cells than tTreg expansion according to methods known in the art, for example, more than 500 times, more than 600 times, more than 700 times, more than 800 times, more than 900 times, or more than 1000 times, as measured on days 8, 9, 10, 11, 12, 13, 14, and 15 after the first day of editing.

[0358] III. Delivery Method

[0359] The guide RNA, RNA-guided DNA binder (e.g., Cas nuclease), and nucleic acid sequence disclosed herein may be delivered in vitro or in vitro to cells or cell populations using various suitable methods known and available in the art for the production of engineered T cells, comprising: insertion of a sequence encoding dmTGFB1; optionally, further comprising a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown, IFNG, or a modification of an endogenous nucleic acid sequence encoding IL17A, e.g., knockdown; insertion of sequence(s) encoding regulatory T cell promoter molecules, e.g., IL10, CTLA4; optionally further comprising insertion of sequence(s) encoding a targeting receptor such as a CAR; and optionally further comprising a modification of TCR sequence(s), e.g., knockdown. The guide RNA, RNA-guided DNA binder, and nucleic acid construct may be delivered individually or in any combination using the same or different suitable delivery methods.

[0360] Conventional viral and non-viral-based gene delivery methods can be used to introduce guide RNA, as well as RNA-guided DNA binders and donor constructs, into cells (e.g., mammalian cells) and target tissues. As further provided herein, non-viral vector delivery system nucleic acids, e.g., non-viral vectors, plasmid vectors, and e.g., naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes, lipid nanoparticles (LNPs), or poloxamers. Viral vector delivery systems include DNA and RNA viruses.

[0361] Methods and compositions for the non-viral delivery of nucleic acids include electroporation, lipofection, microinjection, biorhysics, virosomes, liposomes, immunoliposomes, LNPs, polycations or lipid:nucleic acid conjugates, naked nucleic acids (e.g., naked DNA / RNA), artificial virions, and agent-enhanced uptake of DNA. For example, sonoporation using a Sonitron 2000 system (Rich-Mar) may also be used for nucleic acid delivery.

[0362] Various delivery systems containing guide RNA, RNA-guided DNA binder, and donor constructs alone or in combination (e.g., Vectors, liposomes, LNPs) may also be administered to cells or cell cultures in vitro. Administration is carried out by any route commonly used to introduce molecules into blood, body fluids, or cells, and includes, but is not limited to, injection, infusion, topical application, and electroporation. Suitable methods for administering these nucleic acids are available and are well known to those skilled in the art.

[0363] In certain embodiments, the present disclosure comprises any composition disclosed herein, for example, a guide RNA or sequence encoding a dmTGFB1 molecule comprising one or more of the guide sequences described herein for modifying (e.g., knockdown) TGFBR2, IFNG, and TNFA, sequence(s) encoding a regulatory T cell promoter molecule, for example, IL10 or CTLA4, or a targeting receptor, for example, CAR, for example, MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute transporter family 2 member 2 (SCL2A2), glutamate The present invention provides a DNA or RNA vector encoding a donor construct comprising a sequence encoding decarboxylase (GAD2), demoglain 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CAR. In some embodiments, the vector also comprises a sequence encoding an RNA-guided DNA binder. In certain embodiments, the DNA or RNA vector encodes any one or more of the compositions described herein, or any combination thereof. In some embodiments, the vector further comprises, for example, a promoter, an enhancer, and a regulatory sequence. In some embodiments, a vector comprising a guide RNA comprising any one or more guide sequences described herein also comprises one or more nucleotide sequence(s) encoding crRNA, trRNA, or crRNA and trRNA as disclosed herein.

[0364] In certain embodiments, the present disclosure provides a DNA or RNA vector encoding a regulatory T cell promoter molecule and a targeting receptor. Such a vector enables the selection of cells based on the presence of a receptor on a cell that also contains the encoding sequence for the regulatory T cell promoter molecule. Methods for positive and negative selection based on the presence of cell surface molecules are known in the art.

[0365] In some embodiments, the vector comprises a nucleotide sequence encoding the guide RNA described herein. In some embodiments, the vector comprises one copy of the guide RNA. In other embodiments, the vector comprises more than one copy of the guide RNA. In embodiments having more than one guide RNA, the guide RNAs may not be identical to target different target sequences, or they may be identical in that they target the same target sequence. In some embodiments where the vector comprises more than one guide RNA, each guide RNA may have different properties, such as activity or stability, within a complex with an RNA-guided DNA nuclease, e.g., a Cas RNP complex. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to at least one transcription or translation control sequence, e.g., a promoter, a 3' UTR, or a 5' UTR. In one embodiment, the promoter is a tRNA promoter, e.g., a tRNA Lys3 Or it could be a tRNA chimera. Mefferd et al. RNA . 2015 21:1683-9; Scherer et al., Nucleic Acids ResSee . 2007 35: 2620-2628. In some embodiments, the promoter may be recognized by RNA polymerase III (Pol III). Non-limiting examples of Pol III promoters include U6 and H1 promoters. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human U6 promoter. In other embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human H1 promoter. In embodiments having more than one guide RNA, the promoter used to induce expression may be the same or different. In some embodiments, the nucleotide encoding the crRNA of the guide RNA and the nucleotide encoding the trRNA of the guide RNA may be provided in the same vector. In some embodiments, the nucleotide encoding the crRNA and the nucleotide encoding the trRNA may be driven by the same promoter. In some embodiments, crRNA and trRNA may be transcribed into a single transcript. For example, crRNA and trRNA may be processed from a single transcript to form a double-molecule guide RNA. Alternatively, crRNA and trRNA may be transcribed into a single-molecule guide RNA (sgRNA). In other embodiments, crRNA and trRNA may be driven by a corresponding promoter on the same vector. In yet another embodiment, crRNA and trRNA may be encoded by different vectors.

[0366] In some embodiments, the nucleotide sequence encoding the guide RNA may be located on the same vector containing the nucleotide sequence encoding an RNA-guided DNA-binding agent, such as the Cas protein. In some embodiments, the expression of the guide RNA and the RNA-guided DNA-binding agent, e.g., the Cas protein, 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 RNA-guided DNA-binding agent, such as the Cas protein. In some embodiments, the guide RNA and the RNA-guided DNA-binding agent, e.g., the Cas protein transcript, may be contained within a single transcript. For example, the guide RNA may be within the untranslated region (UTR) of the RNA-guided DNA-binding agent, such as the Cas protein transcript. In some embodiments, the guide RNA may be within the 5' UTR of the transcript. In other embodiments, the guide RNA may be within the 3' UTR of the transcript. In some embodiments, the intracellular half-life of the transcript may be reduced by containing the guide RNA within its 3' UTR and shortening the length of its 3' UTR. In additional embodiments, the guide RNA may be located within an intron of the transcript. In some embodiments, a suitable splice site may be added in the intron where the guide RNA is located so that the guide RNA is properly spliced ​​from the transcript. In some embodiments, the expression of an RNA-guided DNA-binding agent, such as a Cas protein, and the guide RNA from the same vector in close transient proximity may facilitate more efficient formation of the CRISPR RNP complex.

[0367] In some embodiments, the nucleotide sequence encoding the guide RNA or RNA-guided DNA-binding agent is the dmTGFB1 molecule, the regulatory T cell promoter molecule, e.g., IL10, CTLA4; Or may be located on the same vector comprising a construct containing a sequence encoding a targeting receptor, e.g., CAR, e.g., MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), demoglain 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CAR. In some embodiments, on the same vector, a dmTGFB1 molecule, a regulatory T cell promoter molecule, e.g., IL10, CTLA4; Or, proximity of a construct comprising a guide RNA (or RNA-guided DNA binder) to a sequence encoding a targeting receptor, e.g., CAR, e.g., MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), demoglain 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CAR can facilitate more effective insertion of the construct into an insertion site created by the guide RNA / RNA-guided DNA binder.

[0368] In certain embodiments, DNA and RNA vectors may contain more than one open read frame for expression under a single promoter present in the vector or at the genome insertion site. In these embodiments, a coding sequence for a self-cleaving peptide may be included between the open read frames. The self-cleaving peptide may be, for example, a 2A peptide, for example, a P2A peptide, an E2A peptide, an F2A peptide, or a T2A peptide.

[0369] In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding sgRNA and mRNA encoding an RNA-guided DNA binder, which may be a Cas protein, e.g., Cas9 or Cpf1. In some embodiments, the vector comprises crRNA, trRNA, and one or more nucleotide sequence(s) encoding mRNA encoding an RNA-guided DNA binder, which may be a Cas protein, e.g., Cas9 or Cpf1. In one embodiment, Cas9 is Streptococcus phyogenes It originates from (i.e., Spy Cas9). In some embodiments, Cas nuclease is Neisseria meningitidis It is a Cas9 nuclease from (i.e., Nme Cas9, e.g., Nme1, Nme2, or Nme3 Cas9). In some embodiments, a nucleotide sequence encoding crRNA, trRNA, or crRNA and trRNA (which may be sgRNA) comprises or consists of a guide sequence having all or part of a repeat sequence from a naturally occurring CRISPR / Cas system side by itself. 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 not naturally found together with crRNA, trRNA, or crRNA and trRNA.

[0370] In some embodiments, crRNA and trRNA are encoded by non-continuous nucleic acids within a single vector. In other embodiments, crRNA and trRNA may be encoded by continuous nucleic acids. In some embodiments, crRNA and trRNA are encoded by opposing strands of a single nucleic acid. In other embodiments, crRNA and trRNA are encoded by the same strand of a single nucleic acid.

[0371] In some embodiments, the vector comprises a donor construct comprising a sequence encoding a dmTGFB1 molecule, a regulatory T cell promoter molecule, e.g., IL10, or CTLA4, or a targeting receptor, e.g., CAR, e.g., MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), demoglain 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CAR as disclosed herein. In some embodiments, in addition to the donor construct disclosed herein, the vector may further comprise a nucleic acid encoding the guide RNA described herein or a nucleic acid encoding an RNA-guided DNA-binding agent (e.g., a Cas nuclease such as Cas9). In some embodiments, the nucleic acid encoding the RNA-guided DNA-binding agent is on a separate vector from the vector containing the donor construct disclosed herein, either individually or both. In any embodiment, the vector may comprise 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 a regulatory T cell promoter molecule of the donor construct, e.g., IL10 or CTLA4, or a targeting receptor, e.g., MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), demoglain 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CAR. In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding crRNA, trRNA, or both crRNA and trRNA. In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding sgRNA and mRNA encoding an RNA-guided DNA nuclease that may be a Cas nuclease (e.g., Cas9). In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding crRNA, trRNA, and mRNA encoding an RNA-guided DNA nuclease that may be a Cas nuclease such as Cas9. In some embodiments, Cas9 is. Streptococcus phyogenes It is derived from (i.e., Spy Cas9). In some embodiments, Cas nuclease is Neisseria meningitidisIt is a Cas9 nuclease from. In some embodiments, a nucleotide sequence encoding crRNA, trRNA, or crRNA and trRNA (which may be sgRNA) comprises or consists of a guide sequence having all or part of a repeat sequence from a naturally occurring CRISPR / Cas system on its side. A 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 not naturally found together with crRNA, trRNA, or crRNA and trRNA.

[0372] In some embodiments, the vector may be circular. In other embodiments, the vector may be linear. In some embodiments, the vector may be surrounded by lipid nanoparticles, liposomes, non-lipid nanoparticles, or viral capsids. Non-limiting exemplary vectors include plasmids, phagemids, cosmids, artificial chromosomes, minichromosomes, transposons, viral vectors, and expression vectors.

[0373] 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 facilitate cloning or to change one or more properties of the vector. These properties may include packaging ability, transduction efficiency, immunogenicity, genomic integration, replication, transcription, and translation. In some embodiments, a portion of the viral genome may be deleted so that the virus can package an exogenous sequence having a larger size. In some embodiments, the viral vector may have enhanced transduction efficiency. In some embodiments, the immune response induced by the virus may be reduced. In some embodiments, a viral gene that promotes the integration of a viral sequence into the genome (e.g., integrase) may be mutated so that the virus does not integrate. In some embodiments, the viral vector may be a replication defect. In some embodiments, the viral vector may include an exogenous transcription or translation control sequence that drives the expression of a coded sequence on the vector. In some embodiments, the virus may be helper-dependent. For example, the virus may require one or more helper viruses to supply viral components (e.g., viral proteins) necessary to amplify the vector and package it into a viral particle. In such cases, one or more helper components, comprising one or more vectors encoding viral components, may be introduced into a cell or cell population together with the vector system described herein. In other embodiments, the virus may be helper-free. For example, the virus may amplify and package the vector without helper viruses. In some embodiments, the vector system described herein may encode viral components necessary for viral amplification and packaging.

[0374] Non-limiting exemplary viral vectors include adeno-associated virus (AAV) vectors, lentivirus vectors, adenovirus vectors, helper-dependent adenovirus vectors (HDAd), herpes simplex virus (HSV-1) vectors, bacteriophage T4, baculovirus vectors, and retrovirus vectors. In some embodiments, the viral vector may be an AAV vector. In other embodiments, the viral vector may be a lentivirus vector.

[0375] In some embodiments, “AAV” refers to all serotypes, subtypes, and naturally occurring AAVs as well as recombinant AAVs. “AAV” may be used to refer to the virus itself or a derivative thereof. The term “AAV” includes AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAVrh.64R1, AAVhu.37, AAVrh.8, AAVrh.32.33, AAV8, AAV9, AAV-DJ, AAV2 / 8, AAVrh10, AAVLK03, AV10, AAV11, AAV12, rh10, and their hybrids, avian AAV, bovine AAV, canid AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV. In certain embodiments, AAVs are acceptable for use in in vitro applications on human cells. In certain embodiments, the AAV is AAV6. The genomic sequences of various serotypes of AAV, as well as the sequences of natural terminal repeats (TRs), Rep proteins, and capsid subunits, are known in the art. These sequences may be found in the literature or in public databases such as GenBank. As used herein, "AAV vector" refers to an AAV vector containing a heterogeneous sequence that is not of AAV origin (i.e., a nucleic acid sequence heterogeneous to AAV) and typically contains a sequence encoding the heterogeneous polypeptide of interest. The composition may include AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAVrh.64R1, AAVhu.37, AAVrh.8, AAVrh.32.33, AAV8, AAV9, AAV-DJ, AAV2 / 8, AAVrh10, AAVLK03, AV10, AAV11, AAV12, rh10, and their hybrids, avian AAV, bovine AAV, canid AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV capsid sequences.Generally, the heterologous nucleic acid sequence (transplant gene) is flanked by at least one, typically two, AAV inversion terminal repeat sequences (ITRs). The AAV vector can be single-stranded (ssAAV) or self-complementary (scAAV).

[0376] In some embodiments, a lentivirus may be incorporated. In some embodiments, a lentivirus may not be incorporated. In some embodiments, the virus vector may be an adenovirus vector. In some embodiments, the adenovirus may be a high-cloning ability or "gutless" adenovirus, wherein all cryptic virus regions except the 5' and 3' inversion terminal repeats (ITRs) and the packaging signal ('I') are deleted from the virus to increase its packaging capacity. In another embodiment, the virus vector may be an HSV-1 vector. In some embodiments, the HSV-1-based vector is helper-dependent, and in other embodiments, helper-independent. For example, an amplicon vector containing only the packaging sequence requires a helper virus having structural components for packaging, whereas a 30kb-deleted HSV-1 vector removing non-essential virus functions does not require a helper virus. In additional embodiments, the viral vector may be a bacteriophage T4. In some embodiments, the bacteriophage T4 may package any linear or circular DNA or RNA molecule when the head of the virus is empty. In additional embodiments, the viral vector may be a baculovirus vector. In another embodiment, the viral vector may be a retrovirus vector. In embodiments using an AAV or other vector having a smaller cloning capacity, more than one vector may be used to deliver all components of the vector system as disclosed herein. For example, one AAV vector may contain a sequence encoding an RNA-guided DNA-binding agent such as a Cas protein (e.g., Cas9), while a second AAV vector may contain one or more guide sequences.

[0377] In some embodiments, the vector system may induce the expression of one or more nuclease components in a cell. In some embodiments, the vector does not contain a promoter that drives the expression of one or more coding sequences once incorporated into a cell (e.g., using the cell's endogenous promoter when inserted into a specific genomic locus of the cell as illustrated herein). Promoters suitable for driving expression in different types of cells, e.g., CD4+ T cells, are known in the art. In some embodiments, the promoter may be wild-type. In other embodiments, the promoter may be modified for more efficient or effective expression. In yet another embodiment, the promoter may be truncated but retain its function. For example, the promoter may have a normal size or a reduced size suitable for properly packaging the vector within a virus.

[0378] In some embodiments, the vector may comprise a nucleotide sequence encoding an RNA-guided DNA-binding agent, such as the Cas protein (e.g., Cas9) described herein. In some embodiments, the nuclease encoded by the vector may be a Cas protein. In some embodiments, the vector system may comprise one copy of the nucleotide sequence encoding the nuclease. In other embodiments, the vector system may comprise 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.

[0379] In some embodiments, the vector may comprise any one or more constructs comprising a sequence encoding a dmTGFB1 molecule as described herein; a regulatory T cell promoter molecule, e.g., IL10, CTLA4; or a targeting receptor, e.g., a CAR, e.g., MAdCAM-1 CAR. In some embodiments, the dmTGFB1 molecule, the regulatory T cell promoter molecule, e.g., IL10, CTLA4; Or a sequence of a targeting receptor, e.g., CAR, e.g., MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), demoglain 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CAR, may be operably linked to at least one transcription or translation control sequence. In some embodiments, dmTGFB1 molecule, regulatory T cell promoter molecule, e.g., IL10, CTLA4; Or a sequence of a targeting receptor, e.g., CAR, e.g., MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), demoglain 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CAR, may be operably linked to at least one promoter.In some embodiments, the sequence of a dmTGFB1 molecule, a regulatory T cell promoter molecule, e.g., IL10, CTLA4; or a targeting receptor, e.g., CAR, e.g., MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), demoglain 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CAR is not linked to a promoter that drives the expression of a heterologous gene.

[0380] In some embodiments, the promoter may be constitutive, inductive, or tissue-specific. In some embodiments, the promoter may be a constitutive promoter. Non-limiting exemplary constitutive promoters include cytomegalovirus immediate early promoter (CMV), primate virus (SV40) promoter, adenovirus major late (MLP) promoter, roose sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor-alpha (EF1a) promoter, ubiquitin promoter, actin promoter, tubulin promoter, immunoglobulin promoter, functional fragments thereof, or any combination of the foregoing. In some embodiments, the promoter may be a CMV promoter. In some embodiments, the promoter may be a truncated CMV promoter. In other embodiments, the promoter may be an EF1a promoter. In some embodiments, the promoter may be an inducible promoter. Non-limiting exemplary inducible promoters include those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter is, for example, Tet-On ® It may have low basal (non-induced) expression levels, such as the promoter (Clontech).

[0381] In certain embodiments, the promoter is suitable for use in in vitro application in human cells.

[0382] In some embodiments, the promoter may be a tissue-specific promoter, for example, a promoter specific to expression in T cells.

[0383] In some embodiments, the composition comprises a vector system. In some embodiments, the vector system may comprise a single vector. In other embodiments, the vector system may comprise two vectors. In additional embodiments, the vector system may comprise three vectors. When different guide RNAs are used for multiplexing or when multiple copies of guide RNA are used, the vector system may comprise more than three vectors.

[0384] In some embodiments, the vector system may include an inducible promoter that begins expression only after being delivered to a target cell. Non-limiting exemplary inducible promoters include those induced by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter is, for example, Tet-On ® It may have low basal (non-induced) expression levels, such as the promoter (Clontech).

[0385] In an additional embodiment, the vector system may include a tissue-specific promoter.

[0386] A non-restrictive exemplary viral vector sequence is provided below:

[0387] CTLA4 insert (nucleotide sequence)

[0388]

[0389] CTLA4 insert (amino acid sequence)

[0390]

[0391] IL10 insert (nucleotide sequence)

[0392]

[0393] IL10 insert (amino acid sequence)

[0394]

[0395] Empty Lenticular

[0396]

[0397]

[0398]

[0399]

[0400] Empty Lenticular

[0401]

[0402]

[0403]

[0404]

[0405]

[0406] Other sequences are provided throughout this specification and in the table below, and the list of sequences submitted herein constitutes part of this specification.

[0407] Ranking table

[0408] In the table below, the terms "mA", "mC", "mU", or "mG" are used to denote nucleotides modified to 2'-O-Me.

[0409] In the table below, "N" is used to independently represent any nucleotide (e.g., A, U, T, C, G). In a given embodiment, the nucleotide is an unmodified RNA nucleotide residue, namely a ribose sugar and a phosphodiester backbone.

[0410] In the table below, "*" is used to indicate PS variations. In this application, term A * , C * , U * or G * It can be used to represent a nucleotide connected to the next (e.g., 3') nucleotide by a PS bond.

[0411] When a DNA sequence (including T) is mentioned in relation to RNA, T should be replaced with U (which may be modified or unmodified depending on the context), and it is understood that the same applies in the reverse case.

[0412] In the table below, a single amino acid character code is used to provide the peptide sequence.

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425] A vector comprising, individually or in any combination, a donor construct comprising a sequence encoding a guide RNA, an RNA-binding DNA binder, or a dmTGFB1 molecule, a regulatory T cell promoter molecule, e.g., IL10, CTLA4; or a targeting receptor, e.g., CAR, may be delivered by liposomes, nanoparticles, exosomes, or microvesicles. The vector may also be delivered by lipid nanoparticles (LNPs). A donor construct comprising one or more guide RNAs, RNA-binding DNA binders (e.g., mRNA), or sequences encoding a heterologous protein may be delivered individually or in any combination by LNPs, liposomes, nanoparticles, exosomes, or microvesicles. A donor construct comprising one or more guide RNAs, RNA-binding DNA binders (e.g., mRNA), or sequences encoding a heterologous protein may be delivered individually or in any combination by LNPs. In some embodiments, one or more guide RNAs and RNA-guided DNA binders (e.g., mRNA) are delivered by an LNP. The donor construct may be delivered by a viral vector.

[0426] Lipid nanoparticles (LNPs) are a well-known means for the delivery of nucleotide and protein cargoes and can be used for the delivery of any guide RNA, RNA-guided DNA binder, or donor construct disclosed herein.

[0427] As used herein, lipid nanoparticles (LNPs) refer to particles comprising a plurality (i.e., more than one) of lipid molecules that are physically associated with one another by intermolecular forces. LNPs may be, for example, microspheres (monolayer and multilayer vesicles, e.g., “liposomes”—layered lipid bilayers, which are substantially spherical in some embodiments and, in more specific embodiments, may comprise an aqueous core containing, for example, a substantial portion of RNA molecules), the dispersed phase of an emulsion, micelles, or the internal phase of a suspension (see, for example, WO2017173054, the contents of which are incorporated herein by reference in their entirety). Any LNP known to those skilled in the art that is capable of delivering nucleotides to a target may be utilized. Exemplary LNP formulations and methods for delivering an agent to T cells for modification are provided, for example, in WO2021222287.

[0428] In some embodiments, the present invention provides a method for delivering any of the guide RNAs described herein or any of the donor constructs disclosed herein, either alone or in combination, to a cell or a cell population or a target, wherein any one or more components are associated with an LNP. In some embodiments, the method further comprises an RNA-guided DNA-binding agent (e.g., Cas9 or a sequence encoding Cas9).

[0429] In some embodiments, the present invention provides a composition comprising, alone or in combination with an LNP, any of the guide RNA described herein or the donor constructs disclosed herein. In some embodiments, the composition further comprises an RNA-guided DNA binder (e.g., Cas9 or a sequence encoding Cas9).

[0430] In some embodiments, the LNP comprises cationic lipids. In some embodiments, the LNP comprises (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also known as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate or other ionic lipids. For example, refer to the lipids in WO2019067992, WO2017173054, WO2015095340, and WO2014136086, as well as the references provided herein. 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 used interchangeably in the context of the LNP lipid, for example, where the ionizable lipid is cationic depending on pH.

[0431] In some embodiments, the LNP associated with the composition disclosed herein is intended for use in manufacturing cell-based agents for suppressing an immune response. Methods for manufacturing cell-based therapies and reagents for use in cell-based therapies are known in the art.

[0432] In some embodiments, any of the guide RNA, RNA-guided DNA binder, or donor construct disclosed herein, whether naked or part of a vector, is formulated into or administered via lipid nanoparticles, alone or in combination; see, for example, WO2019067992, WO2017173054, or WO2021222287, the contents of which are incorporated herein by reference in whole.

[0433] In some embodiments, the LNP composition comprises an RNA component and a lipid component, wherein the lipid component comprises amine lipids such as biodegradable, ionizable lipids. In some cases, the lipid component comprises biodegradable, ionizable lipids, cholesterol, DSPC (distearoylphosphatidylcholine), and PEG-DMG (1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene glycol 2000 (PEG2k-DMG)). In a specific embodiment, the lipid nucleic acid assembly contained ionizable lipid A ((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate), also known as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, cholesterol, DSPC (distearoylphosphatidylcholine), and PEG2k-DMG (1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene glycol 2000(PEG2k-DMG)). In a specific embodiment, the components were each 50 lipid A:38 Cholesterol:9 DSPC:3 PEG-DMG molar ratio. In certain embodiments, the components are present in a 35 lipid A:47.5 cholesterol:15 DSPC:2.5 PEG-DMG molar ratio, respectively. The lipid nucleic acid assembly may be formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 6 and a gRNA to mRNA weight ratio of 2:1, 1:1, or 1:2.

[0434] It will be apparent that a donor construct comprising a guide RNA, an RNA-guided DNA binder (e.g., a Cas nuclease or a nucleic acid encoding a Cas nuclease), and a dmTGFB1 molecule; and a sequence encoding a regulatory T cell promoter molecule, e.g., IL10, or a targeting receptor such as a CAR, can be delivered using the same or different systems. For example, the guide RNA, Cas nuclease, and the construct can be carried by the same vector (e.g., AAV). Alternatively, the Cas nuclease (as a protein or mRNA) or gRNA can be carried by a plasmid or LNP, while the donor construct can be carried by a vector such as AAV.

[0435] Different delivery systems may be delivered simultaneously or in any sequential order. In some embodiments, the donor construct, guide RNA, and Cas nuclease may be delivered simultaneously, for example, as one vector, two vectors, individual vectors, one LNP, two LNPs, individual LNPs, or a combination thereof. In some embodiments, the donor construct may be delivered as a vector or associated with an LNP before delivering the guide RNA or Cas nuclease as a vector or associated with an LNP alone or together or as a ribonucleoprotein (RNP) (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more). As an additional example, guide RNA and Cas nuclease may be delivered as a vector or associated with LNP alone or together or as a ribonucleoprotein (RNP) prior to delivery of the construct as a vector or associated with LNP (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more).

[0436] IV. Methods for Manipulating T Cells

[0437] The present disclosure provides a method for manipulating T cells to include modifications by intracellular insertion of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence and modifications to an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown. The present disclosure provides a method for manipulating T cells further comprising modifications to an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modifications to an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; and / or intracellular insertion of heterologous sequence(s) encoding regulatory T cell promoter molecules under the control of a promoter sequence. The present disclosure provides a method for manipulating T cells to include modifications by intracellular insertion of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence and modifications to an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown. The present disclosure provides modifications to an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; The present disclosure provides a method for manipulating T cells further comprising: a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; and / or the insertion into the cell of a heterologous sequence(s) encoding IL10 under the control of a promoter sequence. The present disclosure provides a method for manipulating T cells to include: a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; and / or; and the insertion into the cell of a heterologous sequence(s) encoding CTLA4 under the control of a promoter sequence. The present disclosure provides a method for manipulating T cells to include a modification by the insertion into the cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence and a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown.The present disclosure provides a method for manipulating T cells further comprising: a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; and / or the insertion into the cell of heterologous sequences encoding IL10 and CTLA4, each under the control of a promoter sequence.

[0438] In some embodiments, the method comprises the step of manipulating a T cell to include modification by insertion into the cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence, and knockdown of an endogenous nucleic acid sequence encoding TGFBR2, etc., and optionally, modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; and / or insertion into the cell of heterologous sequence(s) encoding a regulatory T cell promoter molecule, e.g., IL10 or CTLA4.

[0439] In some embodiments, the method comprises the step of manipulating a T cell to include: a modification by insertion into a cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; insertion into a cell of heterologous sequence(s) encoding a regulatory T cell promoter molecule, e.g., IL10 or CTLA4; and further including the insertion into a cell of heterologous sequence(s) encoding a targeting receptor such as a CAR, and further including a modification of the TCR sequence(s), e.g., knockdown.

[0440] In some embodiments, the method comprises the step of manipulating a T cell to include modifications by insertion into the cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence, modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; insertion into the cell of heterologous sequence(s) encoding a regulatory T cell promoter molecule, e.g., IL10 or CTLA4, modification of TCR sequence(s), e.g., knockdown; and insertion into the cell of heterologous sequence(s) encoding a targeting receptor such as a CAR.

[0441] In some embodiments, the method comprises modification by insertion into a cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence, modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; insertion into a cell of heterologous sequence(s) encoding regulatory T cell promoter molecules, selective knockdown of a TCR gene, and selective insertion into a cell of a targeting receptor such as a CAR, and is operated using a CRISPR / Cas system and the guide RNA disclosed herein.

[0442] In these embodiments, the regulatory T cell promoter molecule to be inserted may be provided through a donor construct. The regulatory T cell promoter molecule provided through a donor construct may be selected from IL10, CTLA4, TIGIT, IDO1, ENTPD1, ​​NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, BACH2, and IL2RA; and modifications of TCR gene sequence(s), e.g., knockdowns.

[0443] In these embodiments, the targeting receptor to be inserted may be provided through a donor construct. In some embodiments, the targeting receptor may be a chimeric antigen receptor (CAR), a T-cell receptor (TCR), or a receptor for a cell surface molecule operably linked through at least a transmembrane domain to an internal signaling domain that may be activated upon binding of an extracellular receptor portion to a target. In some embodiments, the targeting receptor may be a receptor present on the surface of a cell, e.g., a T cell, that has been engineered to allow the cell to bind to a target site, e.g., a specific cell or tissue within an organism. In some embodiments, the targeting receptor may allow the engineered T cell to bind to a specific target cell, wherein the engineered T cell migrates to a disease site (e.g., mucosal cells at an inflamed site) and acts directly on the target cell (e.g., to kill or inhibit the target cell); and / or the targeting receptor may allow the binding of engineered T cells to specific target cells that are not specific to the disease site (e.g., activated immune cells of the lymphatic system) and act directly on the target cells (e.g., kill or inhibit target cells).

[0444] In some of these embodiments, the targeting receptor is a CAR capable of targeting MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), demoglain 3 (DSG3), or MHC class I HLA-A (HLA-A*02).

[0445] Gene editing systems suitable for manipulating T cells to include insertions and modifications, e.g., knockdowns, are disclosed herein and are known in the art. In some embodiments, gene editing systems include, but are not limited to, CRISPR / Cas systems; zinc finger nuclease (ZFN) systems; and transcription activator-like effector nuclease (TALEN) systems. Generally, gene editing systems involve the use of a cleavage system engineered to induce double-strand breaks (DSBs) or nicks (e.g., single-strand breaks or SSBs) in a target DNA sequence. Cleavage or nicking may occur through the use of specific nucleases, such as engineered ZFNs or TALENs, or by using a CRISPR / Cas system in conjunction with a guide RNA engineered to guide specific cleavage or nicking of the target DNA sequence, such as a CRISPR / Cas9 system. Furthermore, an Argonaute system (e.g., known as 'TtAgo') T . Thermophilus from, Swarts et al. (2014) Nature Targeted nucleases based on (see 507(7491): 258-261) are being developed and may also have potential for genome editing and gene therapy.

[0446] Transcriptional activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences of DNA. They are created by fusing a TAL effector DNA-binding domain to a DNA cleaving domain (a nuclease that cleaves DNA strands). Transcriptional activator-like effectors (TALEs) can be engineered to bind to a desired DNA sequence to promote DNA cleavage at a specific location (see, e.g., Boch, TALEs of genome targeting Nature Biotech. 29:135-136 (2011)). Restriction enzymes can be introduced into cells for use in in situ genome editing, a technique known as genome editing using engineered nucleases, or for gene editing. Such methods and compositions for use herein are known in the art. See, for example, WO2019147805, WO2014040370, and WO2018073393, the contents of which are incorporated herein in their entirety.

[0447] Zinc-finger nucleases (ZFNs) are artificial restriction enzymes produced by fusing a zinc finger DNA-binding domain to a DNA-cleaving domain. The zinc finger domain can be engineered to target specific desired DNA sequences so that the zinc-finger nuclease can target specific sequences within the complex genome. Non-specific cleaving domains derived from the type II restriction endonuclease FokI are typically used as the cleaving domains of ZFNs. The cleavage is repaired by endogenous DNA repair mechanisms, enabling ZFNs to precisely alter the genomes of higher organisms. Methods and compositions for such use are known in the art. For example, refer to WO2011091324, the contents of which are incorporated herein in their entirety.

[0448] RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression or translation by neutralizing targeted mRNA molecules. Small interfering RNA (siRNA) is the core of RNA interference. RNA is a direct product of genes, and these small RNAs (typically each strand being 19–23 nucleotides long and forming a 19–21 nucleotide double chain) can induce RNA-induced silencing (RISC) complexes to degrade messenger RNA (mRNA) molecules, thereby reducing activity by preventing translation through post-transcriptional gene silencing. Short hairpin RNA (shRNA) is siRNA in which the strand forming the double chain region is a single RNA strand with a hairpin structure often generated by transcription from an expression vector. RNAi can also be achieved by longer RNA double chain structures, referred to as Dicer substrate molecules, which are cleaved by the enzyme Dicer before being loaded into RISC to facilitate targeted mRNA cleavage. Methods of such use and compositions are known in the art. In the compositions and methods provided herein, the RNA molecule promoting RNA interference is preferably provided as an expression vector for durability, for example, see WO2018208837, the contents of which are incorporated herein in their entirety. In some embodiments, RNAi is used together with the expression vector.

[0449] It will be recognized that the present disclosure considers an insertion method performed with or without the guide RNA disclosed herein (e.g., using a ZFN system to cause disruption in a target DNA sequence to create an insertion site for the construct). In the case of a method using the guide RNA disclosed herein, the present method comprises modifying, e.g., knocking down a nucleic acid sequence encoding TGFBR2, TNFA, IFNG, or TCR using a CRISPR / Cas system. It will also be recognized that the present disclosure considers a method of modifying, e.g., knocking down TGFBR2, TNFA, IFNG, or TCR that can be performed without the guide RNA disclosed herein (e.g., using a ZFN system to cause disruption in a target DNA sequence to create an insertion site for the construct).

[0450] In some embodiments, a donor construct comprising a sequence for insertion, e.g., dmTGFB1 or a sequence encoding a regulatory T cell promoter, e.g., IL10 or CTLA4, is inserted into a genomic locus at a sequence targeted for modification, e.g., knockdown, e.g., a TCR gene.

[0451] In some embodiments, a CRISPR / Cas system (e.g., guide RNA and RNA-guided DNA binder) may be used to create an insertion site at a desired locus within the genome, and at this site, a donor construct comprising a sequence encoding dmTGFB1 disclosed herein; a regulatory T cell promoter, e.g., IL10, CTLA4, or a targeting receptor, e.g., CAR, e.g., MAdCAM-1 CAR, comprises dmTGFB1; a regulatory T cell promoter, e.g., IL10, CTLA4; Or it may be inserted to express a CAR, e.g., MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullized vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), demoglain 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CAR.Targeting receptors, e.g., CARs, e.g., MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), demoglain 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CARs; Or the inserted sequence, dmTGFB1 or a regulatory T cell promoter, e.g., IL10, or CTLA4 may be heterogeneous with respect to its insertion site or locus, e.g., Safe Haver locus or TCR locus, from which dmTGFB1; a regulatory T cell promoter, e.g., IL10, CTLA4; Or targeting receptors, e.g., CARs, e.g., MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), demoglain 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CARs are not normally expressed as described herein.In some embodiments, the guide RNA described herein may be used according to the present method together with an RNA-guided DNA binder (e.g., Cas nuclease) to create an insertion site, and at this site dmTGFB1; a regulatory T cell promoter, e.g., IL10, CTLA4; or a donor construct containing a sequence encoding a targeting receptor, e.g., CAR, e.g., MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), demoglain 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CAR, inserted into dmTGFB1; regulatory T cell promoter molecules, e.g., IL10, CTLA4; Or, CARs, for example, MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), demoglain 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CARs may be expressed.Guide RNAs for inserting dmTGFB1; regulatory T cell promoter molecules, e.g., IL10, CTLA4; or targeting receptors, e.g., CARs, e.g., MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), demoglain 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CARs into specific genomic loci are illustrated and described herein. In an alternative embodiment, the targeting receptor is a TNFA targeting receptor.

[0452] In some embodiments, CD4+ T cells are engineered by transduction using gRNA (e.g., gRNA targeting TGFBR2, IFNG, TNFA, or TCR for knockdown), an RNA-guided DNA binder (e.g., Cas nuclease), and a donor construct (e.g., using viral or non-viral delivery). In some embodiments, the engineered T cells are transduced with 1) gRNA, an RNA-guided DNA binder (e.g., Cas nuclease) that targets TGFBR2 and a nucleic acid sequence encoding a pro-inflammatory cytokine, e.g., IFNG, or TNFA, and 2) a donor construct comprising a nucleic acid sequence(s) encoding dmTGFB1 and a regulatory T cell promoter molecule, e.g., IL10 or CTLA4; and targeting receptors, e.g., CAR, e.g., MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), demoglain 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CAR; or are transduced with a donor construct comprising a TNFA targeting receptor. In certain embodiments, the engineered cells are selected for the expression of the targeting receptor.

[0453] In some embodiments, CD4+ T cells are engineered by transduction with gRNA (e.g., gRNA targeting TGFBR2, IFNG, TNFA, or TCR for knockdown), RNA-guided DNA-binding agents (e.g., Cas nucleases), and donor constructs. In some embodiments, the engineered T cells are 1) dmTGFB1 and optionally regulatory T cell promoter molecules, e.g., IL10 or CTLA4; and / or transduced with a donor construct comprising a nucleic acid sequence(s) encoding a targeting receptor, e.g., CAR, e.g., MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), demoglain 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CAR, and 2) TGFBR2, and optionally a pro-inflammatory cytokine, e.g., IFNG or The nucleic acid sequence encoding TNFA is transduced with a gRNA, RNA-guided DNA binder (e.g., Cas nuclease) that targets the TNFA. In certain embodiments, the engineered cells are selected for the expression of the targeting receptor.

[0454] In some embodiments, CD4+ T cells are engineered by transducing gRNA (e.g., gRNA targeting TGFBR2, IFNG, TNFA, or TCR for knockdown), RNA-guided DNA binders (e.g., Cas nucleases), and donor constructs. In some embodiments, the engineered T cells are 1) dmTGFB1 and optionally regulatory T cell promoter molecules, e.g., IL10 or CTLA4; and / or transduced with a donor construct comprising a nucleic acid sequence(s) encoding a targeting receptor, e.g., CAR, e.g., MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), demoglain 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CAR, and 2) TGFBR2, and optionally a pro-inflammatory cytokine, e.g., IFNG or gRNA that targets a nucleic acid sequence encoding TNFA; and transduced with an RNA-guided DNA binder (e.g., Cas nuclease).

[0455] As described herein, a donor construct comprising a sequence encoding dmTGFB1; regulatory T cell promoter molecules such as IL10, CTLA4; or a targeting receptor such as CAR, a guide RNA (e.g., gRNA targeting TGFBR2, IFNG, TNFA, or TCR for knockdown), and an RNA-guided DNA binder may be delivered using any suitable delivery system and method known in the art. In some embodiments, the guide RNA and Cas nuclease are associated with an LNP and delivered to a cell or cell population prior to delivering the donor construct comprising a sequence encoding dmTGFB1; regulatory T cell promoter molecules such as IL10, CTLA4; or a targeting receptor such as CAR. In some embodiments, the guide RNA and Cas nuclease are associated with an LNP and dmTGFB1; regulatory T cell promoter molecules such as IL10, CTLA4; Alternatively, a donor construct containing a sequence encoding a targeting receptor such as a CAR is delivered to a cell or cell population.

[0456] In some embodiments, administration of the gRNA, donor construct, and RNA-guided DNA binder described herein to naturally occurring T cells can convert naturally occurring T cells, e.g., CD4+ T cells, into cells exhibiting regulatory T cell characteristics, e.g., immunosuppressive characteristics.

[0457] Modifying TGFBR2, IFNG, TNFA, or TCR gene expression, e.g., knocking it down, or dmTGFB1; Regulatory T cell promoter molecules, e.g., IL10, CTLA4, or targeting receptors, e.g., CARs, e.g., MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipid protein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamate decarboxylase (GAD2), demoglain 3 (DSG3), or MHC class I HLA-A (HLA-A*02) gRNA for inserting a sequence encoding a CAR, donor constructs, and RNA-induced DNA binders are conventional T cells or conventional T cells It can be introduced into a population to generate the engineered T cells or T cell population described herein.

[0458] Methods using various RNA-guided DNA-binding agents, e.g., nucleases, e.g., Cas nucleases, e.g., Cas9, are also well known in the art. While the use of a CRISPR / Cas system is exemplified herein, it will be recognized that suitable modifications to the system may be used. Depending on the context, it will be recognized that the RNA-guided DNA-binding agent may be provided as a nucleic acid (e.g., DNA or mRNA), e.g., as mRNA encoding the provided RNA-guided DNA-binding agent, or as a protein. In some embodiments, the method may be carried out in cells that already contain or express the RNA-guided DNA-binding agent.

[0459] In some embodiments, an RNA-guided DNA-binding agent, such as a Cas9 nuclease, has cleavage enzyme activity, which may also be referred to as double-stranded endonuclease activity. In some embodiments, an RNA-guided DNA-binding agent, such as a Cas9 nuclease, has nikase activity, which may also be referred to as single-stranded endonuclease activity. In some embodiments, the RNA-guided DNA-binding agent comprises a Cas nuclease. Examples of Cas nucleases include S. Phyogenes , S. Aureus , Neisseria meningitidis Includes Cas nucleases of the type II CRISPR system of , and other prokaryotes (e.g., see the list in the following paragraph), and variants or mutant versions thereof (e.g., engineered, non-natural, natural, or other variants). See, for example, US20160312198; US 20160312199.

[0460] Non-restrictive exemplary species from which Cas nuclease may originate are Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus species, Staphylococcus aureus, Listeria inoculae, Lactobacillus gasseri, Francisela novicida, Valinella succinogenes, Suterella wadswottensis, Gammaproteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogenes, Rhodospirillum rubrum, Nocardiopsis d'Asonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alliccyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenityreducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus boucneri, Treponema denticola, Microcystis marinana, Burgholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas species, Crocosphaera wassoni, Cyanotheca species, Microcystis aeruginosa, Synecococcus species, Acetohalobium arabaticum, Ammonifex degencii, Caldicellulose syrupter vexcii, Candidatus desulforudis, Clostridium botulinum, Clostridium difficile, Panegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropioncum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter species, Nitrosococcus halopillus, Nitrosococcus wassoni, Pseudoalteromonas haloplanchthys, Ctedonobacter racemifer, Methanohalovium bestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc species, Arthrospira maxima, Arthrospira platensis, Arthrospira species, Lingvia species, Microcoleus etonoplasmes, Oscillatoria species, Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari,Includes Parvivaculum ravamentiborans, Corynebacterium diphtheria, Ashidaminococcus species, Lachnospirasis bacterium ND2006, and Acariochloris marinana.

[0461] In some embodiments, Cas nuclease is Streptococcus phyogenes It is a Cas9 nuclease from. In some embodiments, the Cas nuclease is Streptococcus thermophilus It is a Cas9 nuclease from. In some embodiments, the Cas nuclease is Neisseria meningitidis It is a Cas9 nuclease from. In some embodiments, the Cas nuclease is Staphilococcus aureus It is a Cas9 nuclease from. In some embodiments, the Cas nuclease is Francisela Novicida It is a Cpf1 nuclease from. In some embodiments, the Cas nuclease is Ashidaminococcus It is a Cpf1 nuclease from the species. In some embodiments, Cas nuclease is Lachnospirasi It is a Cpf1 nuclease from Bacterium ND2006. In additional embodiments, the Cas nuclease is Francisela tularensis , Lachnospirasis bacterium, Butyrivibrio proteocladicus, Peregrinii bacterium, Parcubacterium bacterium, Smitella, Acidaminococcus, Candidatus methanoplusma thermitum, Eubacterium elegans, Moraxella boboculi, Leptospira inadai, Porphyromonas crevioricanis, Prevotella diciens, or Porphyromonas macacae It is a Cpf1 nuclease from. In certain embodiments, the Cas nuclease is from Ashidaaminococcus or Lachnospirasi It is a Cpf1 nuclease from.

[0462] In some embodiments, the gRNA together with the RNA-guided DNA binder is called a ribonucleoprotein complex (RNP). In some embodiments, the RNA-guided DNA binder is a Cas nuclease. In some embodiments, the gRNA together with the Cas nuclease is called a Cas RNP. In some embodiments, the RNP contains a type I, type II, or type III component. In some embodiments, the Cas nuclease is a Cas9 protein from the type II CRISPR / Cas system. In some embodiments, the gRNA together with Cas9 is called a Cas9 RNP.

[0463] Wild-type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domain cleaves a non-target DNA strand, and the HNH domain cleaves a target DNA strand. In some embodiments, the Cas9 protein comprises one or more RuvC domains or one or more HNH domains. In some embodiments, the Cas9 protein is wild-type Cas9. In each embodiment of the composition, use, and method, Cas induces a double-strand cleavage in the target DNA.

[0464] In some embodiments, a chimeric Cas nuclease is used, wherein one domain or region of the protein is replaced with part of another protein. In some embodiments, the Cas nuclease domain may be replaced with a domain from a different nuclease, such as Fok1. In some embodiments, the Cas nuclease may be a modified nuclease.

[0465] In other embodiments, the Cas nuclease may be derived 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 derived from a Type III CRISPR / Cas system. In some embodiments, the Cas nuclease may have RNA cleavage activity.

[0466] In some embodiments, the RNA-guided DNA-binding agent has single-strand nickase activity, that is, it can cleave one DNA strand to produce a single-strand break, also known as a "nick." In some embodiments, the RNA-guided DNA-binding agent comprises Cas nickase. Nickase is an enzyme that produces a nick in dsDNA, that is, it cleaves one strand of the DNA double helix but does not cleave the other strand. In some embodiments, Cas nickase is a version of Cas nuclease (e.g., the Cas nuclease discussed above) in which the endonucleoritic active site is inactivated by one or more modifications (e.g., point mutations) within the catalytic domain. For example, see U.S. Patent No. 8889356 for a discussion of Cas nickase and exemplary catalytic domain modifications. In some embodiments, Cas nickase, such as Cas9 nickase, has an inactivated RuvC or HNH domain.

[0467] In some embodiments, the RNA-guided DNA-binding agent is modified to contain only one functional nuclease domain. For example, the agent protein may be modified so that one of the nuclease domains is mutated or completely or partially deleted to reduce nucleic acid cleavage activity. In some embodiments, a nikkase having a reduced-activity RuvC domain is used. In some embodiments, a nikkase having an inactive RuvC domain is used. In some embodiments, a nikkase having an HNH domain with reduced activity is used. In some embodiments, a nikkase having an inactive HNH domain is used.

[0468] In some embodiments, a conserved amino acid within the Cas protein nuclease domain is substituted to reduce or alter nuclease activity. In some embodiments, the Cas nuclease may include amino acid substitutions in the RuvC or RuvC-like nuclease domain. An exemplary amino acid substitution in the RuvC or RuvC-like nuclease domain is D10A(S. Phyogenes Includes Cas9 protein-based. For example, Zetsche et al. (2015) Cell See Oct 22:163(3): 759-771. In some embodiments, the Cas nuclease may include amino acid substitutions in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions within the HNH or HNH-like nuclease domain are E762A, H840A, N863A, H983A, and D986A (S. Phyogenes Includes Cas9 protein-based. For example, Zetsche et al. (2015) Refer to Additional exemplary amino acid substitutions are D917A, E1006A, and D1255A ( Francisela Novicida It includes the U112 Cpf1(FnCpf1) sequence (based on UniProtKB - A0Q7Q2(CPF1_FRATN)). N. meningitidis Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain or RuvC or RuvC-like domain include Nme2Cas9D16A (HNH nikcaase) and Nme2Cas9H588A (RuvC nikcaase).

[0469] In some embodiments, the nickase is provided in combination with a pair of guide RNAs that are complementary to the sense and antisense strands of the target sequence, respectively. In this embodiment, the guide RNAs introduce a DSB by guiding the nickase to the target sequence and generating a nick on the opposing strand of the target sequence (i.e., double nicking). In some embodiments, the nickase is used with two separate guide RNAs that target the opposing strands of DNA to generate a double nick on the target DNA. In some embodiments, the nickase is used with two separate guide RNAs selected to be very close to generate a double nick on the target DNA.

[0470] In some embodiments, the RNA-guided DNA-binding agent comprises one or more heterogeneous functional domains (e.g., a fusion polypeptide or comprises such).

[0471] In some embodiments, a heterofunctional domain may facilitate the transport of an RNA-guided DNA-binder into the cell nucleus. For example, the heterofunctional domain may be a nuclear localization signal (NLS). In some embodiments, the RNA-guided DNA-binder may be fused with 1 to 5 NLS(s). In some embodiments, the RNA-guided DNA-binder may be fused with 2, 3, or 4 NLS(s). In some embodiments, the RNA-guided DNA-binder may be fused with 2 NLS(s). In some embodiments, the RNA-guided DNA-binder may be fused with a single NLS. When a single NLS is used, the NLS may be attached to the N-terminus or C-terminus of the RNA-guided DNA-binder sequence. In some embodiments, the NLS is not attached to the C-terminus. It may be inserted into the RNA-guided DNA-binder sequence. In other embodiments, the RNA-guided DNA-binding agent may be fused with more than one NLS. In certain circumstances, at least two NLS are identical (e.g., two SV40 NLS). In certain embodiments, at least two different NLS are present in the RNA-guided DNA-binding agent. In some embodiments, the RNA-guided DNA-binding agent is fused with two SV40 NLS sequences linked at the carboxyl terminus. In some embodiments, the RNA-guided DNA-binding agent may be fused with two NLS, one linked at the N-terminus and one linked at the C-terminus. In some embodiments, the RNA-guided DNA-binding agent may be fused with three NLS. In some embodiments, the RNA-guided DNA-binding agent may be fused without NLS. In some embodiments, the NLS may be a monopartite sequence, e.g., SV40 NLS, PKKKRKV (SEQ ID NO. 143), or PKKKRRV (SEQ ID NO. 144).In some embodiments, the NLS may be a dipartial sequence, e.g., the NLS of nucleoplasmin, KRPAATKKAGQAKKKK (SEQ No. 145). In certain embodiments, a single PKKKRKV (SEQ No. 143) NLS may be linked at the C-terminus of an RNA-guided DNA-linker. One or more linkers are optionally included in the fusion site.

[0472] V. Treatment Methods

[0473] The present disclosure provides a method for suppressing an immune response in a subject, comprising the step of administering engineered T cells comprising the intracellular insertion of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence and a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown. The engineered T cells may optionally further comprise a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; or the intracellular insertion of heterologous sequence(s) encoding a regulatory T cell promoter molecule under the control of a promoter sequence. The present disclosure comprises the intracellular insertion of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; The present disclosure provides a method for suppressing an immune response in a subject, comprising the step of administering engineered T cells comprising insertion into the cell of a heterologous sequence(s) encoding a regulatory T cell promoter molecule under the control of a promoter sequence. The present disclosure provides a method for suppressing an immune response in a subject, comprising the step of administering engineered T cells comprising a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; and insertion into the cell of a heterologous sequence encoding IL10 under the control of a promoter sequence.The present disclosure provides a method for suppressing an immune response in a subject, comprising the step of administering engineered T cells comprising: insertion into a cell a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; and insertion into a cell a heterologous sequence encoding CTLA4 under the control of a promoter sequence. The present disclosure provides insertion into a cell a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; The present invention provides a method for suppressing the immune response of a subject, comprising the step of administering engineered T cells comprising insertion into the cells heterogeneous sequences encoding IL10 and CTLA4 under the control of each promoter sequence.

[0474] The present disclosure provides a method for treating an autoimmune disorder in a subject, comprising the step of administering engineered T cells comprising insertion into a cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence and modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown. The engineered T cells may optionally further comprise modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; or insertion into a cell of heterologous sequence(s) encoding a regulatory T cell promoter molecule under the control of a promoter sequence. The present disclosure provides a method for treating an autoimmune disorder in a subject, comprising insertion into a cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; The present disclosure provides a method comprising the step of administering engineered T cells comprising a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown, and the insertion into a cell of a heterologous sequence(s) encoding a regulatory T cell promoting molecule under the control of a promoter sequence. The present disclosure provides a method for treating an autoimmune disorder in a subject, comprising the step of administering engineered T cells comprising: the insertion into a cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; and the 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 the step of administering engineered T cells comprising: insertion into a cell a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; and insertion into a cell a heterologous sequence(s) encoding CTLA4 under the control of a promoter sequence. The present disclosure provides a method for treating an autoimmune disorder in a subject, comprising: insertion into a cell a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; The present invention provides a method comprising the step of administering engineered T cells comprising a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown, a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown, and the insertion into the cell of heterologous sequences encoding IL10 and CTLA4, respectively, under the control of a promoter sequence.

[0475] The present disclosure provides a method for treating GvHD in a subject, comprising the step of administering engineered T cells comprising insertion into a cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence and modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown. The engineered T cells may optionally further comprise modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; or insertion into a cell of heterologous sequence(s) encoding a regulatory T cell promoter molecule under the control of a promoter sequence. The present disclosure provides a method for treating GvHD in a subject, comprising: insertion into a cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; The present disclosure provides a method comprising the step of administering engineered T cells comprising: a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; and the insertion into the cell of a heterologous sequence(s) encoding a regulatory T cell promoter molecule under the control of a promoter sequence. The present disclosure provides a method for treating GvHD in a subject comprising the step of administering engineered T cells comprising: the insertion into the cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding INFG, e.g., knockdown; and the insertion into the cell of a heterologous sequence(s) encoding IL10 under the control of a promoter sequence.The present disclosure provides a method for treating GvHD in a subject, comprising the step of administering engineered T cells comprising: insertion into a cell a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; and insertion into a cell a heterologous sequence(s) encoding CTLA4 under the control of a promoter sequence. The present disclosure provides a method for treating GvHD in a subject, comprising: insertion into a cell a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding INFG, e.g., knockdown; The present invention provides a method comprising the step of administering engineered T cells comprising inserting heterogeneous sequences encoding IL10 and CTLA4 into the cells under the control of each promoter sequence.

[0476] The present disclosure provides a method for treating a subject receiving a transplant or blood transfusion, comprising the step of administering engineered T cells—including the insertion into a cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence and a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown—optionally, before, simultaneously with, or after receiving the transplant or blood transfusion. The engineered T cells may optionally further comprise a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; or the insertion into a cell of heterologous sequence(s) encoding a regulatory T cell promoter molecule under the control of a promoter sequence. The present disclosure provides a method for treating a subject receiving a transplant or blood transfusion, comprising the insertion into a cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; The present invention provides a method comprising the step of administering engineered T cells—selectively, before, simultaneously with, or after receiving a transplant or blood transfusion—comprising a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; and the insertion into the cell of a heterologous sequence(s) encoding a regulatory T cell promoting molecule under the control of a promoter sequence.The present disclosure provides a method for treating a subject receiving a transplant or transfusion, comprising the step of administering an engineered T cell—selectively, before, simultaneously with, or after receiving the transplant or transfusion—comprising the insertion into a cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding INFG, e.g., knockdown; and the insertion into a cell of heterologous sequence(s) encoding IL10 under the control of a promoter sequence. The present disclosure provides a method for treating a subject receiving a transplant or transfusion, comprising the step of: insertion into a cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; The present invention provides a method comprising the step of administering engineered T cells—including modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; and insertion into the cell of a heterologous sequence(s) encoding CTLA4 under the control of a promoter sequence—optionally, before, simultaneously with, or after receiving a transplant or blood transfusion.The present disclosure provides a method for treating a subject receiving a transplant or blood transfusion, comprising the step of administering engineered T cells—selectively, before, simultaneously with, or after receiving the transplant or blood transfusion—comprising insertion into a cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence; modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding INFG, e.g., knockdown; and insertion into a cell of heterologous sequences encoding IL10 and CTLA4, respectively, under the control of a promoter sequence.

[0477] In some embodiments, the method comprises the step of administering engineered T cells further comprising the insertion into the cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence, a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown, and the insertion of sequence(s) encoding a targeting receptor such as a CAR. The engineered T cells may optionally further comprise a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; or the insertion into the cell of heterologous sequence(s) encoding a regulatory T cell promoter molecule under the control of a promoter sequence. In some embodiments, the method comprises a modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; a modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; The method includes the step of administering engineered T cells comprising the insertion of sequence(s) encoding a regulatory T cell promoter molecule and further comprising the insertion of sequence(s) encoding a targeting receptor such as a CAR.

[0478] In some embodiments, the targeting receptor, e.g., CAR, may target engineered T cells to the gastrointestinal system, e.g., the targeting receptor is a CAR that targets MAdCAM-1, intended to suppress immune responses including inflammation in disorders such as inflammatory bowel disease, ulcerative colitis, or Crohn's disease. In some embodiments, the targeting receptor, e.g., CAR, may target engineered T cells to tissues including endothelial cells, e.g., the targeting receptor is a CAR that targets VCAM-1, intended to suppress immune responses including inflammation in disorders such as Crohn's disease and multiple sclerosis. In some embodiments, the targeting receptor, e.g., CAR, may target engineered T cells to endothelial cells, e.g., the targeting receptor is a CAR that targets CEACAM6, intended to suppress immune responses in disorders such as Crohn's disease. In some embodiments, the targeting receptor, e.g., CAR, can target engineered T cells against B cells, e.g., the targeting receptor is a CAR that targets CD19 to suppress an immune response in disorders such as multiple sclerosis and systemic lupus erythematosus. In some embodiments, the targeting receptor, e.g., CAR, can target engineered T cells against B cells, e.g., the targeting receptor is a CAR that targets CD20 to suppress an immune response in disorders such as multiple sclerosis and systemic lupus erythematosus. In some embodiments, the targeting receptor, e.g., CAR, can target engineered T cells against inflammatory tissue, e.g., the targeting receptor is a CAR that targets TNFA to suppress an immune response in disorders such as inflammatory bowel disease, ulcerative colitis, or Crohn's disease.In some embodiments, the targeting receptor, e.g., CAR, may target engineered T cells to neural tissue, e.g., the targeting receptor is a CAR that targets MBP, MOG, or PLP to suppress an immune response in disorders such as multiple sclerosis. In some embodiments, the targeting receptor, e.g., CAR, may target engineered T cells to immune cells expressing TNFSF7, including but not limited to activated T lymphocytes, B lymphocytes, dendritic cells, and / or NK cells, e.g., the CAR targets TNFSF7 to suppress an immune response in disorders such as Crohn's disease, ulcerative colitis, systemic lupus erythematosus, or multiple sclerosis. In some embodiments, the targeting receptor, e.g., CAR, may target engineered T cells to tissues containing mature B lymphocytes, e.g., the targeting receptor is a CAR that targets TNFRSF17 to suppress an immune response in disorders such as systemic lupus erythematosus. In some embodiments, the targeting receptor, e.g., CAR, may target engineered T cells to synovial tissues, e.g., the targeting receptor is a CAR that targets citrullinated vimentin to suppress an immune response in disorders such as rheumatoid arthritis.

[0479] In some embodiments, the targeting receptor is a CAR that targets CD70 to suppress the immune response in disorders such as GvHD, for example. CD70 is a transmembrane protein that is transiently expressed on the surface of CD4+ and CD8+ T cells, regulatory T cells (Treg), antigen-presenting cells such as B cells and dendritic cells, and natural killer (NK) cells in response to immune activation. Without being bound by theory, CD70 may be upregulated in immune cells such as T cells of patients with autoimmune diseases. In some embodiments, engineered T cells as described herein may suppress the activity of CD70-expressing immune cells and / or kill these cells.

[0480] In some embodiments, the targeting receptor is a CAR that targets DPP6, SCL2A2, glutamate decarboxylase (GAD2), demoglain 3 (DSG3) and MHC class I HLA-A (HLA-A*02).

[0481] In some embodiments, the method comprises the step of administering engineered T cells comprising insertion into the cell of a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence and modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown, and further comprising modification of TCR sequence(s), e.g., knockdown. The engineered T cells may optionally further comprise modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; or insertion into the cell of heterologous sequence(s) encoding a regulatory T cell promoter molecule under the control of a promoter sequence. In some embodiments, the method comprises modification of an endogenous nucleic acid sequence encoding TGFBR2, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding TNFA, e.g., knockdown; modification of an endogenous nucleic acid sequence encoding IFNG, e.g., knockdown; The method includes the step of administering engineered T cells comprising the insertion of sequence(s) encoding regulatory T cell promoter molecules and further comprising modifications of TCR sequence(s), e.g., knockdown.

[0482] In some embodiments, the sequence(s) to be inserted are inserted into a sequence to be modified, e.g., knocked down, e.g., a CAR sequence is inserted into a TNFA genome sequence to modify, e.g., knock down the TNFA sequence.

[0483] In some embodiments, the method comprises the step of administering a T cell population comprising T cells engineered as described above. In some embodiments, 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 as evaluated, for example, by sequencing, for example, NGS.

[0484] In some embodiments, the autoimmune disorder is selected from ulcerative colitis, Crohn's disease, rheumatoid arthritis, psoriasis, multiple sclerosis, systemic lupus erythematosus, type 1 diabetes, and graft-versus-host disease (GvHD). In some embodiments, the engineered T cells are for autologous or allogeneic use.

[0485] In some embodiments, the efficacy of the treatment using the engineered T cells described above can be evaluated 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 following the administration of the engineered T cells (wherein the animal is sacrificed after a significant loss of body weight, e.g., 20% of the starting body weight). In some embodiments, the effective treatment results in a statistically significant increase in survival rate compared to a suitable control, e.g., an animal treated with PBMCs.

[0486] Examples

[0487] The following examples are provided to illustrate specific disclosed embodiments and should not be construed as limiting the scope of the disclosure in any way.

[0488] Example 1: General method

[0489] Example 1.1 Preparation of lipid nanoparticles

[0490] Generally, lipid components were dissolved in 100% ethanol at various molar ratios. RNA products (e.g., Cas9 mRNA and sgRNA) were dissolved in 25 mM citrate buffer, 100 mM NaCl, pH 5.0 to produce RNA product concentrations of approximately 0.45 mg / mL.

[0491] Unless otherwise specified, the lipid-nucleic acid assembly contained ionizable lipid A ((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate), also known as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, cholesterol, DSPC, and PEG2k-DMG, respectively, in a molar ratio of lipid A:47.5, cholesterol:15, and DSPC:2.5. The lipid-nucleic acid assembly had a lipid amine to RNA phosphate (N:P) molar ratio of about 6, and a weight ratio of 1:1. It was formulated with a gRNA to mRNA ratio.

[0492] LNP was prepared using a cross-flow technique utilizing collision jet mixing of lipids in ethanol containing 2 volumes of RNA solution and 1 volume of water. First, the lipids in ethanol were mixed with 2 volumes of RNA solution via a mixing cross. Subsequently, a fourth water stream was mixed with the outlet stream of the cross via an inline tee (see WO 2016010840, Fig. 2). The LNP was maintained at room temperature for 1 hour and then further diluted with water (approximately 1:1 v / v). The LNP was concentrated using tangential flow filtration on a flat sheet cartridge (Sartorius, 100 kD MWCO) and buffered with 50 mM Tris, 45 mM NaCl, 5% (w / v) sucrose, and pH 7.5 (TSS) using a PD-10 desalination column (GE). Alternatively, the LNP was optionally concentrated using a 100 kDa Amicon spin filter and buffered with TSS using a PD-10 desalination column (GE). The resulting mixture was then filtered using a 0.2 μm sterile filter. The final LNP was stored at 4°C or -80°C until future use.

[0493] Example 1.2. In vitro transcription of mRNA ("IVT")

[0494] Capped and polyadenylated mRNA containing N1-methyl pseudo-U was generated by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase. The linearized plasmid DNA containing the T7 promoter and sequence for transcription was linearized by restriction endonuclease digestion followed by thermal inactivation of the reaction mixture, and then purified from the enzyme and buffer salt. Messenger RNA was synthesized and purified using standard techniques known in the art.

[0495] Messenger RNA was generated from plasmid DNA encoding an open read frame according to SEQ ID NO. 218 in Table 4. It is understood that where a sequence cited in this paragraph is mentioned below in relation to RNA, T should be replaced with U (which may be a modified nucleoside as described above). The messenger RNA used in the examples contains a 5' cap and a 3' polyadenylation sequence, e.g., up to 100 nts. Guide RNA was chemically synthesized using standard in vitro synthesis techniques with modified nucleotides or from commercial vendors.

[0496] Example 2. Inhibitory activity of engineered T cells overexpressing Treg-associated factors

[0497] Example 2.1. T cell preparation

[0498] Human PBMCs were prepared from fresh Leucopec (AllCells) and cryopreserved for future use by a method known in the art. CD4+ T cells were isolated from PBMCs by negative selection using a human CD4+ T cell isolation kit (Miltenyi; catalog number 130-096-533) according to the manufacturer's protocol. CD4+ T cells were activated by plating at a density of 5 x 10^6 cells / mL in OpTimizer basal medium containing 200 U / mL IL-2, 5 ng / mL IL-7, 5 ng / mL IL-15, and 25 UL / mL Immunocult human CD3 / CD28 T cell activator (Stemcell Technologies, Cat. 10991) in CTS OpTmizer T cell expansion SFM (Gibco, Cat. A3705001), 1% penicillin-streptomycin, 1X Glutamax, 10 mM HEPES, and 2.5% human AB serum (Gemini, Cat. 100-512), and cultured at 37°C for 24 hours prior to lentivirus transduction.

[0499] Natural regulatory T cells (nTreg), also referred to as thymic Treg (tTreg) in this institution, were produced using methods known in the art. CD4+ CD25+ T cell populations are known to be rich in natural Treg (nTreg). CD25+ cells were isolated from CD4+ T cells using human CD25 Microbeads II (Miltenyi; Cat No. 130-092-983) according to the manufacturer's instructions. Isolated CD25+ CD4+ T cells were cultured in 6-well Grex containing 5 x 10^ per well, 30 ml of OpTmizer basal medium supplemented every other day with 1000 U / mL recombinant human interleukin-2 (Miltenyi Biotec; Catalog No. 130-097-746), 1% penicillin-streptomycin, 1X Glutamax, 10 mM HEPES, 2.5% human AB serum (Gemini, Catalog 100-512). 6Cells were plated at a cell density. Cells were activated by adding 25 µL / mL ImmunoCult human CD3 / CD28 T cell activator (Stemcell Technologies, Catalog 10991) and cultured at 37°C for 2 days until lentivirus transduction. After an additional 3 days, activated CD25+ T cells were harvested and labeled with 50 µg / mL biotinylated anti-LAP antibody (Miltenyi; Catalog 130-095-213) for 15 minutes at 4°C. Latent-associated peptide (LAP) is a marker for activated Treg cells. Labeled cells were washed twice and further labeled with anti-biotin micro beads (Miltenyi; Catalog 130-090-485) according to the manufacturer's instructions. LAP+ cells were isolated using an LS column (Miltenyi; Catalog 130-042-401). Isolated natural Tregs were cultured every other day until injection in 6-well Grex plates containing 30 mL of OpTmizer basal medium supplemented with 1000 U / mL IL-2 and containing CTS OpTmizer T cell expansion SFM (Gibco, catalog A3705001), 1% penicillin-streptomycin, 1X Glutamax, 10 mM HEPES, and 2.5% human AB serum (Gemini, catalog 100-512).

[0500] Example 2.2. T cell transduction and cell classification

[0501] To manipulate suppressor T cells, activated CD4+ T cells Table 5As described in [reference], lentiviral constructs were transduced sequentially with a one-day interval. One lentiviral construct encoded both IL10DE (SEQ No. 229) and CTLA4 (SEQ No. 130), as well as GFP and mScarlet as markers. The second lentiviral construct encoded the constitutively active double mutant TGFB1 (caTGFB1; R218H C225R; SEQ No. 214). CD25+ T cells were transduced with a lentiviral construct encoding GFP. For transduction, the concentrated viral supernatant was added to the T cells, and the samples were centrifuged at 37 °C for 60 minutes. The T cells were resuspended in the cell / viral supernatant mixture and incubated in OpTmizer medium. Transfected CD4+ cell samples were supplemented with 200 U / mL IL-2, 5 ng / mL IL-7, and 5 ng / mL IL-15, and transfected CD25+ samples were supplemented with 1000 U / mL IL-2. Cells were divided, and medium or cytokines were regularly replenished.

[0502]

[0503] T cells use LNP Table 5 Edited according to [the following]. Each LNP species was co-formulated with Cas9-encoding mRNA and the following dosage guidelines: INF-gamma (G019753) with 0.67 µg / ml total RNA product, TNF-alpha (G019757) with 0.67 µg / ml total RNA product, and TGFBR2 (G029528) with 1 µg / ml total RNA product. The LNPs were prepared as described in Example 1 using a molar ratio of 35 lipid A / 47.5 cholesterol / 15 DSPC / 2.5 PEG2k-DMG. The LNPs were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of approximately 6. The LNPs were prepared with a weight ratio of gRNA to Cas9 mRNA product of 1:1.

[0504] To briefly explain, T cells in serum-free medium supplemented with 1000 U / mL IL-2, 10 ng / mL IL-7, and 10 ng / mL IL-15 at a concentration of 1 x 10^ 6 After resuspending at a concentration of cells / mL, the cells were transferred to a T-75 flask and incubated at 37°C. Each LNP preparation was incubated in T cell basal medium supplemented with 5 µg / ml recombinant human ApoE3 (Peprotech, Catalog 350-02). The LNP mixture was incubated at 37°C for 15 minutes. An equal volume of the pre-incubated LNP mixture was added to each T cell sample to double the original volume.

[0505] Twenty-four hours after LNP treatment, T cells were collected, washed, and suspended in OpTmizer basal medium supplemented with 500 U / mL recombinant human IL-2, 5 ng / ml recombinant human IL-7, and 5 ng / ml recombinant human IL-15. C cells were proliferated in 6-well Grex plates, and cytokines were supplemented every other day.

[0506] Example 2.3. Confirmation of cell manipulation

[0507] The manipulated samples were classified by flow cytometry to obtain an enriched cell population.

[0508] Engineered T cells were evaluated by flow cytometry to determine phenotype, gene insertion, and disruption. Cells were washed and stained in FACS buffer (PBS + 2% FBS + 2 mM EDTA). Engineered T cells were incubated with antibodies targeting CD4 (Biolegend) or TGFBR2 (Miltenyi). Subsequently, T cells were washed and analyzed using a Cytoflex instrument (Beckman Coulter). Data analysis was performed using the FlowJo software package. T cells were gated based on size, unicellularity, CD4 expression, and marker expression. Targeted expression of TGFBR2, verified by flow cytometry, was Table 6The values ​​presented correspond to the mean percentage of cells positive for TGFBR2 expression and the TGFBR2 expression level measured as the fluorescence median for each cell population.

[0509]

[0510] Example 2.4. Inhibitory ability of engineered T cells

[0511] The suppressive function of classified transduced and engineered T cells was evaluated using the Mixed Lymphocyte Response (MLR) assay. MLR is an inflammatory response that occurs when T cells recognize other leukocytes (allogeneic leukocytes) as foreign cells. Tregs suppress the MLR inflammatory response by inhibiting the proliferation of inflammatory T cells. Cell proliferation is measured by labeling the initial population with an intracellular fluorescent dye and diluting it as it is passed to each subsequent generation as part of the cellular contents.

[0512] MLR assays were performed in 96-well U-bottom plates using OpTmizer basal medium containing CTS OpTmizer T cell expansion SFM (Gibco, catalog A3705001), 1% penicillin-streptomycin, 1X Glutamax, 10 mM HEPES, and 2.5% human AB serum (Gemini, catalog 100-512). Transfected T cells (autologous T cells) and untransfected CD3+CD4+ T cells from the same donor were used as reactive cells. These untransfected cells were labeled with CellTrace Violet (CTV) (Thermofisher Scientific; catalog number C34557) according to the manufacturer's instructions. Reactive untransfected T cells were stimulated against transfected T cells using CD3-depleted PBMCs from an allogeneic donor. Cultures were prepared by combining 50,000 CTV-labeled autologous T cells, 50,000 CD3-depleted allogeneic PBMCs, and approximately 50,000 (1:1), 16,666 (3:1), or 5,555 (9:1) sorted transduced CD3+CD4+ T cells per well. After incubation at 37°C for 5 days, the culture plates were centrifuged. The cell pellet was resuspended in FACS buffer containing APC / Fire 750 anti-CD4 and incubated at 4°C for 30 minutes. Subsequently, the cells were washed, processed on a CytoFlex flow cytometer (Beckman Coulter), and analyzed using the FlowJo software package. Cells were gated first for positive CD4 expression, then for CTV signaling, and finally for the undiluted CTV population. The inhibition of CTV dilution was calculated using the following equation:

[0513]

[0514] Here, y = mean fluorescence intensity of the entire CTV-labeled population / mean fluorescence intensity of the undiluted portion of the CTV-labeled population. The data Fig. 1 and Table 7 It is presented in.

[0515]

[0516] Example 3. Analysis of In vivo Functional Characteristics of Engineered Suppressive T Cells

[0517] Example 3.1. In vivo evaluation of inflammatory response in a GvHD model

[0518] The in vivo inhibitory function of engineered T cells was evaluated using a graft-versus-host disease (GvHD) mouse model.

[0519] T cells classified for in vivo injection were harvested and resuspended in CTS OpTmizer T cell expansion medium (Gibco, Cat. A1048501) at a density of 4 x 10^6 cells / mL. Autologous PBMCs were thawed and the cell density was adjusted to 8 x 10^6 cells / mL. PBMCs were added to each assay group at a ratio of 0.5:1, and the cells were resuspended in RPMI supplemented with 10 mM HEPES and 2% FBS at a density of 6 x 10^6 cells / 100 μL. The PBMC-only group was resuspended at 4 x 10^6 cells / 100 μL.

[0520] Female NOG mouse (NOD.Cg- Prkdc scid Il2rg tm1Sug / JicTac; Taconic, catalog number NOG-F) was conditioned for cell transplantation one day prior to injection using an X-ray (RS-2000 irradiator; Rad Source Technologies) at a sublethal dose (150 rad). 100 μL of each aforementioned test cell population was intravenously injected into a cohort of irradiated NOG mice. Eight irradiated mice injected with the injection medium were used as an irradiation-only control (vehicle). Body weight was monitored daily. Mice were sacrificed when body weight decreased by 20%, and the cellular composition of their spleens was evaluated. Survival data Table 8 and Fig. 2 It is floated to.

[0521]

[0522] Example 3.2. Sustainability of engineered T cells

[0523] Spleen composition was evaluated to confirm the persistence of engineered T cells. Treg persistence was determined by assessing the relative number of Treg and total splenocytes in the spleen after euthanasia. At the time of euthanasia, the spleen of each animal was collected in a 15 mL conical tube containing complete Optimizer medium. The spleen was manually dissociated using the rubber side of a 1 mL syringe plunger. The cell suspension was filtered through a 70-micron cell strainer (Corning, Cat. 08-771-2), and cells were counted using a Cellometer K2 (Nexcelom Bioscience). Splenocytes were cryopreserved in CryoStor CS10 medium (Stemcell Technologies; Cat# 07930). After the completion of the study, all splenocytes were thawed and counted using a Cellometer K2 (Nexcelom Bioscience). Approximately one million viable splenocytes were resuspended in Optimizer medium, and 6 x 10^5 cells were stained with an antibody panel containing APC anti-human CD45 (BD Pharmigen, Catalog 561864) and APC-Fire 750 anti-human CD4 (Biolegend, Catalog 300560) at 4°C for 30 minutes. Splenocytes were processed on a Cytoflex flow cytometer (Beckman Coulter) and analyzed using the FlowJo software package. Cells were gated on mouse CD45- and human CD45+. tTregs were gated as GFP+, and engineered T cells were gated as mScarlet+GFP+. To determine the cell count of individual populations, the percentage of each population was applied to the total number of recovered human splenocytes. The data Table 9 and Fig. 3 It is presented in.

[0524]

[0525] Example 3.3. Analysis of In Vivo Cytokine Profiles of Engineered T Cells

[0526] Plasma for cytokine analysis was isolated by blood extraction via cardiac puncture and preserved with the addition of EDTA. The collected blood was centrifuged at 1000xg for 10 minutes, and the plasma was stored at -80°C until further analysis.

[0527] Plasma samples were evaluated for cytokine release levels using MSD. Plasma samples were thawed for subsequent cytokine quantification using a custom U-PLEX biomarker kit (Meso Scale Diagnostics, Catalog K15067L-2) according to the manufacturer's instructions. U-PLEX biomarker plates were read using a Meso Quickplex SQ120 instrument (Meso Scale Discovery), and the data were analyzed using the Discovery Workbench 4.0 software package (Meso Scale Discovery). The results are Table 10 and Fig. 4 It is presented in.

[0528]

[0529] Example 4. In vivo study of CD70-CAR targeting by synTreg using a xenoGvHD mouse model

[0530] The in vivo suppressive function of engineered T cells was evaluated using a graft-versus-host disease (GvHD) mouse model. Specifically, CD70-CAR targeted and non-targeted triple-KI (dmTGFB1 / IL10DE / CTLA4) / triple-KO (TGFBR2 / IFNG / TNFA) synTregs were compared with non-edited tTregs and controls. CD70 is upregulated by activated lymphocytes, including autoreactive T cells and B cells.

[0531] Example 4.1 T cell manipulation

[0532] Generally, T cells were prepared as described in Example 2. CD4+ and CD4+ CD25+ T cells were isolated from human PBMCs using a commercially available kit (Miltenyi) according to the manufacturer's protocol. The cells were activated with an immunocult, and Table 11 Edited according to

[0533]

[0534] On Day 0, CD4+ and CD4+CD25+ cells were activated with an immunocult. CD4+ cells were treated with LNPs carrying gRNA G019753, which targets SpCas9 mRNA and IFNg, and gRNA G019757, which targets TNFα, to destroy these genes. On Day 1, double knockout cells were harvested, resuspended, and treated with a lentivirus encoding CTLA4 and IL10DE. On Day 2, the resulting double knockout and double knocked cells were harvested, resuspended, and treated with a lentivirus encoding dmTGFB1. On day 3, a portion of the resulting double knockout and triple knockout cells were harvested, resuspended, and treated with an LNP carrying mRNA encoding SpCas9, an approximately 500 nucleotide homologous arm with a sequence surrounding the TRAC guide cleavage site, and an AAV encoding CD70 CAR containing gRNA G013006 targeting TRAC to insert the CAR into the TRAC locus. The same cells were also treated with an LNP carrying mRNA encoding the NmeCas9 base editor (SEQ No. 365) and gRNA G034199 targeting CD70, and an LNP carrying mRNA encoding UGI for the destruction of the CD70 gene. On day 4, the CD70 edited cells were harvested, resuspended, and cultured overnight. On day 5, double knockout, triple knockout cells and CD70 CAR-targeted double knockout, triple knockout cells were harvested, resuspended, and treated with LNP carrying SpCas9 mRNA and gRNA G029528 targeting TGFBR2 to disrupt TGFBR2. On day 6, thymic Treg, synTreg, and CD70 CAR synTreg cells were washed and cultured with the regular addition of cytokines.

[0535] Example 4.2 Analysis of In vivo Functional Characteristics

[0536] The in vivo suppressive function of engineered T cells was evaluated using a graft-versus-host disease (GvHD) mouse model. Selected T cells and PBMCs for in vivo injection were harvested and resuspended in culture medium. Female NOG mice (NOD.Cg- Prkdc scid Il2rg tm1Sug / JicTac; Taconic, catalog number NOG-F) was conditioned for cell transplantation one day prior to injection using X-rays (RS-2000 irradiator; Rad Source Technologies) at a sub-lethal dose (150 rad). In a cohort of irradiated NOG mice Table 11 Four million PBMC cells and 400,000 T cells as described were injected intravenously. Eight irradiated mice injected with the injection medium were used as an irradiation-only control (vehicle). Body weight was monitored daily. Mice were sacrificed when body weight decreased by 20%, and the cellular composition of the spleen was evaluated. Survival data Table 12 and Fig. 5 It is floated to.

[0537]

[0538] The persistence of Treg cells was generally evaluated as described in Example 3. Table 13 and Fig. 6 Figure 1 shows the average percentage of synTregs among all human CD45+ cells recovered from the mouse spleen at sacrifice. The percentage of tTregs in the spleen was not measured in this experiment, but in similar experiments, it was typically in the 1-5% range.

[0539]

[0540] Mice were evaluated for phenotypic signs of GvHD at sacrifice based on the method described in Lai, et al. Cell Transplantation. 2012;21(9):2033-2045. For each mouse, each phenotypic criterion was Table 14As described in [document], grades were assigned from 0 to 2, and the grades for each mouse were summed to obtain the GvHD score. The average GvHD score for each experimental condition is Table 15 and Fig. 7 Presented in ). Mice treated with untargeted tTreg or synTreg showed a statistically significant improvement in GvHD scores compared to the PBMC-alone control group. . Mice treated with CD70-CAR synTreg were asymptomatic throughout the experiment and were indistinguishable from mice in the vehicle control sample. The GvHD scores for CD70-CAR synTreg-treated mice were significantly different from those of the PBMC-alone control and untargeted synTreg.

[0541]

[0542] The cytokine profile was generally evaluated as described in Example 3. Table 16 and Fig. 8 This shows the average IL-10 serum levels for mice in each treatment group.

[0543]

[0544] Example 4.3. Inhibitory activity of synTreg isolated from the spleen

[0545] The inhibitory function of Tregs isolated from frozen spleen samples of the mice of this example was evaluated using a bead-based inhibition assay. The inhibition assay was performed by a method known in the art, generally as described in Example 2 and using allogeneic APCs and human anti-CD3 / CD28 beads for T cell stimulation. Unmanipulated CD4+ and CD8+ T cells (Tconv) were isolated from frozen PBMCs of the same donor used to manipulate synTregs to be used as negative controls. Tregs were isolated from spleen samples by flow cytometry based on fluorescent labels, GFP for tTregs and GFP / mScarlet for synTregs. CD3+ T cells from frozen PBMCs were isolated using the Miltenyi Biotec kit to be used as responder (Tresp) cells.

[0546] Tconv control cells were labeled with CellTrace Yellow, and Tresp cells were labeled with CellTrace Violet. Co-cultures of Tresp and Treg cells were stimulated for 5 days using anti-human CD3 / CD28 activator beads (Miltenyi Biotec, Cat# 130-091-441). Cells were harvested and analyzed by flow cytometry. The percentage of inhibition of expansion was calculated using the following formula:

[0547]

[0548] The calculated inhibition rate of CTV-labeled cell expansion is Table 17 and Fig. 9 This is presented in [the text]. Splenic-isolated synTregs remained highly repressive and, particularly at low Treg:Tresp ratios, exceeded the activity of splenic-isolated tTregs. Consistent with in vivo persistence data, synTreg survival in vitro was superior to tTreg.

[0549]

[0550] Spleen-isolated synTreg and a control group of Tconv cells cultured without Tresp cells were restimulated using αCD3 / CD28 beads. The supernatant was analyzed using the Meso Scale Discovery MSD -U-PLEX Proinflammatory Combo 1 Human Kit according to the manufacturer's protocol. SynTreg Table 18 and Figs. 10a-10d As shown in [the text], when stimulated by beads, it did not produce pro-inflammatory cytokines but still secreted IL-10.

[0551]

[0552] Example 5. Characterization of mouse MAdCAM-1-CAR synTreg

[0553] Mouse MAdCAM-CAR targeted and non-targeted tri-KI(dmTGFB1 / IL10DE / CTLA4) / tri-KO(TGFBR2 / IFNG / TNFA) synTregs were compared with a normal T cell control group when cytokine secretion was evaluated in vitro in response to cell activation and activation.

[0554] Example 5.1 T cell manipulation

[0555] Generally, T cells were prepared as described in Example 2. CD4+ T cells were isolated from human PBMCs using a commercially available kit (Miltenyi) according to the manufacturer's protocol. The cells were activated with an immunocult, and Table 19 Edited according to

[0556]

[0557] On day 1, CD4+ cells were activated with an immunoculturic acid. On day 2, a portion of the CD4+ cells were treated with a lentivirus encoding a cassette containing CTLA4, IL10DE, and dmTGFB1. On day 3, the treated cells were harvested and resuspended in medium. A portion of the triple-knockout cells were treated with a lentivirus encoding a chimeric antigen receptor based on the monoclonal anti-mouse MAdCAM-1 and MECA-367 antibodies. On day 4, the cells were harvested, resuspended, and treated with LNPs carrying SpCas9 mRNA and gRNAs targeting IFNg and TNF to destroy these genes. The cells were washed and resuspended in medium for continued culture the following day. On day 6, the cells were sorted by flow cytometry to isolate populations of 1) Tconv, 2) triple knockout / double knockout, and 3) CAR+ triple knockout / double knockout cells. After sorting, the edited cells were further treated with LNPs carrying SpCas9-encoding mRNA and gRNA targeting TRAC, and LNPs carrying SpCas9 mRNA and gRNA targeting TGFBR2, to destroy these genes. The cells were washed and resuspended the following day.

[0558] Example 5.2 Analysis of in vitro functional characteristics

[0559] Cell activation was evaluated through co-culture with murine SVEC4-10 endothelial target cells expressing transgenic murine MAdCAM-1 or in wells coated with murine MAdCAM-1. Cells cultured alone were used as a negative control. After 24 hours, cells were harvested, and the expression of CD69, a marker of T cell activation, was evaluated by flow cytometry. Although not Tconv or untargeted synTreg, CAR-synTreg Table 20 and Figs. 11a-c As presented in [the document], it was activated by exposure to murine MAdCAM-1.

[0560]

[0561] Cytokine secretion was evaluated upon restimulation with mouse MAdCAM-1 or Immunocult. Cells were incubated for 48 hours in wells coated with murine MAdCAM-1 or containing Immunocult. Cells were harvested by centrifugation, and the supernatant was collected. The supernatant was analyzed using the Meso Scale Discovery MSD-U-PLEX Proinflammatory Combo 1 Human Kit according to the manufacturer's protocol. Table 21 and Fig. 12 As shown in [the text], only Tconv produced the pro-inflammatory cytokines IFNg and TNFa upon stimulation. Similarly, targeted and untargeted synTregs produce IL-10 and active TGFB upon appropriate stimulation.

[0562]

[0563] Example 6. SynTreg demonstrates potent in vitro inhibition of ulcerative colitis patient-derived T cells.

[0564] The inhibitory ability of synTreg was evaluated in a mixed lymphocyte response using T cells derived from patients with ulcerative colitis as responders. The inhibition of responder cell growth and inflammatory cytokine secretion was measured.

[0565] The inhibitory function of Tregs was measured using the Mixed Lymphocyte Response (MLR) assay. The MLR assay was generally performed as described in Example 2 and according to methods known in the art. CD4+ T cells (Tresp) were isolated from PBMCs of a healthy donor (HD) and three ulcerative colitis (UC) donors using a commercially available kit (e.g., Miltenyi). SynTregs were generally manipula...

Claims

Claim 1 A engineered T cell comprising: i) a heterogeneous nucleic acid sequence encoding a double mutant transforming growth factor beta 1 (dmTGFB1) under the control of a promoter sequence; and ii) a modification of an endogenous nucleic acid sequence encoding transforming growth factor beta receptor 2 (TGFBR2), wherein the modification knocks down the expression of TGFBR2. Claim 2 The engineered T cell according to claim 1, wherein the dmTGFB1 comprises mutations in two or more amino acids selected from F198, D199, V200, L208, F217, L219, R218, H222, C223, and C225 compared to wild-type human TGFB1. Claim 3 An engineered T cell according to claim 1 or 2, wherein the dmTGFB1 comprises mutations in two or more amino acids selected from R218, H222, C223, and C225 compared to wild-type human TGFB1. Claim 4 A engineered T cell according to claim 1, 2, or 3, wherein the dmTGFB1 comprises two or more amino acid mutations selected from R218C / H, H222D, C223S / R / G, and C225R compared to wild-type human TGFB1. Claim 5 A engineered T cell according to any one of claims 1 to 4, wherein the dmTGFB1 comprises amino acid mutations R218C / H and C225R compared to wild-type human TGFB1. Claim 6 A engineered T cell according to any one of claims 1 to 5, wherein the engineered T cell is a CD4+ T cell. Claim 7 A engineered T cell according to any one of claims 1 to 6, further comprising: i) a modification of an endogenous nucleic acid sequence encoding tumor necrosis factor alpha (TNFA), wherein said modification knocks down the expression of TNFA; ii) a modification of an endogenous nucleic acid sequence encoding interferon-gamma (IFNG), wherein said modification knocks down the expression of IFNG; or iii) a heterologous nucleic acid sequence encoding a regulatory T cell promoting molecule under the control of a promoter sequence. Claim 8 A engineered T cell according to any one of claims 1 to 7, comprising: i) a modification of an endogenous nucleic acid sequence encoding tumor necrosis factor alpha (TNFA), wherein the modification knocks down the expression of TNFA; ii) a modification of an endogenous nucleic acid sequence encoding interferon-gamma (IFNG), wherein the modification knocks down the expression of IFNG; and iii) a heterologous nucleic acid sequence encoding a regulatory T cell promoting molecule under the control of a promoter sequence. Claim 9 In claim 7 or 8, the regulatory T cell promoting molecule is interleukin-10 (IL10), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), T cell immune receptor having Ig and ITIM domains (TIGIT), indoleamine 2,3-deoxygenase 1 (IDO1), ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), 5'-nucleotidase ecto (NT5E), interleukin-22 (IL-22), ampyregulin (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), lymphatic enhancer binding factor 1 (LEF1), BTB domain and CNC homologues Engineered T cells selected from 2 (BACH2) and interleukin 2 receptor subunit alpha (IL2RA). Claim 10 In claim 9, the engineered T cell, wherein the regulatory T cell promoting molecule is IL10 or CTLA4. Claim 11 A engineered T cell according to claim 9 or 10, wherein the regulatory T cell promoter is a first regulatory T cell promoter and further comprises a heterogeneous nucleic acid sequence encoding a second regulatory T cell promoter under the control of a promoter sequence. Claim 12 In claim 11, the engineered T cell, wherein the first and second regulatory T cell promoting molecules are IL10 and CTLA4. Claim 13 A engineered T cell according to any one of claims 9 to 12, wherein the IL10 comprises one or more amino acid mutations selected from D25, E96, N21, and R104 compared to mature wild-type human IL10. Claim 14 A engineered T cell according to any one of claims 9 to 13, wherein the IL10 comprises one or more amino acid mutations selected from D25A / K, E96A, N21A, and R104A compared to mature wild-type human IL10. Claim 15 A engineered T cell according to any one of claims 9 to 14, wherein the IL10 comprises a combination of amino acid mutations selected from D25K, D25A, D25A / E96A, D25K / E96A, N21A / R104A, D25A / N21A / R104A, and D25K / N21A / R104A compared to mature wild-type human IL10. Claim 16 In any one of claims 9 to 12, the engineered T cell wherein the IL10 comprises mature wild-type human IL10. Claim 17 A engineered T cell according to any one of claims 1 to 16, further comprising a modification of an endogenous nucleic acid sequence encoding a protein selected from TRAC, HLA-A, HLA-B, and CIITA, wherein said modification knocks down the expression of said protein. Claim 18 In paragraph 17, the above protein is TRAC, and the above modification is a modified T cell that knocks down the expression of TRAC. Claim 19 In claim 17 or 18, the engineered T cell wherein the protein is HLA-A and the modification knocks down the expression of HLA-A. Claim 20 A modified T cell according to any one of claims 17 to 19, wherein the protein is HLA-B and the modification knocks down the expression of HLA-B. Claim 21 A modified T cell according to any one of claims 17 to 20, wherein the protein is CIITA and the modification knocks down the expression of CIITA. Claim 22 An engineered T cell according to any one of claims 1 to 16, further comprising: a) a modification of an endogenous nucleic acid sequence encoding TRAC, wherein said modification knocks down the expression of TRAC; b) a modification of an endogenous nucleic acid sequence encoding HLA-A, wherein said modification knocks down the expression of HLA-A; c) a modification of an endogenous nucleic acid sequence encoding HLA-B, wherein said modification knocks down the expression of HLA-B; and d) a modification of an endogenous nucleic acid sequence encoding CIITA, wherein said modification knocks down the expression of CIITA. Claim 23 A modified T cell comprising: i) a modification of a first endogenous nucleic acid sequence encoding tumor necrosis factor alpha (TNFA), wherein the modification of the first endogenous nucleic acid knocks down the expression of TNFA; ii) a modification of a second endogenous nucleic acid sequence encoding interferon-gamma (IFNG), wherein the modification of the second endogenous nucleic acid knocks down the expression of IFNG; iii) a modification of a third endogenous nucleic acid sequence encoding a protein selected from TRAC, HLA-A, HLA-B, and CIITA, wherein the modification of the third endogenous nucleic acid knocks down the expression of said protein; and iv) a heterogeneous nucleic acid sequence encoding a regulatory T cell promoter molecule under the control of a promoter sequence. Claim 24 In paragraph 23, the above protein is TRAC, and the above third modification is a modified T cell that knocks down the expression of TRAC. Claim 25 A engineered T cell according to claim 23 or 24, wherein the protein is HLA-A and the third modification knocks down the expression of HLA-A. Claim 26 A engineered T cell according to any one of claims 23 to 25, wherein the protein is HLA-B and the third modification knocks down the expression of HLA-B. Claim 27 A engineered T cell according to any one of claims 23 to 26, wherein the protein is CIITA and the third modification knocks down the expression of CIITA. Claim 28 As a modified T cell, i) a modification of a first endogenous nucleic acid sequence encoding tumor necrosis factor alpha (TNFA), wherein said modification knocks down the expression of TNFA; ii) a modification of a second endogenous nucleic acid sequence encoding interferon-gamma (IFNG), wherein said modification knocks down the expression of IFNG; iii) a modification of a third endogenous nucleic acid sequence encoding TRAC, wherein said modification of the third endogenous nucleic acid sequence knocks down the expression of TRAC; iv) a modification of a fourth endogenous nucleic acid sequence encoding HLA-A, wherein said modification of the fourth endogenous nucleic acid sequence knocks down the expression of HLA-A; v) a modification of a fifth endogenous nucleic acid sequence encoding HLA-B, wherein said modification of the fifth endogenous nucleic acid sequence knocks down the expression of HLA-B; vi) a modification of a sixth endogenous nucleic acid sequence encoding CIITA, wherein the modification of the sixth endogenous nucleic acid sequence knocks down the expression of CIITA; and vii) an engineered T cell comprising a heterologous nucleic acid sequence encoding a regulatory T cell promoter molecule under the control of a promoter sequence. Claim 29 In any one of claims 23 to 28, the regulatory T cell promoting molecule comprises interleukin-10 (IL10), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), T cell immune receptor having Ig and ITIM domains (TIGIT), indoleamine 2,3-deoxygenase 1 (IDO1), ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), 5'-nucleotidase ecto (NT5E), interleukin-22 (IL-22), ampyregulin (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), lymphatic enhancer binding factor 1 (LEF1), BTB domain and Engineered T cells selected from CNC homolog 2 (BACH2) and interleukin 2 receptor subunit alpha (IL2RA). Claim 30 In claim 29, the engineered T cell, wherein the regulatory T cell promoting molecule is IL10 or CTLA4. Claim 31 A engineered T cell according to claim 29 or 30, wherein the regulatory T cell promoter is a first regulatory T cell promoter and further comprises a heterogeneous nucleic acid sequence encoding a second regulatory T cell promoter under the control of a promoter sequence. Claim 32 In claim 31, the engineered T cell, wherein the first and second regulatory T cell promoting molecules are IL10 and CTLA4. Claim 33 A engineered T cell according to any one of claims 29 to 32, wherein the IL10 comprises a mutation in one or more amino acids selected from D25, E96, N21, and R104 compared to mature wild-type human IL10. Claim 34 A engineered T cell according to any one of claims 29 to 33, wherein the IL10 comprises one or more amino acid mutations selected from D25A / K, E96A, N21A, and R104A compared to mature wild-type human IL10. Claim 35 A engineered T cell according to any one of claims 29 to 34, wherein the IL10 comprises a combination of amino acid mutations selected from D25K, D25A, D25A / E96A, D25K / E96A, N21A / R104A, D25A / N21A / R104A, and D25K / N21A / R104A compared to mature wild-type human IL10. Claim 36 In any one of claims 29 to 35, the engineered T cell wherein the IL10 comprises mature wild-type human IL10. Claim 37 An engineered T cell according to any one of claims 1 to 36, further comprising a modification of an endogenous nucleic acid sequence encoding interleukin 17A (IL17A), interleukin-2 (IL2), interleukin 6 (IL6), perforin 1 (PRF1), granzyme A (GZMA), granzyme B (GZMB), natural killer cell granule protein 7 (NKG7), Fas ligand (FASL, NF superfamily, member 6), lyanodine receptor 2 (RYR2), and colony-stimulating factor 2 (CSF2), wherein said modification knocks down the expression of said IL17A, said IL2, said IL6, said PRF1, said GZMA, said GZMB, said FASL, said RYR2, or said CSF2. Claim 38 A engineered T cell according to any one of claims 1 to 37, further comprising a modification of an endogenous nucleic acid sequence encoding an endogenous T cell receptor (TCR), wherein said modification knocks down the expression of said endogenous TCR. Claim 39 A engineered T cell according to any one of claims 1 to 38, further comprising a heterologous coding sequence for a targeting receptor under the control of a promoter sequence. Claim 40 In claim 39, the targeting receptor is mucosal vascular addressin cell adhesion molecule 1 (MADCAM1), tumor necrosis factor alpha (TNFA), CD70 molecule (CD70), 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 (ligand) superfamily member 7 (TNFSF7), 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 Engineered T cells targeted to a ligand selected from HLA-A (HLA-A*02). Claim 41 In paragraph 39 or 40, the engineered T cell wherein the targeting receptor comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR). Claim 42 A engineered T cell according to any one of claims 39 to 41, wherein the heterogeneous nucleic acid sequence encoding the targeting receptor is incorporated into an expression construct. Claim 43 A engineered T cell in an expression construct, wherein, in any one of claims 39 to 42, the heterogeneous nucleic acid sequence encoding the targeting receptor does not include a nucleic acid sequence encoding a regulatory T cell promoter molecule. Claim 44 A engineered T cell according to any one of claims 39 to 42, wherein the heterogeneous nucleic acid sequence encoding a targeting receptor is in an expression construct comprising a nucleic acid sequence encoding a regulatory T cell promoter molecule. Claim 45 A engineered T cell according to any one of claims 11 to 22 or 31 to 44, wherein the heterogeneous nucleic acid sequence encoding the first regulatory T cell promoter molecule is incorporated into the expression construct and the heterogeneous nucleic acid sequence encoding the second regulatory T cell promoter molecule is incorporated into the expression construct. Claim 46 A engineered T cell according to any one of claims 11 to 22 or 31 to 45, wherein the heterogeneous nucleic acid sequence encoding the first regulatory T cell promoter molecule and the heterogeneous nucleic acid sequence encoding the second regulatory T cell promoter molecule are incorporated into a separate expression construct. Claim 47 A engineered T cell according to any one of claims 11 to 22 or 31 to 46, wherein the heterogeneous nucleic acid sequence encoding the first regulatory T cell promoter molecule and the heterogeneous nucleic acid sequence encoding the second regulatory T cell promoter molecule are incorporated into a single expression construct. Claim 48 A engineered T cell according to any one of claims 1 to 47, wherein at least one heterogeneous coding sequence is present in the episome expression construct. Claim 49 A engineered T cell in which at least one heterogeneous nucleic acid sequence is inserted into the genome, in any one of claims 1 to 48. Claim 50 In paragraph 49, a engineered T cell in which the insertion into the genome is a non-targeted insertion. Claim 51 In paragraph 49, the above insertion is a targeted insertion, a engineered T cell. Claim 52 In claim 51, the engineered T cell, wherein the targeted insertion is an insertion into a selected site from the TCR locus, TNF locus, IFNG locus, IL17A locus, IL6 locus, IL2 locus, and adeno-associated virus integration site 1 (AAVS1) locus. Claim 53 In paragraph 52, the engineered T cell in which the TCR locus is the T cell receptor alpha-constant (TRAC) locus. Claim 54 A engineered T cell according to any one of claims 7 to 53, wherein the modification that knocks down gene expression comprises one or more of an insertion, deletion, or substitution. Claim 55 A engineered T cell according to any one of claims 1 to 54, wherein the engineered T cell is a CD4+ T cell. Claim 56 A population of engineered T cells comprising the engineered T cells of any one of claims 1 to 55. Claim 57 A manipulated T cell population comprising a manipulated T cell according to any one of claims 1 to 22 or claims 37 to 56, wherein at least 30% or at least 40% of the cells in the population comprise a heterogeneous nucleic acid sequence encoding dmTGFB1 under the control of a promoter sequence; and at least 50% or at least 70% of the cells in the population comprise a modification of an endogenous nucleic acid sequence encoding TGFBR2. Claim 58 A manipulated T cell population according to claim 56 or 57, wherein at least 50% or at least 70% of the cells in the population comprise a modification of an endogenous nucleic acid sequence encoding a TNFA; at least 50% or at least 70% of the cells in the population comprise a modification of an endogenous nucleic acid sequence encoding an IFNG; or at least 30% or at least 40% of the cells in the population comprise a heterologous nucleic acid sequence encoding a regulatory T cell promoter molecule under the control of a promoter sequence. Claim 59 A manipulated T cell population according to claim 57 or 58, wherein the percentage of cells containing insertions or modifications is determined by the percentage of reads by next-generation sequencing (NGS). Claim 60 A pharmaceutical composition comprising a engineered T cell of any one of claims 1 to 55 or a population of engineered T cells of claims 56 to 59. Claim 61 A method or use of administering to a subject the engineered T cell of any one of claims 1 to 55, the engineered T cell population of any one of claims 56 to 59, or the pharmaceutical composition of claim 60. Claim 62 In paragraph 61, a method or use in which the subject requires immunosuppression. Claim 63 In paragraph 61 or 62, a method or use for treating immune disorders. Claim 64 A method or use for treating an autoimmune disease in any one of paragraphs 61 through 63. Claim 65 In paragraph 64, the method or use wherein the autoimmune disease is selected from ulcerative colitis, Crohn's disease, rheumatoid arthritis, psoriasis, multiple sclerosis, systemic lupus erythematosus, and type 1 diabetes. Claim 66 A method or use for treating graft-versus-host disease (GvHD) in any one of paragraphs 61 to 65. Claim 67 In any one of paragraphs 61, 62, or 66, the method or use in which the subject receives a transplant or blood transfusion. Claim 68 In paragraph 67, the method or use wherein the transplant or transfusion comprises a solid organ or cell population. Claim 69 A method or use according to paragraph 67 or 68, wherein the transplant or transfusion comprises cells from a donor who is not the subject. Claim 70 A method or use in which, in any one of claims 67 to 69, the subject receives the engineered T cells of any one of claims 1 to 55, the engineered T cell population of any one of claims 56 to 59, or the pharmaceutical composition of claim 60 before receiving a transplant or blood transfusion. Claim 71 A method or use according to any one of claims 67 to 70, wherein the subject receives a transplant or blood transfusion while simultaneously receiving the engineered T cells of any one of claims 1 to 55, the engineered T cell population of any one of claims 56 to 59, or the pharmaceutical composition of claim 60. Claim 72 A method or use in which, in any one of claims 67 to 71, the subject receives the engineered T cells of any one of claims 1 to 55, the engineered T cell population of any one of claims 56 to 59, or the pharmaceutical composition of claim 60 after receiving a transplant or blood transfusion.

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