Engineered T cells that conditionally express recombinant receptors, related polynucleotides, and methods

Abstract

Methods, cells, compositions, and kits for manipulating T cells are provided for use in the treatment of cancer and other conditions. [Solution] Manipulated T cells are provided, containing a modified T cell stimulation-associated locus encoding a recombinant receptor or a portion thereof. In some aspects, the nucleic acid sequence encoding the recombinant receptor or a portion thereof is functionally linked to an endogenous transcriptional regulatory element of the T cell stimulation-associated locus, and possibly manipulated by targeted incorporation. In some aspects, the manipulated cells conditionally express the recombinant receptor, for example, in response to a stimulation or activation signal in the T cell. Also disclosed are cell compositions, nucleic acids for manipulating cells, and methods and articles for producing manipulated cells. In some aspects, the manipulated cells may be used in connection with cell therapy, for example, in connection with cancer immunotherapy, including adoptive transfer of manipulated cells.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 044,984, filed on 26 June 2020, entitled "ENGINEERED T CELLS CONDITIONALLY EXPRESSING A RECOMBINANT RECEPTOR, RELATED POLYNUCLEOTIDES AND METHODS," the contents of which are incorporated in their entirety by reference.

[0002] Inclusion by referencing sequence listings This application is filed together with an electronic sequence listing. The sequence listing is provided as a file titled 735042013840SeqList.txt, created on June 23, 2021, with a size of 220 kilobytes. The electronic information of the sequence listing is incorporated in its entirety by reference.

[0003] field This disclosure relates to engineered T cells containing a modified T cell stimulation-associated locus encoding a recombinant receptor or a portion thereof. In some aspects, the nucleic acid sequence encoding the recombinant receptor or a portion thereof is functionally linked to an endogenous transcriptional regulatory element of the T cell stimulation-associated locus and, optionally, engineered by targeted incorporation. In some aspects, the engineered cells conditionally express the recombinant receptor, for example, in response to a stimulation or activation signal in the T cell. Also disclosed are cell compositions, nucleic acids for engineering cells, and methods and articles for producing engineered cells. In some aspects, engineered cells may be used in connection with cell therapy, for example, in connection with cancer immunotherapy, including adoptive transfer of engineered cells. [Background technology]

[0004] background Adoptive cell therapy, which utilizes recombinant receptors such as chimeric antigen receptors (CARs) or recombinant T cell receptors (TCRs) that recognize disease-related antigens, constitutes an attractive mode of treatment for cancer and other diseases. For example, improved strategies for manipulating T cells to express recombinant receptors are needed for use in adoptive immunotherapy, for instance, in the treatment of cancer, infectious diseases, and autoimmune diseases. Methods, cells, compositions, and kits are provided for use in methods that satisfy such needs. [Overview of the Initiative]

[0005] overview Engineered T cells comprising a modified T cell stimulation-associated locus containing a transgene encoding a recombinant receptor or a portion thereof, wherein the transgene is functionally linked to an endogenous transcriptional regulatory element of the T cell stimulation-associated locus, and the endogenous transcriptional regulatory element induces or upregulates the expression of the functionally linked transgene following a simulation or activation signal in the T cell.

[0006] In some of the embodiments, the endogenous transcriptional regulatory element is a promoter of an endogenous T cell stimulation-related gene locus. In some of the embodiments, the transgene encoding a recombinant receptor or a portion thereof is located downstream of the promoter.

[0007] In some of the embodiments, the expression of a functionally linked transgene is inducible and is induced following a stimulus or activation signal in the cell. In some of the embodiments, the expression of a functionally linked transgene is upregulated or induced within less than 6 hours or approximately less than 6 hours following a stimulus or activation signal in the T cell. In some of the embodiments, the expression of a functionally linked transgene is upregulated or induced within less than 12 hours or approximately less than 12 hours following a stimulus or activation signal in the T cell. In some of the embodiments, the expression of a functionally linked transgene is upregulated or induced within less than 24 hours or approximately less than 24 hours following a stimulus or activation signal in the T cell. In some of the embodiments, the expression of a functionally linked transgene is upregulated or induced within less than 36 hours or approximately less than 36 hours following a stimulus or activation signal in the T cell. In some of the embodiments, the expression of a functionally linked transgene is upregulated or induced within less than 48 hours or approximately less than 48 hours following a stimulus or activation signal in a T cell.

[0008] In some of the embodiments, the expression of a functionally linked transgene may be reduced, but not permanently, over time or in the absence of T cell stimulation or activation signals. In some of the embodiments, the expression of a functionally linked transgene is reduced or downregulated following upregulation or induction of expression, or following reduction or absence of simulation or activation signals in T cells. In some of the embodiments, the expression of a functionally linked transgene is reduced or downregulated by more than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or by more than approximately 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or more following upregulation or induction of expression, or following reduction or absence of simulation or activation signals in T cells. In some of the embodiments, following upregulation or induction of expression, the expression of a functionally linked transgene is reduced or downregulated 1, 2, 3, 4, 5, 6, 7, or 8 days or more after the stimulation or activation signal in T cells, or approximately 1, 2, 3, 4, 5, 6, 7, or 8 days or more. In some of the embodiments, the expression of a functionally linked transgene is reduced or downregulated within 6, 12, 18, 24, 36, or 48 hours or approximately 6, 12, 18, 24, 36, or 48 hours after the reduction or absence of the simulation or activation signal in T cells.

[0009] In some of the embodiments, the expression of a functionally linked transgene may be re-induced or upregulated following further simulation or activation signals in T cells after reduction or absence of simulation or activation signals. In some of the embodiments, the expression of a functionally linked transgene is upregulated or induced within less than 6, 12, 18, 24, 36, or 48 hours, or within approximately 6, 12, 18, 24, 36, or 48 hours, following further simulation or activation signals in T cells after reduction or absence of simulation or activation signals. In some of the embodiments, the expression of a functionally linked transgene is inducible and induced following stimulation or activation signals in cells. In some of the embodiments, the expression of a functionally linked transgene is upregulated or induced within less than 6 hours, or within approximately 6 hours, following further simulation or activation signals in T cells after reduction or absence of simulation or activation signals. In some of the embodiments, the expression of a functionally linked transgene is upregulated or induced within 12 hours or approximately 12 hours following further simulation or activation signaling in T cells after reduction or absence of simulation or activation signaling. In some of the embodiments, the expression of a functionally linked transgene is upregulated or induced within 24 hours or approximately 24 hours following further simulation or activation signaling in T cells after reduction or absence of simulation or activation signaling. In some of the embodiments, the expression of a functionally linked transgene is upregulated or induced within 36 hours or approximately 36 hours following further simulation or activation signaling in T cells after reduction or absence of simulation or activation signaling.In some of the embodiments, the expression of a functionally linked transgene is upregulated or induced within less than 48 hours or approximately less than 48 hours following further simulation or activation signaling in T cells, after reduction or absence of simulation or activation signaling.

[0010] In some of the embodiments, following a simulation or activation signal in a T cell, the product translated from the open reading frame of the endogenous T cell stimulation-associated locus is not expressed in the cell, or the functional endogenous gene product of the endogenous T cell stimulation-associated locus is not expressed. In some of the embodiments, the modified T cell stimulation-associated locus includes a deletion, insertion, frameshift mutation, or nonsense mutation in the open reading frame of the endogenous T cell stimulation-associated locus.

[0011] In some of the embodiments, the endogenous T cell stimulation-associated locus is selected from the PDCD1, CD69, Nur77, FoxP3, and HLA-DR loci. In some of the embodiments, the endogenous T cell stimulation-associated locus is the PDCD1 locus. In some of the embodiments, the endogenous T cell stimulation-associated locus is the CD69 locus. In some of the embodiments, the endogenous T cell stimulation-associated locus is the Nur77 locus. In some of the embodiments, the endogenous T cell stimulation-associated locus is the FoxP3 locus. In some of the embodiments, the endogenous T cell stimulation-associated locus is the HLA-DR locus. In some of the embodiments, the endogenous transcriptional regulatory element comprises one or more response elements recognized by a transcription factor that is activated following a stimulus or activation signal.

[0012] In some of the embodiments, the recombinant receptor or a portion thereof can induce or transmit a stimulating or activating signal in T cells.

[0013] In some of the embodiments, the recombinant receptor comprises an intracellular region containing an intracellular signaling domain of a component of the T cell receptor (TCR) complex, and the stimulating or activating signal in the T cell comprises a signal through the intracellular signaling domain present in the recombinant receptor. In some of the embodiments, the recombinant receptor comprises an intracellular region containing an intracellular signaling domain containing an immunoreceptor-activated tyrosine motif (ITAM), and the stimulating or activating signal in the T cell comprises a signal through the intracellular signaling domain present in the recombinant receptor. In some of the embodiments, the intracellular signaling domain of the recombinant receptor (e.g., CAR) may contain a CD3 zeta signaling domain, and the stimulating or activating signal in the T cell comprises a signal through the intracellular signaling domain present in the recombinant receptor (e.g., CAR).

[0014] In some of the embodiments, the recombinant receptor is a TCR that can recruit components of the T cell complex to induce or stimulate activation signals in T cells.

[0015] In some of the embodiments, the recombinant receptor includes an extracellular domain comprising a binding domain capable of binding to or recognizing an activator (e.g., a target antigen). In some of the embodiments, a stimulatory or activation signal is induced in the T cell upon binding to or recognition of the activator by the recombinant receptor.

[0016] In some of the embodiments, the active agent is a target antigen. In some of the embodiments, the target antigen is a recombinant protein or an antigen expressed on the surface of a cell. In some of the embodiments, the target antigen is associated with, specific to, or expressed on cells or tissues of a disease, disorder, or condition. In some of the embodiments, the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer. In some of the embodiments, the target antigen is a tumor antigen. In some of the embodiments, the target antigens are αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9; also known as CAIX or G250), cancer testis antigen, cancer / testis antigen 1B (CTAG; also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, CC motif chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), and type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor-like 5 (FCRL5;Also known as Fc receptor homolog 5 or FCRH5, fetal acetylcholine receptor (fetal AchR), folate-binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G protein-coupled receptor class C group 5 member D (GPRC5D), Her2 / neu (receptor tyrosine kinase erb-B2) Her3 (erb-B3), Her4 (erb-B4), erbB dimer, human high molecular weight melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22Rα), IL-13 receptor alpha 2 (IL-13Rα2), kinase insert domain receptor (kdr), κ light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM Leucine-rich repeat-containing 8 family member A (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, mouse cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligand, melan A (MART-1), neuronal cell adhesion molecule (NCAM), tumor embryo antigen, melanoma preferential expression antibody PRAME, progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivorvin, trophoblast glycoprotein (TPBG; also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-related protein 1 (TRP1; also known as TYRP1 or gp75), tyrosinase-related protein 2 (TRP2;Selected from among dopachrome tautomerase (also known as dopachrome delta-isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor 1 (WT-1), pathogen-specific antigens or pathogen-expressed antigens, or antigens associated with a universal tag, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV, or other pathogens.

[0017] In some of the embodiments, the active agent is an anti-idiotype antibody that is specific to the extracellular domain of the recombinant receptor.

[0018] In some of the embodiments, the recombinant receptor is a chimeric antigen receptor (CAR). In some of the embodiments, the CAR comprises an extracellular domain containing a binding domain, a transmembrane domain, and an intracellular domain. In some of the embodiments, the extracellular domain also comprises a spacer. In some of the embodiments, the spacer is functionally linked between the binding domain and the transmembrane domain. In some of the embodiments, the extracellular domain comprises a binding domain which is or contains an antibody or its antigen-binding fragment, e.g., a single-stranded variable fragment (scFv).

[0019] In some of the embodiments, the intracellular domain of a recombinant receptor (e.g., CAR) includes an intracellular signaling domain. In some of the embodiments, the intracellular signaling domain is or includes the intracellular signaling domain of a CD3 chain. In some of the embodiments, the intracellular signaling domain is or includes the CD3-zeta (CD3ζ) chain or its signaling portion. In some of the embodiments, the intracellular domain includes one or more co-stimulatory signaling domains. In some of the embodiments, the intracellular domain includes the CD3-zeta (CD3ζ) chain or its signaling portion, and one or more co-stimulatory signaling domains. In some of the embodiments, one or more co-stimulatory signaling domains include the intracellular signaling domain of CD28, 4-1BB, or ICOS, or its signaling portion. In some of the embodiments, the co-stimulatory signaling domain includes the intracellular signaling domain of 4-1BB.

[0020] In some of the embodiments, the modified T cell stimulation-associated locus encodes a recombinant receptor which is a CAR, the CAR comprising, in order from its N-terminus to its C-terminus, an extracellular binding domain, a spacer, a transmembrane domain, and an intracellular domain.

[0021] In some of the embodiments, the transgene includes, in order, an extracellular binding domain, e.g., scFv; a spacer, e.g., a sequence derived from human immunoglobulin hinges, e.g., IgG1, IgG2, or IgG4, or a modified version thereof, further including, e.g., a CH2 region and / or a CH3 region; a transmembrane domain, e.g., from human CD28; a co-stimulatory signaling domain, e.g., from human 4-1BB; and a sequence of nucleotides encoding an intracellular signaling domain, e.g., a CD3ζ chain or a portion thereof. In some of the embodiments, the modified T cell stimulation-associated locus includes, in order, an extracellular binding domain, e.g., scFv; a spacer, e.g., a sequence derived from human immunoglobulin hinges, e.g., IgG1, IgG2, or IgG4, or a modified version thereof, further including, e.g., a CH2 region and / or a CH3 region; a transmembrane domain, e.g., from human CD28; a co-stimulatory signaling domain, e.g., from human 4-1BB; and a sequence of nucleotides encoding an intracellular signaling domain, e.g., a CD3ζ chain or a portion thereof.

[0022] In some of the embodiments, the transgene encodes a recombinant receptor, e.g., the complete or whole sequence of the recombinant receptor. In some of the embodiments, the recombinant receptor is a single-chain polypeptide. For example, the recombinant receptor may be a chimeric antigen receptor (CAR), and the transgene encodes the chimeric antigen receptor (CAR), e.g., the complete or whole sequence of the CAR. In some of the embodiments, the recombinant receptor is a multi-chain polypeptide, e.g., a double-chain polypeptide. For example, the recombinant receptor may be a T cell receptor (TCR) containing α and β chains, and the transgene encodes both the α and β chains of the TCR, e.g., the complete or whole sequence of the α and β chains of the TCR. In such examples, the separate chains of the TCR may be separated by a multi-cistronic element, such as a ribosome skipping element (e.g., T2A or P2A) or an IRES.

[0023] In some of the embodiments, the transgene encodes a portion of the recombinant receptor. In some embodiments, the portion of the recombinant receptor encoded by the transgene can promote or enable the same or similar functional activity (e.g., antigen-binding activity and receptor signaling activity) of the full-length recombinant receptor when expressed from a T cell, for example, following a simulation or activation signal in the T cell. In some of the embodiments, when expressed from a T cell, the portion of the recombinant receptor can form a complete recombinant receptor or a sub-sequence that retains the functional activity (e.g., antigen-binding activity and receptor signaling activity) of the full-length form of the recombinant receptor. In some of the embodiments, when expressed from a T cell, the portion of the recombinant receptor can promote or enable at least 75%, 85%, 90%, or 100% of the complete activity of the recombinant receptor. In some examples, when expressed from a T cell, the portion of the recombinant receptor encoded by the transgene can form a complete functional receptor together with another component of the recombinant receptor (e.g., another chain of the recombinant receptor) also expressed by the engineered T cell. In other examples, the recombinant receptor may be a single-chain polypeptide (e.g., CAR) in part, which may contain a sequence of recombinant receptor amino acids required for the functional activity of the recombinant receptor when expressed from a T cell. In such embodiments, the portion of the recombinant receptor may have a sequence of recombinant receptor amino acids that comprises a sequence length of at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 95%, or at least about 97% of the length of the recombinant receptor, and which encodes a partial recombinant receptor that retains the activity of the recombinant receptor (e.g., antigen-binding activity and receptor signaling activity).

[0024] In some of the embodiments, the portion of the recombinant receptor is, for example, one polypeptide chain of the recombinant receptor in embodiments where the recombinant receptor is composed of multiple chains (e.g., a TCR containing α and β chains, or a multi-chain CAR). For example, the other chains of the recombinant receptor are further expressed separately by the engineered T cell. In some of the embodiments, the recombinant receptor contains two separate polypeptide chains, where the portion of the recombinant receptor encoded by the transgene is one chain of the recombinant receptor, and the engineered T cell further expresses the other chain of the recombinant receptor. In some of the embodiments, the other chain of the recombinant receptor is encoded by a second transgene that can be separately contained in and expressed by the T cell.

[0025] In some of the embodiments, the recombinant receptor is a CAR that is a multichain CAR. In some examples, the transgene encodes a multichain CAR, e.g., the complete or whole sequence of the multichain CAR, e.g., the first and second chains of a multichain CAR having two chains. In such examples, the separate chains of the multichain CAR may be separated by a multicistronic element such as a ribosome skipping element (e.g., T2A or P2A) or IRES. In other examples, the transgene encodes one chain of the multichain CAR, and the other chain of the multichain CAR is separately encoded by the engineered cell, e.g., by a second transgene. In some of the embodiments provided, the engineered T cells can express a fully functional recombinant multichain CAR that retains or exhibits antigen-binding activity and receptor signaling activity, e.g., following a simulation or activation signal in the T cell.

[0026] In some of the embodiments, the recombinant receptor is a recombinant T cell receptor (TCR). In some of the embodiments, the recombinant TCR comprises an alpha (TCRα) chain and a beta (TCRβ) chain, and the transgene comprises a nucleic acid sequence encoding the TCRα chain and / or a nucleic acid sequence encoding the TCRβ chain. In some examples, the transgene encodes the complete or whole sequence of the TCR, e.g., the TCRα and TCRβ chains. In such examples, the separate chains of the TCR may be separated by a multicistronic element such as a ribosome skipping element (e.g., T2A or P2A) or IRES. In some of the embodiments, the transgene encodes either the TCRα or TCRβ chain, and the other TCRα or TCRβ chain is separately encoded by the engineered cell, e.g., by a second transgene. In some of the embodiments provided, the engineered T cell can express a fully functional recombinant TCR that retains or exhibits antigen-binding activity and receptor signaling activity, e.g., following a simulation or activation signal in the T cell.

[0027] In some of the embodiments, the recombinant receptor is a TCR comprising a TCRα chain and a TCRβ chain. In some of the embodiments, the TCRα chain comprises a constant (Cα) region comprising one or more introduced cysteine ​​residues, and / or the TCRβ chain comprises a Cβ region comprising one or more introduced cysteine ​​residues, the one or more introduced cysteine ​​residues capable of forming one or more non-natural disulfide crosslinks between the α and β chains. In some of the embodiments, the one or more introduced cysteine ​​residues comprise the substitution of a non-cysteine ​​residue with a cysteine ​​residue. In some of the embodiments, the Cα region comprises cysteine ​​at the position corresponding to position 48 in numbering as shown in SEQ ID NO:92; and / or the Cβ region comprises cysteine ​​at the position corresponding to position 57 in numbering as shown in SEQ ID NO:96.

[0028] In some of the embodiments, the transgene comprises a sequence of nucleotides encoding at least one further protein. In some of the embodiments, the at least one further protein is a surrogate marker, such as a marker that monitors or substitutes for the expression of recombinant receptors by engineered T cells. In some of the embodiments, the surrogate marker is a truncated receptor. In some of the embodiments, the truncated receptor lacks an intracellular signaling domain and / or is unable to mediate intracellular signaling when bound to its ligand.

[0029] In some of the embodiments, the transgene further comprises a multicistronic element. In some of the embodiments, the multicistronic element comprises a ribosome skipping element selected from T2A, P2A, E2A, or F2A, or a sequence encoding an intrasequence ribosome entry site (IRES).

[0030] In some of the embodiments, the multicistronic element is located between the sequence of nucleotides encoding the CAR and the sequence of nucleotides encoding at least one further protein. In some of the embodiments, the recombinant receptor is a recombinant TCR, and the multicistronic element is located between the sequence of nucleotides encoding TCRα and the sequence of nucleotides encoding TCRβ. In some of the embodiments, the recombinant receptor is a multichain CAR, and the multicistronic element is located between the sequence of nucleotides encoding one chain of the multichain CAR and the sequence of nucleotides encoding another chain of the multichain CAR. In some of the embodiments, the multicistronic element is upstream of the sequence of nucleotides encoding the recombinant receptor.

[0031] In some of the embodiments, the modified T cell stimulation-related locus is created by, for example, the incorporation of a transgene encoding a recombinant receptor into an endogenous T cell stimulation-related locus by gene editing using homology-directed repair. In some of the embodiments, the incorporation is achieved by a) inducing gene disruption at one or more target sites in or near the endogenous T cell stimulation-related locus; and b) introducing a polynucleotide for homology-directed repair (HDR).

[0032] In some of the embodiments, the transgene encoding the recombinant receptor is integrated at or near at least one target site in a T cell stimulation-associated locus. In some of the embodiments, gene disruption is brought about by a combination of zinc finger nuclease (ZFN), TAL effector nuclease (TALEN), or CRISPR-Cas9 that specifically binds to, recognizes, or hybridizes with the target site. In some of the embodiments, gene disruption is brought about by a combination of CRISPR-Cas9, and the CRISPR-Cas9 combination includes a guide RNA (gRNA) having a targeting domain complementary to at least one target site. In some of the embodiments, the CRISPR-Cas9 combination is a ribonucleoprotein (RNP) complex containing the gRNA and the Cas9 protein. In some of the embodiments, gene disruption is brought about by an RNP introduced into multiple T cells via electroporation.

[0033] In some of the embodiments, the T cell stimulation-associated locus is PDCD1. In some of the embodiments, gene disruption is brought about by a CRISPR-Cas9 combination containing a gRNA, and the gRNA has a targeting domain complementary to the target site in the PDCD1 gene. In some of the embodiments, the gRNA contains the sequence shown in SEQ ID NO: 75 and one of 104-109. In some of the embodiments, the gRNA contains the sequence shown in SEQ ID NO: 75.

[0034] In some of the embodiments, the T cell stimulation-associated locus is CD69. In some of the embodiments, gene disruption is induced by a CRISPR-Cas9 combination containing a gRNA, and the gRNA has a targeting domain complementary to the target site in the CD69 gene. In some of the embodiments, the gRNA contains a sequence represented by one of SEQ ID NO: 116-121.

[0035] In some of the embodiments, the T cell stimulation-associated locus is Nur77. In some of the embodiments, gene disruption is induced by a CRISPR-Cas9 combination containing a gRNA, and the gRNA has a targeting domain complementary to the target site in the Nur77 gene. In some of the embodiments, the gRNA contains sequences shown in SEQ ID NO: 122-127 and 134-136.

[0036] In some of the embodiments, the T cell stimulation-associated locus is FoxP3. In some of the embodiments, gene disruption is brought about by a CRISPR-Cas9 combination containing a gRNA, and the gRNA has a targeting domain complementary to the target site in the FoxP3 gene.

[0037] In some of the embodiments, the T cell stimulation-associated locus is the HLA-DR locus. 9. In some of the embodiments, gene disruption is induced by a CRISPR-Cas9 combination containing a gRNA, and the gRNA has a targeting domain complementary to the target site in the HLA-DR gene.

[0038] In some of the embodiments, the T cells further include gene disruption in the endogenous T cell receptor α constant region (TRAC) gene and / or the endogenous T cell receptor β constant region (TRBC) gene. In some of the embodiments, the T cells further include gene disruption in the endogenous T cell receptor α constant region (TRAC) gene. In some of the embodiments, the T cells further include gene disruption in the endogenous T cell receptor α constant region (TRBC) gene. In some of the embodiments, the T cells further include gene disruption in the endogenous T cell receptor α constant region (TRAC) gene and the endogenous T cell receptor β constant region (TRBC) gene. In some embodiments, gene disruption is induced by a combination of a zinc finger nuclease (ZFN), a TAL effector nuclease (TALEN), or CRISPR-Cas9 that specifically binds to, recognizes, or hybridizes to at least one target site, e.g., at least one target site within either the TRAC gene or the TRBC gene, or either the TRBC1 or TRBC2 gene. In some embodiments, gene disruption is induced by a combination of CRISPR-Cas9, and the CRISPR-Cas9 combination includes a guide RNA (gRNA) having a targeting domain complementary to at least one target site. In some embodiments, the CRISPR-Cas9 combination is a ribonucleoprotein (RNP) complex containing the gRNA and the Cas9 protein. In some embodiments, gene disruption is induced by an RNP introduced into multiple T cells via electroporation.

[0039] In some of the embodiments, gene disruption is induced by a CRISPR-Cas9 combination including a gRNA, and the gRNA has a targeting domain complementary to at least one target site in either the TRAC gene or the TRBC gene, TRBC1 or TRBC2 gene. In some of the embodiments, the gRNA has a targeting domain complementary to a target site in the TRAC gene. In some of the embodiments, the gRNA contains a sequence shown in SEQ ID NO: 77 and one of 188-218. In some of the embodiments, the gRNA contains a sequence shown in SEQ ID NO: 77. In some of the embodiments, the gRNA has a targeting domain complementary to a target site in the TRBC gene. In some of the embodiments, the gRNA contains a sequence shown in SEQ ID NO: 219-276.

[0040] In some of the embodiments, the signaling activity through the intracellular signaling domain of the encoded recombinant receptor in T cells in the absence of simulation or activation signals is reduced by 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more, or by approximately 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more, compared to engineered T cells containing a transgene encoding the same recombinant receptor located at a different location in the T cell genome or at a random location in the T cell genome.

[0041] In some of the embodiments, the T cells are CD8+ T cells or CD4+ T cells or their subtypes. In some of the embodiments, the T cells are primary T cells derived from a subject. In some of the embodiments, the subject is human. In some embodiments, the T cells are primary T cells derived from a human subject. In some embodiments, the T cells are primary human T cells. In some of the embodiments, the T cells are derived from multipotent or pluripotent cells. In some of the embodiments, the T cells are derived from iPSCs.

[0042] Also provided are compositions containing any of a plurality of provided engineered cells. In some of the embodiments, the expression of a functionally linked transgene is upregulated or induced in one or more cells in the composition within 6, 12, 18, 24, 36, or 48 hours or about 6, 12, 18, 24, 36, or 48 hours following a stimulation or activation signal in a T cell. In some of the embodiments, the frequency of cells expressing a functionally linked transgene among the cells in the composition following a simulation or activation signal in a T cell is higher than 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or about 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or higher. In some of the embodiments, the expression of a functionally linked transgene is reduced or downregulated in one or more cells in the composition, following upregulation or induction of expression, or following reduction or absence of simulation or activation signals in T cells.

[0043] In some of any of the aspects, following upregulation or induction of expression, or following reduction or absence of stimulation or activation signals in T cells, the frequency of cells expressing the functionally linked transgene among the cells in the composition is more than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or more, or about 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or more, and is reduced. In some of any of the aspects, following upregulation or induction of expression, the expression of the functionally linked transgene is reduced or downregulated in one or more cells in the composition 1, 2, 3, 4, 5, 6, 7, or 8 days, or more, or about 1, 2, 3, 4, 5, 6, 7, or 8 days, or more, after the stimulation or activation signal in T cells.

[0044] In some of any of the aspects, the expression of the functionally linked transgene in one or more of the cells in the composition is reduced or downregulated within less than 6, 12, 18, 24, 36, or 48 hours, or about within less than 6, 12, 18, 24, 36, or 48 hours, following reduction or absence of stimulation or activation signals in T cells. In some of any of the aspects, following reduction or absence of stimulation or activation signals in T cells, the frequency of cells expressing the recombinant receptor among the cells in the composition is lower than 50%, 40%, 30%, 25%, 20%, 15%, 10%, or 5%, or less, or about lower than 50%, 40%, 30%, 25%, 20%, 15%, 10%, or 5%, or less.

[0045] In some embodiments of any aspect, the composition comprises CD4+ T cells and / or CD8+ T cells. In some embodiments of any aspect, the composition comprises CD4+ T cells and CD8+ T cells, and the ratio of CD4+ T cells to CD8+ T cells is 1:3 to 3:1 or about 1:3 to 3:1. In some embodiments of any aspect, it is 1:1.

[0046] Also provided is a polynucleotide comprising (a) a transgene encoding a recombinant receptor or a portion thereof, and (b) one or more homology arms linked to the transgene, the one or more homology arms comprising sequences homologous to one or more regions of an endogenous T cell-stimulating related locus in a T cell.

[0047] In some embodiments of any aspect, the recombinant receptor or a portion thereof is encoded by a modified T cell-stimulating related locus containing the transgene encoding the recombinant receptor or a portion thereof when the recombinant receptor is expressed from the cell into which the polynucleotide has been introduced. In some embodiments of any aspect, the transgene is a sequence that is foreign or heterologous to the open reading frame of the endogenous T cell-stimulating related locus of a T cell.

[0048] In some embodiments of any aspect, the T cell is a primary human T cell. In some embodiments of any aspect, the T cell is a T cell derived from a subject. In some embodiments of any aspect, the subject is human. In some embodiments of any aspect, the T cell is a human T cell.

[0049] In some embodiments of any aspect, the one or more homology arms comprise a 5' homology arm and / or a 3' homology arm. In some embodiments of any aspect, the 5' homology arm and the 3' homology arm comprise nucleic acid sequences homologous to the nucleic acid sequences surrounding a target site, and the target site is within a T cell-stimulating related locus. In some embodiments of any aspect, the target site is downstream of an endogenous transcriptional control element of a T cell-stimulating related locus.

[0050] In some of the embodiments, the polynucleotide comprises the structure [5' homology arm]-[transgene]-[3' homology arm]. In some of the embodiments, the 5' homology arm and the 3' homology arm contain nucleic acid sequences homologous to the nucleic acid sequence surrounding at least one target site. In some of the embodiments, the 5' homology arm and the 3' homology arm are independently 50 or about 50 to 750 or about 750 nucleotides, 50 or about 50 to 500 or about 500 nucleotides, 50 or about 50 to 250 or about 250 nucleotides, 50 or about 50 to 100 or about 100 nucleotides, 100 or about 100 to 750 or about 750 nucleotides, 100 or about 100 to 500 or about 500 nucleotides, 100 or about 100 to 250 or about 250 nucleotides, 250 or about 250 to 750 or about 750 nucleotides, 250 or about 250 to 500 or about 500 nucleotides in length. In some of the embodiments, the 5' homology arm and the 3' homology arm are independently 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, or 500 nucleotides in length or about 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, or 500 nucleotides, or any value between any of the above. In some of the embodiments, the 5' homology arm and the 3' homology arm are independently 100 or about 100 nucleotides in length. In some of the embodiments, the 5' homology arm and the 3' homology arm are independently 50, 60, 70, 80, or 90 nucleotides in length or about 50, 60, 70, 80, or 90 nucleotides, or any value between any of the above.

[0051] In some of the embodiments, the T cell stimulation-related locus is selected from among the PDCD1, CD69, Nur77, FoxP3, and HLA-DR loci.

[0052] In some of the embodiments, the T cell stimulation-associated locus is PDCD1. In some of the embodiments, the 5' homology arm and the 3' homology arm contain sequences homologous to one or more regions of PDCD1. In some of the embodiments, the 5' homology arm is a) a sequence containing 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 consecutive nucleotides to a sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the sequence shown in SEQ ID NO: 66; b) SEQ ID A sequence containing 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 consecutive nucleotides of the sequence shown in NO:66, or at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 consecutive nucleotides; or c) a sequence showing SEQ ID NO:66.In some of the embodiments, the 3' homology arm is a) a sequence containing 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 consecutive nucleotides to a sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the sequence shown in SEQ ID NO: 67; b) SEQ ID A sequence containing 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 consecutive nucleotides of the sequence shown in NO:67, or at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 consecutive nucleotides; or c) a sequence showing SEQ ID NO:67.

[0053] In some of the embodiments, the T cell stimulation-associated locus is CD69. In some of the embodiments, the 5' homology arm and the 3' homology arm contain sequences homologous to one or more regions of CD69.

[0054] In some of the embodiments, the T cell stimulation-associated locus is Nur77. In some of the embodiments, the 5' homology arm and the 3' homology arm contain sequences homologous to one or more regions of Nur77.

[0055] In some of the embodiments, the T cell stimulation-associated locus is FoxP3. In some of the embodiments, the 5' homology arm and the 3' homology arm contain sequences homologous to one or more regions of FoxP3.

[0056] In some of the embodiments, the T cell stimulation-associated locus is the HLA-DR locus. In some of the embodiments, the 5' homology arm and the 3' homology arm contain sequences homologous to one or more regions of the HLA-DR locus.

[0057] In some of the embodiments, the recombinant receptor or a portion thereof can induce or transmit a stimulating or activating signal in T cells.

[0058] In some of the embodiments, the recombinant receptor comprises an intracellular region containing an intracellular signaling domain of a component of a T cell receptor (TCR) complex, and the stimulating or activating signal in the T cell comprises a signal through the intracellular signaling domain present in the recombinant receptor, or the recombinant receptor comprises an intracellular region containing an intracellular signaling domain containing an immunoreceptor-activated tyrosine motif (ITAM), and the stimulating or activating signal in the T cell comprises a signal through the intracellular signaling domain present in the recombinant receptor.

[0059] In some of the embodiments, the recombinant receptor includes an extracellular domain comprising a binding domain capable of binding to or recognizing an activator. In some of the embodiments, a stimulus or activation signal is induced in the T cell upon binding of the activator.

[0060] In some of the embodiments, the active agent is a target antigen. In some of the embodiments, the target antigen is a recombinant protein or an antigen expressed on the surface of a cell. In some of the embodiments, the target antigen is associated with, specific to, or expressed on cells or tissues of a disease, disorder, or condition. In some of the embodiments, the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer. In some of the embodiments, the target antigen is a tumor antigen. In some of the embodiments, the target antigens are αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9; also known as CAIX or G250), cancer testis antigen, cancer / testis antigen 1B (CTAG; also known as NY-ESO-11 and LAGE-12), carcinoembryonic antigen (CEA), cyclin, cyclin A2, CC motif chemokine ligand 1 (CCL-11), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), and type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-12), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor-like 5 (FCRL5;Also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), folate-binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G protein-coupled receptor class C group 5 member D (GPRC5D), Her2 / neu (receptor tyrosine kinase erb-B2), H er3 (erb-B3), Her4 (erb-B4), erbB dimer, human high molecular weight melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-122 receptor alpha (IL-122Rα), IL-113 receptor alpha 2 (IL-113Rα2), kinase insert domain receptor (kdr), κ light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitome of L1-CAM This product contains leucine-rich repeat-containing 8 family member A (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, mouse cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligand, melan A (MART-11), neuronal adhesion molecule (NCAM), tumor embryo antigen, and melanoma-preferential expression. Antigen (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivorvin, trophoblast glycoprotein (TPBG; also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-related protein 1 (TRP1; also known as TYRP1 or gp75), tyrosinase-related protein 2 (TRP2;Selected from among dopachrome tautomerase (also known as dopachrome delta-isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor 1 (WT-11), pathogen-specific antigens or pathogen-expressed antigens, or antigens associated with a universal tag, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV, or other pathogens.

[0061] In some of these embodiments, the active ingredient is an anti-idiotype antibody.

[0062] In some of the embodiments, the recombinant receptor is a chimeric antigen receptor (CAR). In some of the embodiments, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In some of the embodiments, the extracellular domain comprises a spacer. In some of the embodiments, the spacer is functionally linked between the binding domain and the transmembrane domain. In some of the embodiments, the extracellular domain comprises a binding domain which is or contains an antibody or an antigen-binding fragment thereof.

[0063] In some of the embodiments, the intracellular region includes an intracellular signaling domain. In some of the embodiments, the intracellular signaling domain is or includes an intracellular signaling domain of the CD3 chain. In some of the embodiments, it is the CD3-zeta (CD3ζ) chain or its signaling portion. In some of the embodiments, the intracellular region includes one or more co-stimulatory signaling domains. In some of the embodiments, one or more co-stimulatory signaling domains include an intracellular signaling domain of CD28, 4-1BB, or ICOS, or its signaling portion. In some of the embodiments, the co-stimulatory signaling region includes an intracellular signaling domain of 4-1BB.

[0064] In some of the embodiments, the modified T cell stimulation-associated locus encodes a recombinant receptor which is a CAR, the CAR comprising, in order from its N-terminus to its C-terminus, an extracellular binding domain, a spacer, a transmembrane domain, and an intracellular domain.

[0065] In some of the embodiments, the transgene includes, in order, an extracellular binding domain, e.g., scFv; a spacer, e.g., a sequence derived from human immunoglobulin hinges, e.g., IgG1, IgG2, or IgG4, or a modified version thereof, further including, e.g., a CH2 region and / or a CH3 region; and a transmembrane domain, e.g., from human CD28; a co-stimulatory signaling domain, e.g., from human 4-1BB; and a sequence of nucleotides encoding an intracellular signaling domain, e.g., a CD3ζ chain or a portion thereof; and / or, the modified T cell stimulation-associated locus includes, in order, an extracellular binding domain, e.g., scFv; a spacer, e.g., a sequence derived from human immunoglobulin hinges, e.g., IgG1, IgG2, or IgG4, or a modified version thereof, further including, e.g., a CH2 region and / or a CH3 region; and a transmembrane domain, e.g., from human CD28; a co-stimulatory signaling domain, e.g., from human 4-1BB; and a sequence of nucleotides encoding an intracellular signaling domain, e.g., a CD3ζ chain or a portion thereof.

[0066] In some of these embodiments, the transgene encodes a recombinant receptor.

[0067] In some of the embodiments, the transgene encodes a portion of the recombinant receptor. In some of the embodiments, the recombinant receptor comprises two separate polypeptide chains, where the portion of the recombinant receptor encoded by the transgene is one chain of the recombinant receptor. In some of the embodiments, the other chain of the recombinant receptor is encoded by a second transgene.

[0068] In some embodiments of any aspect, the CAR is a multi-chain CAR. In some embodiments of any aspect, the transgene encodes one chain of the multi-chain CAR.

[0069] In some embodiments of any aspect, the recombinant receptor is a recombinant T cell receptor (TCR). In some embodiments of any aspect, the recombinant TCR comprises an alpha (TCRα) chain and a beta (TCRβ) chain, and the transgene comprises a nucleic acid sequence encoding the TCRα chain and / or a nucleic acid sequence encoding the TCRβ chain. In some embodiments of any aspect, the transgene encodes one of the TCRα chain or the TCRβ chain.

[0070] In some embodiments of any aspect, the TCRα chain comprises a constant (Cα) region comprising one or more introduced cysteine residues, and / or the TCRβ chain comprises a Cβ region comprising one or more introduced cysteine residues, and the one or more introduced cysteine residues can form one or more non-natural disulfide bridges between the α chain and the β chain. In some embodiments of any aspect, the one or more introduced cysteine residues comprise substitution of a non-cysteine residue with a cysteine residue. In some embodiments of any aspect, the Cα region comprises cysteine at a position corresponding to position 48 in the numbering as shown in SEQ ID NO:92; and / or the Cβ region comprises cysteine at a position corresponding to position 57 in the numbering as shown in SEQ ID NO:96.

[0071] In some embodiments of any aspect, the transgene comprises a nucleotide sequence encoding at least one additional protein. In some embodiments of any aspect, the at least one additional protein is a surrogate marker. In some embodiments of any aspect, the surrogate marker is a truncated receptor. In some embodiments of any aspect, the truncated receptor lacks an intracellular signaling domain and / or cannot mediate intracellular signaling when its ligand binds.

[0072] In some of the embodiments, the transgene further comprises a multicistronic element. In some of the embodiments, the multicistronic element comprises a ribosome skipping element selected from T2A, P2A, E2A, or F2A, or a sequence encoding an intrasequence ribosome entry site (IRES).

[0073] In some of the embodiments, the multicistronic element is located between the sequence of nucleotides encoding a CAR and the sequence of nucleotides encoding at least one further protein; the recombinant receptor is a recombinant TCR, and the multicistronic element is located between the sequence of nucleotides encoding TCRα and the sequence of nucleotides encoding TCRβ; the recombinant receptor is a multichain CAR, and the multicistronic element is located between the sequence of nucleotides encoding one chain of the multichain CAR and the sequence of nucleotides encoding another chain of the multichain CAR; and / or the multicistronic element is upstream of the sequence of nucleotides encoding the recombinant receptor.

[0074] In some of the embodiments, the polynucleotide is a linear polynucleotide. In some of the embodiments, the polynucleotide is a double-stranded polynucleotide. In some of the embodiments, the polynucleotide is a single-stranded polynucleotide. In some of the embodiments, the polynucleotide is contained in a viral vector. In some of the embodiments, the viral vector is an AAV vector. In some of the embodiments, the viral vector is a retroviral vector. In some of the embodiments, the viral vector is a lentiviral vector.

[0075] In some of the embodiments, the polynucleotide is 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,250, 3,500, 3,750, or 4,000 nucleotides in length or about 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,250, 3,500, 3,750, or 4,000 nucleotides in length, or any value between any of the above. In some of the embodiments, the polynucleotide is 1,500 or about 1,500 to 2,500 or about 2,500 nucleotides in length, or 1,750 or about 1,750 to 2,250 or about 2,250 nucleotides in length.

[0076] Also provided is a method for producing genetically engineered T cells, comprising the steps of (a) introducing one or more active agents into a T cell that can induce gene disruption at a target site within an endogenous T cell stimulation-related locus of a T cell; and (b) introducing one of the provided polynucleotides into a T cell that includes gene disruption at a T cell stimulation-related locus, wherein the method produces a modified T cell stimulation-related locus, the modified T cell stimulation-related locus contains a transgene encoding a recombinant receptor or a portion thereof. In some of the embodiments, the transgene encoding a recombinant receptor or a portion thereof is incorporated into the endogenous T cell stimulation-related locus via homology-directed repair (HDR).

[0077] The present invention also provides a method for producing genetically modified T cells, comprising the step of introducing a polynucleotide containing a transgene encoding a recombinant receptor or a portion thereof into T cells, wherein the T cells have a gene disruption within the T cell stimulation-related locus of the T cell, and the transgene encoding the recombinant receptor or a portion thereof is incorporated into the endogenous T cell stimulation-related locus via homology-directed repair (HDR).

[0078] In some of the embodiments, gene disruption is carried out by introducing one or more activators into a T cell that can induce gene disruption at a target site within an endogenous T cell stimulation-associated locus on the T cell. In some of the embodiments, the method involves creating a modified T cell stimulation-associated locus, which contains a transgene encoding a recombinant receptor or a portion thereof.

[0079] In some of the embodiments, the polynucleotide further comprises one or more homology arms ligated to a nucleic acid sequence, the one or more homology arms comprising a sequence homologous to one or more regions of an endogenous T cell stimulation-associated gene locus in a T cell. In some of the embodiments, the one or more homology arms comprises a 5' homology arm and / or a 3' homology arm. In some of the embodiments, the 5' homology arm and the 3' homology arm comprise a nucleic acid sequence homologous to a nucleic acid sequence surrounding a target site, the target site being located within a T cell stimulation-associated gene locus.

[0080] In some of the embodiments, the target site is located downstream of an endogenous transcriptional regulatory element of a T cell stimulation-related gene locus.

[0081] In some of the embodiments, the polynucleotide comprises the structure [5' homology arm]-[transgene]-[3' homology arm]. In some of the embodiments, the 5' homology arm and the 3' homology arm contain nucleic acid sequences homologous to the nucleic acid sequence surrounding at least one target site. In some of the embodiments, the 5' homology arm and the 3' homology arm are independently 50 or about 50 to 750 or about 750 nucleotides, 50 or about 50 to 500 or about 500 nucleotides, 50 or about 50 to 250 or about 250 nucleotides, 50 or about 50 to 100 or about 100 nucleotides, 100 or about 100 to 750 or about 750 nucleotides, 100 or about 100 to 500 or about 500 nucleotides, 100 or about 100 to 250 or about 250 nucleotides, 250 or about 250 to 750 or about 750 nucleotides, 250 or about 250 to 500 or about 500 nucleotides in length. In some of the embodiments, the 5' homology arm and the 3' homology arm are independently 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, or 500 nucleotides in length or about 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, or 500 nucleotides, or any value between any of the above. In some of the embodiments, the 5' homology arm and the 3' homology arm are independently 100 or about 100 nucleotides in length. In some of the embodiments, the 5' homology arm and the 3' homology arm are independently 50, 60, 70, 80, or 90 nucleotides in length or about 50, 60, 70, 80, or 90 nucleotides, or any value between any of the above.

[0082] In some of the embodiments, the T cell stimulation-related locus is selected from among the PDCD1, CD69, Nur77, FoxP3, and HLA-DR loci.

[0083] In some of the embodiments, the T cell stimulation-associated locus is PDCD1. In some of the embodiments, the 5' homology arm and the 3' homology arm contain sequences homologous to one or more regions of PDCD1. In some of the embodiments, the 5' homology arm is a) a sequence containing 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 consecutive nucleotides to a sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the sequence shown in SEQ ID NO: 66; b) SEQ ID A sequence containing 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 consecutive nucleotides of the sequence shown in NO:66, or at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 consecutive nucleotides; or c) a sequence showing SEQ ID NO:66.In some of the embodiments, the 3' homology arm is a) a sequence containing 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 consecutive nucleotides to a sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the sequence shown in SEQ ID NO: 67; b) SEQ ID A sequence containing 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 consecutive nucleotides of the sequence shown in NO:67, or at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 consecutive nucleotides; or c) a sequence showing SEQ ID NO:67.

[0084] In some of the embodiments, the T cell stimulation-associated locus is CD69. In some of the embodiments, the 5' homology arm and the 3' homology arm contain sequences homologous to one or more regions of CD69.

[0085] In some of the embodiments, the T cell stimulation-associated locus is Nur77. In some of the embodiments, the 5' homology arm and the 3' homology arm contain sequences homologous to one or more regions of Nur77.

[0086] In some of the embodiments, the T cell stimulation-associated locus is FoxP3. In some of the embodiments, the 5' homology arm and the 3' homology arm contain sequences homologous to one or more regions of FoxP3.

[0087] In some of the embodiments, the T cell stimulation-associated locus is the HLA-DR locus. In some of the embodiments, the 5' homology arm and the 3' homology arm contain sequences homologous to one or more regions of the HLA-DR locus.

[0088] In some of the embodiments, the recombinant receptor or a portion thereof can induce or transmit a stimulating or activating signal in T cells.

[0089] In some of the embodiments, the recombinant receptor comprises an intracellular region containing an intracellular signaling domain of a component of a T cell receptor (TCR) complex, and the stimulating or activating signal in the T cell comprises a signal through the intracellular signaling domain present in the recombinant receptor, or the recombinant receptor comprises an intracellular region containing an intracellular signaling domain containing an immunoreceptor-activated tyrosine motif (ITAM), and the stimulating or activating signal in the T cell comprises a signal through the intracellular signaling domain present in the recombinant receptor.

[0090] In some of the embodiments, the recombinant receptor includes an extracellular domain comprising a binding domain capable of binding to or recognizing an activator. In some of the embodiments, a stimulus or activation signal is induced in the T cell upon binding of the activator.

[0091] In some of the embodiments, the active agent is a target antigen. In some of the embodiments, the target antigen is a recombinant protein or an antigen expressed on the surface of a cell. In some of the embodiments, the target antigen is associated with, specific to, or expressed on cells or tissues of a disease, disorder, or condition. In some of the embodiments, the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer.

[0092] In some of the embodiments, the target antigen is a tumor antigen, a pathogen-specific antigen or pathogen-expressing antigen, an inflammatory antigen, or an autoantigen. In some of the embodiments, the target antigen is a tumor antigen. In some of the embodiments, the target antigens are αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9; also known as CAIX or G250), cancer testis antigen, cancer / testis antigen 1B (CTAG; also known as NY-ESO-11 and LAGE-12), carcinoembryonic antigen (CEA), cyclin, cyclin A2, CC motif chemokine ligand 1 (CCL-11), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), and type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-12), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor-like 5 (FCRL5;Also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), folate-binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G protein-coupled receptor class C group 5 member D (GPRC5D), Her2 / neu (receptor tyrosine kinase erb-B2), H er3 (erb-B3), Her4 (erb-B4), erbB dimer, human high molecular weight melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-122 receptor alpha (IL-122Rα), IL-113 receptor alpha 2 (IL-113Rα2), kinase insert domain receptor (kdr), κ light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitome of L1-CAM This product contains leucine-rich repeat-containing 8 family member A (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, mouse cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligand, melan A (MART-11), neuronal adhesion molecule (NCAM), tumor embryo antigen, and melanoma-preferential expression. Antigen (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivorvin, trophoblast glycoprotein (TPBG; also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-related protein 1 (TRP1; also known as TYRP1 or gp75), tyrosinase-related protein 2 (TRP2;Selected from among dopachrome tautomerase (also known as dopachrome delta-isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor 1 (WT-11), pathogen-specific antigens or pathogen-expressed antigens, or antigens associated with a universal tag, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV, or other pathogens.

[0093] In some embodiments, gene disruption is induced by a combination of zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), or CRISPR-Cas9 that specifically bind to, recognize, or hybridize with a target site. In some embodiments, gene disruption is induced by a CRISPR-Cas9 combination, which includes a guide RNA (gRNA) having a targeting domain complementary to at least one target site. In some embodiments, the CRISPR-Cas9 combination is a ribonucleoprotein (RNP) complex containing the gRNA and the Cas9 protein. In some embodiments, gene disruption is induced by an RNP introduced into multiple T cells via electroporation.

[0094] In some of the embodiments, the T cell stimulation-associated locus is PDCD1. In some of the embodiments, gene disruption is brought about by a CRISPR-Cas9 combination containing a gRNA, and the gRNA has a targeting domain complementary to the target site in the PDCD1 gene. In some of the embodiments, the gRNA contains the sequence shown in SEQ ID NO: 75 and one of 104-109. In some of the embodiments, the gRNA contains the sequence shown in SEQ ID NO: 75.

[0095] In some of the embodiments, the T cell stimulation-associated locus is CD69. In some of the embodiments, gene disruption is induced by a CRISPR-Cas9 combination containing a gRNA, and the gRNA has a targeting domain complementary to the target site in the CD69 gene. In some of the embodiments, the gRNA contains a sequence represented by one of SEQ ID NO: 116-121.

[0096] In some of the embodiments, the T cell stimulation-associated locus is Nur77. In some of the embodiments, gene disruption is induced by a CRISPR-Cas9 combination containing a gRNA, and the gRNA has a targeting domain complementary to the target site in the Nur77 gene. In some of the embodiments, the gRNA contains sequences shown in SEQ ID NO: 122-127 and 134-136.

[0097] In some of these embodiments, the T cell stimulation-related locus is FoxP3.

[0098] In some of these embodiments, the T cell stimulation-related locus is the HLA-DR locus.

[0099] In some embodiments, the T cell further involves gene disruption in the endogenous T cell receptor α constant region (TRAC) gene and / or the endogenous T cell receptor β constant region (TRBC) gene. In some embodiments, the gene disruption is brought about by a combination of zinc finger nuclease (ZFN), TAL effector nuclease (TALEN), or CRISPR-Cas9 that specifically binds to, recognizes, or hybridizes to at least one target site within the TRAC, TRBC1, and / or TRBC2 genes. In some embodiments, the gene disruption is brought about by a combination of CRISPR-Cas9, and the CRISPR-Cas9 combination includes a guide RNA (gRNA) having a targeting domain complementary to at least one target site. In some embodiments, the CRISPR-Cas9 combination is a ribonucleoprotein (RNP) complex containing the gRNA and the Cas9 protein. In some of the embodiments, gene disruption is induced by an RNP introduced into multiple T cells via electroporation. In some of the embodiments, gene disruption is induced by a CRISPR-Cas9 combination containing a gRNA, the gRNA having a targeting domain complementary to at least one target site in the TRAC, TRBC1, and / or TRBC2 genes.

[0100] In some of the embodiments, the gRNA has a targeting domain complementary to the target site in the TRAC gene. In some of the embodiments, the gRNA contains the sequence shown in SEQ ID NO: 77 and one of 188-218. In some of the embodiments, the gRNA contains the sequence shown in SEQ ID NO: 77. In some of the embodiments, the gRNA has a targeting domain complementary to the target site in the TRBC gene. In some of the embodiments, the gRNA contains the sequence shown in SEQ ID NO: 219-276.

[0101] In some of the embodiments, the recombinant receptor is a chimeric antigen receptor (CAR). In some of the embodiments, the encoded recombinant receptor is or includes a recombinant T cell receptor (TCR).

[0102] In some of the embodiments, RNP is introduced via electroporation, particle gun, calcium phosphate transfection, or cell compression or compression. In some of the embodiments, RNP is introduced via electroporation. In some of the embodiments, RNP is introduced into multiple T cells via electroporation. In some of the embodiments, the concentration of RNP is from 1 μM or about 1 μM to 5 μM or about 5 μM. In some of the embodiments, the concentration of RNP is 2 μM or about 2 μM.

[0103] In some of the embodiments, T cells include CD8+ T cells and / or CD4+ T cells or their subtypes. In some of the embodiments, T cells are self to the subject. In some of the embodiments, T cells are primary T cells derived from the subject. In some of the embodiments, the subject is human. In some of the embodiments, T cells are allogeneic to the subject. In some of the embodiments, T cells are derived from pluripotent or multipotent cells. In some of the embodiments, T cells are derived from iPSCs.

[0104] In some of the embodiments, the polynucleotide is a linear polynucleotide. In some of the embodiments, the polynucleotide is a double-stranded polynucleotide. In some of the embodiments, the polynucleotide is a single-stranded polynucleotide. In some of the embodiments, the polynucleotide is contained in a viral vector. In some of the embodiments, the viral vector is an AAV vector. In some of the embodiments, the viral vector is a retroviral vector. In some of the embodiments, the viral vector is a lentiviral vector.

[0105] In some of the embodiments, the polynucleotide is 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,250, 3,500, 3,750, or 4,000 nucleotides in length or about 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,250, 3,500, 3,750, or 4,000 nucleotides in length, or any value between any of the above. In some of the embodiments, the polynucleotide is 1,500 or about 1,500 to 2,500 or about 2,500 nucleotides in length, or 1,750 or about 1,750 to 2,250 or about 2,250 nucleotides in length.

[0106] In some of the embodiments, one or more active substances and polynucleotides are introduced simultaneously or sequentially in any order. In some of the embodiments, one or more active substances and polynucleotides are introduced simultaneously. In some of the embodiments, the polynucleotide is introduced after the introduction of one or more active substances. In some of the embodiments, the polynucleotide is introduced immediately after the introduction of the active substances, or within approximately 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 6 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, or 4 hours after such introduction.

[0107] In some of the embodiments, prior to the introduction of one or more active agents and / or polynucleotides, the method includes the step of incubating cells in vitro with an stimulant under conditions for stimulating or activating one or more immune cells. In some of the embodiments, the stimulant comprises an anti-CD3 antibody and / or an anti-CD28 antibody. In some of the embodiments, the stimulant comprises an oligomeric particle reagent comprising an anti-CD3 antibody and / or an anti-CD28 antibody. In some of the embodiments, the stimulant comprises beads coated with an anti-CD3 antibody and / or an anti-CD28 antibody.

[0108] In some of the embodiments, the method further includes the step of incubating cells with one or more recombinant cytokines before, during, or after the introduction of one or more active agents and / or polynucleotides. In some of the embodiments, the one or more recombinant cytokines are selected from the group consisting of IL-2, IL-7, and IL-15. In some of the embodiments, the one or more recombinant cytokines are added at concentrations selected from IL-2 concentrations of 10 U / mL or about 10 U / mL to 200 U / mL or about 200 U / mL. In some of the embodiments, the one or more recombinant cytokines are added at concentrations of 50 IU / mL or about 50 IU / mL to 100 U / mL or about 100 U / mL; IL-7 is added at concentrations of 0.5 ng / mL to 50 ng / mL. In some of the embodiments, one or more recombinant cytokines are added at concentrations ranging from 5 ng / mL or about 5 ng / mL to 10 ng / mL or about 10 ng / mL, and / or IL-15 is added at concentrations ranging from 0.1 ng / mL to 20 ng / mL. In some of the embodiments, one or more recombinant cytokines are added at concentrations ranging from 0.5 ng / mL or about 0.5 ng / mL to 5 ng / mL or about 5 ng / mL.

[0109] In some of the embodiments, incubation is carried out for up to 24 hours, 36 hours, 48 ​​hours, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days, or approximately 24 hours, 36 hours, 48 ​​hours, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days, after the introduction of one or more active substances and polynucleotides. In some of the embodiments, incubation is carried out for up to 7 days or approximately 7 days.

[0110] Also provided are engineered T cells or multiple engineered T cells generated using one of the provided methods.

[0111] Also provided are compositions comprising one or more of the provided manipulated cells.

[0112] Furthermore, compositions containing any of the multiple provided manipulated cells are also provided.

[0113] In some of the embodiments, the expression of a functionally linked transgene is upregulated or induced in one or more cells in the composition within 6, 12, 18, 24, 36, or 48 hours, or approximately within 6, 12, 18, 24, 36, or 48 hours, following a stimulation or activation signal in a T cell. In some of the embodiments, the frequency of cells expressing a functionally linked transgene among the cells in the composition, following a simulation or activation signal in a T cell, is higher than 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or approximately 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or higher. In some of the embodiments, the expression of a functionally linked transgene is reduced or downregulated in one or more cells in the composition, following upregulation or induction of expression, or following reduction or absence of simulation or activation signals in T cells.

[0114] In some of the embodiments, following upregulation or induction of expression, or following reduction or absence of simulation or activation signals in T cells, the frequency of cells expressing a functionally linked transgene among the cells in the composition is reduced by more than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or by more than approximately 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or by more than approximately 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or by more. In some of the embodiments, following upregulation or induction of expression, the expression of a functionally linked transgene is reduced or downregulated in one or more cells in the composition 1, 2, 3, 4, 5, 6, 7, or 8 days or more after stimulation or activation signals in T cells.

[0115] In some of the embodiments, the expression of a functionally linked transgene in one or more cells in the composition is reduced or downregulated within less than 6, 12, 18, 24, 36, or 48 hours, or within approximately 6, 12, 18, 24, 36, or 48 hours, following the reduction or absence of simulation or activation signals in T cells. In some of the embodiments, following the reduction or absence of simulation or activation signals in T cells, the frequency of cells expressing recombinant receptors among the cells in the composition is lower than 50%, 40%, 30%, 25%, 20%, 15%, 10%, or 5%, or less, or approximately 50%, 40%, 30%, 25%, 20%, 15%, 10%, or 5%, or less.

[0116] In some of the embodiments, the composition comprises CD4+ T cells and / or CD8+ T cells. In some of the embodiments, the composition comprises CD4+ T cells and CD8+ T cells, with a CD4+ T cell to CD8+ T cell ratio of 1:3 to 3:1 or approximately 1:3 to 3:1. In some of the embodiments, the ratio is 1:1.

[0117] Also provided are methods of treatment, which include administering one of the provided manipulated cells or one of the provided manipulated compositions to a subject having a certain disease or disorder.

[0118] The use of any of the provided manipulated cells or any of the provided manipulated compositions for the treatment of a certain disease or disorder is also provided.

[0119] The use of any of the provided engineered cells or any of the provided engineered compositions in the manufacture of a drug for treating a certain disease or disorder is also provided.

[0120] Also provided are any of the provided engineered cells or any of the provided engineered compositions for use in the treatment of a certain disease or disorder.

[0121] In some of the embodiments, the disease or disorder is cancer or a tumor. In some of the embodiments, the cancer or tumor is a hematological malignancy. In some of the embodiments, the cancer or tumor is a lymphoma, leukemia, or plasma cell malignancy. In some of the embodiments, cancer is lymphoma, and lymphoma is Burkitt lymphoma, non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, Waldenström macroglobulinemia, follicular lymphoma, small non-incisional nuclear cell lymphoma, mucosa-associated lymphoid tissue lymphoma (MALT), marginal zone lymphoma, splenic lymphoma, nodular monocytic B-cell lymphoma, immunoblastic lymphoma, large cell lymphoma, diffuse mixed-cell lymphoma, pulmonary B-cell angiocentral lymphoma, small lymphocytic lymphoma, primary mediastinal B-cell lymphoma, lymphoplasmacytic lymphoma (LPL), or mantle cell lymphoma (MCL). In some of the embodiments, cancer is leukemia, and leukemia is chronic lymphocytic leukemia (CLL), plasma cell leukemia, or acute lymphocytic leukemia (ALL). In some of these embodiments, cancer is a plasma cell malignancy, and plasma cell malignancy is multiple myeloma (MM).

[0122] In some of the embodiments, the tumor is a solid tumor. In some of the embodiments, the solid tumor is non-small cell lung cancer (NSCLC) or head and neck squamous cell carcinoma (HNSCC).

[0123] Also provided are kits comprising one or more active agents capable of inducing gene disruption at target sites within T cell stimulation-related gene loci; and one of the provided polynucleotides.

[0124] Also provided are a kit comprising: one or more activators capable of inducing gene disruption at a target site within a T cell stimulation-related locus; a polynucleotide comprising a nucleic acid sequence encoding a recombinant receptor or a portion thereof, wherein a transgene encoding the recombinant receptor or its antigen-binding fragment or chain is targeted for integration at or near the target site via homology-directed repair (HDR); and instructions for carrying out any of the provided methods. [Brief explanation of the drawing]

[0125] [Figure 1] A schematic diagram illustrating engineered T cells that conditionally express a recombinant receptor is shown. In some cases, recombinant receptor expression is under manipulable regulation of T cell stimulation-associated loci. In exemplary engineered T cells provided herein, which include a modified T cell stimulation-associated locus containing a transgene encoding a recombinant receptor or a portion thereof functionally linked to an endogenous transcriptional regulatory element of the T cell stimulation-associated locus, the encoded recombinant receptor may be expressed following a stimulation or activation signal in the T cell. In some cases, endogenous TCR expression is reduced or repressed by introducing gene disruption to loci encoding components of the endogenous TCR, e.g., the endogenous T cell receptor α constant region (TRAC) gene and / or the endogenous T cell receptor β constant region (TRBC) gene. In some cases, if signaling through the recombinant receptor is reduced or eliminated by the elimination of target cells expressing a target antigen, recombinant receptor expression is reduced or eliminated. [Figure 2] The percentage of cells expressing various markers, including gene products from various T cell stimulation-related loci, is shown over time after stimulation with a reagent containing anti-CD3 and anti-CD28 antibodies on day 0. Marker expression was evaluated by flow cytometry. Around day 7 after the initial stimulation, the cells were re-stimulated with the same reagent, and the percentage of cells expressing markers was evaluated over time. [Figure 3A]Figures 3A-3B illustrate flow cytometry plots for the expression of CD3, PD-1, and chimeric antigen receptors (CARs) in cells introduced with a ribonucleoprotein (RNP) complex containing PDCD1 targeting gRNA (PD-1 KO), an RNP complex containing TRAC targeting gRNA (TRAC KO), a polynucleotide containing a sequence encoding a CAR for HDR targeting at the PDCD1 locus (PD-1 KI CAR), a polynucleotide containing a sequence encoding a CAR for HDR targeting at the TRAC locus (TRAC KI CAR), or a combination thereof. [Figure 3B] See the explanation in Figure 3A. [Figure 4A] Figures 4A–4C show flow cytometry plots of CD3, PD-1, CD69, and chimeric antigen receptor (CAR) expression after the rest period following initial stimulation, or after restimulation following initial stimulation and rest. These plots contain a CAR-coding sequence under manipulable regulation at the endogenous PDCD1 locus (PD-1 KI CAR) or a CAR-coding sequence under manipulable regulation at the TRAC transcriptional regulatory element (TRAC KI CAR). [Figure 4B] See the explanation in Figure 4A. [Figure 4C] See the explanation in Figure 4A. [Figure 5] Figures 5A-5B illustrate the percentage of CAR+ cells over time, with or without restimulation, after the rest period following initial stimulation, or after restimulation following initial stimulation and rest period, in engineered T cells containing a CAR-coding sequence under manipulable regulation at the endogenous PDCD1 locus (PD-1 KI CAR) or a CAR-coding sequence under manipulable regulation at the TRAC transcriptional regulatory element (TRAC KI CAR). [Figure 6]Figure 6A illustrates flow cytometry plots for the expression of exemplary anti-CD19 CAR (detected using an anti-idiotype antibody) (PD1 KI CAR) and CD8 under PDCD1 promoter regulation in cells expressing exemplary anti-CD19 CAR under manipulable regulation at the endogenous PDCD1 locus (PD1 KI CAR) after a first round of stimulation by co-culturing with irradiated CD19-expressing lymphoblastoid cell lines (LCLs) for 7 days, 7 days after electroporation, and after a second round of stimulation by co-culturing with irradiated CD19-expressing LCLs. Figure 6B illustrates the cell growth ratios after the first and second rounds of cell growth in PD1KI CAR cells, PDCD1 KO cells (electroporation with only the PDCD1-targeting RNP complex and without the polynucleotide encoding the exemplary CAR; PD1 KO), mock-treated cells (negative control), and / or cells expressing the same exemplary CAR manipulated with a lentiviral vector (LV control). [Figure 7-1] Figure 7A illustrates flow cytometry plots of exemplary anti-CD19 CAR (detected using anti-idiotype antibody; aID) (PD1 K1 CAR) and CD8 expression under PDCD1 promoter regulation after the first round of stimulation (first stimulation) by co-culturing with irradiated CD19-expressing LCL in quiescent cells (quiescent) or cells subjected to restimulation (second stimulation). Figure 7B shows the mean fluorescence intensity (MFI) (detected using anti-idiotype antibody; left), percentage of CAR-expressing cells (center), and percentage of CD25+CD69+ cells (right) of CAR expression in PD1 KO cells, PD1 KI CAR cells quiescent without restimulation (PD1 KI CAR quiescent), and PD1 KI CAR cells subjected to restimulation (PD1 KI CAR restimulation). [Figure 7-2]Figure 7C shows the integrated mean fluorescence intensity (MFI) of CAR expression in PD1 KO cells, PD1 KI CAR cells that were quiescent without restimulation (PD1 KI CAR quiescent), and PD1 KI CAR cells that were restimulated (PD1 KI CAR restimulated). Figure 7D illustrates the cytolytic activity of PD1 KO cells, PD1 KI CAR cells that were quiescent without restimulation (PD1 KI CAR quiescent), and PD1 KI CAR cells that were restimulated (PD1 KI CAR restimulated) against target cells expressing CD19. Mock-treated cells (negative control), LV control, and human embryonic kidney (HEK) cells were used as controls. [Figure 8-1] Figure 8A illustrates a schematic time series for evaluating in vivo antitumor activity in mouse models injected with Raji lymphoma tumor cells transfected with firefly luciferase, cells expressing exemplary anti-CD19 CARs under manipulable regulation at the endogenous PDCD1 locus (PD-1 KI CARs), or cells delivered by lentiviral delivery (LV controls). Figure 8B illustrates tumor growth over time, as indicated by measuring mean radiance by bioluminescence. Figure 8C illustrates the survival of each mouse group over time. [Figure 8-2] Figure 8D illustrates the results of bioluminescence imaging in mice, showing the presence of tumors on days 1, 7, 14, and 28. [Modes for carrying out the invention]

[0126] Detailed explanation Engineered cells, such as engineered T cells, containing a modified locus, such as a modified T cell stimulation-related locus, are provided herein. In some aspects, the engineered cells contain a heterologous or exogenous nucleic acid sequence (e.g., a transgene) encoding a receptor (e.g., a chimeric antigen receptor or recombinant T cell receptor) or a portion thereof, functionally ligated to an endogenous transcriptional regulatory element of the T cell stimulation-related locus. In some aspects, the endogenous transcriptional regulatory element induces or upregulates the expression of the functionally ligated nucleic acid sequence following a simulation or activation signal in the T cell.

[0127] In some aspects, expression from transcriptional regulatory elements of T cell stimulation-associated loci is responsive to signals through intracellular signaling domains of recombinant receptors, such as primary activation signals in T cells, signaling domains of T cell receptor (TCR) components, and / or signaling domains containing immunoreceptor-activated tyrosine motifs (ITAMs). In some embodiments, exemplary T cell stimulation-associated loci include, but are not limited to, PDCD1 (encoding PD-1), CD69, Nur77 (encoding NR4A1), FoxP3, or HLA-DR loci.

[0128] Also provided are methods for producing genetically engineered cells containing a modified T cell stimulation-related locus expressing a recombinant receptor or a portion thereof. The provided embodiments specifically involve targeting an endogenous T cell stimulation-related locus with a nucleic acid sequence encoding the recombinant receptor or a portion thereof. In some circumstances, the provided embodiments involve inducing target gene disruption, e.g., the generation of a DNA break, using gene editing techniques and homology-directed repair (HDR) for targeted incorporation of the nucleic acid sequence encoding the recombinant receptor at the endogenous T cell stimulation-related locus. Also provided are related cell compositions, nucleic acids, and kits for the production of the engineered cells provided herein and / or for use in the methods provided herein.

[0129] T cell-based therapies, such as adoptive T cell therapy (including those involving the administration of recombinant receptors, engineered receptors, or chimeric receptors specific to the disease or disorder of interest, such as chimeric antigen receptors (CARs), recombinant T cell receptors (TCRs), or engineered cells expressing other recombinant receptors, engineered receptors, or chimeric receptors), may be effective in treating cancer, as well as other diseases and disorders. In certain circumstances, other approaches to designing and generating engineered cells for adoptive cell therapy may not always be completely satisfactory. In some aspects, engineered cells containing recombinant receptors may, in some cases, target healthy cells expressing antigens recognized by the recombinant receptors. In some aspects, recombinant receptor-expressing cells may not be able to distinguish between disease cells, such as tumor cells, and normal cells expressing antigens. In some aspects, after engineered cells have targeted and eliminated disease cells, such as tumor cells expressing the target antigen, the continued persistence of recombinant receptor-expressing cells may target healthy cells expressing the antigen, resulting in undesirable effects. For example, in some cases, the continued persistence of anti-CD19 CAR-expressing cells after tumor clearance may result in B cell dysplasia due to attack by CAR-expressing cells on healthy CD19-expressing B cells.

[0130] In some aspects, the provided embodiments involve inducing target gene disruption and incorporation of a transgene encoding a recombinant receptor or a portion thereof by HDR at an endogenous T cell stimulation-associated locus. In some aspects, the transgene encoding a recombinant receptor or a portion thereof is functionally ligated to an endogenous transcriptional regulatory element of the T cell stimulation-associated locus. For example, the expression of a functionally ligated transgene encoding a recombinant receptor or a portion thereof is regulated by an endogenous transcriptional regulatory element of the T cell stimulation-associated locus, e.g., a promoter. In some aspects, the endogenous transcriptional regulatory element induces or upregulates the expression of a functionally ligated transgene encoding a recombinant receptor, e.g., following a simulation or activation signal in a T cell. In some aspects, the endogenous transcriptional regulatory element reduces or downregulates the expression of a functionally ligated transgene encoding a recombinant receptor, e.g., following a reduction or absence of a simulation or activation signal in a T cell. Thus, the expression of a recombinant receptor can be regulated based on the presence of a stimulation or activation-associated signal in a T cell.

[0131] In some aspects, the provided embodiments are based on the observation that T cells containing recombinant receptors, such as CAR, expressed under the regulation of T cell stimulation-associated loci, e.g., PDCD1, are transiently induced or upregulated in response to stimulation or activation signals. Recombinant receptors were observed to be expressed in response to restorative signals after a period of restorative activity. Expression of exemplary T cell stimulation-associated gene products was induced in response to the initial stimulation signal, e.g., via anti-CD3 and anti-CD28 antibodies, and this was observed to be reduced for a period after the initial stimulation. Following restorative stimulation after a certain period, expression of exemplary T cell stimulation-associated gene products increased after the reduction. The provided embodiments are also based on the observation that, following restorative stimulation, recombinant receptors, such as CAR, expressed under the regulation of T cell stimulation-associated loci, e.g., PDCD1, are again induced or again upregulated, and that T cells expressing recombinant receptors can efficiently kill target cells.

[0132] In some aspects, gene expression under manipulable regulation of transcriptional regulatory elements at T cell stimulation-associated loci is responsive to T cell stimulation or activation signals. Such responsiveness allows for the control of recombinant receptor expression, which helps minimize undesirable effects on healthy cells expressing target antigens or undesirable functions of recombinant receptor-expressing cells, without further drawbacks to the function of recombinant receptor-expressing cells.

[0133] In some cases, the fully functional or unmodified endogenous gene product of a T cell stimulation-related locus is expressed in the cell in such a way that the endogenous gene product and its recombinant receptor are co-expressed. In other cases, the endogenous gene product of a T cell stimulation-related locus is not expressed or is knocked out.

[0134] In some aspects, the engineered cells further involve genetic disruption of genes encoding the endogenous T cell receptor in the cell. For example, the endogenous T cell receptor α constant region (TRAC) gene and / or the endogenous T cell receptor β constant region (TRBC) gene are disrupted in T cells. In some aspects, such disruption also inhibits antigen-independent, tonic signaling within recombinant receptor-expressing cells and minimizes unregulated expression of the endogenous T cell receptor in T cells. In some embodiments, antigen-independent signaling of the encoded recombinant receptor is reduced by 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more compared to engineered cells containing randomly incorporated transgenes encoding the same recombinant receptor, or by approximately 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more.

[0135] In some aspects, the provided embodiment offers the advantage that disruption of the gene encoding the endogenous T cell receptor in a cell, along with the expression of the recombinant receptor under the regulation of transcriptional regulatory elements of T cell stimulation-related loci, generates a feedback loop for recombinant receptor expression. In such cases, T cell stimulation or activation is regulated by or dependent on a stimulating signal transmitted or transmitted through the signaling region of the recombinant receptor, for example, by binding or recognition of a target antigen by the recombinant receptor, and the stimulating or activating signal is reduced or eliminated in the absence of the antigen-specific signal. In some situations, the recombinant receptor and the cells expressing the recombinant receptor retain their cytotoxic function against target cells until the target cells are eliminated or the target cells are no longer available. In some aspects, following the removal of target cells, e.g., diseased cells expressing a target antigen, the stimulating or activating signal in recombinant receptor-expressing cells is reduced, and the cells become less responsive or unresponsive, which allows a healthy normal cell population to remain unaffected by recombinant receptor-expressing cells. In some cases, the absence of endogenous T cell receptor expression prevents the undesirable reactivation of recombinant receptor-expressing cells. In some embodiments, the cells, compositions, and methods provided may result in improved cell therapies, particularly for cell therapies that target or are specific to antigens in the tumor microenvironment. In some situations, the cells, compositions, and methods provided also offer advantages in regulating and controlling the expression of recombinant receptors on cells in cell therapy, such as CARs. In some aspects, the manipulated cells and methods provided allow for sophisticated temporal control of recombinant receptor-expressing cells and minimize undesirable antigen-independent effects of recombinant receptor-expressing cells. In some cases, the embodiments provided allow for the recovery of healthy cells after administration of recombinant receptor-expressing cells.

[0136] In some circumstances, recombinant receptors encoded from modified T cell stimulation-associated loci in the manipulated cells provided herein may be encoded under the regulation of cis-regulatory elements, such as endogenous regulatory elements of the T cell stimulation-associated loci, e.g., the promoter of the endogenous T cell stimulation-associated loci, or the 5' and / or 3' untranslated region (UTR). In some aspects, such embodiments allow recombinant receptors, e.g., CARs, or a portion thereof, to be expressed, and / or their expression to be regulated in a similar manner to that of the endogenous T cell stimulation-associated loci, in terms of time, duration, level, and similar expression kinetics.

[0137] In some cases, the entire recombinant receptor or the full-length recombinant receptor is encoded from a modified T cell stimulation-related locus in the engineered cell. In some cases, a portion of the recombinant receptor, such as a domain or region of the recombinant receptor, or one or more chains of a recombinant receptor containing multiple chains (e.g., a multi-chain CAR or a recombinant T cell receptor (TCR) containing two or more chains), is encoded from a modified T cell stimulation-related locus in the engineered cell. In some cases, the remaining portion (e.g., another chain or another domain of the recombinant receptor) is encoded by a second transgene present in the engineered cell.

[0138] In some situations, the optimal potency of manipulated cells may depend on the ability of the administered cells to express recombinant receptors, including the homogeneous, consistent, and / or controlled expression of receptors among cells such as immune cells and / or populations of cells in a therapeutic cell composition, as well as the ability of the recombinant receptors to recognize and bind targets, e.g., target antigens, within the subject, tumor, and its environment. In some cases, methods available for introducing recombinant receptors, such as CARs, into cells involve the random insertion of sequences encoding recombinant receptors. In certain respects, such methods are not entirely satisfactory. In some aspects, random insertion may result in possible insertional mutagenesis and / or disruption of another gene locus in the cell, including those that may be important to the function and activity of the cell. In some cases, semi-random or random insertion of a transgene encoding a receptor into the cell's genome may result in harmful and / or unwanted effects, in some cases, due to the insertion of nucleic acid sequences into undesirable locations in the genome, e.g., essential genes or genes important in the regulation of cell activity.

[0139] In some cases, random incorporation may result in fluctuating incorporation of the recombinant receptor-coding sequence, which may result in inconsistent or uncontrolled expression, fluctuating nucleic acid copy number, and / or variability in receptor expression within cells of cell compositions such as therapeutic cell compositions. In some cases, random incorporation of the receptor-coding nucleic acid sequence may result in heteromorphic, heterogeneous, non-uniform, uncontrolled, and / or suboptimal expression or antigen binding, oncogenic transformation, and transcriptional silencing of the nucleic acid sequence, depending on the site of incorporation and / or the copy number of the nucleic acid sequence. In some aspects, heterogeneous and non-uniform expression in a cell population may result in mismatch or instability in expression and / or antigen binding by the recombinant receptor, unpredictability or reduced function of the engineered cells, and / or heterogeneous drug products, thereby reducing the potency of the engineered cells. In some aspects, the use of certain random incorporation vectors, such as certain lentiviral vectors, requires confirmation that the engineered cells do not contain replicable viruses. Improved strategies are needed to achieve consistent expression levels and function of recombinant receptors while minimizing random incorporation of nucleic acids and / or heterogeneous expression in populations.

[0140] In some aspects, the embodiments provided relate to manipulating cells to have nucleic acids encoding recombinant receptors that are incorporated into endogenous T cell stimulation-associated loci by homology-directed repair (HDR) in cells, for example, T cells. In some aspects, HDR can mediate site-directed integration of a transgene (e.g., a transgene encoding a recombinant receptor or a portion thereof) at or near a target site, such as an endogenous T cell stimulation-associated locus. In some embodiments, the presence of a template polynucleotide containing a gene disruption (e.g., at an endogenous T cell stimulation-associated locus) and one or more homologous arms (e.g., containing homologous sequences of nucleic acid sequences around the gene disruption) can induce or lead to HDR, where the homologous sequences act as templates for DNA repair. Based on the homology between the endogenous gene sequence surrounding the gene disruption and the homology arms contained in the template polynucleotide, the cell's DNA repair mechanism can use the template polynucleotide to repair the DNA break at the site of the gene disruption and resynthesize the genetic information, thereby effectively inserting or incorporating the sequence between the homology arms (e.g., a transgene encoding a recombinant receptor or a portion thereof) at or near the site of the gene disruption. The provided embodiment can generate cells containing a modified T cell stimulation-related locus encoding a recombinant receptor or a portion thereof, in which the transgene encoding a recombinant receptor or a portion thereof is incorporated into the endogenous T cell stimulation-related locus by HDR.

[0141] In some aspects, the embodiments provided may offer advantages in the creation of engineered cells with improved and / or more efficient targeting of nucleic acids encoding recombinant receptors into cells, and may result in improved activity and / or function of the engineered cells. In some cases, the embodiments provided may minimize possible semi-random or random integration and / or heterogeneous, uncontrolled, or atypiomorphic expression, and result in improved, homogeneous, homogeneous, consistent, controlled, and / or stable expression of recombinant receptors, or a reduced, low, or absent possibility of insertional mutagenesis.

[0142] In some aspects, compared to other methods for producing genetically engineered immune cells expressing recombinant receptors, e.g., TCRs or CARs, the embodiments provided enable more stable, physiological, regulatory, controlled, homogeneous, consistent, and / or homogeneous expression of recombinant receptors. In some cases, the method results in the production of more consistent and predictable drug products, e.g., cell compositions containing engineered cells, which may result in safer therapies for patients being treated. In some aspects, the embodiments provided also enable predictable and consistent incorporation at a single or multiple loci of interest. In some embodiments, the embodiments provided can also result in the production of cell populations with a consistent (typically 1 or 2) copy number of nucleic acids incorporated in the population of cells, which in some aspects provides consistency in recombinant receptor expression and endogenous receptor gene expression within the cell population. In some cases, the embodiments provided do not involve the use of viral vectors for incorporation, thus reducing the need to confirm that engineered cells do not contain replicable viruses, thereby improving the safety of the cell composition.

[0143] Also provided are methods for manipulating, preparing, and producing manipulated cells, as well as kits and devices for generating or producing manipulated cells. Cells and cell compositions produced by the methods are also provided. Furthermore, polynucleotides containing nucleic acid sequences encoding recombinant receptors or portions thereof, such as linear polynucleotides, and methods for introducing such polynucleotides into cells, for example, by transduction or physical delivery, such as electroporation, are also provided. Compositions containing manipulated cells, as well as methods, kits, and devices for administering cells and compositions to a target, for example, for adoptive cell therapy, are also provided. In some aspects, cells are isolated from one target, manipulated, and administered to the same target. In other aspects, cells are isolated from one target, manipulated, and administered to another target. In some embodiments, the polynucleotides, transgenes, and / or vectors provided result in the expression of recombinant receptors, such as TCRs or CARs, which, when delivered into immune cells, can modulate T cell activity and, in some cases, T cell differentiation or homeostasis. The resulting genetically modified cells or cell compositions can be used in adoptive cell therapy methods.

[0144] All publications, including patent documents, scientific articles, and databases, referenced in this application are incorporated by reference in whole for all purposes to the same extent as each individual publication is incorporated by reference individually. If any definition provided herein conflicts with or is otherwise inconsistent with any definition provided herein in any patent, application, published application, or other publication incorporated by reference, the definition provided herein shall prevail over the definition incorporated by reference.

[0145] The section headings used herein are for organizational purposes only and should not be construed as limiting the subjects described.

[0146] I. Cells that conditionally express recombinant receptors Engineered immune cells, such as engineered T cells, are provided herein, which contain a transgene encoding a recombinant receptor or a portion thereof, located at a genomic locus such as a T cell stimulation-associated locus. In some embodiments, the engineered cells contain a modified T cell stimulation-associated locus, such as PDCD1, CD69, Nur77, FoxP3, or HLA-DR locus, which contains a transgene (i.e., a heterologous or exogenous nucleic acid sequence) containing a nucleic acid sequence encoding a recombinant receptor (e.g., a chimeric antigen receptor or recombinant T cell receptor) or a portion thereof. In some embodiments, the nucleic acid sequence encoding a recombinant receptor or a portion thereof is under the manipulable regulation of an endogenous regulatory element of the T cell stimulation-associated locus. In some embodiments, the endogenous transcriptional regulatory element induces or upregulates, for example transiently, the expression of a functionally linked transgene encoding a recombinant receptor or a portion thereof, following a simulation or activation signal in the T cell. In some circumstances, in the provided cells containing a modified T cell stimulation-related locus, the expression of the encoded recombinant receptor or a portion thereof is similar to, or mimics, the expression of the endogenous gene product of the T cell stimulation-related locus in unmodified cells. In some aspects, the temporal control, level, and kinetics of the expression of the encoded recombinant receptor or a portion thereof are similar to those of the endogenous gene product of the T cell stimulation-related locus. For example, in some aspects, the encoded recombinant receptor is transiently induced or upregulated following a stimulation or activation signal in a T cell, and reduced or downregulated in the absence of such a signal. In some aspects, the provided embodiment allows for sophisticated and conditional control of the expression of the encoded recombinant receptor.

[0147] Provided herein are engineered T cells comprising a modified T cell stimulation-associated locus containing a transgene encoding a recombinant receptor or a portion thereof, functionally linked to an endogenous transcriptional regulatory element of the T cell stimulation-associated locus, wherein the endogenous transcriptional regulatory element induces or upregulates the expression of the functionally linked transgene following a simulation or activation signal in the T cell. In some embodiments, the endogenous transcriptional regulatory element is the promoter of the endogenous T cell stimulation-associated locus. In some embodiments, the transgene encoding a recombinant receptor or a portion thereof is located downstream of the promoter.

[0148] Also provided are engineered cells, such as engineered T cells, which include a nucleic acid sequence encoding a recombinant receptor or a portion thereof, functionally linked to an endogenous transcriptional regulatory element of a T cell stimulation-related locus. In some embodiments, the endogenous transcriptional regulatory element of the T cell stimulation-related locus induces or upregulates the expression of the functionally linked nucleic acid sequence in response to a stimulation or activation signal in the T cell, following the simulation or activation signal. In some aspects, the transcriptional regulatory element of the endogenous T cell stimulation-related locus is responsive to a stimulation or activation signal in the T cell.

[0149] Also provided are engineered T cells comprising nucleic acids encoding a recombinant receptor or a portion thereof, functionally linked to a transcriptional regulatory element of an endogenous T cell stimulation-related gene locus, wherein the endogenous transcriptional regulatory element of the T cell stimulation-related gene locus induces or upregulates the expression of a functionally linked nucleic acid sequence in the T cell in response to a stimulation or activation signal, following the simulation or activation signal. Also provided are engineered T cells comprising nucleic acids encoding a recombinant receptor or a portion thereof, functionally linked to a transcriptional regulatory element of an endogenous T cell stimulation-related gene locus, wherein the transcriptional regulatory element of the endogenous T cell stimulation-related gene locus is responsive to a stimulation or activation signal in the T cell.

[0150] In some embodiments, the expression of a functionally linked transgene is transient following a stimulating or activating signal in T cells. In some embodiments, expression is reduced or downregulated in the absence of further stimuli or activation, or following a reduction or absence of a simulation or activating signal in T cells, after upregulation or induction of expression. In some embodiments, the expression of a functionally linked transgene may be re-induced or upregulated following a reduction or absence of signal, and then following a further simulation or activating signal in T cells.

[0151] A. Loci and gene expression associated with stimulation or activation In some aspects, engineered cells are provided that conditionally express a recombinant receptor. In some embodiments, the engineered cells contain a nucleic acid sequence, such as a transgene or heterologous sequence, that encodes the recombinant receptor. In some aspects, the transgene encoding the recombinant receptor is conditionally expressed, for example, in the presence of specific conditions or signals in the engineered cell or environment. In some aspects, the recombinant receptor is not expressed or its expression is reduced in the absence of specific conditions or signals in the engineered cell or environment. In some aspects, the expression of the recombinant receptor is regulated by transcriptional regulatory elements of T cell stimulation-related loci, such as PDCD1 (encoding PD-1), CD69, Nur77 (encoding NR4A1), FoxP3, or the HLA-DR locus. In some aspects, the engineered cells contain a modified T cell stimulation-related locus containing a transgene encoding the recombinant receptor or a portion thereof. In some aspects, a transgene encoding a recombinant receptor or a portion thereof is functionally ligated to an endogenous transcriptional regulatory element of a T cell stimulation-related locus, which induces or upregulates the expression of the functionally ligated transgene following a simulation or activation signal in a T cell. In some aspects of any one aspect, the endogenous transcriptional regulatory element is the promoter of the endogenous T cell stimulation-related locus.

[0152] In some aspects, expression from T cell stimulation-related loci, for example, under the regulation of endogenous transcriptional regulatory elements of the T cell stimulation-related loci, is induced or upregulated following the presence of a stimulating or activating signal in engineered cells, e.g., engineered T cells. In some aspects, the stimulating or activating signal includes signaling through the signaling domain of a T cell receptor (TCR) component and / or signaling through a signaling domain containing an immunoreceptor-activated tyrosine motif (ITAM). For example, in some aspects, a signal from the ligation of an encoded recombinant receptor, e.g., a CAR or TCR, can provide an activating or stimulating signal that induces expression from T cell stimulation-related loci. In some aspects, a signal from the ligation of a receptor containing a signaling domain containing a motif such as an ITAM can provide an activating or stimulating signal that induces expression from T cell stimulation-related loci.

[0153] In some embodiments, the presence of a T cell stimulating or activating signal in engineered T cells can induce the expression of an encoded recombinant receptor, and may result in further induction, expression, or upregulation of the encoded recombinant receptor. In some embodiments, such conditional regulation of the expression of an encoded recombinant receptor may result in a positive feedback loop or feedforward loop, which can allow for increased or amplified expression of the recombinant receptor following a stimulating or activating signal in engineered T cells. In some embodiments, the binding of an activator to the extracellular binding domain of a recombinant receptor results in the induction or transmission of a stimulating or activating signal in the cell. In some embodiments, the stimulating or activating signal in T cells is transmitted through the intracellular signaling domain of the recombinant receptor upon binding of an activator to the extracellular binding domain of the recombinant receptor.

[0154] In some aspects, the expression of a functionally linked transgene, such as a transgene encoding a recombinant receptor or a portion thereof, functionally linked to an endogenous transcriptional regulatory element of a T cell stimulation-associated locus, is upregulated or induced within 6, 12, 18, 24, 36, or 48 hours, or approximately 6, 12, 18, 24, 36, or 48 hours, following a stimulation or activation signal in a T cell. In some aspects, the expression of a functionally linked transgene is upregulated or induced within 24 hours, or approximately 24 hours, following a stimulation or activation signal in a T cell. In some aspects, the expression of functionally linked transgenes is upregulated or induced by more than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or by approximately more than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or by more than 95%, or by more than 95%, or by more than 95%, or more. In some aspects, the upregulation or induction is compared to the expression of functionally linked transgenes before or in the absence of a stimulating or activating signal in T cells, or to the minimum expression of functionally linked transgenes before a stimulating or activating signal in T cells.

[0155] In some aspects, the induction or upregulation of expression is repeatedly regulated in the presence of a stimulating or activating signal in T cells. In some aspects, such regulated expression ensures that recombinant receptors, such as CARs, are expressed at high levels only in the environment in which T cells are activated, for example, at sites where the antigen recognized by the CAR is present. In some aspects, when the activating or stimulating signal terminates, for example, by the disappearance of the antigen, which may occur after cytolytic killing of a tumor, CAR expression is downregulated or reduced. In some aspects, the induction or upregulation of transgene expression is transient over the duration of the stimulating or activating signal, and is then reduced or downregulated.

[0156] In some embodiments, the expression of a functionally linked transgene is reduced or downregulated following a stimulating or activating signal in a T cell, e.g., an initial stimulating or activating signal in a T cell (e.g., via a recombinant receptor), and subsequent upregulation or induction of the expression of the functionally linked transgene (e.g., encoding a recombinant receptor or a portion thereof). In some embodiments, the expression of a functionally linked transgene is reduced or downregulated following upregulation or induction of expression, or following reduction or absence of a simulation or activating signal in a T cell.

[0157] In some embodiments, the expression of a functionally linked transgene is reduced or downregulated after a certain period of upregulation or induction of expression following a stimulating or activating signal in a T cell (e.g., the initial stimulating or activating signal). In some embodiments, the stimulating or activating signal in a T cell is the initial stimulating or activating signal in a T cell, and the expression of the functionally linked transgene is reduced or downregulated 1, 2, 3, 4, 5, 6, 7, or 8 days or more after the initial stimulating or activating signal in a T cell, or about 1, 2, 3, 4, 5, 6, 7, or 8 days or more after the initial stimulating or activating signal in a T cell. In some embodiments, the expression of a functionally linked transgene is reduced or downregulated following upregulation or induction of expression following a stimulating or activating signal in a T cell, 1, 2, 3, 4, 5, 6, 7, or 8 days or more after the initial stimulating or activating signal in a T cell, or about 1, 2, 3, 4, 5, 6, 7, or 8 days or more after the initial stimulating or activating signal in a T cell. In some embodiments, the expression of a functionally linked transgene is reduced or downregulated two, three, or four days or more after the initial stimulation or activation signal in T cells. In some embodiments, the expression of a functionally linked transgene is reduced or downregulated two days or approximately two days after the initial stimulation or activation signal in T cells.

[0158] In some embodiments, following a reduction or absence of simulation or activation signals in T cells, the expression of a functionally linked transgene encoding a recombinant receptor or a portion thereof is reduced or downregulated. In some embodiments, the expression of a functionally linked transgene is reduced or downregulated within less than 6, 12, 18, 24, 36, or 48 hours, or within approximately 6, 12, 18, 24, 36, or 48 hours, following a reduction or absence of simulation or activation signals in T cells. In some aspects, following a stimulus or activation signal in a T cell, or following a reduction or absence of a simulation or activation signal in a T cell, the expression of a functionally linked transgene encoding a recombinant receptor or a portion thereof is reduced by more than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or by approximately more than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or by more than approximately 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or more. In some aspects, the reduction is compared to the maximum expression of a functionally linked transgene following, for example, an initial stimulus or activation signal in a T cell.

[0159] In some aspects, such conditional control of the expression of encoded recombinant receptors can result in a feedback loop in which the expression of encoded recombinant receptors can be limited or blocked in the absence of a stimulating or activating signal, for example, in the absence of a target antigen or target cells. Such conditional control can reduce nonspecific activation of engineered cells or activation of engineered cells in the absence of a target antigen. In some aspects, following the removal of target cells, for example, malignant or tumor cells expressing a target antigen, engineered T cells may express less recombinant receptors and become less reactive, which allows the healthy cell population to recover and reduces nonspecific activation or stimulation of engineered cells.

[0160] In some embodiments, the expression of a functionally linked transgene may be re-induced or upregulated following further simulation or activation signals in T cells after signal reduction or absence. In some embodiments, the expression of a functionally linked transgene may be upregulated or induced within less than 6, 12, 18, 24, 36, or 48 hours, or within approximately 6, 12, 18, 24, 36, or 48 hours, following further simulation or activation signals in T cells after signal reduction or absence. In some embodiments, the expression of a functionally linked transgene may be upregulated or induced within less than 24 hours, or within approximately 24 hours, following further simulation or activation signals in T cells after signal reduction or absence. In some aspects, the expression of functionally linked transgenes is upregulated or induced by more than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or by approximately more than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or by more than 95%, or by more. In some aspects, upregulation or induction is compared to the expression of functionally linked transgenes before or in the absence of further stimulation or activation signals in T cells, or to the minimum expression of functionally linked transgenes before further stimulation or activation signals in T cells. In some embodiments, following further simulation or activation signaling in T cells, the mean level of recombinant receptor expression is increased by 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more, or approximately 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more.

[0161] In some embodiments, T cell stimulation-associated loci encode molecules that are transiently upregulated or induced on T cells. In some embodiments, exemplary T cell stimulation-associated loci include the PDCD1 (encoding PD-1), CD69, Nur77 (encoding NR4A1), FoxP3, or HLA-DR loci. In some aspects, a transgene (e.g., encoding a recombinant receptor or a portion thereof) is functionally linked to a transcriptional regulatory element, such as a promoter, at the endogenous PDCD1 (encoding PD-1), CD69, Nur77 (encoding NR4A1), FoxP3, or HLA-DR loci. In some embodiments, a transgene (e.g., encoding a recombinant receptor or a portion thereof) is incorporated into or near the endogenous PDCD1 (encoding PD-1), CD69, Nur77 (encoding NR4A1), FoxP3, or HLA-DR loci. In some cases, the transgene is functionally linked to and integrated downstream of the promoter of a T cell stimulation-related locus. In some cases, the transgene is functionally linked to and integrated downstream of the endogenous PDCD1 promoter. In some cases, the transgene is functionally linked to and integrated downstream of the endogenous CD69 promoter. In some cases, the transgene is functionally linked to and integrated downstream of the endogenous Nur77 promoter. In some cases, the transgene is functionally linked to and integrated downstream of the endogenous FoxP3 promoter. In some cases, the transgene is functionally linked to and integrated downstream of the endogenous HLA-DR promoter.

[0162] In some embodiments, for example, a transgene encoding a recombinant receptor or a portion thereof is associated with, under the manipulable regulation of, and / or controlled by a T cell stimulation-associated locus. In some embodiments, the recombinant receptor is encoded by a manipulable regulated nucleic acid sequence of an endogenous T cell stimulation-associated locus. In some aspects, for example, the expression of a transgene encoding a recombinant receptor or a portion thereof is controlled by regulatory elements that are responsive to the quality and / or intensity of signals through intracellular signaling regions, and / or to the binding and / or recognition of the recombinant receptor to a target antigen or epitope. In some embodiments, the “T cell stimulation-associated locus” is an endogenous locus that is responsive to signals transmitted through components of the T cell TCR complex, or through a recombinant receptor that includes an intracellular signaling region containing components or a portion thereof of the TCR complex, and / or to receptors present on or expressed on T cells, such as the T cell receptor (TCR) or antigen or epitope binding by the recombinant receptor. In some embodiments, the T cell stimulation-associated locus may be controlled by standard factors that are part of the normal downstream signaling pathway of T cells. In some embodiments, antigen or epitope binding, and / or signaling or activity through the intracellular signaling domain of a recombinant receptor, such as a CAR or TCR, induces signaling, for example, by inducing a T cell stimulation-related locus to express a transgene encoding a recombinant receptor. Detectable expression of endogenous gene products and / or transgenes can then be monitored as an indicator of T cell activation.

[0163] In some embodiments, a T cell stimulation-associated locus contains one or more regulatory elements, such as one or more transcriptional regulatory elements, whose expression depends on or is related to the activation of components of the TCR complex, thereby the regulatory domain or element being recognized by a transcription factor to drive the expression of such gene. In some embodiments, a T cell stimulation-associated locus contains a promoter, enhancer, or other response element, or a portion thereof, which is recognized by a transcription factor and whose activity drives the expression of a gene whose activity is typically turned on or activated by T cell stimulation or activation. In some embodiments, a T cell stimulation-associated locus may contain a regulatory domain or region of a transcription factor (e.g., a promoter, enhancer, or other response element) whose activity is turned on by T cell stimulation or activation. In some embodiments, a T cell stimulation-associated locus is responsive to one or more of the quality and / or intensity of signaling through an intracellular signaling region, and / or the binding and / or recognition of a recombinant receptor to a target antigen, ligand, or epitope. In some embodiments, the regulatory element is responsive to one or more of the following: the state of the endogenous TCR bound to the antigen or epitope, T cell stimulation or activation, the signal intensity through the TCR, and / or the quality of signaling through the intracellular signaling region of the endogenous TCR.

[0164] In some embodiments, the endogenous T cell stimulation-associated locus contains one or more transcriptional regulatory elements, such as promoters, enhancers, or response elements, which include one or more binding sites to T cell transcription factors and thereby relate to the downstream activity of the T cell transcription factors. In some embodiments, the transcription factors are activated T cell nuclear factor (NFAT), C / EBP, STAT1, STAT2, or NF-κB. In some embodiments, the T cell stimulation-associated locus contains one or more response elements recognized by activated T cell nuclear factor (NFAT), C / EBP, STAT1, STAT2, or NF-κB. In some embodiments, the T cell stimulation-associated locus may contain one or more regulatory elements that are recognized by or responsive to one or two, and possibly three or more, specific transcription factors.

[0165] In some embodiments, a T cell stimulation-related locus includes one or more response elements recognized by a transcription factor activated upon T cell stimulation via the endogenous TCR complex. In some embodiments, a gene regulatory region includes numerous regulatory elements that can be responsive to more than one signaling pathway in the cell. In some embodiments, a T cell stimulation-related locus includes one or more regulatory elements recognized by NFAT. In some embodiments, a T cell stimulation-related locus includes one or more regulatory elements recognized by NF-κB.

[0166] In some embodiments, T cell stimulation-associated loci are associated with NFAT activity and / or NFAT regulatory signaling. The NFAT family of transcription factors plays a role in the transcriptional regulation of cytokine genes and other genes involved in immune responses, including responses to T cell activation. In some embodiments, T cell stimulation-associated loci contain regulatory elements such as promoters, enhancers, or response elements that contain binding sites and / or are recognized by NFAT and functionally linked thereto, which can drive the expression of transgenes, such as those encoding recombinant receptors.

[0167] In some embodiments, T cell stimulation-associated loci are associated with NF-κB activity and / or NF-κB-mediated signaling. NF-κB activation depends on TCR stimulation (i.e., via CD3 signaling) and CD28-mediated co-stimulation, and can be regulated by ligation of both CD3 and CD28. CD28 or CD3 signaling can induce NF-κB transcription, but co-ligation of CD28 with TCR signaling (i.e., CD3 signaling) can result in greater transcriptional activity (Thaker et al. (2015) Immunology Letters, 163:113-119). In some embodiments, T cell stimulation-associated loci can be transcriptional regulatory elements such as promoters, enhancers, or response elements that contain one or more binding sites and / or are recognized by NF-κB and functionally linked thereto, capable of driving the expression of a transgene, for example, encoding a recombinant receptor. In some cases, T cell stimulation-related loci containing regulatory elements responsive to NF-κB signaling can be indicators of the quality of T cell signaling, as well as the presence of both TCR-mediated and co-stimulatory signaling.

[0168] In some embodiments, the expression controlled by regulatory elements of T cell stimulation-related loci is dependent on, induced by, and / or upregulated by, T cell signaling. For example, the regulatory domain or element may be the promoter or a portion of the endogenous T cell stimulation-related locus. In some embodiments, the promoter or a portion of the promoter may contain a binding site and / or be recognized by one or more transcription factors.

[0169] In some embodiments, the T cell stimulation-associated locus contains transcriptional regulatory elements such as promoters or enhancers or other response elements, which are endogenous loci that control the expression of T cell transcription factors, whose expression may be induced by T cell signaling or activation. In some embodiments, the transcription factors are activated T cell nuclear factor (NFAT), nerve growth factor IB (Nur77, also known as NR4A1), C / EBP, STAT1, STAT2, and NFκB.

[0170] In some aspects, recombinant receptors are encoded by manipulable, regulated nucleic acid sequences at T cell stimulation-associated loci, such as regulatory elements that are responsive to the quality and / or intensity of signaling through antigen receptors, such as TCR complexes. In some aspects, T cell stimulation-associated loci are responsive to the quality and / or intensity of signaling through the intracellular signaling region of recombinant receptors (such as receptors functionally linked to endogenous T cell stimulation-associated loci) and / or in response to binding to and / or recognition of their target antigens or epitopes.

[0171] In some of the embodiments provided, the T cell stimulation-associated locus is PDCD1. In some embodiments, the engineered cells provided contain a modified PDCD1 locus containing a transgene encoding a recombinant receptor or a portion thereof, functionally linked to an endogenous transcriptional regulatory element of PDCD1. PDCD1 encodes inhibitory receptor programmed cell death-1 (PD-1, PDCD1; CD279; PD-1; PD1; SLEB2; hPD-1; hPD-1; or hSLE1), a mediator of central and peripheral immune tolerance and immunodepletion. In some aspects, PD-1 expression on CD8 T cells correlates with the presence and / or intensity of signaling through the T cell receptor (TCR). In some aspects, TCR stimulation initiates a signaling cascade through the calcineurin pathway, resulting in the activation and transposition of the transcription factor NFATc1 (also known as NFAT2). In some aspects, NFATc1 is involved in the expression induced by the initial activation of PD-1 in CD4 and CD8 T cells, and other regulatory mechanisms are also involved in the maintenance and enhancement of expression during chronic antigen exposure (see, for example, Bally et al., J Immunol (2016) 196 (6) 2431-2437; Simon et al., Oncoimmunology. 2018; 7(1): e1364828; Arasanz et al., Oncotarget. 2017 Aug 1; 8(31): 51936-51945).

[0172] In some of the embodiments provided, the T cell stimulation-associated locus is CD69. In some embodiments, the engineered cells provided contain a modified CD69 locus containing a transgene encoding a recombinant receptor or a portion thereof, functionally linked to an endogenous transcriptional regulatory element of CD69. CD69 encodes Cluster of Differentiation 69 (CD69, AIM; BL-AC / P26; CLEC2C; EA1; GP32 / 28; or MLR-3), a transmembrane type C lectin protein. CD69 is an early activation marker expressed in T cells, hematopoietic stem cells, natural killer (NK) cells, dendritic cells (DCs), and other immune cells. CD69 is an early inducible cell surface glycoprotein acquired during lymphocyte activation, involved in lymphocyte proliferation, and functions as a signaling receptor in lymphocytes, including natural killer (NK) cells, and platelets. CD69 expression is rapidly induced on the surface of T lymphocytes after TCR / CD3 binding and can be detected, for example, early after activation (30-60 minutes), activating cytokine and polyclonal pro-mitotic stimuli. Transcriptional expression of the CD69 gene rapidly decreases after 4-6 hours. CD69 protein expression can be detected as early as 2-3 hours after stimulation (see, for example, Alari-Pahissa et al., PLoS One. 2012; 7(10): e48593; Cibrian et al., Eur J Immunol. 2017 Jun; 47(6): 946-953).

[0173] In some of the embodiments provided, the T cell stimulation-associated locus is Nur77. In some embodiments, the engineered cells provided contain a modified Nur77 locus containing a transgene encoding a recombinant receptor or a portion thereof, functionally linked to an endogenous transcriptional regulatory element of Nur77. Nur77 is a member of the Nur nuclear receptor family of intracellular transcription factors, including nerve growth factor IB (NGFIB: Nr4a1, GFRP1; Gfrp; HMR; Hbr-1; Hbr1; Hmr; N10; NAK-1; NGFI-B; NGFIB; NP10; Ngfi-b; orphan nuclear receptor HMR; ST-59; TIS1; TR3; TR3 orphan receptor; early response protein NAK1; growth factor-inducible nucleoprotein N10; hormone receptor; and very early gene transcription factor NGF I-B; nerve growth factor IB nuclear receptor variant 1; nerve growth factor-inducing protein IB; nerve growth factor-inducing protein IB; neuronal orphan nuclear receptor NUR77; nhr-6; nr4a1; nuclear hormone receptor NUR / 77; nuclear protein N10; nuclear receptor subfamily 4 group A member 1; orphan nuclear receptor NGFI-B; orphan nuclear receptor NR4A1; orphan nuclear receptor TR3; steroid receptor TR3; testicular receptor 3; or also known as zgc:92434). In some situations, Nur77 expression is sensitive to signals from signaling domains including primary activation signals in T cells, signaling domains of T cell receptor (TCR) components, and / or immunoreceptor-activated tyrosine motifs (ITAMs). In some situations, Nur77 expression is dose-responsive to signals through signaling domains.

[0174] Nur77 is an early response gene expressed in T cells within hours of TCR stimulation and can be induced by phytohemagglutinin in human lymphocytes and by serum stimulation of arrested fibroblasts. Nur77 is induced in response to the endogenous TCR complex, signaling through binding to the endogenous TCR, or activation of signals from there, and / or via molecules containing the immunoreceptor-activated tyrosine motif (ITAM), which is involved in signaling from the TCR complex, e.g., the CD3-zeta signaling region. The Nur77 gene product itself can generally induce downstream gene expression by binding to regulatory elements associated with the promoters of several genes. The level or degree of Nur77 expression can serve as an indicator of the intensity of T cell signaling, e.g., TCR signaling (Moran et al. (2011) JEM, 208:1279-1289). Therefore, in some embodiments, the expression of a reporter molecule functionally linked to one or more transcriptional regulatory elements of the Nur77 locus, or a portion thereof, may provide an indicator of the intensity of T cell signaling. Furthermore, Nur77 expression may generally act in an extracellular manner and may not be dependent on other signaling pathways such as cytokine signaling or Toll-like receptor (TLR) signaling (see, for example, Ashouri et al., (2017) J. Immunol. 198:657-668), which may act in an extracellular manner and may not be dependent on signaling via recombinant receptors.

[0175] In some of the embodiments provided, the T cell stimulation-associated locus is FOXP3. In some embodiments, the engineered cells provided contain a modified FOXP3 locus containing a transgene encoding a recombinant receptor or a portion thereof, functionally linked to an endogenous transcriptional regulatory element of FOXP3. The FOXP3 gene encodes forkhead box P3 (Foxp3; also known as scurfin, AIID, DIETER, IPEX, JM2, PIDX, XPID), a protein involved in immune system responses, which in some cases is considered a regulator of regulatory pathways in regulatory T cell development and function. FoxP3 expression is induced by T cells, including in activated non-suppressor T cells or suppressor T cells following stimulation or activation. Expression is transient in CD8+CD25+ T cells, but in some cases, expression may be more stable, for example, in CD4+CD25+ regulatory T cells (see, for example, Kmieciak et al., J Transl Med. 2009; 7: 89; Wang et al., Eur J Immunol. 2007 Jan; 37(1):129-38; Yu et al., Oncol Lett. 2018 Jun; 15(6): 8187-8194).

[0176] In some of the embodiments provided, the T cell stimulation-associated locus is the HLA-DR locus. In some embodiments, the engineered cells provided contain a modified HLA-DR locus comprising a transgene encoding a recombinant receptor or a portion thereof, functionally linked to an endogenous transcriptional regulatory element of the HLA-DR locus. HLA-DR is a human class II major histocompatibility complex (MHC) antigen that is constitutively expressed on the surface of B lymphocytes, monocytes, and macrophages, and appears on T cells and NK cells in the late stages of activation. In some contexts, HLA-DR is a late-activation marker (see, for example, Bajnok et al., Mediators of Inflammation (2017) Article ID 8045161; Revenfeld et al., Int J Mol Sci. 2017 Jul; 18(7): 1603; Reddy et al., J. Immun. Methods. 2004, 293(1-2): 127-142). HLA-DR is an MHC class II cell surface receptor encoded by the human leukocyte antigen complex on chromosome 6p21.31. HLA-DR is encoded by several loci and several genes with different functions at each locus. The DRα chain is encoded by the HLA-DRA locus. The DRβ chain is encoded by several different loci, including HLA-DRB1~HLA-DRB9, only some of which are present in each individual. The HLA-DRB1 locus is ubiquitous and encodes a very large number of functionally variable gene products (HLA-DR1 to HLA-DR17) (see, for example, Marsh et al., Tissue Antigens. 2010 Apr; 75(4): 291-455).

[0177] In some cases, the level, amount, pattern, and timing of expression of T cell stimulation-related loci and / or, for example, a transgene functionally linked to a T cell stimulation-related locus, can be determined by using assays that assess gene expression in the presence of T cell stimulation or activation signals. In some cases, detecting the level, amount, pattern, and timing of expression of T cell stimulation-related loci and / or, for example, a transgene functionally linked to a T cell stimulation-related locus, involves performing in vitro assays or in vivo assays.

[0178] In some embodiments, the assay includes assays that detect the level of expressed gene products (e.g., polypeptides or proteins encoded by T cell stimulation-associated loci), such as immunoassays, aptamer-based assays, histological or cytological assays, or assays that detect the level of expressed ribonucleic acid (RNA), such as mRNA expression level assays. In some embodiments, the level, amount, pattern, and timing of expression of T cell stimulation-associated loci and / or, for example, a transgene functionally linked to a T cell stimulation-associated locus, are detected by immunocytochemistry or immunohistochemistry, enzyme-linked immunosorbent assays (ELISA; direct, indirect, sandwich, competitive, multiplex, and portable ELISA (see, for example, U.S. Patent No. 7,510,687)), Western blotting (optionally including peptide sequencing, one-dimensional, two-dimensional, or higher-dimensional blotting or other chromatographic means), immunoblotting, immunoprecipitation. These are detected by assays such as radioimmunoassays (RIA), immunohistochemistry, flow cytometry assays, surface plasmon resonance (SPR), chemiluminescence assays, lateral flow immunoassays, inhibition assays, binding affinity assays, nucleic acid-based or protein-based aptamer techniques, high-performance liquid chromatography (HPLC), peptide sequencing (optionally coupled to HPLC, such as Edman degradation sequencing or mass spectrometry (e.g., MS / MS)), and any of the aforementioned microarray adaptations (including nucleic acid, antibody, or protein-protein (i.e., non-antibody) arrays). In some embodiments, the level, amount, pattern, and timing of expression of a T cell stimulation-associated locus and / or, for example, a transgene functionally linked to a T cell stimulation-associated locus, are determined using a binding reagent that specifically binds to the gene product of the recombinant receptor it encodes. In some cases, the binding reagent is an antibody or its antigen-binding fragment, aptamer, or nucleic acid probe.

[0179] In some embodiments, the level, amount, pattern, and timing of expression of T cell stimulation-associated loci and / or, for example, a transgene functionally linked to a T cell stimulation-associated locus, are determined using methods for detecting the amount, level, or expression of nucleic acids, such as messenger RNA. In certain embodiments, the assay includes detecting, measuring, evaluating, and / or quantifying the level of polynucleotides, such as mRNA from a T cell stimulation-associated locus or mRNA produced from a transgene. In some aspects, the amount or level of polynucleotides includes reverse transcriptase (rt) PCR, droplet digital PCR, real-time and quantitative PCR methods (e.g., TAQMAN®, Molecular Beacon, LIGHTUP®, SCORPION®, SIMPLEPROBES®; see, for example, U.S. Patent Nos. 5,538,848; 5,925,517; 6,174,670; 6,329,144; 6,326,145 and 6,635,427), poly It can be evaluated, measured, determined, and / or quantified by any other method known in the art, such as Melase Chain Reaction (PCR); Northern blotting; Southern blotting of reverse transcripts and derivatives, for example; array-based methods, including blot arrays, microarrays, or in-situ synthetic arrays; and sequencing, for example, synthetic sequencing, pyrosequencing, dideoxysequencing, or ligation sequencing, or by any other method known in the art, such as Shendure et al., Nat. Rev. Genet. 5:335-44 (2004) or Nowrousian, Eukaryotic Cell 9(9): 1300-1310 (2010), including specific platforms such as HELICOS®, ROCHE® 454, ILLUMINA® / SOLEXA®, ABI SOLiD®, and POLONATOR® sequencing. In some of these embodiments, the level of polynucleotides is measured by qRT-PCR.In some embodiments, qRT-PCR uses three sets of nucleic acids for each gene, the three nucleic acids comprising primer pairs, along with probes that bind between regions of the target nucleic acid to which the primers bind.

[0180] In some embodiments, the level, amount, pattern, and timing of expression of T cell stimulation-associated loci and / or transgenes functionally linked to, for example, T cell stimulation-associated loci are determined by sequencing polynucleotides. In some embodiments, sequencing is performed by non-Sanger sequencing methods and / or next-generation sequencing (NGS) techniques. Examples of next-generation sequencing techniques include, but are not limited to, Massively Parallel Signature Sequencing (MPSS), Polony sequencing, pyrosequencing, reversible dye-terminator sequencing, SOLiD sequencing, Ion semiconductor sequencing, DNA nanoball sequencing, Helioscope single-molecule sequencing, single-molecule real-time (SMRT) sequencing, single-molecule real-time (RNAP) sequencing, and Nanopore DNA sequencing. In some embodiments, the NGS technique is RNA sequencing (RNA-Seq). RNA sequencing is compatible with the most common DNA sequencing platforms, such as the HiSeq system (Illumina), the 454 Genome Sequencer FLX System (Roche), Applied Biosystems SOLiD (Life Technologies), and IonTorrent (Life Technologies). These platforms generally require the reverse transcription of the initial RNA into cDNA. Conversely, the single-molecule sequencer HeliScope (Helicos BioSciences) can use RNA as a template for sequencing. Proof of principle for direct RNA sequencing on the PacBio RS platform has also been demonstrated (Pacific Bioscience). In some embodiments, one or more RNA gene products are evaluated, measured, determined, and / or quantified by RNAseq.

[0181] In some cases, the level, amount, pattern, and timing of expression of T cell stimulation-associated loci and / or, for example, a transgene functionally linked to a T cell stimulation-associated loci can be determined in cells after exposure to a stimulating or activating signal. In some cases, the expression of T cell stimulation-associated loci and / or a transgene is determined by incubating cells with an active agent that provides a stimulating or activating signal. In some cases, the level, amount, and pattern of expression of T cell stimulation-associated loci and / or, for example, a transgene functionally linked to a T cell stimulation-associated loci can be assessed at various times after exposure to a stimulating or activating signal. In some cases, the level, amount, and pattern of expression of T cell stimulation-associated loci and / or, for example, a transgene functionally linked to a T cell stimulation-associated loci can be determined after restimulation, repeated stimulation, or sequential stimulation. In some embodiments, transgene expression may be evaluated after T cell incubation in the presence or absence of an activator that binds to the binding domain of the recombinant receptor and / or an activator that induces or can induce signaling through the intracellular signaling region of the recombinant receptor.

[0182] In some aspects, exemplary activators that can provide a stimulating or activating signal to assess the level, amount, or pattern of expression include antigen-independent stimuli, such as activators containing anti-CD3 and / or anti-CD28 antibodies, such as beads conjugated with anti-CD3 and anti-CD28 antibodies, or soluble multimeric or oligomeric reagents loaded with anti-CD3 / anti-CD28 antibodies or antibody fragments; or antigen-specific stimuli to recombinant receptors, such as purified or recombinant antigens to which recombinant receptors bind or recognize, such as target antigens or ligands of the antigen-binding or ligand-binding domains of recombinant receptors. Other exemplary activators that can provide a stimulating or activating signal to T cells to assess the level, amount, or pattern of expression include phorbol 12-myrisstart 13-acetate (PMA), also known as 12-O-tetradecanoylphorbol 13-acetate (TPA), ionomisins, and / or concanavalin A (Con A).

[0183] II. Method for creating cells that conditionally express recombinant receptors by homology-directed repair (HDR) Methods for generating or producing genetically engineered cells comprising a modified T cell stimulation-related locus (e.g., PDCD1 (encoding PD-1), CD69, Nur77 (encoding NR4A1), FoxP3, or HLA-DR locus), wherein the modified T cell stimulation-related locus comprises a transgene (e.g., a heterologous or exogenous nucleic acid sequence) encoding a recombinant receptor such as a chimeric antigen receptor (CAR) or T cell receptor (TCR). In some aspects, the modified T cell stimulation-related locus in the genetically engineered cell comprises a transgene encoding a recombinant receptor or a portion thereof, incorporated into an endogenous T cell stimulation-related locus. In some embodiments, methods are provided that involve using a template polynucleotide containing a transgene encoding a recombinant receptor or a portion thereof to induce target gene disruption and homology-dependent repair (HDR), thereby targeting the incorporation of the transgene into the T cell stimulation-related locus. Furthermore, cells and cell compositions produced by the method, as well as polynucleotides for use in the method, such as template polynucleotides, and kits are also provided.

[0184] In some aspects, the provided embodiments utilize HDR for targeted incorporation of recombinant or heterologous sequences into T cell stimulation-related loci. In some cases, the method involves introducing one or more target gene disruptions, e.g., DNA breaks, into endogenous T cell stimulation-related loci by gene editing techniques, in combination with targeted incorporation of a transgene encoding a recombinant receptor or a portion thereof by HDR. In some aspects, the one or more target gene disruptions are carried out by introducing one or more activators capable of introducing gene disruptions. In some aspects, the HDR step requires disruption or breaks in DNA at a target site in the genome, e.g., double-strand breaks. In some aspects, DNA breaks are induced by gene editing techniques, e.g., targeted nucleases. In some aspects, the method generates engineered cells in which expression of the T cell stimulation-related loci is knocked out. In some aspects, the method generates engineered cells that retain expression of the T cell stimulation-related loci. In some aspects, after the method is performed, the engineered T cells contain a transgene encoding a recombinant receptor or a portion thereof that is functionally linked to an endogenous transcriptional regulatory element of the T cell stimulation-related locus. In some aspects, endogenous transcriptional regulatory elements induce or upregulate the expression of functionally linked transgenes following simulation or activation signals in T cells.

[0185] In some aspects, the provided method includes the steps of introducing one or more activators into T cells that can induce gene disruption at a target site within a T cell stimulation-associated locus; and introducing a polynucleotide, e.g., a template polynucleotide, comprising the transgene and one or more homology arms into T cells. In some aspects, the transgene contains a sequence of nucleotides encoding a recombinant receptor or a portion thereof. In some embodiments, the nucleic acid sequence is targeted for integration within a T cell stimulation-associated locus via homology-directed repair (HDR).

[0186] In some aspects, the provided method comprises the step of introducing a polynucleotide containing a transgene sequence encoding a recombinant receptor or a portion thereof into a T cell having a gene disruption within a T cell stimulation-associated locus, wherein the gene disruption is introduced by one or more activators capable of inducing gene disruption at one or more target sites within the T cell stimulation-associated locus, and the nucleic acid sequence is targeted for incorporation within the T cell stimulation-associated locus via HDR. In some aspects, also provided is a composition comprising genetically engineered immune cells produced by any of the provided methods, comprising a population of cells engineered to express a recombinant receptor, e.g., CAR or TCR, such that the cell population exhibits improved, homogeneous, homogeneous, controlled, and / or stable expression and / or antigen binding by the recombinant receptor. In some aspects, the provided embodiments enable control of expression, such as conditional expression of a linked transgene encoding a recombinant receptor upon T cell stimulation, and reduction or downregulation of expression following reduction or absence of simulation or activation signals in the T cell.

[0187] In some aspects, the embodiment includes generating a target DNA cleavage using a gene editing method and / or a targeted nuclease, and subsequently, an HDR based on one or more template polynucleotides, e.g., a transgene encoding a recombinant receptor or a portion thereof, and possibly other molecules, linked to nucleic acid sequences homologous to the sequence of an endogenous T cell stimulation-associated locus, for specifically targeting and incorporating the transgene at or near the DNA cleavage. Thus, in some aspects, the method includes the steps of inducing target gene disruption (e.g., gene editing) and introducing a polynucleotide, e.g., a template polynucleotide containing the transgene, into a cell (e.g., HDR).

[0188] In some embodiments, targeted gene disruption and HDR-mediated targeted integration of a transgene occur at one or more target sites of an endogenous T cell stimulation-related locus. In some aspects, targeted integration occurs within the open reading frame sequence of the endogenous T cell stimulation-related locus. In some aspects, targeted integration of a transgene results in knockout of a gene at an endogenous T cell stimulation-related locus, for example, so that the expression of the endogenous gene is eliminated.

[0189] In some cases, the transgene is integrated into the T cell stimulation-associated locus, for example, by homology-directed repair (HDR) within the exon or a portion of the open reading frame of the endogenous T cell stimulation-associated locus, such that the sequence encoding the chimeric receptor or a portion thereof is in-frame with the exon sequence. In some cases, all or part of the endogenous T cell stimulation-associated locus, such as a portion upstream of the integrated transgene, and the recombinant receptor or a portion thereof, may be separated by a multicistronic element and expressed in the modified T cell stimulation-associated locus.

[0190] In some embodiments, a template polynucleotide is introduced into the engineered cell either simultaneously with or after the introduction of one or more active agents capable of inducing disruption of one or more target genes. In the presence of one or more target gene disruptions, e.g., DNA breaks, the template polynucleotide may be used as a DNA repair template to effectively incorporate the transgene at or near the site of the target gene disruption by HDR, based on the homology between the endogenous gene sequence surrounding the gene disruption and one or more homology arms, such as the 5' and / or 3' homology arms, contained in the template polynucleotide.

[0191] In some cases, a template polynucleotide and one or more active agents capable of inducing disruption of one or more target genes are introduced simultaneously. In some cases, the template polynucleotide and one or more active agents capable of inducing disruption of one or more target genes are introduced using any delivery method as described herein, for example, in sections II.A.3 and II.B.3. In some cases, the template polynucleotide and one or more active agents capable of inducing disruption of one or more target genes are introduced via a physical delivery method, for example, by electroporation, particle gun, calcium phosphate transfection, or cell compression or pressure. In some cases, the template polynucleotide and one or more active agents capable of inducing disruption of one or more target genes are introduced simultaneously via electroporation.

[0192] In some aspects, the two steps may be performed sequentially. In some embodiments, the gene editing and HDR steps are performed simultaneously and / or in a single experimental reaction. In some embodiments, the gene editing and HDR steps are performed sequentially or sequentially in a single or consecutive experimental reaction. In some embodiments, the gene editing and HDR steps are performed simultaneously or at different times in separate experimental reactions.

[0193] Immune cells may include a population of cells containing T cells. Such cells may be obtained from a subject, for example, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or cells obtained from a leukocyte apheresis product. In some embodiments, T cells may be isolated or selected to enrich the T cells in the population using positive or negative selection and enrichment methods. In some embodiments, the population may contain CD4+ T cells, CD8+ T cells, or CD4+ T cells and CD8+ T cells. In some embodiments, the steps of introducing a polynucleotide template and introducing an active agent (e.g., Cas9 / gRNA RNP) may be performed simultaneously, sequentially, or in any order. In some embodiments, the polynucleotide template is introduced into the immune cells after gene disruption has been induced by the step of introducing an active agent (e.g., Cas9 / gRNA RNP). In some embodiments, cells are cultured or incubated under conditions that stimulate cell growth and / or proliferation, before, during, and / or after the introduction of a polynucleotide template and one or more active agents (e.g., Cas9 / gRNA RNP).

[0194] In some aspects of the methods provided, the introduction of a template polynucleotide is performed concurrently with the introduction of one or more active agents capable of inducing gene disruption. Depending on the specific active agent used to induce gene disruption, any method for introducing one or more active agents can be used as described. In some aspects, disruption is carried out by gene editing using an RNA-inducible nuclease, such as a clustered regularly interspersed short palindromic nucleic acid (CRISPR)-Cas system, e.g., the CRISPR-Cas9 system, which is specific to the T cell stimulation-related locus to be disrupted. In some aspects, an active agent containing Cas9 and a guide RNA (gRNA) containing a targeting domain, targeting a region of the T cell stimulation-related locus, is introduced into the cell. In some aspects, the active agent is or comprises a ribonucleoprotein (RNP) complex (Cas9 / gRNA RNP) of Cas9 and a gRNA containing a targeting domain that targets the T cell stimulation-related locus. In some embodiments, introduction involves in vitro contact of the active substance or a portion thereof with cells, which may include culturing or incubating the cells and the active substance for up to 24, 36, or 48 hours, or 3, 4, 5, 6, 7, or 8 days. In some embodiments, introduction may further include delivery into cells of a polynucleotide containing the active substance and / or a transgene such as a template for HDR. In various embodiments, the methods, compositions, and cells according to this disclosure utilize direct delivery of a ribonucleoprotein (RNP) complex of Cas9 and gRNA and / or a template polynucleotide into cells, for example, by electroporation. In some cases, electroporation of the cells to be modified includes subjecting the cells to a cold shock, for example at 32°C, after electroporation and before plating.

[0195] Depending on the specific method used for delivering the template polynucleotide to the cell, any method for introducing the template polynucleotide can be used as described. In some embodiments, the template polynucleotide is a linear polynucleotide. In some embodiments, the template polynucleotide is a single-stranded linear polynucleotide. In some embodiments, the template polynucleotide is a double-stranded linear polynucleotide. In some aspects, the template polynucleotide is delivered into the cell separately or together with one or more agents to induce gene disruption at one or more target sites in the genome by physical delivery means such as electroporation.

[0196] Exemplary methods include those for the transfer of receptor-encoding nucleic acids, including via viruses, such as retroviruses or lentiviruses, transduction, transposons, and electroporation. In some embodiments, viral transduction methods are used. In some embodiments, template polynucleotides may be transferred or introduced into cells using recombinant infectious viral particles, such as vectors derived from Simian virus 40 (SV40), adenoviruses, or adeno-associated viruses (AAVs). In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr 3. doi: 10.1038 / gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November 29(11): 550-557). In some embodiments, the viral vector is an AAV such as AAV2 or AAV6.

[0197] In such aspects of the method provided, the template polynucleotide is introduced into the cell after the introduction of one or more active agents, such as Cas9 / gRNA RNP, which are introduced, for example, via electroporation. In some embodiments, the template polynucleotide is introduced immediately after the introduction of one or more active agents that can induce gene disruption. In some embodiments, the template polynucleotide is introduced into the cell within 30 seconds or about 30 seconds, within 1 minute or about 1 minute, within 2 minutes or about 2 minutes, within 3 minutes or about 3 minutes, within 4 minutes or about 4 minutes, within 5 minutes or about 5 minutes, within 6 minutes or about 6 minutes, within 6 minutes or about 6 minutes, within 8 minutes or about 8 minutes, within 9 minutes or about 9 minutes, within 10 minutes or about 10 minutes, within 15 minutes or about 15 minutes, within 20 minutes or about 20 minutes, within 30 minutes or about 30 minutes, within 40 minutes or about 40 minutes, within 50 minutes or about 50 minutes, within 60 minutes or about 60 minutes, within 90 minutes or about 90 minutes, within 2 hours or about 2 hours, within 3 hours or about 3 hours, or within 4 hours or about 4 hours, after the introduction of one or more active substances capable of inducing gene disruption. In some embodiments, the template polynucleotide is subjected to the introduction of one or more active substances for a period of time from 15 minutes or about 15 minutes to 4 hours or about 4 hours, for example, from 15 minutes or about 15 minutes to 3 hours or about 3 hours, from 15 minutes or about 15 minutes to 2 hours or about 2 hours, from 15 minutes or about 15 minutes to 1 hour or about 1 hour, from 15 minutes or about 15 minutes to 30 minutes or about 30 minutes, from 30 minutes or about 30 minutes to 4 hours or about 4 hours, and from 30 minutes or about 30 minutes to 3 hours or about 3 hours. It is introduced into the cells over a period of time between 30 minutes or approximately 30 minutes and 2 hours or approximately 2 hours, between 30 minutes or approximately 30 minutes and 1 hour or approximately 1 hour, between 1 hour or approximately 1 hour and 4 hours or approximately 4 hours, between 1 hour or approximately 1 hour and 3 hours or approximately 3 hours, between 1 hour or approximately 1 hour and 2 hours or approximately 2 hours, between 2 hours or approximately 2 hours and 4 hours or approximately 4 hours, between 2 hours or approximately 2 hours and 3 hours or approximately 3 hours, or between 3 hours or approximately 3 hours and 4 hours or approximately 4 hours.In some embodiments, the template polynucleotide is introduced into the cell 2 hours or approximately 2 hours after the introduction of one or more active agents, such as Cas9 / gRNA RNP, which are introduced, for example, via electroporation.

[0198] In some embodiments, the provided method involves incubating cells in the presence of cytokines, stimulating agents, and / or agents capable of inducing proliferation, stimulation, or activation of immune cells (e.g., T cells), before, during, or following contact with the active agent, and / or before, during, or following delivery (e.g., electroporation). In some embodiments, at least a portion of the incubation is in the presence of a CD3-specific antibody, a CD28-specific antibody, and / or cytokines, such as anti-CD3 / anti-CD28 beads, or stimulating agents containing these. In some embodiments, at least a portion of the incubation is in the presence of one or more cytokines, such as recombinant IL-2, recombinant IL-7, and / or recombinant IL-15. In some embodiments, incubation may last up to 8 days, for example, up to 24, 36, or 48 hours, or 3, 4, 5, 6, 7, or 8 days, before or after the introduction of one or more active substances, such as Cas9 / gRNA RNPs and template polynucleotides, via electroporation, for example.

[0199] In some embodiments, the method involves activating or stimulating cells with a stimulating agent (e.g., an anti-CD3 / anti-CD28 antibody) prior to introducing an active agent, e.g., Cas9 / gRNA RNP, and a polynucleotide template. In some embodiments, incubation in the presence of the stimulating agent (e.g., anti-CD3 / anti-CD28) extends for 6 to 96 hours, e.g., 24 to 48 hours or 24 to 36 hours, prior to the introduction of one or more active agents, e.g., Cas9 / gRNA RNP, e.g., via electroporation. In some embodiments, incubation with the stimulating agent may further include the presence of one or more cytokines, e.g., recombinant IL-2, recombinant IL-7, and / or recombinant IL-15. In some embodiments, incubation is carried out in the presence of recombinant cytokines, e.g., IL-2 (e.g., 1 U / mL to 500 U / mL, e.g., 10 U / mL to 200 U / mL, e.g., at least or about 50 U / mL or 100 U / mL), IL-7 (e.g., 0.5 ng / mL to 50 ng / mL, e.g., 1 ng / mL to 20 ng / mL, e.g., at least or about 5 ng / mL or 10 ng / mL), or IL-15 (e.g., 0.1 ng / mL to 50 ng / mL, e.g., 0.5 ng / mL to 25 ng / mL, e.g., at least or about 1 ng / mL or 5 ng / mL). In some embodiments, the stimulant (e.g., anti-CD3 / anti-CD28 antibody) is washed or removed from the cells before introducing or delivering the activator Cas9 / gRNA RNP and / or polynucleotide template into the cells, which can induce gene disruption. In some embodiments, the cells are rested before the introduction of the active substance, for example, by the removal of any stimulant or activating agent. In some embodiments, the stimulant or activating agent and / or cytokines are not removed before the introduction of the active substance.

[0200] In some embodiments, cells are incubated, cultivated, or cultured in the presence of one or more recombinant cytokines, such as recombinant IL-2, recombinant IL-7, and / or recombinant IL-15, following the introduction of an active agent, e.g., Cas9 / gRNA, and / or a polynucleotide template. In some embodiments, incubation is carried out in the presence of recombinant cytokines, e.g., IL-2 (e.g., 1 U / mL to 500 U / mL, e.g., 10 U / mL to 200 U / mL, e.g., at least or about 50 U / mL or 100 U / mL), IL-7 (e.g., 0.5 ng / mL to 50 ng / mL, e.g., 1 ng / mL to 20 ng / mL, e.g., at least or about 5 ng / mL or 10 ng / mL), or IL-15 (e.g., 0.1 ng / mL to 50 ng / mL, e.g., 0.5 ng / mL to 25 ng / mL, e.g., at least or about 1 ng / mL or 5 ng / mL). Cells can be incubated or cultured under conditions to induce cell proliferation or growth. In some embodiments, cells can be incubated or cultured until a threshold cell number for harvesting, e.g., a therapeutically effective dose, is achieved.

[0201] In some embodiments, incubation between any part of the process or the entire process may be at temperatures of 30°C±2°C to 39°C±2°C, for example, at least or about at least 30°C±2°C, 32°C±2°C, 34°C±2°C, or 37°C±2°C. In some embodiments, at least part of the incubation is at 30°C±2°C, and at least part of the incubation is at 37°C±2°C.

[0202] In some aspects, the provided embodiments allow recombinant receptors to be expressed under the regulation of endogenous transcriptional regulatory elements of T cell stimulation-related loci, e.g., endogenous promoters of T cell stimulation-related loci. In some aspects, the provided embodiments allow nucleic acids encoding recombinant receptors to be functionally ligated to endogenous regulatory or regulatory elements of endogenous T cell stimulation-related loci, e.g., promoters, or cis-regulatory elements such as 5' and / or 3' untranslated regions (UTRs). Thus, in some aspects, the provided embodiments allow recombinant receptors, e.g., CARs, to be expressed, and / or their expression to be conditionally, temporally, and / or quantitatively regulated, as with endogenous T cell stimulation-related loci. In some aspects, the expression of functionally ligated transgenes is upregulated or induced following a stimulating or activating signal in T cells. In some aspects, the expression of functionally ligated transgenes is reduced or downregulated following a reduction or absence of a simulation or activating signal in T cells. In some aspects, the expression of functionally linked transgenes can be re-induced or upregulated following further simulation or activation signals in T cells after signal reduction or absence.

[0203] A. Genetic disruption In some embodiments, disruption of one or more target genes is induced at one or more endogenous T cell stimulation-related loci. In some embodiments, the disruption of target genes is induced in or near the exons of the endogenous T cell stimulation-related loci. In some embodiments, the disruption of target genes is induced in or near the introns of the endogenous T cell stimulation-related loci. In some embodiments, the disruption of target genes is induced at or near the promoter of the endogenous T cell stimulation-related loci. In some aspects, the disruption of one or more target genes and the presence of a template polynucleotide, such as a transgene encoding a recombinant receptor or a portion thereof, may result in targeted integration of the transgene at or near the disruption of one or more genes at the endogenous T cell stimulation-related loci.

[0204] In some embodiments, gene disruption results in DNA breaks, such as double-strand breaks (DSBs) or cleavage, or nicks, such as single-strand breaks (SSBs), at one or more target sites in the genome. In some embodiments, at the site of gene disruption, e.g., DNA break or nick, the cellular DNA repair mechanism may result in knockout, insertion, missense mutation or frameshift mutation, e.g., biallelic frameshift mutation, deletion of all or part of the gene, or modification of the DNA sequence based on a repair template, e.g., in the presence of a template polynucleotide, the incorporation or insertion of nucleic acid sequences contained in a template polynucleotide (e.g., any of those described in Section II.B.2 of this specification). In some embodiments, gene disruption may be targeted to one or more exons or portions thereof of a gene. In some embodiments, gene disruption may be targeted near a desired site for targeted incorporation of an exogenous sequence, e.g., an exogenous sequence encoding a recombinant receptor. In some aspects, after integration of a transgene encoding a recombinant receptor or a portion thereof, the modified T cell stimulation-associated locus contains deletions, insertions, frameshift mutations, or nonsense mutations in the open reading frame of the endogenous T cell stimulation-associated locus. In some aspects, the endogenous gene product of the T cell stimulation-associated locus is not produced in the cell, or is truncated or nonfunctional. In some aspects, the endogenous gene product of the T cell stimulation-associated locus is produced in full length or is functional in the cell.

[0205] In some embodiments, a DNA-binding protein or DNA-binding nucleic acid that specifically binds to or hybridizes to a sequence in a region close to at least one of the target sites is used for targeted disruption. In some embodiments, a template polynucleotide, for example, a template polynucleotide comprising a nucleic acid sequence encoding a recombinant receptor or a portion thereof and a homologous sequence, can be introduced for HDR-mediated targeted incorporation of a recombinant receptor-encoding sequence at or near the site of gene disruption, for example, as described herein, for example, in Section II.A.

[0206] In some embodiments, gene disruption is carried out by introducing one or more active agents capable of inducing gene disruption. In some embodiments, such active agents include DNA-binding proteins or DNA-binding nucleic acids that specifically bind to or hybridize with genes. In some embodiments, the active agent includes a variety of components, such as a fusion protein or RNA-inducible nuclease containing a DNA-targeting protein and a nuclease. In some embodiments, the active agent can target one or more target sites or locations. In some aspects, a pair of single-strand breaks (e.g., nicks) may be generated on each side of the target site.

[0207] In the embodiments provided, the term “introduce” encompasses a variety of methods for introducing DNA into cells, either in vitro or in vivo, such methods including transformation, transduction, transfection (e.g., electroporation), and infection. Vectors are useful for introducing DNA encoding molecules into cells. Possible vectors include plasmid vectors and viral vectors. Viral vectors include retroviral vectors, lentiviral vectors, or other vectors, such as adenovirus vectors or adeno-associated vectors. Methods such as electroporation may also be used to introduce or deliver proteins, or ribonucleoproteins (RNPs) containing, for example, the Cas9 protein in complex with a targeting gRNA, into cells of interest.

[0208] In some embodiments, gene disruption occurs at a target site (also known as the “target location,” “target DNA sequence,” or “target site”), for example, at an endogenous T cell stimulation-associated locus. In some embodiments, the target site includes a site on target DNA (e.g., genomic DNA) that is modified by one or more active agents capable of inducing gene disruption, for example, a Cas9 molecule complexed with a gRNA that identifies the target site. For example, the target site may include a location in the DNA of an endogenous T cell stimulation-associated locus where cleavage or DNA break occurs. In some embodiments, the incorporation of nucleic acid sequences by HDR may occur at or near the target site or target sequence. In some embodiments, the target site may be two nucleotides on DNA to which one or more nucleotides are added, for example, a site between adjacent nucleotides. The target site may include one or more nucleotides that are modified by a template polynucleotide. In some embodiments, the target site is within a target sequence (e.g., a sequence to which the gRNA binds). In some embodiments, the target site is upstream or downstream of the target sequence.

[0209] 1. Exemplary target sites of endogenous T cell stimulation-related gene loci In some embodiments, the incorporation of a transgene encoding a recombinant receptor or a portion thereof via gene disruption and / or homology-directed repair (HDR) is targeted to endogenous or genomic T cell stimulation-associated loci as described herein. In some aspects, the resulting engineered T cells, due to gene disruption at the target site described herein and the presence of a template polynucleotide for targeted incorporation of the transgene at the T cell stimulation-associated locus, contain a transgene encoding a recombinant receptor or a portion thereof, functionally ligated to an endogenous transcriptional regulatory element of the T cell stimulation-associated locus. In some aspects, the endogenous transcriptional regulatory element induces or upregulates the expression of the functionally ligated transgene following a simulation or activation signal in the T cell.

[0210] In some embodiments, gene disruption is targeted to, near, or within a T cell stimulation-associated locus. In some embodiments, the T cell stimulation-associated locus encodes a molecule that is transiently upregulated or induced on T cells. In some embodiments, exemplary T cell stimulation-associated loci include PDCD1 (encoding PD-1), CD69, Nur77 (encoding NR4A1), FoxP3, or HLA-DR loci. In some aspects, the target site is at or near the PDCD1 (encoding PD-1), CD69, Nur77 (encoding NR4A1), FoxP3, or HLA-DR locus. Expression of exemplary T cell stimulation-associated loci is described herein, for example, in Section IA.

[0211] An exemplary human PD-1 precursor polypeptide sequence is shown at SEQ ID NO:79 (the mature polypeptide includes residues 24–288 of SEQ ID NO:79; see Uniprot accession number Q15116-1; mRNA sequence shown at SEQ ID NO:80, NCBI reference sequence: NM_005018.3). In humans, PDCD1, the genomic locus encoding PD-1, contains an open reading frame with five exons and four introns. An exemplary mRNA transcript of PDCD1 spans the sequence corresponding to the reverse strand of chromosome 2:241,849,884–241,858,894, referring to the human genome version GRCh38 (UCSC Genome Browser on Human Dec. 2013 (GRCh38 / hg38) Assembly). Table 1 shows the coordinates of the exons, introns, and untranslated regions of the open reading frame of the exemplary transcript encoding PD-1.

[0212] (Table 1) Coordinates of exons and introns of the human PDCD1 gene locus (GRCh38, chromosome 2, reverse strand) TIFF2026090319000002.tif47165

[0213] An exemplary human CD69 polypeptide sequence is shown at SEQ ID NO:81 (Uniprot accession number Q07108-1; see mRNA sequence shown at SEQ ID NO:82, NCBI reference sequence: NP_001772.1). In humans, the locus encoding CD69 contains an open reading frame with five exons and four introns. The exemplary CD69 mRNA transcript spans the sequence corresponding to the reverse strand of chromosome 12:9,752,486–9,760,901, referring to the human genome version GRCh38 (UCSC Genome Browser on Human Dec. 2013 (GRCh38 / hg38) Assembly). Table 2 shows the coordinates of the exons, introns, and untranslated regions of the open reading frame of the exemplary CD69 transcription.

[0214] (Table 2) Coordinates of exons and introns of the human CD69 locus (GRCh38, chromosome 12, reverse strand) TIFF2026090319000003.tif47165

[0215] An exemplary human NR4A1 polypeptide sequence is shown at SEQ ID NO:83 (Isoform 1; Uniprot accession number P22736-1; refer to the mRNA sequence shown at SEQ ID NO:84, NCBI reference sequence: NP_002126.2). In humans, several different mRNA and protein isoforms exist for Nur77. Nur77 (also known as NR4A1), the exemplary genomic locus encoding NR4A1, contains an open reading frame with eight exons and seven introns for the transcript variant encoding isoform 1. The exemplary mRNA transcript of Nur77 encoding isoform 1 spans the sequence corresponding to the forward strand of chromosome 12:52,051,402–52,059,506, referring to the human genome version GRCh38 (UCSC Genome Browser on Human Dec. 2013 (GRCh38 / hg38) Assembly). Table 3 shows the coordinates of the exons, introns, and untranslated regions of the open reading frame of an exemplary transcript isoform 1 encoding NR4A1.

[0216] (Table 3) Coordinates of exons and introns of the human Nur77 locus (GRCh38, chromosome 12, forward strand) TIFF2026090319000004.tif75165

[0217] An exemplary human FoxP3 polypeptide sequence is shown at SEQ ID NO:85 (Isoform 1; Uniprot accession number Q9BZS1-1; mRNA sequence shown at SEQ ID NO:86, see NCBI reference sequence:NM_014009.3). In humans, several different mRNA and protein isoforms exist for FoxP3. FOXP3, the exemplary genomic locus encoding FoxP3, contains an open reading frame with 12 exons and 11 introns for the transcript variant encoding isoform 1. The exemplary mRNA transcript of FOXP3 encoding isoform 1 spans the X chromosome:49,250,436–49,264,924 reverse strand, referring to the human genome version GRCh38 (UCSC Genome Browser on Human Dec. 2013 (GRCh38 / hg38) Assembly). Table 4 shows the coordinates of the exons, introns, and untranslated regions of the open reading frame of an exemplary transcript isoform 1 encoding FoxP3.

[0218] (Table 4) Coordinates of exons and introns of the human FOXP3 gene locus (GRCh38, X chromosome, reverse strand) TIFF2026090319000005.tif112165

[0219] HLA-DR is a heterodimeric protein containing alpha (α) and beta (β) chains. Each subunit contains two extracellular domains, a transmembrane domain, and a cytoplasmic tail. Both the α and β chains are anchored to the membrane. HLA-DR is encoded by several loci and several genes with different functions at each locus. The DRα chain is encoded by the HLA-DRA locus. The DRβ chain is encoded by several different loci, including HLA-DRB1 to HLA-DRB9, only some of which are present in each individual. The HLA-DRB1 locus is ubiquitous and encodes a very large number of functionally variable gene products (HLA-DR1 to HLA-DR17) (see, e.g., Marsh et al., Tissue Antigens. 2010 Apr; 75(4): 291-455).

[0220] An exemplary precursor human HLA-DRα chain polypeptide sequence is shown at SEQ ID NO:87 (the mature polypeptide includes residues 26-254 of SEQ ID NO:87; see Uniprot accession number P01903-1; mRNA sequence shown at SEQ ID NO:88, NCBI reference sequence: NM_019111.4). In humans, HLA-DRA, the locus encoding the HLA-DRα chain, contains an open reading frame with five exons (four of which encode exons) and four introns. An exemplary mRNA transcript of HLA-DRA spans the sequence corresponding to the forward strand of chromosome 6:32,439,887-32,445,046, referring to the human genome version GRCh38 (UCSC Genome Browser on Human Dec. 2013 (GRCh38 / hg38) Assembly). Table 5 shows the coordinates of exons, introns, and untranslated regions in the open reading frame of an exemplary transcript encoding an HLA-DRα chain.

[0221] (Table 5) Coordinates of exons and introns of the human HLA-DRA gene locus (GRCh38, chromosome 6, forward strand) TIFF2026090319000006.tif52165

[0222] An exemplary precursor human HLA-DRβ chain polypeptide sequence is shown at SEQ ID NO:89 (the mature polypeptide includes residues 30–266 of SEQ ID NO:89; see Uniprot accession number P04229-1; mRNA sequence shown at SEQ ID NO:90, GenBank: X03069.1). In humans, HLA-DRB1, the exemplary locus encoding the HLA-DRβ chain, contains an open reading frame with six exons and five introns. The exemplary mRNA transcript of HLA-DRB1 spans the sequence corresponding to the reversed strand on chromosome 6:32,578,769–32,589,848, referring to the human genome version GRCh38 (UCSC Genome Browser on Human Dec. 2013 (GRCh38 / hg38) Assembly). Table 6 shows the coordinates of the exons, introns, and untranslated regions of the open reading frame of the exemplary transcript encoding the HLA-DRβ chain.

[0223] (Table 6) Coordinates of exons and introns of the human HLA-DRB1 gene locus (GRCh38, chromosome 6, forward strand) TIFF2026090319000007.tif56165

[0224] In some embodiments, engineered cells containing a modified T cell stimulation-related locus, for example, a transgene encoding a recombinant receptor or a portion thereof functionally linked to an endogenous transcriptional regulatory element of the T cell stimulation-related locus, also contain gene disruption in the endogenous T cell receptor α constant region (TRAC) gene and / or the endogenous T cell receptor β constant region (TRBC) gene. In some aspects, additional gene disruption at the endogenous TRAC and / or TRBC loci inhibits the expression of the endogenous TCR in the engineered cells, thereby inhibiting the expression of a transgene encoding a recombinant receptor or a portion thereof, for example, in the absence of recombinant receptor-mediated stimulation or activation signals. In some aspects, additional gene disruption at the endogenous TRAC and / or TRBC loci inhibits the re-expression of a transgene encoding a recombinant receptor or a portion thereof, in the absence of antigen-specific stimulation or activation signals, for example, in the absence of tumor-specific antigens after tumor elimination.

[0225] In some embodiments, endogenous TCR Cα is encoded by the TRAC gene (IMGT nomenclature). Exemplary human TCR Cα polypeptide sequences are shown in SEQ ID NO: 91 or 92 (see UniProtKB accession number P01848 or Genbank accession number CAA26636.1; mRNA sequence shown in SEQ ID NO: 93, GenBank: X02592.1). In humans, the exemplary genomic locus of TRAC contains an open reading frame with four exons and three introns. Exemplary mRNA transcripts of TRAC can span sequences corresponding to coordinates on the forward strand of chromosome 14: 22,547,506–22,552,154, referring to the human genome version GRCh38 (UCSC Genome Browser on Human Dec. 2013 (GRCh38 / hg38) Assembly). Table 7 shows the coordinates of exons, introns, and untranslated regions of the open reading frame of an exemplary human TRAC locus transcript.

[0226] (Table 7) Exemplary human TRAC locus exon and intron coordinates (GRCh38, chromosome 14, forward strand) TIFF2026090319000008.tif38165

[0227] In some embodiments, endogenous TCR Cβ is encoded by the TRBC1 or TRBC2 gene (IMGT nomenclature). Exemplary human TCR Cβ polypeptide sequences are shown in SEQ ID NO: 94, 95, or 96 (UniProtKB accession number P01850, A0A5B9, or A0A0G2JNG9; mRNA sequence shown in SEQ ID NO: 97; see GenBank: X00437.1).

[0228] In humans, the exemplary genomic locus of TRBC1 contains an open reading frame with four exons and three introns. The exemplary TRBC1 mRNA transcript can span sequences corresponding to coordinates on the forward strand of chromosome 7:142,791,694–142,793,368, referring to the human genome version GRCh38 (UCSC Genome Browser on Human Dec. 2013 (GRCh38 / hg38) Assembly). Table 8 shows the coordinates of the exons, introns, and untranslated regions of the open reading frame of the exemplary human TRBC1 locus transcript.

[0229] (Table 8) Exemplary coordinates of exons and introns of the human TRBC1 locus (GRCh38, chromosome 7, forward strand) TIFF2026090319000009.tif38165

[0230] In humans, the exemplary genomic locus of TRBC2 contains an open reading frame with four exons and three introns. The exemplary mRNA transcript of TRBC2 can span sequences corresponding to coordinates on the forward strand of chromosome 7:142,801,041–142,802,748, referring to the human genome version GRCh38 (UCSC Genome Browser on Human Dec. 2013 (GRCh38 / hg38) Assembly). Table 9 shows the coordinates of the exons, introns, and untranslated regions of the open reading frame of the transcript of the exemplary human TRBC2 locus.

[0231] (Table 9) Exemplary coordinates of exons and introns of the human TRBC2 locus (GRCh38, chromosome 7, forward strand) TIFF2026090319000010.tif38165

[0232] In some of the embodiments, gene disruption is targeted to, near, or within the open reading frame of a T cell stimulation-related locus, TRAC, and / or TRBC (as listed, for example, in Tables 1-9 herein). In certain embodiments, gene disruption is targeted to, near, or within the open reading frame encoding the TCRα constant domain. In some embodiments, gene disruption is targeted to, all or part of, a T cell stimulation-related locus, TRAC, and / or TRBC (as listed, for example, in Tables 1-9 herein), e.g., 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, or 4,000, or at least 500, 1,0 Targeting is performed near, or within, sequences having 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to 00, 1,500, 2,000, 2,500, 3,000, 3,500, or 4,000 consecutive nucleotides, or at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity.

[0233] In some aspects, the target site is located within an exon of the open reading frame of an endogenous T cell stimulation-related locus, TRAC, and / or TRBC. In some aspects, the target site is located within an intron of the open reading frame of an endogenous T cell stimulation-related locus, TRAC, and / or TRBC. In some aspects, the target site is located within a regulatory or regulatory element of an T cell stimulation-related locus, TRAC, and / or TRBC, such as a promoter, 5' untranslated region (UTR), or 3' UTR. In some embodiments, the target site is located within the T cell stimulation-related locus, TRAC, and / or TRBC genomic region sequence listed in Tables 1-9 herein, or within any exon or intron of a T cell stimulation-related locus, TRAC, and / or TRBC genomic region sequence contained therein. In some cases, the target site is at or near the junction or boundary between an exon and an intron, or between an exon and a regulatory or regulatory element, such as a promoter, 5' untranslated region (UTR), or 3' UTR, of an endogenous T cell stimulation-related locus, TRAC, and / or TRBC. In some cases, the target site is within an intron of the open reading frame of the T cell stimulation-related locus, TRAC, and / or TRBC.

[0234] In some embodiments, target sites for gene disruption are selected such that, after the integration of the transgene, the cell is knocked out, reduced, and / or eliminated from the expression of endogenous T cell stimulation-associated loci, TRAC, and / or TRBC.

[0235] In some embodiments, gene disruption, such as DNA breaks, is targeted within the exons of the T cell stimulation-associated locus, TRAC, and / or TRBC, or its open reading frame. In certain embodiments, gene disruption is located within the first, second, third, or fourth exon of the T cell stimulation-associated locus, TRAC, and / or TRBC, or its open reading frame. In some embodiments, gene disruption is located within the first exon of the T cell stimulation-associated locus, TRAC, and / or TRBC, or its open reading frame. In some embodiments, gene disruption is located within 500 base pairs (bp) downstream of the 5' end of the first exon of the T cell stimulation-associated locus, TRAC, and / or TRBC, or its open reading frame. In some embodiments, gene disruption is located between the 5' nucleotide of exon 1 and the upstream of the 3' nucleotide of exon 1. In certain embodiments, the gene disruption is located within 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100 bp, or 50 bp downstream from the 5' end of the first exon in the T cell stimulation-related locus, TRAC, and / or TRBC, or its open reading frame. In some of the embodiments, the gene disruption is located within 1 bp to 400 bp, 50 to 300 bp, 100 bp to 200 bp, or 100 bp to 150 bp downstream from the 5' end of the first exon in the T cell stimulation-related locus, TRAC, and / or TRBC, or its open reading frame, including the values ​​at both ends. In certain embodiments, the gene disruption is located within 100 bp to 150 bp downstream from the 5' end of the first exon in the T cell stimulation-related locus, TRAC, and / or TRBC, or its open reading frame, including the values ​​at both ends.

[0236] In some aspects, the target site is located within an exon, such as an exon corresponding to the initial coding region. In some embodiments, the target site is located within or very close to an exon corresponding to the initial coding region, for example, exons 1, 2, 3, 4, or 5 of the open reading frame of an endogenous T cell stimulation-related locus, TRAC, and / or TRBC (as listed in Tables 1-9 herein), or within or within exons 1, 2, 3, 4, or 5 immediately following the transcription start site, or within sequences of less than 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 bp in exons 1, 2, 3, 4, or 5. In some aspects, the target site is located within a regulatory or regulatory element of a T cell stimulation-related locus, TRAC, and / or TRBC, such as a promoter.

[0237] In certain embodiments, gene disruption is targeted to, near, or within T cell stimulation-related loci, TRACs, and / or TRBCs. In some of the embodiments, gene disruption is targeted to, near, or within the open reading frames of T cell stimulation-related loci, TRACs, and / or TRBCs (as described, for example, in Tables 1-9 herein). In certain embodiments, gene disruption is targeted to, near, or within the open reading frames encoding T cell stimulation-related loci, TRACs, and / or TRBCs. In some embodiments, gene disruption is performed on T cell stimulation-related loci, TRACs, and / or TRBCs (as listed in Tables 1-9 of this specification), or on all or part of T cell stimulation-related loci, TRACs, and / or TRBCs (as listed in Tables 1-9 of this specification), for example, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, or 4,000, or at least 500, 1,0 Targeting is performed near, or within, sequences having 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to 00, 1,500, 2,000, 2,500, 3,000, 3,500, or 4,000 consecutive nucleotides, or at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity.

[0238] In some aspects, a transgene (e.g., an exogenous nucleic acid sequence) within a template polynucleotide can be used to guide the location of the target site and / or homology arms. In some aspects, the target site of gene disruption can be used as a guide for designing the template polynucleotide and / or homology arms used in HDR. In some embodiments, gene disruption can be targeted near the desired site of targeted integration of the transgene (e.g., encoding a recombinant receptor or a portion thereof). In some embodiments, one or more homology arm sequences of the template polynucleotide are designed to surround the site of gene disruption (target site). In some aspects, gene disruption is targeted so that the expression of endogenous T cell stimulation-associated loci, TRAC, and / or TRBCs is reduced or eliminated upon integration of the transgene encoding a recombinant receptor. In some aspects, gene disruption is targeted so that all or a portion of the endogenous T cell stimulation-associated loci, TRAC, and / or TRBCs are expressed upon integration of the transgene encoding a recombinant receptor. In some aspects, the target site is located within or near the exons of the endogenous T cell stimulation-associated loci, TRAC, and / or TRBC, so that the transgene encoding the recombinant receptor can be incorporated in frame with the coding sequences of the expressed T cell stimulation-associated loci, TRAC, and / or TRBC.

[0239] 2. Methods of gene disruption In some aspects, methods for generating genetically modified cells include the step of introducing gene disruption to one or more target sites, e.g., one or more target sites of T cell stimulation-associated loci, TRACs and / or TRBCs. Methods for generating gene disruption, including those described herein, may include the use of an engineered system that induces gene disruption, e.g., one or more active agents capable of inducing gene disruption, e.g., gene disruption, cleavage and / or double-strand breaks (DSBs) or nicks (e.g., single-strand breaks (SSBs)) at or near the target site or location, resulting in repair of the breaks by processes that produce errors such as non-homologous end joining (NHEJ), or repair by HDR using a repair template, which may result in the insertion of a sequence of interest (e.g., an exogenous nucleic acid sequence or transgene encoding a recombinant receptor or a portion thereof) at or near the target site or location. Also provided herein are one or more active agents capable of inducing gene disruption for use in the methods provided herein. In some aspects, one or more activators can be used in combination with the template nucleotides provided herein for homology-directed repair (HDR)-mediated target incorporation of transgenes.

[0240] In some embodiments, one or more active agents capable of inducing gene disruption include DNA-binding proteins or DNA-binding nucleic acids that specifically bind to or hybridize to a particular site or location in the genome, e.g., a target site or location. In some embodiments, targeted gene disruption, e.g., DNA cleavage or cleavage, at endogenous T cell stimulation-associated loci, TRACs and / or TRBCs is achieved using proteins or nucleic acids, where the proteins or nucleic acids bind to or complex with gene-editing nucleases, e.g., in chimeric or fusion proteins. In some embodiments, one or more active agents capable of inducing gene disruption include RNA-inducible nucleases or fusion proteins comprising a DNA-targeting protein and a nuclease.

[0241] In some embodiments, the active agent comprises various components, such as RNA-induced nucleases, or fusion proteins comprising a DNA-targeting protein and a nuclease. In some embodiments, target gene disruption is performed using a DNA-targeting molecule comprising a nuclease, such as an endonuclease, fused with a DNA-binding protein, such as one or more zinc finger proteins (ZFPs) or a transcription activator-like effector (TALE). In some embodiments, target gene disruption is performed using an RNA-induced nuclease, such as a clustered regularly interspaced short palindromic nucleic acid (CRISPR)-associated nuclease (Cas) system (including Cas and / or Cfp1). In some embodiments, targeted gene disruption is performed using active agents capable of inducing gene disruption, such as sequence-specific or targeted nucleases, which include DNA-binding targeted nucleases and gene-editing nucleases, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), as well as RNA-inducible nucleases, such as CRISPR-related nuclease (Cas) systems, which are specifically designed to target at least one target site, gene sequence, or portion thereof. Exemplary ZFNs, TALENs, and TALENs are described, for example, in Lloyd et al., Frontiers in Immunology, 4(221): 1-7 (2013).

[0242] Zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs), and CRISPR system binding domains can be "engineered" to bind to a given nucleotide sequence, for example, by manipulating the recognition helix region (modification of one or more amino acids) of a naturally occurring ZFP or TALE protein. The engineered DNA-binding protein (ZFP or TALE) is a protein that does not exist naturally. Reasonable criteria for design include substitution rules, as well as the application of computerized algorithms for processing information in databases storing information on the design and binding of existing ZFPs and / or TALEs. See, for example, U.S. Patents 6,140,081; 6,453,242; and 6,534,261. See also WO98 / 53058; WO98 / 53059; WO98 / 53060; WO02 / 016536, and WO03 / 016496, as well as U.S. Patent Application Publication 20110301073.

[0243] In some embodiments, one or more activators specifically target at least one target site in or near a T cell stimulation-associated locus, TRAC and / or TRBC. In some embodiments, the activator includes a combination of ZFN, TALEN, or CRISPR / Cas9 that specifically binds to, recognizes, or hybridizes to the target site. In some embodiments, the CRISPR / Cas9 system includes an engineered crRNA / tracr RNA ("single guide RNA") to guide specific cleavage. In some embodiments, the active ingredient comprises an Argonaut system-based nuclease (e.g., derived from T. thermophilus, known as "TtAgo" (Swarts et al. (2014) Nature 507(7491): 258-261)). By leveraging targeted cleavage using any of the nuclease systems described herein, the sequence of the transgene, e.g., a nucleic acid sequence encoding a recombinant receptor or a portion thereof, can be inserted into specific target sites of endogenous T cell stimulation-associated loci, TRACs and / or TRBCs, using either an HDR or NHEJ-mediated process.

[0244] In some embodiments, a “zinc finger DNA-binding protein” (or binding domain) is a protein or a domain within a larger protein that binds sequence-specifically to DNA via one or more zinc fingers, which are regions of amino acid sequences within a binding domain whose structure is stabilized via the coordination of zinc ions. The term zinc finger DNA-binding protein is often abbreviated as zinc finger protein or ZFP. Among ZFPs are artificial ZFP domains, typically 9–18 nucleotides long, that target specific DNA sequences, generated by the assembly of individual fingers. ZFPs include single-finger domains that are approximately 30 amino acids long and contain an alpha-helix with two immutable histidine residues coordinated with two cysteine ​​cysteines in a single beta turn via zinc, and may have two, three, four, five, or six fingers. Generally, the sequence specificity of a ZFP can be modified by making amino acid substitutions at four helical positions (-1, 2, 3, and 6) on the zinc finger recognition helix. Therefore, for example, ZFPs or ZFP-containing molecules do not exist in nature and are, for example, engineered to bind to a selective target site.

[0245] In some cases, the DNA targeting molecule is a zinc finger DNA-binding domain fused to a DNA cleavage domain to form a zinc finger nuclease (ZFN), or contains such a zinc finger DNA-binding domain. For example, the fusion protein contains a cleavage domain (or cleavage half-domain) derived from at least one IIS-type restriction enzyme, and one or more zinc finger-binding domains, which may or may not be manipulated. In some cases, the cleavage domain is derived from the IIS-type restriction endonuclease FokI, which generally catalyzes double-strand cleavage of DNA at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand. See, for example, U.S. Patent Nos. 5,356,802; 5,436,150 and 5,487,994; Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994) J. Biol. Chem. 269: 978-982. Several gene-specific engineered zinc fingers are commercially available. For example, a platform called CompoZr for zinc finger construction is available, which provides zinc fingers specifically targeted to thousands of targets. See, for example, Gaj et al., Trends in Biotechnology, 2013, 31(7), 397-405. In some cases, commercially available zinc fingers are used or specially designed. In some embodiments, for example, one or more target sites within T cell stimulation-associated loci, TRAC and / or TRBC may be targeted for gene disruption by the engineered ZFN.

[0246] Transcription activator-like effectors (TALEs) are proteins derived from the bacterial species Xanthomonas and contain multiple repeat sequences, each repeat containing two residues (RVDs) at positions 12 and 13 that are specific to each nucleotide base of the nucleic acid target sequence. Binding domains with similar modular base-per-base nucleic acid binding properties (MBBBDs) may also originate from different bacterial species. Novel modular proteins have the advantage of exhibiting more sequence diversity than TAL repeats. In some embodiments, the RVDs associated with the recognition of different nucleotides are HD for C recognition, NG for T recognition, NI for A recognition, NN for G or A recognition, NS for A, C, G, or T recognition, HG for T recognition, IG for T recognition, NK for G recognition, HA for C recognition, ND for C recognition, HI for C recognition, HN for G recognition, NA for G recognition, SN for G or A recognition, and YG for T recognition, TL for A recognition, VT for A or G recognition, and SW for A recognition. In some embodiments, key amino acids 12 and 13 can be mutated into other amino acid residues in order to modulate their specificity for nucleotides A, T, C, and G, and in particular to enhance this specificity.

[0247] In some embodiments, a “TALE DNA-binding domain” or “TALE” is a polypeptide comprising one or more TALE repeat domains / units. Each repeat domain, containing two repeat variable residues (RVDs), is involved in the binding of the TALE to its homogeneous target DNA sequence. A single “repeat unit” (also referred to as a “repeat”) is typically 33–35 amino acids long and exhibits at least some degree of sequence homology to other TALE repeat sequences within naturally occurring TALE proteins. TALE proteins can be designed to bind to target sites using standard or non-standard RVDs within the repeat unit. See, for example, U.S. Patents 8,586,526 and 9,458,205.

[0248] In some embodiments, a "TALE nuclease" (TALEN) is a fusion protein typically comprising a nucleic acid-binding domain derived from a transcription activator-like effector (TALE) and a nuclease catalytic domain that cleaves nucleic acid target sequences. The catalytic domain includes a nuclease domain, or a domain with endonuclease activity, such as I-TevI, ColE7, NucA, and Fok-I. In certain embodiments, the TALE domain can be fused to meganucleases, such as I-CreI and I-OnuI or their functional variants. In some embodiments, the TALEN is a monomeric TALEN. A monomeric TALEN is a TALEN that does not require dimerization for specific recognition and cleavage, e.g., fusion of an engineered TAL repeat with the catalytic domain of I-TevI ​​as described in WO2012138927. TALENs have been described and used for gene targeting and gene modification (see, for example, Boch et al. (2009) Science 326(5959): 1509-12; Moscou and Bogdanove (2009) Science 326(5959): 1501; Christian et al. (2010) Genetics 186(2): 757-61; Li et al. (2011) Nucleic Acids Res 39(1): 359-72). In some embodiments, one or more sites in T cell stimulation-related loci, TRACs and / or TRBCs may be targeted for gene disruption by manipulated TALENs.

[0249] In some aspects, "TtAgo" is a prokaryotic Argonaut protein thought to be involved in gene silencing. TtAgo originates from the bacterium Thermus thermophilus. See, for example, Swarts et al (2014) Nature 507(7491): 258-261; G. Sheng et al., (2013) Proc. Natl. Acad. Sci. USA 111, 652. The "TtAgo system" refers to all the necessary components, including the guide DNA for cleavage by the TtAgo enzyme.

[0250] In some embodiments, engineered zinc finger proteins, TALE proteins, or CRISPR / Cas systems are not found in nature, and their production arises primarily from experimental processes such as phage display, interaction trapping, or hybrid selection. See, for example, U.S. Patents 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,200,759; WO95 / 19431; WO96 / 06166; WO98 / 53057; WO98 / 54311; WO00 / 27878; WO01 / 60970; WO01 / 88197 and WO02 / 099084.

[0251] Zinc finger and TALE DNA-binding domains can be manipulated to bind to a given nucleotide sequence, for example, by manipulating the recognition helix region of a naturally occurring zinc finger protein (by changing one or more amino acids), or by manipulating the amino acids involved in DNA binding (a repeating variable 2-residue or RVD region). Thus, a manipulated zinc finger protein or TALE protein is a protein that does not exist in nature. Non-limiting examples of methods for manipulating zinc finger proteins and TALEs are design and selection. A designed protein is a protein that does not exist in nature, whose design / composition is derived from reasonable criteria. Reasonable criteria for design include substitution rules, as well as the application of computerized algorithms for processing information in databases storing information on existing ZFP or TALE designs (standard and non-standard RVDs) and binding data. See, for example, U.S. Patents 9,458,205; 8,586,526; 6,140,081; 6,453,242; and 6,534,261. See also WO98 / 53058;WO98 / 53059;WO98 / 53060;WO02 / 016536, and WO03 / 016496.

[0252] Various methods and compositions for targeted cleavage of genomic DNA are described. Such targeted cleavage events can be used, for example, to induce targeted mutagenesis, to induce targeted deletions of cellular DNA sequences, and to promote targeted recombination at a given chromosomal locus. For example, the disclosure is incorporated in its entirety by reference in U.S. Patents 9,255,250; 9,200,266; 9,045,763; 9,005,973; 9,150,847; 8,956,828; 8,945,868; 8,703,489; and 8,586,526. No. 6,534,261; No. 6,599,692; No. 6,503,717; No. 6,689,558; No. 7,067,317; No. 7,262, No. 054; No. 7,888,121; No. 7,972,854; No. 7,914,796; No. 7,951,925; No. 8,110,379; No. 8,4 09,861; U.S. Patent Publication No. 20030232410; No. 20050208489; No. 20050026157; No. 20050064474; No. 20060063231; No. 20080159996; No. 201000218264; No. 20120017290; No. 20110265198 See issues No. 20130137104; No. 20130122591; No. 20130177983; No. 20130196373; No. 20140120622; No. 20150056705; No. 20150335708; No. 20160030477, and No. 20160024474.

[0253] a. CRISPR / Cas9 In some embodiments, target gene disruption, e.g., DNA cleavage, of endogenous T cell stimulation-associated loci, TRAC and / or TRBC, in humans is carried out using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-related (Cas) proteins. See Sander and Joung (2014) Nature Biotechnology, 32(4): 347-355.

[0254] Generally, the “CRISPR system” refers collectively to the transcripts and other elements involved in directing the expression or activity of CRISPR-related (“Cas”) genes, including the Cas gene encoding sequence, tracr (trans-activated CRISPR) sequences (e.g., tracr RNA or active partial tracr RNA), tracr mate sequences (including “serial repeat sequences” and partially serial repeat sequences processed by tracr RNA in the context of an endogenous CRISPR system), guide sequences (also referred to as “spacers” in the context of an endogenous CRISPR system), and / or other sequences and transcripts derived from the CRISPR locus.

[0255] In some aspects, a CRISPR / Cas nuclease or CRISPR / Cas nuclease system comprises a sequence-specific non-coding guide RNA (gRNA) that binds to DNA and a Cas protein (e.g., Cas9) that has nuclease functionality.

[0256] Also provided are one or more active substances capable of introducing gene disruption. Furthermore, polynucleotides (e.g., nucleic acid molecules) encoding one or more components of one or more active substances capable of inducing gene disruption are also provided.

[0257] (i) Guide RNA (gRNA) In some embodiments, one or more active agents capable of inducing gene disruption include at least one of guide RNA (gRNA) or at least one nucleic acid encoding gRNA having a targeting domain complementary to the target sites of T cell stimulation-associated loci, TRAC and / or TRBC.

[0258] In some aspects, a “gRNA molecule” is a nucleic acid that facilitates the specific targeting or homing of a gRNA molecule / Cas9 molecule complex to a target nucleic acid, such as a gene locus on the cellular genomic DNA. A gRNA molecule can be monomolecule (having a single RNA molecule) or modular (containing more than one, typically two, separate RNA molecules), sometimes referred to herein as a “chimeric” gRNA. Generally, a guide sequence, e.g., a guide RNA, is any polynucleotide sequence that includes at least a sequence portion having sufficient complementarity with the target polynucleotide sequence, for example, the T cell stimulation-associated gene locus, TRAC and / or TRBC in humans, to hybridize with the target sequence at the target site and lead to sequence-specific binding of the CRISPR complex to the target sequence. In some aspects, in the context of CRISPR complex formation, the “target sequence” is a sequence in which the guide sequence is designed to be complementary, and hybridization between the target sequence and the domain of the guide RNA, e.g., the targeting domain, facilitates the formation of the CRISPR complex. Complete complementarity is not necessarily required, provided that there is sufficient complementarity to induce hybridization and promote the formation of the CRISPR complex. Generally, guide sequences are selected to reduce the degree of secondary structure within the guide sequence. The secondary structure can be determined by any suitable polynucleotide folding algorithm.

[0259] In some embodiments, a guide RNA (gRNA) specific to a target locus of interest (e.g., T cell stimulation-associated loci, TRAC and / or TRBC in humans) is used for an RNA-induced nuclease, such as Cas, to induce DNA cleavage at a target site or location. Methods for designing gRNAs and exemplary targeting domains include, for example, those described in International PCT Publication Nos. WO2015 / 161276, WO2017 / 193107, and WO2017 / 093969.

[0260] Several exemplary gRNA structures in which domains are shown are described in WO2015 / 161276, for example in Figures 1A–1G therein. While we do not wish to be constrained by theory, the three-dimensional morphology of the active form of gRNA, or regions of high complementarity with respect to intra-chain or inter-chain interactions, are sometimes shown as double helixes in WO2015 / 161276, for example in Figures 1A–1G therein, and in other depictions provided herein.

[0261] In some cases, gRNAs are monomolecular or chimeric gRNAs comprising, from 5' to 3', a targeting domain complementary to the target nucleic acid, such as a sequence derived from a T cell stimulation-associated locus, TRAC and / or TRBC gene; a first complementarity domain; a ligation domain; a second complementarity domain (complementary to the first complementarity domain); a proximal domain; and optionally, a tail domain.

[0262] In some cases, the gRNA is a modular gRNA comprising first and second strands. In these cases, the first strand preferably comprises, 5' to 3', a targeting domain (complementary to the target nucleic acid, such as a sequence derived from a T cell stimulation-associated locus, TRAC, and / or TRBC gene) and a first complementarity domain. The second strand generally comprises, 5' to 3', optionally a 5' extension domain; a second complementarity domain; a proximal domain; and optionally a tail domain.

[0263] (a) Targeting domains A targeting domain includes a nucleotide sequence that is complementary to the target sequence on the target nucleic acid, for example, at least 80, 85, 90, 95, 98, or 99% complementary, for example, perfectly complementary. The strand of target nucleic acid containing the target sequence is referred to herein as the “complementary strand” of the target nucleic acid. Guidance on the selection of targeting domains can be found, for example, in Fu et al., Nat Biotechnol 2014 Mar;32(3):279-284) and Sternberg et al., Nature 2014, 507:62-67. Examples of targeting domain configurations include those described in Figures 1A-1G, for example, in WO2015 / 161276.

[0264] The targeting domain is part of the RNA molecule and therefore contains the base uracil (U), while any DNA encoding a gRNA molecule contains the base thymine (T). While we do not wish to be bound by theory, in some embodiments, the complementarity between the targeting domain and the target sequence is thought to contribute to the specificity of the interaction between the gRNA / Cas9 molecule complex and the target nucleic acid. In the pairing of the targeting domain and the target sequence, it is understood that the uracil base in the targeting domain pairs with the adenine base in the target sequence. In some embodiments, the target domain itself contains any secondary and core domains in the 5' to 3' direction. In some embodiments, the core domain is perfectly complementary to the target sequence. In some embodiments, the targeting domain is 5 to 50 nucleotides long. The chain of target nucleic acid to which the targeting domain is complementary is referred to herein as the complementary chain. Some or all of the nucleotides in the domain may have modifications, such as making them less susceptible to degradation or improving biocompatibility. As a non-limiting example, the backbone of the target domain can be modified with phosphorothioates or other modifications. In some cases, the nucleotides of the targeting domain may include 2' modifications, such as 2-acetylation, 2'-methylation, or other modifications.

[0265] In various embodiments, the targeting domain is 16 to 26 nucleotides long (i.e., it is 16 nucleotides long, or 17 nucleotides long, or 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides long).

[0266] (b) Exemplary targeting domains In some aspects, targeting domain sequences or gRNA sequences containing such sequences are designed or identified to target specific target sites in genes, such as T cell stimulation-associated loci, TRAC, and / or TRBC. Genome-wide gRNA databases for CRISPR genome editing are publicly available and contain exemplary single guide RNA (sgRNA) sequences targeting constitutive exons of genes in the human or mouse genome (see, for example, genescript.com / gRNA-database.html; also see Sanjana et al. (2014) Nat. Methods, 11:783-4). In some aspects, gRNA sequences are sequences that have minimal off-target binding to non-target sites or locations, or contain such binding.

[0267] In some embodiments, the target sequence (target domain) is located at or near a T cell stimulation-associated locus, TRAC, and / or TRBC, such as any part of a T cell stimulation-associated locus, TRAC, and / or TRBC. In some embodiments, a target nucleic acid complementary to the targeting domain is located in the early coding region of the gene of interest, such as a T cell stimulation-associated locus, TRAC, and / or TRBC. Targeting the early coding region can be used to disrupt the gene of interest (i.e., eliminate its expression). In some embodiments, the early coding region of the gene of interest contains a sequence immediately following the start codon (e.g., ATG) or within 500 bp of the start codon (e.g., 500, 450, 400, 350, 300, 250, 200, 150, 100, 50 bp, 40 bp, 30 bp, 20 bp, or less than 10 bp). In certain cases, the target nucleic acid is within 200 bp, 150 bp, 100 bp, 50 bp, 40 bp, 30 bp, 20 bp, or 10 bp of the start codon. In some cases, the targeting domain of the gRNA is complementary to the target sequence on the target nucleic acid, such as the target nucleic acid in T cell stimulation-associated loci, TRAC, and / or TRBC, e.g., at least 80, 85, 90, 95, 98, or 99% complementary, e.g., perfectly complementary. In some embodiments, the targeting domain is located downstream and / or near an endogenous transcriptional regulatory element of an endogenous T cell stimulation-associated locus, TRAC, and / or TRBC, e.g., a promoter.

[0268] In some aspects, gRNA can target sites of T cell stimulation-related loci, TRACs, and / or TRBCs near the desired site of targeted integration of a transgene encoding a recombinant receptor, for example. In some aspects, gRNA can target sites based on the amount of sequences encoding T cell stimulation-related loci, TRACs, and / or TRBCs, which are desirable for controlling recombinant receptor expression in a manner, time, and degree similar to the control of endogenous T cell stimulation-related loci, TRACs, and / or TRBCs. In some aspects, gRNA can target sites based on the amount of sequences encoding T cell stimulation-related loci, TRACs, and / or TRBCs, which are desirable for expression in cells expressing recombinant receptors. In some aspects, gRNA can target sites such that, upon integration of a transgene sequence encoding a recombinant receptor, the resulting T cell stimulation-related loci, TRACs, and / or TRBCs maintain the expression of endogenous gene products encoded by T cell stimulation-related loci, TRACs, and / or TRBCs. In some cases, endogenous gene products are not expressed (e.g., knocked out) following gRNA targeting and subsequent HDR. In some cases, gRNA can target sites within the exons of the open reading frames of endogenous T cell stimulation-associated loci, TRAC, and / or TRBC. In some cases, gRNA can target sites within the introns of the open reading frames of T cell stimulation-associated loci, TRAC, and / or TRBC. In some cases, gRNA can target regulatory or regulatory elements of T cell stimulation-associated loci, TRAC, and / or TRBC, such as sites within or downstream of the promoter. In some cases, the target sites of T cell stimulation-associated loci, TRAC, and / or TRBC targeted by gRNA may be any target sites described herein, for example, in Section II.A.1.In some embodiments, the gRNA can target exons corresponding to the initial coding region, such as exons 1, 2, 3, 4, or 5 of the open reading frame of the endogenous T cell stimulation-associated locus, TRAC, and / or TRBC, or sites within or very close to them, or sites within or containing sequences of less than 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 bp immediately following the transcription start site in exons 1, 2, 3, 4, or 5. In some embodiments, the gRNA can target exon 2 or nearby sites of the endogenous T cell stimulation-associated locus, TRAC, and / or TRBC, or sites within or containing less than 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 bp of exon 2.

[0269] Exemplary target sequences for the T cell stimulation-associated locus PDCD1 include sequences shown in SEQ ID NO: 74, 78, or 98-103. Exemplary gRNAs may include ribonucleic acid sequences that can bind to, target, complement to, or bind to the target site sequences shown in SEQ ID NO: 74, 78, or 98-103, or to their complementary strand sequences. Exemplary PDCD1 gRNA sequences include sequences shown in SEQ ID NO: 75 or 104-109. Exemplary PDCD1 gRNA sequences include sequences shown in SEQ ID NO: 75. Any known method can be used to target and generate endogenous PDCD1 gene disruption, and can be used in the embodiments provided herein. Exemplary target sequences or targeting domains contained within gRNAs for targeting gene disruption at the human PDCD1 locus include, for example, those described in WO2015 / 161276, WO2017 / 093969, Schumann et al., PNAS August 18, 2015 112 (33) 10437-10442, and Xu et al., Sci Rep. 2018; 8: 11649, which are incorporated herein by reference.

[0270] Exemplary target sequences for the exemplary T cell stimulation-associated locus CD69 include sequences shown in SEQ ID NO: 110-115. Exemplary gRNAs may include ribonucleic acid sequences that can bind to, target, complement to, or bind to the target site sequences shown in SEQ ID NO: 110-115, or their complementary strand sequences. Exemplary CD69 gRNA sequences include sequences shown in SEQ ID NO: 116-121. Any known method can be used to target and generate gene disruption of endogenous CD69, and can be used in the embodiments provided herein. Exemplary target sequences or targeting domains contained within gRNAs for targeting gene disruption of the human CD69 locus include, for example, those described in Simenov et al., Nature. 2017 Sep 7; 549(7670): 111-115, which are incorporated herein by reference.

[0271] Exemplary target sequences for the exemplary T cell stimulation-associated locus Nur77(NR4A1) include sequences shown in SEQ ID NO: 122-127 or 134-136. Exemplary gRNAs may include ribonucleic acid sequences that can bind to, target, complement to, or bind to the target site sequences shown in SEQ ID NO: 122-127 or 134-136. Exemplary Nur77(NR4A1) gRNA sequences include sequences shown in SEQ ID NO: 128-133 or 136-138. Any known method can be used to target and generate endogenous Nur77(NR4A1) gene disruption, and can be used in the embodiments provided herein. Exemplary target sequences or targeting domains contained within gRNAs for targeting gene disruption at the human Nur77(NR4A1) locus include, for example, those described in WO 2019 / 089982, WO 2019 / 104245, and Munnur et al., Cell Reports (2019) 26, 2028-2036, which are incorporated herein by reference.

[0272] Exemplary target sequences for the exemplary T cell stimulation-associated locus FoxP3 include sequences shown in SEQ ID NO: 140-147. Exemplary gRNAs may include ribonucleic acid sequences that can bind to, target, complement, or bind to the target site sequences shown in SEQ ID NO: 140-147. Exemplary FoxP3 gRNA sequences include sequences shown in SEQ ID NO: 148-155. Any known method can be used to target and generate endogenous FoxP3 gene disruption, and can be used in the embodiments provided herein. Exemplary target sequences or targeting domains contained within gRNAs for targeting gene disruption at the human FoxP3 locus include, for example, those described in Okada et al., Epigenetics Chromatin. 2017; 10: 24 and Holohan et al., bioRxiv 644229, which are incorporated herein by reference.

[0273] Exemplary target sequences for exemplary T cell stimulation-associated loci HLA-DRA include sequences shown in SEQ ID NO: 156-161. Exemplary gRNAs may include ribonucleic acid sequences that can bind to, target, complement, or bind to the target site sequences shown in SEQ ID NO: 156-161. Exemplary HLA-DRA gRNA sequences include sequences shown in SEQ ID NO: 162-167. Any known method can be used to target and generate gene disruption of endogenous HLA-DRA, and can be used in the embodiments provided herein. Exemplary target sequences or targeting domains contained within gRNAs for targeting gene disruption of human HLA-DRA loci include, for example, those described in WO 2016 / 021972 and WO 2017 / 093969, which are incorporated herein by reference.

[0274] Exemplary target sequences for the exemplary T cell stimulation-associated locus HLA-DRB1 include sequences shown in SEQ ID NO: 168-177. Exemplary gRNAs may include ribonucleic acid sequences that can bind to, target, complement, or bind to the target site sequences shown in SEQ ID NO: 168-177. Exemplary HLA-DRB1 gRNA sequences include sequences shown in SEQ ID NO: 178-187. Any known method can be used to target and generate gene disruption of endogenous HLA-DRB1 and can be used in the embodiments provided herein. Exemplary target sequences or targeting domains contained within gRNAs for targeting gene disruption of the human HLA-DRB1 locus include, for example, those described in WO 2016 / 021972 and WO 2017 / 093969, which are incorporated herein by reference.

[0275] Exemplary targeting domains contained in gRNA names for targeting gene disruption of human TRAC, TRBC1, or TRBC2 include, for example, those described in WO2015 / 161276, WO2017 / 193107, WO2017 / 093969, WO2019 / 195492, US2016 / 272999, and US2015 / 056705, or targeting domains capable of binding to the aforementioned target sequences. Exemplary targeting domains contained in gRNAs for targeting gene disruption of the human TRAC locus using Cas9 of Streptococcus pyogenes or Staphylococcus aureus may include any of those shown in SEQ ID NO: 77 and 188-218. Exemplary targeting domains contained within gRNAs for targeting gene disruption of the human TRBC1 or TRBC2 locus using Cas9 of Streptococcus pyogenes or Staphylococcus aureus may include any of those shown in SEQ ID NO:219-276.

[0276] In some embodiments, the gRNA for targeting TRAC, TRBC1, and / or TRBC2 includes a targeting domain that can bind to any of the target sequences described herein or elsewhere, e.g., WO2015 / 161276, WO2017 / 193107, WO2017 / 093969, WO2019 / 195492, US2016 / 272999, and US2015 / 056705, or the target sequences described above. In some embodiments, the gRNA for targeting the TRAC locus includes a targeting domain that can bind to any of the target sequences described herein or elsewhere, e.g., WO2015 / 161276, WO2017 / 193107, WO2017 / 093969, WO2019 / 195492, US2016 / 272999, and US2015 / 056705, or the target sequences described above. The gRNA can be obtained by in vitro transcription of TIFF2026090319000011.tif17165 (shown as SEQ ID NO:277; the bolded and underlined portion is complementary to the target site in the TRAC gene locus) or by chemical synthesis, and the gRNA is sequence It had TIFF2026090319000012.tif17157 (shown as SEQ ID NO:278; see Osborn et al., Mol Ther. 24(3):570-581 (2016)). Other exemplary gRNA sequences for generating gene disruption of endogenous genes encoding TCR domains or regions, e.g., TRAC, TRBC1, and / or TRBC2, are described, for example, WO2015 / 161276, WO2017 / 193107, WO2017 / 093969, WO2019 / 195492, US2016 / 272999, and US2015 / 056705.

[0277] Exemplary methods for gene editing of endogenous TCR loci include, for example, U.S. Publication numbers US2011 / 0158957, US2014 / 0301990, US2015 / 0098954, US2016 / 0208243, US2016 / 272999, and US2015 / 056705; International PCT Publication numbers WO2014 / 191128, WO2015 / 136001, WO2015 / 161276, WO2016 / 069283, WO2016 / 016341, WO2017 / 193107, and WO2017 / 093969; and those described in Osborn et al. (2016) Mol. Ther. 24(3):570-581. Any known method can be used to generate gene disruption of an endogenous gene encoding a TCR domain or region, and can be used in the embodiments provided herein.

[0278] In some embodiments, the targeting domain includes one for introducing gene disruption at the TRAC, TRBC1, and / or TRBC2 loci using Streptococcus pyogenes Cas9 or Neisseria meningitidis Cas9. In some embodiments, the targeting domain includes one for introducing gene disruption at the TRAC, TRBC1, and / or TRBC2 loci using Streptococcus pyogenes Cas9. Any of the targeting domains can be used with Streptococcus pyogenes Cas9 molecules that produce double-strand breaks (Cas9 nuclease) or single-strand breaks (Cas9 nickase).

[0279] In some embodiments, dual targeting is used to create two nicks on the reverse DNA strand by using a Cas9 nickase from Streptococcus pyogenes having two targeting domains complementary to the reverse DNA strand, for example, a gRNA containing any minus-strand targeting domain can be paired with any gRNA containing a plus-strand targeting domain. In some embodiments, the two gRNAs are oriented on the DNA such that the PAM faces outward and the distance between the 5' ends of the gRNAs is 0-50 bp. In some embodiments, the two gRNAs are used to target two Cas9 nucleases or two Cas9 nickases using a pair of Cas9 molecule / gRNA molecule complexes guided by two different gRNA molecules to cleave the target domain with two single-strand breaks on the reverse strand of the target domain, for example. In some embodiments, the two Cas9 nickase molecules may include a molecule having HNH activity, for example, a Cas9 molecule with inactivated RuvC activity, for example, a Cas9 molecule with a mutation at D10, for example, a D10A mutation; a molecule having RuvC activity, for example, a Cas9 molecule with inactivated HNH activity, for example, a Cas9 molecule with a mutation at H840, for example, a H840A mutation; or a molecule having RuvC activity, for example, a Cas9 molecule with inactivated HNH activity, for example, a Cas9 molecule with a mutation at N863, for example, a N863A mutation. In some embodiments, each of the two gRNAs is complexed with D10A Cas9 nickase.

[0280] In some embodiments, the target sequence (target domain) is located at or near the T cell stimulation-associated locus, TRAC, TRBC1, and / or TRBC2 locus, such as any part of the T cell stimulation-associated locus, TRAC, TRBC1, and / or TRBC2 coding sequences, as listed in Tables 1-9 herein. In some embodiments, the target nucleic acid complementary to the targeting domain is located in the initial coding region of the gene of interest, such as the T cell stimulation-associated locus, TRAC, TRBC1, and / or TRBC2. Targeting the initial coding region can be used to disrupt the gene of interest (i.e., eliminate its expression). In some embodiments, the initial coding region of the gene of interest contains a sequence immediately following the start codon (e.g., ATG) or within 500 bp of the start codon (e.g., 500, 450, 400, 350, 300, 250, 200, 150, 100, 50 bp, 40 bp, 30 bp, 20 bp, or less than 10 bp). In specific examples, the target nucleic acid is within 200 bp, 150 bp, 100 bp, 50 bp, 40 bp, 30 bp, 20 bp, or 10 bp of the start codon. In some examples, the targeting domain of the gRNA is complementary to the target sequence on the target nucleic acid, such as the target nucleic acid in T cell stimulation-associated loci, TRAC, TRBC1, and / or TRBC2, e.g., at least 80, 85, 90, 95, 98, or 99% complementary, e.g., perfectly complementary.

[0281] In some cases, gRNAs can target sites within the exons of the open reading frames of endogenous T cell stimulation-associated loci, TRAC, TRBC1, and / or TRBC2. In some cases, gRNAs can target sites within the introns of the open reading frames of T cell stimulation-associated loci, TRAC, TRBC1, and / or TRBC2. In some cases, gRNAs can target sites within the regulatory or moduloconstrictive elements of T cell stimulation-associated loci, TRAC, TRBC1, and / or TRBC2, such as the promoter. In some cases, the target sites of T cell stimulation-associated loci, TRAC, TRBC1, and / or TRBC2 targeted by gRNAs may be any target sites described herein, for example, in Section II.A.1. In some embodiments, the gRNA can target exons corresponding to the initial coding region, for example, exons 1, 2, or 3 of the open reading frame of endogenous T cell stimulation-related loci, TRAC, TRBC1, and / or TRBC2, or sites within or very close to them, or sites within or containing sequences of less than 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 bp in exons 1, 2, or 3 immediately after the transcription start site. In some embodiments, the gRNA can target exon 2 or nearby sites of endogenous T cell stimulation-related loci, TRAC, TRBC1, and / or TRBC2, or sites within or less than 50 bp in exon 2.

[0282] In some embodiments, the targeting domain includes one for introducing gene disruption to T cell stimulation-related loci, TRAC, and / or TRBC genes using Streptococcus pyogenes Cas9 or Neisseria meningitidis Cas9. In some embodiments, the targeting domain includes one for introducing gene disruption to T cell stimulation-related loci, TRAC, and / or TRBC genes using Streptococcus pyogenes Cas9. Any of the targeting domains can be used with Streptococcus pyogenes Cas9 molecules that produce double-strand breaks (Cas9 nuclease) or single-strand breaks (Cas9 nickase).

[0283] In some embodiments, dual targeting is used to create two nicks on the reverse DNA strand by using a Cas9 nickase from Streptococcus pyogenes having two targeting domains complementary to the reverse DNA strand, for example, a gRNA containing any minus-strand targeting domain can be paired with any gRNA containing a plus-strand targeting domain. In some embodiments, the two gRNAs are oriented on the DNA such that the PAM faces outward and the distance between the 5' ends of the gRNAs is 0-50 bp. In some embodiments, the two gRNAs are used to target two Cas9 nucleases or two Cas9 nickases using a pair of Cas9 molecule / gRNA molecule complexes guided by two different gRNA molecules to cleave the target domain with two single-strand breaks on the reverse strand of the target domain, for example. In some embodiments, the two Cas9 nickase molecules may include a molecule having HNH activity, for example, a Cas9 molecule with inactivated RuvC activity, for example, a Cas9 molecule with a mutation at D10, for example, a D10A mutation; a molecule having RuvC activity, for example, a Cas9 molecule with inactivated HNH activity, for example, a Cas9 molecule with a mutation at H840, for example, a H840A mutation; or a molecule having RuvC activity, for example, a Cas9 molecule with inactivated HNH activity, for example, a Cas9 molecule with a mutation at N863, for example, a N863A mutation. In some embodiments, each of the two gRNAs is complexed with D10A Cas9 nickase.

[0284] Other domains of gRNA, such as the complementary domain, ligation domain, 5' extension domain, proximal domain, and tail domain, as well as their structures, are described, for example, in WO2015 / 161276, specifically in Figures 1A-1G.

[0285] Methods for designing gRNAs, including methods for selecting, designing, and validating targeting domains, are described herein. Exemplary targeting domains are also provided herein. The targeting domains discussed herein can be incorporated into the gRNAs described herein.

[0286] Methods for selecting and validating target sequences, as well as for off-target analysis, are described, for example, in Mali et al., 2013 Science 339(6121): 823-826; Hsu et al. Nat Biotechnol, 31(9): 827-32; Fu et al., Nat Biotechnol 2014 Mar; 32(3): 279-284; Heigwer et al., 2014 Nat Methods 11(2): 122-3; Bae et al., Bioinformatics. 2014 May 15; 30(10): 1473-5; Xiao A et al., Bioinformatics. 2014 Apr 15; 30(8): 1180-1182.

[0287] In some embodiments, software tools can be used to optimize the selection of gRNAs within the user's target sequence, for example, to minimize overall off-target activity across the genome. Off-target activity may not be limited to cleavage. For example, using Cas9 in Streptococcus pyogenes, for each possible gRNA selection, the software tool can identify all potential off-target sequences (preceding either NAG or NGG PAM) containing up to a certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base pairs across the genome. The cleavage efficiency at each off-target sequence can be predicted, for example, using an experimentally obtained weighting scheme. Each possible gRNA can then be ranked according to its overall predicted off-target cleavage; the top-ranked gRNA corresponds to the one that is likely to have the largest on-target and smallest off-target cleavage. Other functions, such as automated reagent design for gRNA vector construction, primer design for on-target Surveyor assays, and primer design for high-throughput detection and quantification of off-target cleavage via next-generation sequencing, may also be included in the tool. Candidate gRNA molecules can be evaluated by methods known in the art or as described herein.

[0288] In some aspects, gRNAs for use with Cas9 in Streptococcus pyogenes, Staphylococcus aureus, and Neisseria meningitidis are identified using DNA sequence search algorithms, for example, custom gRNA design software based on the public tool cas-offinder (Bae et al. Bioinformatics. 2014;30(10): 1473-1475). The custom gRNA design software scores the guides after calculating their genome-wide off-target tendencies. Typically, matches ranging from perfect matches to 7 mismatches are considered for guides spanning 17–24 lengths. In some aspects, once off-target sites are computationally determined, a total score is calculated for each guide and compiled into a tabular output using a web interface. In addition to identifying potential gRNA sites adjacent to PAM sequences, the software can also identify all PAM-adjacent sequences that differ from selected gRNA sites by one, two, three, or more nucleotides. In some embodiments, the genomic DNA sequence for each gene can be obtained from the UCSC Genome browser, and the sequence can be screened for repeat elements using the publicly available RepeatMasker program. RepeatMasker searches the input DNA sequence for repeat elements and low-complexity regions. The output is a detailed annotation of repeats present in a given query sequence.

[0289] After identification, gRNAs can be ranked hierarchically based on one or more of their distance to the target site, their orthogonality, and the presence of 5'G (based on the identification of exact matches in the human genome, including relevant PAMs, e.g., NGG PAM for Streptococcus pyogenes, NNGRR (e.g., NNGRRT or NNGRRV) PAM for Staphylococcus aureus, and NNNNGATT or NNNNGCTT PAM for Neisseria meningitidis). Orthogonality refers to the number of sequences in the human genome that contain the minimum number of mismatches to the target sequence. "High level of orthogonality" or "good orthogonality" can refer to, for example, a 20-mer targeting domain that has no identical sequences in the human genome except for the intended target, and does not contain any sequences that contain one or two mismatches to the target sequence. Targeting domains with good orthogonality are selected to minimize off-target DNA cleavage. It should be understood that this is a non-limiting example, and that various strategies can be employed to identify gRNAs for use with Streptococcus pyogenes, Staphylococcus aureus, and Neisseria meningitidis or other Cas9 enzymes.

[0290] In some aspects, gRNAs for use with Cas9 of Streptococcus pyogenes can be identified using a publicly available web-based ZiFiT server (Fu et al., Nat Biotechnol 2014 Mar;32(3):279-284; see Sander et al., 2007, NAR 35:W599-605; Sander et al., 2010, NAR 38:W462-8 for original references). In addition to identifying potential gRNA sites adjacent to PAM sequences, the software also identifies all PAM-adjacent sequences that differ from the selected gRNA site by one, two, three, or more nucleotides. In some aspects, genomic DNA sequences for each gene can be obtained from the UCSC Genome browser and the sequences can be screened for repeat elements using the publicly available Repeat-Masker program. RepeatMasker searches the input DNA sequence for repeat elements and low-complexity regions. The output is a detailed annotation of the iterations present in the given query array.

[0291] (ii) Cas9 Various species of Cas9 molecules can be used in the methods and compositions described herein. While Cas9 molecules from Streptococcus pyogenes, Staphylococcus aureus, Neisseria meningitidis, and S. thermophilus are the subject of much of the disclosure herein, Cas9 molecules from other species of Cas9 proteins listed herein, Cas9 molecules derived from said Cas9 proteins, and Cas9 molecules based on said Cas9 proteins can also be used. In other words, while much of the description herein uses Cas9 molecules from Streptococcus pyogenes, Staphylococcus aureus, Neisseria meningitidis, and S. thermophilus, Cas9 molecules from other species can be substituted for them. Such species include: Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., Cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, and Bacteroides species. (sp.), Blastopirellula marina, Bradyrhizobium sp.), Brevibacillus laterosporus, Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Candidatus puniceispirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter shibae), Eubacterium dolichum, Gammaproteobacterium, Gluconacetobacter diazotrophicus, Haemophilus parainfluenzae, Haemophilus sputorum, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, Ilyobacter polytropus, Kingella kingae, Lactobacillus crispatus, Listeria ibanobii Listeria monocytogenes (ivanovii), Listeriaceae bacterium, Methylocystis sp.), Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria meningitidis, Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans, Pasteurella maltosida multocida), Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus aureus, Staphylococcus lugdunensis, Streptococcus sp., Subdoligranulum (sp.), Tistrella mobilis, Treponema sp.), or Verminephrobacter eiseniae. Examples of Cas9 molecules include those described in WO2015 / 161276, WO2017 / 193107, WO2017 / 093969, US2016 / 272999, and US2015 / 056705.

[0292] When these terms are used herein, a Cas9 molecule or Cas9 polypeptide refers to a molecule or polypeptide that can interact with a gRNA molecule and, in cooperation with the gRNA molecule, directs to or localizes to a site containing a target domain and PAM sequence. When these terms are used herein, a Cas9 molecule and a Cas9 polypeptide refer to a naturally occurring Cas9 molecule and an engineered, altered, or modified Cas9 molecule or Cas9 polypeptide that differs by at least one amino acid residue from a reference sequence, e.g., the most similar naturally occurring Cas9 molecule.

[0293] The crystal structures have been determined for two different naturally occurring bacterial Cas9 molecules (Jinek et al., Science, 343(6176):1247997, 2014), and for pyogenic streptococcal Cas9 that possesses guide RNA (e.g., a synthetic fusion of crRNA and tracrRNA) (Nishimasu et al., Cell, 156:935-949, 2014; and Anders et al., Nature, 2014 Sep 25;513(7519):569-73).

[0294] Exemplary Cas9 molecules, their structures, and variants include, for example, those described in WO2015 / 161276, e.g., Figures 2A-2G and 8A-8B therein, as well as those described in WO2017 / 193107, WO2017 / 093969, US2016 / 272999, and US2015 / 056705.

[0295] In any aspect of the embodiments provided herein, a Cas9 molecule or Cas9 polypeptide, such as a nucleic acid encoding an eaCas9 molecule or eaCas9 polypeptide, may be used.

[0296] Exemplary nucleic acids encoding the Cas9 molecule or Cas9 polypeptide are shown in Cong et al., Science 2013, 399(6121):819-823; Wang et al., Cell 2013, 153(4):910-918; Mali et al., Science 2013, 399(6121):823-826; Jinek et al., Science 2012, 337(6096):816-821, and WO2015 / 161276, for example, in Figure 8.

[0297] In some embodiments, the nucleic acid encoding the Cas9 molecule or Cas9 polypeptide may be a synthetic nucleic acid sequence. For example, the synthetic nucleic acid molecule may be chemically modified. In some embodiments, the Cas9 mRNA has one or more (e.g., all) of the following properties: the Cas9 mRNA is capped, polyadenylated, or substituted with 5-methylcytidine and / or pseudouridine. Furthermore, or instead, the synthetic nucleic acid sequence can be codon-optimized, for example, by replacing at least one non-common or less common codon with a common codon. For example, the synthetic nucleic acid can lead to the synthesis of optimized messenger mRNA, optimized for expression in mammalian expression systems, for example, as described herein. Furthermore, or instead, the nucleic acid encoding the Cas9 molecule or Cas9 polypeptide may include a nuclear localization sequence (NLS). Nuclear localization sequences are known.

[0298] In some embodiments, the Cas9 molecule includes a sequence that is or contains any of SEQ ID NO: 279-287, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to any of SEQ ID NO: 112-117. Exemplary Cas9 molecules include Cas9 molecules from Streptococcus pyogenes, Staphylococcus aureus, or Neisseria meningitidis. In some embodiments, the Cas9 molecule or Cas9 polypeptide includes regions 1-5, along with additional Cas9 molecular sequences sufficient to provide a biologically active molecule, e.g., a Cas9 molecule having at least one activity described herein. In some embodiments, each of regions 1 to 6 independently has 50%, 60%, 70%, or 80% homology with the corresponding residues of the Cas9 molecules or Cas9 polypeptides described herein, for example, those shown in SEQ ID NO: 279 to 287, or the sequences disclosed in WO2015 / 161276, for example, those derived from Figures 2A to 2G or Figures 7A to 7B.

[0299] It will be understood that if any of the aforementioned Cas9 sequences are fused with a peptide or polypeptide at the C-terminus, the stop codon will be removed.

[0300] Various types of Cas molecules or Cas polypeptides can be used to carry out the invention disclosed herein. In some embodiments, Cas molecules of type II Cas systems are used. In other embodiments, Cas molecules of other Cas systems are used. For example, type I or type III Cas molecules may be used. Exemplary Cas molecules (and Cas systems) are described, for example, in Haft et al., PLoS Computational Biology 2005, 1(6): e60 and Makarova et al., Nature Review Microbiology 2011, 9:467-477, the contents of both references are incorporated herein by reference in their entirety. Exemplary Cas molecules (and Cas systems) include, for example, those described in WO2015 / 161276, WO2017 / 193107, WO2017 / 093969, US2016 / 272999, and US2015 / 056705.

[0301] (iii) Cpf1 In some embodiments, guide RNA or gRNA facilitates the specific binding targeting of RNA-induced nucleases, such as Cas9 or Cpf1, to target sequences, such as cellular genomic sequences or episomal sequences. Generally, gRNAs can be monomolecular (containing a single RNA molecule, or referred to as a chimera) or modular (containing more than one, typically two separate RNA molecules, e.g., crRNA and tracrRNA, which are usually linked to each other by double-stranding in some embodiments). gRNAs and their components are described throughout the literature in Briner et al. Molecular Cell (2014) 56(2), 333-339, which is incorporated by reference in some embodiments.

[0302] Guide RNAs, whether monomolecular or modular, generally contain a targeting domain that is fully or partially complementary to the target, typically 10–30 nucleotides long, and in certain embodiments, 16–24 nucleotides long (in some embodiments, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides long). In some aspects, the targeting domain is located at or near the 5' end of the gRNA in the case of Cas9 gRNA, and at or near the 3' end in the case of Cpf1 gRNA. While the above description has focused on gRNAs for use with Cas9, it should be understood that other RNA-inducible nucleases have been discovered or invented (or may be discovered or invented in the future) that utilize gRNAs that differ in some respects from those described so far. In some aspects, Cpf1 ("CRISPR from Prevotella and Franciscella 1") is a recently discovered RNA-inducible nuclease that does not require tracrRNA to function. (Zetsche et al., 2015, Cell 163, 759-771, incorporated herein by reference). gRNAs for use with the Cpf1 genome editing system generally contain a targeting domain and a complementation domain (or, referred to as, a "handle"). Note that in gRNAs for use with Cpf1, the targeting domain is usually located at or near the 3' end, rather than at the 5' end as described above for Cas9 gRNA (the handle is at or near the 5' end of Cpf1 gRNA).

[0303] While structural differences may exist between gRNAs from different prokaryotic species, or between Cpf1 and Cas9 gRNAs, the principle by which gRNAs operate is generally consistent. Due to this consistency of operation, gRNAs can be broadly defined by their targeting domain sequence, and those skilled in the art will recognize that a given targeting domain sequence can be incorporated into any suitable gRNA, including monomolecular or chimeric gRNAs, or gRNAs containing one or more chemical and / or sequence modifications (substitutions, nucleotide additions, truncations, etc.). Therefore, in some aspects of this disclosure, gRNAs may be described solely in terms of their targeting domain sequence.

[0304] More generally, several aspects of this disclosure relate to systems, methods, and compositions that can be carried out using multiple RNA-inducible nucleases. Unless otherwise specified, the term gRNA should be understood to encompass not only gRNAs adapted to specific species of Cas9 or Cpf1, but also any suitable gRNAs that can be used with any RNA-inducible nuclease. Exemplarily, in certain embodiments, the term gRNA may include gRNAs for use with class 2 CRISPR systems, e.g., type II or type V, or any RNA-inducible nuclease present in a CRISPR system, or RNA-inducible nucleases derived from or adapted from them.

[0305] While Cas9 and Cpf1 share structural and functional similarities, it should be understood that certain Cpf1 activities are mediated by structural domains that are not similar to any Cas9 domain. In some embodiments, the cleavage of the complementary strand of target DNA appears to be mediated by a Nuc domain that is sequence- and spatially distinct from the HNH domain of Cas9. Furthermore, the non-targeting portion (handle) of Cpf1 gRNA adopts a pseudoknot structure rather than a stem-loop structure formed by the repeat:anti-repeat double helix of Cas9 gRNA.

[0306] RNA-induced nucleases, such as Cas9, Cpf1, or nucleic acids encoding functional fragments thereof, are provided herein. Exemplary nucleic acids encoding RNA-induced nucleases include, for example, those described in Cong et al., Science 2013, 399(6121):819-823; Wang et al., Cell 2013, 153(4):910-918; Mali et al., Science 2013, 399(6121):823-826; and Jinek et al., Science 2012, 337(6096):816-821.

[0307] b. Genome editing approaches In general, it should be understood that any genetic modification by the methods described herein may be mediated by any mechanism, and that no method is limited to any particular mechanism. Exemplary mechanisms that may be involved in genetic modification include, but are not limited to, non-homologous end joining (e.g., classical or alternative), microhomology-mediated end joining (MMEJ), homology-directed repair (e.g., endogenous donor template-mediated), synthesis-dependent strand annealing (SDSA), single-strand annealing, single-strand entry, single-strand break repair (SSBR), mismatch repair (MMR), base excision repair (BER), interstrand crosslinking (ICL), damage-over-synthesis (TLS), or error-free post-replication repair (PRR). Exemplary methods for targeted knockout of one or both alleles of T cell stimulation-associated loci, TRAC and / or TRBC, are described herein. Exemplary mechanisms include, for example, those described in U.S. Patent Application Publication Nos. US20170349894, US20180362943, and US20180245079.

[0308] In some embodiments, for the purpose of inducing NHEJ-mediated indels, gRNA and Cas9 nuclease generate double-strand breaks, the gRNA, e.g., a single molecule (or chimeric) or modular gRNA, is configured to position a single double-strand break very close to a nucleotide at the target site. In some embodiments, the cleavage site is located 0 to 30 bp away from the target site (e.g., less than 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 bp away from the target site).

[0309] In some embodiments, two gRNAs complexed with Cas9 nickase induce two single-strand breaks for the purpose of inducing NHEJ-mediated indels. In these embodiments, the two gRNAs, for example, independently, unimolecular (or chimeric) or modular gRNAs, are configured to position the two single-strand breaks at target nucleotide sites to provide NHEJ repair. In some embodiments, the gRNAs are configured to essentially mimic double-strand breaks, positioning the breaks on different strands, at the same location or within a few nucleotides of each other. In some embodiments, the closer nick is 0–30 bp away from the target location (e.g., less than 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 bp away from the target location), and the two nicks are within 25–55 bp of each other (e.g., 25–50, 25–45, 25–40, 25–35, 25–30, 50–55, 45–55, 40–55, 35–55, 30–55, 30–50, 35–50, 40–50, 45–50, 35–45, or 40–45 bp) and less than 100 bp away from each other (e.g., less than 90, 80, 70, 60, 50, 40, 30, 20, or 10 bp). In some embodiments, the gRNA is configured to place a single-strand break on either side of the nucleotide at the target site.

[0310] In the methods and compositions described herein, a double-strand cleavage eaCas9 molecule and a single-strand cleavage, or both a nickas and an eaCas9 molecule, can be used to generate cleavage on both sides of a target site. Double-strand breaks or paired single-strand breaks may be generated on both sides of the target site to remove the nucleic acid sequence between the two breaks (e.g., a deletion of the region between the two breaks). In some embodiments, two gRNAs, e.g., independently, monomolecule (or chimeric) or modular gRNAs, are configured to position double-strand breaks on both sides of the target site. In alternative embodiments, three gRNAs, e.g., independently, monomolecule (or chimeric) or modular gRNAs, are configured to position double-strand breaks (i.e., a complex of one gRNA and Cas9 nuclease) and two single-strand breaks or paired single-strand breaks (i.e., a complex of two gRNAs and Cas9 nickas) on either side of the target site. In another embodiment, four gRNAs, for example, independently, single (or chimeric) or modular gRNAs, are configured to generate two pairs of single-strand breaks (i.e., two pairs of complexes of two gRNAs and Cas9 nicks) on either side of the target site. The closer of the two single-strand breaks or the double-strand breaks in the pair would ideally be within 0 to 500 bp of the target site (e.g., 450, 400, 350, 300, 250, 200, 150, 100, 50, or 25 bp or less from the target site). When nickase is used, the two nicks in a pair are within 25-55 bp of each other (e.g., 25-50, 25-45, 25-40, 25-35, 25-30, 50-55, 45-55, 40-55, 35-55, 30-55, 30-50, 35-50, 40-50, 45-50, 35-45, or 40-45 bp) and are less than 100 bp apart from each other (e.g., less than 90, 80, 70, 60, 50, 40, 30, 20, or 10 bp).

[0311] Cas molecules, gRNA molecules, or Cas9 molecule / gRNA complexes can be evaluated by methods known in the art or as described herein. For example, exemplary methods for evaluating the endonuclease activity of Cas9 molecules are described, for example, in Jinek et al., Science 2012, 337(6096):816-821, WO2015 / 161276, WO2017 / 193107, WO2017 / 093969, US2016 / 272999, and US2015 / 056705.

[0312] 3. Delivery of agents for gene disruption In some embodiments, target gene disruption, e.g., DNA cleavage, of human endogenous T cell stimulation-associated loci, TRAC and / or TRBC, is carried out by delivering or introducing one or more active agents, e.g., Cas9 and / or gRNA components, into cells using any of several known delivery methods or vehicles for introducing or transferring them into cells, e.g., a virus, e.g., a lentiviral delivery vector, or any known method or vehicle for delivering Cas9 molecules and gRNAs. Exemplary methods are described, for example, in Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505. In some embodiments, nucleic acid sequences encoding one or more components of one or more active agents capable of inducing gene disruption, e.g., DNA cleavage, are introduced into cells by any method for introducing nucleic acids into cells, e.g., described herein or known. In some embodiments, components of one or more active agents capable of inducing gene disruption, e.g., vectors encoding CRISPR guide RNA and / or Cas9 enzymes, can be delivered into cells.

[0313] In some embodiments, one or more activators capable of inducing gene disruption, such as Cas9 / gRNA, are introduced into cells as a ribonucleoprotein (RNP) complex. The RNP complex comprises a ribonucleotide sequence, such as an RNA or gRNA molecule, and a protein, such as the Cas9 protein or a variant thereof. For example, the Cas9 protein is delivered as an RNP complex containing the Cas9 protein and a gRNA molecule targeting a target sequence, for example, by electroporation or other physical delivery methods. In some embodiments, RNPs are delivered into cells via electroporation or other physical means, such as a particle gun, calcium phosphate transfection, cell compression, or cell pressure. In some embodiments, RNPs can cross the cell plasma membrane without the need for further delivery activators (e.g., small molecule activators, lipids, etc.). In some embodiments, delivery of one or more activators capable of inducing gene disruption, such as CRISPR / Cas9 as an RNP, offers the advantage that targeted disruption occurs transiently in the cells to which the RNP is introduced, without the activators being passed on to the cell's offspring. For example, RNP delivery minimizes the inheritance of the activator to its offspring, thereby reducing the opportunity for off-target gene disruption in the offspring. In such cases, gene disruption and transgene integration may be inherited by the offspring cells, but they are transmitted to the offspring cells even without the activator itself, which could potentially introduce further off-target gene disruption.

[0314] In some embodiments, the RNP complex contains a modified gRNA that includes a 3' poly-A tail and a 5' anti-reverse cap analog (ARCA) cap.

[0315] Active agents and components capable of inducing gene disruption, such as Cas9 molecules and gRNA molecules, can be introduced into target cells in various forms using the various delivery methods and formulations described in Tables 10 and 11, or by methods described, for example, WO 2015 / 161276; WO2017 / 193107, WO2017 / 093969, US 2015 / 0056705, US 2016 / 0272999, US 2017 / 0211075; or US 2017 / 0016027. As further described herein, the delivery methods and formulations can be used to deliver template polynucleotides and / or other active agents (e.g., those required to manipulate the cells) to cells in a pre- or post-step of the methods described herein. If the Cas9 or gRNA component is encoded as DNA for delivery, the DNA may, but typically, include regulatory regions, such as promoters, for expression. Useful promoters for the Cas9 molecular sequence include, for example, the CMV, EF-1α, EFS, MSCV, PGK, or CAG promoters. Useful promoters for gRNA include, for example, the H1, EF-1α, tRNA, or U6 promoters. Promoters with similar or dissimilar intensities can be selected to modulate the expression of the components. The sequence encoding the Cas9 molecule may include a nuclear localization signal (NLS), e.g., the SV40 NLS. In some embodiments, the promoter for the Cas9 molecule or the gRNA molecule may be independently inducible, tissue-specific, or cell-specific. In some embodiments, an activator capable of inducing gene disruption is introduced into the RNP complex.

[0316] (Table 10) Exemplary delivery methods TIFF2026090319000013.tif93165

[0317] (Table 11) Comparison of exemplary delivery methods TIFF2026090319000014.tif156165

[0318] In some embodiments, DNA encoding a Cas9 molecule and / or a gRNA molecule, or an RNP complex containing a Cas9 molecule and / or a gRNA molecule, can be delivered into cells by known methods or as described herein. For example, DNA encoding Cas9 and / or gRNA can be delivered, for example, by a vector (e.g., a viral or non-viral vector), by a non-vector-based method (e.g., using naked DNA or a DNA complex), or a combination thereof. In some embodiments, polynucleotides containing the active agent and / or their components are delivered by a vector (e.g., a viral vector / virus or plasmid). The vector may be any of those described herein.

[0319] In some cases, CRISPR enzymes (e.g., Cas9 nucleases) combined with (and optionally complexed with) guide sequences are delivered to cells. For example, one or more elements of a CRISPR system may originate from a type I, type II, or type III CRISPR system. For example, one or more elements of a CRISPR system may originate from certain organisms that contain endogenous CRISPR systems, such as Streptococcus pyogenes, Staphylococcus aureus, or Neisseria meningitidis.

[0320] In some embodiments, a Cas9 nuclease (e.g., one encoded by mRNA derived from Staphylococcus aureus or Streptococcus pyogenes; e.g., pCW-Cas9, Addgene #50661, Wang et al. (2014) Science, 3:343-80-4; or a nuclease or nicker lenticviral vector available from Applied Biological Materials (ABM; Canada) as catalog numbers K002, K003, K005, or K006) and guide RNA specific to a target locus (e.g., T cell stimulation-related loci, TRAC and / or TRBC) are introduced into the cell.

[0321] In some embodiments, polynucleotides or RNP complexes containing the active substance and / or its components are delivered by vector-free methods (e.g., using naked DNA or DNA complexes). For example, DNA or RNA or proteins or combinations thereof, such as ribonucleoprotein (RNP) complexes, can be delivered by, for example, organically modified silica or silicate (Ormosil), electroporation, transient cell compression or compression (as described, e.g., Lee, et al. (2012) Nano Lett 12: 6322-27, Kollmannsperger et al (2016) Nat Comm 7, 10372), gene guns, sonoporation, magnetofection, lipid-mediated transfection, dendrimers, inorganic nanoparticles, calcium phosphate, or combinations thereof.

[0322] In some embodiments, electroporation-mediated delivery involves mixing cells with DNA or RNP complexes encoding Cas9 and / or gRNA in a cartridge, chamber, or cuvette, and applying one or more electrical impulses of a predetermined duration and amplitude. In some embodiments, electroporation-mediated delivery is performed using a system in which DNA encoding Cas9 and / or gRNA and cells are mixed in a container connected to a device (e.g., a pump), which delivers the mixture to a cartridge, chamber, or cuvette, where one or more electrical impulses of a predetermined duration and amplitude are applied, and the cells are then delivered to a second container.

[0323] In some embodiments, the delivery vehicle is a non-viral vector. In some embodiments, the non-viral vector is an inorganic nanoparticle. Exemplary inorganic nanoparticles include, for example, magnetic nanoparticles (e.g., Fe3MnO2) and silica. The outer surface of the nanoparticle can be conjugated with a positively charged polymer (e.g., polyethyleneimine, polylysine, polyserine), thereby enabling the attachment (e.g., conjugation or capture) of the payload. In some embodiments, the non-viral vector is an organic nanoparticle. Exemplary organic nanoparticles include, for example, SNALP liposomes containing cationic lipids along with neutral helper lipids coated with polyethylene glycol (PEG), and lipid-coated protamine-nucleic acid complexes. Exemplary lipids for gene transfer include, for example, those described in WO2015 / 161276, WO2017 / 193107, WO2017 / 093969, US2016 / 272999, and US2015 / 056705.

[0324] In some embodiments, the vehicle has targeting modifications to increase targeted cell reactivation of nanoparticles and liposomes, e.g., cell-specific antigens, monoclonal antibodies, single-chain antibodies, aptamers, polymers, sugars, and cell-permeable peptides. In some embodiments, the vehicle uses fusionable and endosomal destabilizing peptides / polymers. In some embodiments, the vehicle undergoes acid-induced conformational changes (e.g., to accelerate cargo endosomal escape). In some embodiments, cleavable polymers are used, for example, for release in intracellular compartments. For example, disulfide-based cationic polymers that are cleaved in a reducing cellular environment may be used.

[0325] In some embodiments, the delivery vehicle is a biological nonviral delivery vehicle. In some embodiments, the vehicle is an attenuated bacterium (e.g., invasive but attenuated to prevent disease development and engineered naturally or artificially to express a transgene (e.g., Listeria monocytogenes, certain Salmonella strains, Bifidobacterium longum, and modified Escherichia coli), a bacterium with trophic and tissue-specific targeting to target specific cells, or a bacterium with modified surface proteins to alter target cell specificity). In some embodiments, the vehicle is a genetically modified bacteriophage (e.g., an engineered phage with large packaging capacity, low immunogenicity, containing mammalian plasmid maintenance sequences, and incorporating targeting ligands). In some embodiments, the vehicle is a mammalian virus-like particle. For example, modified viral particles can be generated (e.g., by purification of "empty" particles, followed by ex vivo assembly of the virus with the desired cargo). The vehicle can also be manipulated to modify target tissue specificity by incorporating targeting ligands. In some embodiments, the vehicle is a biological liposome. For example, a biological liposome is a phospholipid-based particle derived from a human cell, e.g., a erythrocyte ghost, which is a erythrocyte broken into spherical structures derived from the target (e.g., tissue targeting can be achieved by attachment of various tissue or cell-specific ligands), or a target-derived membrane-bound nanovesicle (30-100 nm) of secretory exosome-endocytosis origin (e.g., which can be generated from various cell types and thus can be taken up by cells without the need for targeting ligands).

[0326] In some embodiments, RNA encoding the Cas9 molecule and / or gRNA molecule can be delivered into cells, e.g., target cells as described herein, by known methods or as described herein. For example, RNA encoding Cas9 and / or gRNA can be delivered by, for example, microinjection, electroporation, transient cell compression or pressure (as described, e.g., Lee, et al. (2012) Nano Lett 12: 6322-27), lipid-mediated transfection, peptide-mediated delivery, e.g., cell-permeable peptides, or a combination thereof.

[0327] In some embodiments, electroporation-mediated delivery involves mixing RNA encoding Cas9 molecules and / or gRNA molecules with cells in a cartridge, chamber, or cuvette, and applying one or more electrical impulses of a predetermined duration and amplitude. In some embodiments, electroporation-mediated delivery is performed using a system in which RNA encoding Cas9 molecules and / or gRNA molecules with cells are mixed in a container connected to a device (e.g., a pump), which delivers the mixture to a cartridge, chamber, or cuvette, where one or more electrical impulses of a predetermined duration and amplitude are applied, and the cells are then delivered to a second container.

[0328] In some embodiments, the Cas9 molecule can be delivered into cells by known methods or as described herein. For example, the Cas9 protein molecule can be delivered by, for example, microinjection, electroporation, transient cell compression or pressure (as described, e.g., Lee, et al. (2012) Nano Lett 12: 6322-27), lipid-mediated transfection, peptide-mediated delivery, or a combination thereof. Delivery may be accompanied by DNA or gRNA encoding the gRNA.

[0329] In some embodiments, one or more active agents capable of inducing cleavage, such as a Cas9 / gRNA system, are introduced into a cell as a ribonucleoprotein (RNP) complex. The RNP complex comprises a ribonucleotide, such as an RNA or gRNA molecule, and a protein, such as a Cas9 protein or a variant thereof. For example, the Cas9 protein is delivered as an RNP complex comprising the Cas9 protein and a gRNA molecule targeting a target sequence, for example, by electroporation or other physical delivery methods. In some embodiments, the RNP is delivered into the cell by electroporation or other physical means, such as a particle gun, calcium phosphate transfection, or cell compression or pressure.

[0330] In some embodiments, electroporation delivery involves mixing the Cas9 molecule with the gRNA molecule or as is with the cells in a cartridge, chamber, or cuvette, and applying one or more electrical impulses of a predetermined duration and amplitude. In some embodiments, electroporation delivery is performed using a system in which the Cas9 molecule with the gRNA molecule or as is with the cells is mixed in a container connected to a device (e.g., a pump), which delivers the mixture to a cartridge, chamber, or cuvette, where one or more electrical impulses of a predetermined duration and amplitude are applied, and the cells are then delivered to a second container.

[0331] In some embodiments, electroporation-mediated delivery involves mixing cells with or without gRNA molecules with Cas9 molecules (e.g., eaCas9 molecules, eiCas9 molecules, or eiCas9 fusion proteins) in a cartridge, chamber, or cuvette, and applying one or more electrical impulses of a predetermined duration and amplitude. In some embodiments, electroporation-mediated delivery is carried out using a system in which Cas9 molecules (e.g., eaCas9 molecules, eiCas9 molecules, or eiCas9 fusion proteins) are mixed with cells.

[0332] In some embodiments, polynucleotides containing the active substance and / or its components are delivered by a combination of vector and non-vector-based methods. For example, virosoms include liposomes combined with an inactivated virus (e.g., HIV or influenza virus), which can result in more efficient gene transfer than either the viral method alone or the liposome method alone.

[0333] In some embodiments, more than one active agent or its components are delivered to the cell. For example, in some embodiments, an active agent capable of inducing gene disruption at two or more locations in the genome, e.g., two or more sites within T cell stimulation-associated loci, TRAC, and / or TRBC, is delivered to the cell. In some embodiments, the active agent and its components are delivered using a single method. For example, in some embodiments, the active agent for inducing gene disruption at T cell stimulation-associated loci, TRAC, and / or TRBC is delivered as a polynucleotide encoding the component for gene disruption. In some embodiments, one polynucleotide may encode an active agent targeting T cell stimulation-associated loci, TRAC, and / or TRBC. In some embodiments, two or more different polynucleotides may encode an active agent targeting T cell stimulation-associated loci, TRAC, and / or TRBC. In some embodiments, an active agent capable of inducing gene disruption may be delivered as a ribonucleoprotein (RNP) complex, and two or more different RNP complexes may be delivered together as a mixture or separately.

[0334] In some embodiments, one or more active agents and / or components thereof capable of inducing gene disruption, e.g., one or more nucleic acid molecules other than the Cas9 molecular component and / or gRNA molecular component, e.g., a template polynucleotide for HDR-inducible incorporation (e.g., any template polynucleotide as described herein, e.g., in Section II.B.2), are delivered. In some embodiments, the nucleic acid molecule, e.g., the template polynucleotide, is delivered simultaneously with one or more components of the Cas system. In some embodiments, the nucleic acid molecule is delivered before or after the delivery of one or more components of the Cas system (e.g., about 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, or less than 4 weeks). In some embodiments, the nucleic acid molecule, e.g., the template polynucleotide, is delivered by means different from one or more components of the Cas system, e.g., the Cas9 molecular component and / or gRNA molecular component. Nucleic acid molecules, such as template polynucleotides, can be delivered by any of the delivery methods described herein. For example, nucleic acid molecules, such as template polynucleotides, can be delivered by viral vectors, such as retroviruses or lentiviruses, and Cas9 molecular components and / or gRNA molecular components can be delivered by electroporation. In some embodiments, the nucleic acid molecule, such as template polynucleotides, includes one or more exogenous sequences, such as sequences encoding recombinant receptors or portions thereof and / or other exogenous gene nucleic acid sequences.

[0335] B. Target embedding via homology-oriented restoration (HDR) In some aspects, the provided embodiments involve the targeted incorporation of a specific portion of a polynucleotide, e.g., a template polynucleotide containing a transgene encoding a recombinant receptor or a portion thereof, at a specific location in the genome (e.g., a target site or target location) of an endogenous T cell stimulation-associated gene locus. In some aspects, homology-directed repair (HDR) can mediate site-specific incorporation of a transgene at a target site. In some aspects, the presence of a template polynucleotide containing a gene disruption (e.g., a DNA break as described in Section II.A) and one or more homology arms (e.g., a homologous sequence of nucleic acid sequence around the gene disruption) can induce or lead to HDR using the homologous sequence acting as a template for DNA repair. Based on homology between the endogenous gene sequence around the gene disruption and the 5' and / or 3' homology arms contained in the template polynucleotide, the cell's DNA repair mechanism can use the template polynucleotide to repair the DNA break at the site of the gene disruption, resynthesize the genetic information, and thereby effectively insert or incorporate the transgene in the template polynucleotide at or near the site of the gene disruption. In some embodiments, gene disruption at endogenous T cell stimulation-related loci can be generated by any of the methods for generating target gene disruption described herein.

[0336] Polynucleotides, such as template polynucleotides described herein, and kits containing such polynucleotides are also provided. In some embodiments, the polynucleotides and / or kits provided may be used in a manner described herein, including, for example, HDRs, to target an endogenous T cell stimulation-associated locus with a transgene encoding a recombinant receptor or a portion thereof.

[0337] In some embodiments, the template polynucleotide is a polynucleotide comprising, or includes, a transgene encoding a recombinant receptor or a portion thereof (e.g., one or more regions or domains of a recombinant receptor), such as an exogenous or heterologous nucleic acid sequence, and a homologous sequence (e.g., a homology arm) homologous to an endogenous genomic site of an endogenous T cell stimulation-associated locus, or a sequence near it. In some aspects, the transgene in the template polynucleotide comprises a sequence of nucleotides encoding a recombinant receptor or a portion thereof. In some aspects, upon targeted integration of the transgene, the T cell stimulation-associated locus of the manipulated cell is modified, and as a result, the modified T cell stimulation-associated locus comprises a transgene encoding a recombinant receptor, such as a chimeric antigen receptor (CAR).

[0338] In some cases, the template polynucleotide is introduced as a linear DNA fragment or contained within a vector. In some cases, the steps to induce gene disruption and the steps for target integration (e.g., by introducing the template polynucleotide) are performed simultaneously or sequentially.

[0339] 1. Homology Directed Repair (HDR) In some embodiments, homology-directed repair (HDR) can be used for the targeted incorporation or insertion of one or more nucleic acid sequences, such as transgenes, at one or more target sites of T cell stimulation-related gene loci. In some embodiments, nuclease-inducible HDR can be used to modify target sequences, incorporate transgenes at specific target sites, and / or edit or repair mutations in specific target genes, such as T cell stimulation-related gene loci.

[0340] Modification of the nucleic acid sequence at a target site can be induced by HDR using an exogenously provided template polynucleotide (also referred to as a "donor polynucleotide" or "template sequence"). For example, a template polynucleotide can result in a modification of the target sequence, such as the insertion of a transgene contained within the template polynucleotide. In some embodiments, plasmids or vectors can be used as templates for homologous recombination. In some embodiments, linear DNA fragments can be used as templates for homologous recombination. In some embodiments, a single-stranded template polynucleotide can be used as a template for modification of the target sequence by an alternative method of homology-directed repair between the target sequence and the template polynucleotide (e.g., single-strand annealing). The modification of the target sequence induced by the template polynucleotide depends on cleavage by a nuclease, such as a target nuclease like CRISPR / Cas9. Nuclease cleavage can include a double-strand break or two single-strand breaks.

[0341] In some embodiments, “recombination” involves the process of exchanging genetic information between two polynucleotides. In some embodiments, “homologous recombination (HR)” involves a specialized form of such exchange, for example, occurring during the repair of double-strand breaks in cells via homology-directed repair mechanisms. This process requires homology of nucleotide sequences and uses a template polynucleotide to repair the template of target DNA (i.e., the DNA that has suffered a double-strand break, e.g., a target site in an endogenous gene), leading to the transfer of genetic information from the template polynucleotide to the target, and is therefore known in various ways as “non-crossover gene conversion” or “short tract gene conversion.” In some embodiments, such transfer may involve “synthesis-dependent strand annealing,” and / or related processes, in which the template polynucleotide is used to correct a hetero-double-strand DNA mismatch that occurs between the broken target and the template polynucleotide, and / or to resynthesize the genetic information that is to be part of the target. Such specialized HRs often result in alterations of the target molecule's sequence, such that some or all of the sequence of the template polynucleotide is incorporated into the target polynucleotide.

[0342] In some embodiments, a template polynucleotide, e.g., a polynucleotide containing a transgene, is incorporated into the cell's genome via a homology-independent mechanism. This method involves creating a double-strand break (DSB) in the cell's genome and using a nuclease to cleave the template polynucleotide molecule, resulting in the integration of the template polynucleotide into the DSB site. In some embodiments, the template polynucleotide is incorporated via a non-homologous-dependent method (e.g., NHEJ). Upon in vivo cleavage, the template polynucleotide may be incorporated into the cell's genome in a targeted manner at the DSB site. The template polynucleotide may contain one or more identical target sites for one or more nucleases used to create the DSB. Thus, the template polynucleotide may be cleaved by one or more of the same nucleases used to cleave the endogenous gene to be incorporated. In some embodiments, the template polynucleotide contains nuclease target sites different from those of the nucleases used to induce the DSB. As described herein, gene disruption at a target site or location can be produced by any known method or any method described herein, such as ZFN, TALEN, CRISPR / Cas9 system, or TtAgo nuclease.

[0343] In some embodiments, the DNA repair mechanism may be induced by a nuclease after (1) a single double-strand break, (2) two single-strand breaks, (3) two double-strand breaks occurring on both sides of the target site, (4) one double-strand break and two single-strand breaks occurring on both sides of the target site, (5) four single-strand breaks occurring with a pair of single-strand breaks on both sides of the target site, or (6) one single-strand break. In some embodiments, a single-strand template polynucleotide is used, and the target site may be modified by an alternative HDR.

[0344] Modification of the target site induced by the template polynucleotide depends on cleavage by a nuclease molecule. Nuclease cleavage can include nicks, double-strand breaks, or two single-strand breaks, for example, one on each strand of DNA at the target site. After introducing a break at the target site, excision occurs at the break ends, resulting in a single-stranded overhanging DNA region.

[0345] In standard HDR, a double-stranded template polynucleotide containing a homologous sequence to the target site is introduced, either directly integrated into the target site, used as a template for introducing a transgene, or modified for the target site's sequence. After excision at the break point, repair can proceed via various pathways, such as the double Holliday junction model (or double-strand break repair, DSBR pathway) or the synthesis-dependent strand annealing (SDSA) pathway.

[0346] In the double Holliday junction model, strand intrusion occurs due to two single-strand protrusions of the target site into a homologous sequence in the template polynucleotide, resulting in the formation of an intermediate with two Holliday junctions. New DNA is synthesized from the end of the intrusion strand, filling the gap caused by excision, and the junction shifts. The end of the newly synthesized DNA is ligated to the excised end, the junction is degraded, and as a result, insertion at the target site occurs, for example, insertion of a transgene into the template polynucleotide. Crossover with the template polynucleotide can occur during junction degradation.

[0347] In the SDSA pathway, only one single-stranded overhang enters the template polynucleotide, and new DNA is synthesized from the end of the overhanging strand to fill the gap caused by the excision. The newly synthesized DNA then anneals to the remaining single-stranded overhang, synthesizing new DNA to fill the gap, the strands are ligated, and a modified DNA double-stranded structure is produced.

[0348] In alternative HDRs, a single-stranded template polynucleotide, e.g., a template polynucleotide, is introduced. Nicks, single-strand breaks, or double-strand breaks at the target site are mediated by a nuclease molecule to modify the desired target site, resulting in excision at the break point and the appearance of a single-strand overhang. The incorporation of the template polynucleotide sequence to modify or alter the target site of the DNA typically occurs via the SDSA pathway, as described herein.

[0349] "Alternative HDR" or alternative homology-directed repair refers, in some embodiments, to a process that repairs DNA damage using homologous nucleic acids (e.g., endogenous homologous sequences, e.g., sister chromatids, or exogenous nucleic acids, e.g., template polynucleotides). Alternative HDR differs from standard HDR in that the process utilizes a different pathway and can be inhibited by the mediators of standard HDR, RAD51 and BRCA2. Furthermore, alternative HDR uses single-stranded or nicked homologous nucleic acids for repairing breaks. "Standard HDR" or standard homology-directed repair refers, in some embodiments, to a process that repairs DNA damage using homologous nucleic acids (e.g., endogenous homologous sequences, e.g., sister chromatids, or exogenous nucleic acids, e.g., template nucleic acids). Standard HDR typically acts when there is significant excision in a double-strand break, forming at least one single-stranded portion of the DNA. In normal cells, HDR typically involves a series of steps including cleavage recognition, cleavage stabilization, excision, single-stranded DNA stabilization, DNA crossover intermediate formation, crossover intermediate degradation, and ligation. This process requires RAD51 and BRCA2, and homologous nucleic acids are typically double-stranded. Unless otherwise indicated, the term "HDR" in some aspects encompasses both standard HDR and alternative HDR.

[0350] In some embodiments, double-strand cleavage is mediated by a nuclease, for example, a Cas9 molecule having cleavage activity associated with an HNH-like domain and cleavage activity associated with a RuvC-like domain, for example, an N-terminal RuvC-like domain, such as wild-type Cas9. Such embodiments require only a single gRNA.

[0351] In some embodiments, a single-strand break or nick is introduced by a nuclease molecule with nickase activity, such as Cas9 nickase. DNA with a nick at the target site can serve as a substrate for alternative HDRs.

[0352] In some embodiments, two single-strand breaks or nicks are mediated by a Cas9 molecule having a nuclease, e.g., nickase activity, e.g., cleavage activity associated with an HNH-like domain or cleavage activity associated with an N-terminal RuvC-like domain. Such embodiments typically require two gRNAs, one for the arrangement of each single-strand break. In some embodiments, a Cas9 molecule with nickase activity cleaves the strand into which the gRNA hybridizes, but not the strand complementary to the strand into which the gRNA hybridizes. In some embodiments, a Cas9 molecule with nickase activity cleaves the strand complementary to the strand into which the gRNA hybridizes, rather than the strand into which the gRNA hybridizes. In some embodiments, the nickase is an HNH-active Cas9 molecule, e.g., one with inactivated RuvC activity, e.g., a Cas9 molecule with a mutation at D10, e.g., a D10A mutation. D10A inactivates RuvC; therefore, Cas9 nickase is thought to have HNH activity (only) and cleave at the strand to which the gRNA hybridizes (e.g., the complementary strand without NGG PAM). In some embodiments, a Cas9 molecule having the H840 mutation, e.g., H840A, can be used as nickase. H840A inactivates HNH; therefore, Cas9 nickase has RuvC activity (only) and cleaves at the non-complementary strand (e.g., the strand having NGG PAM and whose sequence is identical to that of the gRNA). In some embodiments, the Cas9 molecule is an N-terminal RuvC-like domain nickase, e.g., the Cas9 molecule contains the N863 mutation, e.g., N863A.

[0353] In some embodiments, nickase and two gRNAs are used to position two single-stranded nicks, one nick is on the + strand of the target DNA and the other on the - strand. The PAM is oriented outward. The gRNAs can be selected so that they are separated by approximately 0-50, 0-100, or 0-200 nucleotides. In some embodiments, there is no overlap between target sequences complementary to the targeting domains of the two gRNAs. In some embodiments, the gRNAs do not overlap and are separated by approximately 50, 100, or 200 nucleotides. In some embodiments, the use of two gRNAs can increase specificity, for example, by reducing off-target binding (Ran et al., Cell. 2013 Sep 12;154(6):1380-9).

[0354] In some embodiments, a single nick can be used to induce an HDR, for example, an alternative HDR. It is intended herein that a single nick can be used to increase the HR to NHEJ ratio at a given cleavage site, for example, a target site. In some embodiments, the single-strand break is formed on the DNA strand at the target site where the targeting domain of the gRNA is complementary. In some embodiments, the single-strand break is formed on the DNA strand at a target site other than the strand where the targeting domain of the gRNA is complementary.

[0355] In some embodiments, other DNA repair pathways, such as single-strand annealing (SSA), single-strand break repair (SSBR), mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER), intra-strand crosslinking (ICL), damage overcome synthesis (TLS), and error-free post-replication repair (PRR), may be used by cells to repair double-strand or single-strand breaks produced by nucleases.

[0356] Targeted integration integrates a transgene, such as a sequence between homologous arms, into a T cell stimulation-related locus in the genome. The transgene can be integrated into at least one target site or one of several sites in the genome, or anywhere near it. In some embodiments, the transgene is integrated into at least one target site, or near it, for example, within 300, 250, 200, 150, 100, 50, 10, 5, 4, 3, 2, 1 or fewer base pairs upstream or downstream of a cleavage site, for example, within 100, 50, 10, 5, 4, 3, 2, 1 base pairs on either side of the target site, for example, within 50, 10, 5, 4, 3, 2, 1 base pairs on either side of the target site. In some embodiments, the integrated sequence containing the transgene does not include any vector sequence (e.g., a viral vector sequence). In some embodiments, the integrated sequence includes a portion of a vector sequence (e.g., a viral vector sequence).

[0357] A double-strand or single-strand break (e.g., a target site) in one strand must be close enough to the target integration site, e.g., the site for target integration, so that a modification occurs in the desired region, e.g., an insertion of a transgene or modification of a mutation. In some embodiments, the distance is 10, 25, 50, 100, 200, 300, 350, 400, or 500 nucleotides or less. In some embodiments, the break is considered to be close enough to the target integration site so that the break lies within a region subject to exonuclease-mediated removal during end excision. In some embodiments, the targeting domain is configured such that a cleavage event, e.g., a double-strand or single-strand break, is located within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 350, 400, or 500 nucleotides of the region to be modified, e.g., the site for target insertion. The cleavage, e.g., a double-strand or single-strand break, may be located upstream or downstream of the region to be modified, e.g., the site for target insertion. In some embodiments, the cleavage is located within the region to be modified, e.g., within a region defined by at least two mutated nucleotides. In some embodiments, the cleavage is located immediately adjacent to the region to be modified, e.g., immediately upstream or downstream of the target insertion site.

[0358] In some embodiments, a single-strand break is accompanied by a further single-strand break positioned by a second gRNA molecule. For example, the targeting domain is configured such that the cleavage event, e.g., two single-strand breaks, are positioned within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 350, 400, or 500 nucleotides of the target integration site. In some embodiments, the first and second gRNA molecules are configured such that, when guiding Cas9 nickase, the single-strand break is accompanied by a further single-strand break positioned by the second gRNA and located close together, resulting in modification of the desired region. In some embodiments, the first and second gRNA molecules are configured such that, for example, if Cas9 is a nickase, the single-strand break placed by the second gRNA is within 10, 20, 30, 40, or 50 nucleotides of the break placed by the first gRNA molecule. In some embodiments, the two gRNA molecules are configured to place breaks on different strands at the same position or within a few nucleotides of each other, essentially mimicking, for example, a double-strand break.

[0359] In some embodiments, a gRNA (monomolecule, chimeric, or modular gRNA) and Cas9 nuclease induce double-strand breaks for the purpose of inducing HDR-mediated insertion or modification of a transgene, the cleavage site, e.g., the target site, is located between 0 and 200 bp (e.g., 0-175, 0-150, 0-125, 0-100, 0-75, 0-50, 0-25, 25-200, 25-175, 25-150, 25-125, 25-100, 25-75, 25-50, 50-200, 50-175, 50-150, 50-125, 50-100, 50-75, 75-200, 75-175, 75-150, 75-125, 75-100 bp) away from the target integration site. In some embodiments, the target site, such as the cleavage site, is located between 0 and 100 bp (e.g., 0 to 75, 0 to 50, 0 to 25, 25 to 100, 25 to 75, 25 to 50, 50 to 100, 50 to 75, or 75 to 100 bp) away from the site for target integration.

[0360] In some embodiments, HDR can be promoted by inducing breaks with protrusions using nickase. In some embodiments, the single-strand nature of the protrusions can enhance the cell's ability to repair the breaks by HDR, as opposed to NHEJ, for example.

[0361] In particular, in some embodiments, HDR is facilitated by selecting a first gRNA that targets a first nickase to a first target site, and a second gRNA that targets a second nickase to a second target site located on the opposite DNA strand from the first target site and offset from the first nick. In some embodiments, the targeting domain of the gRNA molecule is configured to position the cleavage event well away from pre-selected nucleotides, such as nucleotides in the coding region, so that the nucleotide remains unchanged. In some embodiments, the targeting domain of the gRNA molecule is configured to position the intron cleavage event well away from intron / exon boundaries or naturally occurring splice signals to avoid exon sequencing or unwanted splicing events. In some embodiments, the targeting domain of the gRNA molecule is configured to be positioned in an early exon to enable in-frame transgene integration at or near one of at least one target sites.

[0362] In some embodiments, a double-strand break may be accompanied by a further double-strand break positioned by a second gRNA molecule. In some embodiments, a double-strand break may be accompanied by two further single-strand breaks positioned by a second gRNA molecule and a third gRNA molecule. In some embodiments, two gRNAs, for example, independently, monomolecule, chimeric, or modular gRNAs, are configured to position double-strand breaks on either side of a target integration site, for example, a site for target integration.

[0363] 2. Template polynucleotides In some embodiments, a template polynucleotide, for example, a transgene containing a sequence of nucleotides encoding a recombinant receptor or a portion thereof, e.g., an exogenous or heterologous nucleic acid sequence, and a homologous sequence (e.g., homology arm) homologous to a sequence of an endogenous genomic site or a nearby sequence for target insertion, can be used as a repair template in cellular DNA repair processes, e.g., homologous recombination. In some aspects, a template polynucleotide homologous to a sequence of one or more target sites in endogenous DNA or a nearby sequence can be used to modify the structure of target DNA, e.g., a target site of an endogenous T cell stimulation-related gene locus, for targeted insertion of a transgenic or exogenous sequence, e.g., an exogenous nucleic acid sequence encoding a recombinant receptor or a portion thereof. Polynucleotides for use in the manner provided herein, e.g., template polynucleotides, are also provided, for use as a template for homology-directed repair (HDR)-mediated target insertion of a transgene. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a recombinant receptor or a portion thereof; and one or more homology arms ligated to the nucleic acid sequence, the one or more homology arms comprising a sequence homologous to one or more regions of the open reading frame of a T cell stimulation-associated locus.

[0364] In some embodiments, the template polynucleotide includes one or more homologous sequences (e.g., homology arms) linked to and / or adjacent to a transgene (exogenous or heterologous nucleic acid sequ...

Claims

1. Engineered T cells comprising a modified T cell stimulation-related locus containing a transgene encoding a recombinant receptor incorporated into the endogenous T cell stimulation-related locus of a T cell, wherein the transgene is functionally ligated to an endogenous transcriptional regulatory element of the endogenous T cell stimulation-related locus, and the endogenous transcriptional regulatory element induces or upregulates the expression of the functionally ligated transgene following a simulation or activation signal in the T cell.

2. The manipulated T cell according to claim 1, wherein the endogenous transcriptional regulatory element is a promoter of an endogenous T cell stimulation-related gene locus, and the transgene encoding the recombinant receptor or a portion thereof is located downstream of the promoter.

3. The endogenous transcriptional regulatory element is One or more response elements recognized by a transcription factor that is activated following the aforementioned stimulus or activation signal. The manipulated T cells according to claim 1 or 2, comprising:

4. The manipulated T cell according to any one of claims 1 to 3, wherein the expression of the functionally linked transgene is induced or upregulated within 6, 12, 18, 24, 36, or 48 hours or about 6, 12, 18, 24, 36, or 48 hours following the stimulus or activation signal in the T cell; optionally, the expression of the functionally linked transgene is upregulated or induced by more than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or about 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or more.

5. The engineered T cell according to any one of claims 1 to 4, wherein the induction or upregulation of the expression of the transgene is transient over the duration of the stimulus or activation signal, and is subsequently reduced or downregulated.

6. The engineered T cell according to any one of claims 1 to 5, wherein, optionally, the expression of the functionally linked transgene is reduced or downregulated, following induction or upregulation of expression, and subsequently, following reduction or absence of simulation or activation signals in the T cell.

7. The manipulated T cell according to claim 6, wherein the expression of the functionally linked transgene is induced or upregulated in the T cell following further simulation or activation signaling after reduction or absence of simulation or activation signaling.

8. The manipulated T cell according to any one of claims 1 to 7, wherein the endogenous T cell stimulation-related gene locus is selected from PDCD1, CD69, Nur77, FoxP3, and HLA-DR gene locus.

9. The engineered T cell according to any one of claims 1 to 8, wherein the recombinant receptor comprises an extracellular binding domain, and the binding of an active agent to the extracellular binding domain of the recombinant receptor results in the induction or transmission of the stimulus or activation signal in the cell.

10. The recombinant receptor comprises an intracellular region containing an intracellular signaling domain of a component of the T cell receptor (TCR) complex, and the stimulation or activation signal in the T cell comprises a signal through the intracellular signaling domain present in the recombinant receptor, and / or The recombinant receptor comprises an intracellular region including an intracellular signaling domain containing an immune receptor-activating tyrosine motif (ITAM), and the stimulation or activation signal in T cells comprises a signal transmitted through the intracellular signaling domain present in the recombinant receptor. The manipulated T cell according to any one of claims 1 to 9.

11. The engineered T cell according to claim 10, wherein the intracellular signaling region comprises an intracellular signaling domain of a CD3 chain, optionally a CD3-zeta (CD3ζ) chain, or a signaling portion thereof.

12. The manipulated T cell according to any one of claims 9 to 11, wherein the active substance is a target antigen, and optionally, the target antigen is a recombinant protein or an antigen expressed on the surface of a cell.

13. The manipulated T cell according to claim 12, wherein the target antigen is a tumor antigen, a pathogen-specific antigen or a pathogen-expressing antigen, an inflammatory antigen, or an autoantigen.

14. The manipulated T cell according to any one of claims 9 to 11, wherein the active substance is an anti-idiotype antibody.

15. The engineered T cell according to any one of claims 1 to 14, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

16. The engineered T cell according to any one of claims 1 to 15, wherein the introduced gene encodes the recombinant receptor or a portion of the recombinant receptor.

17. The engineered T cell according to claim 16, wherein the recombinant receptor comprises two separate polypeptide chains, a portion of the recombinant receptor encoded by the transgene is one chain of the recombinant receptor, and the engineered T cell further comprises the other chain of the recombinant receptor, optionally, the other chain of the recombinant receptor is encoded by a second transgene.

18. The engineered T cell according to any one of claims 1 to 14, 16, and 17, wherein the recombinant receptor is a recombinant T cell receptor (TCR), and optionally the recombinant TCR comprises an alpha (TCRα) chain and a beta (TCRβ) chain, and the transgene comprises a nucleic acid sequence encoding the TCRα chain and a nucleic acid sequence encoding the TCRβ chain.

19. The engineered T cell according to any one of claims 1 to 18, wherein the transgene further comprises one or more multicistronic elements, optionally the multicistronic elements comprising a ribosome skipping element selected from T2A, P2A, E2A, or F2A, or a sequence encoding an intrasequence ribosome entry site (IRES).

20. The recombinant receptor is a recombinant TCR, and the multicistronic element is positioned between the sequence of a nucleotide encoding TCRα and the sequence of a nucleotide encoding TCRβ; The recombinant receptor is a multichain CAR, and the multicistronic element is positioned between the sequence of nucleotides encoding one chain of the multichain CAR and the sequence of nucleotides encoding another chain of the multichain CAR; and / or The multicistronic element is located upstream of the sequence of the nucleotide encoding the recombinant receptor. The manipulated T cell according to claim 19.

21. The modified T cell stimulation-related gene locus is modified in the following steps: a) a step of inducing gene disruption at or near one or more target sites of an endogenous T cell stimulation-related gene locus, wherein the gene disruption is optionally brought about by a combination of zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), or CRISPR-Cas9 that specifically bind to, recognize, or hybridize with the target site; and b) Steps to introduce polynucleotides for homology-directed repair (HDR). This is produced by incorporating the transgene encoding the recombinant receptor into the cell stimulation-related gene locus, The manipulated T cell according to any one of claims 1 to 20.

22. The engineered T cell according to claim 21, wherein the transgene encoding the recombinant receptor is incorporated at or near one target site in a T cell stimulation-related locus.

23. The manipulated T cell according to any one of claims 1 to 22, wherein the T cell stimulation-related gene locus is PDCD1, the gene disruption is brought about by a CRISPR-Cas9 combination including a gRNA, and the gRNA has a targeting domain complementary to a target site in the PDCD1 gene, and optionally the gRNA includes one of SEQ ID NO: 75 and 104-109, and optionally the sequence shown in SEQ ID NO:

75.

24. The manipulated T cell according to any one of claims 1 to 22, wherein the T cell stimulation-related gene locus is CD69, the gene disruption is brought about by a CRISPR-Cas9 combination including a gRNA, and the gRNA has a targeting domain complementary to a target site in the CD69 gene, and optionally the gRNA includes a sequence shown in any one of SEQ ID NO: 116 to 121.

25. The manipulated T cell according to any one of claims 1 to 22, wherein the T cell stimulation-associated gene locus is Nur77, the gene disruption is brought about by a CRISPR-Cas9 combination including a gRNA, and the gRNA has a targeting domain complementary to a target site in the Nur77 gene, and optionally the gRNA includes sequences shown in SEQ ID NO: 122-127 and 134-136.

26. The manipulated T cell according to any one of claims 1 to 22, wherein the T cell stimulation-related gene locus is FoxP3.

27. The manipulated T cell according to any one of claims 1 to 22, wherein the T cell stimulation-related gene locus is the HLA-DR gene locus.

28. The engineered T cell according to any one of claims 1 to 27, wherein the T cell further comprises gene disruption in the endogenous T cell receptor α constant region (TRAC) gene and / or the endogenous T cell receptor β constant region (TRBC) gene, optionally, the gene disruption is brought about by a CRISPR-Cas9 combination, and the CRISPR-Cas9 combination comprises a guide RNA (gRNA) having a targeting domain complementary to at least one target site in the TRAC, TRBC1, and / or TRBC2 genes, optionally, the CRISPR-Cas9 combination is a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas9 protein, and optionally, the gene disruption is brought about by the RNP introduced into a plurality of T cells via electroporation.

29. The gRNA has a targeting domain complementary to a target site in the TRAC gene, and optionally the gRNA contains one of SEQ ID NO:77 and 188-218, optionally the sequence shown in SEQ ID NO:77; and / or The gRNA has a targeting domain complementary to a target site in the TRBC gene, and optionally, the gRNA contains a sequence represented by any one of SEQ ID NO: 219 to 276. The manipulated T cell according to claim 28.

30. The engineered T cell according to any one of claims 1 to 29, wherein the signaling activity through the intracellular signaling domain of the encoded recombinant receptor in the absence of simulation or activation signal in the T cell is reduced by 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more, or by about 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more, compared to engineered T cells comprising a transgene encoding the same recombinant receptor located at different locations in the T cell genome or at random locations in the T cell genome.

31. The manipulated T cell according to any one of claims 1 to 30, wherein the T cell is a CD8+ T cell or a CD4+ T cell, or a subtype thereof.

32. The manipulated T cells according to any one of claims 1 to 31, wherein the T cells are T cells derived from a subject, and optionally the subject is human.

33. (a) a transgene encoding a recombinant receptor or a portion thereof, and (b) One or more homologous arms linked to the transgene, each containing a sequence homologous to one or more regions of an endogenous T cell stimulation-related gene locus in a T cell. Polynucleotides, including [specifically, polynucleotides].

34. The recombinant receptor is expressed from cells into which the polynucleotide has been introduced, and the recombinant receptor or a portion thereof is encoded by a modified T cell stimulation-associated locus containing a transgene encoding the recombinant receptor or a portion thereof; and / or The aforementioned transgene is a sequence that is exogenous or heterologous to the open reading frame of the endogenous T cell stimulation-related gene locus of T cells, optionally human T cells. The polynucleotide according to claim 33.

35. The polynucleotide according to claim 33 or 34, wherein the one or more homology arms include a 5' homology arm and / or a 3' homology arm, and optionally the 5' homology arm and / or 3' homology arm include a nucleic acid sequence homologous to the nucleic acid sequence surrounding the target site, and the target site is located within a T cell stimulation-associated gene locus.

36. The polynucleotide according to claim 35, wherein the target site is located downstream of an endogenous transcriptional regulatory element of a T cell stimulation-related gene locus.

37. The polynucleotide according to claim 35 or 36, comprising the structure [5' homology arm]-[introduced gene]-[3' homology arm].

38. The 5' homology arm and the 3' homology arm independently consist of 50 or about 50 to 750 or about 750 nucleotides, 50 or about 50 to 500 or about 500 nucleotides, 50 or about 50 to 250 or about 250 nucleotides, 50 or about 50 to 100 or about 100 nucleotides, 100 or about 100 to 750 or about 750 nucleotides, 100 or about 100 to 500 or about 500 nucleotides, 100 or about 100 to 250 or about 250 nucleotides, 250 or about 250 to 750 or about 750 nucleotides, 250 or about 250 to 500 or about 500 nucleotides. The polynucleotide according to any one of claims 33 to 37, having a length of; independently, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, or 500 nucleotides or about 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, or 500 nucleotides, or any value between any of the aforementioned; or independently, a length of less than 100 nucleotides or about less than 100 nucleotides, optionally 50, 60, 70, 80, or 90 nucleotides or about 50, 60, 70, 80, or 90 nucleotides, or any value between any of the aforementioned.

39. The polynucleotide according to any one of claims 33 to 38, wherein the T cell stimulation-related gene locus is selected from PDCD1, CD69, Nur77, FoxP3, and HLA-DR gene locus.

40. The polynucleotide according to claim 39, wherein the T cell stimulation-related gene locus is PDCD1, and optionally the 5' homology arm and the 3' homology arm contain sequences homologous to one or more regions of PDCD1.

41. The aforementioned 5' homology arm, a) A sequence containing 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 consecutive nucleotides to a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the sequence shown in SEQ ID NO:66; b) A sequence containing 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 consecutive nucleotides of the sequence shown in SEQ ID NO:66, or at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 consecutive nucleotides; or c) Sequence shown in SEQ ID NO:66 Including; and / or The 3' homology arm is d) A sequence containing 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 consecutive nucleotides to a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the sequence shown in SEQ ID NO:67; e) A sequence containing 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 consecutive nucleotides of the sequence shown in SEQ ID NO:67, or at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 consecutive nucleotides; or f) Sequence shown in SEQ ID NO:67 including, The polynucleotide according to claim 40.

42. The polynucleotide according to claim 39, wherein the T cell stimulation-associated gene locus is CD69, and optionally the 5' homology arm and the 3' homology arm contain sequences homologous to one or more regions of CD69.

43. The polynucleotide according to claim 39, wherein the T cell stimulation-associated gene locus is Nur77, and optionally the 5' homology arm and the 3' homology arm contain sequences homologous to one or more regions of Nur77.

44. The polynucleotide according to claim 39, wherein the T cell stimulation-associated gene locus is FoxP3, and optionally the 5' homology arm and the 3' homology arm contain sequences homologous to one or more regions of FoxP3.

45. The polynucleotide according to claim 39, wherein the T cell stimulation-related gene locus is an HLA-DR gene locus, and optionally the 5' homology arm and the 3' homology arm contain sequences homologous to one or more regions of the HLA-DR gene locus.

46. The polynucleotide according to any one of claims 33 to 45, wherein the recombinant receptor comprises an extracellular binding domain, and the binding of an active agent to the extracellular binding domain of the recombinant receptor results in the induction or transmission of the stimulus or activation signal in the cell.

47. The recombinant receptor comprises an intracellular region containing an intracellular signaling domain of a component of the T cell receptor (TCR) complex, and the stimulation or activation signal in the T cell comprises a signal through the intracellular signaling domain present in the recombinant receptor, and / or The recombinant receptor comprises an intracellular region including an intracellular signaling domain containing an immune receptor-activating tyrosine motif (ITAM), and the stimulation or activation signal in T cells comprises a signal transmitted through the intracellular signaling domain present in the recombinant receptor. The polynucleotide according to any one of claims 33 to 46.

48. The polynucleotide according to claim 47, wherein the intracellular signaling region comprises an intracellular signaling domain of a CD3 chain, optionally a CD3-zeta (CD3ζ) chain, or a signaling portion thereof.

49. The polynucleotide according to any one of claims 46 to 48, wherein the active substance is a target antigen, and optionally, the target antigen is a recombinant protein or an antigen expressed on the surface of a cell.

50. The polynucleotide according to claim 49, wherein the target antigen is a tumor antigen, a pathogen-specific antigen or a pathogen-expressing antigen, an inflammatory antigen, or an autoantigen.

51. The polynucleotide according to any one of claims 46 to 48, wherein the active substance is an anti-idiotype antibody.

52. The polynucleotide according to any one of claims 33 to 51, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

53. The polynucleotide according to any one of claims 33 to 52, wherein the introduced gene encodes the recombinant receptor or a portion of the recombinant receptor.

54. The polynucleotide according to claim 53, wherein the recombinant receptor comprises two separate polypeptide chains, a portion of the recombinant receptor encoded by the transgene is one chain of the recombinant receptor, and optionally the other chain of the recombinant receptor is encoded by a second transgene.

55. The polynucleotide according to any one of claims 33 to 51, 53, and 54, wherein the recombinant receptor is a recombinant T cell receptor (TCR), and optionally the recombinant TCR comprises an alpha (TCRα) chain and a beta (TCRβ) chain, and the transgene comprises a nucleic acid sequence encoding the TCRα chain and a nucleic acid sequence encoding the TCRβ chain.

56. The polynucleotide according to any one of claims 33 to 55, wherein the transgene further comprises one or more multicistronic elements, optionally the multicistronic elements comprising a ribosome skipping element selected from T2A, P2A, E2A, or F2A, or a sequence encoding an intra-sequence ribosome entry site (IRES).

57. The recombinant receptor is a recombinant TCR, and the multicistronic element is positioned between the sequence of a nucleotide encoding TCRα and the sequence of a nucleotide encoding TCRβ; The recombinant receptor is a multichain CAR, and the multicistronic element is positioned between the sequence of nucleotides encoding one chain of the multichain CAR and the sequence of nucleotides encoding another chain of the multichain CAR; and / or The multicistronic element is located upstream of the sequence of the nucleotide encoding the recombinant receptor. The polynucleotide according to claim 56.

58. A linear polynucleotide, as described in any one of claims 33 to 57.

59. A polynucleotide according to any one of claims 33 to 57, contained in a viral vector.

60. The polynucleotide according to claim 59, wherein the viral vector is an AAV vector.

61. A polynucleotide according to any one of claims 33 to 60, having a length of 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,250, 3,500, 3,750, or 4,000 nucleotides, or a length of approximately 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,250, 3,500, 3,750, or 4,000 nucleotides, or any value between any of the above; or a length of 1,500 or approximately 1,500 to 2,500 or approximately 2,500 nucleotides, or 1,750 or approximately 1,750 to 2,250 or approximately 2,250 nucleotides.

62. (a) the step of introducing one or more active agents into T cells that can induce gene disruption at a target site within an endogenous T cell stimulation-related gene locus on T cells; and (b) A step of introducing a polynucleotide according to any one of claims 33 to 61 into a T cell. A method for producing genetically modified T cells, including, The method is Modified T cell stimulation-related loci, including transgenes encoding recombinant receptors or parts thereof. To create, The transgene encoding the recombinant receptor or a portion thereof is incorporated into an endogenous T cell stimulation-related locus via homology-directed repair (HDR). The aforementioned method.

63. A method for producing genetically modified T cells, comprising the step of introducing a polynucleotide according to any one of claims 33 to 61 into T cells, wherein the T cells have a gene disruption within the T cell stimulation-related gene locus of the T cells, and the transgene encoding the recombinant receptor or a portion thereof is incorporated into the endogenous T cell stimulation-related gene locus via homology-directed repair (HDR).

64. The method according to claim 63, wherein the gene disruption is carried out by introducing one or more active substances into a T cell that can induce gene disruption at a target site within an endogenous T cell stimulation-related gene locus of a T cell.

65. Modified T cell stimulation-related loci, including transgenes encoding recombinant receptors or parts thereof. A method for producing according to any one of claims 62 to 64.

66. The method according to any one of claims 62 to 65, wherein the target site is downstream of an endogenous transcriptional regulatory element of an endogenous T cell stimulation-related gene locus.

67. The method according to any one of claims 62 to 66, wherein the T cell stimulation-related gene locus is selected from among PDCD1, CD69, Nur77, FoxP3, and HLA-DR gene locus.

68. The method according to any one of claims 62 to 67, wherein the gene disruption is brought about by a combination of zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), or CRISPR-Cas9 that specifically bind to, recognize, or hybridize with a target site.

69. The method according to any one of claims 62 to 68, wherein the gene disruption is brought about by a combination of CRISPR-Cas9, and the combination of CRISPR-Cas9 comprises a guide RNA (gRNA) having a targeting domain complementary to at least one target site, optionally the combination of CRISPR-Cas9 is a ribonucleoprotein (RNP) complex comprising gRNA and Cas9 protein, and optionally the gene disruption is brought about by RNP introduced into a plurality of T cells via electroporation.

70. The method according to any one of claims 62 to 69, wherein the T cell stimulation-related gene locus is PDCD1, the gene disruption is brought about by a combination of CRISPR-Cas9 including a gRNA, and the gRNA has a targeting domain complementary to a target site in the PDCD1 gene, and optionally the gRNA includes one of SEQ ID NO: 75 and 104-109, and optionally the sequence shown in SEQ ID NO:

75.

71. The method according to any one of claims 62 to 69, wherein the T cell stimulation-related gene locus is CD69, the gene disruption is brought about by a CRISPR-Cas9 combination including a gRNA, and the gRNA has a targeting domain complementary to a target site in the CD69 gene, and optionally the gRNA includes a sequence shown in any one of SEQ ID NO: 116 to 121.

72. The method according to any one of claims 62 to 69, wherein the T cell stimulation-related gene locus is Nur77, the gene disruption is brought about by a CRISPR-Cas9 combination including a gRNA, and the gRNA has a targeting domain complementary to a target site in the Nur77 gene, and optionally the gRNA includes sequences shown in SEQ ID NOs: 122-127 and 134-136.

73. The method according to any one of claims 62 to 69, wherein the T cell stimulation-related gene locus is FoxP3.

74. The method according to any one of claims 62 to 69, wherein the T cell stimulation-related gene locus is the HLA-DR gene locus.

75. The method according to any one of claims 62 to 74, wherein the T cells further comprise gene disruption in the endogenous T cell receptor α constant region (TRAC) gene and / or the endogenous T cell receptor β constant region (TRBC) gene, optionally, the gene disruption is brought about by a CRISPR-Cas9 combination, and the CRISPR-Cas9 combination comprises a guide RNA (gRNA) having a targeting domain complementary to at least one target site in the TRAC, TRBC1, and / or TRBC2 genes, optionally, the CRISPR-Cas9 combination is a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas9 protein, and optionally, the gene disruption is brought about by an RNP introduced into a plurality of T cells via electroporation.

76. The gRNA has a targeting domain complementary to a target site in the TRAC gene, and optionally the gRNA contains one of SEQ ID NO:77 and 188-218, optionally the sequence shown in SEQ ID NO:77; and / or The gRNA has a targeting domain complementary to a target site in the TRBC gene, and optionally, the gRNA contains a sequence represented by any one of SEQ ID NO: 219 to 276. The method according to claim 75.

77. The method according to any one of claims 69 to 76, wherein the RNP is optionally introduced via electroporation, particle gun, calcium phosphate transfection, cell compression or compression, and optionally the RNP is introduced into a plurality of T cells via electroporation.

78. The method according to any one of claims 69 to 77, wherein the concentration of the RNP is 1 μM or about 1 μM to 5 μM or about 5 μM, and optionally, the concentration of the RNP is 2 μM or about 2 μM.

79. The method according to any one of claims 62 to 78, wherein the T cells include CD8+ T cells and / or CD4+ T cells or their subtypes.

80. The method according to any one of claims 62 to 79, wherein the T cells are human T cells, and optionally, primary T cells derived from a human subject.

81. The method according to any one of claims 62 and 64-80, wherein the one or more active substances and the polynucleotide are introduced simultaneously.

82. The method according to any one of claims 62 and 64-80, wherein the polynucleotide is introduced after the introduction of the one or more active substances.

83. The method according to claim 82, wherein the polynucleotide is introduced immediately after the introduction of the active substance, or within approximately 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 6 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, or 4 hours after the introduction.

84. The method according to any one of claims 62 and 64-83, wherein, prior to the introduction of the one or more active agents and / or the polynucleotide, the method comprises in vitro incubation of the cells with a stimulant under conditions for stimulating or activating one or more immune cells, wherein the stimulant optionally comprises an anti-CD3 antibody and / or an anti-CD28 antibody, and optionally comprises an oligomeric particle reagent comprising an anti-CD3 antibody and / or an anti-CD28 antibody, or beads coated with an anti-CD3 antibody and / or an anti-CD28 antibody.

85. The method further comprises the step of incubating the cells with one or more recombinant cytokines before, during, or after the introduction of the one or more active substances and / or the introduction of the polynucleotide, wherein the one or more recombinant cytokines are optionally selected from the group consisting of IL-2, IL-7, and IL-15, and optionally the one or more recombinant cytokines are present in concentrations of IL-2 from 10 U / mL or about 10 U / mL to 200 U / mL or about 200 U / mL, optionally from 50 IU / mL or about 50 IU / mL, and 100 U / mL or about 100 U / mL; concentrations of IL-7 from 0.5 ng / mL to 50 ng / mL, optionally from 5 ng / mL or about 5 ng / mL, and / or from 0.1 ng / mL to 20 ng / mL, optionally from 0.5 ng / mL or about 0.5 ng / mL, and 5 The method according to any one of claims 62 and 64-84, wherein IL-15 is added at a concentration selected from ng / mL or about 5 ng / mL.

86. The method according to claim 84 or 85, wherein incubation is carried out for a maximum of 24 hours, 36 hours, 48 ​​hours, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days, or optionally for a maximum of 7 days or about 7 days, after the introduction of the one or more active substances and the introduction of the polynucleotide.

87. Engineered T cells produced using the method described in any one of claims 62 to 86.

88. A composition comprising manipulated cells according to any one of claims 1 to 32 or a plurality of manipulated cells according to any one of claims 1 to 32.

89. A composition comprising the manipulated T cells described in claim 87 or a plurality of the manipulated T cells described in claim 87.

90. The composition according to claim 88 or 89, wherein the expression of the functionally linked transgene is induced or upregulated in one or more cells in the composition within 6, 12, 18, 24, 36, or 48 hours or about 6, 12, 18, 24, 36, or 48 hours following the stimulation or activation signal in T cells, and optionally, the frequency of cells expressing the functionally linked transgene among the cells in the composition following the simulation or activation signal in T cells is higher than 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or about 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or higher.

91. The composition according to any one of claims 88 to 90, wherein the expression of the functionally linked transgene is reduced or downregulated in one or more cells in the composition, following upregulation or induction of expression, or following reduction or absence of simulation or activation signals in T cells.

92. Following upregulation or induction of expression, the expression of the functionally linked transgene is reduced or downregulated in one or more cells in the composition 1, 2, 3, 4, 5, 6, 7, or 8 days or more after the stimulus or activation signal in T cells; or The expression of the functionally linked transgene in one or more cells in the composition is reduced or downregulated within 6, 12, 18, 24, 36, or 48 hours, or within approximately 6, 12, 18, 24, 36, or 48 hours, following the reduction or absence of simulation or activation signals in T cells, and optionally, the frequency of cells expressing the functionally linked transgene in the cells of the composition is reduced by more than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or more, or approximately 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or more; and / or Following a reduction or absence of simulation or activation signals in T cells, the frequency of cells expressing the recombinant receptor in the composition is lower than 50%, 40%, 30%, 25%, 20%, 15%, 10%, or 5%, or about 50%, 40%, 30%, 25%, 20%, 15%, 10%, or 5%, or lower. The composition according to any one of claims 88 to 91.

93. The composition according to any one of claims 88 to 92, comprising CD4+ T cells and / or CD8+ T cells.

94. The composition according to any one of claims 88 to 93, comprising CD4+ T cells and CD8+ T cells, wherein the ratio of CD4+ T cells to CD8+ T cells is 1:3 to 3:1 or about 1:3 to 3:1, optionally 1:

1.

95. A method of treatment comprising the step of administering manipulated cells according to any one of claims 1 to 32 and 87, or a composition according to any one of claims 88 to 94, to a subject having a disease or disorder.

96. The method according to claim 95, wherein the disease or disorder is cancer or a tumor.

97. The method according to claim 96, wherein the cancer or tumor is a hematological malignancy, optionally a lymphoma, leukemia, or plasma cell malignancy, and optionally the cancer is a lymphoma, and the lymphoma is Burkitt lymphoma, non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, Waldenström macroglobulinemia, follicular lymphoma, small non-incisional nuclear cell lymphoma, mucosa-associated lymphoid tissue lymphoma (MALT), marginal zone lymphoma, splenic lymphoma, nodular monocytic B-cell lymphoma, immunoblastic lymphoma, large cell lymphoma, diffuse mixed cell lymphoma, pulmonary B-cell angiocentral lymphoma, small lymphocytic lymphoma, primary mediastinal B-cell lymphoma, lymphoplasmacytic lymphoma (LPL), or mantle cell lymphoma (MCL).

98. The method according to claim 96 or 97, wherein the cancer is leukemia, and the leukemia is chronic lymphocytic leukemia (CLL), plasma cell leukemia, or acute lymphocytic leukemia (ALL).

99. The method according to claim 96 or 97, wherein the cancer is a plasma cell malignancy, and the plasma cell malignancy is multiple myeloma (MM).

100. The method according to claim 96, wherein the tumor is a solid tumor, and optionally, the solid tumor is non-small cell lung cancer (NSCLC) or head and neck squamous cell carcinoma (HNSCC).

101. The method according to any one of claims 96 to 100, wherein the volume or size of cancer or tumors is reduced and / or the survival of the subject is extended compared to that of the manipulated cells or the subject not administered the composition.

102. One or more activators capable of inducing gene disruption at target sites within T cell stimulation-related gene loci; and Polynucleotide according to any one of claims 33 to 61 A kit that includes this.

103. One or more activators capable of inducing gene disruption at target sites within T cell stimulation-related gene loci; and A polynucleotide comprising a nucleic acid sequence encoding a recombinant receptor or a portion thereof, wherein a transgene encoding the recombinant receptor or its antigen-binding fragment or chain is targeted for integration at or near a target site via homology-directed repair (HDR); and Instructions for carrying out the method described in any one of claims 62 to 86. A kit that includes this.

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