TW202542303A - Engineered t cells - Google Patents

Abstract

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

Description

Engineered T cells

Adaptive immunity is a defense mechanism through which the body eliminates foreign pathogens. T cells are immune cells that mediate this immune response. T cell receptors (TCRs) are protein complexes on the surface of T cells that recognize antigens. T cell diversity arises from rearrangements at the TCR α and β loci.

One characteristic of adaptive immunity is the ability to distinguish between "self" and "non-self" antigens. Autoimmunity and autoinflammatory diseases are characterized by pathogenic immune responses against "self" antigens. Some rearrangements at the TCR α and β loci produce autoreactive T cells. Owen et al., Regulatory T Cell Development in the Thymus, J Immunol 203(8) (2019). Many autoreactive T cells are eliminated due to pure lineage loss in the thymus, but other T cells can evade pure lineage loss and elicit harmful immune responses. Ibid. Specialized T cells, called regulatory T cells (Tregs), are used for "self" tolerance. Ibid. Tregs can suppress excessive immune responses, autoimmune responses, and unwanted immune responses, such as in graft-versus-host disease. Ibid. An imbalance of Tregs (e.g., insufficient Treg numbers or abnormal Treg function) can lead to an autoimmune response. (Source: same as above)

Current treatments for autoimmune diseases 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 therapy has been used to suppress antigen-specific immune responses in various diseases, including graft-versus-host disease (GvHD), in which donor cells mediate the immune attack on host tissues after hematopoietic stem cell transplantation. (Pierini et al., T Cells Expressing Chimeric Antigen Receptor Promoter Immune Tolerance, JCI Insight 2(20) (2017).) However, "significant challenges remain in the clinical implementation of Treg-based therapies." (Ibid.) Therefore, the industry still needs effective T-cell therapies, including Treg therapy, to suppress immune responses (including inflammation and autoimmunity).

One aspect of this disclosure relates to engineered T cells comprising i) a heterologous nucleic acid sequence encoding a double mutant transformant growth factor β1 (dmTGFB1) under the control of a promoter sequence; and ii) a modification of an endogenous nucleic acid sequence encoding transformant growth factor β receptor 2 (TGFBR2), wherein the modification knocks down the expression of TGFBR2.

In some embodiments, relative to wild-type human TGFB1, dmTGFB1 includes mutations at two or more amino acids selected from the following: F198, D199, V200, L208, F217, L219, R218, H222, C223, and C225. In some embodiments, relative to wild-type human TGFB1, dmTGFB1 includes mutations at two or more amino acids selected from the following: R218C/H, H222D, C223S/R/G, and C225R. In some embodiments, dmTGFB1 contains amino acid mutations R218C/H and C225R, as opposed to wild-type human TGFB1.

In some implementations, the engineered T cells are CD4+ T cells.

In some embodiments, the engineered T cells further comprise: i) a modification of an endogenous nucleic acid sequence encoding tumor necrosis factor α (TNFA), wherein the modification knocks down TNFA expression; ii) a modification of an endogenous nucleic acid sequence encoding interferon-γ (IFNG), wherein the modification knocks down IFNG expression; or iii) a heterologous nucleic acid sequence encoding a regulatory T cell-promoting molecule under the control of a promoter sequence.

In some embodiments, the engineered T cells comprise: i) a modification of an endogenous nucleic acid sequence encoding tumor necrosis factor α (TNFA), wherein the modification knocks down TNFA expression; ii) a modification of an endogenous nucleic acid sequence encoding interferon-γ (IFNG), wherein the modification knocks down IFNG expression; and iii) a heterologous nucleic acid sequence encoding a regulatory T cell-promoting molecule under the control of a promoter sequence.

In some embodiments, the regulatory T-cell stimulating molecules are selected from interleukin-10 (IL10), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), T-cell immune receptor with Ig and ITIM domains (TIGIT), indoleamine 2,3-dioxygenase 1 (IDO1), extracellular nucleoside triphosphate diphosphate hydrolase 1 (ENTPD1), extracellular 5'-nucleotidase (NT5E), interleukin-22 (IL-22), amphotericin G (AREG), interleukin-35 (IL35), GARP, CD274 molecule (CD274), forkhead box P3 (FOXP3), IKAROS family zinc finger 2 (IKZF2), familial eosinophilia (EOS), interferon regulator 4 (IRF4), lymphoid enhancement binding factor 1 (LEF1), BTB domain and CNC homologue 2. (BACH2) and interleukin-2 receptor subunit α (IL2RA).

In some embodiments, the regulatory T-cell stimulating molecule is IL10 or CTLA4.

In some embodiments, the regulatory T cell stimulating molecule is a first regulatory T cell stimulating molecule, and further includes a heterologous nucleic acid sequence encoding a second regulatory T cell stimulating molecule under the control of a promoter sequence. In some embodiments, the first and second regulatory T cell stimulating molecules are IL10 and CTLA4.

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

In some embodiments, the engineered T cells include modifications to the 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 cells include modifications to the endogenous nucleic acid sequence encoding a protein selected from TRAC, HLA-A, HLA-B, and CIITA, wherein the modification knocks down protein expression. In some embodiments, the protein is TRAC, and the modification knocks down TRAC expression. In some embodiments, the protein is HLA-A, and the modification knocks down HLA-A expression. In some embodiments, the protein is HLA-B, and the modification knocks down HLA-B expression. In some embodiments, the protein is CIITA, and the modification knocks down CIITA expression.

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

Another aspect of this disclosure relates to engineered T cells comprising: i) a modification of a first endogenous nucleic acid sequence encoding tumor necrosis factor α (TNFA), wherein the modification of the first endogenous nucleic acid knocks down TNFA expression; ii) a modification of a second endogenous nucleic acid sequence encoding interferon-γ (IFNG), wherein the modification of the second endogenous nucleic acid knocks down IFNG expression; 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-promoting molecule under the control of a promoter sequence.

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

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

In some embodiments, the regulatory T-cell stimulating molecules are selected from interleukin-10 (IL10), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), T-cell immune receptor with Ig and ITIM domains (TIGIT), indoleamine 2,3-dioxygenase 1 (IDO1), extracellular nucleoside triphosphate diphosphate hydrolase 1 (ENTPD1), extracellular 5'-nucleotidase (NT5E), interleukin-22 (IL-22), amphotericin G (AREG), interleukin-35 (IL35), GARP, CD274 molecule (CD274), forkhead box P3 (FOXP3), IKAROS family zinc finger 2 (IKZF2), familial eosinophilia (EOS), interferon regulator 4 (IRF4), lymphoid enhancement binding factor 1 (LEF1), BTB domain and CNC homologue 2. (BACH2) and interleukin-2 receptor subunit α (IL2RA).

In some embodiments, the regulatory T-cell stimulating molecule is IL10 or CTLA4.

In some embodiments, the regulatory T cell stimulating molecule is a first regulatory T cell stimulating molecule, and further includes a heterologous nucleic acid sequence encoding a second regulatory T cell stimulating molecule under the control of a promoter sequence.

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

In some embodiments, the engineered T cells further include 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 granzyme 7 (NKG7), Fas ligand (FASL, NF superfamily member 6), ryanodine receptor 2 (RYR2), and community stimulating factor 2 (CSF2), wherein the modifications knock down the expression of IL17A, IL2, IL6, PRF1, GZMA, GZMB, FASL, RYR2, or CSF2, respectively.

In some embodiments, the engineered T cells further include modifications to the endogenous nucleic acid sequence encoding the endogenous T cell receptor (TCR), wherein the modifications knock down the expression of the endogenous TCR.

In some embodiments, the engineered T cells further include a heterologous encoding sequence targeting the receptor under the control of the promoter sequence. In some embodiments, the target receptor is selected from the following ligands: mucosal vascular inotropic cell adhesion molecule 1 (MADCAM1), tumor necrosis factor α (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), protein lipoprotein 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), glutamic acid decarboxylase (GAD2), and bridgene core protein 3. (DSG3) and MHC Class I HLA-A (HLA-A*02).

In some embodiments, the target receptor includes a chimeric antigen receptor (CAR) or a T-cell receptor (TCR).

In some embodiments, a heterologous nucleic acid sequence encoding a target receptor is incorporated into the phenotype. In some embodiments, the phenotype containing the heterologous nucleic acid sequence encoding the target receptor does not contain a nucleic acid sequence encoding a regulatory T cell-stimulating molecule. In some embodiments, the phenotype containing the heterologous nucleic acid sequence encoding the target receptor contains a nucleic acid sequence encoding a regulatory T cell-stimulating molecule.

In some embodiments, a heterologous nucleic acid sequence encoding a first regulatory T cell-stimulating molecule is incorporated into the expression building, and a heterologous nucleic acid sequence encoding a second regulatory T cell-stimulating molecule is incorporated into the expression building. In some embodiments, both the heterologous nucleic acid sequences encoding the first and second regulatory T cell-stimulating molecules are incorporated into a single expression building. In some embodiments, both the heterologous nucleic acid sequences encoding the first and second regulatory T cell-stimulating molecules are incorporated into a single expression building.

In some embodiments, at least one heterologous coding sequence is in a free phenotype 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 at a site selected from the following loci: TCR locus, TNF locus, IFNG locus, IL17A locus, IL6 locus, IL2 locus, and adeno-associated virus integration site 1 (AAVS1) locus. In some embodiments, the TCR locus is the T cell receptor α-constancy (TRAC) locus.

In some embodiments, the modification to knock down gene expression includes one or more of insertions, deletions, or substitutions. In some embodiments, the engineered T cells are CD4+ T cells.

Another aspect of this disclosure relates to an engineered T cell population comprising engineered T cells as described herein. In some embodiments, the engineered T cell population comprises engineered T cells as described herein, wherein at least 30% or at least 40% of the cells in the population contain 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 contain a modification of an endogenous nucleic acid sequence encoding TGFBR2. In some embodiments, the engineered T cell population comprises engineered T cells as described herein, wherein at least 50% or at least 70% of the cells in the population contain modifications to an endogenous nucleic acid sequence encoding TNFA; at least 50% or at least 70% of the cells in the population contain modifications to an endogenous nucleic acid sequence encoding IFNG; or at least 30% or at least 40% of the cells in the population contain a heterologous nucleic acid sequence encoding a regulatory T cell-promoting molecule under the control of a promoter sequence. In some embodiments, the engineered T cell population comprises engineered T cells as described herein, wherein the percentage of cells containing insertions or modifications is determined based on the percentage of reads from next-generation sequencing (NGS).

One aspect of this disclosure relates to pharmaceutical compositions that include engineered T cells or engineered T cell populations as described herein.

Another aspect of this disclosure relates to methods or uses of administering engineered T cells, engineered T cell populations, or pharmaceutical compositions, as described herein, to an individual. In some embodiments, the individual requires immunosuppression. In some embodiments, the methods or uses described herein are used to treat immune disorders. In some embodiments, the methods or uses described herein are used to treat autoimmune diseases.

In some embodiments, the autoimmune diseases selected are ulcerative colitis, Crohn's disease, rheumatoid arthritis, psoriasis, multiple sclerosis, systemic lupus erythematosus, and type 1 diabetes. In some embodiments, the methods or uses described herein are used to treat graft-versus-host disease (GvHD).

In some embodiments, an individual receives a transplant or blood transfusion. In some embodiments, an individual will receive a transplant or blood transfusion. In some embodiments, an individual is receiving a transplant or blood transfusion. In some embodiments, an individual has already received a transplant or transfusion. In some embodiments, the transplant or blood transfusion comprises a solid organ or cell population. In some embodiments, the transplant or blood transfusion comprises cells from a donor who is not the individual.

In some embodiments, the individual is given engineered T cells, engineered T cell populations, or pharmaceutical combinations as described herein prior to receiving a transplant or transfusion. In some embodiments, the individual is given engineered T cells, engineered T cell populations, or pharmaceutical combinations as described herein concurrently with receiving a transplant or transfusion. In some embodiments, the individual is given engineered T cells, engineered T cell populations, or pharmaceutical combinations as described herein after receiving a transplant or transfusion.

Related Applications This application claims the interests and priorities of U.S. Provisional Application No. 63/612,847, filed December 20, 2023, and U.S. Provisional Application No. 63/727,648, filed December 3, 2024, the entire contents of which are expressly incorporated herein by reference in their entirety.

This application is filed together with an electronically submitted sequence list. The sequence list file, titled 12793_0041-00304_SL, was created on December 18, 2024, and is 800,521 bytes in size. The information contained in the electronically submitted sequence list is incorporated herein by reference in its entirety.

Reference will now be made to certain embodiments of this disclosure, examples of which are illustrated in the accompanying drawings. Although this disclosure will be described in conjunction with the illustrated embodiments, it should be understood that such embodiments are not intended to limit this disclosure to them. On the contrary, this disclosure is intended to cover all alternatives, modifications, and equivalents that may be included in this disclosure as defined in the appended embodiments.

The chapter headings used in this document are for organizational purposes only and should not be construed as limiting the intended subject matter in any way. In the event of any conflict between any material incorporated herein by reference and any terminology defined herein or any other representation herein, this document shall prevail.

I. Definition

Before detailing the teachings of this invention, it should be understood that this disclosure is not limited to specific compositions or process steps, as these elements are subject to variation. It should be noted that, unless the context clearly indicates otherwise, as used in this specification and accompanying embodiments, the singular forms "a" and "the" include a plurality of indicators. Thus, for example, a reference to "compound" includes a plurality of compounds, and a reference to "cell" includes a plurality of cells or groups of cells, and so on. As used herein, the term "comprising" and its grammatical variations are intended to be non-restrictive, such that the listing of items in the list does not exclude other similar items that may be substituted for or added to the listed items.

A range of values includes the values that define that range. Taking into account significant figures and measurement-related errors, measured and measurable values should be understood as approximate values. Similarly, the use of "comprise," "comprises," "comprising," "contain," "contains," and "include" is not intended to be restrictive. It should be understood that both the foregoing general and detailed descriptions are illustrative and explanatory only and do not limit the teaching content.

Unless expressly stated in this specification, embodiments in this specification that "comprise" various components shall be considered as "consisting of" or "substantially composed of" the listed components; embodiments in this specification that "comprise" various components shall be considered as "comprising" or "substantially composed of" the listed components; and embodiments in this specification that "substantially consist of various components" shall be considered as "comprising" or "comprising" the listed components (this interchangeability does not apply to the use of these terms within the scope of the patent application).

Unless the context clearly indicates otherwise, the term "or" is used in an inclusive sense, that is, equivalent to "and/or".

When used before a list or range, the term "about" modifies each member of the list or each endpoint of the range. The term "about" or "approximately" means the acceptable error of a particular value as determined by someone skilled in the art, depending in part on how that value is measured or determined. In this document, the term "about" refers to a typical tolerance range in the art. For example, "about" can be understood as approximately 2 standard deviations from the mean. In some embodiments, "about" means +/- 10%. In some embodiments, "about" means +/- 5%.

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

As is clear from the context, the term "at least" preceding a numerical value or series of numerical values should be understood to include the numerical value adjacent to the term "at least," as well as all subsequent numerical values or integers that are logically permissible. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 17 nucleotides in a nucleic acid molecule of 20 nucleotides" means that 17, 18, 19, or 20 nucleotides have the indicated property. When "at least" appears before a series of numerical values or ranges, it should be understood that "at least" modifies each numerical value within that series or range.

As used herein, "not more than" or "less than" should be understood as the lowest logically valid value or integer up to zero that is adjacent to the phrase and that is logically consistent with the context. For example, a doublet region that is "not more than 2 nucleotide base pairs" has 2, 1, or 0 nucleotide base pairs. When "not more than" or "less than" appears before a series of values or ranges, it should be understood that each value in that series or range is modified.

As used in this article, the range includes both the upper and lower limits.

As used herein, it should be understood that when the maximum value is expressed as 100% (e.g., 100% inhibition or 100% encapsulation), that value is limited by the detection method. For example, 100% inhibition should be understood as inhibition below the level of analytical detection, and 100% encapsulation should be understood as the material intended for encapsulation not being detectable outside the vesicle.

If any sequence in this application conflicts with the instruction registration number or the position within the registration number, the sequence in this application shall prevail.

Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings.

As used herein, "knockdown" refers to the reduction of expression of a specific gene product (e.g., full-length or wild-type mRNA, protein, or both) in a cell, cell population, tissue, or organ through gene editing. In some embodiments, gene editing can be evaluated by sequence, such as next-generation sequencing (NGS). Compared to a suitable control (e.g., where the gene sequence is unmodified), expression can be reduced by at least 70%, 75%, 80%, 85%, 90%, 95%, or reduced to below the detection level of the analysis. Protein knockdown can be measured by detecting the amount of protein from the tissue, cell population, or fluid of interest. Methods for measuring mRNA knockdown are known and include sequencing of mRNA isolated from the tissue or cell population of interest. Flow cytometry analysis is a known method for measuring protein knockdown. For secreted proteins, knockdown can be evaluated in fluids such as tissue culture media, blood, or serum or plasma derived from blood. In some embodiments, "knockdown" can refer to some loss of expression of a specific gene product, such as a reduction in the amount of full-length wild-type mRNA transcribed or translated into full-length protein, or a reduction in the amount of protein expressed by a cell population. It is well known which changes in the mRNA sequence will lead to a reduction in the expression of wild-type or full-length protein. In some embodiments, "knockdown" can refer to some loss of expression of a specific gene product (e.g., TGFBR2, IFNG, or TNFA gene products in body fluids or tissue culture media). Modifications to endogenous nucleic acid sequences that encode, for example, TGFBR2, IFNG, or TNFA can lead to knockdown.

As used herein, "T cell receptor" or "TCR" refers to the receptor in T cells. Generally, a TCR is a heterodimeric receptor molecule containing two TCR polypeptide chains, α and β. The α and β TCR polypeptide chains can complex with various CD3 molecules and trigger an immune response, including inflammation and autoimmunity, upon antigen binding. As used herein, TCR knockdown refers to the partial or complete knockdown of any TCR gene, such as a partial deletion of the TRBC1 gene alone or in combination with partial or complete knockdown of other TCR genes.

"TRAC" refers to the T cell receptor α chain. The human wild-type TRAC sequence can be obtained from NCBI Gene ID: 28755; Ensembl: ENSG00000277734. T cell receptor α constant region, TCRA, IMD7, TRCA, and TRA are genetic synonyms for TRAC.

"TRBC" refers to the T cell receptor β chain, such as TRBC1 and TRBC2. "TRBC1" and "TRBC2" are two homologous genes that encode the T cell receptor β chain, and are gene products of the TRBC1 or TRBC2 genes.

The human wild-type TRBC1 sequence can be obtained from NCBI Gene ID: 28639; Ensembl: ENSG00000211751. T cell receptor β-constant region, V_ region translation product, BV05S1J2.2, TCRBC1, and TCRB are genetic synonyms of TRBC1.

The human wild-type TRBC2 sequence can be obtained from NCBI Gene ID: 28638; Ensembl: ENSG00000211772. The T cell receptor β-constant region, V_ region translation products, and TCRBC2 are genetic synonyms for TRBC2.

As used herein, "immune response" refers to one or more immune system responses, such as an increase in the production or activity of immune system cells compared to an unstimulated control immune system, including (but not limited to) T cells, B cells, natural killer cells, monocytes, neutrophils, eosinophils, basophils, fat cells, erythrocytes, dendritic cells, antigen-presenting cells, macrophages, or phagocytes. Exposure of the immune system to antigens can trigger an immune response. These antigens can be foreign or autoantigens, such as (but not limited to) pathogens (microorganisms, viruses, prion proteins, fungi, etc.), allergens (dust, pollen, dust mites, etc.), toxins (chemicals, drugs, etc.), or physiological changes (hypercholesterolemia, obesity, organ transplantation, etc.). Immune responses can also include the donor-mediated immune attack on host tissues following hematopoietic stem cell transplantation in GvHD. Immune responses can lead to inflammation. Alternatively, immune responses can also include the host-mediated immune attack on donor tissues following solid organ transplantation or the introduction of other non-host cells via transplantation or transfusion. Immune responses can target, attack, remove, or neutralize antigens, such as foreign or autoantigens. Immune responses may be desirable or undesirable. Immune responses can be acute or chronic. Immune responses can damage the cells, tissues, or organs targeted by the immune response.

As used herein, "autoimmune response" refers to an immune system response to one or more autoantigens produced by an individual's own cells, tissues, or organs. An autoimmune response can result in an increased production or activity of immune system cells, such as (but not limited to) T cells, B cells, natural killer cells, monocytes, neutrophils, eosinophils, basophils, obesity cells, erythrocytes, dendritic cells, antigen-presenting cells, macrophages, or phagocytes, compared to a suitable control (e.g., a healthy control). Autoimmune responses can lead to inflammation, such as chronic inflammation, or to autoimmune diseases. Autoimmune responses can target, attack, remove, or neutralize autoantigens produced by an individual's own cells, tissues, or organs, which can lead to autoimmune diseases.

As used herein, “suppressive” immune response means a reduction or inhibition of one or more immune system responses (e.g., the production or activity of immune system cells) compared to a suitable control (e.g., before or after treatment with engineered T cells as described herein). “Suppressive” immune response may also refer to a reduction in the production or activity of immune system cells compared to a suitable control (e.g., before or after treatment with engineered T cells as described herein). “Suppressive” immune response may also refer to increased immune tolerance. For example, the production or activity of immune system cells can be measured by: cell count, such as lymphocyte count or spleen cell count; cell activity, such as T cell analysis; or gene or protein expression, such as biomarker expression; wherein the production or activity is reduced by 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or below the detection level of the analysis compared to a suitable control (e.g., before or after treatment with engineered T cells as described herein).

As used herein, "autoimmune disease" or "autoimmune syndrome" refers to a disorder characterized by a pathological immune response to an individual's own antigens, cells, tissues, or organs. Examples of autoimmune diseases and syndromes include (but are not limited to): ulcerative colitis, Crohn's disease, rheumatoid arthritis, psoriasis, multiple sclerosis, systemic lupus erythematosus, and type 1 diabetes. In some embodiments, engineered T cells have autologous or allogeneic uses.

As used herein, "immunodisorder" should be understood as a disease or ailment characterized by a pathological or undesirable immune response in an individual. In some embodiments, an immunodisorder is an autoimmune disease. In some embodiments, an immunodisorder is GvHD. In some embodiments, an individual with an immunodisorder requires suppression of the immune response. In some embodiments, an individual with an immunodisorder requires enhancement of immune tolerance.

"T cells" play a central role in the immune response following exposure to antigens. T cells can be naturally occurring or non-natural, for example, when T cells are generated through engineering (e.g., autologous stem cells) or transdifferentiation (e.g., reprogramming of somatic cells). T cells can be distinguished from other lymphocytes by the presence of T cell receptors on their cell surface. This definition includes adaptive T cells, including helper CD4+ T cells, cytotoxic CD8+ T cells, memory T cells, and regulatory CD4+ T cells, as well as innate-like T cells, including natural killer T cells, mucosa-associated invariant T cells, and γδ T cells. In some embodiments, T cells are CD4+. In some embodiments, T cells are CD3+/CD4+. In some embodiments, 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-); the T cell population may contain one or more of these T cell subtypes.

"Regulatory T cells" or "Tregs" refer to specialized T cells that play a central role in suppressing excessive immune responses, including inflammation and autoimmunity. Tregs can be naturally occurring (here they may be referred to interchangeably as native Tregs ("nTregs") and thymic Tregs ("tTregs")) or non-natural, such as when they are engineered (including by inserting a sequence encoding the dmTGFB1 molecule). When engineered, Tregs may include other modifications, such as by modifying (e.g., knocking down) endogenous nucleic acid sequences encoding TGFBR2, IFNG, and TNFA, and by inserting at least one sequence encoding a regulatory T cell-promoting molecule. Naturally occurring Tregs, or native Tregs or nTregs, are sometimes also referred to as thymic Tregs or tTregs, which are specialized T cells that normally develop in the thymus and function by suppressing excessive immune responses. In some embodiments, cells can be engineered by inserting a sequence encoding the dmTGFB1 molecule and, where appropriate, modifying endogenous nucleic acid sequences encoding, for example, TGFBR2, TNFA, and IFNG, such as knocking down nucleic acid sequences encoding TGFBR2, TNFA, and IFNG, and inserting sequences encoding regulatory T cell-promoting molecules into cells to exhibit the phenotypic characteristics and inhibitory functions of regulatory T cells, such as known T cells or known T cell populations, for example, T cell populations that do not enrich the presence of nTreg cells, and such cells can be referred to as transduced or "engineered" T cells. In some embodiments, the engineered T cells contain an insertion sequence encoding the dmTGFB1 molecule; modifications to the endogenous nucleic acid sequence encoding TGFBR2, the endogenous nucleic acid sequence encoding IFNG, and the endogenous nucleic acid sequence encoding TNFA; and the insertion of heterologous regulatory T cell-promoting molecules, such as IL10 and CTLA4. Modifications to the endogenous nucleic acid sequence (e.g., modifications that knock down endogenous gene expression) may include or consist of one or more insertions, deletions, or substitution mutations in the genome sequence.

As used herein, dmTGFB1 is a mutant TGFB1 based on wild-type TGFB1 from any species, including, for example, human, mouse, rat, or cynomolgus monkey TGFB1. In some embodiments, dmTGFB1 is human dmTGFB1, that is, a mutant TGFB1 relative to wild-type human TGFB1. The coding and amino acid sequences of wild-type TGFB1 are readily available in sequence databases including NCBI, and illustrative sequences can be found in accessions NM_000660.7 and NP_000651.3 (human), NM_011577.2 and NP_035707.1 (mouse), NM_021578.2 and NP_067589.1 (rat), and XM_005589339.3 and XP_005589396.1 (cynomolgus monkey). Each accession is incorporated by reference into the version available as of the filing date of this application. The ability to locate the mutation to the wild-type sequence is entirely within the capabilities of a person skilled in the art.

Transgenic growth factor β-1 (TGFB1) is synthesized into a large precursor molecule. The TGFB1 precursor protein contains a 29-amino acid signal peptide, which is cleaved via proteolysis. TGFB1 is further cleaved after amino acid 278 to form a latent related peptide (LAP) and active TGFB1. The LAP dimers at C223 and C225 via interchain disulfide bonds. TGFB1 can be secreted as an inactive small latent complex, which is formed by the non-covalent bonding of the mature TGF-β1 homodimer with the LAP homodimer at LAP residues I53-L59. The LAP shields the type II receptor binding site in mature TGFB1. Most cells secrete TGFB1 as the large potential complex (LLC) of TGF-β1/LAP, which covalently binds to the potential TGFB binding protein (LTBP) at C33 of the LAP chain. LTBP facilitates TGFB1 folding and secretion and may target the extracellular matrix. Activation of LLC occurs via the binding of the N-terminal domain of LTBP to the extracellular matrix.

Camurati-Engelmann disease (CED) is caused by a specific, inversely zygotic mutation in a domain of the transgrowth factor-β-1 gene (TGFB1; OMIM entry 190180, incorporated herein by reference in the version available at the filing date of this application) on chromosome 19q13. Mutations reported in the Camurati-Engelmann disease family include (Janssen et al., 2006. J Med Genet. 43:1.): Exons DNA mutation protein mutation 1 28_36 repeated L10-L12 repeat 1 241T → C Y81H 2 463C → T R156C 4 652C → T R218C 4 653G → A R218H 4 664C → G H222D 4 ? C223S 4 667T → C C223R 4 667T → G C223G 4 673T → C C225R

As shown in the table, most pathogenic variants in individuals with CED exhibit a single amino acid substitution at the carboxyl terminus of the TGFB1 latent related peptide (LAP). This substitution occurs near the interchain disulfide bond site between LAP homodimers. These pathogenic variants disrupt LAP dimerization and binding to active TGFB1 (Walton et al., 2010. J Biol Chem. 285:17029-37), leading to increased release of active TGFB1 from the cell. Walton et al. further demonstrated that the stability of the resulting large potential complex depends on the covalent dimerization of LAP, which is promoted by key residues (F198, D199, V200, L208, F217, and L219) at the dimer interface. Compared to normal fibroblasts, fibroblasts with the R218H mutation from individuals with CED showed increased TGFB1 activity in cell culture medium (Saito et al., 2001. J Biol Chem. 276:11469-72). In vitro analysis of the R218C, H222D, and C225R mutant constructs also showed increased TGF-β1 activity in transfected cell culture medium. In contrast, the pathogenic variants Leu11_Leu13dup and Tyr81His resulted in decreased TGFB1 secretion. However, in the analysis of luciferase reporter genes specific to the TGFB1-induced transcriptional response, mutant cells showed increased luciferase activity, indicating intracellular activation of the receptor (Janssens et al., 2003. J Biol Chem. 278:7718-24).

As used herein, in some embodiments, dmTGFB1 refers to TGFB1 containing mutations at two or more amino acid sites relative to wild-type TGFB1. These mutations reduce the stability of the resulting high-potential complex, which depends on the covalent dimerization of LAP relative to each individual TGFB1 mutant. Mutations at two or more amino acid sites relative to wild-type human TGFB1 may include mutations selected from the following sites: F198, D199, V200, L208, F217, L219, R218, H222, C223, and C225.

In some embodiments, dmTGFB1 is a TGFB1 containing mutations at two or more amino acid sites relative to wild-type TGFB1, wherein the mutations are selected from the following sites relative to wild-type human TGFB1: F198, D199, V200, L208, F217, L219, R218, H222, C223, and C225, wherein when dmTGFB1 is produced, it is secreted from cells that normally express TGFB1. In some embodiments, dmTGFB1 contains naturally occurring mutations, such as those naturally present in CED. In some embodiments, dmTGFB1 contains non-naturally occurring mutations, such as those not naturally present in CED.

In some embodiments, human dmTGFB1 contains mutations at two or more amino acid sites selected from R218, H222, C223 and C225, as opposed to wild-type human TGFB1.

In some embodiments, human dmTGFB1 contains two or more mutations selected from R218C/H, H222D, C223S/R/G, and C225R, relative to wild-type human TGFB1.

In some embodiments, human dmTGFB1 contains two or more mutations selected from R218C/H and C225R, depending on whether they are R218H or C225R, relative to wild-type human TGFB1.

The dmTGFB1 described herein exhibits greater free activity than any single mutant alone. In some embodiments, the increase in dmTGFB1 activity compared to the activity of a single mutant alone is at least additive. In some embodiments, the increase in dmTGFB1 activity compared to the activity of a single mutant alone is significantly greater than additive. The methods for determining total TGFB1 and active TGFB1 levels using commercially available kits (e.g., the LEGEND MAX Total TGF-β1 ELISA kit (BioLegend, catalog number 436707) and the LEGEND MAX Free Active TGF-β1 ELISA kit) and as provided in the examples below are known in the art.

As used herein, "regulatory T cell-promoting molecules" refers to molecules that promote the conversion of learned T cells into regulatory T cells, including immunosuppressive molecules and Treg transcription factors. Furthermore, regulatory T cell-promoting molecules also refer to molecules that endow learned T cells with regulatory activity, including Treg-related immunosuppressive molecules and transcription factors. Regulatory T cell-promoting molecules can be used in combination with dmTGFB1 to promote the conversion of learned T cells into regulatory T cells or to endow learned T cells with regulatory activity. Examples of immunosuppressive molecules to be used in combination with dmTGFB1 include (but are not limited to) interleukin-10 (IL10), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), T-cell immune receptor with Ig and ITIM domains (TIGIT), indoleamine 2,3-dioxygenase 1 (IDO1), extracellular nucleoside triphosphate diphosphate hydrolase 1 (ENTPD1), extracellular 5'-nucleotidase (NT5E), interleukin-22 (IL22), amphotericin G (AREG), interleukin-35 (IL35), leucine-rich 32-repetitive sequence (GARP), CD274 (CD274), forkhead box P3 (FOXP3), IKAROS family zinc finger 2 (IKZF2), familial eosinophilia (EOS), interferon regulator 4 (IRF4), and lymphoid enhancement binding factor 1. (LEF1), BTB domain and CNC homologue 2 (BACH2), and interleukin-2 receptor subunit α (IL2RA, CD25). In some embodiments, regulatory T-cell activating molecules can be used in specific combinations, such as IL10 with CTLA4, ENTPD1 with NT5E, and IL22 with AREG. Specifically, this article provides the combination of IL10 and CTLA4 used in conjunction with dmTGFB1. In some embodiments, the expression of immunosuppressive molecules can be promoted by the expression of transcription factors such as FoxP3, Helios, Eos, IRF4, Lef1, or BACH2.

In some embodiments, it is known that T cells can be engineered to modify, insert, or delete sequences in the genome, and that "engineered" T cells exhibit one or more phenotypic characteristics and inhibitory functions of naturally occurring regulatory T cells. For example, "engineered" T cells exhibit inhibitory activity in the mixed lymphocyte reaction analysis provided in Example 2.4 below, or may be able to inhibit graft-versus-host disease in the mouse model presented in Example 3.2 below, for example, in a statistically significant manner (e.g., see also Parmar et al., Ex vivo fucosylation of third-party human regulatory T cells enhances anti–graft-versus-host disease potency in vivo, Blood 125(9) (2015)). In some embodiments, the "engineered" T cells are learned T cells that have been modified by inserting the coding sequence of a regulatory T cell-promoting molecule and by modifying (e.g., knocking down) the expression of pro-inflammatory interstitials (e.g., TNFA and one or a combination of IFNG and IL17A). In some embodiments, the engineered initiator T cell population is not enriched with naturally occurring Tregs; for example, the initiator T cell population has less than 20% naturally occurring Tregs.

As used herein, "pro-inflammatory" molecules (such as intercytokines) enhance immune responses as described herein, for example, by dose-responsively reducing the efficacy of Tregs in the graft-versus-host disease mouse model 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 granzyme 7 (NKG7), Fas ligand (FasL, NF superfamily, member 6), ranotide receptor 2 (RYR2), and community-stimulating factor 2 (CSF2).

As used herein, "targeted receptor" refers to a receptor that is present on the surface of cells (e.g., T cells) to allow the cells to bind to a target site (e.g., a specific cell or tissue in an organism). Targeted receptors include (but are not limited to) chimeric antigen receptors (CARs), T cell receptors (TCRs), and receptors of cell surface molecules, which are operatively linked via at least one transmembrane domain in an internal signaling domain that can activate T cells after binding to the extracellular receptor portion of a protein, such as mucosal indices 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), proteolipoprotein 1 (PLP1), CD19, CD20, and TNF. (Ligand) superfamily member 7 (TNFSF7), TNF receptor superfamily member 17 (TNFRSF17), dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), stromal core protein 3 (DSG3), and MHC class I HLA-A (HLA-A*02).

As used herein, a "chimeric antigen receptor" refers to an extracellular antigen recognition domain operatively linked to an intracellular signaling domain, such as scFv, VHH, or nanoantibodies, which activates T cells upon antigen binding. A CAR consists of four domains: 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 portions of which are incorporated herein by reference). Reverse universal CARs that facilitate the binding of immune cells to target cells via connector molecules are also considered (e.g., see WO2019238722, the contents of which are incorporated herein by reference in their entirety). CARs can target any antigen that can produce antibodies and are typically molecules displayed on the surface of the cells or tissues to be targeted. In some embodiments, CARs can enable engineered T cells to target the gastrointestinal tract; for example, CARs targeting MAdCAM-1. In some embodiments, CARs can enable engineered T cells to target activated immune cells (e.g., T cells or B cells that upregulate their targets); for example, CARs targeting CD70. In some embodiments, CARs can enable engineered T cells to target tissues containing endothelial cells; for example, CARs targeting VCAM-1 can be used to suppress immune responses in diseases such as Crohn's disease and multiple sclerosis. In some embodiments, CARs can enable engineered T cells to target endothelial cells; for example, CARs targeting CEACAM6 can be used to suppress immune responses in diseases such as Crohn's disease. In some embodiments, CARs can enable engineered T cells to target pre-B cells; for example, CARs targeting CD19 can be used to suppress immune responses in diseases such as multiple sclerosis and systemic lupus erythematosus. In some embodiments, CARs can target engineered T cells to B lymphocytes, for example, CARs targeting CD20, for example, to suppress immune responses in diseases such as multiple sclerosis and systemic lupus erythematosus. In some embodiments, CARs can target engineered T cells to inflamed tissues, for example, CARs targeting TNFA, for example, to suppress immune responses in diseases such as rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, or Crohn's disease. In some embodiments, CARs can target engineered T cells to inflamed tissues, for example, CARs targeting TGF-β1, for example, to suppress immune responses in diseases such as inflammatory bowel disease, ulcerative colitis, or Crohn's disease. In some embodiments, CARs can enable engineered T cells to target neural tissue, for example, CARs targeting MBP, MOG, or PLP1, for example, to suppress immune responses in diseases such as multiple sclerosis. In some embodiments, CARs can enable engineered T cells to target immune cells expressing TNFSF7, including (but not limited to) activated T lymphocytes, B lymphocytes, dendritic cells, and/or NK cells, for example, CARs targeting TNFSF7, for example, to suppress immune responses in diseases such as Crohn's disease, ulcerative colitis, systemic lupus erythematosus, or multiple sclerosis. In some embodiments, CARs can enable engineered T cells to target tissues containing mature B lymphocytes; for example, CARs targeting TNFRSF17 can be used to suppress immune responses in conditions such as systemic lupus erythematosus. In some embodiments, CARs can enable engineered T cells to target synovial tissue; for example, CARs targeting citrullinated vimentin can be used to suppress immune responses in conditions such as rheumatoid arthritis. In some embodiments, CARs target dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), stromal core protein 3 (DSG3), or MHC class I HLA-A (HLA-A*02). Other CAR targets (e.g., inflammatory antigens) are known in this art. For example, see WO2020092057A1, the content of which is incorporated into this article in its entirety.

As used herein, "treatment" means any administration or application of a therapeutic agent to an individual's disease or condition, including suppressing the disease, preventing its development, alleviating one or more symptoms of the disease, curing the disease, preventing one or more symptoms of the disease, or preventing the recurrence of one or more symptoms of the disease. Treatment of autoimmune or inflammatory responses or conditions may include alleviating inflammation associated with a specific condition, thereby alleviating disease-specific symptoms. Treatment with engineered T cells as described herein may be used before, after, or in combination with other therapeutic agents (e.g., anti-inflammatory agents, immunosuppressants, or biologics, such as Remicade and Humira) used to treat autoimmune conditions.

A "starter" is a regulatory region that controls the expression of genes linked to that region.

"Polynucleotide" and "nucleic acid" as used herein refer to polymeric compounds containing nucleosides or nucleoside analogs, having nitrogen-containing heterocyclic bases or base analogs linked together along a backbone, including known RNA, DNA, mixed RNA-DNA, and polymers as analogs. The nucleic acid "backbone" can be composed of various bonds, including one or more of the following: sugar-phosphodiester bonds, peptide-nucleic acid bonds ("peptide nucleic acid" or PNA; PCT No. WO 95/32305), thiophosphate bonds, methylphosphonate bonds, or combinations thereof. The sugar moiety of the nucleic acid can be ribose, deoxyribose, or analogs with substitutions (e.g., 2'-methoxy, 2'-halogenated, or 2'-O-(2-methoxyethyl) (2'-O-moe) substitutions). The nitrogenous base can be a known base (A, G, C, T, U), its analogues (e.g., modified uridine, such as 5-methoxyuridine, pseudouridine, or N1-methylpseudouridine, or other uridines); inosine; purine or pyrimidine derivatives (e.g., ^N4 -methyldeoxyguanidine, denitrified purine or azapurine, denitrified pyrimidine or azapyrimidine, pyrimidine bases with substituents at the 5 or 6 position (e.g., 5-methylcytosine), purine bases with substituents at the 2, 6, or 8 position, 2-amino-6-methylaminopurine, ^O6 -methylguanine, 4-thiopyrimidine, 4-aminopyrimidine, 4-dimethylhydrazine-pyrimidine, and ^O4 -alkylpyrimidine; US Patent No. 5,378,825 and PCT No. WO (93/13121). For general discussion, see * The Biochemistry of the Nucleic Acids * 5-36, eds. Adams et al., 11th edition, 1992. Nucleic acids may include one or more "base-free" residues, wherein the backbone does not include nitrogenous bases targeting polymer positions (US Patent No. 5,585,481). Nucleic acids may contain only known RNA or DNA sugars, bases, and bonds, or may include known components and substitutions for both (e.g., known bases with 2'-methoxy bonds, or polymers containing known bases and one or more base analogues). Nucleic acids include locked nucleic acids (LNAs), which are analogues containing one or more LNA nucleotide monomers, with bicyclic furanose units locked in a sugar conformation mimicking RNA. This enhances hybrid affinity for complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). Nucleic acids also include unlocked nucleic acids (UNAs). RNA and DNA can have different sugar moieties, and these can vary depending on the presence of uracil or its analogues in RNA and thymine or its analogues in DNA.

As used herein, "peptide" refers to a polymeric compound containing amino acid residues, which may take a three-dimensional configuration. Peptides include (but are not limited to) enzymes, enzyme precursor proteins, regulatory proteins, structural proteins, receptors, nucleic acid-binding proteins, antibodies, etc. Peptides may (but do not necessarily) contain post-translational modifications, non-natural amino acids, coenzymes, and the like.

As used herein, a gene's "open reading frame" or "ORF" refers to a sequence of codons that specify the amino acid sequence of the protein encoded by the gene. An ORF typically 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).

The terms "guide RNA," "gRNA," and "guide" are used interchangeably in this article to refer to crRNA (also known as CRISPR RNA) or a combination of crRNA and trRNA (also known as tracrRNA). crRNA and trRNA can combine to form a single RNA molecule (single guide RNA, sgRNA) or two separate RNA molecules (dual guide RNA, dgRNA). "Guide RNA" or "gRNA" refers to each type. trRNA can be a naturally occurring sequence or a modified or altered trRNA sequence compared to a naturally occurring sequence.

As used herein, "guide sequence," "guide region," "target sequence," "septum," or "septum sequence," and similar terms refer to a sequence within the gRNA that complements the target sequence and is used to guide the gRNA to the target sequence for binding or modification (e.g., cleavage) by an RNA-guided nickase. The guide sequence can be 20 nucleotides long, for example in the case of Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologues/orthologs. Shorter or longer sequences can also be used as guides, for example, 15, 16, 17, 18, 19, 21, 22, 23, 24, or 25 nucleotides in length. The length of the lead sequence can be 20-25 nucleotides, for example, in the case of Nme Cas9, the length can be 20, 21, 22, 23, 24 or 25 nucleotides. For example, a lead sequence of 24 nucleotides can be used with Nme Cas9 (e.g. Nme2 Cas9).

In some embodiments, the target sequence is located, for example, at a locus in the genome or on a chromosome, and is complementary to the guide sequence. In some embodiments, the complementarity or similarity 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 target region may be 100% complementary or similar. In other embodiments, the guide sequence and target region may contain at least one mismatch. For example, the guide sequence and target sequence may contain 1, 2, 3, or 4 mismatches, wherein the total length of the target sequence is at least 17, 18, 19, 20, or more base pairs. In some embodiments, the guide sequence and target region may contain 1-4 mismatches, wherein the guide sequence contains at least 17, 18, 19, 20, or more nucleotides. In some embodiments, the lead sequence and target region may contain 1, 2, 3, or 4 mismatches, wherein the lead sequence comprises 20 nucleotides. In some embodiments, for example, when the lead sequence comprises a sequence of 24 adjacent nucleotides, the complementarity or similarity between the lead sequence and its corresponding target sequence is at least 80%, 85%, 90%, or 95%. In some embodiments, the lead sequence and target region may be 100% complementary or similar. In other embodiments, the lead sequence and target region may contain at least one mismatch, i.e., one nucleotide that is inconsistent or non-complementary, depending on the reference sequence. For example, the lead and target sequences may contain one or two mismatches, such as no more than one mismatch, where the total length of the target sequence is 19, 20, 21, 22, 23, or 24 or more nucleotides. In some embodiments, the lead and target regions may contain one or two mismatches, where the lead sequence contains at least 24 or more nucleotides. In some embodiments, the lead and target regions may contain one or two mismatches, where the lead sequence contains 24 nucleotides. Mismatch locations are known in this art, such as tolerance for distal mismatches in PAMs often being better than that provided by proximal PAM matches. Mismatch tolerance at other locations is known in this art (e.g., see Sternberg et al., 2015, Nature:527:110-113).

As used herein, "target sequence" or "genosome target sequence" refers to the nucleic acid sequence at the locus of the target gene in the positive or negative strand, which is complementary to the lead sequence of the gRNA, that is, fully complementary to the lead sequence of the gRNA to allow the lead sequence to bind specifically to the target sequence. The interaction between the target sequence and the lead sequence guides the RNA-guided DNA binder to bind within the target sequence and potentially cleave or break down (depending on the activity of the binder). The specific length of the target sequence and the possible number of mismatches between the target sequence and the lead sequence depend on, for example, the identity of the Cas9 nuclease guided by the gRNA. The target sequence of the Cas protein includes both the positive and negative strands of the genosome DNA (i.e., the given sequence and its inverse complementary sequence), because the nucleic acid receptor of the Cas protein is a double-stranded nucleic acid. Therefore, when the guide sequence is referred to as "complementary to the target sequence," it should be understood that the guide sequence can guide an RNA-guided DNA binder (e.g., dCas9 or damaged Cas9) to bind to the inverse complement of the target sequence. Thus, in some embodiments, where the guide sequence binds to the inverse complement of the target sequence, certain nucleotides of the guide sequence are identical to those of the target sequence (e.g., a target sequence excluding PAM), except that U replaces T in the guide sequence.

The target sequence of an RNA-guided DNA binder includes both the positive and negative strands of the genomic DNA (i.e., the given sequence and its inverse complement), because the nucleic acid receptor of the RNA-guided DNA binder is a double-stranded nucleic acid. Therefore, when the guide sequence is referred to as "complementary to the target sequence," it should be understood that the guide sequence can guide the guide RNA to bind to the inverse complement of the target sequence. Thus, in some embodiments, where the guide sequence binds to the inverse complement of the target sequence, certain nucleotides of the guide sequence are identical to those of the target sequence (e.g., a target sequence excluding PAM), except that U replaces T in the guide sequence.

As used herein, "RNA-induced DNA binder" means a polypeptide or polypeptide complex having both 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 RNA sequence. The term RNA-induced DNA binder also includes nucleic acids encoding such polypeptides. Exemplary RNA-induced DNA binders include Cas lyases/nicking enzymes. Exemplary RNA-induced DNA binders may include their inactive form ("dCas DNA binders"), for example, if they are modified to allow DNA cleavage, for example, by fusing with a FokI lyase domain. As used herein, "Cas nuclease" encompasses both Cas lyases and Cas nicking enzymes. Cas lyases and Cas nickases include the Csm or Cmr complex of the type III CRISPR system, its Cas10, Csm1, or Cmr2 subunits, the cascade complex of the type I CRISPR system, its Cas3 subunit, and class II Cas nucleases. As used herein, "class II Cas nucleases" are single-stranded polypeptides with RNA-guided DNA-binding activity. Class II Cas nucleases include class II Cas lyases/nickases (e.g., H840A, D10A, or N863A variants) that further possess RNA-guided DNA lyase or nickase activity, and class II dCas DNA binders, wherein the lyase/nickase activity is inactive, for example, if the binder is modified to allow DNA cleavage, or has C-to-T deaminase or A-to-G deaminase activity. Cas cleavage enzymes include one of the RuvC or HNH domains of the Cas protein, which is a nuclease that is cleaved by a single-stranded nuclease. In some embodiments, RNA-guided DNA binders include a deaminerase region and an RNA-guided DNA cleavage enzyme, such as the Cas9 cleavage enzyme. Class 2 Cas nucleases include proteins and their modified forms, such as 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). The Cpf1 protein (Zetsche et al., Cell , 163: 1-13 (2015)) also contains a RuvC-like nuclease domain. Zetsche's Cpf1 sequence is incorporated in whole. For example, see Zetsche, Tables S1 and S3. For example, see Makarova et al., Nat Rev Microbiol , 13(11): 722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015). As used herein, RNA-delivering DNA binders (e.g., Cas nucleases, Cas9 nucleases, or Streptococcus pustulosis Cas9 nucleases or Neisseria meningitidis Cas9 nucleases) include those that deliver polypeptides or mRNA.

As used herein, the term "editor" or "base editor" refers to an agent containing 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 can deaminate bases within nucleic acids. In some embodiments, the editor can deaminate bases within DNA molecules. In some embodiments, the editor can deaminate cytosine (C) in DNA. In some embodiments, the editor is a fusion protein containing an RNA-guided nickase fused to a cytidine deaminase domain. In some embodiments, the editor is a fusion protein containing an RNA-guided nickase fused to APOBEC3A deaminase (A3A). In some embodiments, the editor comprises a Cas9 nickase fused to APOBEC3A deaminase (A3A). In some embodiments, the editor is a fusion protein comprising an enzyme-inactive RNA-guided DNA-binding protein fused to a cytidine deaminase domain.

As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to a guide RNA along with an RNA-guided DNA binder, such as a Cas nuclease, such as a Cas lyase, a Cas nickase, or a dCas DNA binder (e.g., Cas9). In some embodiments, the guide RNA guides the RNA-guided DNA binder (e.g., Cas9) to a target sequence, and the guide RNA hybridizes with the target sequence and the binder binds to the target sequence; in the case of the binder being a lyase or a nickase, cleavage or nicking can occur after binding.

As used herein, the terms "uracil glycosidase inhibitor," "uracil-DNA glycosidase inhibitor," or "UGI" refer to proteins that can inhibit uracil-DNA glycosidase (UDG) base excision repair enzymes (e.g., UniPROT ID: P14739; MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSKRTADGSEFESPKKKRKVE (SEQ ID NO: 226); or TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO: 227)).

The following provides exemplary nucleotide and polypeptide sequences of Cas9 molecules. Methods for identifying alternative nucleotide sequences (including alternative naturally occurring variants) encoding Cas9 polypeptide sequences are known in this art. Sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any Cas9 nucleic acid sequence or amino acid sequence provided herein are also considered. In some embodiments, the nucleotide sequence encoding the 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 some embodiments, the Cas9 amino acid sequence is not a naturally occurring Cas9 sequence.

Spy Exemplary open reading frame of Cas9 AUGGACAAGAAGUACUCCAUCGGCCUGGACAUCGGCACCAACUCCGUGGGCUGGGCCGUGAUCACCGACGAGUACAAGGUGCCCUCCAAGAAGUUCAAGGUGCUGGGCAACACCGA CCGGCACUCCAUCAAGAAGAACCUGAUCGGCGCCCUGCUGUUCGACUCCGGCGAGACCGCCGAGGCCACCCGGCUGAAGCGGACCGCCCGCCGGCGGUACACCCGGCGGAAGAACCGGAUCUGCUACCUG CAGGAGAUCUUCUCCAACGAGAUGGCCAAGGUGGACGACUCCUUCUUCCACCGGCUGGAGGAGUCCUUCCUGGUGGAGGAGGACAAGAAGCACGAGCGGCACCCAUCUUCGGCAACAUCGUGGACGAGG UGGCCUACCACGAGAAGUACCCACCAUCUACCACCUGCGGAAGAAGCUGGUGGACUCCACCGACAAGGCCGACCUGCGGCUGAUCUACCUGGCCCUGGCCCACAUGAUCAAGUUCCGGGGCCACUUCCU GAUCGAGGGCGACCUGAACCCCGACAACUCCGACGUGGACAAGCUGUUCAUCCAGCUGGUGCAGACCUACAACCAGCUGUUCGAGGAGAACCCCAUCAACGCCUCCGGCGUGGACGCCAAGGCCAUCCU GUCCGCCCGCCUGUCCAAGUCCCGGCGGCUGGAGAACCUGAUCGCCCAGCUGCCCGGCGAGAAGAAGAACGGCCUGUUCGGCAACCUGAUCGCCUGUCCCUGGGCCUGACCCCCAACUUCAAGUCCAAC UUCGACCUGGCCGAGGACGCCAAGCUGCAGCUGUCCAAGGACACCUACGACGACGACCUGGACAACCUGCUGGCCCAGAUCGGCGACCAGUACGCCGACCUGUUCCUGGCCGCCAAGAACCUGUCCGACG CCAUCCUGCUGUCCGACAUCCUGCGGGUGAACACCGAGAUCACCAAGGCCCCCCUGUCCGCCUCCAUGAUCAAGCGGUACGACGAGCACCACCAGGACCUGACCCUGCUGAAGGCCCUGGUGCGGCAGCA GCUGCCCGAGAAGUACAAGGAGAUCUUCUUCGACCAGUCCAAGAACGGCUACGCCGGCUACAUCGACGGCGGCGCCUCCCAGGAGGAGUUCUACAAGUUCAUCAAGCCCAUCCUGGAGAAGAUGGACGG CACCGAGGAGCUGCUGGUGAAGCUGAACCGGGAGGACCUGCUGCGGAAGCAGCGGACCUUCGACAACGGGCUCCAUCCCCCACCAGAUCCACCUGGGCGAGCUGCACGCCAUCCUGCGGCGGCAGGAGGAC UUCUACCCCUUCCUGAAGGACAACCGGGAGAAGAUCGAGAAGAUCCUGACCUUCCGGAUCCCCUACUACGUGGGCCCCCUGGCCCGGGGCAACUCCCGGUUCGCCUGGAUGACCCGGAAGUCCGAGGAGA CCAUCACCCCCUGGAACUUCGAGGAGGUGGUGGACAAGGGCGCCCCGCCCAGUCCUUCAUCGAGCGGAUGACCAACUUCGACAAGAACCUGCCCAACGAGAAGGUGCUGCCCAAGCACUCCCUGCUGUA CGAGUACUUCACCGUGUACAACGAGCUGACCAAGGUGAAAGUACGUGACCGAGGGCAUGCGGAAGCCCGCCUUCCUGUCCGGCGAGCAGAAGAAGGCCAUCGUGGACCUGCUGUUCAAGACCAACCGGAA GGUGACCGUGAAGCAGCUGAAGGAGGACUACUUCAAGAAGAUCGAGUGCUUCGACUCCGUGGAGAUCUCCGGCGUGGAGGACCGGUUCAACGCCUCCCUGGGCACCUACCACGACCUGCUGAAGAUCAUC AAGGACAAGGACUUCCUGGACAACGAGGAGAACGAGGACAUCCUGGAGGACAUCGUGCUGACCCUGACCCUGUUCGAGGACCGGGAGAUGAUCGAGGAGCGGCUGAAGACCUACGCCCACCUGUUCGACG ACAAGGUGAUGAAGCAGCUGAAGCGGCGGCGGUACACCGGCUGGGGCCGGCUGUCCCGGAAGCUGAUCAACGGCAUCCGGGACAAGCAGUCCGGCAAGACCAUCCUGGACUUCCUGAAGUCCGACGGCUU CGCCAACCGGAACUUCAUGCAGCUGAUCCACGACGACUCCCUGACCUUCAAGGAGGACAUCCAGAAGGCCCAGGUGUCCGGCCAGGGCGACUCCCUGCACGAGCACAUCGCCAACCUGGCCGGCUCCCC CGCCAUCAAGAAGGGCAUCCUGCAGACCGUGAAGGUGGUGGACGAGCUGGUGAAGGUGAUGGGCCGGCACAAGCCCGAGAACAUCGUGAUCGAGAUGGCCCGGGAGAACCAGACCACCCAGAAGGGCCAG AAGAACUCCCGGGAGCGGAUGAAGCGGAUCGAGGAGGGCAUCAAGGAGCUGGGCUCCCAGAUCCUGAAGGAGCACCCCGUGGAGAACACCCAGCUGCAGAACGAGAAGCUGUACCUGUACUACCUGCAGA ACGGCCCGGGACAUGUACGUGGACCAGGAGCUGGACAUCAACCGGCUGUCCGACUACGACGUGGACCACAUCGUGCCCCAGUCCUUCCUGAAGGACGACUCCAUCGACAACAAGGUGCUGACCCGGUCCGA CAAGAACCGGGGCAAGUCCGACAACGUGCCCUCCGAGGAGGUGGUGAAGAAGAUGAAGAACUACUGGCGGCAGCUGCUGAACGCCAAGCUGAUCACCCAGCGGAAGUUCGACAACCUGACCAAGGCCGA GCGGGGCGGCCUGUCCGAGCUGGACAAGGCCGGCUUCAUCAAGCGGCAGCUGGUGGAGACCCGGCAGAUCACCAAGCACGUGGCCCAGAUCCUGGACUCCCGGAUGAACACCAAGUACGACGAGAACGAC AAGCUGAUCCGGGAGGUGAAGGUGAUCACCCUGAAGUCCAAGCUGGUGUCCGACUUCCGGAAGGACUUCCAGUUCUACAAGGUGCGGGAGAUCAACAACUACCACCACGCCCACGACGCCUACCUGAACG CCGUGGUGGGCACCGCCCUGAUCAAGAAGUACCCCAAGCUGGAGUCCGAGUUCGUGUACGGCGACUACAAGGUGUACGACGUGCGGAAGAUGAUCGCCAAGUCCGAGCAGGAGAUCGGCAAGGCCACCGC CAAGUACUUCUUCUACUCCAACAUCAUGAACUUCUUCAAGACCGAGAUCACCCUGGCCAACGGCGAGAUCCGGAAGCGGCCCCUGAUCGAGACCAACGGCGAGACCGGCGAGAUCGUGUGGGACAAGGG CCGGGACUUCGCCACCGUGCGGAAGGUGCUGUCCAUGCCCCAGGUGAACAUCGUGAAGAAGACCGAGGUGCAGACCGGCGGCUUCUCCAAGGAGUCCAUCCUGCCCAAGCGGAACUCCGACAAGCUGAUC GCCCGGAAGAAGGACUGGGACCCCAAGAAGUACGGCGGCUUCGACUCCCCCACCGUGGCCUACUCCGUGCUGGUGGUGGCCAAGGUGGAGAAGGGCAAGUCCAAGAAGCUGAAGUCCGUGAAGGAGCUGC UGGGCAUCACCAUCAUGGAGCGGUCCUCCUUCGAGAAGAACCCCAUCGACUUCCUGGAGGCCAAGGGCUACAAGGAGGUGAAGAAGGACCUGAUCAUCAAGCUGCCCAAGUACUCCCUGUUCGAGCUGGA GAACGGCCGGAAGCGGAUGCUGGCCUCCGCCGGCGAGCUGCAGAAGGGCAACGAGCUGGCCCUGCCCUCCAAGUACGUGAACUUCCUGUACCUGGCCUCCCACUACGAGAAGCUGAAGGGCUCCCCCGAG GACAACGAGCAGAAGCAGCUGUUCGUGGAGCAGCACAAGCACUACCUGGACGAGAUCAUCGAGCAGAUCUCCGAGUUCCAAGCGGGUGAUCCUGGCCGACGCCAACCUGGACAAGGUGCUGUCCGCCU ACAACAAGCACCGGGACAAGCCCAUCCGGGAGCAGGCCGAGAACAUCAUCCACCUGUUCACCCUGACCAACCUGGGCGCCCCCGCCGCCUUCAAGUACUUCGACACCACCAUCGACCGGAAGCGGUACAC CUCCACCAAGGAGGUGCUGGACGCCACCCUGAUCCACCAGUCCAUCACCGGCCUGUACGAGACCCGGAUCGACCUGUCCCAGCUGGGCGGCGACGGCGGCGGCUCCCCCAAGAAGAAGCGGAAGGUGUGA (SEQ ID NO: 218)

Spy Exemplary amino acid sequence of Cas9 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHM IKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLL KALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFL KSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGGSPKKKRKV (SEQ ID NO: 219)

Spy Exemplary open reading frame of Cas9 AUGGACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCCCGGGAAACACAGA CAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCGACGAAG UCGCAUACCACGAAAAGUACCCGACAAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCU GAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCU GAGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAAC UUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACG CAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCA GCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGG AACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGAC UUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAA CAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCUGCUGUA CGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAA GGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUC AAGGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACG ACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGAGCGACGGAUU CGCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCC GGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAG AAGAACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGA ACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGA CAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGA GAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACGAAAACGAC AAGCUGAUCAGAGAAGUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACG CAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGC AAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGG AAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUC GCAAGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGC UGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGA AAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAA GACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCAU ACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACAC AAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGUCUAG (SEQ ID NO: 116)

Spy with Hibit tag Exemplary open reading frame of Cas9 AUGGACAAGAAGUACUCCAUCGGCCUGGACAUCGGCACCAACUCCGUGGGCUGGGCCGUGAUCACCGACGAGUACAAGGUGCCCUCCAAGAAGUUCAAGGUGCUGGGCAACACCGACC GGCACUCCAUCAAGAAGAACCUGAUCGGCGCCCUGCUGUGUUCGACUCCGGCGAGACCGCCGAGGCCACCCGGCUGAAGCGGACCGCCCGCCGGCGGUACACCCGGCGGAAGAACCGGAUCUGCUACCUGCAGG AGAUCUUCUCCAACGAGAUGGCCAAGGUGGACGACUCCUUCUUCCACCGGCUGGAGGAGUCCUUCCUGGUGGAGGAGGACAAGAAGCACGAGCGGCACCCAUCUUCGGCAACAUCGUGGACGAGGUGGCC UACCACGAGAAGUACCCCACCAUCUACCACCUGCGGAAGAAGCUGGUGGACUCCACCGACAAGGCCGACCUGCGGCUGAUCUACCUGGCCCUGGCCCACAUGAUCAAGUUCCGGGGCCACUUCCUGAUCGAG GGCGACCUGAACCCCGACAACUCCGACGUGGACAAGCUGUUCAUCCAGCUGGUGCAGACCUACAACCAGCUGUUCGAGGAGAACCCCAUCAACGCCUCCGGCGUGGACGCCAAGGCCAUCCUGUCCGCCCG GCUGUCCAAGUCCCGGCGGCUGGAGAACCUGAUCGCCCAGCUGCCCGGCGAGAAGAAGAACGGCCUGUUCGGCAACCUGAUCGCCCUGUCCCUGGGCCUGACCCCCAACUUCAAGUCCAACUUCGACCUGGC CGAGGACGCCAAGCUGCAGCUGUCCAAGGACACCUACGACGACGACCUGGACAACCUGCUGGCCCAGAUCGGCGACCAGUACGCCGACCUGUUCCUGGCCGCCAAGAACCUGUCCGACGCCAUCCUGCUGU CCGACAUCCUGCGGGUGAACACCGAGAUCACCAAGGCCCCCCUGUCCGCCUCCAUGAUCAAGCGGUACGACGAGCACCACCAGGACCUGACCCUGCUGAAGGCCCUGGUGCGGCAGCAGCCGCCCGAGAAGU ACAAGGAGAUCUUCUUCGACCAGUCCAAGAACGGCUACGCCGGCUACAUCGACGGCGGCGCCUCCCAGGAGGAGUUCUACAAGUUCAUCAAGCCCAUCCUGGAGAAGAUGGACGGCACCGAGGAGCUGCUG GUGAAGCUGAACCGGGAGGACCUGCUGCGGAAGCAGCGGACCUUCGACAACGGGCUCCAUCCCCCACCAGAUCCACCUGGGCGAGCUGCACGCCAUCCUGCGGCGGCAGGAGGACUUCUACCCCUUCCUGAAG GACAACCGGGAGAAGAUCGAGAAGAUCCUGACCUUCCGGAUCCCCUACUACGUGGGCCCCCUGGCCCGGGGCAACUCCCGGUUCGCCUGGAUGACCCGGAAGUCCGAGGAGACCAUCACCCCCUGGAACUU CGAGGAGGUGGUGGACAAGGGCGCCUCCGCCCAGUCCUUCAUCGAGCGGAUGACCAACUUCGACAAGAACCUGCCCAACGAGAAGGUGCUGCCCAAGCACUCCCUGCUGUACGAGUACUUCACCGUGUACAA CGAGCUGACCAAGGUGAAAGUACGUGACCGAGGGCAUGCGGAAGCCCGCCUUCCUGUCCGGCGAGCAGAAGAAGGCCAUCGUGGACCUGCUGUUCAAGACCAACCGGAAGGUGACCGUGAAGCAGCUGAAGG AGGACUACUUCAAGAAGAUCGAGUGCUUCGACUCCGUGGAGAUCUCCGGCGUGGAGGACCGGUUCAACGCCUCCCUGGGCACCUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACG AGGAGAACGAGGACAUCCUGGAGGACAUCGUGCUGACCCUGACCCUGUUCGAGGACCGGGAGAUGAUCGAGGAGCGGCUGAAGACCUACGCCCACCUGUUCGACGACAAGGUGAUGAAGCAGCUGAAGCGGC GGCGGUACACCGGCUGGGGCCGGCUGUGUCCCGGAAGCUGAUCAACGGCAUCCGGGACAAGCAGUCCGGCAAGACCAUCCUGGACUUCCUGAAGUCCGACGGCUUCGCCAACCGGAACUUCAUGCAGCUGAUCC ACGACGACUCCCUGACCUUCAAGGAGGACAUCCAGAAGGCCCAGGUGUCCGGCCAGGGCGACUCCCUGCACGAGCACAUCGCCAACCUGGCCGGCUCCCCGCCAUCAAGAAGGGCAUCCUGCAGACCGUG AAGGUGGUGGACGAGCUGGUGAAGGUGAUGGGCCGGCACAAGCCCGAGAACAUCGUGAUCGAGAUGGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAACUCCCGGGAGCGGAUGAAGCGGAUCGAG GAGGGCAUCAAGGAGCUGGGCUCCCAGAUCCUGAAGGAGCACCCCGUGGAGAACACCCAGCUGCAGAACGAGAAGCUGUACCUGUACUACCUGCAGAACGGCCGGGACAUGUACGUGGACCAGGAGCUGGA CAUCAACCGGCUGUCCGACUACGACGUGGACCACAUCGUGCCCCAGUCCUUCCUGAAGGACGACUCCAUCGACAACAAGGUGCUGACCCGGUCCGACAAGAACCGGGGCAAGUCCGACAACGUGCCCUCCGA GGAGGUGGUGAAGAAGAUGAAGAACUACUGGCGGCAGCUGCUGAACGCCAAGCUGAUCACCCAGCGGAAGUUCGACAACCUGACCAAGGCCGAGCGGGGCGGCCUGUCCGAGCUGGACAAGGCCGGCUUCA UCAAGCGGCAGCUGGUGGAGACCCGGCAGAUCACCAAGCACGUGGCCCAGAUCCUGGACUCCCGGAUGAACACCAAGUACGACGAGAACGACAAGCUGAUCCGGGAGGUGAAGGUGAUCACCCUGAAGUCCA AGCUGGUGUCCGACUUCCGGAAGGACUUCCAGUUCUACAAGGUGCGGGAGAUCAACAACUACCACCACGCCCACGACGCCUACCUGAACGCCGUGGUGGGCACCGCCCUGAUCAAGAAGUACCCCAAGCUG GAGUCCGAGUUCGUGUACGGCGACUACAAGGUGUACGACGUGCGGAAGAUGAUCGCCAAGUCCGAGCAGGAGAUCGGCAAGGCCACCGCCAAGUACUUCUUACUCCAACAUCAUGAACUUCUUCAAGACC GAGAUCACCCUGGCCAACGGCGAGAUCCGGAAGCGGCCCCUGAUCGAGACCAACGGCGAGACCGGCGAGAUCGUGUGGGACAAGGGCCGGGACUUCGCCACCGUGCGGAAGGUGCUGUCCAUGCCCCAGGU GAACAUCGUGAAGAAGACCGAGGUGCAGACCGGCGGCUUCUCCAAGGAGUCCAUCCUGCCCAAGCGGAACUCCGACAAGCUGAUCGCCCGGAAGAAGGACUGGGACCCCAAGAAGUACGGCGGCUUCGACUC CCCCACCGUGGCCUACUCCGUGCUGGUGGUGGCCAAGGUGGAGAAGGGCAAGUCCAAGAAGCUGAAGUCCGUGAAGGAGCUGCUGGGCAUCACCAUCAUGGAGCGGUCCUCCUUCGAGAAGAACCCCAUCG ACUUCCUGGAGGCCAAGGGCUACAAGGAGGUGAAGAAGGACCUGAUCAUCAAGCUGCCCAAGUACUCCCUGUUCGAGCUGGAGAACGGCCGGAAGCGGAUGCUGGCCUCCGCCGGCGAGCUGCAGAAGGGCA ACGAGCUGGCCCUGCCCUCCAAGUACGUGAACUUCCUGUACCUGGCCUCCCACUACGAGAAGCUGAAGGGCUCCCCCGAGGACAACGAGCAGAAGCAGCUGUUCGUGGAGCAGCACAAGCACUACCUGGAC GAGAUCAUCGAGCAGAUCUCCGAGUUCUCCAAGCGGGUGAUCCUGGCCGACGCCAACCUGGACAAGGUGCUGUCCGCCUACAACAAGCACCGGGACAAGCCCAUCCGGGAGCAGGCCGAGAACAUCAUCCAC CUGUUCACCCUGACCAACCUGGGCGCCCCCGCCGCCUUCAAGUACUUCGACACCACCAUCGACCGGAAGCGGUACACCUCCACCAAGGAGGUGCUGGACGCCACCCUGAUCCACCAGUCCAUCACCGGCCUG UACGAGACCCGGAUCGACCUGUCCCAGCUGGGCGGCGACGGCGGCGGCUCCCCCAAGAAGAAGCGGAAGGUGUCCGAGUCCGCCACCCCCGAGUCCGUGUCCGGCUGGCGGCUGUUCAAGAAGAUCUCCUGA (SEQ ID NO: 117)

Spy with Hibit tag Exemplary amino acid sequence of Cas9 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALV RQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN FMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSD NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNF FKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQ KGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGGSPKKKRKVSESATPESVSGWRLFKKIS (SEQ ID NO: 118)

Spy Exemplary amino acid sequence of Cas9 nickase MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKL VDSTDKADLRLIYLALAHMIKFRGHFLIEGDLTGGGGSVDVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQ EDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVV DELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK AERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP LIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGGSPKKKRKV (SEQ ID NO: 220)

Exemplary open reading frame of Nme2Cas9 atgGACGGCTCCGGCGGCGGCTCCCCCAAGAAGAAGCGGAAGGTGGAGGACAAGCGGCCCGCCGCCACCAAGAAGGCCGGCCAGGCCAAGAAGAAGAAGGGCGGCTCCGGCGGCGGCgccgccttcaagcccaaccccatcaactacatcctgggcctggacatcggcatcgcctccgtgggctgggccatggt ggagatcgacgaggaggagaaccccatccggctgatcgacctgggcgtgcgggtgttcgagcgggccgaggtgcccaagaccggcgactccctggccatggcccg gcggctggcccggtccgtgcggcggctgacccggcggcgggcccaccggctgctgcgggcccggcggctgctgaagcggggagggcgtgctgcaggccgccgacttc gacgagaacggcctgatcaagtccctgcccaacaccccctggcagctgcgggccgccgccctggaccggaagctgacccccctggagtggtccgccgtgctgctg cacctgatcaagcaccggggctacctgtcccagcggaagaacgagggcgagaccgccgacaaggagctgggcgccctgctgaagggcgtggccaacaacgcccacg ccctgcagaccggcgacttccggacccccgccgagctggccctgaacaagttcgagaaggagtccggccacatccggaaccagcggggcgactactcccacacctt ctcccggaaggacctgcaggccgagctgatcctgctgttcgagaagcagaaggagttcggcaacccccacgtgtccggcggcctgaaggagggcatcgagaccctg ctgatgacccagcggcccgccctgtccggcgacgccgtgcagaagatgctgggccactgcaccttcgagcccgccgagcccaaggccgccaagaacacctacacc gccgagcggttcatctggctgaccaagctgaacaacctgcggatcctggagcagggctccgagcggcccctgaccgacaccgagcgggccaccctgatggacgagc cctaccggaagtccaagctgacctacgcccaggcccggaagctgctgggcctggaggacaccgccttcttcaagggcctgcggtacggcaaggacaacgccgagg cctccaccctgatggagatgaaggcctaccacgccatctcccgggccctggagaaggagggcctgaaggacaagaagtcccccctgaacctgtcctccgagctgca ggacgagatcggcaccgccttctccctgttcaagaccgacgaggacatcaccggccggctgaaggaccgggtgcagcccgagatcctggaggccctgctgaagca catctccttcgacaagttcgtgcagatctccctgaaggccctgcggcggatcgtgcccctgatggagcagggcaagcggtacgacgaggcctgcgccgagatctac ggcgaccactacggcaagaagaacaccgaggagaagatctacctgccccccatccccgccgacgagatccggaaccccgtggtgctgcgggccctgtcccaggccc ggaaggtgatcaacggcgtggtgcggcggtacggctcccccgcccggatccacatcgagaccgcccgggaggtgggcaagtccttcaaggaccggaaggagatcga gaagcggcaggaggagaaccggaaggaccgggagaaggccgccgccaagttccgggagtacttccccaacttcgtgggcgagcccaagtccaaggacatcctgaa gctgcggctgtacgagcagcagcacggcaagtgcctgtactccggcaaggagatcaacctggtgcggctgaacgagaagggctacgtggagatcgaccacgccctg cccttctcccggacctgggacgactccttcaacaacaaggtgctggtgctgggctccgagaaccagaacaagggcaaccagaccccctacgagtacttcaacggc aaggacaactcccgggagtggcaggagttcaaggcccgggtggagacctcccggttcccccggtccaagaagcagcggatcctgctgcagaagttcgacgaggacg gcttcaaggagtgcaacctgaacgacacccggtacgtgaaccggttcctgtgccagttcgtggccgaccacatcctgctgaccggcaagggcaagcggcgggtgt tcgcctccaacggccagatcaccaacctgctgcggggcttctggggcctgcggaaggtgcgggccgagaacgaccggcaccacgccctggacgccgtggtggtggc ctgctccaccgtggccatgcagcagaagatcacccggttcgtgcggtacaaggagatgaacgccttcgacggcaagaccatcgacaaggagaccggcaaggtgctg caccagaagacccacttcccccagccctgggagttcttcgcccaggaggtgatgatccgggtgttcggcaagcccgacggcaagcccgagttcgaggaggccgaca cccccgagaagctgcggaccctgctggccgagaagctgtcctcccggcccgaggccgtgcacgagtacgtgacccccctgttcgtgtcccgggcccccaaccgga agatgtccggcgcccacaaggacaccctgcggtccgccaagcggttcgtgaagcacaacgagaagatctccgtgaagcgggtgtggctgaccgagatcaagctggc cgacctggagaacatggtgaactacaagaacggccggggagatcgagctgtacgaggccctgaaggcccggctggaggcctacggcggcaacgccaagcaggcctt cgaccccaaggacaaccccttctacaagaagggcggccagctggtgaaggccgtgcgggtggagaagacccaggagtccggcgtgctgctgaacaagaagaacgcc tacaccatcgccgacaacggcgacatggtgcgggtggacgtgttctgcaaggtggacaagaagggcaagaaccagtacttcatcgtgcccatctacgcctggcag gtggccgagaacatcctgcccgacatcgactgcaagggctaccggatcgacgactcctacaccttctgcttctccctgcacaagtacgacctgatcgccttccaga aggacgagaagtccaaggtggagttcgcctactacatcaactgcgactcctccaacggccggttctacctggcctggcacgacaagggctccaaggagcagcagtt ccggatctccacccagaacctggtgctgatccagaagtaccaggtgaacgagctgggcaaggagatccggccctgccggctgaagaagcggccccccgtgcggtag (SEQ ID NO: 221)

Exemplary mRNAGGGAAGCUCAGAAUAAACGCUCAACUUUGGCCGGAUCUGCCACCAUGGACCGGCUCCGGCGGCGGCUCCCCCAAGAAGAAGCGGAAGGUGGAGGACAAGCGGCCC for Nme2Cas9 nickase GCCGCCACCAAGAAGGCCGGCCAGGCCAAGAAGAAGAAGGGCGGCUCCGGCGGCGGCGCCGCCUUCAAGCCCAACCCCAUCAACUACAUCCUGGGCCUGGACAUCGGCAUCGCCUCCGUGGGC UGGGCCAUGGUGGAGAUCGACGAGGAGGAGAACCCCAUCCGGCUGAUCGACCUGGGCGUGCGGGUGUUCGAGCGGGCCGAGGUGCCCAAGACCGGCGACUCCCUGGCCAUGGCCCGGCGGCUG GCCCGGUCCGUGCGGCGGCUGACCCGGCGGCGGGCCCACCGGCUGCUGCGGGCCCGGCGGCUGCUGAAGCGGGAGGGCGUGCUGCAGGCCGCCGACUUCGACGAGAACGGCCUGAUCAAGUCC CUGCCACACACCCCCUGGCAGCUGCGGCCGCCGCCCUGGACCGGAAGCUGACCCCCCUGGAGUGGUCCGCCGUGCUGCUGCACCUGAUCAAGCACCGGGGCUACCUGUCCCAGCGGAAGAACGAGGGCGAGACCGCCGACAAGGAGCUGGGCGCCCUGCUGAAGGGCGUGGCCAACAACGCCCACGCCCUGCAGACCGGCGACUUCCGGACCCCCGCCGAGCUGGCCCUGAACAAGUUCGAGAAG GAGUCCGGCCACAUCCGGAACCAGCGGGGCGACUACUCCCACACCUUCUCCCGGAAGGACCUGCAGGCCGAGCUGAUCCUGCUGUUCGAGAAGCAGAAGGAGUUCGGCAACCCCCACGUGUCC GGCGGCCUGAAGGAGGGCAUCGAGACCCUGCUGAUGACCCAGCGGCCCGCCCUGUCCGGCGACGCCGUGCAGAAGAUGCUGGGCCACUGCACCUUCGAGCCCGCCGAGCCCAAGGCCGCCAAGA ACACCUACACCGCCGAGCGGUUCAUCUGGCUGACCAAGCUGAACAACCUGCGGAUCCUGGAGCAGGGCUCCGAGCGGCCCUGACCGACACCGAGCGGGCCACCCUGAUGGACGAGCCCUACC GGAAGUCCAAGCUGACCUACGCCCAGGCCCGGAAGCUGCUGGGCCUGGAGGACACCGCCUUCUUCAAGGGCCUGCGGUACGGCAAGGACAACGCCGAGGCCUCCACCCUGAUGGAGAUGAAGG CCUACCACGCCAUCUCCCGGGCCCUGGAGAAGGAGGGCCUGAAGGACAAGAAGUCCCCCUGAACCUGUCCUCCGAGCUGCAGGACGAGAUCGGCACCGCCUUCUCCCUGUUCAAGACCGACG AGGACAUCACCGGCCGGCUGAAGGACCGGGUGCAGCCCGAGAUCCUGGAGGCCCUGCUGAAGCACAUCUCCUUCGACAAGUUCGUGCAGAUCUCCCUGAAGGCCCUGCGGCGGAUCGUGCCCC UGAUGGAGCAGGGCAAGCGGUACGACGAGGCCUGCGCCGAGAUCUACGGCGACCACUACGGCAAGAAGAACACCGAGGAGAAGAUCUACCUGCCCCCCAUCCCCGCCGACGAGAUCCGGAACCCCGUGGUGCUGCGGGCCCUGUCCCAGGCCCGGAAGGUGAUCAACGGCGUGGUGCGGCGGUACGGCUCCCCCGCCCGGAUCCACAUCGAGACCGCCCGGGAGGUGGGCAAGUCCUUCAAGGACC GGAAGGAGAUCGAGAAGCGGCAGGAGGAGAACCGGAAGGACCGGGAGAAGGCCGCCGCCAAGUUCCGGGAGUACUUCCCCAACUUCGUGGGCGAGCCCAAGUCCAAGGACAUCCUGAAGCUGC GGCUGUACGAGCAGCAGCACGGCAAGUGCCUGUACUCCGGCAAGGAGAUCAACCUGGUGCGGCUGAACGAGAAGGGCUACGUGGAGAUCGACCACGCCCUGCCCUUCUCCCGGACCUGGGACGA CUCCUUCAACAACAAGGUGCUGGUGCUGGGCUCCGAGAACCAGAACAAGGGCAACCAGACCCCCUACGAGUACUUCAACGGCAAGGACAACUCCCGGGAGUGGCAGGAGUUCAAGGCCCGGGU GGAGACCUCCCGGUUCCCCGUCCAAGAAGCAGCGGAUCCUGCUGCAGAAGUUCGACGAGGACGGCUUCAAGGAGUGCAACCUGAACGACACCCGGUACGUGAACCGGUUCCUGUGCCAGUU CGUGGCCGACCACAUCCUGCUGACCGGCAAGGGCAAGCGGCGGUGUUCGCCUCCAACGGCCAGAUCACCAACCUGCUGCGGGGCUUCUGGGGCCUGCGGAAGGUGCGGGCCGAGAACGACCG GCACCACGCCCUGGACGCCGUGGUGGUGGCCUGCUCCACCGUGGCCAUGCAGCAGAAGAUCACCCGGUUCGUGCGGUACAAGGAGAUGAACGCCUUCGACGGCAAGACCAUCGACAAGGAGAC CGGCAAGGUGCUGCACCAGAAGACCCACUUCCCCCAGCCCUGGGAGUUCUUCGCCCAGGAGGUGAUGAUCCGGGUGUUCGGCAAGCCCGACGGCAAGCCCGAGUUCGAGGAGGCCGACACCCCCGAGAAGCUGCGGACCCUGCUGCCGAAAGCUGUCCUCCCGGCCCGAGGCCGUGCACGAGUACGUGACCCCCCUGUUCGUGUCCCGGGCCCCCAACCGGAAGAUGUCCGGCGCCCACAAGGA CACCCUGCGGUCCGCCAAGCGGUUCGUGAAGCACAACGAGAAGAUCUCCGUGAAGCGGGUGUGGCUGACCGAGAUCAAGCUGGCCGACCUGGAGAACAUGGUGAACUACAAGAACGGCCGGGA GAUCGAGCUGUACGAGGCCCUGAAGGCCCGGCUGGAGGCCUACGGCGGCAACGCCAAGCAGGCCUUCGACCCCAAGGACAACCCCUUCUACAAGAAGGGCGGCCAGCUGGUGAAGGCCGUGCGG GUGGAGAAGACCCAGGAGUCCGGCGUGCUGCUGAACAAGAAGAACGCCUACACCAUCGCCGACAACGGCGACAUGGUGCGGGUGGACGUGUUCUGCAAGGUGGACAAGAAGGGCAAGAACCAG UACUUCAUCGUGCCCAUCUACGCCUGGCAGGUGGCCGAGAACAUCCUGCCCGACAUCGACUGCAAGGGCUACCGGAUCGACGACUCCUACACCUUCUGCUUCUCCCUGCACAAGUACGACCUG AUCGCCUUCCAGAAGGACGAGAAGUCCAAGGUGGAGUUCGCCACUACAUCAACUGCGACUCCUCCAACGGCCGGUUCUACCUGGCCUGGCACGACAAGGGCUCCAAGGAGCAGCAGUUCCGG AUCUCCACCCAGAACCUGGUGCUGAUCCAGAAGUACCAGGUGAACGAGCUGGGCAAGGAGAUCCGGCCCUGCCGGCUGAAGAAGCGGCCCCCCGUGCGGUAGCUAGCACCAGCCUCAAGAACA CCCGAAUGGAGUCUAAGCUACAUAAUACCAACUUACACUUUACAAAAUGUUGUCCCCAAAAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAGAAAGUUUCUUCACAUUCUCUCGAGAAAAAAAAAAAAUGGAAAAAAAAAAAACGGAAAAAAAAAAAAGGUAAAAAAAAAAAAUAUAAAAAAAAACAUAAAAAAAAAAAACGAAAAAAAAACGUAAAAAAAAAAAAAAAAAA AAAAGAUAAAAAAAAAAAACCUAAAAAAAAAUGUAAAAAAAAAAAAGGGAAAAAAAAAAAACGCAAAAAAAAAAAACACAAAAAAAAAUGCAAAAAAAAAUCGAAAAAAAAAAUCUAAAAAAAAACGAAAAAAAAAAAACCCAAAAAAAAAAAAGACAAAAAAAAAAAAUAGAAAAAAAAAAGUAAAAAAAAAAAACUGAAAAAAAAAUUUAAAAAAAAAAAAUCUAG (SEQ ID NO: 222)

Exemplary amino acid sequence of Nme2Cas9 MDGSGGGSSPKKKRKVEDKRPAATKKAGQAKKKKGGSGGGAAFKPNPINYILGLDIGIASVGWAMVEIDEEEENPIRLIDLGVRVFERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRARRLLK REGVLQAADFDENGLIKSLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVANNAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYSHTFSRKDLQAELILLFEKQKEFGNPHVSG GLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRALEKEGLKDKKSP LNLSSELQDEIGTAFSLFKTDEDITGRLKDRVQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTEEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSF KDRKEIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLVRLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQRILLQ KFDEDGFKECNLNDTRYVNRFLCQFVADHILLTGKGKRRVFASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFDGKTIDKETGKVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEF EEADTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGAHKDTLRSAKRFVKHNEKISVKRVWLTEIKLADLENMVNYKNGREIELYEALKARLEAYGGNAKQAFDPKDNPFYKKGGQLVKAVRVEKTQESGVLLNK KNAYTIADNGDMVRVDVFCKVDKKGKNQYFIVPIYAWQVAENILPDIDCKGYRIDDSYTFCFSLHKYDLIAFQKDEKSKVEFAYYINCDSSNGRFYLAWHDKGSKEQQFRISTQNLVLIQKYQVNELGKEIRPCRLKKRPPVR (SEQ ID NO: 223)

Exemplary mRNA encoding UGI GGGAGACCCAAGCUGGCUAGCUCCCGCAGUCGGCGUCCAGCGGCUCUGCUUGUUCGUGUGUGUGUCGUUGCAGGCCUUAUUCGGAUCCGCCACCAUGGGACCGAAGAAGAAGAGAAAGGUCGGAGGAGGAAGCACAA ACCUGUCGGACAUCAUCGAAAAGGAAACAGGAAAGCAGCUGGUCAUCCAGGAAUCGAUCCUGAUGCUGCCGGAAGAAGUCGAAGAAGUCAUCGGAAACAAGCCGGAAUCGGGACAUCCUGGUCCACACAGCAUACGACGAAUCGACAGACGA AAACGUCAUGCUGCUGACAUCGGACGCACCGGAAUACAAGCCGUGGGCACUGGUCAUCCAGGACUCGAACGGAGAAAACAAGAUCAAGAUGCUGUGAUAGUCUAGACAUCACAUUUAAAAGCAUCUCAGCCUACCAUGAGAAUAAGAGAA AGAAAAUGAAGAUCAAUAGCUUAUUCAUCUCUUUUUCUUUUUCGUUGGUGUAAAGCCAACACCCUGUCUAAAAAACAUAAAUUUCUUUAAUCAUUUUGCCUCUUUUCUCUGUGCUUCAAUUAAUAAAAAAUGGAAAGAACCUCGAGUCUAG (SEQ ID NO: 224)

UGI open reading frame AUGGGACCGAAGAAGAAGAGAAAGGUCGGAGGAGGAAGCACAAACCUGUCGGACAUCAUCGAAAAGGAAACAGGAAAGCAGCUGGUCAUCCAGGAAUCGAUCCUGAUGCUGCCGGAAGAAGUCGAAGAAGUCAUCGGAAAC AAGCCGGAAUCGGACAUCCUGGUCCACACAGCAUACGACGAAUCGACAGACGAAAACGUCAUGCUGCUGACAUCGGACGCACCGGAAUACAAGCCGUGGGCACUGGUCAUCCAGGACUCGAACGGAGAAAACAAGAUCAAGAUGCUGUGA (SEQ ID NO: 225)

Example of UGI: Amino acid sequence MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSKRTADGSEFESPKKKRKVE (SEQ ID NO: 226)

Example of a UGI amino acid sequence: TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO: 227)

As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to a guide RNA along with an RNA-guided DNA binder, such as a Cas nuclease, such as a Cas lyase, Cas nickase, or a dCas DNA binder (e.g., Cas9). In some embodiments, the guide RNA guides the RNA-guided DNA binder (e.g., Cas9) to a target sequence, and the guide RNA hybridizes with the target sequence and the binder binds to the target sequence; in the case of the binder being a lyase or nickase, binding can result in double-stranded or single-stranded DNA cleavage.

As used herein, if an alignment of a first sequence with a second sequence shows that X% or more positions in the entire second sequence match the first sequence, then the first sequence is considered to "contain a sequence that has at least X% similarity to the second sequence". For example, the sequence AAGA contains a sequence that has 100% similarity to the sequence AAG, because the alignment will result in 100% similarity since it matches all three positions in the second sequence. Differences between RNA and DNA (usually replacing uridine with thymidine, or vice versa) and the presence of nucleoside analogues (such as modified uridine) do not cause differences in identity or complementarity in polynucleotides, as long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complementary sequence (e.g., all thymidine, uridine, or modified uridine are adenosine; another example is cytosine and 5-methylcytosine, both of which use guanosine or modified guanosine as complementary sequences). Therefore, 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 because both are completely complementary to the same sequence (5'-CAU). Exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well-known in this technique. Those familiar with this technique will understand which algorithm and parameter settings are suitable for the given sequence pairs to be aligned; for sequences that are generally of similar length and expected consistency (>50% for amino acids or >75% for nucleotides), the Needleman-Wunsch algorithm with its default interface provided by EBI on the www.ebi.ac.uk web server is generally appropriate.

As used herein, if X% of the bases in the first sequence pair with bases in the second sequence, then 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', and the second sequence is 50% complementary to the first sequence.

As used herein, "mRNA" refers to a polynucleotide that is entirely or primarily RNA or modified RNA and contains an open reading frame (i.e., a recipient for ribosomes and aminoglycosides of tRNA) that can be translated into polypeptides. mRNA may contain a phosphodiester-glycosidic backbone, including ribose residues or analogues thereof, such as 2'-methoxyribose residues. In some embodiments, the sugars in the mRNA phosphodiester-glycosidic backbone are substantially composed of ribose residues, 2'-methoxyribose residues, or combinations thereof.

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 a target nucleic acid. As used herein, when an insertion/deletion results in an insertion, the insertion is a random insertion at the double-strand break site that is not induced by or based on the template sequence.

As used herein, "target sequence" refers to a nucleic acid sequence in a target gene that is complementary to the guide sequence of the gRNA. The interaction between the target sequence and the guide sequence guides an RNA-guided DNA binder to bind within the target sequence and potentially cleave or break down the DNA (depending on the activity of the binder).

As used herein, "peptide" refers to a wild-type or variant protein (e.g., a mutant, fragment, fusion, or combination thereof). A variant peptide 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 peptide. In some embodiments, the sequence of the variant is at least 70%, 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to that of the wild-type peptide. In some embodiments, the variant peptide may be an overactive variant. In some cases, variants possess approximately 80% to approximately 120%, 140%, 160%, 180%, 200%, 300%, 400%, 500%, or more of the functional activity of the wild-type polypeptide. As used herein, a "heterogeneous gene" refers to a gene introduced into a cell as an exogenous source (e.g., inserted at a gene locus, such as a safe harbor locus, including the TCR locus). That is, the introduced gene is heterologous with respect to its insertion site. The polypeptide expressed by this heterogeneous gene is then called a "heterogeneous polypeptide." Heterogeneous genes can be naturally occurring or engineered, and can be wild-type or variants. Heterogeneous genes may include nucleotide sequences (e.g., internal ribosome entry sites) that differ from the sequence encoding the heterogeneous polypeptide. A heterologous gene can be a gene that is naturally present in the genome as a wild type or a variant (e.g., a mutant). For example, although a cell contains the gene of interest (as a wild type or as a variant), the same gene or a variant thereof can be introduced as a foreign source to express, for example, at a highly expressed locus. A heterologous gene can also be a gene that is not naturally present in the genome, or a gene that expresses a heterologous polypeptide that is not naturally present in the genome. The terms "heterologous gene," "exogenous gene," and "transgenic gene" are used interchangeably. In some embodiments, a heterologous gene or transgenic gene includes a foreign nucleic acid sequence, such as a nucleic acid sequence that is not endogenous to the recipient cell. In some embodiments, a heterologous gene or transgenic gene includes a foreign nucleic acid sequence, such as a nucleic acid sequence that is not naturally present in the recipient cell. For example, a heterologous gene can be heterologous in terms of its insertion site and in terms of its recipient cell.

"Safe harbor" loci are intragenomic loci where genes can be inserted without causing significant harmful effects on cells. Non-limiting examples of safe harbor loci targeted by nucleases for use herein include AAVS1 (PPP1 R12C), TCR, B2M, and any of the targeted knockdown loci described herein, such as TNFA, IFNG, IL17A, and IL6 genomic loci. In some embodiments, insertion at one or more targeted knockdown loci (such as TRC genes, e.g., TRAC genes) is advantageous for allogeneic cell lines. Other suitable safe harbor loci are known in this art.

II. Composition A. Targeting Receptor In some embodiments, engineered T cells comprising a heterologous sequence encoding a dmTGFB1 molecule and a modified (e.g., knockdown) endogenous nucleic acid sequence encoding TGFBR2 further comprising the insertion of a heterologous sequence encoding a targeting receptor into the cells, and, where applicable, further comprising a modified (e.g., knockdown) endogenous nucleic acid sequence encoding TNFA, a modified (e.g., knockdown) endogenous nucleic acid sequence encoding IFNG, and/or the insertion of a heterologous sequence encoding a regulatory T cell-promoting molecule into the cells, each under the control of a promoter sequence. The sequence encoding the targeting receptor is under the control of a promoter sequence (e.g., an endogenous promoter or a heterologous promoter). In some embodiments, the regulatory T cell-promoting molecule comprises IL10 or CTLA4. In some embodiments, the regulatory T-cell stimulating molecules include IL10 and CTLA4.

In some embodiments, the targeting receptor is a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a receptor of a cell surface molecule operatively linked via at least one transmembrane domain in an internal signaling domain that, upon binding to an extracellular receptor portion, can activate T cells. In some embodiments, the targeting receptor may be a receptor present on the surface of cells (e.g., T cells) to allow the cell to bind to a target site (e.g., a specific cell or tissue in an organism). The targeting receptor need not be an antigen receptor; for example, it may be an RGD peptide capable of targeting integrin. In some embodiments, the target receptor is selected from the group of molecules including: MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), protein lipoprotein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), stromal core protein 3 (DSG3), and MHC class I HLA-A (HLA-A*02).

In some embodiments, the engineered T cells include modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding TGFBR2; modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding TNFA; modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding IFNG; and insert sequences encoding the following: dmTGFB1, IL10, CTLA4, and targeting receptors (e.g., CARs targeting MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipoprotein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), and solute carrier family 2 members. (SCL2A2), glutamic acid decarboxylase (GAD2), stromal core protein 3 (DSG3) or MHC class I HLA-A (HLA-A*02) CAR).

In some embodiments, the sequence encoding the target receptor is incorporated into the phenotype. In some embodiments, the phenotype containing the sequence encoding the target receptor further includes a sequence encoding a regulatory T cytokinetic molecule; for example, the sequences encoding the target receptor and the regulatory T cytokinetic molecule are incorporated into the same phenotype. In some embodiments, the phenotype containing the sequence encoding the target receptor does not further include a sequence encoding a regulatory T cytokinetic molecule; for example, the sequence encoding a regulatory T cytokinetic molecule is incorporated into a separate phenotype. In some embodiments, the phenotype containing the sequence encoding the target receptor is a free phenotype. In some embodiments, the sequence encoding the target receptor is inserted into the genome, for example, through targeted or non-targeted insertion.

In some embodiments, the sequence encoding the target receptor may be inserted into a site selected from the following: 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.

In some embodiments, such as those evaluated by sequencing (e.g., NGS), engineered T cells contain sequences that encode target receptors through gene editing insertion.

In some embodiments, the T cell population comprises T cells engineered to include: an insert sequence encoding dmTGFB1; a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TGFBR2; 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; an insert sequence encoding a regulatory T cell-promoting molecule; and an insert sequence encoding a target receptor (e.g., CAR). In some embodiments, for example, as evaluated by sequencing (e.g., NGS), 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 an insert sequence encoding a target receptor. It should be understood that selection methods known in this art can be used to enrich T cell populations from cell populations with targeted receptors.

In some embodiments, this document discloses engineered T cells by introducing or inserting the target receptor (e.g., CAR) nucleic acid into T cells (e.g., within the genome locus of a T cell or T cell population) using, for example, a guide RNA and an RNA-guided DNA binder and a construct (e.g., a donor) containing a target receptor (e.g., CAR) nucleic acid. In some embodiments, this document discloses engineered T cells by expressing the target receptor (e.g., CAR) at the genome locus of a T cell or T cell population, for example, using a guide RNA and an RNA-guided DNA binder and a construct (e.g., a donor) containing a target receptor (e.g., CAR) nucleic acid. In some embodiments, this document discloses engineered T cells by inducing intragenomic breakage (e.g., double-strand breakage (DSB) or single-strand breakage (notch)) in T cells or T cell populations, for example, using guide RNA and RNA-guided DNA binders (e.g., CRISPR/Cas systems), for insertion into a targeted receptor (e.g., CAR). Cells and cell populations prepared by such methods are also provided.

In some embodiments, a targeting receptor (e.g., CAR) can impart targeting specificity to engineered T cells containing the targeting receptor (e.g., CAR), such as specific cells, tissues, or organs.

In some embodiments, targeting receptors (such as CARs) can enable engineered T cells to target the gastrointestinal system. For example, the targeting receptor is a CAR that targets MAdCAM-1, which can be used to suppress immune responses in conditions such as inflammatory bowel disease, ulcerative colitis, or Crohn's disease.

In some embodiments, targeting receptors (such as CARs) can enable engineered T cells to target inflamed tissues. For example, the targeting receptor is a CAR that targets TNFA, which can be used to suppress immune responses in conditions such as inflammatory bowel disease, ulcerative colitis, or Crohn's disease.

In some embodiments, a target receptor (e.g., a CAR) can enable engineered T cells to target inflamed tissues and/or activated immune cells. For example, the target receptor is a CAR that targets CD70, which may be used to suppress immune responses in conditions such as inflammatory bowel disease, ulcerative colitis, or Crohn's disease.

In some embodiments, targeting receptors (such as CARs) can enable engineered T cells to target endothelial cells. For example, the targeting receptor is a CAR that targets CEACAM6, which can be used to suppress immune responses, including inflammation, in conditions such as Crohn's disease.

In some embodiments, targeting receptors (such as CARs) can enable engineered T cells to target tissues containing endothelial cells. For example, the targeting receptor is a CAR that targets VCAM-1, which may be used to suppress immune responses in diseases such as Crohn's disease and multiple sclerosis.

In some embodiments, CARs can enable engineered T cells to target synovial tissue, for example, by targeting receptors such as CARs that target citrullinated vimentin, for example, to suppress immune responses in conditions such as rheumatoid arthritis.

In some embodiments, targeting receptors (such as CARs) can enable engineered T cells to target neural tissue. For example, the targeting receptor is a CAR that targets MBP, MOG, or PLP1, which can be used to suppress immune responses in diseases such as multiple sclerosis.

In some embodiments, targeting receptors (such as CARs) can enable engineered T cells to target B cells. For example, the targeting receptor is a CAR that targets CD19, which can be used to suppress immune responses in diseases such as multiple sclerosis and systemic lupus erythematosus.

In some embodiments, targeting receptors (such as CARs) can enable engineered T cells to target B cells. For example, the targeting receptor is a CD20-targeting CAR, which can be used to suppress immune responses in diseases such as multiple sclerosis and systemic lupus erythematosus.

In some embodiments, targeting receptors (e.g., CARs) can enable engineered T cells to target immune cells expressing TNFSF7, including (but not limited to) activated T lymphocytes, B lymphocytes, dendritic cells, and/or NK cells. For example, CARs targeting TNFSF7 can be used to suppress immune responses in conditions such as Crohn's disease, ulcerative colitis, systemic lupus erythematosus, or multiple sclerosis.

In some embodiments, a target receptor (e.g., a CAR) can enable engineered T cells to target tissues containing mature B lymphocytes. For example, the target receptor is a CAR that targets TNFRSF17, which may be used to suppress immune responses in diseases such as systemic lupus erythematosus.

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

Other CAR targets (e.g., inflammatory antigens) are known in this art. For example, see WO2020092057A1, the contents of which are incorporated herein by reference in their entirety. In some embodiments, insertions can be evaluated by detecting the amount of protein or mRNA in engineered T cells, engineered T cell populations, the tissue of interest, body fluids, or tissue culture media containing engineered T cells. In some embodiments, insertions by gene editing can be evaluated by sequence analysis, such as next-generation sequencing (NGS). Analysis of protein and mRNA expression targeting receptors (e.g., CARs) is described herein and is known in this art.

In embodiments disclosed herein, where the target of the target receptor (e.g., CAR) is also present on the surface of cells (e.g., immune cells or T cells), the engineered T cells or T cell population may further include modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding the target.

In some embodiments, the engineered T cells or T cell populations contain two or more heterologous coding sequences under promoter control, such as dmTGFB1, regulatory T cell-promoting molecules, and targeting receptors. In some embodiments, each heterologous coding sequence is under the control of a separate promoter. In some embodiments, two heterologous coding sequences are under the control of the same promoter. In some embodiments, two or more heterologous coding sequences are under the control of the same promoter. In some embodiments, when the engineered T cells or T cell populations contain three or more heterologous coding sequences (e.g., dmTGFB1, regulatory T cell stimulating molecules, and target receptors) under promoter control, each heterologous sequence is independently under the control of a single promoter or under the control of a promoter that controls the expression of more than one heterologous coding sequence.

In some embodiments, the engineered T cells or T cell populations do not include heterologous targeting receptors.

B. Engineered T Cells This article provides engineered T cells and T cell populations that include modifications, such modifications including the insertion of a heterologous sequence encoding dmTGFB1 under promoter control into the cells and modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding TGFBR2.

In some embodiments, T cells comprising a heterologous sequence encoding dmTGFB1 under promoter control and a modified (e.g., knockdown) endogenous nucleic acid sequence encoding TGFBR2 are further engineered to include modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding IFNG, modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding TNFA, and/or the insertion of a heterologous sequence encoding a regulatory T cell-promoting molecule under promoter sequence control into the cells, as well as combinations thereof and uses. In some embodiments, the regulatory T cell-promoting molecule is selected from IL10, CTLA4, TIGIT, IDO1, ENTPD1, NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, BACH2, and IL2RA.

In some embodiments, T cells or T cell populations containing a heterologous sequence encoding a dmTGFB1 molecule under promoter control are further engineered to include modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding TGFBR2, modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA, and the insertion of heterologous sequences encoding two or more regulatory T cytokines, each under promoter sequence control, into the cells. For example, the engineered T cells contain a first heterologous sequence encoding a first regulatory T cytokinetic molecule under the control of a first promoter and a second heterologous sequence encoding a second regulatory T cytokinetic molecule under the control of a second promoter. The first and second promoters may be the same or different promoters. In some embodiments, the heterologous sequence encoding the dmTGFB1 molecule is under the control of a promoter sequence that controls the expression of regulatory T cell-promoting molecules. In some embodiments, the heterologous sequence encoding the dmTGFB1 molecule is not under the control of a promoter sequence that controls the expression of regulatory T cell-promoting molecules. In some embodiments, the heterologous sequence encoding the dmTGFB1 molecule is under the control of a promoter sequence that controls the expression of a target receptor. In some embodiments, the heterologous sequence encoding the dmTGFB1 molecule is not under the control of a promoter sequence that controls the expression of a target receptor.

In some embodiments, T cells or T cell populations containing a promoter-controlled heterologous sequence encoding dmTGFB1 and a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TGFBR2 are further engineered to include modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG; insertion of a promoter-controlled heterologous sequence encoding IL10 into the cells; and insertion of a promoter-controlled heterologous sequence encoding CTLA4 into the cells.

In some embodiments, T cells or T cell populations comprising a heterologous sequence encoding dmTGFB1 under promoter control and a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TGFBR2 are further engineered to include a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG; insertion of a heterologous sequence encoding IL10 under promoter control into the cells; and insertion of a heterologous sequence encoding CTLA4 under promoter control into the cells, and exhibiting at least one inhibitory activity of naturally occurring regulatory T cells (nTregs), such as inhibiting immune responses or biomarkers in in vitro or in vivo analyses (e.g., animal models of GvHD).

In some embodiments, a heterologous sequence encoding dmTGFB1 or one or more regulatory T cell-stimulating molecules is incorporated into the expression architecture. In some embodiments, heterologous sequences encoding two or more molecules may be incorporated into two or more separate expression architectures. For example, a first heterologous sequence encoding dmTGFB1 is provided in a first expression architecture, a second heterologous sequence encoding a regulatory T cell-stimulating molecule is provided in a second separate expression architecture, and a third heterologous sequence encoding a regulatory T cell-stimulating molecule is provided in a third separate expression architecture. In alternative examples, a first heterologous sequence encoding dmTGFB1, a second heterologous sequence encoding a regulatory T cell-stimulating molecule, and a third heterologous sequence encoding a regulatory T cell-stimulating molecule are provided in the same phenotype. In some embodiments, one or more phenotypes are free phenotypes. In some embodiments, one or more heterologous sequences are inserted into the genome, for example, through targeted or non-targeted insertion.

In some embodiments, modification (e.g., knockdown) of the endogenous nucleic acid sequence encoding TGFBR2 includes modification of TGFBR2. In some embodiments, modification (e.g., knockdown) of the endogenous nucleic acid sequence encoding IFNG includes modification of IFNG. In some embodiments, modification (e.g., knockdown) of the endogenous nucleic acid sequence encoding TNFA includes modification of TNFA.

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

In some embodiments, engineered T cells or T cell populations are engineered to include modifications (e.g., the insertion of a sequence encoding dmTGFB1), for example by gene editing, such as 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 contain the insertion sequence encoding the dmTGFB1 molecule.

In some embodiments, the engineered T cell population includes modifications to an insertion sequence encoding dmTGFB1, for example, generated by gene editing, and, for example, as evaluated by sequencing (e.g., NGS), at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells contain an insertion sequence encoding the dmTGFB1 molecule, further including modifications to the TGFBR2 sequence by gene editing (e.g., knockdown), for example, as evaluated by sequencing (e.g., NGS), where at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells contain insertions, deletions, or substitutions in the endogenous TGFBR2 sequence. In some embodiments, such as those measured by ELISA or flow cytometry, 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%, or 95%, or reduced to below the detection limit of the analysis, compared to a suitable control (e.g., in which the TGFBR2 gene is unmodified). Analysis of TGFBR2 protein and mRNA expression in, for example, T cell populations is known in this technique.

In some embodiments, the engineered T cell population includes modifications to an insertion sequence encoding dmTGFB1, for example, generated by gene editing, and, for example, as evaluated by sequencing (e.g., NGS), at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells contain an insertion sequence encoding the dmTGFB1 molecule, further including modifications to the TNFA sequence by gene editing (e.g., knockdown), and, for example, as evaluated by sequencing (e.g., NGS), at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells contain insertions, deletions, or substitutions in the endogenous TNFA sequence. In some embodiments, such as those measured by ELISA or flow cytometry, 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%, or 95%, or reduced to below the detection limit of the analysis, compared to a suitable control (e.g., in which the TNFA gene is not modified). Analysis of TNFA protein and mRNA expression in, for example, T cell populations is known in this technique. In some embodiments, such as those measured using a custom U-PLEX biomarker kit (Meso Scale Diagnostics, catalog number K15067L-2) according to the manufacturer's instructions, TNFA is knocked down to a level of 2500 pg/ml or lower.

In some embodiments, the engineered T cell population includes modifications (e.g., insertion of a sequence encoding dmTGFB1), for example by gene editing, such as 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 contain an insertion sequence encoding the dmTGFB1 molecule, further including modifications (e.g., knockdown) in the IFNG sequence by gene editing, such as as evaluated by sequencing (e.g., NGS), wherein at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells contain insertions, deletions, or substitutions in the endogenous IFNG sequence. In some embodiments, such as those measured by ELISA or flow cytometry, the expression of IFNG (full-length wild-type protein or mRNA) is reduced by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or reduced to below the detection limit of the analysis, compared to a suitable control (e.g., in which the IFNG gene is not modified). Analysis of IFNG protein and mRNA expression in, for example, T cell populations is known in this art. In some embodiments, such as those measured using a custom U-PLEX biomarker kit (Meso Scale Diagnostics, catalog number K15067L-2) according to the manufacturer's instructions, IFNG knockdown is achieved to an IFNG level of 300,000 pg/ml or lower.

In some implementations, the modification of the expression of knockdown genes (such as TGFBR2, TNFA, or IFNG) is one or more of insertion, deletion, or substitution.

In some embodiments, the engineered T cells or T cell populations contain modifications (e.g., the insertion of a sequence encoding dmTGFB1), for example by gene editing, such as 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 contain the insertion sequence encoding the dmTGFB1 molecule, and further contain the insertion sequence encoding a regulatory T cell-promoting molecule, for example by gene editing, such as 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 contain the insertion sequence encoding the regulatory T cell-promoting molecule. In some embodiments, such as those measured by ELISA or flow cytometry, the insertion of a regulatory T cell-promoting molecule (e.g., IL10) results in a statistically significant increase in protein or mRNA expression compared to a suitable control (e.g., in which no regulatory T cell-promoting molecule gene is inserted). In some embodiments, engineered T cells contain a sequence encoding IL10 inserted by gene editing, for example, 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 contain the inserted sequence encoding IL10. In some embodiments, the insertion sequence encoding IL10 results in a statistically significant increase in protein or mRNA expression compared to a suitable control (e.g., a regulatory T cell-promoting molecule). Analysis of IL10 protein and mRNA expression in, for example, T cell populations is known in this technique, using methods such as ELISA and flow cytometry. In some embodiments, such as using a custom-designed U-PLEX biomarker kit (Meso Scale Diagnostics, catalog number K15067L-2) according to the manufacturer's instructions, the IL10 level is at least 300 pg/ml.

In some embodiments, the engineered T cells or T cell populations contain modifications (e.g., the insertion of a sequence encoding dmTGFB1), for example by gene editing, such as 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 contain the insertion sequence encoding the dmTGFB1 molecule, and further contain the insertion sequence encoding CTLA4, for example by gene editing, such as 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 contain the insertion sequence encoding CTLA4. In some embodiments, the insertion sequence encoding CTLA4 results in a statistically significant increase in protein or mRNA expression compared to a suitable control (e.g., a regulatory T cell-promoting molecule). Analysis of CTLA4 protein and mRNA expression in, for example, T cell populations is described herein and is known in this art, such as ELISA and flow cytometry.

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

In some embodiments, engineered T cells or T cell populations are engineered to include modifications (e.g., insertion of a sequence encoding dmTGFB1), for example by gene editing, such as 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 contain the code for dmTGF. The insertion sequence of the B1 molecule further includes modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding TGFBR2; modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding TNFA; modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding IFNG; and the inserted heterologous sequence encoding a regulatory T cell-promoting molecule under the control of the promoter sequence, further including modifications of the endogenous nucleic acid sequence encoding the following: interleukin-17A (IL17A), interleukin-6 (IL6), interleukin-2 (IL2), perforin-1 (PRF1), granzyme A (GZMA), granzyme B (GZMB), natural killer cell granzyme 7 (NKG7), Fas ligand (FasL, NF superfamily, member 6), ranotin receptor 2 (RYR2), and community stimulating factor 2 (CSF2), wherein the modification knocks down the expression of IL17A, IL6, IL2, PRF1, GZMA, GZMB, NKG7, FASL, RYR2, or CSF2, and further modification of endogenous nucleic acid sequences includes modification of IL17A.

In some embodiments, T cells or T cell populations are engineered using gene editing systems, such as RNA-guided DNA binders. In some embodiments, T cells are engineered using CRISPR/Cas gene editing systems. In some embodiments, T cells are engineered using CRISPR/Cas type II gene editing systems, such as Cpf1. In some embodiments, T cells are engineered using CRISPR/Cas9 gene editing systems, such as SpyCas9. An illustrative Cas9 sequence is provided herein.

In some embodiments, T cells or T cell populations are engineered using guide RNAs that specifically target sites within the TGFBR2, IFNG, and TNFA genes to provide knockdown of these genes. Tables 1 and 2 provide exemplary sequences for knockdown of IFNG and TNFA, respectively, along with the genomic coordinates of the target for each listed guide sequence. Table 3 provides exemplary sequences for knockdown of TGFBR2, along with the genomic coordinates of the target for each listed guide sequence.

In some embodiments, the engineered T cells or T cell populations comprise TGFBR2, IFNG, and TNFA genes knocked down using the guide RNA and RNA-guided DNA binder disclosed herein. In some embodiments, this paper discloses T cells engineered by inducing breakage (e.g., double-strand breakage (DSB) or single-strand breakage (notch)) within the TGFBR2, IFNG, and TNFA genes of T cells, for example, using the guide RNA and RNA-guided DNA binder disclosed herein (e.g., a CRISPR/Cas system). These methods can be used in vitro or in vivo, for example, to manufacture cell products for suppressing immune responses, including inflammation and autoimmunity. In some embodiments, the guide RNA disclosed herein mediates target-specific cleavage of an RNA-guided DNA binder (e.g., Cas nuclease) at the site described herein within the TGFBR2 gene. In some embodiments, the guide RNA disclosed herein mediates target-specific cleavage of an RNA-guided DNA binder (e.g., Cas nuclease) at the site described herein within the IFNG gene. In some embodiments, the guide RNA disclosed herein mediates target-specific cleavage of an RNA-guided DNA binder (e.g., Cas nuclease) at the site described herein within the TNFA gene. It should be understood that in some embodiments, the guide RNA comprises a guide sequence that binds to or is capable of binding to such regions.

Engineered T cells or T cell populations containing genetically modified genomic coordinates selected from those listed in Table 1 are provided, for example, cells containing insertion, deletion, or substitution mutations within any of the listed genomic regions in IFNG. Engineered T cells containing genetically modified genomic coordinates selected from those listed in Table 2 are also provided, for example, cells containing insertion, deletion, or substitution mutations within any of the listed genomic regions in TNFA. In some embodiments, the engineered T cells will include modifications within the genomic coordinate regions selected from Table 1 and modifications within the genomic coordinate regions selected from Table 2. Provide genetically modified T cells or T cell populations containing genetically modified genomic coordinates selected from those listed in Table 3 , for example, cells containing insertion, deletion, or substitution mutations within any of the listed genomic ranges in TGFBR2.

In some embodiments, the lead RNA disclosed herein contains a lead sequence that is 95%, 90%, 85%, 80%, or 75% identical to the sequences selected from the groups of sequences in Tables 1, 2, or 3 .

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

In some embodiments, the guide RNA disclosed herein comprises a guide sequence having at least 15, 16, 17, 18, 19, or 20 adjacent nucleotides of a sequence selected from the following groups: sequences that are 95%, 90%, 85%, 80%, or 75% identical to sequences selected from the groups 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 adjacent nucleotides of a sequence selected from the groups 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 sequences selected from the groups of sequences in Table 2. In some embodiments, the guide RNA disclosed herein comprises a guide sequence of 17, 18, 19, or 20 adjacent nucleotides as a sequence selected from the sequence group in Table 2.

In some embodiments, the guide RNA disclosed herein comprises a guide sequence having at least 15, 16, 17, 18, 19, or 20 adjacent nucleotides of a sequence selected from the following groups: sequences that are 95%, 90%, 85%, 80%, or 75% identical to sequences selected from the groups of sequences in Table 3. In some embodiments, the guide RNA disclosed herein comprises a guide sequence having at least 17, 18, 19, or 20 adjacent nucleotides of a sequence selected from the groups of sequences in Table 3. In some embodiments, the guide RNA disclosed herein comprises a guide sequence that is 95%, 90%, 85%, 80%, or 75% identical to sequences selected from the groups of sequences in Table 2. In some embodiments, the guide RNA disclosed herein comprises a guide sequence of 17, 18, 19, or 20 adjacent nucleotides as a sequence selected from the sequence group in Table 3. In some embodiments, the guide RNA disclosed herein comprises a guide sequence selected from the sequence group in Table 3 .

Unless otherwise noted, the genomic coordinates are always based on the human reference genomic body hg38.

In some embodiments, this includes a guide RNA containing a guide sequence targeting IFNG and a guide RNA containing a guide sequence targeting TNFA.

In some embodiments, the engineered T cells may be engineered autologous T cells or engineered allogeneic T cells. In autologous T cell therapy, T cells may be derived from T cells of the same patient who has been treated with engineered T cells. In some embodiments, engineered autologous T cells may be generated by extracting T cells from the patient and modifying those T cells according to the modifications described herein, and then reintroducing those engineered autologous T cells into the same patient. In some embodiments, engineered autologous T cells may be generated for each individual patient and administered on a patient-by-patient basis.

In some implementations, 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 delivered to one or more patients using an "off-the-shelf" method.

In some embodiments, the engineered T cells described herein refer to engineered allogeneic T cells. Therefore, this document provides compositions and methods for modifying (e.g., inhibiting or reducing) the expression and/or function (e.g., the level of expression of a functional form) of a gene target or a protein encoded by that gene target, thereby improving the efficacy (e.g., by reducing or eliminating undesirable immunogenicity (such as host-graft or graft-versus-host response)), function, proliferation, stimulation, or survival of transplanted cells, such as engineered T cells described herein, such as allogeneic engineered T cells used in immunotherapy, to generate "ready-made" T cells.

"Off-the-shelf" therapies using donor (allogeneic) cells eliminate the need to recycle and modify the patient's own lymphocytes. However, allogeneic T cells can elicit undesirable immune responses or have a shorter lifespan. Typically, immune rejection of allogeneic cells is caused by a mismatch of major histocompatibility complex (MHC) molecules between the donor and recipient. For example, slight differences in MHC alleles between individuals can lead to T cell activation in the recipient. During T cell development, an individual's T cell lineage is tolerant to its own MHC molecules, but T cells that recognize another individual's MHC molecules can persist in circulation and are called allogeneic reactive T cells. Allogeneic reactive T cells can be activated, for example, by the presence of another somatic cell expressing MHC molecules, thereby causing, for example, graft-versus-host disease and transplant rejection. See also, for example, WO/2023/245108, the contents of which are incorporated herein by reference in their entirety.

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, it is believed that inhibiting or eliminating the level of allogeneic T cell targets or the expression level of allogeneic T cell target gene targets (e.g., through gene modification) can improve cell function (e.g., transplanted cells, such as engineered allogeneic T cells) by reducing or eliminating graft-versus-host response or host-versus-graft response, or will render the transplanted cells resistant to immunosuppressive therapy.

In some embodiments, the compositions and methods described herein can be used to improve the function (e.g., by reducing or eliminating undesirable immunogenicity (such as host-graft or graft-versus-host response)), survival, proliferation and/or efficacy of cells (e.g., allogeneic engineered T cells described herein) by altering the genes of components of the major histocompatibility complex (e.g., HLA proteins, such as HLA-A and/or HLA-B). While not wishing to be bound by theory, it is believed that reducing or eliminating the expression of mismatched (e.g., mismatches with the individual receiving cell therapy) HLA proteins (or components) by decreasing or eliminating the expression of these proteins can reduce or eliminate host resistance to graft disease by eliminating the recognition and response of host T cell receptors to mismatched (e.g., allogeneic) graft tissue. Therefore, this method could be used to generate "ready-made" T cells (Torikai et al., 2012 Blood 119, 5697-5705).

In some embodiments, the compositions and methods described herein can be used to improve the function (e.g., by reducing or eliminating undesirable immunogenicity (such as host-versus-graft or graft-versus-host disease)), survival, proliferation, and/or efficacy of cells (e.g., allogeneic engineered T cells described herein) by altering the genes of T cell receptor (TCR) components (e.g., TRAC). While not wishing to be theoretically constrained, it is believed that reducing or eliminating the expression of functional T cell receptor components reduces or eliminates the presence of TCRs on the cell surface, thereby reducing or preventing graft-versus-host disease by eliminating T cell receptor recognition and response to host tissues. Therefore, this method can be used to generate "ready-made" T cells.

In some embodiments, the compositions and methods described herein can be used to improve the function (e.g., by reducing or eliminating undesirable immunogenicity (such as host-graft or graft-versus-host response)), survival, proliferation, and/or efficacy of cells (e.g., allogeneic engineered T cells described herein) by altering genes that encode proteins that regulate the expression of one or more components of the major histocompatibility complex (e.g., CIITA). While not wishing to be theoretically constrained, it is believed that reducing or eliminating the expression of MHC class II expression regulators (such as CIITA) will reduce or eliminate the expression of MHC class II molecules on allogeneic cells, thereby reducing or eliminating the expression of mismatched (e.g., mismatches with the individual receiving cell therapy) MHC class II proteins (or components). This, in turn, reduces or eliminates host resistance to grafts by, for example, eliminating the recognition and response of host T cell receptors to mismatched (e.g., allogeneic) graft tissues (e.g., allogeneic engineered T cells as described herein). Therefore, this method can be used to generate "ready-made" T cells.

In some embodiments, reducing or eliminating the expression of one or more MHC class I molecules and one or more MHC II molecules, for example in T cells, such as in allogeneic engineered T cells as described herein, may be beneficial to further reduce or eliminate host resistance to graft-versus-graft disease after cell administration. Therefore, in embodiments of the cells and methods disclosed herein, cells may be brought into contact with a combination of the present disclosure (e.g., a combination containing gRNA and Cas9 molecules) containing gRNA molecules targeting HLA-A or HLA-B (e.g., as described herein) (e.g., causing a reduction or elimination of the expression of one or more MHC class I molecules in the cell), and with a combination of the present disclosure (e.g., a combination containing gRNA and Cas9 molecules) containing gRNA molecules targeting CIITA (e.g., as described herein) (e.g., causing a reduction or elimination of the expression of one or more MHC class II molecules). In embodiments of the cells and methods disclosed herein, cells may also be brought into contact with a combination of the present disclosure (e.g., a combination containing gRNA and Cas9 molecules) containing a gRNA molecule targeting a component of TCR (e.g., targeting TRAC) (e.g., as described herein) (e.g., resulting in a reduction or elimination of the expression of T cell receptors (e.g., one or more components of TCR)). In some embodiments, the TCR expression of the cells disclosed herein is reduced or eliminated (e.g., as detected by flow cytometry), the expression of one or more MHC class I molecules is reduced or eliminated (e.g., as detected by flow cytometry), and the expression of one or more MHC class II molecules is reduced or eliminated (e.g., as detected by flow cytometry).

In some embodiments, "off-the-shelf" allogeneic T cells and allogeneic T cell populations are engineered to include modifications including: insertion of a heterologous sequence encoding IL10 under promoter control into the cells; insertion of a heterologous sequence encoding CTLA4 under promoter control into the cells; modification (e.g., knockdown) of endogenous nucleic acid sequences encoding TNFA; modification (e.g., knockdown) of endogenous nucleic acid sequences encoding IFNG; modification (e.g., knockdown) of endogenous nucleic acid sequences encoding HLA-A; modification (e.g., knockdown) of endogenous nucleic acid sequences encoding HLA-B; modification (e.g., knockdown) of endogenous nucleic acid sequences encoding CIITA; and modification (e.g., knockdown) of endogenous nucleic acid sequences encoding TRAC. In some embodiments, “ready-to-use” allogeneic T cells and allogeneic T cell populations further include targeting receptors as described herein.

In some embodiments, "off-the-shelf" allogeneic T cells and allogeneic T cell populations are engineered to include modifications including: inserting a heterologous sequence encoding dmTGFB1 under promoter control into the cells; inserting a heterologous sequence encoding IL10 under promoter control into the cells; inserting a heterologous sequence encoding CTLA4 under promoter control into the cells; and modifying the sequence encoding TNFA. Modification of endogenous nucleic acid sequences (e.g., knockdown); modification of endogenous nucleic acid sequences encoding IFNG (e.g., knockdown); modification of endogenous nucleic acid sequences encoding HLA-A (e.g., knockdown); modification of endogenous nucleic acid sequences encoding HLA-B (e.g., knockdown); modification of endogenous nucleic acid sequences encoding CIITA (e.g., knockdown); and modification of endogenous nucleic acid sequences encoding TRAC (e.g., knockdown). In some embodiments, "ready-to-use" allogeneic T cells and allogeneic T cell populations further include targeting receptors as described herein.

In some embodiments, "off-the-shelf" allogeneic T cells and allogeneic T cell populations are engineered to include modifications, such modifications including: inserting a heterologous sequence encoding dmTGFB1 under promoter control into the cells; inserting a heterologous sequence encoding IL10 under promoter control into the cells; inserting a heterologous sequence encoding CTLA4 under promoter control into the cells; and modifying the endogenous nucleic acid sequence encoding TGFBR2 (e.g., ...). Modifications to endogenous nucleic acid sequences encoding TNFA (e.g., knockdown); modifications to endogenous nucleic acid sequences encoding IFNG (e.g., knockdown); modifications to endogenous nucleic acid sequences encoding HLA-A (e.g., knockdown); modifications to endogenous nucleic acid sequences encoding HLA-B (e.g., knockdown); modifications to endogenous nucleic acid sequences encoding CIITA (e.g., knockdown); and modifications to endogenous nucleic acid sequences encoding TRAC (e.g., knockdown). In some embodiments, "ready-to-use" allogeneic T cells and allogeneic T cell populations further include targeting receptors as described herein.

In some embodiments, the "ready-made" allogeneic T cells and the allogeneic T cell population are CD3+ cells. In some embodiments, “off-the-shelf” allogeneic CD3+ T cells and allogeneic CD3+ T cell populations may first be engineered to include modifications including one or more of the following: modifications to endogenous nucleic acid sequences encoding HLA-A (e.g., knockdown); modifications to endogenous nucleic acid sequences encoding HLA-B (e.g., knockdown); modifications to endogenous nucleic acid sequences encoding CIITA (e.g., knockdown); and modifications to endogenous nucleic acid sequences encoding TRAC (e.g., knockdown). These modifications are then selected for CD4+ T cells or CD8+ T cells and further modified to produce the engineered T cells described herein. The order in which different modifications are engineered can be varied. For example, "off-the-shelf" allogeneic CD3+ T cells and allogeneic CD3+ T cell populations can be engineered to include modifications such as: (a) insertion of a heterologous sequence encoding IL10 under promoter control into the cells; insertion of a heterologous sequence encoding CTLA4 under promoter control into the cells; and modification of endogenous nucleic acid sequences encoding TNFA (e.g., knockdown); modification of endogenous nucleic acid sequences encoding IFNG (e.g., knockdown); (b) Insertion of a heterologous sequence encoding dmTGFB1 under promoter control into cells; insertion of a heterologous sequence encoding IL10 under promoter control into cells; insertion of a heterologous sequence encoding CTLA4 under promoter control into cells; and modification (e.g., knockdown) of endogenous nucleic acid sequences encoding TNFA; modification (e.g., knockdown) of endogenous nucleic acid sequences encoding IFNG; or (c) The following steps were performed: inserting a heterologous sequence encoding dmTGFB1 under promoter control into cells; inserting a heterologous sequence encoding IL10 under promoter control into cells; inserting a heterologous sequence encoding CTLA4 under promoter control into cells; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TGFBR2; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA; and modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG, and subsequently engineering the sequence to include the target receptor.

In other embodiments, “off-the-shelf” allogeneic CD3+ T cells and allogeneic CD3+ T cell populations are engineered to include a target receptor and then subsequently engineered to include the modifications described above (a), (b) or (c).

Table 1: Human guide sequences and chromosome coordinates used for IFNG knockdown Indicative genomic coordinates (hg38) exons share Guide sequence (5' to 3') SEQ ID NO chr12:68155336-68155356 4 + GCAGGCAGGACAACCAUUAC 1 chr12:68155337-68155357 4 + CAGGCAGGACAACCAUUACU 2 chr12:68155374-68155394 4 - AGGAGUCAGAUGCUGUUUCG 3 chr12:68155394-68155414 4 - CUAAAACAGGGAAGCGAAAA 4 chr12:68155398-68155418 4 + CGCUUCCCUGUUUUAGCUGC 5 chr12:68155406-68155426 4 - UGUCGCCAGCAGCUAAAACA 6 chr12:68155407-68155427 4 - CUGUCGCCAGCAGCUAAAAC 7 chr12:68155420-68155440 4 + GCGACAGUUCAGCCAUCACU 8 chr12:68155435-68155455 4 - ACAUGAACUCAUCCAAGUGA 9 chr12:68155447-68155467 4 + GUUCAUGUAUUGCUUUGCGU 10 chr12:68157972-68157992 3 + GACAUUCAUGUCUUCCUUGA 11 chr12:68157989-68158009 3 + UGAUGGUCUCCACACUCUUU 12 chr12:68157989-68158009 3 - AAAGAGUGUGGAGACCAUCA 13 chr12:68158001-68158021 3 - CCAGAGCAUCCAAAAGAGUG 14 chr12:68158215-68158235 2 - GAUAAUGGAACUCUUUUCUU 15 chr12:68158230-68158250 2 - CAUUCAGAUGUAGCGGAUAA 16 chr12:68158237-68158257 2 - UGCAGGUCAUUCAGAUGUAG 17 chr12:68159524-68159544 1 + CUUCUUUUACAUAUGGGUCC 18 chr12:68159559-68159579 1 - UGCAUCGUUUUGGGUUCUCU 19 chr12:68159568-68159588 1 - UUUCAGCUCUGCAUCGUUUU 20 chr12:68159569-68159589 1 - UUUUCAGCUCUGCAUCGUUU twenty one

Table 2: Human guide sequences and chromosomal coordinates used for knocking down T NFA Indicative genomic coordinates (hg38) Exons share Guide sequence (5' to 3') SEQ ID NO chr6:31575742-31575762 1 + UGAGCACUGAAAGCAUGAUC twenty two chr6:31575749-31575769 1 + UGAAAGCAUGAUCCGGGACG twenty three chr6:31575755-31575775 1 + CAUGAUCCGGGACGUGGAGC twenty four chr6:31575781-31575801 1 - CUUCUUGGGGAGCGCCUCCU 25 chr6:31575783-31575803 1 + GAGGCGCUCCCCAAGAAGAC 26 chr6:31575784-31575804 1 + AGGCGCUCCCCAAGAAGACA 27 chr6:31575785-31575805 1 + GGCGCUCCCCAAGAAGACAG 28 chr6:31575786-31575806 1 + GCGCUCCCCAAGAAGACAGG 29 chr6:31575794-31575814 1 + CAAGAAGACAGGGGGGCCCC 30 chr6:31575794-31575814 1 - GGGGCCCCCCUGUCUUCUUG 31 chr6:31575796-31575816 1 - CUGGGGCCCCCCUGUCUUCU 32 chr6:31575813-31575833 1 - AAGCACCGCCUGGAGCCCUG 33 chr6:31575814-31575834 1 - CAAGCACCGCCUGGAGCCCU 34 chr6:31575823-31575843 1 - GCUGAGGAACAAGCACCGCC 35 chr6:31575842-31575862 1 + CCUCUUCUCCUUCCUGAUCG 36 chr6:31575846-31575866 1 + UUCUCCUUCCUGAUCGUGGC 37 chr6:31575853-31575873 1 - GGCGCCUGCCACGAUCAGGA 38 chr6:31575857-31575877 1 - UGGUGGCGCCUGCCACGAUC 39 chr6:31575888-31575908 1 + CUGCUGCACUUUGGAGUGAU 40 chr6:31575890-31575910 1 - CGAUCACUCCAAAGUGCAGC 41 chr6:31575898-31575918 1 + UUGGAGUGAUCGGCCCCCAG 42 chr6:31575899-31575919 1 + UGGAGUGAUCGGCCCCCAGA 43 chr6:31575917-31575937 1 - AGGCACUCACCUCUUCCCUC 44 chr6:31576538-31576558 2 - UGAUUAGAGAGAGGUCCCUG 45 chr6:31576539-31576559 2 - CUGAUUAGAGAGAGGUCCCU 46 chr6:31576540-31576560 2 - GCUGAUUAGAGAGAGGUCCC 47 chr6:31576544-31576564 2 + CCUCUCUCUAAUCAGCCCUC 48 chr6:31576547-31576567 2 - CCAGAGGGCUGAUUAGAGAG 49 chr6:31576550-31576570 2 + UCUAAUCAGCCCUCUGGCCC 50 chr6:31576562-31576582 2 - UACUGACUGCCUGGGCCAGA 51 chr6:31576563-31576583 2 - UUACUGACUGCCUGGGCCAG 52 chr6:31576570-31576590 2 - GAGACACUUACUGACUGCCU 53 chr6:31576571-31576591 2 - GGAGACACUUACUGACUGCC 54 chr6:31576785-31576805 3 - GGGCUACAGGCUUGUCACUC 55 chr6:31576786-31576806 3 - UGGGCUACAGGCUUGUCACU 56 chr6:31576798-31576818 3 - UUACCUACAACAUGGGCUAC 57 chr6:31576805-31576825 3 - AGAGCUCUUACCUACAACAU 58 chr6:31576806-31576826 3 - CAGAGCUCUUACCUACAACA 59 chr6:31577110-31577130 4 + UCCAGCAAACCCUCAAGCUG 60 chr6:31577112-31577132 4 + CAGCAAACCCUCAAGCUGAG 61 chr6:31577122-31577142 4 - GGAGCUGCCCCUCAGCUUGA 62 chr6:31577123-31577143 4 - UGGAGCUGCCCCUCAGCUUG 63 chr6:31577136-31577156 4 + AGCUCCAGUGGCUGAACCGC 64 chr6:31577137-31577157 4 + GCUCCAGUGGCUGAACCGCC 65 chr6:31577143-31577163 4 - UGGCCCGGCGGUUCAGCCAC 66 chr6:31577152-31577172 4 + CCGCCGGGCCAAUGCCCUCC 67 chr6:31577155-31577175 4 - CCAGGAGGGCAUUGGCCCGG 68 chr6:31577159-31577179 4 + GCCAAUGCCCUCCUGGCCAA 69 chr6:31577163-31577183 4 - GCCAUUGGCCAGGAGGGCAU 70 chr6:31577164-31577184 4 + UGCCCUCCUGGCCAAUGGCG 71 chr6:31577170-31577190 4 - GCUCCACGCCAUUGGCCAGG 72 chr6:31577173-31577193 4 - UCAGCUCCACGCCAUUGGCC 73 chr6:31577178-31577198 4 - AUCUCUCAGCUCCACGCCAU 74 chr6:31577185-31577205 4 + GGAGCUGAGAGAUAACCAGC 75 chr6:31577188-31577208 4 + GCUGAGAGAUAACCAGCUGG 76 chr6:31577200-31577220 4 + CCAGCUGGUGGUGCCAUCAG 77 chr6:31577201-31577221 4 + CAGCUGGUGGUGCCAUCAGA 78 chr6:31577216-31577236 4 - AUGAGGUACAGGCCCUCUGA 79 chr6:31577227-31577247 4 - CCUGGGAGUAGAUGAGGUAC 80 chr6:31577237-31577257 4 + UACUCCCAGGUCCUCUUCAA 81 chr6:31577245-31577265 4 - CUUGGCCCUUGAAGAGGACC 82 chr6:31577295-31577315 4 - GGCGAUGCGGCUGAUGGUGU 83 chr6:31577296-31577316 4 - CGGCGAUGCGGCUGAUGGUG 84 chr6:31577301-31577321 4 - GGAGACGGCGAUGCGGCUGA 85 chr6:31577308-31577328 4 - UCUGGUAGGAGACGGCGAUG 86 chr6:31577311-31577331 4 + CGCCGUCUCCUACCAGACCA 87 chr6:31577322-31577342 4 - GAGGUUGACCUUGGUCUGGU 88 chr6:31577331-31577351 4 - GGCAGAGAGGAGGUUGACCU 89 chr6:31577349-31577369 4 + CCAUCAAGAGCCCCUGCCAG 90 chr6:31577350-31577370 4 + CAUCAAGAGCCCCUGCCAGA 91 chr6:31577362-31577382 4 - CUGGGGUCUCCCUCUGGCAG 92 chr6:31577363-31577383 4 - UCUGGGGUCUCCCUCUGGCA 93 chr6:31577397-31577417 4 - GAUGGGCUCAUACCAGGGCU 94 chr6:31577401-31577421 4 + CUGGUAUGAGCCCAUCUAUC 95 chr6:31577402-31577422 4 - AGAUAGAUGGGCUCAUACCA 96 chr6:31577402-31577422 4 + UGGUAUGAGCCCAUCUAUCU 97 chr6:31577403-31577423 4 - CAGAUAGAUGGGCUCAUACC 98 chr6:31577406-31577426 4 + AUGAGCCCAUCUAUCUGGGA 99 chr6:31577407-31577427 4 + UGAGCCCAUCUAUCUGGGAG 100 chr6:31577414-31577434 4 - AAGACCCCUCCCAGAUAGAU 101 chr6:31577437-31577457 4 - GUCGGUCACCCUUCUCCAGC 102 chr6:31577454-31577474 4 + GACUCAGCGCUGAGAUCAAU 103 chr6:31577455-31577475 4 - GAUUGAUCUCAGCGCUGAGU 104 chr6:31577480-31577500 4 - UCGGCAAAGUCGAGAUAGUC 105 chr6:31577481-31577501 4 - CUCGGCAAAGUCGAGAUAGU 106 chr6:31577483-31577503 4 + UAUCUCGACUUUGCCGAGUC 107 chr6:31577484-31577504 4 + AUCUCGACUUUGCCGAGUCU 108 chr6:31577488-31577508 4 + CGACUUUGCCGAGUCUGGGC 109 chr6:31577498-31577518 4 + GAGUCUGGGCAGGUCUACUU 110 chr6:31577499-31577519 4 + AGUCUGGGCAGGUCUACUUU 111 chr6:31577499-31577519 4 - AAAGUAGACCUGCCCAGACU 112 chr6:31577532-31577552 4 - GGAUGUUCGUCCUCCUCACA 113

Table 3: Human guide sequences and chromosomal coordinates used to knock down T GFBR2 Indicative genomic coordinates (hg38) Guide sequence (5' to 3') SEQ ID NO chr3:30672267-30672287 ACAGUGAUCACACUCCAUGU 235 chr3:30644743-30644763 UAUCAUGUCGUUAUUAACUG 236 chr3:30671764-30671784 GCAGAAGCUGAGUUCAACCU 237 chr3:30672177-30672197 ACCUACAGGAGUACCUGACG 238 chr3:30606958-30606978 CCCGACUUCUGAACGUGCGG 239 chr3:30606870-30606890 AGCGGGGUCUGCCAUGGGUC 240 chr3:30606961-30606981 UCACCCGACUUCUGAACGUG 241 chr3:30606911-30606931 GGCCGCUGCACAUCGUCCUG 242 chr3:30606909-30606929 GGACGAUGUGCAGCGGCCAC 243 chr3:30606954-30606974 CCCACCGCACGUUCAGAAGU 244 chr3:30606865-30606885 CGAGCAGCGGGGUCUGCCAU 245 chr3:30606947-30606967 AACGUGCGGUGGGAUCGUGC 246 chr3:30606864-30606884 ACGAGCAGCGGGGUCUGCCA 247 chr3:30606916-30606936 GUCCACAGGACGAUGUGCAG 248 chr3:30606871-30606891 GCGGGGUCUGCCAUGGGUCG 249 chr3:30606930-30606950 UGCUGGCGAUACGCGUCCAC 250 chr3:30606957-30606977 CCGACUUCUGAACGUGCGGU 251 chr3:30623239-30623259 UGGGCAGUCCUAUUACAGCU 252 chr3:30623238-30623258 GGGCAGUCCUAUUACAGCUG 253 chr3:30623240-30623260 AUGGGCAGUCCUAUUACAGC 254 chr3:30623263-30623283 GAUACUUUACUAUGUCUCAG 255 chr3:30644843-30644863 AGUUGCUCAUGCAGGAUUUC 256 chr3:30644757-30644777 AUGAUAGUCACUGACAACAA 257 chr3:30650418-30650438 AUUGCACUCAUCAGAGCUAC 258 chr3:30650323-30650343 CCCCUACCAUGACUUUAUUC 259 chr3:30650327-30650347 UCCAGAAUAAAGUCAUGGUA 260 chr3:30650317-30650337 AGUCAUGGUAGGGGAGCUUG 261 chr3:30650326-30650346 CCAGAAUAAAGUCAUGGUAG 262 chr3:30650318-30650338 AAGUCAUGGUAGGGGAGCUU 263 chr3:30650319-30650339 AAAGUCAUGGUAGGGGAGCU 264 chr3:30650393-30650413 CACAUGAAGAAAGUCUCACC 265 chr3:30672152-30672172 AUCACCGCCUUCCACGCCAA 266 chr3:30672410-30672430 GUGGAUGACCUGGCUAACAG 267 chr3:30672296-30672316 CUGUGCACGAUGGGCAUCUU 268 chr3:30671871-30671891 GUUGAUGUUGUUGGCACACG 269 chr3:30672193-30672213 AGCUGAUGACAUGCCGCGUC 270 chr3:30671791-30671811 CGGCAAGACGCGGAAGCUCA 271 chr3:30672024-30672044 CCAAGAGGCAUACUCCUCAU 272 chr3:30671784-30671804 CCGCGUCUUGCCGGUUUCCC 273 chr3:30672322-30672342 CGAGGAUAUUGGAGCUCUUG 274 chr3:30672192-30672212 UGACGCGGCAUGUCAUCAGC 275 chr3:30672270-30672290 GUGAUCACACUCCAUGUGGG 276 chr3:30671941-30671961 UCGCUUUGCUGAGGUCUAUA 277 chr3:30671752-30671772 GCUUCUGCUGCCGGUUAACG 278 chr3:30672387-30672407 CAGAGUAGGGUCCAGACGCA 279 chr3:30671929-30671949 CAAAGCGACCUUUCCCCACC 280 chr3:30672266-30672286 CACAGUGAUCACACUCCAUG 281 chr3:30672295-30672315 CAAGAUGCCCAUCGUGCACA 282 chr3:30672021-30672041 CCUAUGAGGAGUAUGCCUCU 283 chr3:30671932-30671952 GGGGAAAGGUCGCUUUGCUG 284 chr3:30671835-30671855 GCGGUCAUCUUCCAGGAUGA 285 chr3:30672023-30672043 CAAGAGGCAUACUCCUCAUA 286 chr3:30671908-30671928 GCCCAUUGAGCUGGACACCC 287 chr3:30672212-30672232 UGGGAGGACCUGCGCAAGCU 288 chr3:30671854-30671874 ACGUGGAGCUGAUGUCAGAG 289 chr3:30671701-30671721 UGGCAACUCCCAGUGGUGGC 290 chr3:30672294-30672314 CCAAGAUGCCCAUCGUGCAC 291 chr3:30672193-30672213 GACGCGGCAUGUCAUCAGCU 292 chr3:30671821-30671841 CGAGCACUGUGCCAUCAUCC 293 chr3:30672268-30672288 CACAUGGAGUGUGAUCACUG 294 chr3:30672250-30672270 UGUGGAGGUGAGCAAUCCCC 295 chr3:30672421-30672441 UUACCUGCCCACUGUUAGCC 296 chr3:30672400-30672420 UACUCUGUCUGUGGAUGACC 297 chr3:30672249-30672269 GUGGAGGUGAGCAAUCCCCC 298 chr3:30672196-30672216 GCGGCAUGUCAUCAGCUGGG 299 chr3:30672340-30672360 AGGUUAGGUCGUUCUUCACG 300 chr3:30671842-30671862 UGUCAGAGCGGUCAUCUUCC 301 chr3:30671739-30671759 UCUACUGCUACCGCGUUAAC 302 chr3:30674221-30674241 ACAUCUCGCUGUAAUGCAGU 303 chr3:30674178-30674198 GCAGACCGAUGUCUACUCCA 304 chr3:30674085-30674105 CUGUCUGUUUUUGCUAUAGG 305 chr3:30674136-30674156 CCUAGAAUCCAGGAUGAAUU 306 chr3:30674220-30674240 GACAUCUCGCUGUAAUGCAG 307 chr3:30674184-30674204 CGAUGUCUACUCCAUGGCUC 308 chr3:30688451-30688471 GACAACGUGUUGAGAGAUCG 309 chr3:30688403-30688423 CGCACCUUGGAACCAAAUGG 310 chr3:30688434-30688454 GUCCUUCAUGCUUUCGACAC 311 chr3:30688432-30688452 CCUUCAUGCUUUCGACACAG 312 chr3:30688402-30688422 UCCAUUUGGUUCCAAGGUGC 313 chr3:30688388-30688408 AAAGAUUAUGAGCCUCCAUU 314 chr3:30688429-30688449 CCCCUGUGUCGAAAGCAUGA 315 chr3:30688510-30688530 GUAAACACUCACUCCUUACC 316 chr3:30688416-30688436 ACAGGGGUGCUCCCGCACCU 317 chr3:30691489-30691509 GAAGCGUUCUGCCACACACU 318 chr3:30691522-30691542 AUCUGGACAGGCUCUCGGGG 319 chr3:30691427-30691447 UCAACGUCUCACACACCAUC 320 chr3:30691519-30691539 AGCAUCUGGACAGGCUCUCG 321 chr3:30691435-30691455 GUGAGACGUUGACUGAGUGC 322 chr3:30691594-30691614 CCUGCCCCAGAAGAGCUAUU 323 chr3:30691409-30691429 CCCGCUACAGGGCAUCCAGA 324 chr3:30691463-30691483 UGAGACGGGCCUCUGGGUCG 325 chr3:30606962-30606982 ACGUUCAGAAGUCGGGUGAG 326 chr3:30606974-30606994 CGGGUGAGUGGUCCCCAGCC 327 chr3:30606975-30606995 GGGUGAGUGGUCCCCAGCCC 328 chr3:30644885-30644905 GAAGCCACAGGAAGUCUGUG 329 chr3:30644893-30644913 AGGAAGUCUGUGUGGCUGUA 330 chr3:30650250-30650270 CCUAGAGUGAAGAGAUUCAU 331 chr3:30671618-30671638 UCUGAUGGGGAAACAAAACA 332 chr3:30671763-30671783 AGCAGAAGCUGAGUUCAACC 333 chr3:30671983-30672003 UUCAGAGCAGUUUGAGACAG 334 chr3:30672088-30672108 GAACAUACUCCAGUUCCUGA 335 chr3:30672094-30672114 ACUCCAGUUCCUGACGGCUG 336 chr3:30672099-30672119 AGUUCCUGACGGCUGAGGAG 337 chr3:30674083-30674103 UAUAGCAAAAACAGACAGUG 338 chr3:30674198-30674218 UUCCCAGAGCACCAGAGCCA 339 chr3:30688476-30688496 GACCAGAAAUUCCCAGCUUC 340 chr3:30688481-30688501 AGCCAGAAGCUGGGAAUUUC 341 chr3:30688490-30688510 UGGUGGUUGAGCCAGAAGCU 342 chr3:30688491-30688511 CUGGUGGUUGAGCCAGAAGC 343 chr3:30688507-30688527 AACACUCACUCCUUACCUGG 344 chr3:30691396-30691416 UAGCGGGAAAGAGAUCCAAA 345 chr3:30691397-30691417 GUAGCGGGAAAGAGAUCCAA 346 chr3:30691412-30691432 CCAUCUGGAUGCCCUGUAGC 347 chr3:30691413-30691433 ACCAUCUGGAUGCCCUGUAG 348 chr3:30691582-30691602 GAGCUAUUUGGUAGUGUUUA 349 chr3:30691583-30691603 AGAGCUAUUUGGUAGUGUUU 350

The non-restrictive modified lead sequence used to knock down TNFA is shown below (hg38 coordinates chr12:68158001-68158021, G019757): mC*mC*mA*GAGCAUCCAAAAGAGUGGUUUUAGAmGmCmUmAmGmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmGmGmCmAmCmGmGmGmUmGmGmCmU*mU*mU*mU (SEQ ID NO: 119), where m is a 2'-OMe modified nucleotide/nucleoside residue, * indicates a phosphate thioester bond between residues, and uppercase letters indicate residues, such as residues containing ribose.

The non-restrictive modified lead sequence used to knock down IFNG is shown below (hg38 coordinates chr6:31576805-31576825, G019753): mA*mG*mA*GCUCUUACCUACAACAUGUUUUAGAmGmCmUmAmGmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmGmGmCmGmGmGmGmGmCmU*mU*mU*mU (SEQ ID NO: 120).

The non-restrictive modified lead sequence used to knock down IL17A is shown below (hg38 coordinates chr6:52189069-52189089): mU*mC*mA*CAGAGGGAUAUCUCUCAGUUUUAGAmGmCmUmAmGmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmGmUmGmGmGmCmAmCmCmGmAmGmUmGmGmGmCmGmGmGmGmGmCmU*mU*mU*mU (SEQ ID NO: 217).

An illustrative modified simulation guide is shown below (hg38 coordinates chr1:0-20): mG*mA*mU*CACGUCGGCCGUUGGCGGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmGmGmCmAmCmGmAmGmUmGmGmGmCmU*mU*mU*mU (SEQ ID NO: 121).

The non-restrictive modified lead sequence used to knock down TGFBR2 is shown below (hg38 coordinates chr3:30671941-30671961): mU*mC*mG*CUUUGCUGAGGUCUAUAGUUUUAGAmGmCmUmAmGmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCACGAAAGGGCACCGAGUCGGmU*mG*mC*mU (SEQ ID NO: 351).

The non-restrictive modified lead sequence used to knock down HLA-A is shown below (hg38 coordinates chr6:29942891-29942915, G034202): mG*mC*mU*mCmUAUmCmCAmCGmGCGCCmCGCGmGCUmGUUGmUmAmGmCUCCCmUmGmAmAmAmCmCGUUmGmCUAmCAAUAAGmGmCCmGmUmCmGmAmAmAmGmUGUGCmCGmCAAmCGCUCUmGmCCmUmUmCmUGGCAUCG*mU*mU (SEQ ID NO: 366), where m is a 2'-OMe modified nucleotide/nucleoside residue, * indicates a phosphate thioester bond between residues, and uppercase letters indicate residues, such as residues containing ribose.

The non-restrictive modified lead sequence used to knock down HLA-B is shown below (hg38 coordinates chr6:31355222-31355246, G034208): mU*mC*mU*mGmGGAmAmAGmGAmGGGGAmAGAUmGAGmGUUGmUmAmGmCUCCCmUmGmAmAmAmCmCGUUmGmCUAmCAAUAAGmGmCCmGmUmCmGmAmAmAmGmUGUGCmCGmCAAmCGCUCUmGmCCmUmUmCmUGGCAUCG*mU*mU (SEQ ID NO: 367), where m is a 2'-OMe modified nucleotide/nucleoside residue, * indicates a phosphate thioester bond between residues, and uppercase letters indicate residues, such as residues containing ribose.

The non-restrictive modified lead sequence used to knock down CIITA is shown below (hg38 coordinates chr16:10906643-10906667, G034619): mA*mG*mC*mUmGCCmGmUUmCUmGCCCAmGUCCmGGGmGUUGmUmAmGmCUCCCmUmGmAmAmAmCmCGUUmGmCUAmCAAUAAGmGmCCmGmUmCmGmAmAmAmGmUGUGCmCGmCAAmCGCUCUmGmCCmUmUmCmUGGCAUCG*mU*mU (SEQ ID NO: 368), where m is a 2'-OMe modified nucleotide/nucleoside residue, * indicates a phosphate thioester bond between residues, and uppercase letters indicate residues, such as residues containing ribose.

The non-restrictive modified lead sequence used to knock down TRAC is shown below (hg38 coordinates Chr14: 22547524-22547544, G025420): mC*mU*mC*UCAGCUGGUACACGGCAGUUUUAGAmGmCmUmAmGmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCACGAAAGGGCACCGAGUCGGGmU*mG*mC*mU (SEQ ID NO: 369), where m is a 2'-OMe modified nucleotide/nucleoside residue, * indicates a phosphate thioester bond between residues, and uppercase letters indicate residues, such as residues containing ribose; or mC*mU*mC*UCAGCUGGUACACGGCAGUUUUAGAmGmCmUmAmGmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmGmGmCmAmCmGmGmGmGmGmGmCmU*mU*mU*mU (SEQ ID NO: 370), where m is a nucleotide/nucleoside residue modified with 2'-OMe, * indicates a phosphate thioester bond between residues, and uppercase letters indicate residues, such as residues containing ribose.

In some embodiments, this paper discloses engineered T cells by introducing or inserting heterologous dmTGFB1 nucleic acid into the genome locus of T cells or T cell populations using a guide RNA, an RNA-guided DNA binder, and a construct (e.g., a donor construct or template) containing heterologous dmTGFB1 nucleic acid (e.g., for the creation of engineered T cells). In some embodiments, this paper discloses engineered T cells by expressing heterologous dmTGFB1 from the genome locus of T cells or T cell populations, for example, using a guide RNA, an RNA-guided DNA binder, and a construct (e.g., a donor) containing heterologous dmTGFB1 nucleic acid. In some embodiments, this document discloses engineered T cells by inducing intragenomic fragmentation (e.g., double-strand fragmentation (DSB) or single-strand fragmentation (notch)) in T cells or T cell populations using, for example, guide RNA and RNA-guided DNA binders (e.g., CRISPR/Cas systems) for insertion into the dmTGFB1 gene. Cells and cell populations prepared by such methods are also provided.

In some embodiments, this document discloses engineered T cells by introducing or inserting heterologous dmTGFB1 nucleic acid into the genome locus of T cells. These T cells are further engineered by introducing or inserting heterologous IL10 nucleic acid into the genome locus of T cells or T cell populations using a guide RNA and an RNA-guided DNA binder, and a construct (e.g., a donor construct or template) containing heterologous IL10 nucleic acid (e.g., for creating engineered T cells). In some embodiments, this document discloses engineered T cells by expressing heterologous IL10 from the genome locus of T cells or T cell populations, for example, using a guide RNA and an RNA-guided DNA binder, and a construct (e.g., a donor) containing heterologous IL10 nucleic acid. In some embodiments, this document discloses engineered T cells by inducing intragenomic fragmentation (e.g., double-strand fragmentation (DSB) or single-strand fragmentation (notch)) in T cells or T cell populations for insertion into the IL10 gene, for example using guide RNA and RNA-guided DNA binders (e.g., CRISPR/Cas systems). Cells and cell populations prepared by such methods are also provided.

In some embodiments, this paper discloses T cells engineered by introducing or inserting heterologous dmTGFB1 nucleic acid into the genome locus of T cells. These T cells are further engineered by introducing or inserting heterologous CTLA4 nucleic acid into the genome locus of T cells or T cell populations using a guide RNA and an RNA-guided DNA binder, and a construct (e.g., a donor construct or template) containing heterologous CTLA4 nucleic acid (e.g., for creating engineered T cells). In some embodiments, this paper discloses T cells engineered by expressing heterologous CTLA4 from the genome locus of T cells or T cell populations, for example, using a guide RNA and an RNA-guided DNA binder, and a construct (e.g., a donor) containing heterologous CTLA4 nucleic acid. In some embodiments, this document discloses engineered T cells by inducing intragenomic fragmentation (e.g., double-strand fragmentation (DSB) or single-strand fragmentation (notch)) in T cells or T cell populations for insertion into the CTLA4 gene, for example using guide RNA and RNA-guided DNA binders (e.g., CRISPR/Cas systems). Cells and cell populations prepared by such methods are also provided.

In some embodiments, this article discloses T cells engineered by introducing or inserting heterologous dmTGFB1 nucleic acid into the genome locus of T cells. These T cells are further engineered by introducing or inserting heterologous CTLA4 nucleic acid and heterologous IL10 nucleic acid into the genome locus of T cells or T cell populations using guide RNA and RNA-guided DNA binders and one or more constructs (e.g., donor constructs or templates) containing heterologous CTLA4 nucleic acid and heterologous IL10 nucleic acid (e.g., to create engineered T cells). In some embodiments, this paper discloses T cells engineered to express heterologous CTLA4 and heterologous IL10 at the genome loci of T cells or T cell populations, for example, using guide RNA and RNA-guided DNA binders and constructs (e.g., donor constructs or templates) containing heterologous CTLA4 and heterologous IL10 nucleic acids. In some embodiments, this paper discloses T cells engineered to induce intragenomic fragmentation (e.g., double-strand fragmentation (DSB) or single-strand fragmentation (notch)) in T cells or T cell populations for insertion into the CTLA4 and IL10 genes, for example, using guide RNA and RNA-guided DNA binders (e.g., CRISPR/Cas systems). In some embodiments, the guide RNA mediates target-specific cleavage of an RNA-guided DNA binder (e.g., a Cas nuclease) at the sites described herein, to allow insertion of sequences encoding two or more regulatory T cell-promoting molecules (e.g., IL10 and CTLA4). It should be understood that in some embodiments, the guide RNA comprises or is capable of binding to such regions. Cells and cell populations prepared by these methods are also provided.

The following provides exemplary nucleotide and polypeptide sequences of regulatory T cell stimulating molecules. Methods for identifying alternative nucleotide sequences (including alternative naturally occurring variants and non-human homologues) encoding polypeptide sequences are known in this art. The following provides exemplary nucleic acid sequences encoding dmTGFB1, IL10, and CTLA4. Other suitable dmTGFB1, IL10, and CTLA4 sequences are known in this art or can be designed based on the disclosures provided herein. For example, see 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 (specifically IL-10 variants), and sequences of these documents are incorporated herein by reference. Methods for identifying alternative IL10 and CTLA4 sequences are also known in this art. For example, see ibid. Gorby, for instance, revealed the anti-inflammatory and pro-cytotoxic activities of IL-10. In addition to its anti-inflammatory activity, recent studies have also shown that IL-10 can increase the cytotoxic function of CD8 T cells, thereby enhancing their ability to target tumors and strengthening anti-cancer responses (Oft, 2019). This seems contradictory, as IL-10 in the tumor microenvironment is associated with tumor evasion of the immune response, most likely due to the inhibitory effect of IL-10 on antigen presentation (Mannino et al., 2015; Yue et al., 1997). Despite this contradiction, several studies have demonstrated that IL-10 can enhance the production of CD8 effector molecules granzyme B and interferon-γ both in vitro and in vivo (Emmerich et al., 2012; Mumm et al., 2011; Mumm and Oft, 2013). Currently, several clinical trials testing the antitumor properties of IL-10 have yielded promising preliminary results (Naing et al., 2019). These trials used high doses of PEGylated IL-10 (pegilodekakin), which prolonged IL-10 retention in circulation to ensure efficacy, again emphasizing that effective in vivo IL-10 responses require high concentrations and sustained levels of IL-10. Saxton et al. used yeast-display-based directed evolution to engineer IL-10 variants. IL-10 is an immunomodulatory interferon with anti-inflammatory and immunostimulatory properties, and it is frequently dysregulated in disease. Mechanistically, IL-10 functions as a secreted homodimer, binding to a two-copy heterodimer receptor complex comprising a specific receptor subunit IL-10Rα and a shared subunit IL-10Rβ. IL-10-dependent dimerization of IL-10Rα and IL-10Rβ initiates activation of the transcription factor STAT3, which mediates a wide range of IL-10 biological effects. Saxton et al. taught a series of IL-10 variants with varying IL-10Rβ binding strengths, revealing substantial differences in response thresholds among immune cell populations, providing a means of manipulating IL-10 cell type selectivity. Saxton et al. have identified a “super 10” variant (D25A/E96A, based on amino acid designation of SEQ ID NO: 231) with enhanced affinity for IL-10Rβ, enabling the assembly of the hexameric IL-10-IL-10Rα-IL-10Rβ complex. Other variants (e.g., D25K, D25A, N21A/R104A, and D25A/N21A/R104A, based on amino acid designation of SEQ ID NO: 231) uncouple the primary opposite function of IL-10 by inhibiting macrophage activation without stimulating inflammatory CD8+ T cells and exhibiting myeloid-like biased activity. In some embodiments, the engineered T cells provided herein use IL-10 variants with impaired immunostimulatory properties (referred to herein as "inhibitory IL-10 variants"), such as variants that maintain myeloid-like activity by inhibiting macrophage activation and exhibit impaired stimulation of inflammatory CD8+ T cells. In some embodiments, the inhibitory IL-10 variants used in the engineered T cells provided herein include substitutions selected from D25K, D25A, D25A/E96A, N21A/R104A, and D25A/N21A/R104A, such as D25A/E96A. The results of Saxton et al. provide a mechanistic blueprint for regulating the pleiotropic effects of IL-10. WO2021243057 provides other inhibitory IL-10 variants, which are incorporated herein by reference, and provides various IL-10 sequences, including peptides with altered binding affinity for IL-10Rβ compared to reference IL-10 peptides lacking one or more amino acid substitutions, as well as methods for characterizing activity. Other variants (including inhibitory IL-10 variants) are revealed and predicted to exhibit myeloid-like biased activity by inhibiting macrophage activation without stimulating inflammatory CD8+ T cells, similar to the D25K, D25A, D25A/E96A, N21A/R104A, and D25A/N21A/R104A variants. The provided embodiments include inhibitory IL-10 variants having one or more amino acid substitutions at the positions corresponding to amino acid residues selected from D25, H14, N18, R24, D28, E74, H90, N92, E96, T100, and R104, and, where appropriate, further substitutions at one or more amino acids selected from N21, M22, R32, and S93. In some embodiments, D25 is substituted with an amino acid selected from K, A, N, H, I, K, or V. In some embodiments, E96 is substituted with an amino acid selected from A, N, D, Q, H, K, or S. Exemplary alternative combinations may include (a) N18Y/N92Q/T100D/R104W; (b) N18Y/N21H/N92Q/E96D/T100V/R104W; (c) N18Y/N21H/E96H/T100V/R104W; (d) N18Y/D25A/N92Q/T100D/R104W; (e) N18Y/D25K/N92Q/T100D/R104W; and (f) N18Y/D25A/N92Q/E96A/T100D/R104W. Exemplary substitutions include (a) D25A; (b) D25K; (c) E96A; (d) E96K; (e) D25A/E96A; (f) N21A/R104A; (g) N21A/D25A; (h) N21A/D25A/E96A; and (i) N21A/M22A/D25A. Sequences that, for example, due to mutation or truncation, have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any of the nucleic acid sequences, amino acid sequences, or nucleic acid sequences encoding amino acid sequences described herein are also considered. In some embodiments, nucleic acid sequences encoding any of the amino acid sequences provided herein are also provided.

Non-limiting exemplary nucleic acid sequences encoding TGFB1 are provided: wild-type TGFB1Atgccgccctccgggctgcggctgctgctgctgctgctaccgctgctgtggctactggtgctgacgcctggccggccggccgcgggactatccacctgcaagactatcgacatggagctg gtgaagcggaagcgcatcgaggccatccgcggccagatcctgtccaagctgcggctcgccagccccccgagccagggggaggtgccgcccggcccgctgcccgaggccgtgctcgccctgtacaacagcacccgcgaccgggtggccggg gagagtgcagaaccggagcccgagcctgaggccgactactacgccaaggaggtcacccgcgtgctaatggtggaaacccacaacgaaatctatgacaagttcaagcagagtacacacagcatatatatgttcttcaacacatcagagctc cgagaagcggtacctgaacccgtgttgctctcccgggcagagctgcgtctgctgaggctcaagttaaaagtggagcagcacgtggagctgtaccagaaatacagcaacaattcctggcgatacctcagcaaccggctgctggcacccagc gactcgccagagtggttatcttttgatgtcaccggagttgtgcggcagtggttgagccgtggaggggaaattgagggctttcgccttagcgcccactgctcctgtgacagcagggataacacactgcaagtggacatcaacgggttcact accggccgccgaggtgacctggccaccattcatggcatgaaccggcctttcctgcttctcatggccaccccgctggagaggggcccagcatctgcaaagctcccggcaccgccgagccctggacaccaactattgcttcagctccacggag aagaactgctgcgtgcggcagctgtacattgacttccgcaaggacctcggctggaagtggatccacgagcccaagggctaccatgccaacttctgcctcgggcctgcccctacatttggagcctggacacgcagtacagcaaggtcctg gccctgtacaaccagcataacccgggcgcctcggcggcgccgtgctgcgtgccgcaggcgctggagccgctgcccatcgtgtactacgtgggccgcaagcccaaggtggagcagctgtccaacatgatcgtgcgctcctgcaagtgcagc (SEQ ID NO: 204)

Double mutant (dm) TGFB1 (R218H, C225R)Atgccgccctccgggctgcggctgctgctgctgctgctaccgctgctgtggctactggtgctgacgcctggccggccggccgcgggactatccacctgcaagactatcgacatggagctggtgaagcggaagcg catcgaggccatccgcggccagatcctgtccaagctgcggctcgccagccccccgagccagggggaggtgccgcccggcccgctgcccgaggccgtgctcgccctgtacaacagcacccgcgaccgggtggccggggagagtgcagaa ccggagcccgagcctgaggccgactactacgccaaggaggtcacccgcgtgctaatggtggaaacccacaacgaaatctatgacaagttcaagcagagtacacacagcatatatatgttcttcaacacatcagagctccgagaagcgg tacctgaacccgtgttgctctcccgggcagagctgcgtctgctgaggctcaagttaaaagtggagcagcacgtggagctgtaccagaaatacagcaacaattcctggcgatacctcagcaaccggctgctggcacccagcgactcgcc agagtggttatcttttgatgtcaccggagttgtgcggcagtggttgagccgtggaggggaaattgagggctttCACcttagcgcccactgctccAGAgacagcagggataacacactgcaagtggacatcaacgggttcactaccggc cgccgaggtgacctggccaccattcatggcatgaaccggcctttcctgcttctcatggccaccccgctggagaggggcccagcatctgcaaagctcccggcaccgccgagccctggacaccaactattgcttcagctccacggagaaga actgctgcgtgcggcagctgtacattgacttccgcaaggacctcggctggaagtggatccacgagcccaagggctaccatgccaacttctgcctcgggccctgcccctacatttggagcctggacacgcagtacagcaaggtcctggc cctgtacaaccagcataacccgggcgcctcggcggcgccgtgctgcgtgccgcaggcgctggagccgctgcccatcgtgtactacgtgggccgcaagcccaaggtggagcagctgtccaacatgatcgtgcgctcctgcaagtgcagc (SEQ ID NO: 208)

Non-limiting exemplary amino acid sequence of human TGFB1 wild type MPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPSQGEVPPGPLPEAVLAL YNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLS NRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCF SSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEEPLPIVYYVGRKPKVEQLSNMIVRSCKCS (SEQ ID NO: 210)

dmTGFB1 R218C, C225RMPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDS PEWLSFDVTGVVRQWLSRGGEIEGFHLSAHCSRDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS (SEQ ID NO: 214)

Provides a non-restrictive illustrative nucleic acid sequence encoding IL10: Wild-type IL10: ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCT GAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCC TGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAAC (SEQ ID NO: 122)

High affinity IL10 (N36I, N110I, K117N, F129L): ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCATCCTGCCT AACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGA GATGATCCAGTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGatcTCCCTGGGGGAGAACCTGAATACCCTCAGGCTGAGGCTACGGCGCTGTCATCGActcCTTCCCT GTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAAC (SEQ ID NO: 123)

Provides a non-restrictive illustrative amino acid sequence for IL10: Wild-type IL10: MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 124)

High affinity IL10 (N36I, N110I, K117N, F129L): MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGILPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKG YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVISLGENLNTLRLRLRRCHRLLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 125)

Mature wild-type IL10SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 231)

IL10 (D25K)SPGQGTQSENSCTHFPGNLPNMLRKLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 232)

IL10 (D25E)SPGQGTQSENSCTHFPGNLPNMLRELRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 233)

IL10 (D25K/E96A)SPGQGTQSENSCTHFPGNLPNMLRKLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGANLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 234)

Provided non-restrictive exemplary nucleic acid sequences encoding TGFB1: wild-type TGFB1 (see paragraph [000145] above and SEQ ID NO: 204) double mutant TGFB1 (R218C, C225R) Atgccgccctccgggctgcggctgctgctgctgctgctaccgctgctgtggctactggtgctgacgcctggccggccggccgcgggactatccacctgcaagactatcgacatggagctggtgaagcggaagcgcatcgaggccatccgcggccagatcctgtccaagctgcggctcgccagccccccgagccagggggaggtgccgcccggcccgctgcccgaggccgtgctcgccctgtacaacagcacccgcgaccaccgggtggccggggagagtgcagaa ccggagcccgagcctgaggccgactactacgccaaggaggtcacccgcgtgctaatggtggaaacccacaacgaaatctatgacaagttcaagcagagtacacacagcatatatatgttcttcaacacatcagagctccgagaagcgg tacctgaacccgtgttgctctcccgggcagagctgcgtctgctgaggctcaagttaaaagtggagcagcacgtggagctgtaccagaaatacagcaacaattcctggcgatacctcagcaaccggctgctggcacccagcgactcgcc agagtggttatcttttgatgtcaccggagttgtgcggcagtggttgagccgtggaggggaaattgagggctttTGCcttagcgcccactgctccAGAgacagcagggataacacactgcaagtggacatcaacgggttcactaccggc cgccgaggtgacctggccaccattcatggcatgaaccggcctttcctgcttctcatggccaccccgctggagaggggcccagcatctgcaaagctcccggcaccgccgagccctggacaccaactattgcttcagctccacggagaaga actgctgcgtgcggcagctgtacattgacttccgcaaggacctcggctggaagtggatccacgagcccaagggctaccatgccaacttctgcctcgggccctgcccctacatttggagcctggacacgcagtacagcaaggtcctggc cctgtacaaccagcataacccgggcgcctcggcggcgccgtgctgcgtgccgcaggcgctggagccgctgcccatcgtgtactacgtgggccgcaagcccaaggtggagcagctgtccaacatgatcgtgcgctcctgcaagtgcagc (SEQ ID NO: 352)

The double mutant TGFB1 (R218H, C225R) (see paragraph [000347] above and SEQ ID NO: [214]).

Non-limiting exemplary nucleic acid sequences encoding CTLA4 are provided: Wild-type CTLA4: ATGGCCTGCTTGGGCTTCCAAAGGCATAAAGCCCAGCTTAATCTTGCTACTCGCACGTGGCCCTGCACATTGCTCTTTTTCCTCCTGTTCATTCCCGTGTTTTGCAAGGCGATGCATGTGGCACAACCTGCCGTCGTTCTGGCA TCATCAAGAGGTATTGCTAGCTTCGTTTGTGAGTACGCCTCCCCTGGAAAAGCGACGGAGGTGCGCGTCACTGTATTGCGGCAAGCCGACAGCCAAGTTACTGAAGTCTGCGCGGCAACGTATATGATGGGCAATGAGCTGACATTCCTTGACGATTCAATCTGCACGGGAACAA GTAGTGGTAACCAGGTGAATCTCACTATTCAAGGTCTGAGAGCCATGGACACCGGCCTCTACATTTGTAAGGTGGAGCTGATGTATCCTCCCCCATATTATCTGGGGATCGGAAATGGGACACAGATATATGTTATTGATCCCGAGCCATGTCCCGATAGTGACTTCCTCTTGTG GATACTTGCCGCTGTGAGCAGTGGTTTGTTTTTTTCATTCCCTCTTACGGCAGTATCACTTTCAAAAATGCTCAAGAAGCGAAGTCCTTTGACAACTGGCGTATATGTCAAAATGCCACCAACAGAGCCCGAATGTGAGAAACAGTTCCAGCCGTACTTTATTCCTATAAAC (SEQ ID NO: 126)

High affinity CTLA4 (belatacept; binding domain: A29Y, L104E): ATGGCCTGCTTGGGCTTCCAAAGGCATAAAGCCCAGCTTAATCTTGCTACTCGCACGTGGCCCTGCACATTGCTCTTTTTCCTCCTGTTCATTCCCGTGTTTTGCAAGGCGATGCATGTGGCACAACCTGCCGTCGTTCT GGCATCATCAAGAGGTATTGCTAGCTTCGTTTGTGAGTACGCCTCCCCTGGAAAATACACGGAGGTGCGCGTCACTGTATTGCGGCAAGCCGACAGCCAAGTTACTGAAGTCTGCGCGGCAACGTATATGATGGGCAATGAGCTGACATTCCTTGACGATTCAATCTGCACGGGAA CAAGTAGTGGTAACCAGGTGAATCTCACTATTCAAGGTCTGAGAGCCATGGACACCGGCCTCTACATTTGTAAGGTGGAGCTGATGTATCCTCCCCCATATTATGAGGGGATCGGAAATGGGACACAGATATATGTTATTGATCCCGAGCCATGTCCCGATAGTGACTTCCTCTTG TGGATACTTGCCGCTGTGAGCAGTGGTTTGTTTTTTTATTCATTCCTCCTTACGGCAGTATCACTTTCAAAAATGCTCAAGAAGCGAAGTCCTTTGACAACTGGCGTATATGTCAAAATGCCACCAACAGAGCCCGAATGTGAGAAACAGTTCCAGCCGTACTTTATTCCTATAAAC (SEQ ID NO: 127)

High affinity CTLA4 (Binding domain: A29H): ATGGCCTGCTTGGGCTTCCAAAGGCATAAAGCCCAGCTTAATCTTGCTACTCGCACGTGGCCCTGCACATTGCTCTTTTTCCTCCTGTTCATTCCCGTGTTTTGCAAGGCGATGCATGTGGCACAACCTGCCGTCGTTCTGGCATCATCAAGAGGTA TTGCTAGCTTCGTTTGTGAGTACGCCTCCCCTGGAAAACATACGGAGGTGCGCGTCACTGTATTGCGGCAAGCCGACAGCCAAGTTACTGAAGTCTGCGCGGCAACGTATATGATGGGCAATGAGCTGACATTCCTTGACGATTCAATCTGCACGGGAACAAGTAGTGGTA ACCAGGTGAATTCACTATTCAAGGTCTGAGAGCCATGGACACCGGCCTCTACATTTGTAAGGTGGAGCTGATGTATCCTCCCCCATATTATCTGGGGATCGGAAATGGGACACAGATATATGTTATTGATCCCGAGCCATGTCCCGATAGTGACTTCCTTCTGTGGATA CTTTGCCGCTGTGAGCAGTGGTTTGTTTTTTTCATTCCTCCTACGGCAGTATCACTTTCAAAAATGCTCAAGAAGCGAAGTCCTTTGACAACTGGCGTATATGTCAAAATGCCACCAACAGAGCCCGAATGTGAGAAACAGTTCCAGCCGTACTTTATTCCTATAAAC (SEQ ID NO: 128)

High affinity CTLA4 (Binding domain: K28H, A29H): ATGGCCTGCTTGGGCTTCCAAAGGCATAAAGCCCAGCTTAATCTTGCTACTCGCACGTGGCCCTGCACATTGCTCTTTTTCCTCCTGTTCATTCCCGTGTTTTGCAAGGCGATGCATGTGGCACAACCTGCCGTCGTTCTGGCATCATCAAGA GGTATTGCTAGCTTCGTTTGTGAGTACGCCTCCCCTGGACATCACACGGAGGTGCGCGTCACTGTATTGCGGCAAGCCGACAGCCAAGTTACTGAAGTCTGCGCGGCAACGTATATGATGGGCAATGAGCTGACATTCCTTGACGATTCAATCTGCACGGGAACAAGTAGTG GTAACCAGGTGAATCTCACTATTCAAGGTCTGAGAGCCATGGACACCGGCCTCTACATTTGTAAGGTGGAGCTGATGTATCCTCCCCCATATTATCTGGGGATCGGAAATGGGACACAGATATATGTTATTGATCCCGAGCCATGTCCCGATAGTGACTTCCTTCTTGTGGAT ACTTGCCGCTGTGAGCAGTGGTTTGTTTTTTTATTCATTCCTCCTTACGGCAGTATCACTTTCAAAAATGCTCAAGAAGCGAAGTCCTTTGACAACTGGCGTATATGTCAAAATGCCACCAACAGAGCCCGAATGTGAGAAACAGTTCCAGCCGTACTTTATTCCTATAAAC (SEQ ID NO: 129)

Provided is a non-restrictive illustrative amino acid sequence of CTLA4: Wild-type CTLA4: MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO: 130)

High affinity CTLA4 (Belatacept; binding domain: A29Y, L104E): MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLD DSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO: 131)

High affinity CTLA4 (Binding domain: A29H): MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKHTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTG TSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO: 132)

High affinity CTLA4 (Binding domain: K28H, A29H): MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGHHTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSI CTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO: 133)

In some embodiments, the engineered T cells or T cell populations containing a heterologous sequence encoding dmTGFB1 under promoter control; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TGFBR2, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; and the engineered T cells or T cell populations with a heterologous sequence encoding a regulatory T cell-promoting molecule under promoter sequence control inserted into the cells exhibit at least one inhibitory activity of naturally occurring regulatory T cells (nTregs), for example, inhibiting immune responses or biomarkers in in vitro or in vivo analyses (e.g., animal models of GvHD). For example, in a mouse GvHD model, mice receiving engineered T cells containing the following components exhibited improved survival compared to controls (e.g., mice receiving PBMCs): a heterologous sequence encoding dmTGFB1 under promoter control; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TGFBR2; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; and insertion of a heterologous sequence encoding a regulatory T cell-promoting molecule under promoter sequence control into the cells. In some embodiments, the regulatory T cell-promoting molecule includes IL10 or CTLA4. In some embodiments, the regulatory T cell-promoting molecule includes both IL10 and CTLA4.

In some embodiments, the engineered T cells or T cell populations contain two or more heterologous coding sequences under promoter control, such as dmTGFB1 and regulatory T cell-promoting molecules. In some embodiments, each heterologous coding sequence is under the control of a separate promoter. In some embodiments, two heterologous coding sequences are under the control of the same promoter. In some embodiments, two or more heterologous coding sequences are under the control of the same promoter. In some embodiments, when the engineered T cells or T cell populations contain three or more heterologous coding sequences (e.g., dmTGFB1 and regulatory T cell stimulating molecules) under promoter control, each heterologous sequence is independently under the control of a single promoter or under the control of a promoter that controls the expression of more than one heterologous coding sequence.

C. T cell receptor (TCR) In some embodiments, it includes engineered T cells or T cell populations that have been modified (e.g., inserted into cells with a heterologous sequence encoding the dmTGFB1 molecule under the control of a promoter sequence), and further include modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding the TCR gene sequence and modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding the TGFBR2 gene sequence.

In some embodiments, engineered T cells or T cell populations may include modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding a dmTGFB1 molecule inserted into cells under the control of a promoter sequence, further including modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG; and/or modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding a regulatory T cell-promoting molecule inserted into cells under the control of a promoter sequence.

In some embodiments, the engineered T cells or T cell populations include modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding TGFBR2; modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding TNFA; modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding IFNG; and the insertion of heterologous sequences encoding dmTGFB1 molecules and regulatory T cell-promoting molecules, each under the control of a promoter sequence, into the cells, further including modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding the TCR gene sequence.

In some embodiments, the engineered T cells or T cell populations include modifications such as inserting a heterologous sequence encoding a dmTGFB1 molecule under the control of a promoter sequence into the cells; modifications such as (e.g., knockdown) of endogenous nucleic acid sequences encoding TGFBR2; modifications such as (e.g., knockdown) of endogenous nucleic acid sequences encoding TNFA; modifications such as (e.g., knockdown) of endogenous nucleic acid sequences encoding IFNG; inserting heterologous sequences encoding dmTGFB1 molecules and regulatory T cell-promoting molecules, each under the control of a promoter sequence, into the cells; inserting heterologous sequences encoding target receptors into the cells; and further modifications such as (e.g., knockdown) of endogenous nucleic acid sequences encoding TCR gene sequences.

Typically, TCRs are heterodimeric receptor molecules containing two TCR polypeptide chains (i.e., α and β). Suitable α and β gene sequences or loci for targeted knockdown are known in this art. In some embodiments, engineered T cells contain modifications (e.g., knockdown) of the TCR α-chain gene sequence (e.g., TRAC). See, for example, NCBI gene ID: 28755; Ensembl: ENSG00000277734 (T cell receptor α-constancy), US 2018/0362975, and WO2020081613.

In some embodiments, the engineered T cells or T cell populations contain modifications that include a sequence containing the insertion code dmTGFB1.

In some embodiments, engineered T cells or T cell populations that include the insertion of a heterologous sequence encoding the dmTGFB1 molecule under the control of a promoter sequence and modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding TGFBR2 further include modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding TNFA; modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding IFNG; insertion of sequences encoding regulatory T cell-promoting molecules selected from IL10, CTLA4, TIGIT, IDO1, ENTPD1, NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, BACH2, and IL2RA; and modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding the TCR gene sequence.

In some embodiments, the engineered T cells or T cell populations comprising inserting a heterologous sequence encoding the dmTGFB1 molecule under the control of a promoter sequence and modifying (e.g., knocking down) the endogenous nucleic acid sequence encoding TGFBR2 further comprising a combination of modifying (e.g., knocking down) the endogenous nucleic acid sequence encoding TNFA and modifying (e.g., knocking down) the endogenous nucleic acid sequence encoding IFNG; inserting sequences encoding regulatory T cell-promoting molecules selected from IL10, CTLA4, TIGIT, IDO1, ENTPD1, NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, BACH2 and others; and modifying (e.g., knocking down) the endogenous nucleic acid sequence encoding the TCR gene sequence.

In some embodiments, engineered T cells or T cell populations that include the insertion of a heterologous sequence encoding the dmTGFB1 molecule under the control of a promoter sequence and modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding TGFBR2 further include modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG; insertion of a sequence encoding IL10 or CTLA4; and modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding the TCR gene sequence.

In some embodiments, the engineered T cells or T cell populations include modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding TGFBR2; modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding TNFA; modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding IFNG; insertion of sequences encoding dmTGFB1, IL10, and CTLA4; and modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding the TCR gene sequence.

In some embodiments, the engineered T cells or T cell populations include modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding TGFBR2; modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding TNFA; modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding IFNG; insertion of a sequence encoding the dmTGFB1 molecule; insertion of a sequence encoding a regulatory T cell-promoting molecule; and modifications (e.g., knockdown) of the endogenous TCR gene (e.g., the TRAC gene sequence).

In some embodiments, the engineered T cells or T cell populations include modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding TGFBR2; modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding TNFA; modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding IFNG; insertion of a sequence encoding the dmTGFB1 molecule; insertion of sequences encoding regulatory T cell-promoting molecules selected from IL10, CTLA4, TIGIT, IDO1, ENTPD1, NT5E, IL22, AREG, IL35, GARP, CD274, FOXP3, IKZF2, EOS, IRF4, LEF1, BACH2, and IL2RA; and modifications (e.g., knockdown) of the endogenous TCR gene (e.g., the TRAC gene sequence).

In some embodiments, the engineered T cells or T cell populations include modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding TGFBR2; modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding TNFA; modifications (e.g., knockdown) of the endogenous nucleic acid sequence encoding IFNG; insertion of a sequence encoding dmTGFB1, insertion of a sequence encoding IL10 or CTLA4, and modifications (e.g., knockdown) of TCR genes (e.g., TRAC gene sequences).

In any of these embodiments, the engineered T cells or T cell population may further include an insert sequence encoding a target receptor as described herein, such as a CAR, targeting, for example, the following CARs: MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), protein lipoprotein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), stromal core protein 3 (DSG3), or MHC class I HLA-A (HLA-A*02).

In some embodiments, engineered T cells or T cell populations comprising inserting a heterologous sequence encoding a dmTGFB1 molecule under the control of a promoter sequence into cells further comprise modifications (e.g., knockdown) of the TRC gene sequence by gene editing, such as 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 contain insertions, deletions, or substitutions in the endogenous TRC gene sequence. In some embodiments, compared to a suitable control (e.g., where the TRC gene is not modified), the TRC is reduced by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or reduced to below the detection limit of the analysis. Analysis of TRC protein and mRNA expression is known in this technique.

In some embodiments, such as those evaluated by sequencing (e.g., NGS), engineered T cells or T cell populations contain sequences that encode target receptors through gene editing insertion.

In some embodiments, the T cell population comprises T cells containing a heterologous sequence encoding a dmTGFB1 molecule inserted into the cell under the control of a promoter sequence, and these T cells are further engineered to include modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding TGFBR2. In some embodiments, the T cell population further comprises T cells that: modify (e.g., knockdown) an endogenous nucleic acid sequence encoding TNFA; modify (e.g., knockdown) an endogenous nucleic acid sequence encoding an insert sequence, wherein the insert sequence(e.g.) encodes a regulatory T cell-promoting molecule; and/or modify (e.g., knockdown) at least one TCR gene sequence. In some embodiments, such as those evaluated by sequencing (e.g., NGS), 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 modifications (e.g., knockdown) of at least one TCR gene sequence.

In some embodiments, the T cell population comprises T cells containing a heterologous sequence encoding a dmTGFB1 molecule inserted into the cell under the control of a promoter sequence and a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TGFBR2. These T cells are further engineered to include modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG; an inserted sequence encoding a regulatory T cell-promoting molecule; and modifications (e.g., knockdown) of at least one TCR gene sequence. In some embodiments, such as those evaluated by sequencing (e.g., NGS), 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 modifications (e.g., knockdown) of at least one TCR gene sequence.

In some embodiments, guide RNAs that specifically target sites within TCR genes (e.g., TRAC genes) are used to provide modifications to the TCR genes (e.g., knockdown).

In some embodiments, guide RNA and RNA-guided DNA binders are used to modify (e.g., knock down) the TCR gene in T cells. In some embodiments, this paper discloses T cells engineered by inducing breakage (e.g., double-strand breakage (DSB) or single-strand breakage (notch)) within the TCR gene of T cells, for example, using guide RNA and RNA-guided DNA binders (e.g., CRISPR/Cas systems). These methods can be used in vitro or in vitro, for example, to manufacture cell products for suppressing immune responses.

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

D. Guide RNA In any embodiment herein, the guide RNA may further comprise trRNA. In each of the compositions and methods described herein, crRNA and trRNA may be coupled as a single RNA (sgRNA) or may be coupled to a single RNA (dgRNA). In the context of sgRNA, the crRNA and trRNA components may be covalently linked, for example, via phosphodiester bonds or other covalent bonds. In some embodiments, the sgRNA contains one or more non-phosphodiester bonds between nucleotides.

In each of the compositions, uses, and method embodiments described herein, the guide RNA may comprise two RNA molecules as "double guide RNAs" or "dgRNAs". The dgRNA comprises a first RNA molecule containing crRNA (containing, for example, the guide sequence shown herein) and a second RNA molecule containing trRNA. The first and second RNA molecules may not be covalently linked, but may form an RNA doublet through base pairing between portions of the crRNA and trRNA.

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

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

In some embodiments, the target sequence or region may be complementary to the lead sequence of the guide RNA. In some embodiments, the complementarity or similarity between the lead 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 lead sequence of the gRNA may be 100% complementary or similar. In other embodiments, the target sequence and the lead sequence of the gRNA may contain one mismatch. For example, the target sequence and the lead sequence of the gRNA may contain 1, 2, 3, 4, or 5 mismatches, wherein the total length of the lead sequence is about 20 or 20 nucleotides. In some embodiments, the target sequence and the lead sequence of the gRNA may contain 1-4 mismatches, wherein the lead sequence is about 20 or 20 nucleotides. In some embodiments, such as when the lead sequence comprises a sequence of 24 adjacent nucleotides, the complementarity or similarity between the lead sequence and its corresponding target sequence is at least 80%, 85%, 90%, or 95%. In some embodiments, the lead sequence and target region may be 100% complementary or similar. In other embodiments, the lead sequence and target region may contain at least one mismatch, i.e., one nucleotide that is inconsistent or non-complementary, depending on the reference sequence. For example, the lead sequence and target sequence may contain 1-2, for example, no more than 1, mismatches, wherein the total length of the target sequence is 19, 20, 21, 22, 23, or 24 or more nucleotides. In some embodiments, the lead sequence and target region may contain 1-2 mismatches, wherein the lead sequence comprises at least 24 or more nucleotides. In some embodiments, the lead sequence and target region may contain 1-2 mismatches, wherein the lead sequence contains 24 nucleotides.

In any embodiment of this document, each guide sequence may further include additional nucleotides to form crRNA or guide RNA, for example having the following exemplary nucleotide sequence after the 3' end of the guide sequence: GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 134), oriented from 5' to 3'.

In the case of sgRNA, the aforementioned lead sequence may further include additional nucleotides to form sgRNA, for example, having the following exemplary nucleotide sequences after the 3' end of the lead sequence: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 135); GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 136); GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGCACCGAGUCGGUGC (SEQ ID NO: 136). 200);GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCACGAAAGGGCACCGAGUCGGUGC (SEQ ID NO: 201); (SEQ ID NO: 202); GUUGUAGCUCCCUGAAACCGUUGCUACAAUAAGGCCGUCGAAAGAUGUGCCGCAACGCUCUGCCUUCUGGCAUCGUU (SEQ ID NO: 203).

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

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

In some embodiments, the guide RNA sequence comprises a sequence complementary to a sequence selected from the gene body region in the table below, based on coordinates in the human reference gene body 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 the gene body region selected from the table below. 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 genosome region selected from the tables herein.

This article reveals a guide RNA-mediated target-specific cleavage that produces double-stranded (DSB) cuts. This article also reveals a guide RNA-mediated target-specific cleavage that produces single-stranded (SSB) cuts.

E. Chemically Modified gRNA In some embodiments, the gRNA is chemically modified. A gRNA containing one or more modified nucleosides or nucleotides is called a "modified" gRNA or "chemically modified" gRNA to describe the presence of one or more non-natural or naturally occurring components or configurations that replace or supplement the normative A, G, C, and U residues. In some embodiments, the modified gRNA is synthesized from non-normative nucleosides or nucleotides; this is referred to herein as "modified". Modified nucleosides and nucleotides may include one or more of the following: (i) alteration (e.g., substitution) of one or two non-linked phosphodiester oxygens or one or more linked phosphodiester oxygens in the phosphodiester backbone (illustrated backbone modification); (ii) alteration (e.g., substitution) of the ribose (e.g., the 2' hydroxyl group on the ribose) (illustrated sugar modification); (iii) modification or substitution of native nucleobases, including the use of non-standard nucleobases (illustrated base modification); and (iv) modification of the 3' or 5' end of oligonucleotides to provide external nuclease stability, such as ribose modification with 2' O-me, 2' halogen, or 2' deoxygenated substitution; or reverse abase-free terminal nucleotides, or substitution of phosphodiester with thiophosphate.

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

In some embodiments, the lead RNA disclosed herein contains one of the modification patterns disclosed in WO2018107028, the contents of which are incorporated herein by reference. In some embodiments, the lead RNA disclosed herein contains one of the structural/modification patterns disclosed in US20170114334, which is incorporated herein by reference. In some embodiments, the lead RNA disclosed herein contains one of the structural/modification patterns disclosed in WO2017136794, WO2017004279, WO2019237069, US2018187186, US2019048338, WO2021119275, or WO2022125968, which are incorporated herein by reference.

F. mRNA encoding RNA-guided DNA binders In some embodiments, the cell or method comprises mRNA containing an open reading frame (ORF) encoding an RNA-guided DNA binder (such as a Cas nuclease as described herein). A Cas9 ORF is provided herein and is known in the art. As an example, the 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 codon" refers to a variation in the codon usage for a given amino acid and may or may not be a better or optimized codon for a given expression system (codon optimization). Better codon usage or codons that are well-tolerant in a given expression system are known in the art. The Cas9 coding sequences, Cas9 mRNA, and Cas9 protein sequences of WO2013/176772, WO2014/065596, WO2016/106121, WO2019/067910, and WO2022/125968 are incorporated herein by reference. Specifically, the ORF and Cas9 amino acid sequences in the table of paragraph [0449] of WO2019/067910, and the Cas9 mRNA and ORF in paragraphs [0214]-[0234] of WO2019/067910 are incorporated herein by reference.

In some embodiments, the modified ORF may contain modified uridine at at least one, a plurality of, or all of the uridine positions. In some embodiments, the modified uridine is a uridine modified at the 5-position, for example, by a halogen, methyl, or ethyl group. In some embodiments, the modified uridine is a pseudouridine modified at the 1-position, for example, by a halogen, methyl, or ethyl group. The modified uridine may be, for example, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine, or combinations thereof. In some embodiments, the modified uridine is 5-methoxyuridine. In some embodiments, the modified uridine is 5-iodouridine. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of N1-methyl-pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5-methoxyuridine.

In some embodiments, the mRNA disclosed herein contains a 5' cap, such as cap 0, cap 1, or cap 2. The 5' cap is typically a 7-methylguanine ribonucleotide (which may be further modified, for example, ARCA (anti-reverse cap analog; Thermo Fisher Scientific catalog number AM8045) is a cap analog containing 7-methylguanine 3'-methoxy-5'-triphosphate linked to the 5' position of the guanine ribonucleotide), which is linked via the 5'-triphosphate to the 5' position of the first nucleotide in the 5' to 3' chain of the mRNA, i.e., the proximal nucleotide of the first cap. In cap 0, both the first and second proximal nucleotides of the mRNA contain a 2'-hydroxyl group in their ribose. In cap 1, the first and second transcription nucleotides of the mRNA contain a 2'-methoxyl and a 2'-hydroxyl group in their ribose, respectively. For example, see CleanCap ^™ AG (m7G(5')ppp(5')(2'OMeA)pG; TriLink Biotechnologies, catalog number N-7113) or CleanCap ^™ GG (m7G(5')ppp(5')(2'OMeG)pG; TriLink Biotechnologies, catalog number N-7133). In cap 2, the ribose of both the first and second cap proximal nucleotides of the mRNA contains a 2'-methoxy group. For example, see Katibah et al. (2014) Proc Natl Acad Sci USA 111(33):12025-30; Abbas et al. (2017) Proc Natl Acad Sci USA 114(11):E2106-E2115.

In some embodiments, the mRNA further includes a polyadenylated (poly-A) tail. In some embodiments, the poly-A tail contains 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenine nucleotides (SEQ ID NO: 147), and in some cases up to 300 adenine nucleotides (SEQ ID NO: 148). In some embodiments, the poly-A tail contains 95, 96, 97, 98, 99, or 100 adenine nucleotides (SEQ ID NO: 149).

G. Engineered T Cells The engineered cells provided herein are prepared from a T cell-rich cell population. In some embodiments, the T cells are CD4+ T cells. CD4+ T cells can be autologous or allogeneic cells. These cells can be readily obtained from fresh leukopak samples, which are commercially available from various sources, including, for example, StemCell Technologies. CD4+ T cells can be isolated using commercially available kits using conventional methods, such as using a human CD4+ T cell isolation kit with negative selection. However, methods for preparing CD4+ T cells from other sources are also known in this art. For example, pluripotent cells such as hematopoietic stem cells (HSCs, such as those isolated from bone marrow or umbilical cord blood), hematopoietic progenitor cells (e.g., lymphoid progenitor cells), or mesenchymal stem cells (MSCs) can be used to obtain CD4+ T cells. Pluripotent cells can develop into more than one cell type, but their differentiation range is more limited than that of highly fertile cells. Pluripotent cells can be derived from established cell lines or isolated from human bone marrow or umbilical cord blood. For example, HSCs can be isolated from patients or healthy donors after G-CSF-induced motility, plerixafor-induced motility, or a combination thereof. To isolate HSCs from blood or bone marrow, cells in the blood or bone marrow can be pan-selected using antibodies conjugated to unwanted cells, such as antibodies against CD4 and CD8 (T cells), CD45 (B cells), GR-I (granulocytes), and Iad (differentiation antigen-presenting cells) (e.g., see Inaba et al. (1992) J Exp Med. 176: 1693-1702). Methods for promoting differentiation into CD4+ T cells are known in this technique. Compared to amplifying tTreg using methods known in this technique, such as measurements taken at 8, 9, 10, 11, 12, 13, 14, and 15 days after the first day of editing, the CD4+ T cell-engineered T cells described herein can produce, for example, more than 500, 600, 700, 800, 900, or 1000 times more cells.

In some embodiments, the engineered cells are a population rich in CD8+ T cells. CD8+ T cells can be autologous or allogeneic cells. Like CD4+ T cells, CD8+ T cells can be isolated from a sample (e.g., a commercially available sample or a patient sample) or prepared from other sources using methods known in this art. For example, sources may include pluripotent cells, such as hematopoietic stem cells (HSCs, such as those 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 this art. Compared to amplifying tTreg using methods known in this technique, such as measurements taken at 8, 9, 10, 11, 12, 13, 14, and 15 days after the first day of editing, the CD8+ T cell-engineered T cells described herein can produce, for example, more than 500, 600, 700, 800, 900, or 1000 times more cells.

III. Delivery Methods The guide RNA, RNA-guided DNA binder (e.g., Cas nuclease), and nucleic acid sequence disclosed herein can be delivered in vitro or in vitro to cells or cell populations using various known and suitable methods available in this technique to generate engineered T cells containing an insert sequence encoding dmTGFB1; and, where applicable, further containing modifications (e.g., knockdown) of endogenous nucleic acid sequences encoding TNFA, modifications (e.g., knockdown) of endogenous nucleic acid sequences encoding IFNG or IL17A, insert sequences encoding regulatory T cell-promoting molecules (e.g., IL10, CTLA4); and, where applicable, further containing insert sequences encoding target receptors (e.g., CAR), and, where applicable, further containing modifications (e.g., knockdown) of TCR sequences. Guide RNA, RNA-guided DNA binders, and nucleic acid constructs can be delivered individually or in any combination, using the same or different delivery methods as appropriate.

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

Non-viral methods and combinations used for nucleic acid delivery include electroporation, liposome transfection, microinjection, gene gun, virions, liposomes, immunoliposomes, LNPs, polycationic or lipid-nucleic acid conjugates, naked nucleic acids (e.g., naked DNA/RNA), artificial viral particles, and agent-enhanced DNA uptake. Sonoporation using systems such as the Sonitron 2000 (Rich-Mar) can also be used for nucleic acid delivery.

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

In some embodiments, this disclosure provides DNA or RNA vectors encoding any of the compositions disclosed herein, such as lead RNAs containing one or more lead sequences described herein, for example, for modifying (e.g., knocking down) TGFBR2, IFNG, and TNFA, or donor constructs containing sequences encoding the dmTGFB1 molecule, sequences encoding regulatory T cell stimulators such as IL10 or CTLA4, or targeting receptors such as CARs such as MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), and protein lipoprotein 1. The vector may contain PLP1, CD19 (CD19), CD20 (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute vector family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), stromal core protein 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CAR. In some embodiments, the vector may also contain a sequence encoding an RNA-guided DNA binder. In some embodiments, the DNA or RNA vector encodes any one or more of the compounds or any combination thereof described herein. In some embodiments, the vector may further contain, for example, promoters, enhancers, and regulatory sequences. In some embodiments, the vector containing a guide RNA having one or more of the guide sequences described herein also contains one or more nucleotide sequences encoding crRNA, trRNA, or crRNA and trRNA as disclosed herein.

In some embodiments, this disclosure provides DNA or RNA vectors that encode regulatory T cell-stimulating molecules and targeting receptors. Such vectors allow cell selection based on the presence of receptors in cells that also contain the coding sequence of the regulatory T cell-stimulating molecule. Positive and negative selection methods based on the presence of molecules on the cell surface are known in this art.

In some embodiments, the vector contains a nucleotide sequence encoding the guide RNA described herein. In some embodiments, the vector contains one copy of the guide RNA. In other embodiments, the vector contains more than one copy of the guide RNA. In embodiments having more than one guide RNA, the guide RNAs may be non-uniform, targeting different target sequences, or they may be uniform, targeting the same target sequence. In some embodiments where the vector contains more than one guide RNA, each guide RNA may have other different properties, such as activity or stability within RNA-guided DNA nuclease complexes (such as Cas RNP complexes). In some embodiments, the nucleotide sequence encoding the guide RNA may be operatively linked to at least one transcriptional or translational control sequence, such as a promoter, 3' UTR, or 5' UTR. In one embodiment, the promoter may be a tRNA promoter, such as tRNA ^Lys3 , or a tRNA chimera. See Mefferd et al., RNA . 2015 21:1683-9; Scherer et al., Nucleic Acids Res . 2007 35: 2620-2628. In some embodiments, the promoter may be recognized by RNA polymerase III (Pol III). Non-restrictive examples of Pol III promoters include the U6 and H1 promoters. In some embodiments, the nucleotide sequence encoding the lead RNA may be operatively linked to the mouse or human U6 promoter. In other embodiments, the nucleotide sequence encoding the lead RNA may be operatively linked to the mouse or human H1 promoter. In embodiments having more than one guide RNA, the promoters used to drive expression may be the same or different. In some embodiments, the nucleotides encoding the crRNA and the nucleotides encoding the trRNA may be provided on the same vector. In some embodiments, the nucleotides encoding the crRNA and the nucleotides encoding the trRNA may be driven by the same promoter. In some embodiments, the crRNA and trRNA may be transcribed into a single transcript. For example, the crRNA and trRNA may be processed from a single transcript to form a bimolecular guide RNA. Alternatively, the crRNA and trRNA may be transcribed into a single-molecule guide RNA (sgRNA). In other embodiments, the crRNA and trRNA may be driven by their respective promoters on the same vector. In other embodiments, the crRNA and trRNA may be encoded by different vectors.

In some embodiments, the nucleotide sequence encoding the guide RNA may be located on the same vector as the nucleotide sequence encoding the RNA-guided DNA binder (such as the Cas protein). In some embodiments, the expression of the guide RNA and the RNA-guided DNA binder (such as the Cas protein) may be driven by their respective promoters. In some embodiments, the expression of the guide RNA may be driven by the same promoter that drives the expression of the RNA-guided DNA binder (such as the Cas protein). In some embodiments, the guide RNA and the RNA-guided DNA binder (such as the Cas protein) transcript may be contained within a single transcript. For example, the guide RNA may be located within the untranslated region (UTR) of the RNA-guided DNA binder (such as the Cas protein) transcript. In some embodiments, the guide RNA is located within the 5' UTR of the transcript. In other embodiments, the guide RNA is located within the 3' UTR of the transcript. In some embodiments, the intracellular half-life of the transcript can be reduced by containing the guide RNA within its 3' UTR, thereby shortening the length of the 3' UTR. In other embodiments, the guide RNA is located within an intron of the transcript. In some embodiments, a suitable splicing site can be added at the intron where the guide RNA is located, allowing the guide RNA to be correctly spliced from the transcript. In some embodiments, the temporal closeness of RNA-guided DNA binders (such as Cas proteins) and guide RNA from the same vector can promote more efficient formation of CRISPR RNP complexes.

In some embodiments, the nucleotide sequence encoding a guide RNA or RNA-guided DNA binder may be located on the same carrier comprising a structure containing sequences encoding the following: dmTGFB1 molecule; regulatory T cell stimulating molecules, such as IL10, CTLA4; or targeting receptors, such as CARs, such as MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipoprotein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), bridgene core protein 3. (DSG3) or MHC Class I HLA-A (HLA-A*02) CAR. In some embodiments, the proximity of a construct containing sequences encoding the following to a guide RNA (or RNA-guided DNA binder) on the same vector facilitates more efficient insertion of the construct into an insertion site generated by the guide RNA/RNA-guided DNA binder: dmTGFB1 molecule; regulatory T cell stimulating molecules, such as IL10, CTLA4; or targeting receptors, such as CARs, such as MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipoprotein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), and solute carrier family 2 member 2. (SCL2A2), glutamic acid decarboxylase (GAD2), ceramide core protein 3 (DSG3) or MHC class I HLA-A (HLA-A*02) CAR.

In some embodiments, the DNA and RNA vectors may include more than one open reading frame (ORF) expressed under a single promoter, which is present in the vector or at a gene body insertion site. In such embodiments, the coding sequence of the self-cleaving peptide may be included between the ORFs. The self-cleaving peptide may be, for example, a 2A peptide, such as a P2A peptide, E2A peptide, F2A peptide, or T2A peptide.

In some embodiments, the vector contains one or more nucleotide sequences encoding sgRNA and mRNA, wherein the mRNA encodes an RNA-guided DNA binder, which may be a Cas protein, such as Cas9 or Cpf1. In some embodiments, the vector contains one or more nucleotide sequences encoding crRNA, trRNA, and mRNA, wherein the mRNA encodes an RNA-guided DNA binder, which may be a Cas protein, such as Cas9 or Cpf1. In one embodiment, Cas9 is derived from *Streptococcus brevis* (i.e., Spy Cas9). In some embodiments, the Cas nuclease is a Cas9 nuclease derived from *Neisseria meningitidis* (i.e., Nme Cas9, such as Nme1, Nme2, or Nme3 Cas9). In some embodiments, the nucleotide sequence encoding crRNA, trRNA, or crRNA and trRNA (which may be sgRNA) includes or consists of a lead sequence that is laterally adjacent to all or part of a repeating sequence from a naturally occurring CRISPR/Cas system. The nucleic acid containing crRNA, trRNA, or crRNA and trRNA, or consisting thereof, may further include a vector sequence, wherein the vector sequence includes or consists of nucleic acids not naturally found with crRNA, trRNA, or crRNA and trRNA.

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

In some embodiments, as disclosed herein, the vector comprises a donor construct containing sequences encoding the following: a dmTGFB1 molecule; a regulatory T-cell stimulating molecule, such as IL10 or CTLA4; or a targeting receptor, such as a CAR, such as MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipoprotein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), stromal core protein 3 (DSG3), or MHC class I HLA-A (HLA-A*02). CAR. In some embodiments, in addition to the donor constructs disclosed herein, the vector may further comprise nucleic acids encoding the guide RNA described herein or nucleic acids encoding RNA-guided DNA binders (e.g., Cas nucleases, such as Cas9). In some embodiments, the nucleic acids encoding the RNA-guided DNA binders, individually or both, are located on a vector different from the vector containing the donor constructs disclosed herein. In any embodiment, as described herein, the vector may include other sequences, including (but not limited to) promoters, enhancers, and regulatory sequences. In some embodiments, the promoter does not drive the expression of regulatory T cell-promoting molecules (e.g., IL10 or CTLA4) or targeting receptors (e.g., CARs such as MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipoprotein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), stromal core protein 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CARs). In some embodiments, the vector contains one or more nucleotide sequences encoding crRNA, trRNA, or a combination of crRNA and trRNA. In some embodiments, the vector contains one or more nucleotide sequences encoding sgRNA and mRNA, wherein the mRNA encodes an RNA-guided DNA nuclease, which may be a Cas nuclease (e.g., Cas9). In some embodiments, the vector contains one or more nucleotide sequences encoding crRNA, trRNA, and mRNA, wherein the mRNA encodes an RNA-guided DNA nuclease, which may be a Cas nuclease, such as Cas9. In some embodiments, Cas9 is derived from *Streptococcus brevis* (i.e., Spy Cas9). In some embodiments, the Cas nuclease is a Cas9 nuclease derived from *Neisseria meningitidis*. In some embodiments, the nucleotide sequence encoding crRNA, trRNA, or crRNA and trRNA (which may be sgRNA) includes or consists of a lead sequence that is laterally adjacent to all or part of a repeating sequence from a naturally occurring CRISPR/Cas system. The nucleic acid containing crRNA, trRNA, or crRNA and trRNA, or consisting thereof, may further include a vector sequence, wherein the vector sequence includes or consists of nucleic acids not naturally found with crRNA, trRNA, or crRNA and trRNA.

In some embodiments, the vector may be circular. In other embodiments, the vector may be linear. In some embodiments, the vector may be encapsulated in lipid nanoparticles, liposomes, non-liposome nanoparticles, or viral capsids. Non-limiting illustrative vectors include plasmids, phages, granules, artificial chromosomes, microchromosomes, transposons, viral vectors, and expression vectors.

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 contain insertions, deletions, or substitutions of one or more nucleotides to promote selection or alter one or more properties of the vector. Such properties may include packaging capacity, transduction efficiency, immunogenicity, genome integration, replication, transcription, and translation. In some embodiments, a portion of the viral genome may be deleted, allowing the virus to package a larger exogenous sequence. In some embodiments, the viral vector may have enhanced transduction efficiency. In some embodiments, the virus-induced immune response may be reduced. In some embodiments, viral genes (such as integrases) that promote viral sequence integration into the genome may be mutated, rendering the virus non-integrative. In some embodiments, the viral vector may be replication-defective. In some embodiments, the viral vector may contain exogenous transcriptional or translational control sequences to drive the expression of the encoded sequences on the vector. In some embodiments, the virus may be helper-dependent. For example, the virus may require one or more helper viruses to provide the viral components (such as viral proteins) needed to amplify the vector and package it into viral particles. In this case, one or more helper components (including vectors encoding one or more viral components) may be introduced into cells or cell populations together with the vector system described herein. In other embodiments, the virus may be helpless. For example, the virus may be able to amplify and package the vector without helper viruses. In some embodiments, the carrier system described herein can also encode the viral components required for viral amplification and packaging.

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

In some implementations, "AAV" refers to all serotypes, subtypes, naturally occurring AAVs, and recombinant AAVs. "AAV" can also refer to the virus itself or its derivatives. The term "AAV" includes AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAVrh.64R1, AAVhu.37, AAVrh.8, AAVrh.32.33, AAV8, AAV9, AAV-DJ, AAV2/8, AAVrh10, AAVLK03, AV10, AAV11, AAV12, rh10 and its hybrids, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV. In some embodiments, AAV is acceptable for in vitro applications in human cells. In some embodiments, the AAV is AAV6. The genomic sequences of various serotypes of AAV, as well as the sequences of native terminal repeats (TRs), Rep proteins, and capsid subunits, are known in this art. These sequences can be found in literature or public databases (such as GenBank). As used herein, "AAV vector" refers to an AAV vector containing a heterologous sequence not of AAV origin (i.e., a nucleic acid sequence heterologous to AAV), which typically contains a sequence encoding the heterologous polypeptide of interest. The structure may contain 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, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV capsid sequences. Generally, the heterologous nucleic acid sequence (transgenic gene) is laterally attached to at least one, and usually two, AAV inverted terminal repeat (ITR) sequences. AAV carriers can be monostranded (ssAAV) or self-complementary (scAAV).

In some embodiments, the lentivirus may be integrative. In some embodiments, the lentivirus may be non-integrative. In some embodiments, the viral vector may be an adenovirus vector. In some embodiments, the adenovirus may be a highly selective or gutless adenovirus in which all coding viral regions except for the 5' and 3' inverted terminal repeats (ITRs) and the packaging signal ('I') are deleted from the virus to increase its packaging capacity. In other embodiments, the viral vector may be an HSV-1 vector. In some embodiments, the HSV-1-based vector is co-dependent, and in other embodiments, it is co-independent. For example, an amplicon vector that retains only the packaging sequence requires an auxiliary virus with the structural components for packaging, while an HSV-1 vector with a 30 kb deletion removing non-essential viral functions does not require an auxiliary virus. In other embodiments, the viral vector may be bacteriophage T4. In some embodiments, bacteriophage T4 may be able to package any linear or circular DNA or RNA molecule when the viral head is emptied. In other embodiments, the viral vector may be a rod-virus vector. In other embodiments, the viral vector may be a retrotransfer virus vector. In embodiments using AAV or other vectors with lower selectivity, more than one vector may be used to deliver all components of the vector system disclosed herein. For example, one AAV vector may contain a sequence that encodes an RNA-guided DNA binder (such as a Cas protein (e.g., Cas9)), while a second AAV vector may contain one or more guide sequences.

In some embodiments, the vector system may be able to drive the expression of one or more nuclease components in cells. In some embodiments, the vector does not contain a promoter that drives the expression of one or more coding sequences upon integration into the cell (e.g., using an endogenous cellular promoter, such as when inserted into a specific genomic locus of the cell, as illustrated herein). Suitable promoters for driving expression in different types of cells (e.g., CD4+ T cells) are known in this art. In some embodiments, the promoter may be wild-type. In other embodiments, the promoter may be modified to obtain more efficient or effective expression. In other embodiments, the promoter may be truncated but retain its function. For example, the promoter can be of normal size or reduced size to properly package the vector into the virus.

In some embodiments, the vector may contain a nucleotide sequence encoding an RNA-guided DNA binder (such as a Cas protein, e.g., Cas9) as described herein. In some embodiments, the nuclease encoded by the vector may be a Cas protein. In some embodiments, the vector system may contain one copy of the nucleotide sequence encoding the nuclease. In other embodiments, the vector system may contain more than one copy of the nucleotide sequence encoding the nuclease. In some embodiments, the nucleotide sequence encoding the nuclease may be operatively linked to at least one transcriptional or translational control sequence. In some embodiments, the nucleotide sequence encoding the nuclease may be operatively linked to at least one promoter.

In some embodiments, the vector may comprise any one or more structures that contain sequences encoding the following: dmTGFB1 molecules; regulatory T cell stimulating molecules, such as IL10, CTLA4; or targeting receptors, such as CARs, such as MAdCAM-1 CAR as described herein. In some embodiments, the sequence of dmTGFB1 molecule, regulatory T cell stimulating molecule (e.g., IL10, CTLA4) or targeting receptor (e.g., CAR, such as MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipoprotein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), stromal core protein 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CAR) can be operatively linked to at least one transcriptional or translational control sequence. In some embodiments, the sequence of dmTGFB1 molecule, regulatory T cell stimulating molecule (e.g., IL10, CTLA4) or targeting receptor (e.g., CAR, such as MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipoprotein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), stromal core protein 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CAR) can be operatively linked to at least one promoter. In some embodiments, the sequences of dmTGFB1 molecules, regulatory T cell stimulating molecules (e.g., IL10, CTLA4) or targeting receptors (e.g., CARs such as MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipoprotein 1 (PLP1), CD19 molecules (CD19), CD20 molecules (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute vector family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), stromal core protein 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CARs) are not linked to promoters that drive heterologous gene expression.

In some embodiments, the promoter may be genotypic, inducible, or tissue-specific. In some embodiments, the promoter may be a genotypic promoter. Non-limiting illustrative genotypic promoters include the cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor-α (EF1a) promoter, ubiquitin promoter, actin promoter, tubulin promoter, immunoglobulin promoter, functional fragments thereof, or any combination thereof. 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 induced promoter. Non-limiting illustrative induced promoters include promoters that can be induced by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohols. In some embodiments, the induced promoter may be a promoter with low baseline (non-induced) performance levels, such as the Tet- ^On® promoter (Clontech).

In some embodiments, the promoter is acceptable for in vitro applications in human cells.

In some embodiments, the promoter can be a tissue-specific promoter, for example, a promoter that exhibits specificity in T cells.

In some embodiments, the combination 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 other embodiments, the vector system may comprise three vectors. When different lead RNAs are used for multiplexing, or when multiple lead RNAs are used for copying, the vector system may comprise three or more vectors.

In some embodiments, the vector system may include an inducible promoter that begins to express only after it has been delivered to the target cells. Non-limiting illustrative inducible promoters include those induced by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohols. In some embodiments, the inducible promoter may be a promoter with low baseline (non-inducible) performance levels, such as the Tet- ^On® promoter (Clontech).

In other embodiments, the carrier system may include a tissue-specific promoter.

The following provides a non-restrictive illustrative viral vector sequence: CTLA4 insertion (nucleotide sequence) ATGGCCTGCTTGGGCTTCCAAAGGCATAAAGCCCAGCTTAATCTTGCTACTCGCACGTGGCCCTGCACATTGCTCTTTTTCCTCCTGTTCATTCCCGTGTTTTGCAAGGCGATGCATGTGGCACAACCTGCCGTCGTTCTGGCATCATCAAGAGGTATTGCTAGCTTCGTTTGTGAGTACGCCTCCCCTGGAAAAGCGACGGAGGTGCGCGTCACTGTATTGCGGCAAGCCGACAGCCAAGTTACTGAAGTCTGCGCGGCAACGTATATGATGGGCAATGAGCTGACATTCCTTGACGATTCAATCTGCACGGGAACAAG TAGTGGTAACCAGGTGAATCTCACTATTCAAGGTCTGAGAGCCATGGACACCGGCCTCTACATTTGTAAGGTGGAGCTGATGTATCCTCCCCCATATTATCTGGGGATCGGAAATGGGACACAGATATATGTTATTGATCCCGAGCCATGTCCCGATAGTGACTTCCTCTTGTGGA TACTTGCCGCTGTGAGCAGTGGTTTGTTTTTTTATTCATTCCTCCTTACGGCAGTATCACTTTCAAAAATGCTCAAGAAGCGAAGTCCTTTGACAACTGGCGTATATGTCAAAATGCCACCAACAGAGCCCGAATGTGAGAAACAGTTCCAGCCGTACTTTATTCCTATAAACTGA (SEQ ID NO: 137)

CTLA4 insertion (amino acid sequence) MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICT GTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO: 130)

IL10 insert (nucleotide sequence) ATGCACAGTCCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTC GAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCC AGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAA AACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAACTGA (SEQ ID NO: 138)

IL10 insert (amino acid sequence)MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 124)

Empty lentiviral vectorACCGTGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGG TACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACATAAACGGGTCTCTCTGG TTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTA GAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGC ACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGA AAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACA AATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCACTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATAAATATAAAGTA GTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTAT GGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGG CATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGA ATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAG AGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGATGGCCGCCCCCTTCACCGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATG CTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGAGTCTTCTTTTTTGAAGACACTTCGGACTGTAGAACTCTGAACCTCGAGCAATTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTCTGCGTTGTTGTCGG TGCTCGTTCTCTGCTCTTCACGCTACTGAATTCATCACCGGTTCTTCGAAGGCCTCCGCGCCGGGTTTTTGGCGCCTCCCGCGGGCGCCCCCCTCCTCACGGCGAGCCGTCGCCACGTCAGACGAAG GGCGCAGCGAGCGTCCTGATCCTTCCGCCCGGACGCTCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAGTATCAGCAGAAGGACATTTTAGGACGGGACTTGGGTGACTCTA GGGCACTGGTTTTCTTTCCAGAGAGCGGAACAGGCGAGGAAAAGTAGTCCCTTTCTCGGCGATTCTGCGGAGGGATCTCCGTGGGGCGGTGAACGCCGATGATTATAAGGACGCGCCGGGTGTG GCACAGCTAGTTCCGTCGCAGCCGGGATTTGGGTCGCGGTTCTTGTTTGTGGATCGCTGTGATCGTCACTTGGTCTAGACGCCACCATGAGCGGGGGCGAGGAGCTGTTCGCCGGCATCGTGCCCG TGCTGATCGAGCTGGACGGCGACGTGCACGGCCACAAGTTCAGCGTGCGCGGCGAGGGCGAGGGCGACGCCGACTACGGCAAGCTGGAGATCAAGTTCATCTGCACCACCGGCAAGCTGCCCGTG CCCTGGCCCACCCTGGTGACCACCCTCTGCTACGGCATCCAGTGCTTCGCCCGCTACCCCGAGCACATGAAGATGAACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACATCCAGGAGCGCACC ATCCAGTTCCAGGACGACGGCAAGTACAAGACCCGCGGCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCAAGGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAGCTTCAACAGCCACAACGTGTACATCCGCCCCGACAAGGCCAACAACGGCCTGGAGGCTAACTTCAAGACCCGCCACAACATCGAGGGCGGCGGCGTGCAGCTGGCCGACCA CTACCAGACCAACGTGCCCCTGGGCGACGGCCCCGTGCTGATCCCCATCAACCACTACCTGAGCACTCAGACCAAGATCAGCAAGGACCGCAACGAGGCCCGCGACCACATGGTGCTCCTGGAGTCCTTCAGCGCCTGCTGCCACACCCACGGCATGGACGAGCTGTACAGGGGATCCGAGGGCAGAGGAAGCCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTTCCGGGATGACCGAGTACA AGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCAGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCACACCGTCGATCCGGACCGCCACATCGAGCGGGTC ACCGAGCTGCAAGAACTCTTCCTCACGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCGGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTC GCCGAGATCGGCCCGCGCATGGCCGAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGCCCAAGGAGCCCGCGTGTTTCCTGGCCACCGTCGGCGTC TCGCCCGACCACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAGCGCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTTCTACGAG CGGCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCGAAGGACCGCGCACCTGGTGCATGACCCGCAAGCCCGGTGCCTGAATCTAGGTCGACAATCAACCTCTGGATTACAAAATTTGTGA AAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAAATCCTG GTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGA CTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCT TTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTT CCGCGTCTCCGCCTTCGCCCTCAGACGAGTCGGATCTCTCTTTGGGCCGCCTCCCCGCCTGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGG GGGACTGGAAGGGCTAATTCACTCCCAACGAAGATAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTA AGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCAT GTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCC ATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTGGAGGCCTAGACTTTTGCAGAGACCAAATTCGTAATCATGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATT AATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCA CTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAA AGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACC AGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATC TCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCG CCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTG CTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAAATGAAGTTT TAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGT AGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAA GTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCA CGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAG TTGGCCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCT GTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTC CGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCG GCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGA TCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTG (SEQ ID NO: 141)

Empty lentiviral vector GCGATCGCAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAG TACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCAT TGACGTCAATGGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCT TGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACCTGAAAGCGAAAGGGAAACCAGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATT TTGACTAGCGGAGGCTAGAAGGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTAC AACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATA AATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCCTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTAT TGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGA GAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAG TTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACAT ACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTGGCTCCCGATCGTTGCGTTACACACACAATTACTGCTGATCGAGTGTAGCCTTCGAATGAAAGACCCCACCTGTAGGTTTTGGCAAGATAGCTGCAGTAACGCCATTTTGCAAGGCATGGAAAAATACCAAACCAAGAATAGAGAAGTTCAGATCAAGGGCGGGTACATGAAAATAGC TAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCGGCCCGGGGCCAAGAACAGATGGTCACCGCAGTTTCGGCCCCGGCCCGAGGCCAAGAACAGATGGTCCCCAGATATGGCCCAACCCTCAGCAGTTTCTTAAGACCCATCAGATGTTTCCAGGCTCCCCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTAACCAATCAGCCT GCTTCTCGCTTCTGTTCGCGCTTCTGCTTCCCGAGCTCTATAAAAGAGCTCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATTGACTGAGTCGCCCTGATCATTGTCGATCCTACCATCCACTCGACACACCCGCCAGGGCCCTGCCAAGCTTCCGAGCTCTCGATATCAAAGGAGGTACCCAACATGGTCAGCAAGGGCGAGGAACTGTTCACCGGGG TGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGTACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAATGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAA GTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAAC GGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACCGGCAGCGTGCAACTCGCCGACCACTACCAGCAGAACACCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGG ACGAGCTGTACAAGTAGAAGTTGTCTCCTCCTGCACTGACTGACTGATACAATCGATTTCTGGATCCGCAGGCCTCTGCTAGAAGTTGTCTCCTCCTGCACTGACTGACTGATACAATCGATTTCTGGATCCGCAGGCCTCTGCTAGCTTGACTGACTGAGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGT TGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGG ACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCC GCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGT ACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTCCTGGCCAACGTGAGCACCGTGCTGACCTCCAAATAT CGTTAAGCTGGAGCCTGGGAGCCGGCCTGGCCCTCCGCCCCCCCCCACCCCCGCAGCCCACCCCTGGTCTTTGAATAAAGTCTGAGTGAGTGGCCGACAGTGCCCGTGGAGTTCTCGTGACCTGAGGTGCAGGGCCGGCGCTAGGGACACGTCCGTGCACGTGCCGAGGCCCCCTGTGCAGCTGCAAGGGACAGGCCTAGCCCTGCAGGCCTAACTCCGCCCAT CCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCTCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGACGCTTTTTTGGAGGCCGAGGCTTTTGCAAAGATCGAACAAGAGACAGGACCTGCAGGTTAATTAAATTTAAATCATGTGAGCAAAAGGCCAGCAAAAGGCCAGGA ACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCA TAGCTCCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGG CCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACT CACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCATTTAAATGGCCGGCCTGGCGCGCCGTTTAAACCTAGAT ATTGATAGTCTGATCGGTCAACGTATAATCGAGTCCTAGCTTTGCAAACATCTATCAAGAGACAGGATCAGCAGGAGGCTTTCGCATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTCCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCGCGAGTGGGTTACATCGA ACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGCTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTATTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTA TGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATTGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACCTTGCGTAAACTATTAACTGGCG AACTACTTACTCTAGCTTCCCGGCAACAGTTGATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGA TGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACCGATTCTAGGTGCATTGGCGCAGAAAAAAATGCCTGATGCGACGCTGCCGTCTTATACTCCCACATATGCCAGATTCAGCAACGGATACGGCTTCCCCAACTTGCCCACTTCCATACGTGTCCTCCTTACCAGAAATTTATCCTTAAGATCCCGAATCGTTAAAC (SEQ ID NO: 142)

Other sequences are provided throughout the specification and in the table below; and the list of sequences submitted with this document forms part of the specification.

The sequence listing is in the table below. The terms "mA", "mC", "mU" or "mG" are used to denote nucleotides modified with 2'-O-Me.

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

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

It should be understood that if RNA refers to a DNA sequence (containing T), then T should be replaced by U (which may be considered as modified or unmodified in the context), and vice versa.

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

Table 4: Sequence List SEQ ID describe sequence 204 Nucleic acid sequence encoding TGFB1 atgccgccctccgggctgcggctgctgctgctgctgctaccgctgctgtggctactggtgctgacgcctggccggccggccgcgggactatccacctgcaagactatcgacatggagctggtgaagcggaagcgcatcgaggccat ccgcggccagatcctgtccaagctgcggctcgccagccccccgagccagggggaggtgccgcccggcccgctgcccgaggccgtgctcgccctgtacaacagcacccgcgaccgggtggccggggagagtgcagaaccggagcccg agcctgaggccgactactacgccaaggaggtcacccgcgtgctaatggtggaaacccacaacgaaatctatgacaagttcaagcagagtacacacagcatatatatgttcttcaacacatcagagctccgagaagcggtacctgaa cccgtgttgctctcccgggcagagctgcgtctgctgaggctcaagttaaaagtggagcagcagtggagctgtaccagaaatacagcaacaattcctggcgatacctcagcaaccggctgctggcacccagcgactcgccagagtgg ttatcttttgatgtcaccggagttgtgcggcagtggttgagccgtggaggggaaattgagggctttcgccttagcgcccactgctcctgtgacagcagggataacacactgcaagtggacatcaacgggttcactaccggccgccg aggtgacctggccaccattcatggcatgaaccggcctttcctgcttctcatggccaccccgctggagaggggcccagcatctgcaaagctcccggcaccgccgagccctggacaccaactattgcttcagctccacggagaagaact gctgcgtgcggcagctgtacattgacttccgcaaggacctcggctggaagtggatccacgagcccaagggctaccatgccaacttctgcctcgggccctgcccctacatttggagcctggacacgcagtacagcaaggtcctggcc ctgtacaaccagcataacccgggcgcctcggcggcgccgtgctgcgtgccgcaggcgctggagccgctgcccatcgtgtactacgtgggccgcaagcccaaggtggagcagctgtccaacatgatcgtgcgctcctgcaagtgcagc 205 Nucleic acid sequence encoding TGFB1 R218H atgccgccctccgggctgcggctgctgctgctgctgctaccgctgctgtggctactggtgctgacgcctggccggccggccgcgggactatccacctgcaagactatcgacatggagctggtgaagcggaagcgcatcgaggccat ccgcggccagatcctgtccaagctgcggctcgccagccccccgagccagggggaggtgccgcccggcccgctgcccgaggccgtgctcgccctgtacaacagcacccgcgaccgggtggccggggagagtgcagaaccggagcccg agcctgaggccgactactacgccaaggaggtcacccgcgtgctaatggtggaaacccacaacgaaatctatgacaagttcaagcagagtacacacagcatatatatgttcttcaacacatcagagctccgagaagcggtacctgaa cccgtgttgctctcccgggcagagctgcgtctgctgaggctcaagttaaaagtggagcagcagtggagctgtaccagaaatacagcaacaattcctggcgatacctcagcaaccggctgctggcacccagcgactcgccagagtgg ttatcttttgatgtcaccggagttgtgcggcagtggttgagccgtggaggggaaattgagggctttCACcttagcgcccactgctcctgtgacagcagggataacacactgcaagtggacatcaacgggttcactaccggccgccg aggtgacctggccaccattcatggcatgaaccggcctttcctgcttctcatggccaccccgctggagaggggcccagcatctgcaaagctcccggcaccgccgagccctggacaccaactattgcttcagctccacggagaagaact gctgcgtgcggcagctgtacattgacttccgcaaggacctcggctggaagtggatccacgagcccaagggctaccatgccaacttctgcctcgggccctgcccctacatttggagcctggacacgcagtacagcaaggtcctggcc ctgtacaaccagcataacccgggcgcctcggcggcgccgtgctgcgtgccgcaggcgctggagccgctgcccatcgtgtactacgtgggccgcaagcccaaggtggagcagctgtccaacatgatcgtgcgctcctgcaagtgcagc 206 Nucleic acid sequence encoding TGFB1 R218C atgccgccctccgggctgcggctgctgctgctgctgctaccgctgctgtggctactggtgctgacgcctggccggccggccgcgggactatccacctgcaagactatcgacatggagctggtgaagcggaagcgcatcgaggccat ccgcggccagatcctgtccaagctgcggctcgccagccccccgagccagggggaggtgccgcccggcccgctgcccgaggccgtgctcgccctgtacaacagcacccgcgaccgggtggccggggagagtgcagaaccggagcccg agcctgaggccgactactacgccaaggaggtcacccgcgtgctaatggtggaaacccacaacgaaatctatgacaagttcaagcagagtacacacagcatatatatgttcttcaacacatcagagctccgagaagcggtacctgaa cccgtgttgctctcccgggcagagctgcgtctgctgaggctcaagttaaaagtggagcagcagtggagctgtaccagaaatacagcaacaattcctggcgatacctcagcaaccggctgctggcacccagcgactcgccagagtgg ttatcttttgatgtcaccggagttgtgcggcagtggttgagccgtggaggggaaattgagggctttTGCcttagcgcccactgctcctgtgacagcagggataacacactgcaagtggacatcaacgggttcactaccggccgccg aggtgacctggccaccattcatggcatgaaccggcctttcctgcttctcatggccaccccgctggagaggggcccagcatctgcaaagctcccggcaccgccgagccctggacaccaactattgcttcagctccacggagaagaact gctgcgtgcggcagctgtacattgacttccgcaaggacctcggctggaagtggatccacgagcccaagggctaccatgccaacttctgcctcgggccctgcccctacatttggagcctggacacgcagtacagcaaggtcctggcc ctgtacaaccagcataacccgggcgcctcggcggcgccgtgctgcgtgccgcaggcgctggagccgctgcccatcgtgtactacgtgggccgcaagcccaaggtggagcagctgtccaacatgatcgtgcgctcctgcaagtgcagc 207 Nucleic acid sequence encoded TGFB1 C225R atgccgccctccgggctgcggctgctgctgctgctgctaccgctgctgtggctactggtgctgacgcctggccggccggccgcgggactatccacctgcaagactatcgacatggagctggtgaagcggaagcgcatcgaggccat ccgcggccagatcctgtccaagctgcggctcgccagccccccgagccagggggaggtgccgcccggcccgctgcccgaggccgtgctcgccctgtacaacagcacccgcgaccgggtggccggggagagtgcagaaccggagcccg agcctgaggccgactactacgccaaggaggtcacccgcgtgctaatggtggaaacccacaacgaaatctatgacaagttcaagcagagtacacacagcatatatatgttcttcaacacatcagagctccgagaagcggtacctgaa cccgtgttgctctcccgggcagagctgcgtctgctgaggctcaagttaaaagtggagcagcagtggagctgtaccagaaatacagcaacaattcctggcgatacctcagcaaccggctgctggcacccagcgactcgccagagtgg ttatcttttgatgtcaccggagttgtgcggcagtggttgagccgtggaggggaaattgagggctttcgccttagcgcccactgctccAGAgacagcagggataacacactgcaagtggacatcaacgggttcactaccggccgccg aggtgacctggccaccattcatggcatgaaccggcctttcctgcttctcatggccaccccgctggagaggggcccagcatctgcaaagctcccggcaccgccgagccctggacaccaactattgcttcagctccacggagaagaact gctgcgtgcggcagctgtacattgacttccgcaaggacctcggctggaagtggatccacgagcccaagggctaccatgccaacttctgcctcgggccctgcccctacatttggagcctggacacgcagtacagcaaggtcctggcc ctgtacaaccagcataacccgggcgcctcggcggcgccgtgctgcgtgccgcaggcgctggagccgctgcccatcgtgtactacgtgggccgcaagcccaaggtggagcagctgtccaacatgatcgtgcgctcctgcaagtgcagc 208 Nucleic acid sequence encoded TGFB1 R218H C225R atgccgccctccgggctgcggctgctgctgctgctgctaccgctgctgtggctactggtgctgacgcctggccggccggccgcgggactatccacctgcaagactatcgacatggagctggtgaagcggaagcgcatcgaggccat ccgcggccagatcctgtccaagctgcggctcgccagccccccgagccagggggaggtgccgcccggcccgctgcccgaggccgtgctcgccctgtacaacagcacccgcgaccgggtggccggggagagtgcagaaccggagcccg agcctgaggccgactactacgccaaggaggtcacccgcgtgctaatggtggaaacccacaacgaaatctatgacaagttcaagcagagtacacacagcatatatatgttcttcaacacatcagagctccgagaagcggtacctgaa cccgtgttgctctcccgggcagagctgcgtctgctgaggctcaagttaaaagtggagcagcagtggagctgtaccagaaatacagcaacaattcctggcgatacctcagcaaccggctgctggcacccagcgactcgccagagtgg ttatcttttgatgtcaccggagttgtgcggcagtggttgagccgtggaggggaaattgagggctttCACcttagcgcccactgctccAGAgacagcagggataacacactgcaagtggacatcaacgggttcactaccggccgccg aggtgacctggccaccattcatggcatgaaccggcctttcctgcttctcatggccaccccgctggagaggggcccagcatctgcaaagctcccggcaccgccgagccctggacaccaactattgcttcagctccacggagaagaact gctgcgtgcggcagctgtacattgacttccgcaaggacctcggctggaagtggatccacgagcccaagggctaccatgccaacttctgcctcgggccctgcccctacatttggagcctggacacgcagtacagcaaggtcctggcc ctgtacaaccagcataacccgggcgcctcggcggcgccgtgctgcgtgccgcaggcgctggagccgctgcccatcgtgtactacgtgggccgcaagcccaaggtggagcagctgtccaacatgatcgtgcgctcctgcaagtgcagc 209 Nucleic acid sequence encoding GFP atgtcgaagggagaagaactgttcacaggagtcgtcccgatcctggtcgaactggacggagacgtcaacggaccaaagttctcggtcagaggagaaggagaaggagacgcaacaaacggaaagctgacactgaagttcatctgcacaacaggaaagctgccggtcccgtggccgacac tggtcacaacactgacatacggagtccagtgcttttcgagatacccggaccacatgaagagacacgacttcttcaagtcggcaatgccggaaggatacgtccaggaaagaacaatctcgttcaaggacgacggaacatacaagacaagagcagaagtcaagttcgaaggagacacactg gtcaacagaatcgaactgaagggaatcgacttcaaggaagacggaaacatcctgggacacaagctggaatacaacttcaactcgcacaacgtctacatcacagcagacaagcagaagaacggaatcaaggcaaacttcaagatcagacacaacgtcgaagacggatcggtccagctggc agaccactaccagcagaacacaccgatcggagacggaccggtcctgctgccggacaaccactacctgtcgacacagtcggtcctgtcgaaggacccgaacgaaaagagagaccacatggtcctgctggaattcgtcacagcagcaggaatcacacacggaatggacgaactgtacaagt 210 amino acid sequence of TGFB1 MPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEW LSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS 211 The amino acid sequence of TGFB1 R218H MPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEW LSFDVTGVVRQWLSRGGEIEGFHLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS 212 amino acid sequence of TGFB1 R218C MPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEW LSFDVTGVVRQWLSRGGEIEGFCLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS 213 The amino acid sequence of TGFB1 C225R MPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEW LSFDVTGVVRQWLSRGGEIEGFRLSAHCSRDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS 214 The amino acid sequence of TGFB1 R218H C225R MPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEW LSFDVTGVVRQWLSRGGEIEGFHLSAHCSRDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS 215 GFP amino acid sequence MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK 216 Lentiviral vectors tatgagtaaacttggtctgacagTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGG GAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACT TTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTT GGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAG TGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGA GATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGA CACGGAAATGTTGAATACTCATactcttcctttttcaatattattgaagcatttatcagggttatattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttcc gcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtga aaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaacta tgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttggga agggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagt gccaagctgacgcgtgtagtctttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaag gtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtatttaagtgcctagctcgatacataaacgggtct ctctggttagaccagatctgagcctggggagctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactct ggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgc tgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcggggggagaattag atcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatc agaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaa agacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccactgatcttcagacctggaggaggagatatgagggacaattggagaagtga attataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttctt gggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcat ctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcacc actgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattg aagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatga tagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgaca ggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgatttaaaagaaaaggggggattggggggtacagtgcagggg aaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccagtttgctagaccataga gcccaccgcatccccagcatgcctgctattgtcttcccaatcctcccccttgctgtcctgccccaccccaccccccagaatagaatgacacctactcagacaatgcgatgcaatttcctcatt ttattaggaaaggacagtggggagtggcaccttccagggtcaaggaaggcacgggggaggggcaaacaacagatggctggcaactagaaggcacaggctagctaggtggatccGAATAAGGCCTG CAACGACACACACACGAACAAGCAGAGCCGCTGGACGCCGACTGCGGGAcgaaaggcccggagatgaggaagaggagaacagcgcggcagacgtgcgcttttgaagcgtgcagaatgccgggc ctccggaggaccttcgggcgcccgccccgcccctgagcccgcccctgagcccgcccccggacccaccccttcccagcctctgagcccagaaagcgaaggagcaaagctgctattggccgctgcc ccaaaggcctacccgcttccattgctcagcggtgctgtccatctgcacgagactagtgagacgtgctacttccatttgtcacgtcctgcacgacgcgagctgcggggcgggggggaacttcct gactaggggaggagtagaaggtggcgcgaaggggccaccaaagaacggagccggttggcgcctaccggtggatgtggaatgtgtgcgaggccagaggccacttgtgtagcgccaagtgcccag cggggctgctaaagcgcatgctccagactgccttgggaaaagcgcctcccctacccatcgatGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGG GAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGT CGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCAC CTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTG GGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGT AAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGGGCGGCGACGGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATC GGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGTCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGG CCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCaGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTCCGTCCTCAGCCGTCGCTTCATGTGAC TCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAG ACTGAAGTTAGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGG TGTCGTGAtctagatgccaccGATGCATgtcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgcttt aatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgc actgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcc cgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgtgttgccacctggattctgcgcgggacgtccttct gctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtctccgccttcgccctcagacgagtcggatctctctttgggccgcct ccccgcctggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagataagatctgcttttt gcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgt ctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatat cagagagtgagaggaacttgtttatgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactca tcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgca gaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagacttttgcagagaccaaattcgtaatcatgtcatagctgtttcctgtgtgaaattg ttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcggga aacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcga gcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaaggccaggaaccgtaaaaaggccgcgttgctg gcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtg cgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctcca agctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaaca ggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaa aagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctac ggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtata 119 G019753 mC*mC*mA*GAGCAUCCAAAAGAGUGGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 120 G019757 mA*mG*mA*GCUCUUACCUACAACAUGUUUUAGAmGmCmUmAmGmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmGmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 217 G027259 mU*mC*mA*CAGAGGGAUAUCUCUCAGUUUUAGAmGmCmUmAmGmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 218 SpCas9 Open Reading Box AUGGACAAGAAGUACUCCAUCGGCCUGGACAUCGGCACCAACUCCGUGGGCUGGGCCGUGAUCACCGACGAGUACAAGGUGCCCUCCAAGAAGUUCAAGGUGCUGGGCAACACCGACCGGCACUCCAUC AAGAAGAACCUGAUCGGCGCCCUGCUGUUCGACUCCGGCGAGACCGCCGAGGCCACCCGGCUGAAGCGGACCGCCCGCCGGCGGUACACCCGGCGGAAGAACCGGAUCUGCUACCUGCAGGAGAUCUUC UCCAACGAGAUGGCCAAGGUGGACGACUCCUUCUUCCACCGGCUGGAGGAGUCCUUCCUGGUGGAGGAGGACAAGAAGCACGAGCGGCACCCAUCUUCGGCAACAUCGUGGACGAGGUGGCCUACCAC GAGAAGUACCCCACCAUCUACCACCUGCGGAAGAAGCUGGUGGACUCCACCGACAAGGCCGACCUGCGGCUGAUCUACCUGGCCCUGGCCCACAUGAUCAAGUUCCGGGGCCACUUCCUGAUCGAGGGCG ACCUGAACCCCGACAACUCCGACGUGGACAAGCUGUUCAUCCAGCUGGUGCAGACCUACAACCAGCUGUUCGAGGAGAACCCCAUCAACGCCUCCGGCGUGGACGCCAAGGCCAUCCUGUCCGCCCGGC UGUCCAAGUCCCGGCGGCUGGAGAACCUGAUCGCCCAGCUGCCCGGCGAGAAGAAGAACGGCCUGUUCGGCAACCUGAUCGCCCUGUCCCUGGGCCUGACCCCCAACUUCAAGUCCAACUUCGACCUGGC CGAGGACGCCAAGCUGCAGCUGUCCAAGGACACCUACGACGACGACCUGGACAACCUGCUGGCCCAGAUCGGCGACCAGUACGCCGACCUGUUCCUGGCCGCCAAGAACCUGUCCGACGCCAUCCUGCU GUCCGACAUCCUGCGGGUGAACACCGAGAUCACCAAGGCCCCCCUGUCCGCCUCCAUGAUCAAGCGGUACGACGAGCACCACCAGGACCUGACCCUGCUGAAGGCCCUGGUGCGGCAGCAGCCAGCCCGAG AAGUACAAGGAGAUCUUCUUCGACCAGUCCAAGAACGGCUACGCCGGCUACAUCGACGGCGGGCCUCCCAGGAGGAGUUCUACAAGUUCAUCAAGCCCAUCCUGGAGAAGAUGGACGGCACCGAGGAG CUGCUGGUGAAGCUGAACCGGGAGGACCUGCUGCGGAAGCAGCGGACCUUCGACAACGGGCUCCAUCCCCCACCAGAUCCACCUGGGCGAGCUGCACGCCAUCCUGCGGCGGCAGGAGGACUUCUACCCC UUCCUGAAGGACAACCGGGAGAAGAUCGAGAAGAUCCUGACCUUCCGGAUCCCCUACUACGUGGGCCCCCUGGCCCGGGGCAACUCCCGGUUCGCCUGGAUGACCCGGAAGUCCGAGGAGACCAUCACC CCCUGGAACUUCGAGGAGGUGGUGGACAAGGGCGCCCCGCCCAGUCCUUCAUCGAGCGGAUGACCAACUUCGACAAGAACCUGCCCAACGAGAAGGUGCUGCCCAAGCACUCCCUGCUGUACGAGUACU UCACCGUGUACAACGAGCUGACCAAGGUGAAAGUACGUGACCGAGGGCAUGCGGAAGCCCGCCUUCCUGUCCGGCGAGCAGAAGAAGGCCAUCGUGGACCUGCUGUUCAAGACCAACCGGAAGGUGACCG UGAAGCAGCUGAAGGAGGACUACUUCAAGAAGAUCGAGUGCUUCGACUCCGUGGAGAUCUCCGGCGUGGAGGACCGGUUCAACGCCUCCCUGGGCACCUACCACGACCUGCUGAAGAUCAUCAAGGACAA GGACUUCCUGGACAACGAGGAGAACGAGGACAUCCUGGAGGACAUCGUGCUGACCCUGACCCUGUUCGAGGACCGGGAGAUGAUCGAGGAGCGGCUGAAGACCUACGCCCACCUGUUCGACGACAAGGU GAUAAGCAGCUGAAGCGGCGGCGGUACACCGGCUGGGGCCGGCUGUCCCGGAAGCUGAUCAACGGCAUCCGGGACAAGCAGUCCGGCAAGACCAUCCUGGACUUCCUGAAGUCCGACGGCUUCGCCAAC CGGAACUUCAUGCAGCUGAUCCACGACGACUCCCUGACCUUCAAGGAGGACAUCCAGAAGGCCCAGGUGUCCGGCCAGGGCGACUCCCUGCACGAGCACAUCGCCAACCUGGCCGGCUCCCCCGCCAUC AAGAAGGGCAUCCUGCAGACCGUGAAGGUGGUGGACGAGCUGGUGAAGGUGAUGGGCCGGCACAAGCCCGAGAACAUCGUGAUCGAGAUGGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAAC UCCCGGGAGCGGAUGAAGCGGAUCGAGGAGGGCAUCAAGGAGCUGGGCUCCCAGAUCCUGAAGGAGCACCCCGUGGAGAACACCCAGCUGCAGAACGAGAAGCUGUACCUGUACUACCUGCAGAACGGC CGGGACAUGUACGUGGACCAGGAGCUGGACAUCAACCGGCUGUCCGACUACGACGUGGACCACAUCGUGCCCCAGUCCUUCCUGAAGGACGACUCCAUCGACAACAAGGUGCUGACCCGGUCCGACAAGA ACCGGGGCAAGUCCGACAACGUGCCCUCCGAGGAGGUGGUGAAGAAGAUGAAGAACUACUGGCGGCAGCUGCUGAACGCCAAGCUGAUCACCCAGCGGAAGUUCGACAACCUGACCAAGGCCGAGCGGG GCGGCCUGUCCGAGCUGGACAAGGCCGGCUUCAUCAAGCGGCAGCUGGUGGAGACCCGGCAGAUCACCAAGCACGUGGCCCAGAUCCUGGACUCCCGGAUGAACACCAAGUACGACGAGAACGACAAGCU GAUCCGGGAGGUGAAGGUGAUCACCCUGAAGUCCAAGCUGGUGUCCGACUUCCGGAAGGACUUCCAGUUCUACAAGGUGCGGGAGAUCAACAACUACCACCACGCCCACGACGCCUACCUGAACGCCGU GGUGGGCACCGCCCUGAUCAAGAAGUACCCCAAGCUGGAGUCCGAGUUCGUGUACGGCGACUACAAGGUGUACGACGUGCGGAAGAUGAUCGCCAAGUCCGAGCAGGAGAUCGGCAAGGCCACCGCCAAG UACUUCUUCUACUCCAACAUCAUGAACUUCUUCAAGACCGAGAUCACCCUGGCCAACGGCGAGAUCCGGAAGCGGCCCCUGAUCGAGACCAACGGCGAGACCGGCGAGAUCGUGUGGGACAAGGGCCGG GACUUCGCCACCGUGCGGAAGGUGCUGUCCAUGCCCCAGGUGAACAUCGUGAAGAAGACCGAGGUGCAGACCGGCGGCUUCUCCAAGGAGUCCAUCCUGCCCAAGCGGAACUCCGACAAGCUGAUCGCC CGGAAGAAGGACUGGGACCCCAAGAAGUACGGCGGCUUCGACUCCCCCACCGUGGCCUACUCCGUGCUGGUGGGCCAAGGUGGAGAAGGGCAAGUCCAAGAAGCUGAAGUCCGUGAAGGAGCUGCUG GGCAUCACCAUCAUGGAGCGGUCCUCCUUCGAGAAGAACCCCAUCGACUUCCUGGAGGCCAAGGGCUACAAGGAGGUGAAGAAGGACCUGAUCAUCAAGCUGCCCAAGUACUCCCUGUUCGAGCUGGAGA ACGGCCGGAAGCGGAUGCUGGCCUCCGCCGGCGAGCUGCAGAAGGGCAACGAGCUGGCCCUGCCCUCCAAGUACGUGAACUUCCUGUACCUGGCCUCCCACUACGAGAAGCUGAAGGGCUCCCCGAGG ACAACGAGCAGAAGCAGCUGUUCGUGGAGCAGCACAAGCACUACCUGGACGAGAUCAUCGAGCAGAUCUCCGAGUUCCAAGCGGGUGAUCCUGGCCGACGCCAACCUGGACAAGGUGCUGUCCGCCUA CAACAAGCACCGGGACAAGCCCAUCCGGGAGCAGGCCGAGAACAUCAUCCACCUGUUCACCCUGACCAACCUGGGCGCCCCCGCCGCCUUCAAGUACUUCGACACCACCAUCGACCGGAAGCGGUACAC CUCCACCAAGGAGGUGCUGGACGCCACCCUGAUCCACCAGUCCAUCACCGGCCUGUACGAGACCCGGAUCGACCUGUCCCAGCUGGGCGGCGACGGCGGCGGCUCCCCCAAGAAGAAGCGGAAGGUGUGA 219 SpCas9 amino acid sequence MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLP EKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYE YFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFA NRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSD KNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATA KYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFEL ENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGGSPKKKRKV 228 G021925 mU*mU*mC*UUUGUAGAACUUGAAGUGUUUUAGAmGmCmUmAmGmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmCmUmUmGmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 229 Amino acid sequence of IL10 D25/E96A MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRALRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGANLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 230 Nucleic acid sequence of IL10 D25/E96A ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGCTCTCCG AGATGCCTTCAGCAGAGTGAAGACTTTCTTTcaaatgaaggatcagctggacaACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTT ACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGCTAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAAAC AAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAACTGA 351 G029528 mU*mC*mG*CUUUGCUGAGGUCUAGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCACGAAAGGGCACCGAGUCGGmU*mG*mC*mU

Vectors, individually or in any combination thereof, containing guide RNA, RNA-DNA binders, or donor constructs, may be delivered via liposomes, nanoparticles, exosomes, or microvesicles. The donor constructs may contain sequences encoding dmTGFB1 molecules, regulatory T-cell stimulating molecules (e.g., IL10, CTLA4), or targeting receptors (e.g., CAR). Vectors may also be delivered via liposome nanoparticles (LNPs). One or more guide RNAs, RNA-DNA binders (e.g., mRNA), or donor constructs containing sequences encoding heterologous proteins, individually or in any combination thereof, may be delivered via LNPs, liposomes, nanoparticles, exosomes, or microvesicles. One or more guide RNAs, RNA-binding DNA binders (e.g., mRNA), or donor constructs containing sequences encoding heterologous proteins, individually or in any combination, can be delivered via LNPs. In some embodiments, one or more guide RNAs and RNA-guided DNA binders (e.g., mRNA) are delivered via LNPs. Donor constructs can be delivered via viral vectors.

Lipid nanoparticles (LNPs) are a well-known method for delivering nucleotide and protein cargoes, and can be used to deliver any of the guiding RNAs, RNA-guided DNA binders, or donor constructs disclosed herein.

As used herein, a lipid nanoparticle (LNP) refers to a particle comprising a plurality (i.e., more than one) lipid molecules physically bound together by intermolecular forces. An LNP can be, for example, a microsphere (including monolayer and multilayer vesicles, such as "liposomes," i.e., a laminar lipid bilayer, which in some embodiments is substantially spherical and in more specific embodiments may contain an aqueous core (e.g., containing the majority of an RNA molecule)), a dispersed phase in an emulsion, a microcell, or an internal phase in a suspension (e.g., see WO2017173054, the contents of which are incorporated herein by reference in their entirety). Any LNP known to those skilled in the art to be capable of delivering nucleotides to an individual can be utilized. For example, WO2021222287 provides an exemplary LNP formulation and method for delivering an agent to T cells for modification.

In some embodiments, this document provides methods for delivering, alone or in combination, any of the guiding RNAs described herein or the donor constructs disclosed herein to a cell, cell population, or individual, wherein one or more of these components are bound to an LNP. In some embodiments, the method further comprises an RNA-guided DNA binder (e.g., Cas9 or a sequence encoding Cas9).

In some embodiments, this document provides compositions comprising, alone or in combination with, any of the guiding RNAs or donor constructs disclosed herein, and LNPs. In some embodiments, the compositions further comprise an RNA-guided DNA binder (e.g., Cas9 or a sequence encoding Cas9).

In some embodiments, the LNP comprises a cationic lipid. In some embodiments, the LNP comprises (9Z,12Z)-octadecyl-9,12-dienoic acid 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester ((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl Octadeca-9,12-dienoate, also known as (9Z,12Z)-octadecyl-9,12-dienoic acid 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate) or another ionizable lipid. See, for example, lipids in WO2019067992, WO2017173054, WO2015095340 and WO2014136086 and the references provided therein. In some embodiments, LNPs comprise cationic lipoamine-p-RNA phosphates with a molar ratio (N:P) of about 4.5, 5.0, 5.5, 6.0, or 6.5. In some embodiments, the terms cationic and ionizable are interchangeable in the context of LNP lipids, for example, where ionizable lipids are defined as cationic depending on pH.

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

In some embodiments, any guiding RNA, RNA-guided DNA binder, or donor construct described herein, whether alone or in combination, whether naked or as part of a carrier, is formulated with or delivered via lipid nanoparticles; for example, see WO2019067992, WO2017173054, or WO2021222287, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the LNP composition comprises an RNA component and a lipid component, wherein the lipid component includes amine lipids, such as biodegradable ionizable lipids. In some cases, the lipid component comprises biodegradable ionizable lipids, cholesterol, DSPC (distearate phospholipid choline), and PEG-DMG (1,2-dimyristyl-racemic-glycerol-3-methylpolyoxyethylene glycol 2000 (PEG2k-DMG)). In some embodiments, the lipoprotein nucleic acid assembly contains an ionizable lipid A ((9Z,12Z)-octadecyl-9,12-dienoic acid 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester ((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl Octadeca-9,12-dienoate, also known as (9Z,12Z)-octadecyl-9,12-dienoic acid 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate)), cholesterol, DSPC (distearate phospholipid choline), and PEG2k-DMG (1,2-dimyristyl-racemic-glycerol-3-methylpolyoxyethylene glycol 2000 (PEG2k-DMG)). In some embodiments, the components are present in a 50 mol ratio of lipid A: 38 cholesterol: 9 DSPC: 3 PEG-DMG. In other embodiments, the components are present in a 35 mol ratio of lipid A: 47.5 cholesterol: 15 DSPC: 2.5 PEG-DMG. The lipoprotein assemblies can be formulated with a mol ratio of lipoamines to RNA phosphates (N:P) of approximately 6 and a gRNA to mRNA weight ratio of 2:1, 1:1, or 1:2.

It is evident that the guide RNA, the RNA-guided DNA binder (e.g., Cas nuclease or nucleic acid encoding Cas nuclease), and the donor construct containing sequences encoding dmTGFB1 molecules, regulatory T cell stimulating molecules (e.g., IL10), or targeting receptors (e.g., CAR) can be delivered using the same or different systems. For example, the guide RNA, Cas nuclease, and 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 plastids or LNPs, while the donor construct can be carried by a vector (e.g., AAV).

Different delivery systems can deliver simultaneously or in any order. In some embodiments, the donor construct, guide RNA, and Cas nuclease can be delivered simultaneously, for example, in one vector, two vectors, individual vectors, one LNP, two LNPs, individual LNPs, or combinations thereof. In some embodiments, the donor construct can be delivered as a vector or in combination with an LNP before delivery as a vector or alone or together with an LNP, or as guide RNA or Cas nuclease for a ribonucleoprotein (RNP) (e.g., approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days). As another example, a carrier RNA or Cas nuclease, either alone or together with LNP, or as a ribonucleoprotein (RNP), may be delivered before delivery of the carrier or the LNP-bound construct (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days).

IV. Methods for Engineering T Cells This disclosure provides methods for engineering T cells, which are implemented by inserting a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence and modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TGFBR2 into the cells. This disclosure provides methods for engineering T cells, which further include modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG; and/or inserting a heterologous sequence encoding a regulatory T cell-promoting molecule under the control of a promoter sequence into the cells. This disclosure provides methods for engineering T cells to include modifications, which are implemented by inserting a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence and modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TGFBR2 into the cells. This disclosure also provides methods for engineering T cells that further include modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG; and/or inserting a heterologous sequence encoding IL10 under the control of a promoter sequence into the cells. This disclosure provides methods for engineering T cells, comprising modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TGFBR2; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG; and/or inserting a heterologous sequence encoding CTLA4 under the control of a promoter sequence into the cells. This disclosure provides methods for engineering T cells to include modifications, which are implemented by inserting a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence into the cells and modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TGFBR2. This disclosure provides methods for engineering T cells, which further include modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TGFBR2; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG; and/or inserting into the cells a heterologous sequence encoding IL10 and CTLA4, respectively, under the control of a promoter sequence.

In some embodiments, such methods include engineering T cells to include modifications performed by: inserting a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence into the cells, and knocking down, for example, an endogenous nucleic acid sequence encoding TGFBR2, and, where appropriate, modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG; and/or inserting a heterologous sequence encoding a regulatory T cell-promoting molecule (e.g., IL10 or CTLA4) into the cells, and further including modification (e.g., knockdown) of the TCR sequence.

In some embodiments, such methods include engineering T cells to include modifications performed by: inserting a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence into the cells; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TGFBR2; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG; inserting a heterologous sequence encoding a regulatory T cell-promoting molecule (e.g., IL10 or CTLA4) into the cells; and further including inserting a heterologous sequence encoding a targeting receptor (e.g., CAR) into the cells.

In some embodiments, such methods include engineering T cells to include modifications performed by: inserting a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence into the cells; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TGFBR2; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG; inserting a heterologous sequence encoding a regulatory T cell-promoting molecule (e.g., IL10 or CTLA4) into the cells; modifying (e.g., knocking down) a TCR sequence; and inserting a heterologous sequence encoding a targeting receptor (e.g., CAR) into the cells.

In some embodiments, such methods include modifications performed by: inserting a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence into cells; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TGFBR2; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG; inserting a heterologous sequence encoding a regulatory T cell-promoting molecule into cells; knocking down the TCR gene, if applicable; and inserting a targeting receptor (e.g., CAR), if applicable, into cells engineered using the CRISPR/Cas system and guide RNA disclosed herein.

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

In these embodiments, the target receptor to be inserted may be provided via a donor construct. In some embodiments, the target receptor may be a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a receptor of a cell surface molecule operatively linked via at least one transmembrane domain in an internal signaling domain that is activated upon binding of the extracellular receptor portion to the target. In some embodiments, the target receptor may be a receptor present on the surface of an engineered cell (e.g., a T cell) to allow the cell to bind to a target site (e.g., a specific cell or tissue in an organism). In some embodiments, the targeting receptor may allow engineered T cells to bind to specific target cells, wherein the engineered T cells are homed to disease sites (e.g., mucosal cells at sites of inflammation) and act directly on the target cells (e.g., killing or inhibiting the target cells); and/or the targeting receptor may allow engineered T cells to bind to specific target cells that are not specific to disease sites (e.g., activated immune cells of the lymphatic system) and act directly on the target cells (e.g., killing or inhibiting the target cells).

In some of these embodiments, the target receptor is a CAR that can target the following: MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), protein lipoprotein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), stromal core protein 3 (DSG3), or MHC class I HLA-A (HLA-A*02).

This article discloses suitable gene editing systems for engineering T cells to include insertion and modification (e.g., knockdown), and such systems 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. Typically, gene editing systems involve the use of engineered cleavage systems to induce double-strand breaks (DSBs) or nicks (e.g., single-strand breaks or SSBs) in a target DNA sequence. Cleavage or nicks can occur by using specific nucleases (such as engineered ZFNs, TALENs) or by using CRISPR/Cas systems with engineered lead RNA, such as the CRISPR/Cas9 system, to induce specific cleavage or nicks in the target DNA sequence. In addition, targeted nucleases based on the Argonaute system (e.g., from T. thermophilus , called 'TtAgo', see Swarts et al. (2014) Nature 507(7491): 258-261) are being developed, which may also have the potential for use in genome editing and gene therapy.

Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific DNA sequences. They are created by fusing the TAL effector DNA-binding domain with a DNA-cleaving domain (a nuclease that cleaves DNA strands). Transcription activator-like effectors (TALEs) can be engineered to bind to a desired DNA sequence, thereby promoting DNA cleavage at a specific location (e.g., see Boch, TALEs of genome targeting Nature Biotech. 29:135-136 (2011)). Restriction enzymes can be introduced into cells for gene editing or for in situ genome editing; this technique is called genome editing using engineered nucleases. Such methods and the combinations used are known in this technology. For example, see WO2019147805, WO2014040370, and WO2018073393, the contents of which are incorporated herein in their entirety.

Zinc finger nucleases (ZFNs) are artificial restriction enzymes created by fusing a zinc finger DNA-binding domain with a DNA cleavage domain. The zinc finger domain can be engineered to target specific desired DNA sequences, enabling the ZFN to target unique sequences within complex genomes. The non-specific cleavage domain from the type IIs restriction endonuclease FokI is commonly used as the cleavage domain in ZFNs. Cleavage is repaired by endogenous DNA repair mechanisms, allowing ZFNs to precisely alter the genome of higher organisms. Such methods and the combinations used are known in this art. For example, see WO2011091324, the contents of which are incorporated herein by reference in their entirety.

RNA interference (RNAi) is a biological process in which RNA molecules suppress gene expression or translation by neutralizing target mRNA molecules. Small interfering RNAs (siRNAs) are key to RNA interference. RNA is a direct product of genes, and these small RNAs (typically 19-23 nucleotides long, forming a 19-21 nucleotide double strand) can guide RNA-induced silencing (RISC) complexes to degrade messenger RNA (mRNA) molecules, thereby reducing the activity of mRNA molecules by preventing translation through post-transcriptional gene silencing. Short hairpin RNAs (shRNAs) are single-stranded siRNAs in which the strands forming the double-stranded region have a hairpin structure, and are usually produced by transcription from self-expression vectors. RNAi can also be achieved using longer RNA double-stranded structures called dicer acceptors, which are cleaved by dicers to promote the cleavage of target mRNA before being loaded into RISC. Such methods and compositions are known in the art. In the compositions and methods provided herein, preferably, the RNA molecule promoting RNA interference is provided as a persistent expression vector, see, for example, WO2018208837, the contents of which are incorporated herein by reference in their entirety. In some embodiments, RNAi is used in conjunction with an expression vector.

It should be understood that this disclosure considers methods for insertion with or without the guide RNA disclosed herein (e.g., using a ZFN system to induce breaks in the target DNA sequence, thereby generating sites for the insertion building blocks). For methods using the guide RNA disclosed herein, these methods include using a CRISPR/Cas system to modify (e.g., knock down) nucleic acid sequences encoding TGFBR2, TNFA, IFNG, or TCR. It should also be understood that this disclosure considers methods for modifying (e.g., knocking down) TGFBR2, TNFA, IFNG, or TCR, which can be performed without the guide RNA disclosed herein (e.g., using a ZFN system to induce breaks in the target DNA sequence, thereby generating sites for the insertion building blocks).

In some embodiments, a donor construct containing the sequence to be inserted (e.g., a sequence encoding dmTGFB1 or a regulatory T cell stimulating molecule (e.g., IL10 or CTLA4)) is inserted at a gene locus of a sequence targeted for modification (e.g., knockdown) (e.g., the TCR gene).

In some embodiments, CRISPR/Cas systems (e.g., guide RNA and RNA-guided DNA binders) can be used to generate insertion sites at desired loci within the genome, where donor constructs containing sequences encoding dmTGFB1, regulatory T cell-stimulating molecules (e.g., IL10, CTLA4), or targeting receptors (e.g., CARs, such as the MAdCAM-1 CAR disclosed herein) can be inserted to express dmTGFB1, regulatory T cell-stimulating molecules (e.g., IL10, CTLA4), or CARs (e.g., MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), or proteolipoprotein 1. (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), stromal core protein 3 (DSG3) or MHC class I HLA-A (HLA-A*02) CAR). Targeting receptors, such as CARs, including MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipoprotein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), stromal core protein 3 (DSG3), or MHC class I HLA-A (HLA-A*02). CAR; or the inserted sequence dmTGFB1 or regulatory T cell stimulating molecule (e.g., IL10 or CTLA4) may be heterologous in relation to its insertion site or locus, for example, as described herein, dmTGFB1, regulatory T cell stimulating molecule (e.g., IL10, CTLA4) or targeting receptor (e.g. CAR, such as MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipoprotein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), bridgene core protein 3) (DSG3) or MHC class I HLA-A (HLA-A*02) CAR) are safe harbor loci or TCR loci that are not usually expressed. In some embodiments, the guide RNA described herein can be used with an RNA-guided DNA binder (e.g., Cas nuclease) according to the method of the present invention to generate an insertion site at which an insertion site can be inserted containing encoding dmTGFB1, regulatory T cell stimulating molecules (e.g., IL10, CTLA4) or targeting receptors (e.g., CARs such as MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipoprotein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), or a member of solute carrier family 2). Donor constructs containing sequences of (SCL2A2), glutamic acid decarboxylase (GAD2), bridgene core protein 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CAR, to express dmTGFB1, regulatory T cell stimulating molecules (e.g., IL10, CTLA4) or CARs (e.g., MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipoprotein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), bridgene core protein 3, etc. (DSG3) or MHC Class I HLA-A (HLA-A*02) CAR). This article illustrates and describes guide RNAs for inserting dmTGFB1, regulatory T cell-promoting molecules (e.g., IL10, CTLA4), or targeting receptors (e.g., CARs such as MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipoprotein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), stromal core protein 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CAR) into specific gene loci. In an alternative implementation, the target receptor is the TNFA target receptor.

In some implementations, CD4+ T cells are engineered using gRNA (e.g., gRNA that targets TGFBR2, IFNG, TNFA, or TCR for knockdown), RNA-guided DNA binders (e.g., Cas nucleases), or donor constructs for transduction (e.g., using viral or non-viral delivery). In some embodiments, engineered T cells are transduced using: 1) gRNAs targeting TGFBR2 and, depending on the situation, pro-inflammatory cytokines (e.g., IFNG or TNFA), RNA-guided DNA binders (e.g., Cas nucleases), and 2) donor constructs containing nucleic acid sequences encoding dmTGFB1 and, depending on the situation, regulatory T cell-promoting molecules (e.g., IL10 or CTLA4) and targeting receptors (e.g., CARs such as MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), and protein lipoprotein 1). (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), stromal core protein 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CAR, or TNFA targeting receptors. In some embodiments, engineered cells are selected to express the targeting receptors.

In some embodiments, CD4+ T cells are engineered by transduction using gRNA (e.g., gRNA that targets TGFBR2, IFNG, TNFA or TCR for knockdown), RNA-guided DNA binders (e.g., Cas nucleases), and donor constructs. In some embodiments, engineered T cells: 1) are transduced using a donor construct containing a nucleic acid sequence encoding the following: dmTGFB1 and, depending on the case, a regulatory T cell-promoting molecule (e.g., IL10 or CTLA4) and/or a target receptor (e.g., CAR, such as MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipoprotein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), bridging core protein 3 (DSG3), or MHC. Class I HLA-A (HLA-A*02) CAR), and 2) transduction with gRNAs targeting the nucleic acid sequences encoding TGFBR2 and, where appropriate, pro-inflammatory cytokines (e.g., IFNG or TNFA), or RNA-guided DNA binders (e.g., Cas nucleases). In some embodiments, engineered cells are selected to express the target receptor.

In some implementations, CD4+ T cells are engineered by transduction using gRNA (e.g., gRNA that targets TGFBR2, IFNG, TNFA, or TCR for knockdown), RNA-guided DNA binders (e.g., Cas nucleases), or donor constructs. In some embodiments, engineered T cells are: 1) transduced with donor constructs containing nucleic acid sequences encoding the following: dmTGFB1 and, depending on the case, regulatory T cell-promoting molecules (e.g., IL10 or CTLA4) and/or targeting receptors (e.g., CARs such as MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipoprotein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute carrier family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), bridgene core protein 3 (DSG3), or MHC class I HLA-A (HLA-A*02) CAR), and 2) transduction with gRNA and RNA-guided DNA binders (e.g., Cas nucleases) that target the nucleic acid sequences encoding TGFBR2 and, depending on the situation, pro-inflammatory interstitial molecules (e.g., IFNG or TNFA).

As described herein, donor constructs comprising sequences encoding dmTGFB1, regulatory T cell stimulating molecules (e.g., IL10, CTLA4) or targeting receptors (e.g., CAR), lead RNA (e.g., gRNA targeting TGFBR2, IFNG, TNFA, or TCR for knockdown), and RNA-guided DNA binders can be delivered using any suitable delivery system and method known in the art. In some embodiments, the lead RNA and Cas nuclease are bound to LNP and delivered to cells or cell populations prior to the delivery of the donor construct comprising sequences encoding dmTGFB1, regulatory T cell stimulating molecules (e.g., IL10, CTLA4), or targeting receptors (e.g., CAR). In some embodiments, the guide RNA and Cas nuclease bind to the LNP and are delivered to the cell or cell population after delivery of a donor construct containing a sequence encoding dmTGFB1, a regulatory T cell stimulating molecule (e.g., IL10, CTLA4) or a target receptor (e.g., CAR).

In some embodiments, administering the gRNA, donor construct and RNA-guided DNA binder described herein to naturally occurring T cells can transform those naturally occurring T cells (e.g., CD4+ T cells) into cells exhibiting characteristics of regulatory T cells (e.g., immunosuppressive characteristics).

This can be used to modify (e.g., knock down) the expression of TGFBR2, IFNG, TNFA, or TCR genes, or to insert encoding dmTGFB1, regulatory T cell-promoting molecules (e.g., IL10, CTLA4), or targeting receptors (e.g., CARs such as MAdCAM-1, TNFA, CD70, CEACAM6, VCAM-1, citrullinated vimentin, myelin basic protein (MBP), MOG (myelin oligodendrocyte glycoprotein), proteolipoprotein 1 (PLP1), CD19 molecule (CD19), CD20 molecule (CD20), TNFSF7, TNFRSF17, dipeptidyl peptidase-like 6 (DPP6), solute vector family 2 member 2 (SCL2A2), glutamic acid decarboxylase (GAD2), stromal core protein 3 (DSG3), or MHC class I HLA-A (HLA-A*02). The CAR sequence of gRNA, donor construct and RNA-guided DNA binder are introduced into learned T cells or learned T cell populations to produce the engineered T cells or T cell populations described herein.

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

In some embodiments, the RNA-guided DNA binder (such as Cas9 nuclease) has cleavage activity, which may also be referred to as double-stranded endonuclease activity. In some embodiments, the RNA-guided DNA binder (such as Cas9 nuclease) has nicking activity, which may also be referred to as single-stranded endonuclease activity. In some embodiments, the RNA-guided DNA binder comprises a Cas nuclease. Examples of Cas nucleases include those of the type II CRISPR system of Streptococcus pyogenes, Staphylococcus aureus, Neisseria meningitidis, and other prokaryotes (see, for example, the list in the next paragraph), as well as their variants or mutants (e.g., engineered, non-natural, naturally occurring, or other variants). For example, see US20160312198; US20160312199.

Non-restrictive illustrative species that can produce Cas nucleases include *Streptococcus pyogenes*, *Streptococcus thermophilus*, *Streptococcus* sp., *Staphylococcus aureus*, *Listeria innocua*, *Lactobacillus gasseri*, *Francisella novicida*, *Wolinella succinogenes*, *Sutterella wadsworthensis*, *Gammaproteobacterium*, *Diplocameningococcus*, *Campylobacter jejuni*, *Pasteurella multocida*, and *Fibrobacter*. *Streptomyces succinogene*, *Rhodospirillum rubrum*, *Nocardiopsis dassonvillei*, *Streptomyces pristinaespiralis*, *Streptomyces viridochromogene*, *Streptomyces viridochromogene*, *Streptosporangium roseum*, *Streptosporangium roseum*, *Alicyclobacillus acidocaldarius*, *Bacillus pseudomycoides*, *Bacillus selenitireducens*, *Exiguobacterium sibiricum*, *Lactobacillus* * *Lactobacillus delbrueckii*, *Lactobacillus salivarius*, *Lactobacillus buchneri*, *Treponema denticola*, *Microscilla marina*, *Burkholderiales bacterium*, *Polaromonas naphthalenivorans*, *Polaromonas sp.*, *Crocosphaera watsonii*, *Cyanothece sp.*, *Microcystis aeruginosa*, *Synechococcus sp.*, *Acetohalobium arabaticum*, *Ammonifex* *Caldicelulosiruptor becscii*, *Candidatus desulforudis*, *Clostridium botulinum*, *Clostridium difficile*, *Finegoldia magna*, *Natranaerobius thermophilus*, *Pelotomaculum thermopropionicum*, *Acidithiobacillus caldus*, *Acidithiobacillus ferrooxidans*, *Allochromatium vinosum*, *Marinobacter sp.*, *Nitrosococcus* halophilus), Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho The following bacteria are listed: *Africanus*, *Streptococcus pasteurianus*, *Neisseria cinerea*, *Campylobacter lari*, *Parvibaculum lavamentivorans*, *Corynebacterium diphtheria*, *Acidaminococcus sp*, *Lachnospiraceae bacterium* ND2006, and *Acaryochloris marina*.

In some embodiments, the Cas nuclease is a Cas9 nuclease from *Streptococcus pyogenes*. In some embodiments, the Cas nuclease is a Cas9 nuclease from *Streptococcus thermophilus*. In some embodiments, the Cas nuclease is a Cas9 nuclease from *Diplococcus meningitidis*. In some embodiments, the Cas nuclease is a Cas9 nuclease from *Staphylococcus aureus*. In some embodiments, the Cas nuclease is a Cpf1 nuclease from *Francisella catarrhalis*. In some embodiments, the Cas nuclease is a Cpf1 nuclease from *Aminococcus* spp. In some embodiments, the Cas nuclease is a Cpf1 nuclease from *Trichophyton* ND2006. In other embodiments, the Cas nuclease is a Cpf1 nuclease derived from the following species: * Francisella tularensis* , *Trichophyton*, *Butyrivibrio proteoclasticus*, *Peregrinibacteria bacterium*, *Parcubacteria bacterium*, *Smithella*, *Aminococcus*, *Candidatus Methanoplasma termitum*, *Eubacterium eligens*, *Moraxella bovoculi*, *Leptospira inadai*, *Porphyromonas crevioricanis*, and *Prevotella disiens*. ) or Porphyromonas macacae . In some embodiments, the Cas nuclease is a Cpf1 nuclease derived from aminococci or spirilla .

In some embodiments, the gRNA and the RNA-guided DNA binder are collectively referred to as a ribonucleoprotein complex (RNP). In some embodiments, the RNA-guided DNA binder is a Cas nuclease. In some embodiments, the gRNA and the Cas nuclease are collectively referred to as a Cas RNP. In some embodiments, the RNP contains type I, type II, or type III components. In some embodiments, the Cas nuclease is the Cas9 protein from the type II CRISPR/Cas system. In some embodiments, the gRNA and Cas9 are collectively referred to as a Cas9 RNP.

Wild-type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domain cleaves non-target DNA strands, and the HNH domain cleaves target DNA strands. In some embodiments, the Cas9 protein contains more than one RuvC domain or more than one HNH domain. In some embodiments, the Cas9 protein is wild-type Cas9. In each of the embodiments of composition, use, and method, Cas induces double-strand breakage in the target DNA.

In some embodiments, chimeric Cas nucleases are used, wherein a domain or region of a protein is partially replaced by a different protein. In some embodiments, the Cas nuclease domain may be replaced by a domain from a different nuclease (such as Fok1). In some embodiments, the Cas nuclease may be a modified nuclease.

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 possess RNA cleavage activity.

In some embodiments, the RNA-guided DNA binder has single-strand nicking activity, meaning it can cleave one strand of DNA to produce a single-strand break, also known as a "nick". In some embodiments, the RNA-guided DNA binder contains a Cas nicking enzyme. A nicking enzyme is an enzyme that creates a nick in dsDNA, that is, it cleaves one strand of the DNA double helix but not the other. In some embodiments, the Cas nicking enzyme is a form of Cas nuclease (e.g., the Cas nuclease discussed above) in which the endonuclease active site (e.g.) is inactive due to one or more changes in the catalytic domain (e.g., point mutation). For a discussion of Cas nicking enzymes and exemplary changes in the catalytic domain, see, for example, U.S. Patent No. 8,889,356. In some embodiments, Cas nicking enzymes (such as Cas9 nicking enzymes) have an inactive RuvC or HNH domain.

In some embodiments, the RNA-guided DNA binder is modified to contain only one functional nuclease domain. For example, a modifier protein may be used to mutate or completely or partially delete one of the nuclease domains, thereby reducing its nucleic acid cleavage activity. In some embodiments, a nickase with a reduced-activity RuvC domain is used. In some embodiments, a nickase with an inactive RuvC domain is used. In some embodiments, a nickase with a reduced-activity HNH domain is used. In some embodiments, a nickase with an inactive HNH domain is used.

In some embodiments, conserved amino acids within the Cas protein nuclease domain are substituted to reduce or alter nuclease activity. In some embodiments, the Cas nuclease may contain amino acid substitutions in the RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the *Streptococcus pustulosa* Cas9 protein). See, for example, Zetsche et al. (2015) Cell Oct 22:163(3):759-771. In some embodiments, the Cas nuclease may contain amino acid substitutions in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the *Streptococcus pustulosa* Cas9 protein). For example, see Zetsche et al. (2015). Other exemplary amino acid substitutions include D917A, E1006A, and D1255A (based on the *F. fraternis* U112 Cpf1 (FnCpf1) sequence (UniProtKB - A0Q7Q2 (CPF1_FRATN)). Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain or the RuvC or RuvC-like domain of *Neisseria meningitidis* include Nme2Cas9D16A (HNH nickase) and Nme2Cas9H588A (RuvC nickase).

In some embodiments, the nicking enzyme is provided in combination with a pair of guide RNAs that complement each other, one sense and one antisense, along with the target sequence. In this embodiment, the guide RNAs guide the nicking enzyme to the target sequence and introduce the DSB by creating nicks (i.e., double nicks) on the opposing strands of the target sequence. In some embodiments, the nicking enzyme is used with two separate guide RNAs targeting opposing DNA strands to create double nicks in the target DNA. In some embodiments, the nicking enzyme is used with two separate guide RNAs selected to be very close together to create double nicks in the target DNA.

In some embodiments, the RNA-guided DNA binder comprises one or more heterologous functional domains (e.g., a fusion polypeptide).

In some embodiments, the heterologous functional domain facilitates the transfer of the RNA-guided DNA binder to the cell nucleus. For example, the heterologous functional domain may be a nuclear localization signal (NLS). In some embodiments, the RNA-guided DNA binder may fuse with 1-5 NLSs. In some embodiments, the RNA-guided DNA binder may fuse with 2, 3, or 4 NLSs. In some embodiments, the RNA-guided DNA binder may fuse with two NLSs. In some embodiments, the RNA-guided DNA binder may fuse with one NLS. When using a single NLS, the NLS may be ligated to the N-terminus or C-terminus of the RNA-guided DNA binder sequence. In some embodiments, the NLS is not ligated to the C-terminus. It may also be inserted within the RNA-guided DNA binder sequence. In other embodiments, the RNA-guided DNA binder may fuse with more than one NLS. In some cases, at least two NLS are identical (e.g., two SV40 NLS). In some embodiments, the RNA-guided DNA binder has at least two different NLS. In some embodiments, the RNA-guided DNA binder fuses with two SV40 NLS sequences linked at the C-terminus. In some embodiments, the RNA-guided DNA binder may fuse with two NLS, one linked at the N-terminus and the other at the C-terminus. In some embodiments, the RNA-guided DNA binder may fuse with three NLS. In some embodiments, the RNA-guided DNA binder may not fuse with any NLS. In some embodiments, the NLS may be a single-component sequence, such as SV40 NLS PKKKRKV (SEQ ID NO: 143) or PKKKRRV (SEQ ID NO: 144). In some embodiments, the NLS may be a two-component sequence, such as the nucleoplasmic protein NLS KRPAATKKAGQAKKKK (SEQ ID NO: 145). In a specific embodiment, a single PKKKRKV (SEQ ID NO: 143) NLS may be ligated to the C-terminus of an RNA-guided DNA binder. One or more ligands may be included at the fusion site, as appropriate.

V. Treatment Methods This disclosure provides methods for suppressing an individual's immune response, including administration of engineered T cells comprising inserting a heterologous sequence encoding dmTGFB1 under promoter sequence control and modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TGFBR2 into the cells. The engineered T cells may, where appropriate, further comprise modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding TNFA; modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding IFNG; or inserting a heterologous sequence encoding a regulatory T cell-promoting molecule under promoter sequence control into the cells. This disclosure provides methods for suppressing an individual's immune response, the methods comprising administering engineered T cells comprising inserting a heterologous sequence encoding dmTGFB1 under the control of a promoter sequence into the cells; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TGFBR2; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA; and inserting a heterologous sequence encoding a regulatory T cell-promoting molecule under the control of a promoter sequence into the cells. This disclosure provides methods for suppressing an individual's immune response, the methods comprising administering engineered T cells containing modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding TGFBR2; modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG; and inserting a heterologous sequence encoding IL10 under the control of a promoter sequence into the cells. This disclosure provides methods for suppressing an individual's immune response, the methods comprising administering engineered T cells comprising inserting a heterologous sequence encoding dmTGFB1 under promoter sequence control into the cells; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TGFBR2; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG; and inserting a heterologous sequence encoding CTLA4 under promoter sequence control into the cells. This disclosure provides methods for suppressing an individual's immune response, the methods comprising administering engineered T cells comprising inserting a heterologous sequence encoding dmTGFB1 under promoter sequence control into the cells; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TGFBR2; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG; and inserting heterologous sequences encoding IL10 and CTLA4, respectively under promoter sequence control, into the cells.

This disclosure provides methods for treating an individual's autoimmune disease, including administering engineered T cells comprising inserting a heterologous sequence encoding dmTGFB1 under promoter sequence control and modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TGFBR2. The engineered T cells may further comprise, as appropriate, modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding TNFA; modification (e.g., knocking down) of an endogenous nucleic acid sequence encoding IFNG; or insertion of a heterologous sequence encoding a regulatory T cell-promoting molecule under promoter sequence control. This disclosure provides methods for treating an individual's autoimmune disease, the methods comprising administering engineered T cells comprising inserting a heterologous sequence encoding dmTGFB1 under promoter sequence control into the cells; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TGFBR2; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA; and inserting a heterologous sequence encoding a regulatory T cell-promoting molecule under promoter sequence control into the cells. This disclosure provides methods for treating an individual's autoimmune disease, the methods comprising administering engineered T cells comprising inserting a heterologous sequence encoding dmTGFB1 under promoter sequence control into the cells; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TGFBR2; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA; and inserting a heterologous sequence encoding IL10 under promoter sequence control into the cells. This disclosure provides methods for treating an individual's autoimmune disease, the methods comprising administering engineered T cells comprising inserting a heterologous sequence encoding dmTGFB1 under promoter sequence control into the cells; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TGFBR2; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA; and inserting a heterologous sequence encoding CTLA4 under promoter sequence control into the cells. This disclosure provides methods for treating an individual's autoimmune disease, the methods comprising administering engineered T cells comprising inserting a heterologous sequence encoding dmTGFB1 under promoter sequence control into the cells; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TGFBR2; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA; and inserting heterologous sequences encoding IL10 and CTLA4, respectively under promoter sequence control, into the cells.

This disclosure provides methods for treating individuals with GvHD, including administering engineered T cells containing a heterologous sequence encoding dmTGFB1 under promoter sequence control and a modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TGFBR2. The engineered T cells may further contain modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG; or the insertion of a heterologous sequence encoding a regulatory T cell-promoting molecule under promoter sequence control. This disclosure provides methods for treating individuals with GvHD, the methods comprising administering engineered T cells comprising inserting a heterologous sequence encoding dmTGFB1 under promoter sequence control into the cells; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TGFBR2; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG; and inserting a heterologous sequence encoding a regulatory T cell-promoting molecule under promoter sequence control into the cells. This disclosure provides methods for treating individuals with GvHD, the methods comprising administering engineered T cells containing the insertion of a heterologous sequence encoding dmTGFB1 under promoter sequence control; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TGFBR2; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding INFG; and insertion of a heterologous sequence encoding IL10 under promoter sequence control into the cells. This disclosure provides methods for treating individuals with GvHD, the methods comprising administering engineered T cells containing the insertion of a heterologous sequence encoding dmTGFB1 under promoter sequence control; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TGFBR2; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG; and insertion of a heterologous sequence encoding CTLA4 under promoter sequence control into the cells. This disclosure provides methods for treating individuals with GvHD, the methods comprising administering engineered T cells comprising inserting a heterologous sequence encoding dmTGFB1 under promoter sequence control into the cells; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TGFBR2; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA; modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding INFG; and inserting heterologous sequences encoding IL10 and CTLA4, respectively under promoter sequence control, into the cells.

This disclosure provides methods for treating individuals receiving transplants or transfusions, including the administration of engineered T cells comprising inserting a heterologous sequence encoding dmTGFB1 under promoter sequence control and modifying (e.g., knocking down) an endogenous nucleic acid sequence encoding TGFBR2, which may be performed before, during, or after the transplant or transfusion. The engineered T cells may further comprise, as appropriate, modification (e.g., knocking down) an endogenous nucleic acid sequence encoding TNFA; modification (e.g., knocking down) an endogenous nucleic acid sequence encoding IFNG; or inserting a heterologous sequence encoding a regulatory T cell-promoting molecule under promoter sequence control into the cells. This disclosure provides methods for treating individuals receiving transplants or transfusions, the methods comprising administering engineered T cells containing the insertion of a heterologous sequence encoding dmTGFB1 under promoter sequence control; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TGFBR2; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG; and insertion of a heterologous sequence encoding a regulatory T cell-promoting molecule under promoter sequence control into the cells, wherein such methods are performed before, during, or after the transplant or transfusion. This disclosure provides methods for treating individuals receiving transplants or transfusions, the methods comprising administering engineered T cells containing the insertion of a heterologous sequence encoding dmTGFB1 under promoter sequence control; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TGFBR2; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding INFG; and insertion of a heterologous sequence encoding IL10 under promoter sequence control into the cells, wherein such methods are performed before, simultaneously with, or after receiving the transplant or transfusion. This disclosure provides methods for treating individuals receiving transplants or transfusions, the methods comprising administering engineered T cells containing the insertion of a heterologous sequence encoding dmTGFB1 under promoter sequence control; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TGFBR2; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG; and insertion of a heterologous sequence encoding CTLA4 under promoter sequence control into the cells, wherein such methods are performed before, simultaneously with, or after receiving the transplant or transfusion. This disclosure provides methods for treating individuals receiving transplants or transfusions, the methods comprising administering engineered T cells containing the insertion of a heterologous sequence encoding dmTGFB1 under promoter sequence control; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TGFBR2; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding INFG; and insertion of heterologous sequences encoding IL10 and CTLA4, respectively under promoter sequence control, wherein such methods are performed before, simultaneously with, or after receiving the transplant or transfusion.

In some embodiments, these methods include administering engineered T cells containing the insertion of a heterologous sequence encoding dmTGFB1 under promoter sequence control and modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TGFBR2, and further including the insertion of a sequence encoding a target receptor (e.g., CAR). The engineered T cells may, where appropriate, further contain modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG; or the insertion of a heterologous sequence encoding a regulatory T cell-promoting molecule under promoter sequence control. In some embodiments, the methods include administering engineered T cells containing modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding TGFBR2; modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG; insertion of a sequence encoding a regulatory T cell-promoting molecule; and further including insertion of a sequence encoding a targeting receptor (e.g., CAR).

In some embodiments, a targeting receptor (e.g., a CAR) enables engineered T cells to target the gastrointestinal system. For example, a CAR targeting MAdCAM-1 can be used to suppress immune responses, including inflammation, in conditions such as inflammatory bowel disease, ulcerative colitis, or Crohn's disease. In some embodiments, a targeting receptor (e.g., a CAR) enables engineered T cells to target tissues containing endothelial cells. For example, a CAR targeting VCAM-1 can be used to suppress immune responses, including inflammation, in conditions such as Crohn's disease and multiple sclerosis. In some embodiments, a targeting receptor (e.g., a CAR) enables engineered T cells to target endothelial cells. For example, a CAR targeting CEACAM6 can be used to suppress immune responses, such as in conditions such as Crohn's disease. In some embodiments, a targeting receptor (e.g., a CAR) enables engineered T cells to target B cells; for example, a CAR targeting CD19 may be used to suppress immune responses in diseases such as multiple sclerosis and systemic lupus erythematosus. In some embodiments, a targeting receptor (e.g., a CAR) enables engineered T cells to target B cells; for example, a CAR targeting CD20 may be used to suppress immune responses in diseases such as multiple sclerosis and systemic lupus erythematosus. In some embodiments, a targeting receptor (e.g., a CAR) enables engineered T cells to target inflamed tissues; for example, a CAR targeting TNFA may be used to suppress immune responses in diseases such as inflammatory bowel disease, ulcerative colitis, or Crohn's disease. In some embodiments, a targeting receptor (e.g., a CAR) enables engineered T cells to target neural tissue. For example, the targeting receptor may be a CAR targeting MBP, MOG, or PLP, used to suppress immune responses in conditions such as multiple sclerosis. In some embodiments, a targeting receptor (e.g., a CAR) enables engineered T cells to target immune cells expressing TNFSF7, including (but not limited to) activated T lymphocytes, B lymphocytes, dendritic cells, and/or NK cells. For example, a CAR targeting TNFSF7 can be used to suppress immune responses in conditions such as Crohn's disease, ulcerative colitis, systemic lupus erythematosus, or multiple sclerosis. In some embodiments, a targeting receptor (e.g., a CAR) enables engineered T cells to target tissues containing mature B lymphocytes. For example, a CAR targeting TNFRSF17 may be used to suppress immune responses in conditions such as systemic lupus erythematosus. In some embodiments, a targeting receptor (e.g., a CAR) enables engineered T cells to target synovial tissue. For example, a CAR targeting citrullinated vimentin may be used to suppress immune responses in conditions such as rheumatoid arthritis.

In some embodiments, the target receptor is a CD70-targeting CAR, for example, used to suppress the immune response in conditions such as GvHD. CD70 is a transmembrane protein that transiently appears on the surface of CD4+ and CD8+ T cells, regulatory T cells (Tregs), B cells, antigen-presenting cells (such as dendritic cells), and natural killer (NK) cells in response to immune activation. Regardless of theoretical constraints, CD70 can be elevated in immune cells (such as T cells in patients with autoimmune diseases). In some embodiments, engineered T cells, as described herein, can suppress the activity of CD70-expressing immune cells and/or kill such cells.

In some embodiments, the target receptor is a CAR that targets DPP6, SCL2A2, glutamic acid decarboxylase (GAD2), stromal core protein 3 (DSG3), and MHC class I HLA-A (HLA-A*02).

In some embodiments, these methods include administering engineered T cells containing the insertion of a heterologous sequence encoding dmTGFB1 under promoter sequence control and modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TGFBR2, and further including modification (e.g., knockdown) of the TCR sequence. The engineered T cells may, where appropriate, further include modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modification (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG; or the insertion of a heterologous sequence encoding a regulatory T cell-promoting molecule under promoter sequence control. In some embodiments, the methods include administering engineered T cells containing modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding TGFBR2; modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding TNFA; modifications (e.g., knockdown) of an endogenous nucleic acid sequence encoding IFNG; insertion of a sequence encoding a regulatory T cell-promoting molecule; and further including modifications (e.g., knockdown) of a TCR sequence.

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

In some embodiments, these methods include administering a T cell population containing engineered T cells as described above. In some embodiments, for example, as evaluated by sequencing (e.g., NGS), 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.

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

In some embodiments, the effectiveness of treatment with the engineered T cells can be evaluated in animal models of graft-versus-host disease (e.g., mouse models) by measuring the body weight or survival of animals after administration of the engineered T cells (where animals are sacrificed after significant weight loss (e.g., 20% of initial body weight)). In some embodiments, effective treatment has resulted in a statistically significant increase in survival compared to suitable controls (e.g., animals treated with PBMCs).

The examples provided below illustrate certain disclosed embodiments and should not be construed as limiting the scope of this disclosure in any way.

Example 1. General Method Example 1.1 Preparation of Lipid Nanoparticles Generally, lipid components are dissolved in 100% ethanol at different molar ratios. RNA products (e.g., Cas9 mRNA and sgRNA) are dissolved in 25 mM citrate buffer, 100 mM NaCl, pH 5.0, to achieve an RNA product concentration of approximately 0.45 mg/mL.

Unless otherwise specified, the lipoprotein nucleic acid assembly contains ionizable lipid A ((9Z,12Z)-octadecyl-9,12-dienoic acid 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester ((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl Octadeca-9,12-dienoate, also known as (9Z,12Z)-octadeca-9,12-dienoic acid 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate), cholesterol, DSPC, and PEG2k-DMG, have a molar ratio of 35 for lipid A: 47.5 for cholesterol: 15 for DSPC: 2.5 for PEG-DMG. The lipoprotein nucleic acid assemblies were formulated with a mortise ratio of lipoamine to RNA phosphate (N:P) of approximately 6 and a gRNA to mRNA weight ratio of 1:1.

LNPs were prepared using a cross-flow technique, employing a collisional jet mixing method involving lipids in ethanol, two volumes of RNA solution, and one volume of water. First, the lipids in ethanol and two volumes of RNA solution were mixed using a cross-flow mixer. Then, a fourth water stream was mixed through an in-line T-tube and the outlet stream of the cross-flow mixer (see WO2016010840, Figure 2). The LNPs were kept at room temperature for 1 hour and further diluted with water (approximately 1:1 v/v). The LNPs were concentrated using a tangential flow filter on a plate (Sartorius, 100 kD MWCO) and exchanged with a PD-10 desalination column (GE) buffer at 50 mM Tris, 45 mM NaCl, 5% (w/v) sucrose, pH 7.5 (TSS). Alternatively, if necessary, concentrate the LNP using a 100 kDa Amicon rotary filter and exchange the buffer in the TSS using a PD-10 desalination column (GE). Then filter the resulting mixture using a 0.2 μm sterile filter. Store the final LNP at 4°C or -80°C until further use.

Example 1.2. In vivo extracellular transcription (“IVT”) of mRNA is performed using a linearized plasso DNA template and T7 RNA polymerase to produce N1-methyl-pseudo-U-capped and polyadenylated mRNA. The linearized plasso DNA containing the T7 promoter and transcription sequence is linearized by restriction endonuclease digestion, followed by heat inactivation of the reaction mixture and purification in enzyme and buffer salt. Messenger RNA is synthesized and purified using standard techniques known in this technique.

The self-coded messenger RNA is generated from plasso DNA within the open reading frame of SEQ ID NO: 218 in Table 4. When the sequence cited in this paragraph is mentioned below in relation to RNA, it should be understood that T should be replaced with U (which may be a modified nucleotide as described above). The messenger RNA used in the examples includes a 5' cap and a 3' polyadenylated sequence, for example, up to 100 nt. The guide RNA is chemically synthesized by a commercial supplier or chemically synthesized using modified nucleotides using standard in vitro synthesis techniques.

Example 2. Inhibitory activity of engineered T cells overexpressing Treg-related factors. Example 2.1. T cells were prepared from fresh leukopak (AllCells) human PBMCs and cryopreserved for future use using methods known in this art. CD4+ T cells were isolated from PBMCs using a human CD4+ T cell isolation kit (Miltenyi; catalog number 130-096-533), following the manufacturer's protocol, by negative selection. CD4+ T cells were activated by ablation at a density of 5 × 10^6 cells/mL in OpTimizer-based medium, which included CTS OpTmizer T cell amplification SFM (Gibco, catalog number A3705001), 1% penicillin-streptomycin, 1× Glutamax, 10 mM HEPES, and 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). 2.5% human AB serum (Gemini, catalog number 100-512) from Technologies (catalog number 10991) was cultured at 37°C for 24 hours before lentivirus transduction.

Naturally occurring regulatory T cells (nTregs), also referred to herein as thymic Tregs (tTregs), are prepared using methods known in this art. The CD4+CD25+ T cell population is known to be rich in natural Tregs (nTregs). According to the manufacturer's instructions, CD25+ cells are isolated from CD4+ T cells using human CD25 microbeads II (Miltenyi; catalog number 130-092-983). The isolated CD25+CD4+ T cells were evenly distributed at a density of 5 × 10^ ⁶ cells per well in 6-well Grex containers containing 30 ml OpTmizer-based medium (including CTS OpTmizer T cell amplification SFM (Gibco, catalog number A3705001), 1% penicillin-streptomycin, 1× Glutamax, 10 mM HEPES, 2.5% human AB serum (Gemini, catalog number 100-512), and supplemented with 1000 U/mL recombinant human interleukin-2 (Miltenyi Biotec; catalog number 130-097-746) every other day). Cells were activated by adding 25 μL/mL ImmunoCult human CD3/CD28 T cell activator (Stemcell Technologies, catalog number 10991) and cultured at 37°C for 2 days until lentiviral transduction. After another 3 days, activated CD25+ T cells were harvested and labeled with 50 μg/mL biotinylated anti-LAP antibody (Miltenyi; catalog number 130-095-213) at 4°C for 15 min. Latent related peptide (LAP) is a marker for activated Treg cells. The labeled cells were washed twice and further labeled with antibiotin beads (Miltenyi; catalog number 130-090-485) according to the manufacturer's instructions. LAP+ cells were isolated using an LS column (Miltenyi; catalog number 130-042-401). Isolated native Tregs were cultured in 6-well Grex plates containing 30 mL of OpTmizer-based medium (including CTS OpTmizer T cell amplification SFM (Gibco, catalog number A3705001), 1% penicillin-streptomycin, 1× Glutamax, 10 mM HEPES, 2.5% human AB serum (Gemini, catalog number 100-512), supplemented every other day with 1000 U/mL IL-2 for amplification) until the day of injection.

Example 2.2. T Cell Transduction and Cell Sorting: Engineered suppressor T cells were continuously transduced using lentiviral constructs at one-day intervals, as described in Table 5. One lentiviral construct encoded IL10DE (SEQ ID NO: 229) and CTLA4 (SEQ ID NO: 130), as well as GFP and mScarlet as markers. A second lentiviral construct encoded the active double mutant TGFB1 (caTGFB1; R218H C225R; SEQ ID NO: 214). CD25+ T cells were transduced using a lentiviral construct encoding GFP. For transduction, concentrated viral supernatant was added to the T cells, and the sample was centrifuged at 37°C for 60 min. T cells were resuspended in a cell/virus supernatant mixture and cultured in OpTmizer medium. Transduced CD4+ cell samples were supplemented with 200 U/mL IL-2, 5 ng/mL IL-7, and 5 ng/mL IL-15, while transduced CD25+ samples were supplemented with 1000 U/mL IL-2. Cell division was induced, and medium or intercytokines were replenished periodically.

Table 5. T-cell editing therapy group. "LV" represents lentivirus, "DKI" represents double knock-in, "DKO" represents double knock-out, "TKI" represents triple knock-in and "TKO" represents triple knock-out. Group Initiator cells LV GFP LV CTLA4 and IL-10DE LV caTGFB1 LNP IFNg LNP TNFa LNP TGFBR2 nTreg CD4+ CD25+ + - - - - - DKI DKO CD4+ - + - + + - TKI DKO CD4+ - + + + + - TKI TKO CD4+ - + + + + +

T cells were edited using LNPs according to Table 5. Each LNP species was co-modified with the mRNA encoding Cas9 and its guide at the following dosages: INF-γ (G019753), 0.67 ug/ml total RNA; TNF-α (G019757), 0.67 ug/ml total RNA; TGFBR2 (G029528), 1 ug/ml total RNA. LNPs were generated using a molar ratio of 35 lipoprotein A/47.5 cholesterol/15 DSPC/2.5 PEG2k-DMG, as described in Example 1. LNPs were modulated with a molar ratio of lipoamines to RNA phosphate (N:P) of approximately 6. LNPs were prepared with a gRNA to Cas9 mRNA weight ratio of 1:1.

In short, T cells were resuspended at a concentration of 1 × 10^ ⁶ cells/mL in serum-free medium supplemented with 1000 U/mL IL-2, 10 ng/mL IL-7, and 10 ng/mL IL-15, and then transferred to T-75 flasks and incubated at 37°C. Each LNP preparation was incubated in T cell-based medium supplemented with 5 μg/mL recombinant human ApoE3 (Peprotech, catalog number 350-02). The LNP mixture was incubated at 37°C for 15 minutes. An equal volume of the pre-cultured LNP mixture was added to each T cell sample, thus doubling the original volume.

Twenty-four hours after LNP treatment, T cells were collected, washed, and suspended in OpTmizer 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, supplemented with intercytokines every other day.

Example 2.3. Confirmation of cell engineering modification : The engineered sample is sorted by flow cytometry to obtain an enriched cell population.

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 then cultured with either an antibody targeting CD4 (Biolegend) or an antibody targeting TGFBR2 (Miltenyi). T cells were subsequently washed and analyzed on a Cytoflex instrument (Beckman Coulter). Data analysis was performed using the FlowJo software package. T cells were gated based on size, single cell count, CD4 expression, and marker expression. Target TGFBR2 expression validated by flow cytometry is shown in Table 6 . The values shown correspond to the average percentage of TGFBR2-positive cells and the TGFBR2 expression level measured by median fluorescence in each cell population.

Table 6. Average fluorescence intensity of TGFBR2 expression in engineered CD4+ T cells TGFBR2 Group Average percentage of TGFBR2+ cells Mean TGFBR2 performance (MFI) n Unstained CD4+ 0.5 108 2 Unedited CD4+ 84.4 1401 2 TKI DKO 74.8 1206 1 TKI TKO 2.8 217 2

Example 2.4. Suppressive Capacity of Engineered T Cells Mixed Lymphocyte Reaction (MLR) analysis was used to analyze the suppressive function of sorted transduced engineered T cells. MLR is an inflammatory response caused by T cells recognizing another leukocyte (allogeneic leukocyte) as a foreign substance. Tregs suppress the MLR inflammatory response by inhibiting the proliferation of inflammatory T cells. Cell proliferation was measured by labeling the initial population with an intracellular fluorescent dye, which became diluted as part of the cellular contents in each subsequent generation.

MLR analysis was performed in 96-well U-plates using OpTmizer-based medium containing CTS OpTmizer T cell amplification SFM (Gibco, catalog number A3705001), 1% penicillin-streptomycin, 1× Glutamax, 10 mM HEPES, and 2.5% human AB serum (Gemini, catalog number 100-512). Untransduced CD3+CD4+ T cells (autologous T cells) from the same donor as the transduced T cells were used as reaction cells. These untransduced cells were labeled with CellTrace Violet (CTV) (Thermofisher Scientific; catalog number C34557) according to the manufacturer's instructions. Untransduced T cells were stimulated using CD3-depleted PBMCs from an allogeneic donor relative to the transduced T cells. 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 culturing at 37°C for 5 days, the culture plates were centrifuged. The cell sediment was resuspended in FACS buffer containing APC/Fire 750 anti-CD4 and incubated at 4°C for 30 min. Cells were then washed and processed using a Cytoflex flow cytometer (Beckman Coulter), and analyzed using the FlowJo software package. Cells were first gated by positive CD4 expression, then by CTV signal, and finally by the undiluted CTV population. Inhibition of CTV dilution was calculated using the following formula: Where y = average fluorescence intensity of the entire CTV marker population / average fluorescence intensity of the undiluted portion of the CTV marker population. The data are shown in Figure 1 and Table 7 .

Table 7. Calculated average percentage of inhibition of autologous T cell proliferation by engineered T cells. 1:1 1:3 1:9 1:27 Sample average value SEM average value SEM average value SEM average value SEM Untransduced T cells (Tconv) 2.74 2.20 3.86 3.86 1.92 1.92 6.09 0.69 tTreg 82.10 0.97 79.05 3.01 70.12 3.31 51.23 5.81 TKI DKO 80.41 2.02 75.64 0.57 46.80 7.56 39.34 3.16 TKI TKO 69.79 0.49 70.71 5.57 64.68 3.61 55.94 1.47

Example 3. In vivo functional characterization of engineered suppressor T cells Example 3.1. In vivo evaluation of inflammatory response in a GvHD model The in vivo suppressor function of engineered T cells was evaluated using a graft-versus-host disease (GvHD) mouse model.

T cells selected for in vivo injection were harvested and resuspended at 4 × 10^6 cells/mL in CTS OpTmizer T cell amplification medium (Gibco, catalog number A1048501). PBMCs, which were autologous for the engineered T cells, were thawed, and the cell density was adjusted to 8 × 10^6 cells/mL. PBMCs were added to each analytical population at a ratio of 0.5:1, and the cells were resuspended in RPMI supplemented with 10 mM HEPES and 2% FBS to achieve a density of 6 × 10^6 cells/100 μL. Only the PBMC group was resuspended at 4 × 10^6 cells/100 μL.

One day prior to injection, female NOG mice (NOD.Cg- Prkdc ^scid Il2rg ^tm1Sug /JicTac; Taconic, catalog number NOG-F) were conditioned for cell transplantation using sublethal X-ray irradiation (150 rads) via an RS-2000 irradiator (Rad Source Technologies). 100 μL of each of the aforementioned test cell populations was intravenously injected into the irradiated NOG mouse population. Eight irradiated mice injected with the injection medium served as irradiation-only controls (medium). Body weight was monitored daily. Mice were sacrificed after a 20% weight loss, and their spleen cell composition was evaluated. Survival data are plotted in Table 8 and Figure 2 .

Table 8. Percentage of mice surviving at each time point after injection in each group. N 8 7 8 8 8 8 sky Mediator Only PBMC tTreg DKI DKO TKI DKO TKI TKO 12 100 85.7 100 100 100 100 13 100 71.4 100 100 100 100 16 100 57.1 100 100 87.5 100 17 87.5 42.9 100 100 87.5 100 19 87.5 28.6 100 100 87.5 100 twenty two 87.5 14.3 100 100 87.5 100 twenty three 87.5 0.0 100 100 87.5 100 twenty four 87.5 0.0 100 87.5 62.5 100 26 87.5 0.0 100 87.5 50 100 28 87.5 0.0 100 75 50 100 28 87.5 0.0 100 75 37.5 100 35 87.5 0.0 100 75 37.5 87.5 44 87.5 0.0 100 62.5 37.5 87.5 49 87.5 0.0 100 62.5 25 87.5 52 87.5 0.0 100 50 25 87.5 55 87.5 0.0 75 37.5 12.5 75 62 87.5 0.0 62.5 37.5 12.5 75 63 87.5 0.0 62.5 37.5 12.5 75

Example 3.2. Persistence of Engineered T Cells To confirm the persistence of engineered T cells, spleen composition was evaluated. Treg persistence was assessed by evaluating the relative cell counts of Tregs and total spleen cells in the spleen after euthanasia. At euthanasia, the spleen of each animal was collected in a 15 mL conical tube containing complete Optimizer medium. The spleen was manually dissected using the rubber side of a 1 mL syringe plunger. The cell suspension was filtered through a 70-micron cell filter (Corning, catalog number 08-771-2), and the cells were counted using a Cellometer K2 (Nexcelom Bioscience). Spleen cells were cryopreserved in CryoStor CS10 medium (Stemcell Technologies; catalog number 07930). After the study, all spleen cells were thawed and counted using a Cellometer K2 (Nexcelom Bioscience). Approximately one million viable spleen cells were resuspended in Optimizer medium, and 6 × 10^5 cells were stained at 4°C with an antibody set including APC anti-human CD45 (BD Pharmigen, catalog number 561864) and APC-Fire 750 anti-human CD4 (Biolegend, catalog number 300560) for 30 min. Spleen cells were processed using a Cytoflex flow cytometer (Beckman Coulter) and analyzed using the FlowJo software package. Cell gating was performed on mouse CD45- and human CD45+ cells. tTreg cells were gated with GFP+, and engineered T cells were gated with mScarlet+GFP+. To determine the cell count of individual populations, the percentage of each population was applied to the total number of recovered human spleen cells. Data are shown in Table 9 and Figure 3 .

Table 9. Quantification of human lymphocytes in mouse spleen % of total human lymphocytes (CD45+) Sample average% SEM n tTreg 5.21 3.39 8 DKI DKO 2.90 2.23 6 TKI DKO 0.54 0.28 3 TKI TKO 40.86 28.07 8

Example 3.3. In vivo intercytokine morphology analysis of engineered T cells: Blood was extracted via cardiac puncture to separate plasma for intercytokine analysis, which was then preserved by adding EDTA. The collected blood was centrifuged at 1000×g for 10 min, and the plasma was stored at -80°C until further analysis.

Intercytokine release levels in plasma samples were evaluated using MSD (Meso Scale Diagnostics). Plasma samples were thawed using a custom U-PLEX biomarker kit (Meso Scale Diagnostics, catalog number K15067L-2) according to the manufacturer's instructions for subsequent intercytokine quantification. U-PLEX biomarker plates were read using a Meso Quickplex SQ120 instrument (Meso Scale Discovery), and data were analyzed using Discovery Workbench 4.0 software (Meso Scale Discovery). Results are shown in Table 10 and Figure 4 .

Table 10. IL-10 levels in plasma (pg/mL) IL10 (pg/mL) Sample average value SEM n Mediator 0 0 1 PBMC 7 7 5 tTreg 11 10 8 DKI DKO 107 80 5 TKI DKO 31 twenty four 6 TKI TKO 924 617 8

Example 4. In vivo study of CD70-CAR-targeted synTregs using a xenoGvHD mouse model. The in vivo inhibitory function of engineered T cells was evaluated using a graft-versus-host disease (GvHD) mouse model. Specifically, CD70-CAR-targeted and non-targeted triple knock-in (dmTGFB1/IL10DE/CTLA4)/triple knockout (TGFBR2/IFNG/TNFA) synTregs were compared with unedited tTregs and controls. CD70 was upregulated by activated lymphocytes, including autoreactive T cells and B cells.

Example 4.1 T Cell Engineering Typically, T cells are prepared as described in Example 2. CD4+ and CD4+CD25+ T cells were isolated from human PBMCs using a commercially available kit (Miltenyi) following the manufacturer's protocol. Cells were activated using Immunocult and edited according to Table 11 .

Table 11. T-cell editing therapy group. "LV" represents lentivirus. "AAV" represents adeno-associated virus. Group Initiator cells LNP IFNg and LNP TNFa LV CTLA4 and IL-10DE LV dmTGFB1 AAV CD70-CAR; LNP TRAC, LNP CD70, LNP UGI LNP TGFBR2 tTreg CD4+ CD25+ - - - - - synTreg CD4+ + + + - + CD70 CAR-synTreg CD4+ + + + + +

On day 0, CD4+ and CD4+CD25+ cells were activated using Immunocult. CD4+ cells were treated with an LNP carrying SpCas9 mRNA and gRNA G019753 targeting IFNγ and gRNA G019757 targeting TNFα to disrupt 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 knock-in cells were harvested, resuspended, and treated with a lentivirus encoding dmTGFB1. On day 3, a portion of the obtained double knockout and triple knock-in cells were harvested, resuspended, and treated with an AAV encoding a CD70 CAR with a homologous arm of approximately 500 nucleotides adjacent to the TRAC-guided cleavage site, an LNP carrying mRNA encoding SpCas9, and 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 ID No. 365), gRNA G034199 targeting CD70, and an LNP carrying mRNA encoding UGI to disrupt the CD70 gene. On day 4, CD70-edited cells were harvested, resuspended, and cultured overnight. On day 5, double knockout triple knock-in cells and CD70 CAR-targeted double knockout triple knock-in cells were harvested, resuspended, and treated with LNPs carrying SpCas9 mRNA and the TGFBR2-targeting gRNA G029528 to disrupt TGFBR2. On day 6, thymic Treg, synTreg, and CD70 CAR synTreg cells were washed and cultured with intercytokines periodically.

Example 4.2 In vivo functional characterization: The in vivo repressive 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. One day prior to injection, female NOG mice (NOD.Cg- Prkdc ^scid Il2rg ^tm1Sug /JicTac; Taconic, catalog number NOG-F) were conditioned for cell transplantation using sublethal X-ray irradiation (150 rads) on an RS-2000 irradiator (Rad Source Technologies). As described in Table 11 , four million PBMCs and four hundred thousand T cells were intravenously injected into the irradiated NOG mouse population. Eight irradiated mice injected with the injection medium served as irradiation-only controls (medium). Body weight was monitored daily. Mice were sacrificed after a 20% weight loss, and their spleen cell composition was evaluated. Survival data are plotted in Table 12 and Figure 5 .

Table 12. Survival percentage of mice at each time point after injection in each group. sky Mediator Only PBMC tTreg synTreg CAR-synTreg 0 100% 100% 100% 100% 100% 14 100% 88% 100% 100% 100% twenty three 100% 75% 100% 88% 100% 26 100% 75% 100% 88% 100% 34 100% 75% 88% 88% 100% 36 100% 63% 88% 88% 100% 39 100% 63% 75% 88% 100% 48 100% 50% 63% 88% 100% 50 100% 13% 63% 88% 100% 54 100% 0% 63% 75% 100% 56 100% 0% 50% 63% 100% 60 100% 0% 50% 50% 100% 63 100% 0% 38% 50% 100% 67 100% 0% 25% 38% 100%

The persistence of Treg cells is typically evaluated as described in Example 3. Table 13 and Figure 6 show the average percentage of synTregs in all human CD45+ cells recovered from mouse spleens at sacrifice. The percentage of tTregs in the spleen was not measured in this experiment, but it is typically in the range of 1%–5% in similar experiments.

Table 13. Mean percentage of synTreg cells in all human CD45+ cells recovered from mouse spleen at sacrifice. treatment average value SD n Only PBMC 0.0 0.0 8 synTreg 10.0 6.7 7 CAR synTreg 37.2 16.6 8

Based on the method described by Lai et al., Cell Transplantation. 2012;21(9):2033-2045, phenotypic characteristics of mice were evaluated at sacrifice. For each mouse, each phenotypic criterion was graded from 0 to 2 as described in Table 14 , and the grades of individual mice were summed to obtain a GvHD score. The mean GvHD scores for each experimental condition are shown in Table 15 and Figure 7. The GvHD scores of mice treated with non-targeted tTreg or synTreg were statistically significantly improved compared with those of the PBMC control alone. Mice treated with CD70-CAR synTreg were asymptomatic throughout the experiment and could not be distinguished from mice in the mediator control sample. GvHD scores in CD70-CAR synTreg-treated mice were significantly different from those in PBMC-only controls and non-targeted synTreg mice.

Table 14. GvHD scoring criteria standard Level 0 Level 1 Level 2 Weight loss <10% 10%-20% >20% Activity normal Mild to moderate reduction Remain still until stimulated Posture normal Only hunchbacked during rest Severe gait, sports injury fur texture normal Mild to moderate wrinkles Severe wrinkles/poor combing Skin integrity normal Claws and/or tail have scales The area of exposed skin is obvious.

Table 15. Average GvHD score at the time of execution. treatment average value SD n Only PBMC 4.6 0.7 8 tTreg 3.4 2.2 8 synTreg 3.0 2.2 8 CAR synTreg 0.0 0.0 8

Intercytokine morphology was typically evaluated as described in Example 3. Table 16 and Figure 8 show the mean serum IL-10 levels in mice in each treatment group.

Table 16. Mean IL-10 levels at sacrifice, expressed as pg/ml serum. treatment average value SD n Mediator 0.3 0.6 7 Only PBMC 9.1 5.7 8 tTreg 7.0 3.7 7 synTreg 98.4 172.7 8 CAR synTreg 5.9 7.4 6

Example 4.3. Inhibitory Activity of synTreg Isolated from Spleen In this example, bead-based inhibition assays were used to analyze the inhibitory function of Tregs isolated from frozen mouse spleen samples. Inhibitory assays were typically performed using allogeneic APCs and human anti-CD3/CD28 beads for T cell stimulation, as described in Example 2 and performed using methods known in this art. Unengineered CD4+ and CD8+ T cells (Tconv) were isolated from frozen PBMCs of the same donor used to engineer synTregs as negative controls. Tregs were isolated from spleen samples by flow cytometry based on fluorescence labeling: GFP for tTregs and GFP/mScarlet for synTregs. CD3+ T cells were isolated from PBMCs using the Miltenyi Biotec kit for use as Tresp cells.

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 five days with anti-human CD3/CD28 activator beads (Miltenyi Biotec, catalog number 130-091-441). Cells were harvested and analyzed by flow cytometry. The percentage of proliferation inhibition was calculated using the following formula:

The calculated inhibition of CTV-labeled cell proliferation is shown in Table 17 and Figure 9. Spleen-isolated synTregs maintained high inhibitory activity, exceeding that of spleen-isolated tTregs, especially at low Treg:Tresp ratios. Consistent with in vivo persistence data, ex vivo synTregs showed superior survival compared to tTregs.

Table 17. Mean percentage inhibition of Tresp cell proliferation Treg:Tresp Tconv comparison tTreg synTreg average value SD n average value SD n average value SD n 1:1 66.7 11.5 3 88.4 3.5 3 99.4 1.0 3 1:3 41.2 9.9 2 82.3 4.0 3 94.8 3.5 2 1:9 29.0 8.8 3 48.2 0.6 2 87.6 4.8 2 1:27 0.0 0.0 3 6.3 10.9 3 56.5 12.9 3

SynTreg cells isolated from the spleen by CD3/CD28 bead restimulation were compared with Tconv cells cultured without Tresp. The supernatant was analyzed using the Meso Scale Discovery MSD-U-PLEX pro-inflammatory combination 1 human kit according to the manufacturer's protocol. As shown in Table 18 and Figures 10A-10D , SynTreg cells did not produce pro-inflammatory cytokines, but still secreted IL-10 after bead-mediated stimulation.

Table 18. Cytokine levels in cell culture supernatant (pg/ml). Intercytokines condition Tconv synTreg average value SD average value SD IFNγ Unstimulated 0 0 0 0 Bead stimulation 2829 1322 14 0 TFNα Unstimulated 0 0 0 0 Bead stimulation 3 1 0 0 IL-2 Unstimulated 0 0 0 0 Bead stimulation 11 2 0 0 IL-10 Unstimulated 0 0 4 0 Bead stimulation 1 0 26 3

Example 5. Characterization of mouse MAdCAM-1-CAR synTregs: When analyzing cell activation and the secretion of intercytokines in response to activation in vivo, mouse MAdCAM-CAR-targeted and non-targeted triple knock-in (dmTGFB1/IL10DE/CTLA4)/triple knockout (TGFBR2/IFNG/TNFA) synTregs were compared with habitual T cells.

Example 5.1 T Cell Engineering T cells are typically prepared as described in Example 2. CD4+ T cells are isolated from human PBMCs using a commercially available kit (Miltenyi) following the manufacturer's protocol. Cells are activated using Immunocult and edited according to Table 19 .

Table 19. T-cell editing therapy group. "LV" represents lentivirus. Group Initiator cells Day 2 Day 3 Day 4 Day 6 LV CTLA4, IL-10DE, dmTGFB1 LV MECA CAR LNP IFNγ and LNP TNFα LNP TGFBR2 and LNP TRAC Tconv CD4+ - - - - synTreg CD4+ + - + + CAR synTreg CD4+ + + + +

On day 1, CD4+ cells were activated using Immunocult. On day 2, a subset of 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 culture medium. A subset of triple knock-in cells were treated with a lentivirus encoding a chimeric antigen receptor based on monoclonal anti-mouse MAdCAM-1 and MECA-367 antibodies. On day 4, the cells were harvested, resuspended, and treated with an LNP carrying SpCas9 mRNA and gRNA targeting IFNg and TNF to disrupt these genes. The cells were washed and resuspended in culture medium for further culture the following day. On day 6, cells were sorted by flow cytometry to separate 1) Tconv populations, 2) triple knock-in/double knockout populations, and 3) CAR + triple knock-in/double knockout cell populations. After sorting, the edited cells were further treated with LNPs carrying SpCas9 mRNA and TRAC-targeting gRNA, as well as LNPs carrying SpCas9 mRNA and TGFBR2-targeting gRNA, to disrupt these genes. The cells were washed and resuspended on the second day.

Example 5.2 In vivo functional characterization : Cell activation was evaluated by co-culturing mouse SVEC4-10 endothelial target cells transfected with mouse MAdCAM-1 or in wells coated with mouse MAdCAM-1. Cells cultured in isolation served as negative controls. After 24 hours, cells were harvested, and the expression of the T cell activation marker CD69 was evaluated by flow cytometry. As shown in Table 20 and Figures 11A-11C , exposure to mouse MAdCAM-1 activated CAR-synTregs but not Tconvs or non-targeted synTregs.

Table 20. Mean percentage of CD69+ T cells. "na" indicates "not analyzed". Sample Individual cultivation mMAdCAM-1 coating Cells exhibiting SVEC mMAdCAM-1 average value SD n average value SD n average value SD n Tconv 3.9 0.3 3 4.2 0.4 3 na synTreg 2.1 0.2 3 2.1 0.1 3 na CAR-synTreg 2.4 0.4 3 47.2 4.4 3 67.5 3.0 3

Intercytokine secretion following restimulation with mouse MAdCAM-1 or Immunocult was evaluated. Cells were cultured for 48 hours in wells containing mouse MAdCAM-1 or Immunocult. Cells were harvested by centrifugation, and the supernatant was retained. The supernatant was analyzed using the Meso Scale Discovery MSD-U-PLEX pro-inflammatory combination 1 human kit according to the manufacturer's protocol. As shown in Table 21 and Figure 12 , only Tconv produced pro-inflammatory intercytokines IFNg and TNFα upon stimulation. Similarly, targeted and non-targeted synTreg produced IL-10 and active TGFB upon appropriate stimulation .

Table 21. Intercytokine secretion after restimulation (pg/ml). Cells were stimulated with mouse MAdCAM-1 (MC-1) or Immunocult (IC). "na" indicates "unanalyzed". Intercytokines condition Tconv comparison synTreg CAR-synTreg average value SD n average value SD n average value SD n IL-10 Unstimulated 1 1 3 113 11 2 4 0 2 MC-1 stimulation na na 2,336 488 3 IC stimulation 510 68 3 642 57 3 868 137 3 TGF-β Unstimulated 0 0 2 98 4 2 0 0 2 MC-1 stimulation nd nd 1,033 96 3 IC stimulation twenty three 34 3 566 29 3 341 57 3 IFNγ Unstimulated 580 94 2 11 2 2 13 6 2 MC-1 stimulation na na 2,363 190 3 IC stimulation 188,353 8,367 3 1,581 91 3 970 165 3 TNFα Unstimulated 2 0 2 0 0 2 0 0 2 MC-1 stimulation na na 3 1 3 IC stimulation 321 98 3 2 1 3 1 1 3

Example 6. SynTreg demonstrates potent in vitro inhibition of T cells derived from patients with ulcerative colitis. The inhibitory capacity of synTreg was evaluated using T cells derived from patients with ulcerative colitis as responders in the mixed lymphocyte response. Inhibition of responder cell growth and inflammatory interferon secretion were measured.

The inhibitory function of Tregs was measured using mixed lymphocyte reaction (MLR) analysis. MLR analysis was typically performed as described in Example 2 using methods known in this technique. CD4+ T cells (Tresp) were isolated from PBMCs from one healthy donor (HD) and three donors with ulcerative colitis (UC) using a commercially available kit (e.g., Miltenyi). SynTregs were typically engineered to resemble the synTreg sample in Example 4, with slight variations in edit time. SynTreg cells were derived from CD4+ cells and edited to insert IL-10DE, CTLA4, and dmTGFB1 while disrupting IFNg, TNFα, and TGFBR2. To evaluate the secretion of inflammatory intercytokines into the culture medium, supernatants were collected at the 72-hour time point in the MLR analysis. Supernatants were analyzed using the Meso Scale Discovery MSD-U-PLEX Pro-inflammatory Combination 1 Human Kit according to the manufacturer's protocol . SynTreg effectively inhibited the proliferation of ulcerative colitis patient-derived T cells ( Table 22 and Figure 13 ) and the release of inflammatory intercytokines ( Table 22 and Figures 14A-14B ).

Table 22. Mean percentage of intercytokine secretion and mean percentage of cell proliferation inhibition in the mixed lymphocyte response using synTreg and T cells derived from patients with ulcerative colitis. HD, healthy donor. UC, ulcerative colitis. analyze Treg: Tresp HD UC No. 1 UC No. 2 UC No. 3 average value SD n average value SD n average value SD n average value SD n TNFa (pg/ml) 1:1 29 7 3 51 8 3 30 6 3 31 2 3 1:3 41 2 3 74 10 3 64 13 3 62 16 3 1:9 60 12 3 84 3 3 54 7 3 65 12 3 IFNg (pg/ml) 1:1 twenty three 6 3 62 twenty one 3 twenty four 10 3 9 2 3 1:3 31 9 3 53 16 3 twenty three 0 3 14 2 3 1:9 twenty two 2 3 66 16 3 20 4 3 13 1 3 inhibition% 1:1 74 2 3 59 2 3 65 2 3 82 3 3 1:3 40 2 3 44 5 3 49 9 3 61 5 3 1:9 51 7 3 7 7 3 46 9 3 50 5 3

Example 7. SynTreg outperforms tTreg in suppressing colonic inflammation in mice. SynTreg and tTreg were typically tested in a humanized mouse model of colonitis using the method described in Cell Mol Gastroenterol Hepatol. 2019 May 9;8(2):193-19 to suppress the gross and histological signs of colonitis. Thymic Treg and synTreg were typically prepared as described in Example 4, but the editing time could vary. SynTreg was engineered using CD4+ starting material, lentiviruses encoding the same sequences as used in Example 4, and the same LNP reagent. SynTreg cells were derived from CD4+ cells and edited to insert IL-10DE, CTLA4, and dmTGFB1 and to disrupt IFNg, TNFα, and TGFBR2.

T cells intended for in vivo injection were harvested and resuspended in culture medium. Two million cells from each test cell population described in Table 23 were intravenously injected into CD34+ humanized NSG mice (Jackson Laboratory, Stock No. 705557). Seven mice were treated in each test group. Two hours after cell injection, mice were anesthetized and subcutaneously injected with PBS containing 1% v/v 2,4,6-trinitrobenzenesulfonic acid (TNBS; Millipore Sigma, 92822) to induce an immune response to this agent. Seven days after the first treatment, mice were challenged again via rectal catheter with 250 μg TNBS to induce colitis. Body weight was monitored daily for the next three days. Mice were sacrificed 10 days after cell injection and 3 days after colitis induction. Colonic tissue was collected for analysis. Colon length was recorded, feces were removed, and tissue weight was recorded. Paraffin-embedded colon sections were stained and analyzed blinded for inflammation, crypt hyperplasia, ulcers, intestinal wall thickening, and edema. Each histological parameter was scored from 0 to 1 in increments of 0.25, resulting in a maximum possible score of 5. The results are shown in Table 23 and Figures 15-16 . As demonstrated by the lower colon weight to length ratio and histological scores of synTreg, multi-strain synTreg is superior to tTreg in suppressing the gross and histological signs of colitis.

Table 23. Tissue analysis in a mouse model of colitis. Colon weight: length (mg/mm) Histological rating Sample average value SD n average value SD n Uninduced control 2.7 0.3 5 0.4 0.2 5 Colitis induction control 4.7 1.1 5 2.8 1.1 6 tTreg 4.5 0.7 6 2.4 1.0 6 synTreg 3.3 0.7 6 1.1 1.1 6

Example 8. Allogeneic editing enhances the persistence of synTreg in humanized models of colitis . This is typically achieved by testing synTregs (prepared "off-the-shelf") with and without additional gene disruption to prevent allogeneicity in humanized mouse models of colitis using the methods described in Cell Mol Gastroenterol Hepatol. 2019 May 9;8(2):193-19.

Typically, T cells are prepared as described in Example 4. CD4 T cells are isolated from human PBMCs using a commercially available kit (e.g., Miltenyi) following the manufacturer's protocol. Cells are activated using Immunocult and edited according to Table 24. All synTreg cells are edited to insert IL-10DE, CTLA4, dmTGFB1, and anti-mouse MAdCAM-1 CAR (“MECA-CAR”) and to destroy IFNg, TNFα, TGFBR2, and TRAC. To avoid rejection by the immune system of humanized mice, the edited cells are also edited to destroy HLA-A, HLA-B, and CIITA. IFNg, TNFα, and TRAC were disrupted using mRNAs encoding SpCas9, with guides G019753, G019757, and G013006, respectively. The MECA-CAR AAV architecture includes homologous arms laterally attached to TRAC-guided cleavage sites. TGFBR2, HLA-A, HLA-B, and CIITA were disrupted using mRNAs encoding the NmeCas9 base editor, with guides G034983, G034202, G034208, and G034619, as well as a separate mRNA encoding UGI, respectively.

Table 24. T-cell editing therapy group. "LV" represents lentivirus. "AAV" represents adeno-associated virus. Group CAR-synTreg Allogeneic CAR-synTreg Initiator cells CD4+ CD4+ LV CTLA4 and IL-10DE + + LV dmTGFB 1 + + LNP IFNg LNP TNFa + + LNP TGFBR2 + + LNP-HLA-B LNP UGI - + AAV MECA-CAR LNP TRAC + + LNP HLA-A LNP CIITA LNP UGI - +

T cells intended for in vivo injection were harvested and resuspended in culture medium. One million cells from each test cell population described in Table 25 were intravenously injected into CD34+ humanized NSG mice (Jackson Laboratory, Stock No. 705557). Seven mice were treated in each test group. Body weight was monitored daily. Two hours after cell injection, mice were anesthetized and subcutaneously injected with 100 μL of PBS containing 2% v/v 2,4-dinitrobenzenesulfonic acid dihydrate (DNBS; Sigma Aldrich, R750735) to induce an immune response to this agent. Six days after the initial treatment, mice were challenged again via rectal cannula with 2.5 mg DNBS to induce colitis. Mice were sacrificed 9 days after cell injection and 3 days after colitis induction. Spleen, mesenteric lymph nodes (MLN), and colon tissue were collected for analysis. Colon length was recorded, feces were removed, and colon weight was recorded. Spleen and MLN tissue were dissected, and residual cells were stained and analyzed by flow cytometry. Results are shown in Table 25 and Figures 17-19 . As demonstrated by the reduced colon weight:length compared to a colitis-induced control, both CAR synTreg and allogeneic-edited CAR synTreg protected mice from colitis ( Table 25, Figure 17 ). Allogeneic editing increased the persistence of engineered T cells in spleen and MLN samples ( Table 25, Figure 18 , and Figure 19 , respectively).

Table 25. Histological analysis in a mouse model of colitis Sample colon Spleen MLN Weight: Length (mg/mm) synTreg% in CD4+ synTreg% in CD4+ average value SD n average value SD n average value SD n Uninduced control 4.1 0.4 7 0.01 0.01 7 0.00 0.00 7 Colitis induction control 4.9 0.5 7 0.00 0.00 7 0.00 0.00 7 CAR synTreg 4.2 0.3 6 0.07 0.06 7 0.04 0.04 7 Allogeneic CAR synTreg 4.1 0.4 7 0.22 0.10 7 0.14 0.09 7

Example 9. SynTreg Suppresses Symptoms in a Chronic Colitis Model . The ability of multiple synTreg lines to suppress colitis induction was tested in a chronic syngeneic mouse colitis model. In this model, colonitis was induced using donor-receptor cell transfer from naïve mouse CD4+ T cells. Multiple mouse synTreg lines were compared with host mouse T cells, including native Tregs, in their ability to suppress inflammatory symptoms in the colon. SynTregs were typically prepared as described in Example 4, but editing times may vary. Mouse CD4+ T cells were isolated from the spleens of C57BL/6 mice using a commercially available kit (Miltenyi) following the manufacturer's protocol. Retrotransfer viruses encoding CTLA4, IL-10DE, and dmTGFB1 are used for gene insertion, and LNP reagents are used to disrupt IFNg, TNFα, and TGFBR2 to engineer SynTreg.

On the day of injection in mice, naïve CD4+ and total CD4+ mouse T cells were isolated from the spleens of C57BL/6 mice using a commercially available kit (Miltenyi) following the manufacturer's protocol. T cells for in vivo injection were harvested and resuspended in culture medium. Immunocompromised RAGN12 mice (Taconic Biosciences) were injected intraperitoneally as described in Table 26. Ten mice were treated in each test group. Mice were sacrificed approximately 48 days after cell injection. Mesenteric lymph nodes (MLNs) and colonic tissue were collected for analysis. Colonic length was recorded, feces were removed, and tissue weight was recorded. Paraffin-embedded colonic sections were stained and scored by a histopathologist in a blinded manner. The morphological criteria used for grading were those recommended by Fabrega et al. (Front Microbiol. July 11, 2017: 8: 1274) and Camuesco et al. (Br J Pharmacol. February 2012; 165(3): 729-40.). Necrosis and ulcers were graded using a 0-4 scale. The broad scoring areas were (1) mucosal epithelium and lamina propria, (2) crypts, (3) submucosa, and (4) muscle layer. The results are shown in Table 26 and Figures 20-21 . As demonstrated by the lower colon weight to length ratio and histological scores in mice treated with synTreg compared to a colitis-induced control, synTreg inhibits the gross and histological signs of colitis.

Table 26. Tissue analysis in a chronic mouse model of colitis. Sample injection Colon weight: length (mg/mm) Histological rating average value SD n average value SD n Uninduced control Mediator (injection medium only) 3.4 0.3 10 3.2 0.4 9 Colitis Induction Control 1.5 M initial CD4+ 5.7 2.3 10 29.6 6.7 10 Colitis Inhibition Control 1 M total CD4+ 3.3 0.2 10 16.1 1.2 10 synTreg 1.5 M initial CD4 + 0.5 M synTreg 4.1 0.8 10 22.7 2.5 10

Other markers of inflammation and colitis were also evaluated. Mesenchymal lymph nodes (MLNs) were dissected, and cells were stained and analyzed by flow cytometry. As shown in Table 27 and Figures 22A-22D , mice injected with synTreg showed a significantly reduced percentage of CD4+ T cells producing inflammatory intercytokines in the colitis drainage lymph nodes.

Table 27. Average percentage of CD4+ cells expressing each inflammatory intercytin in mesenchymal lymph nodes. Sample TNF IFNg IL-22 IL-17 average value SD n average value SD n average value SD n average value SD n Colitis Induction Control 57.8 8.4 8 68.9 16.1 8 19.9 21.1 8 11.5 4.7 8 Colitis Inhibition Control 80.8 9.9 7 36.2 8.1 7 10.9 6.5 7 19.1 2.6 7 synTreg 39.7 16.5 9 48.6 13.2 10 8.5 7.1 10 4.8 4.2 10

Colonic explant analysis was used to evaluate the production of intercytokines by immune cells present in colonic tissue. One cm of freshly harvested colonic tissue was placed in culture medium at 37°C for 5 hours, after which the intercytokine levels in the harvested culture medium were quantified. Intercytokine levels were measured using the MSD-U-PLEX pro-inflammatory combination 1 mouse kit according to the manufacturer's instructions. As shown in Table 28 and Figures 23A-23B , SynTreg treatment reduced the levels of pro-inflammatory intercytokines IFNg and TNF produced by colonic resident immune cells. Lipid transporter-2 is a biomarker produced by neutrophils and colonic epithelial cells in response to inflammation. Human liposome transporter-2 (NGAL) has been reported to be elevated in patients with active colitis. The levels of liposome transporter-2 in fecal samples were assessed using a liposome transporter-2 ELISA (Biolegend). As shown in Table 28 and Figure 24 , the mouse synTreg significantly reduced the amount of liposome transporter-2 in the feces of treated mice.

Table 28. Mean intercytokine levels (pg/ml) in culture medium after colonic explant culture and mean lipid transport protein levels (ng/ml) in fecal samples. Sample IFNg (pg/ml) TNFa (pg/ml) Lipid transport protein (ng/ml) average value SD n average value SD n average value SD n Uninduced control 2.3 3.5 8 10.9 6.2 9 2 1 9 Colitis Induction Control 141.3 156.0 10 178.3 144.0 10 1589 2337 10 Colitis Inhibition Control 1.9 2.0 8 16.5 7.4 10 2 1 10 synTreg 17.9 9.2 10 59.5 40.7 10 18 twenty three 9

Table 29. Sequences describe sequence SEQ ID NO G034199 mC*mU*mC*mAmGCUmAmCGmUCmCCACUmGGAAmGAAmGUUGmUmAmGmCUCCCmUmGmAmAmAmCmCGUUmGmCUAmCAAUAAGmGmCCmGmUmCmGmAmAmGmAmUGUGCmCGmCAAmCGCUCUmGmCCmUmUmCmUGGCAUCG*mU*mU 353 G013006 mC*mU*mC*UCAGCUGGUACACGGCAGUUUUAGAmGmCmUmAmGmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 354 G025420 mC*mU*mC*UCAGCUGGUACACGGCAGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCACGAAAGGGCACCGAGUCGGmU*mG*mC*mU 355 G034983 mC*mG*mA*mGmAUGmUmCAmUUmUCCCAmGAGCmACCmGUUGmUmAmGmCUCCCmUmGmAmAmCmCGUUmGmCUAmCAAUAAGmGmCCmGmUmCmGmAmAmGmAmUGUGCmCGmCAAmCGCUCUmGmCCmUmUmCmUGGCAUCG*mU*mU 356 G034208 mU*mC*mU*mGmGGAmAmAGmGAmGGGGAmAGAUmGAGmGUUGmUmAmGmCUCCCmUmGmAmAmAmCmCGUUmGmCUAmCAAUAAGmGmCCmGmUmCmGmAmAmGmAmUGUGCmCGmCAAmCGCUCUmGmCCmUmUmCmUGGCAUCG*mU*mU 357 G034202 mG*mC*mU*mCmUAUmCmCAmCGmGCGCCmCGCGmGCUmGUUGmUmAmGmCUCCCmUmGmAmAmCmCGUUmGmCUAmCAAUAAGmGmCCmGmUmCmGmAmAmAmGmAmUGUGCmCGmCAAmCGCUCUmGmCCmUmUmCmUGGCAUCG*mU*mU 358 G034619 mA*mG*mC*mUmGCCmGmUUmCUmGCCCAmGUCCmGGGmGUUGmUmAmGmCUCCCmUmGmAmAmCmCGUUmGmCUAmCAAUAAGmGmCCmGmUmCmGmAmAmAmGmAmUGUGCmCGmCAAmCGCUCUmGmCCmUmUmCmUGGCAUCG*mU*mU 359 MECA-CAR ORF 360 MECA-CAR amino acid sequence MDFQVQIFSFLLISASVIMSRMADIQMTQSPSVLSASVGDRVTLSCKTSQNINKNLDWYQQKLGEAPKLLIYFTNNLQTGIPSRFSGSGSGTDYTLTISSLQPEDVATYYCYQYNSGPTFGA GTKLELRGGGGSGGGGSGGGGSQVQLKESGPGLVQPSQTLSLTCTVSGFSITSNGVSWVRQPPGKGLEWMGAIWSGGSRDYNSALKSRLSISRDTSKSQVFLNLNSLQTEDTAIYFCTRSDYH DGTSLYYYVMDAWGQGASVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 361 MECA-CAR amino acid sequence MALPPVTALLLPLALLLHAARPDIQMTQSPSVLSASVGDRVTLSCKTSQNINKNLDWYQQKLGEAPKLLIYFTNNLQTGIPSRFSGSGSGTDYTLTISSLQPEDVATYYCYQYNSGPTFGAGTK LELRGGGGSGGGGSGGGGSQVQLKESGPGLVQPSQTLSLTCTVSGFSITSNGVSWVRQPPGKGLEWMGAIWSGGSRDYNSALKSRLSISRDTSKSQVFLNLNSLQTEDTAIYFCTRSDYHDGTS LYYYVMDAWGQGASVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDPFGFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPP RDFAAYRSLERVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 362 CD70-CAR ORF 363 CD70-CAR amino acid sequence MALPPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFTFSIYAMSWVRQAPGKGLEWVSAISDSGGRTYFADSVRGRFTISRDNSKNTLSLQMNSLRAEDTAVYYCAKVDY SNYLFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSISSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYY CQQYGSSPYTFGQGTKLEIKGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 364 NmeCas9 Base Editor 365

Figure 1 shows the percentage of cell proliferation inhibition by engineered T cells as measured by CTV dilution. Figure 2 shows the survival percentage over time after injection of engineered T cells into NOG mice. Figure 3 shows the persistent morphology of engineered T cells. Figure 4 shows in vivo IL-10 production (pg/ml). Figure 5 shows the in vivo results of CAR-targeted synTreg studies in the xenoGvHD mouse model, plotting the survival percentage of mice surviving at each time point after injection in each group. Figure 6 shows the in vivo results of CAR-targeted synTreg studies in the xenoGvHD mouse model, plotting the mean percentage of synTreg in all human CD45+ cells recovered from the mouse spleen at sacrifice. Figure 7 shows the in vivo results of CAR-targeted synTreg studies in the xenoGvHD mouse model, plotting the mean GvHD score for each experimental condition. Figure 8 shows the in vivo results of CAR-targeted synTreg studies in the xenoGvHD mouse model, plotting the mean serum IL-10 level in mice in each treatment group. Figure 9 shows the inhibitory activity of synTreg isolated from the spleen, as measured by its inhibitory activity against Tresp proliferation. Figures 10A-10D show the pro-inflammatory intercytokine secretion (IFN-γ, Figure 10A; TNF-α, Figure 10B; IL-2, Figure 10C; IL-10, Figure 10D) of Tconv compared to synTreg. Figures 11A-11C show the activation patterns of Tconv (Figure 11A), synTreg (Figure 11B), and the CAR-targeted synTreg (Figure 11C) as measured by CD69 levels after exposure to mouse MAdCAM-1. Figure 12 shows the mean intercytokine secretion (pg/ml) of INFg, TNFα, IL-10, and TFG-b after restimulation. Cells were stimulated with mouse MAdCAM-1 (MC-1) or Immunocult (IC). Figure 13 shows the mean percentage of cell proliferation inhibition in the 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 intercytokine secretion of IFNg (Figure 14A) and TNFα (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 sacrifice. Figure 16 shows the mean histological score of the colon sample. Figure 17 shows the colon weight:length ratio at sacrifice. Figure 18 shows the mean percentage of synTreg in the CD4+ cell population in mouse spleen. Figure 19 shows the mean percentage of synTreg in the CD4+ cell population in mouse mesenteric lymph nodes (MLN). Figure 20 shows the colon weight to length ratio after treatment in the syngeneic colitis model. Figure 21 shows the histological scoring of colon sections after treatment in the syngeneic colitis model. Figures 22A-22D show the intercytokine expression of TNFα (Figure 22A), IFNg (Figure 22B), IL-22 (Figure 22C), and IL-17 (Figure 22D) on CD4+ cells from mesenchymal lymph nodes in the syngeneic colitis model after treatment. Figures 23A-23B show the intercytokine levels of IFNg (Figure 23A) and TNFα (Figure 23B) in the culture medium harvested after colon explant culture. Figure 24 shows the level of lipid transport protein in feces after treatment in a homogenic colitis model.

TW202542303A_113150064_SEQL.xml

Claims (72)

An engineered T cell comprising: i) a heterologous nucleic acid sequence encoding a double mutant transforming growth factor β1 (dmTGFB1) under the control of a promoter sequence; and ii) a modification of an endogenous nucleic acid sequence encoding transforming growth factor β receptor 2 (TGFBR2), wherein the modification knocks down the expression of TGFBR2. The engineered T cells of claim 1, wherein, relative to wild-type human TGFB1, the dmTGFB1 contains mutations at two or more of the following amino acids: F198, D199, V200, L208, F217, L219, R218, H222, C223, and C225. The engineered T cells of claim 1 or 2, wherein, relative to wild-type human TGFB1, the dmTGFB1 contains mutations at two or more amino acids selected from the following: R218, H222, C223, and C225. The engineered T cells of claim 1, 2 or 3, wherein, relative to wild-type human TGFB1, the dmTGFB1 contains two or more amino acid mutations selected from R218C/H, H222D, C223S/R/G and C225R. The engineered T cells of any of claims 1 to 4, wherein the dmTGFB1 contains the amino acid mutations R218C/H and C225R relative to wild-type human TGFB1. The engineered T cells in any of the claims 1 to 5, wherein the engineered T cells are CD4+ T cells. The engineered T cells of any of claims 1 to 6 further include: i) a modification of an endogenous nucleic acid sequence encoding tumor necrosis factor α (TNFA), wherein the modification knocks down the expression of TNFA; ii) a modification of an endogenous nucleic acid sequence encoding interferon-γ (IFNG), wherein the 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. The engineered T cells of any of claims 1 to 7 include: i) a modification of an endogenous nucleic acid sequence encoding tumor necrosis factor α (TNFA), wherein the modification knocks down the expression of TNFA; ii) a modification of an endogenous nucleic acid sequence encoding interferon-γ (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. For example, the engineered T cells requested in item 7 or 8, wherein the regulatory T cell-promoting molecules are selected from interleukin-10 (IL10), cytotoxic T lymphocyte-associated protein 4 (CTLA4), T cell immune receptor with Ig and ITIM domains (TIGIT), indoleamine 2,3-dioxygenase 1 (IDO1), extracellular nucleoside triphosphate diphosphate hydrolase 1 (ENTPD1), extracellular 5'-nucleotidase (NT5E), interleukin-22 (IL-22), amphotericin G (AREG), interleukin-35 (IL35), GARP, CD274 molecule (CD274), forkhead box P3 (FOXP3), IKAROS family zinc finger 2 (IKZF2), familial eosinophilia (EOS), interferon regulator 4 (IRF4), and lymphoid enhancement binding factor 1. (LEF1), BTB domain and CNC homologue 2 (BACH2) and interleukin-2 receptor subunit α (IL2RA). For example, the engineered T cells in claim 9, wherein the regulatory T cell promoting molecule is IL10 or CTLA4. The engineered T cells of claim 9 or 10, wherein the regulatory T cell stimulating molecule is a first regulatory T cell stimulating molecule, and further includes a heterologous nucleic acid sequence encoding a second regulatory T cell stimulating molecule under the control of a promoter sequence. For example, in the engineered T cells of claim 11, the first regulatory T cell stimulating molecule and the second regulatory T cell stimulating molecule are IL10 and CTLA4. The engineered T cells of any of claims 9 to 12, wherein the IL10, relative to mature wild-type human IL10, contains a mutation at one or more amino acids selected from D25, E96, N21, and R104. The engineered T cells of any of claims 9 to 13, wherein the IL10, relative to mature wild-type human IL10, comprises one or more amino acid mutations selected from D25A/K, E96A, N21A, and R104A. The engineered T cells, as described in any of claims 9 to 14, contain, relative to mature wild-type human IL10, combinations of amino acid mutations selected from the following: D25K, D25A, D25A/E96A, D25K/E96A, N21A/R104A, D25A/N21A/R104A, and D25K/N21A/R104A. The engineered T cells, such as those described in any of claims 9 to 12, wherein the IL10 comprises mature wild-type human IL10. The engineered T cells of any of claims 1 to 16 further include modifications to the endogenous nucleic acid sequence encoding proteins selected from TRAC, HLA-A, HLA-B, and CIITA, wherein the modifications knock down the expression of the protein. For example, in claim 17, engineered T cells containing the protein TRAC, and the modification knocks down TRAC expression. For example, the engineered T cells in claim 17 or 18, wherein the protein is HLA-A, and the modification knocks down the expression of HLA-A. For example, the engineered T cells of any of claims 17 to 19, wherein the protein is HLA-B, and the modification knocks down the expression of HLA-B. For example, the engineered T cells of any of the claims 17 to 20, wherein the protein is CIITA, and the modification knocks down the expression of CIITA. The engineered T cells of any of claims 1 to 16 further include: a) modification of the endogenous nucleic acid sequence encoding TRAC, wherein the modification knocks down the expression of TRAC; b) modification of the endogenous nucleic acid sequence encoding HLA-A, wherein the modification knocks down the expression of HLA-A; c) modification of the endogenous nucleic acid sequence encoding HLA-B, wherein the modification knocks down the expression of HLA-B; and d) modification of the endogenous nucleic acid sequence encoding CIITA, wherein the modification knocks down the expression of CIITA. An engineered T cell comprising: i) a modification of a first endogenous nucleic acid sequence encoding tumor necrosis factor α (TNFA), wherein the modification of the first endogenous nucleic acid knocks down TNFA expression; ii) a modification of a second endogenous nucleic acid sequence encoding interferon-γ (IFNG), wherein the modification of the second endogenous nucleic acid knocks down IFNG expression; 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-promoting molecule under the control of a promoter sequence. For example, in claim 23, the engineered T cells contain the protein TRAC, and the third modification knocks down the expression of TRAC. For example, the engineered T cells in claim 23 or 24, wherein the protein is HLA-A, and the third modification knocks down the expression of HLA-A. The engineered T cells of any of claims 23 to 25, wherein the protein is HLA-B, and the third modification knocks down the expression of HLA-B. For example, the engineered T cells of any of claims 23 to 26, wherein the protein is CIITA, and the third modification knocks down the expression of CIITA. An engineered T cell comprising: i) a modification of a first endogenous nucleic acid sequence encoding tumor necrosis factor α (TNFA), wherein the modification knocks down TNFA expression; ii) a modification of a second endogenous nucleic acid sequence encoding interferon-γ (IFNG), wherein the modification knocks down IFNG expression; iii) a modification of a third endogenous nucleic acid sequence encoding TRAC, wherein the modification of the third endogenous nucleic acid sequence knocks down TRAC expression; 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 HLA-A expression; 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 HLA-B expression; vi) Modification of the sixth endogenous nucleic acid sequence encoding CIITA, wherein the modification of the sixth endogenous nucleic acid sequence knocks down the performance of CIITA; and vii) a heterologous nucleic acid sequence encoding a regulatory T cell stimulating molecule under the control of a promoter sequence. For example, the engineered T cells described in any of claims 23 to 28, wherein the regulatory T cell-promoting molecule is selected from interleukin-10 (IL10), cytotoxic T lymphocyte-associated protein 4 (CTLA4), T cell immune receptor with Ig and ITIM domains (TIGIT), indoleamine 2,3-dioxygenase 1 (IDO1), extracellular nucleoside triphosphate diphosphate hydrolase 1 (ENTPD1), extracellular 5'-nucleotidase (NT5E), interleukin-22 (IL-22), amphotericin G (AREG), interleukin-35 (IL35), GARP, CD274 molecule (CD274), forkhead box P3 (FOXP3), IKAROS family zinc finger 2 (IKZF2), familial eosinophilia (EOS), interferon regulator 4 (IRF4), and lymphoid enhancement binding factor 1. (LEF1), BTB domain and CNC homologue 2 (BACH2) and interleukin-2 receptor subunit α (IL2RA). For example, the engineered T cells in claim 29, wherein the regulatory T cell promoting molecule is IL10 or CTLA4. The engineered T cells of claims 29 or 30, wherein the regulatory T cell stimulating molecule is a first regulatory T cell stimulating molecule, and further includes a heterologous nucleic acid sequence encoding a second regulatory T cell stimulating molecule under the control of a promoter sequence. For example, in the engineered T cells of claim 31, the first regulatory T cell stimulating molecule and the second regulatory T cell stimulating molecule are IL10 and CTLA4. The engineered T cells of any of claims 29 to 32, wherein the IL10, relative to mature wild-type human IL10, contains a mutation at one or more amino acids selected from D25, E96, N21 and R104. The engineered T cells of any of claims 29 to 33, wherein the IL10 relative to mature wild-type human IL10 contains one or more amino acid mutations selected from D25A/K, E96A, N21A and R104A. The engineered T cells of any of claims 29 to 34, wherein the IL10 relative to mature wild-type human IL10 comprises a combination of amino acid mutations selected from the following: D25K, D25A, D25A/E96A, D25K/E96A, N21A/R104A, D25A/N21A/R104A, D25K/N21A/R104A. The engineered T cells of any of claims 29 to 35, wherein the IL10 comprises mature wild-type human IL10. The engineered T cells, as described in any of claims 1 to 36, further include 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 granzyme 7 (NKG7), Fas ligand (FASL, NF superfamily, member 6), ryanodine receptor 2 (RYR2), and community stimulating factor 2 (CSF2), wherein the modifications knock down the expression of IL17A, IL2, IL6, PRF1, GZMA, GZMB, FASL, RYR2, or CSF2, respectively. The engineered T cells of any of claims 1 to 37 further include 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. The engineered T cells of any of claims 1 to 38 further include a heterologous encoding sequence targeting a receptor under the control of a promoter sequence. For example, in the engineered T cells of claim 39, the targeting receptor is selected from the following ligands: mucosal vascular indicative cell adhesion molecule 1 (MADCAM1), tumor necrosis factor α (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), protein lipoprotein 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 2. (SCL2A2), glutamic acid decarboxylase (GAD2), ceramide core protein 3 (DSG3) and MHC class I HLA-A (HLA-A*02). For example, the engineered T cells in claims 39 or 40, wherein the targeting receptor includes a chimeric antigen receptor (CAR) or a T cell receptor (TCR). The engineered T cells of any of claims 39 to 41, wherein the heterologous nucleic acid sequence encoding the target receptor is incorporated into the phenotype. For example, the engineered T cells of any of claims 39 to 42, wherein the phenotype containing the heterologous nucleic acid sequence encoding the target receptor does not contain a nucleic acid sequence encoding a regulatory T cell stimulating molecule. The engineered T cells of any of claims 39 to 42, wherein the phenotype containing the heterologous nucleic acid sequence encoding the targeting receptor contains a nucleic acid sequence encoding a regulatory T cell stimulating molecule. The engineered T cells, such as those described in claims 11 to 22 or 31 to 44, wherein the heterologous nucleic acid sequence encoding the first regulatory T cell stimulating molecule is incorporated into the expression building, and the heterologous nucleic acid sequence encoding the second regulatory T cell stimulating molecule is incorporated into the expression building. The engineered T cells, such as those described in any of claims 11 to 22 or 31 to 45, wherein the heterologous nucleic acid sequence encoding the first regulatory T cell stimulating molecule and the heterologous nucleic acid sequence encoding the second regulatory T cell stimulating molecule are incorporated into a separate efficacious building block. The engineered T cells, such as those described in any of claims 11 to 22 or 31 to 46, wherein the heterologous nucleic acid sequence encoding the first regulatory T cell stimulating molecule and the heterologous nucleic acid sequence encoding the second regulatory T cell stimulating molecule are incorporated into a single efficacious building block. For example, the engineered T cells of any of claims 1 to 47, wherein at least one heterologous coding sequence is in a free-form representational structure. For example, the engineered T cells of any of claims 1 to 48, wherein at least one heterologous nucleic acid sequence is inserted into the genome. For example, in the engineered T cells of claim 49, the insertion into the genome is a non-targeted insertion. For example, in request item 49, engineered T cells, where the insertion is a targeted insertion. For example, in the engineered T cells of claim 51, the targeted insertion is selected from the following loci: TCR locus, TNF locus, IFNG locus, IL17A locus, IL6 locus, IL2 locus, and adeno-associated virus integration site 1 (AAVS1) locus. For example, in the engineered T cells of claim 52, the TCR locus is the T cell receptor α-constant (TRAC) locus. For example, the engineered T cells of any of claims 7 to 53, wherein the modification that knocks down gene expression includes one or more of insertion, deletion or substitution. The engineered T cells in any of the claims 1 to 54, wherein the engineered T cells are CD4+ T cells. An engineered population of T cells comprising any of the engineered T cells described in claims 1 to 55. An engineered T cell population comprising engineered T cells as described in any one of claims 1 to 22 or 37 to 56, wherein at least 30% or at least 40% of the cells in the population contain 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 contain a modification of an endogenous nucleic acid sequence encoding TGFBR2. The engineered T cell population of claim 56 or 57, wherein at least 50% or at least 70% of the cells in the population contain a modification of an endogenous nucleic acid sequence encoding TNFA; at least 50% or at least 70% of the cells in the population contain a modification of an endogenous nucleic acid sequence encoding IFNG; or at least 30% or at least 40% of the cells in the population contain a heterologous nucleic acid sequence encoding a regulatory T cell-promoting molecule under the control of a promoter sequence. For example, in the engineered T cell populations of requests 57 or 58, the percentage of inserted or modified cells is determined based on the percentage of reads from next-generation sequencing (NGS). A pharmaceutical composition comprising engineered T cells as described in any of claims 1 to 55 or engineered T cell populations as described in claims 56 to 59. A method or use of administering to an individual an engineered T cell as described in any of claims 1 to 55, an engineered T cell population as described in any of claims 56 to 59, or a pharmaceutical composition as described in claim 60. The method or use described in claim 61, wherein the individual requires immunosuppression. The method or use described in claims 61 or 62 is for the treatment of immune disorders. The method or use described in any of claims 61 to 63 is for the treatment of autoimmune diseases. As in claim 64, the method or use of the autoimmune disease is selected from ulcerative colitis, Crohn's disease, rheumatoid arthritis, psoriasis, multiple sclerosis, systemic lupus erythematosus, and type 1 diabetes. The method or use described in any of claims 61 to 65 is for the treatment of graft-versus-host disease (GvHD). The method or use described in any of the requests 61, 62 or 66, wherein the individual receives a transplant or blood transfusion. The method or use described in claim 67, wherein the transplant or transfusion comprises a solid organ or a population of cells. The method or use of claim 67 or 68, wherein the transplant or transfusion comprises cells from a donor who is not the individual. The method or use of any of claims 67 to 69, wherein, prior to receiving the transplant or transfusion, the individual is given an engineered T cell as described in any of claims 1 to 55, an engineered T cell population as described in any of claims 56 to 59, or a pharmaceutical composition as described in claim 60. The method or use of any of claims 67 to 70, wherein, at the same time as receiving the transplant or blood transfusion, the individual is given an engineered T cell as of any of claims 1 to 55, an engineered T cell population as of any of claims 56 to 59, or a pharmaceutical composition as of claim 60. The method or use of any of claims 67 to 71, wherein, after receiving the transplant or blood transfusion, the individual is given an engineered T cell as described in any of claims 1 to 55, an engineered T cell population as described in any of claims 56 to 59, or a pharmaceutical composition as described in claim 60.