Cells expressing chimeric receptors from an altered CD247 locus, related polynucleotides, and methods

Abstract

Provided herein are engineered immune cells, e.g., T cells, that express a chimeric receptor comprising an intracellular region containing a CD3 zeta (CD3ζ) signaling domain. In some embodiments, the engineered immune cells contain a modified CD247 locus that encodes the chimeric receptor or a portion thereof. In some embodiments, at least a portion of the CD3 zeta chain is encoded by the CD247 genomic locus. Also provided are cell compositions containing the engineered immune cells, nucleic acids for engineering cells, and methods, kits, and articles of manufacture for producing engineered cells, for example, by targeting a transgene encoding a portion of the chimeric receptor for integration into a region of the CD247 genomic locus. In some embodiments, the engineered cells, e.g., T cells, can be used in connection with cell therapy, for example, in connection with cancer immunotherapy, including adoptive transfer of engineered cells. TIFF2022531577000051.tif93154

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 841,578, filed May 1, 2019, entitled "CELLS EXPRESSING A CHIMERIC RECEPTOR FROM A MODIFIED CD247 LOCUS, RELATED POLYNUCLEOTIDES AND METHODS," the contents of which are incorporated by reference in their entirety.

[0002] INCORPORATION-BY-REFERENCE TO SEQUENCE LISTING This application is filed with an electronic Sequence Listing, which is provided as a file entitled 735042015840SeqList.txt, created on April 28, 2020, and having a size of 172 kilobytes. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] Field The present disclosure relates to engineered immune cells, e.g., T cells, that express a chimeric receptor comprising an intracellular region that includes the CD3 zeta (CD3ζ) signaling domain. In some embodiments, the engineered immune cells contain a modified CD247 locus that encodes the chimeric receptor or a portion thereof. In some embodiments, at least a portion of the CD3 zeta chain is encoded by the CD247 genomic locus. Also provided are cell compositions containing the engineered immune cells, nucleic acids for engineering the cells, and methods, kits, and articles of manufacture for producing engineered cells, e.g., by targeting a transgene encoding a portion of the chimeric receptor for integration into a region of the CD247 genomic locus. In some embodiments, the engineered cells, e.g., T cells, can be used in connection with cell therapy, e.g., cancer immunotherapy, including adoptive transfer of the engineered cells. [Background technology]

[0004] background Adoptive cell therapy, utilizing chimeric receptors such as chimeric antigen receptors (CARs) that recognize disease-associated antigens, has become an attractive therapeutic modality for the treatment of cancer and other diseases.For example, there is a need for improved strategies for engineering T cells to express chimeric receptors, for use in adoptive immunotherapy, for example, in the treatment of cancer, infectious diseases, and autoimmune diseases.Methods, cells for use in the methods, compositions, and kits that meet such needs are provided. Summary of the Invention

[0005] overview Provided herein are genetically engineered T cells, as well as compositions, methods, uses, kits, and articles of manufacture related to the genetically engineered T cells. In some of any of the provided embodiments, the genetically engineered T cells comprise a modified cluster of differentiation 247 (CD247) locus. In some of any of the provided embodiments, the modified CD247 locus comprises a transgene sequence encoding a chimeric receptor or a portion thereof. In provided embodiments, the transgene sequence is in-frame with the open reading frame of the endogenous CD247 locus or a subsequence thereof. Thus, in provided embodiments, the modified CD247 locus encodes a chimeric receptor comprising a sequence encoded from the transgene sequence and a sequence encoded from the endogenous CD247 locus. In a specific embodiment, the chimeric receptor contains an intracellular region comprising a CD3 zeta (CD3ζ) signaling domain, and the CD3ζ signaling domain, e.g., the entire CD3ζ signaling domain or at least a portion of the CD3ζ signaling domain, is encoded by the genomic sequence (e.g., open reading frame) of the endogenous CD247 locus (the genomic locus encoding CD3ζ) of the engineered cell, e.g., a T cell.

[0006] Provided herein are genetically engineered T cells containing a modified CD247 locus. In some embodiments, the modified CD247 locus comprises a nucleic acid sequence encoding a chimeric receptor comprising an intracellular region including a CD3 zeta (CD3ζ) signaling domain. In some embodiments, the nucleic acid sequence comprises a transgene sequence encoding a portion of the chimeric receptor, the transgene sequence being integrated into the endogenous CD247 locus. In some embodiments, the integration occurs via homology-directed repair (HDR). In some embodiments, all or a fragment of the CD3ζ signaling domain of the intracellular region of the chimeric receptor is encoded by the open reading frame of the endogenous CD247 locus, or a subsequence thereof. In some embodiments, the nucleic acid sequence comprises an in-frame fusion of (i) a transgene sequence encoding a portion of the chimeric receptor and (ii) an open reading frame of the endogenous CD247 locus, or a subsequence thereof. In a specific embodiment, the modified CD247 locus encodes a chimeric receptor containing an intracellular region comprising a CD3 zeta (CD3ζ) signaling domain, wherein the CD3ζ signaling domain, e.g., the entire CD3ζ signaling domain or at least a portion of the CD3ζ signaling domain, is encoded by the genomic sequence of the endogenous CD247 locus (the genomic locus encoding CD3ζ) of the engineered cell, e.g., a T cell.

[0007] Provided herein are genetically engineered T cells containing a modified CD247 locus, wherein the modified CD247 locus comprises a nucleic acid sequence encoding a chimeric receptor comprising an intracellular region comprising a CD3ζ signaling domain, the nucleic acid sequence comprising an in-frame fusion of (i) a transgene sequence encoding a portion of the chimeric receptor with (ii) an open reading frame of the endogenous CD247 locus encoding the CD3ζ signaling domain, or a subsequence thereof. In a specific embodiment, the modified CD247 locus encodes a chimeric receptor containing an intracellular region comprising the CD3 zeta (CD3ζ) signaling domain, wherein the CD3ζ signaling domain or at least a portion of the CD3ζ signaling domain is encoded by the genomic sequence of the endogenous CD247 locus (a genomic locus encoding CD3ζ) of the engineered cell, e.g., T cell.

[0008] In some of the optional embodiments, the transgene sequence is in frame with one or more exons of the open reading frame of the endogenous CD247 locus or a subsequence thereof.

[0009] In some of the embodiments, the transgene sequence does not include a sequence encoding a 3'UTR. In some of the embodiments, the transgene sequence does not include an intron.

[0010] In some of any of the embodiments, the transgene sequence encodes a fragment of the CD3ζ signaling domain. For example, in specific embodiments, the CD3ζ signaling domain of the chimeric receptor, or a fragment thereof, is encoded by both the sequence of the transgene sequence and the genomic sequence (e.g., open reading frame) of the endogenous CD247 locus (the genomic locus encoding CD3ζ) of the engineered cell, e.g., T cell.

[0011] In some of any of the embodiments, the transgene sequence does not encode the CD3ζ signaling domain or a fragment thereof. For example, in specific embodiments, the entire or full-length CD3ζ signaling domain or a fragment thereof of the chimeric receptor is encoded by the genomic sequence of the endogenous CD247 locus (the genomic locus encoding CD3ζ) of the engineered cell, e.g., T cell.

[0012] In some of the embodiments, the open reading frame or subsequence thereof comprises at least one intron and at least one exon of the endogenous CD247 locus. In some of the embodiments, the open reading frame or subsequence thereof encodes the 3'UTR of the endogenous CD247 locus.

[0013] In some of any of the embodiments, the transgene sequence is downstream of exon 1 and upstream of exon 8 of the open reading frame of the endogenous CD247 locus. In some of any of the embodiments, the transgene sequence is downstream of exon 1 and upstream of exon 3 of the open reading frame of the endogenous CD247 locus.

[0014] In some of the embodiments, at least a fragment of the CD3ζ signaling domain of the encoded chimeric receptor, e.g., the entire CD3ζ signaling domain, is encoded by an open reading frame of the endogenous CD247 locus or a subsequence thereof. In some of the embodiments, the CD3ζ signaling domain is encoded by a sequence of nucleotides that includes at least a portion of exon 2 and exons 3-8 of the open reading frame of the endogenous CD247 locus. In some of the embodiments, the CD3ζ signaling domain is encoded by a sequence of nucleotides that does not include exon 1, does not include the entirety of exon 1, and / or does not include the entirety of exon 2 of the open reading frame of the endogenous CD247 locus.

[0015] In some of the optional embodiments, the encoded chimeric receptor is capable of signaling through the CD3ζ signaling domain.

[0016] In some of the optional embodiments, the encoded CD3 zeta signaling domain comprises a sequence selected from any one of SEQ ID NOs:13-15, or 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 any one of SEQ ID NOs:13-15, or a fragment thereof. In some embodiments, the encoded CD3 zeta signaling domain comprises the sequence set forth in SEQ ID NO:13. In some embodiments, the encoded CD3 zeta signaling domain comprises the sequence set forth in SEQ ID NO:14. In some embodiments, the encoded CD3 zeta signaling domain comprises the sequence set forth in SEQ ID NO:15.

[0017] In some of the optional embodiments, the chimeric receptor is or comprises a functional non-T cell receptor (non-TCR) antigen receptor.

[0018] In some of the embodiments, the chimeric receptor is a chimeric antigen receptor (CAR). In some of the embodiments, the chimeric receptor further comprises an extracellular region and / or a transmembrane domain.

[0019] In some of the embodiments, the transgene sequence comprises a sequence of nucleotides encoding one or more regions of the chimeric receptor. In some of the embodiments, the transgene sequence comprises a sequence of nucleotides encoding one or more of the extracellular region, the transmembrane domain, and / or a portion of the intracellular region. In some of the embodiments, the extracellular region comprises a binding domain. In some of the embodiments, the binding domain is an antibody or an antigen-binding fragment thereof. In some of the embodiments, the binding domain comprises an antibody or an antigen-binding fragment thereof.

[0020] In some of the embodiments, the binding domain is capable of binding to a target antigen associated with, specific for, and / or expressed on cells or tissues of a disease, disorder, or condition, hi some of the embodiments, the target antigen is a tumor antigen. In some of the optional embodiments, the target antigen is selected from the group consisting of 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), epidermal growth factor receptor type III mutant (EGFR), and EGFR fusion protein (EGFR fusion protein). vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor-like 5 (FCRL5;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), kappa light chain, L1 cell adhesion molecule (L1-CAM), and the 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, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligand, melan-A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, melanoma-preferentially expressed antigen Gen (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein G (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;dopachrome tautomerase, 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 or pathogen-expressed antigens, or antigens associated with universal tags, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV, or other pathogens;

[0021] In some of the embodiments, the extracellular region comprises a spacer. In some of the embodiments, the spacer is operably linked between the binding domain and the transmembrane domain. In some of the embodiments, the spacer comprises an immunoglobulin hinge region. In some of the embodiments, the spacer comprises a C H 2 area and C H Includes three areas.

[0022] In some of any of the embodiments, the portion of the intracellular region encoded by the transgene sequence comprises one or more costimulatory signaling domains. In some of any of the embodiments, the one or more costimulatory signaling domains comprise the intracellular signaling domain of CD28, 4-1BB, or ICOS, or a signaling portion thereof. In some embodiments, the costimulatory signaling domain is the signaling domain of human CD28. In some embodiments, the costimulatory signaling domain is the signaling domain of human 4-1BB. In some embodiments, the costimulatory signaling domain is the signaling domain of human ICOS. In some of any of the embodiments, the one or more costimulatory signaling domains comprise the intracellular signaling domain of 4-1BB, e.g., human 4-1BB.

[0023] In some of the embodiments, the modified CD247 locus encodes a chimeric receptor that includes, in N- to C-terminal order, an extracellular binding domain, a spacer, a transmembrane domain, and an intracellular signaling region. In a specific embodiment, the intracellular region contains a CD3 zeta (CD3ζ) signaling domain, and the CD3ζ signaling domain, or at least a portion of the CD3ζ signaling domain, is encoded by the genomic sequence of the endogenous CD247 locus (the genomic locus encoding CD3ζ) of the engineered cell, e.g., T cell.

[0024] In some of the embodiments, the transgene sequence comprises a sequence of nucleotides encoding, in order, an extracellular binding domain; a spacer; a transmembrane domain; and a costimulatory signaling domain. In some of the embodiments, the modified CD247 locus comprises a sequence of nucleotides encoding, in order, an extracellular binding domain; a spacer; a transmembrane domain; and an intracellular region containing a costimulatory signaling domain and a CD3 zeta (CD3ζ) signaling domain. In specific embodiments, the intracellular signaling region contains a costimulatory signaling domain and a CD3 zeta (CD3ζ) signaling domain, and the CD3ζ signaling domain, or at least a portion of the CD3ζ signaling domain, is encoded by a genomic sequence (e.g., an open reading frame) of the endogenous CD247 locus (a genomic locus encoding CD3ζ) of the engineered cell, e.g., a T cell. In some of the embodiments, the transgene sequence comprises, in order, an extracellular binding domain that is an scFv; a sequence derived from a human immunoglobulin hinge derived from IgG1, IgG2, or IgG4, or a modified version thereof, and a C H 2 area and / or C Hand a costimulatory signaling domain derived from human 4-1BB. In some of the embodiments, the modified CD247 locus comprises, in order, an extracellular binding domain that is an scFv; a sequence derived from a human immunoglobulin hinge, such as from IgG1, IgG2, or IgG4, or a modified version thereof, and a sequence of nucleotides encoding the C4 domain. H 2 area and / or C H The engineered T cell comprises a sequence of nucleotides encoding a spacer comprising three regions; a transmembrane domain derived from human CD28; and an intracellular region containing a costimulatory signaling domain and a CD3ζ signaling domain derived from human 4-1BB. In a specific embodiment, the intracellular region contains a costimulatory signaling domain and a CD3 zeta (CD3ζ) signaling domain, and the CD3ζ signaling domain, or at least a portion of the CD3ζ signaling domain, is encoded by a genomic sequence (e.g., an open reading frame) of the endogenous CD247 locus (a genomic locus encoding CD3ζ) of the engineered cell, e.g., T cell.

[0025] In some of the optional embodiments, the chimeric receptor is a CAR that is a multi-chain CAR.

[0026] In some embodiments, the transgene sequence includes a sequence of nucleotides encoding at least one additional protein. For example, the at least one additional protein can be another chain of the CAR. In some examples, the at least one additional protein is a surrogate marker or a truncated receptor for co-expression on cells with the chimeric receptor. In some embodiments, the transgene sequence includes, for example, one or more multicistronic elements separating the chimeric receptor and one or more additional proteins. In some embodiments, the multicistronic element is located between the sequence of nucleotides encoding a portion of the chimeric receptor and the sequence of nucleotides encoding at least one additional protein. In some embodiments, the at least one additional protein is a surrogate marker. In some embodiments, the surrogate marker is a truncated receptor. In some embodiments, the truncated receptor lacks an intracellular signaling domain and / or cannot mediate intracellular signaling when its ligand is bound. In some embodiments, the chimeric receptor is a multi-chain CAR, and a multicistronic element is located between the sequence of nucleotides encoding one chain of the multi-chain CAR and the sequence of nucleotides encoding another chain of the multi-chain CAR.In some embodiments, one or more multicistronic elements are upstream of the sequence of nucleotides encoding a portion of the chimeric receptor.In some embodiments, one or more multicistronic elements are or include a ribosomal skip sequence.In some embodiments, the ribosomal skip sequence is a T2A, P2A, E2A, or F2A element.

[0027] In some of the embodiments, the modified CD247 locus comprises a promoter and / or control or regulatory elements of the endogenous CD247 locus operably linked to regulate expression of the nucleic acid sequence encoding the chimeric receptor. In some of the embodiments, the modified locus comprises one or more heterologous control or regulatory elements operably linked to regulate expression of the nucleic acid sequence encoding the chimeric receptor. In some of the embodiments, the one or more heterologous control or regulatory elements include a promoter, enhancer, intron, polyadenylation signal, Kozak consensus sequence, splice acceptor sequence, and / or splice donor sequence. In some of the embodiments, the heterologous promoter is or comprises the human elongation factor 1 alpha (EF1α) promoter or the MND promoter or variants thereof.

[0028] In some of any of the embodiments, the T cells are primary T cells derived from a subject. In some of any of the embodiments, the subject is human. In some of any of the embodiments, the T cells are CD8+ T cells or subtypes thereof. In some of any of the embodiments, the T cells are CD4+ T cells or subtypes thereof. In some of any of the embodiments, the T cells are derived from multipotent or totipotent cells. In some of any of the embodiments, the totipotent cells are iPSCs. In some of any of the embodiments, the T cells are derived from pluripotent or totipotent cells that are iPSCs.

[0029] Also provided herein are polynucleotides that can be used to integrate a transgene sequence encoding a chimeric receptor into the CD247 locus, e.g., a chimeric receptor. In some embodiments, the polynucleotide comprises: (a) a nucleic acid sequence encoding the chimeric receptor or a portion thereof; and (b) one or more homology arms linked to the nucleic acid sequence, wherein the one or more homology arms comprise a sequence homologous to one or more regions of the open reading frame of the CD247 locus or a subsequence thereof. In some embodiments, the integration of the polynucleotide into the CD247 locus encodes a chimeric receptor comprising an intracellular region (e.g., an intracellular region comprising a CD3ζ signaling domain), wherein the nucleic acid sequence of (a) encodes a portion of the chimeric receptor, the portion not comprising the complete intracellular region of the chimeric receptor. In some embodiments, the complete intracellular region comprises the CD3ζ signaling domain. In some embodiments, the complete intracellular region comprises a costimulatory signaling domain and a CD3ζ signaling domain. In some embodiments, the nucleic acid sequence of (a) encodes a portion of the chimeric receptor that does not include the entire or full length of the sequence encoding the CD3 zeta (CD3ζ) signaling domain. In some embodiments, the nucleic acid sequence of (a) does not contain the sequence encoding the CD3 zeta (CD3ζ) signaling domain. In some embodiments, the nucleic acid sequence of (a) encodes an intracellular region that includes a fragment of the CD3 zeta (CD3ζ) signaling domain. In any of such examples, the nucleic acid sequence of (a) may encode a costimulatory signaling domain of the intracellular region.

[0030] Also provided herein is a polynucleotide containing: (a) a nucleic acid sequence encoding a portion of a chimeric receptor, wherein the chimeric receptor includes an intracellular region (e.g., an intracellular region including a CD3ζ signaling domain), and the portion of the chimeric receptor includes an incomplete intracellular region of the chimeric receptor; and (b) one or more homology arms linked to the nucleic acid sequence, the one or more homology arms including a sequence homologous to one or more regions of the open reading frame of the CD247 locus or a subsequence thereof. In some embodiments, the polynucleotide can be used to integrate a transgene sequence encoding the chimeric receptor into the CD247 locus. In some embodiments, the complete intracellular region includes the CD3 zeta (CD3ζ) signaling domain. In some embodiments, the complete intracellular region includes a costimulatory signaling domain and the CD3 zeta (CD3ζ) signaling domain. In some embodiments, the nucleic acid sequence of (a) encodes a portion of the chimeric receptor that does not include the entire or full-length sequence encoding the CD3 zeta (CD3ζ) signaling domain. In some embodiments, the nucleic acid sequence of (a) does not contain a sequence encoding the CD3 zeta (CD3ζ) signaling domain. In some embodiments, the nucleic acid sequence of (a) encodes an intracellular region comprising a fragment of the CD3 zeta (CD3ζ) signaling domain. In any of such examples, the nucleic acid sequence of (a) may encode a costimulatory signaling domain of the intracellular region.

[0031] In some of any of the embodiments, the complete intracellular region of the chimeric receptor comprises the CD3 zeta (CD3ζ) signaling domain or a fragment thereof, and at least a portion of the intracellular region is encoded by the open reading frame of the endogenous CD247 locus or a subsequence thereof when the chimeric receptor is expressed from a cell into which the polynucleotide has been introduced.

[0032] In some of the optional embodiments, the nucleic acid sequence encoding a portion of the chimeric receptor and the one or more homology arms together comprise at least a fragment of a sequence of nucleotides encoding the intracellular region of the chimeric receptor, wherein at least a portion of the intracellular region comprises the CD3ζ signaling domain or a fragment thereof encoded by the open reading frame of the endogenous CD247 locus or a subsequence thereof when the chimeric receptor is expressed from a cell into which the polynucleotide has been introduced.

[0033] In some of the embodiments, the nucleic acid sequence of (a) does not include a sequence encoding a 3'UTR. In some of the embodiments, the nucleic acid sequence of (a) does not include an intron.

[0034] In some of any of the embodiments, the nucleic acid sequence of (a) encodes a fragment of the CD3ζ signaling domain. In such embodiments, at least a portion of the CD3ζ signaling domain is encoded by the genomic sequence of the endogenous CD247 locus (the genomic locus encoding CD3ζ) of the engineered cell, e.g., a T cell, when the chimeric receptor is expressed from the cell into which the polynucleotide has been introduced. For example, in a specific embodiment, the CD3ζ signaling domain of the chimeric receptor, or a fragment thereof, is encoded by both the sequence of the transgene sequence and the genomic sequence of the endogenous CD247 locus (the genomic locus encoding CD3ζ) of the engineered cell, e.g., a T cell.

[0035] In some of any of the embodiments, the nucleic acid sequence of (a) does not encode the CD3ζ signaling domain or a fragment thereof. In such embodiments, the entire or full-length CD3ζ signaling domain of the chimeric receptor, or a fragment thereof, is encoded by the genomic sequence of the endogenous CD247 locus (the genomic locus encoding CD3ζ) of the engineered cell, e.g., a T cell, when the chimeric receptor is expressed from the cell into which the polynucleotide has been introduced.

[0036] In some of the embodiments, the open reading frame or subsequence thereof of the endogenous CD247 locus comprises at least one intron and at least one exon of the endogenous CD247 locus. In some of the embodiments, the open reading frame or subsequence thereof encodes the 3'UTR of the endogenous CD247 locus.

[0037] In some optional embodiments, at least a fragment of the CD3ζ signaling domain of the encoded chimeric receptor, e.g., the entire CD3ζ signaling domain, is encoded by the open reading frame of the endogenous CD247 locus or a subsequence thereof when the chimeric receptor is expressed from a cell into which the polynucleotide has been introduced.

[0038] In some of any of the embodiments, the nucleic acid sequence of (a) is a sequence that is foreign or heterologous to the open reading frame of the endogenous genomic CD247 locus of the T cell, e.g., a human T cell.

[0039] In some of any of the embodiments, the nucleic acid sequence of (a) comprises a sequence of nucleotides that is in frame with one or more exons of the open reading frame of the CD247 locus, or a subsequence thereof, contained in one or more arms of homology.

[0040] In some of any of the embodiments, one or more regions of the open reading frame or subsequence thereof are or include sequences upstream of exon 8 of the open reading frame of the CD247 locus. In some of any of the embodiments, one or more regions of the open reading frame are or include sequences upstream of exon 3 of the open reading frame of the CD247 locus. In some of any of the embodiments, one or more regions of the open reading frame are or include sequences comprising exon 3 of the open reading frame of the CD247 locus. In some of any of the embodiments, one or more regions of the open reading frame are or include sequences comprising at least a portion of exon 2 of the open reading frame of the CD247 locus. In some of any of the embodiments, one or more homology arms do not include exon 1, the entirety of exon 1, and / or the entirety of exon 2 of the open reading frame of the endogenous CD247 locus.

[0041] In some embodiments, when expressed by a cell into which the polynucleotide has been introduced, the encoded chimeric receptor can signal through the CD3 zeta signaling domain. In some embodiments, the CD3 zeta signaling domain of the complete intracellular region encoded by the chimeric receptor comprises a sequence selected from any one of SEQ ID NOs:13-15, or 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 any one of SEQ ID NOs:13-15, or a fragment thereof. In some embodiments, the CD3 zeta signaling domain has the sequence set forth in SEQ ID NO:13. In some embodiments, the CD3 zeta signaling domain has the sequence set forth in SEQ ID NO:14. In some embodiments, the CD3 zeta signaling domain has the sequence set forth in SEQ ID NO:15.

[0042] In some of the embodiments, the one or more homology arms include a 5' homology arm and a 3' homology arm. In some of the embodiments, the polynucleotide includes the structure [5' homology arm]-[nucleic acid sequence of (a)]-[3' homology arm].

[0043] In some of the embodiments, the 5' homology arm and the 3' homology arm are independently from 50 or about 50 to 2000 or about 2000 nucleotides, from 100 or about 100 to 1000 or about 1000 nucleotides, from 100 or about 100 to 750 or about 750 nucleotides, from 100 or about 100 to 600 or about 600 nucleotides, from 100 or about 100 to 400 or about 400 nucleotides, from 100 or about 100 to 300 or about 300 nucleotides, from 100 or about 100 to 200 or about 200 nucleotides, from 200 or about 200 to 1000 or about 1000 nucleotides, from 200 or about 200 to 750 or about 750 nucleotides, from 200 or about 200 to 600 or about 600 nucleotides, from 200 or about 200 to 400 or about 400 nucleotides. or from about 400 nucleotides, from 200 or about 200 to 300 or about 300 nucleotides, from 300 or about 300 to 1000 or about 1000 nucleotides, from 300 or about 300 to 750 or about 750 nucleotides, from 300 or about 300 to 600 or about 600 nucleotides, from 300 or about 300 to 400 or about 400 nucleotides, from 400 or about 400 to 1000 or about 1000 nucleotides, from 400 or about 400 to 750 or about 750 nucleotides, from 400 or about 400 to 600 or about 600 nucleotides, from 600 or about 600 to 1000 or about 1000 nucleotides, from 600 or about 600 to 750 or about 750 nucleotides, or from 750 or about 750 to 1000 or about 1000 nucleotides. In some of the embodiments, the 5' and 3' homology arms are independently 200, 300, 400, 500, 600, 700, or 800 nucleotides in length, or about 200, 300, 400, 500, 600, 700, or 800 nucleotides in length, or any value between any of these. In some of the embodiments, the 5' and 3' homology arms are independently 300 or greater than about 300 nucleotides in length.In some of the optional embodiments, the 5' homology arm and the 3' homology arm are independently 400, 500, or 600, or about 400, 500, or 600 nucleotides in length, or any value between any of these.

[0044] In some of the optional embodiments, the 5' homology arm comprises the sequence set forth in SEQ ID NO:80, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:80, or a subsequence thereof. In some embodiments, the 5' homology arm comprises the sequence set forth in SEQ ID NO:80. In some embodiments, the 5' homology arm consists of or consists essentially of the sequence set forth in SEQ ID NO:80. In some of the optional embodiments, the 3' homology arm comprises the sequence set forth in SEQ ID NO:81, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:81, or a subsequence thereof. In some embodiments, the 3' homology arm comprises the sequence set forth in SEQ ID NO:81. In some embodiments, the 3' homology arm consists of or consists essentially of the sequence set forth in SEQ ID NO:81.

[0045] In some of the optional embodiments, the chimeric receptor is or comprises a functional non-T cell receptor (non-TCR) antigen receptor.

[0046] In some of the optional embodiments, the chimeric receptor is a chimeric antigen receptor (CAR).

[0047] In some of the embodiments, the nucleic acid sequence of (a) comprises a sequence of nucleotides encoding the extracellular region, a sequence of nucleotides encoding the transmembrane domain and / or a portion of the intracellular region. In some of the embodiments, the nucleic acid sequence of (a) comprises a sequence of nucleotides encoding the extracellular region, a sequence of nucleotides encoding the transmembrane domain, and a sequence of nucleotides encoding a portion of the intracellular region. In some of the embodiments, the extracellular region comprises a binding domain. In some of the embodiments, the binding domain is or comprises an antibody or an antigen-binding fragment thereof.

[0048] In some of the embodiments, the binding domain is capable of binding to a target antigen associated with, specific for, and / or expressed on cells or tissues of a disease, disorder, or condition, hi some of the embodiments, the target antigen is a tumor antigen. In some of the optional embodiments, the target antigen is selected from the group consisting of 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), epidermal growth factor receptor type III mutant (EGFR), and EGFR fusion protein (EGFR fusion protein). vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor-like 5 (FCRL5;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), kappa light chain, L1 cell adhesion molecule (L1-CAM), and the 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, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligand, melan-A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, melanoma-preferentially expressed antigen Gen (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein G (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;dopachrome tautomerase, 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 or pathogen-expressed antigens, or antigens associated with universal tags, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV, or other pathogens;

[0049] In some of the embodiments, the extracellular region comprises a spacer. In some of the embodiments, the spacer is operably linked between the binding domain and the transmembrane domain. In some of the embodiments, the spacer comprises an immunoglobulin hinge region. In some of the embodiments, the spacer comprises a C H 2 area and C H Includes three areas.

[0050] In some of any of the embodiments, the portion of the intracellular region encoded by the nucleic acid of (a) comprises one or more costimulatory signaling domains. In some of any of the embodiments, the one or more costimulatory signaling domains comprise the intracellular signaling domain of CD28, 4-1BB, or ICOS, or a signaling portion thereof. In some embodiments, the costimulatory signaling domain is the signaling domain of human CD28. In some embodiments, the costimulatory signaling domain is the signaling domain of human 4-1BB. In some embodiments, the costimulatory signaling domain is the signaling domain of human ICOS. In some of any of the embodiments, the one or more costimulatory signaling domains comprise the intracellular signaling domain of 4-1BB, e.g., human 4-1BB.

[0051] In some of the embodiments, when the chimeric receptor is expressed from a cell into which the polynucleotide has been introduced, the encoded chimeric receptor comprises, in order from N-terminus to C-terminus, an extracellular binding domain, a spacer, a transmembrane domain, and an intracellular signaling region. In a specific embodiment, when expressed from a cell such as a T cell, the intracellular region of the encoded chimeric receptor contains a CD3 zeta (CD3ζ) signaling domain, and the entire CD3ζ signaling domain, or at least a portion of the CD3ζ signaling domain, is encoded by the genomic sequence of the endogenous CD247 locus (the genomic locus encoding CD3ζ).

[0052] In some of any of the embodiments, the sequence of (a) comprises a sequence of nucleotides encoding, in order, an extracellular binding domain; a spacer; a transmembrane domain; and a costimulatory signaling domain. In some of any of the embodiments, the sequence of (a) comprises a sequence of nucleotides encoding, in order, an extracellular binding domain; a spacer; a transmembrane domain; and an intracellular signaling region containing a costimulatory signaling domain and a fragment of the CD3ζ signaling domain. In specific embodiments, when expressed from a cell, such as a T cell, the polynucleotide encodes a chimeric receptor comprising a costimulatory signaling domain and an intracellular signaling region containing a CD3 zeta (CD3ζ) signaling domain, wherein the CD3ζ signaling domain, or at least a portion of the CD3ζ signaling domain, is encoded by the genomic sequence of the endogenous CD247 locus (the genomic locus encoding CD3ζ) of the engineered cell, e.g., the T cell.

[0053] In some of the optional embodiments, the nucleic acid sequence of (a) comprises, in order: an extracellular binding domain that is an scFv; and a sequence derived from a human immunoglobulin hinge that is derived from IgG1, IgG2, or IgG4, or a modified version thereof; H 2 area and / or C Hand a costimulatory signaling domain derived from human 4-1BB. In some of the optional embodiments, the sequence of (a) includes, in order, an extracellular binding domain that is an scFv; and a sequence derived from a human immunoglobulin hinge, such as from IgG1, IgG2, or IgG4, or a modified version thereof, and C H 2 area and / or C H The polynucleotide comprises a sequence of nucleotides encoding: a spacer comprising a CD3 region; a transmembrane domain derived from CD28; and an intracellular region containing a costimulatory signaling domain derived from 4-1BB and a fragment of the CD3ζ signaling domain. In a specific embodiment, when expressed from a cell such as a T cell, the polynucleotide encodes a chimeric receptor comprising an intracellular signaling region containing a human 4-1BB costimulatory signaling domain and a CD3 zeta (CD3ζ) signaling domain, wherein the CD3ζ signaling domain or at least a portion of the CD3ζ signaling domain is encoded by the genomic sequence of the endogenous CD247 locus (the genomic locus encoding CD3ζ) of the engineered cell, e.g., T cell. In some of the embodiments, after introduction of the polynucleotide into the T cell, the modified CD247 locus comprises, in order: an extracellular binding domain that is an scFv; a sequence derived from a human immunoglobulin hinge, derived from IgG1, IgG2, or IgG4, or modified versions thereof, and a C H 2 area and / or C H It comprises a sequence of nucleotides encoding a spacer comprising three regions; a transmembrane domain derived from human CD28; and a costimulatory signaling domain derived from human 4-1BB.

[0054] In some of any of the embodiments, the CAR is a multi-chain CAR. In some of any of the embodiments, the nucleic acid sequence of (a) comprises a sequence of nucleotides encoding at least one additional protein.

[0055] In some embodiments, the nucleic acid sequence of (a) comprises one or more multicistronic elements. In some embodiments, the multicistronic elements are located between the sequence of nucleotides encoding a portion of the chimeric receptor and the sequence of nucleotides encoding at least one additional protein. In some embodiments, the at least one additional protein is a surrogate marker. In some embodiments, the surrogate marker is a truncated receptor. In some embodiments, the truncated receptor lacks an intracellular signaling domain and / or cannot mediate intracellular signaling when its ligand is bound. In some embodiments, the chimeric receptor is a multi-chain CAR, and the multicistronic element is located between the sequence of nucleotides encoding one chain of the multi-chain CAR and the sequence of nucleotides encoding another chain of the multi-chain CAR. In some embodiments, the one or more multicistronic elements are upstream of the sequence of nucleotides encoding a portion of the chimeric receptor. In some embodiments, the one or more multicistronic elements are or include a ribosomal skip sequence. In some of the optional embodiments, the ribosomal skip sequence is a T2A, P2A, E2A, or F2A element.

[0056] In some of the embodiments, the modified CD247 locus comprises a promoter and / or control or regulatory elements of the endogenous CD247 locus operably linked to regulate expression of the nucleic acid sequence encoding the chimeric receptor after introduction of the polynucleotide into a T cell. In some of the embodiments, the modified locus comprises one or more heterologous control or regulatory elements operably linked to regulate expression of the nucleic acid sequence encoding the chimeric receptor. In some of the embodiments, the one or more heterologous control or regulatory elements include a promoter, enhancer, intron, polyadenylation signal, Kozak consensus sequence, splice acceptor sequence, and / or splice donor sequence. In some of the embodiments, the heterologous promoter is or comprises the human elongation factor 1 alpha (EF1α) promoter or MND promoter or variants thereof.

[0057] In some of the embodiments, the nucleic acid sequence of (a) comprises one or more heterologous control or regulatory elements operably linked to regulate expression of the nucleic acid sequence encoding the chimeric receptor. In some of the embodiments, the one or more heterologous control or regulatory elements comprise a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, a splice acceptor sequence, and / or a splice donor sequence. In some of the embodiments, the heterologous promoter is or comprises the human elongation factor 1 alpha (EF1α) promoter or the MND promoter or a variant thereof.

[0058] 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 AAV vector is selected from AAV1 vector, AAV2 vector, AAV3 vector, AAV4 vector, AAV5 vector, AAV6 vector, AAV7 vector, or AAV8 vector. In some of the embodiments, the AAV vector is an AAV2 vector or an AAV6 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.

[0059] In some of the embodiments, the polynucleotide is a linear polynucleotide, a double-stranded polynucleotide, or a single-stranded polynucleotide.

[0060] In some of the optional embodiments, the polynucleotide is at least or about 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4760, 5000, 5250, 5500, 5750, 6000, 7000, 7500, 8000, 9000, or 10000 nucleotides in length, or any value therebetween. In some of the optional embodiments, the polynucleotide is from at or about 2500 to at or about 5000 nucleotides, from at or about 3500 to at or about 4500 nucleotides, or from at or about 3750 to at or about 4250 nucleotides in length.

[0061] Also provided herein is a method of making a genetically engineered T cell, comprising introducing a polynucleotide of any of the embodiments provided herein into a T cell comprising a gene disruption in the CD247 locus.

[0062] Provided herein are methods of making genetically engineered T cells, comprising: (a) introducing into a T cell one or more agents capable of inducing a gene disruption at a target site within the CD247 locus of the T cell; and (b) introducing any of the polynucleotides described herein into a T cell comprising a gene disruption in the CD247 locus, wherein the method creates a modified CD247 locus, wherein the modified CD247 locus comprises a nucleic acid sequence encoding a chimeric receptor comprising an intracellular region that includes a CD3ζ (CD3ζ) signaling domain.

[0063] In some of the optional embodiments, the polynucleotide comprises a nucleic acid sequence encoding a chimeric receptor or a portion thereof, and the nucleic acid sequence encoding the chimeric receptor or a portion thereof is integrated into the endogenous CD247 locus via homology directed repair (HDR).

[0064] Also provided herein is a method of making a genetically engineered T cell comprising introducing into a T cell a polynucleotide comprising a nucleic acid sequence encoding a chimeric receptor or a portion thereof, wherein the T cell has a gene disruption within the CD247 locus of the T cell, and wherein the nucleic acid sequence encoding the chimeric receptor or a portion thereof is integrated into the endogenous CD247 locus via homology directed repair (HDR).

[0065] In some of the optional embodiments, gene disruption is performed by introducing into the T cell one or more agents capable of inducing gene disruption at a target site within the CD247 locus of the T cell.

[0066] In some of the optional embodiments, the method creates a modified CD247 locus, wherein the modified CD247 locus comprises a nucleic acid sequence encoding a chimeric receptor comprising an intracellular region that includes a CD3ζ (CD3ζ) signaling domain.

[0067] In some of the embodiments, the nucleic acid sequence encoding the chimeric receptor or a portion thereof encodes a portion of the chimeric receptor. In some of the embodiments, the polynucleotide further comprises one or more homology arms linked to the nucleic acid sequence, wherein the one or more homology arms comprise a sequence homologous to one or more regions of an open reading frame of the CD247 locus.

[0068] In some of any of the embodiments, the complete intracellular region of the chimeric receptor comprises the CD3 zeta (CD3ζ) signaling domain or a fragment thereof, and at least a portion of the intracellular region is encoded by the open reading frame of the endogenous CD247 locus or a subsequence thereof in the cells produced by the method.

[0069] In some of the optional embodiments, the nucleic acid sequence encoding a portion of the chimeric receptor and the one or more homology arms together comprise at least a fragment of a sequence of nucleotides encoding the intracellular region of the chimeric receptor, and at least a portion of the intracellular region comprises the CD3ζ signaling domain or a fragment thereof encoded by the open reading frame of the CD247 locus or a subsequence thereof in cells produced by the method.

[0070] In some of any of the embodiments, the nucleic acid sequence encoding the chimeric receptor or portion thereof does not include a sequence encoding a 3'UTR. In some of any of the embodiments, the nucleic acid sequence encoding the chimeric receptor or portion thereof encodes a fragment of the CD3ζ signaling domain in cells produced by the method. In some of any of the embodiments, the nucleic acid sequence encoding the chimeric receptor or portion thereof does not encode the CD3ζ signaling domain or a fragment thereof in cells produced by the method. In some of any of the embodiments, at least a fragment of the CD3ζ signaling domain of the encoded chimeric receptor, e.g., the entire CD3ζ signaling domain, is encoded by an open reading frame of the endogenous CD247 locus or a subsequence thereof in cells produced by the method.

[0071] In some of the optional embodiments, the nucleic acid sequence encoding the chimeric receptor or a portion thereof is a sequence that is foreign or heterologous to the open reading frame of the endogenous genomic CD247 locus of a T cell, e.g., a human T cell.

[0072] In some of the optional embodiments, the nucleic acid sequence encoding the chimeric receptor or a portion thereof comprises a sequence of nucleotides that is in frame with one or more exons of the open reading frame of the CD247 locus or a subsequence thereof contained in one or more arms of homology.

[0073] In some embodiments, the chimeric receptor, when expressed by a cell into which the polynucleotide has been introduced, can signal through the CD3 zeta signaling domain. In some embodiments, the CD3 zeta signaling domain of the complete intracellular region comprises a sequence selected from any one of SEQ ID NOs:13-15, or 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 any one of SEQ ID NOs:13-15, or a fragment thereof. In some embodiments, the CD3 zeta signaling domain comprises the sequence set forth in SEQ ID NO:13. In some embodiments, the CD3 zeta signaling domain comprises the sequence set forth in SEQ ID NO:14. In some embodiments, the CD3 zeta signaling domain comprises the sequence set forth in SEQ ID NO:15.

[0074] In some of the embodiments, the one or more homology arms include a 5' homology arm and a 3' homology arm. In some of the embodiments, the polynucleotide includes the structure [5' homology arm]-[nucleic acid sequence encoding the chimeric receptor or a portion thereof]-[3' homology arm].

[0075] In some of the embodiments, the 5' homology arm and the 3' homology arm are independently from 50 or about 50 to 2000 or about 2000 nucleotides, from 100 or about 100 to 1000 or about 1000 nucleotides, from 100 or about 100 to 750 or about 750 nucleotides, from 100 or about 100 to 600 or about 600 nucleotides, from 100 or about 100 to 400 or about 400 nucleotides, from 100 or about 100 to 300 or about 300 nucleotides, from 100 or about 100 to 200 or about 200 nucleotides, from 200 or about 200 to 1000 or about 1000 nucleotides, from 200 or about 200 to 750 or about 750 nucleotides, from 200 or about 200 to 600 or about 600 nucleotides, from 200 or about 200 to 400 or about 400 nucleotides. or from about 400 nucleotides, from 200 or about 200 to 300 or about 300 nucleotides, from 300 or about 300 to 1000 or about 1000 nucleotides, from 300 or about 300 to 750 or about 750 nucleotides, from 300 or about 300 to 600 or about 600 nucleotides, from 300 or about 300 to 400 or about 400 nucleotides, from 400 or about 400 to 1000 or about 1000 nucleotides, from 400 or about 400 to 750 or about 750 nucleotides, from 400 or about 400 to 600 or about 600 nucleotides, from 600 or about 600 to 1000 or about 1000 nucleotides, from 600 or about 600 to 750 or about 750 nucleotides, or from 750 or about 750 to 1000 or about 1000 nucleotides. In some of the embodiments, the 5' and 3' homology arms are independently 200, 300, 400, 500, 600, 700, or 800 nucleotides in length, or about 200, 300, 400, 500, 600, 700, or 800 nucleotides in length, or any value between any of these. In some of the embodiments, the 5' and 3' homology arms are independently 300 or greater than about 300 nucleotides in length.In some of the optional embodiments, the 5' homology arm and the 3' homology arm are independently 400, 500, or 600, or about 400, 500, or 600 nucleotides in length, or any value between any of these.

[0076] In some of the optional embodiments, the 5' homology arm comprises the sequence set forth in SEQ ID NO:80, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:80, or a subsequence thereof. In some embodiments, the 5' homology arm comprises the sequence set forth in SEQ ID NO:80. In some embodiments, the 5' homology arm consists of or consists essentially of the sequence set forth in SEQ ID NO:80. In some of the optional embodiments, the 3' homology arm comprises the sequence set forth in SEQ ID NO:81, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:81, or a subsequence thereof. In some embodiments, the 3' homology arm comprises the sequence set forth in SEQ ID NO:81. In some embodiments, the 3' homology arm consists of or consists essentially of the sequence set forth in SEQ ID NO:81.

[0077] In some embodiments, the one or more agents that can induce gene disruption include a DNA-binding protein or DNA-binding nucleic acid that specifically binds to or hybridizes with a target site, a fusion protein that includes a DNA targeting protein and a nuclease, or an RNA-guided nuclease.In some embodiments, the one or more agents include a zinc finger nuclease (ZFN), a TAL effector nuclease (TALEN), or a combination of CRISPR-Cas9 that specifically binds to, recognizes, or hybridizes with a target site.

[0078] In some embodiments, each of the one or more agents comprises a guide RNA (gRNA) having a targeting domain complementary to at least one target site. In some embodiments, the one or more agents are introduced as a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas9 protein.

[0079] In some of the embodiments, the RNPs are introduced via electroporation, particle gun, calcium phosphate transfection, compaction or squeezing of the cells. In some of the embodiments, the RNPs are introduced via electroporation.

[0080] In some of the embodiments, the concentration of RNP is 1, 2, 2.5, 5, 10, 20, 25, 30, 40, or 50, or about 1, 2, 2.5, 5, 10, 20, 25, 30, 40, or 50 μM, or a range defined by any two of these values. In some of the embodiments, the concentration of RNP is 25 or about 25 μM.

[0081] In some of the embodiments, the molar ratio of gRNA to Cas9 molecules in the RNP is at or about 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5, or a range defined by any two of these values. In some of the embodiments, the molar ratio of gRNA to Cas9 molecules in the RNP is at or about 2.6:1.

[0082] In some of the optional embodiments, the gRNA comprises: In some of the embodiments, the gRNA has a targeting domain sequence selected from: In some of the embodiments, the gRNA has a targeting domain sequence of: It has the targeting domain sequence TIFF0007754722000003.tif4128.

[0083] In some of any of the embodiments, the T cells are primary T cells derived from a subject. In some of any of the embodiments, the subject is a human. In some of any of the embodiments, the T cells are CD8+ T cells or subtypes thereof. In some of any of the embodiments, the T cells are CD4+ T cells or subtypes thereof. In some of any of the embodiments, the T cells are derived from pluripotent or totipotent cells. In some of any of the embodiments, the pluripotent or totipotent cells are iPSCs. In some of any of the embodiments, the T cells are derived from pluripotent or totipotent cells that are iPSCs.

[0084] 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 AAV vector is selected from AAV1 vector, AAV2 vector, AAV3 vector, AAV4 vector, AAV5 vector, AAV6 vector, AAV7 vector, or AAV8 vector. In some of the embodiments, the AAV vector is an AAV2 vector or an AAV6 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.

[0085] In some of any of the embodiments, the polynucleotide is a linear polynucleotide. In some of any of the embodiments, the linear polynucleotide is a double-stranded polynucleotide or a single-stranded polynucleotide.

[0086] In some of the embodiments, the one or more agents and the polynucleotide are introduced simultaneously or sequentially in any order, hi some of the embodiments, the polynucleotide is introduced after the introduction of the one or more agents.

[0087] In some of the optional embodiments, the polynucleotide is introduced immediately after introduction of the agent or within about 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 thereafter.

[0088] In some of the embodiments, prior to the introduction of the one or more agents, the method includes incubating the cells in vitro with a stimulatory agent under conditions to stimulate or activate one or more immune cells. In some of the embodiments, the stimulatory agent includes an anti-CD3 antibody and / or an anti-CD28 antibody, e.g., anti-CD3 / anti-CD28 beads. In some of the embodiments, the ratio of beads to cells is 1:1 or about 1:1.

[0089] In some of the optional embodiments, the method also includes removing a stimulatory agent from the one or more immune cells prior to introducing the one or more agents.

[0090] In some of the embodiments, the method also includes incubating the cells with one or more recombinant cytokines before, during, or after the introduction of the one or more agents and / or the one or more 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 a concentration selected from IL-2 at or about 10 U / mL to 200 or about 200 U / mL, e.g., 50 or about 50 IU / mL to 100 or about 100 U / mL; IL-7 at or about 0.5 ng / mL to 50 ng / mL, e.g., 5 or about 5 ng / mL to 10 or about 10 ng / mL; and / or IL-15 at or about 0.1 ng / mL to 20 ng / mL, e.g., 0.5 or about 0.5 ng / mL to 5 or about 5 ng / mL.

[0091] In some of any of the embodiments, incubation is carried out for up to or about 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 introduction of the one or more agents and introduction of the polynucleotide, e.g., for up to or about 7 days.

[0092] In some of any of the embodiments, at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells, e.g., T cells, in the plurality of engineered cells produced by the method, or greater than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells, e.g., T cells, comprise a gene disruption of at least one target site within the CD247 locus. In some of any of the embodiments, at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the engineered cells produced by the method, e.g., T cells, or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells, express a chimeric receptor or antigen-binding fragment thereof. In some of any of the embodiments, at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the plurality of engineered cells produced by the method, or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the plurality of engineered cells, express a chimeric receptor or antigen-binding fragment thereof.

[0093] In some of any of the embodiments, a plurality of engineered cells produced by the method, e.g., at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the T cells, or greater than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells, comprise a gene disruption of at least one target site within the CD247 locus. In some of any of the embodiments, at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the engineered cells produced by the method, e.g., T cells, or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells, express a chimeric receptor. In some of any of the embodiments, at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the engineered cells, e.g., T cells, or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells, express a chimeric receptor, which contains an intracellular region containing a CD3 zeta (CD3ζ) signaling domain, and wherein the CD3ζ signaling domain, or at least a portion of the CD3ζ signaling domain, is encoded by the genomic sequence of the endogenous CD247 locus (the genomic locus encoding CD3ζ) of the engineered cell, e.g., T cell. In some embodiments, at least a portion of the CD3ζ signaling domain is encoded by the genomic sequence of the endogenous CD247 locus. In some embodiments, the entire CD3ζ signaling domain or the complete CD3ζ signaling domain of the intracellular region of the chimeric receptor is encoded by the genomic sequence of the endogenous CD247 locus.

[0094] Also provided is an engineered T cell or a plurality of engineered T cells produced using any of the methods described herein.

[0095] Compositions comprising any of the engineered T cells described herein are also provided.

[0096] Also provided are compositions comprising a plurality of T cells, including any of the engineered T cells described herein. In some of any of the embodiments, at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the T cells in the composition, or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells, comprise a gene disruption of at least one target site within the CD247 locus. In some of any of the embodiments, at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the T cells in the composition, or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells, express a chimeric receptor. In some of any of the embodiments, at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the T cells in the composition, or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells, express a chimeric receptor, which contains an intracellular region containing a CD3 zeta (CD3ζ) signaling domain, and wherein the CD3ζ signaling domain, or at least a portion of the CD3ζ signaling domain, is encoded by the genomic sequence of the endogenous CD247 locus (the genomic locus encoding CD3ζ) of the engineered cell, e.g., the T cell. In some embodiments, at least a portion of the CD3ζ signaling domain is encoded by the genomic sequence of the endogenous CD247 locus. In some embodiments, the entire CD3ζ signaling domain or the complete CD3ζ signaling domain of the intracellular region of the chimeric receptor is encoded by the genomic sequence of the endogenous CD247 locus.

[0097] In some of the embodiments, the composition comprises CD4+ T cells and / or 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, e.g., 1:1.

[0098] In some of the optional embodiments, cells expressing the chimeric receptor represent at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of all cells in the composition, or of all CD4+ or CD8+ cells in the composition.

[0099] Also provided herein are methods of treatment that include administering to a subject having a disease or disorder an engineered cell, a plurality of engineered cells, or a composition of any of the aspects provided herein.

[0100] Also provided herein is the use of any of the engineered cells, engineered cells, or compositions described herein for the treatment of a disease or disorder. In provided embodiments, the chimeric receptor expressed by the engineered cell is directed against or targets an antigen associated with or expressed in a cell or tissue of the disease or condition.

[0101] Also provided herein is the use of any of the engineered cells, multiple engineered cells, or compositions described herein in the manufacture of a medicament for the treatment of a disease or disorder. In provided embodiments, the chimeric receptor expressed by the engineered cell is directed against or targets an antigen associated with or expressed in a cell or tissue of the disease or condition.

[0102] Also provided herein is an engineered cell, a plurality of engineered cells, or a composition from any of the embodiments provided herein for use in treating a disease or disorder. In the embodiments provided, the chimeric receptor expressed by the engineered cell is directed against or targets an antigen associated with or expressed in a cell or tissue of the disease or condition.

[0103] In some of the embodiments, the disease or disorder is a cancer or tumor. In some of the embodiments, the cancer or tumor is a hematological malignancy. In some of the embodiments, the hematological malignancy is a lymphoma, leukemia, or plasma cell malignancy. In some embodiments, the cancer is a lymphoma, and the lymphoma is Burkitt's lymphoma, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, Waldenstrom's hypergammaglobulinemia, follicular lymphoma, small non-cleaved cell lymphoma, mucosa-associated lymphoid tissue lymphoma (MALT), marginal zone lymphoma, splenic lymphoma, nodular monocytoid B-cell lymphoma, immunoblastic lymphoma, large cell lymphoma, diffuse mixed cell lymphoma, pulmonary B-cell angiocentric lymphoma, small lymphocytic lymphoma, primary mediastinal B-cell lymphoma, lymphoplasmacytic lymphoma (LPL), or mantle cell lymphoma (MCL). In some embodiments, the cancer is a leukemia, and the leukemia is chronic lymphocytic leukemia (CLL), plasma cell leukemia, or acute lymphocytic leukemia (ALL). In some of any of the embodiments, the cancer is a plasma cell malignancy, and the plasma cell malignancy is multiple myeloma (MM).

[0104] 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).

[0105] Kits are also provided. In some of any of the embodiments, the kits include one or more agents capable of inducing gene disruption at a target site within the CD247 locus; and a polynucleotide of any of the embodiments provided herein.

[0106] Also provided are kits that include one or more agents capable of inducing a gene disruption at a target site within the CD247 locus; a polynucleotide comprising a nucleic acid sequence encoding a chimeric receptor or a portion thereof, wherein a transgene encoding the chimeric receptor or an antigen-binding fragment or chain thereof is targeted for integration at or near the target site via homology-directed repair (HDR); and instructions for practicing any of the methods of the embodiments provided herein. [The present invention 1001] A genetically engineered T cell comprising a modified CD247 locus, wherein the modified CD247 locus comprises a nucleic acid sequence encoding a chimeric receptor comprising an intracellular region that includes a CD3 zeta (CD3ζ) signaling domain. [The present invention 1002] 1001. The genetically engineered T cell of the present invention, wherein the nucleic acid sequence comprises a transgene sequence encoding a portion of a chimeric receptor, the transgene sequence being integrated, optionally via homology directed repair (HDR), into the endogenous CD247 locus of the T cell. [The present invention 1003] The genetically engineered T cell of invention 1001 or invention 1002, wherein the entire CD3ζ signaling domain or a fragment of the CD3ζ signaling domain is encoded by an open reading frame of the endogenous CD247 locus or a subsequence thereof. [The present invention 1004] The genetically engineered T cell of any of claims 1001 to 1003, wherein the nucleic acid sequence encoding the chimeric receptor comprises an in-frame fusion of (i) a transgene sequence encoding a portion of the chimeric receptor and (ii) the open reading frame of the endogenous CD247 locus or a subsequence thereof. [The present invention 1005] A genetically engineered T cell comprising a modified CD247 locus, wherein the modified CD247 locus comprises a nucleic acid sequence encoding a chimeric receptor comprising an intracellular region comprising a CD3 zeta (CD3ζ) signaling domain, the nucleic acid sequence comprising an in-frame fusion of (i) a transgene sequence encoding a portion of the chimeric receptor with (ii) an open reading frame of the endogenous CD247 locus encoding the CD3ζ signaling domain or a subsequence thereof. [The present invention 1006] 1006. The genetically engineered T cell of any of claims 1002 to 1005, wherein the transgene sequence is in frame with one or more exons of the open reading frame of the endogenous CD247 locus or a subsequence thereof. [The present invention 1007] 1007. The genetically engineered T cell of any of claims 1002 to 1006, wherein the transgene sequence does not include a sequence encoding a 3'UTR and / or does not include an intron. [The present invention 1008] The genetically engineered T cell of any of claims 1002 to 1007, wherein the transgene sequence encodes a fragment of the CD3ζ signaling domain or does not encode the CD3ζ signaling domain or a fragment thereof. [The present invention 1009] 9. The genetically engineered T cell of any of claims 1003 to 1008, wherein the open reading frame or a subsequence thereof comprises at least one intron and at least one exon of the endogenous CD247 locus and / or encodes the 3'UTR of the endogenous CD247 locus. [The present invention 1010] 1009. The genetically engineered T cell of any of claims 1002 to 1009, wherein the transgene sequence is downstream of exon 1 and upstream of exon 8 of the open reading frame of the endogenous CD247 locus; optionally downstream of exon 1 and upstream of exon 3 of the open reading frame of the endogenous CD247 locus. [The present invention 1011] 10. The genetically engineered T cell of any of claims 1001 to 1010, wherein at least a fragment of the CD3ζ signaling domain of the encoded chimeric receptor, and optionally the entire CD3ζ signaling domain, is encoded by an open reading frame of an endogenous CD247 locus or a subsequence thereof, and optionally the CD3ζ signaling domain is encoded by a sequence of nucleotides comprising at least a portion of exon 2 and exons 3 to 8 of the open reading frame of the endogenous CD247 locus; or a sequence of nucleotides that does not include exon 1, does not include the entirety of exon 1, and / or does not include the entirety of exon 2 of the open reading frame of the endogenous CD247 locus. [The present invention 1012] 10. The genetically engineered T cell of any of claims 1001 to 1011, wherein the encoded chimeric receptor is capable of signaling through the CD3ζ signaling domain. [The present invention 1013] 10. The genetically engineered T cell of any of claims 1001 to 1012, wherein the encoded CD3 zeta signaling domain comprises a sequence selected from any one of SEQ ID NOs: 13-15, or 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 any one of SEQ ID NOs: 13-15, or a fragment thereof. [The present invention 1014] The genetically engineered T cell of any of claims 1001 to 1013, wherein the chimeric receptor is a chimeric antigen receptor (CAR). [The present invention 1015] 15. The genetically engineered T cell of any one of claims 1001 to 1014, wherein the chimeric receptor comprises an extracellular region comprising a binding domain, a transmembrane domain, and an intracellular region. [The present invention 1016] 1015. The genetically engineered T cell of the present invention, wherein the binding domain comprises an antibody or an antigen-binding fragment thereof. [The present invention 1017] The genetically engineered T cell of any one of claims 1015 to 1016, wherein the binding domain is capable of binding to a target antigen associated with, specific for, and / or expressed on cells or tissues of a disease, disorder, or condition, and optionally the target antigen is a tumor antigen. [The present invention 1018] Target antigens include 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 mutant (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;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), kappa light chain, L1 cell adhesion molecule (L1-CAM), and the 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, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligand, melan-A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, melanoma-preferentially expressed antigen Gen (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein G (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;The genetically engineered T cells of the present invention are selected from among dopachrome tautomerase, dopachrome delta-isomerase, or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms' tumor 1 (WT-1), a pathogen-specific antigen or pathogen-expressed antigen, or an antigen associated with a universal tag, and / or a biotinylated molecule, and / or a molecule expressed by HIV, HCV, HBV, or other pathogens; [The present invention 1019] 19. The genetically engineered T cell of any of claims 1015 to 1018, wherein the extracellular region comprises a spacer, and optionally the spacer is operably linked between the binding domain and the transmembrane domain. [The present invention 1020] The spacer may be an immunoglobulin hinge region and / or a C H 2 area and C H The genetically engineered T cells of the present invention comprise three regions. [The present invention 1021] 1020. The genetically engineered T cell of any of claims 1001 to 1020, wherein the intracellular region comprises one or more costimulatory signaling domains. [The present invention 1022] 1021. The genetically engineered T cell of the present invention, wherein the one or more costimulatory signaling domains comprise the intracellular signaling domain of CD28, 4-1BB, or ICOS, or a signaling portion thereof. [The present invention 1023] The transgene sequence optionally comprises, in order: a binding domain, optionally a single chain Fv fragment (scFv); and a sequence derived from a human immunoglobulin hinge, optionally derived from IgG1, IgG2, or IgG4, or a modified version thereof; H 2nd area and / or C H and / or a sequence of nucleotides encoding a spacer, optionally further comprising three regions; a transmembrane domain, optionally from human CD28; and an intracellular region, optionally from human 4-1BB, including a costimulatory signaling domain; and / or The modified CD247 locus optionally comprises, in order: a binding domain, optionally an scFv; and a sequence derived from a human immunoglobulin hinge, optionally derived from IgG1, IgG2, or IgG4, or a modified version thereof; H 2nd area and / or C H a spacer, optionally further comprising three regions; a transmembrane domain, optionally derived from human CD28; an intracellular region, optionally derived from human 4-1BB, including a costimulatory signaling domain; and a CD3ζ signaling domain, The genetically engineered T cell of any one of 1002 to 1022 of the present invention. [The present invention 1024] 1024. The genetically engineered T cell of any of claims 1002 to 1023, wherein the transgene sequence comprises a sequence of nucleotides encoding at least one additional protein. [The present invention 1025] 1024. The genetically engineered T cell of the present invention, wherein at least one additional protein is a surrogate marker, and optionally said surrogate marker is a truncated receptor, and optionally said truncated receptor lacks an intracellular signaling domain and / or is unable to mediate intracellular signaling when bound by its ligand. [The present invention 1026] 1026. The genetically engineered T cell of any of claims 1002 to 1025, wherein the transgene sequence comprises one or more multicistronic elements. [The present invention 1027] the transgene sequence comprises a sequence of nucleotides encoding a portion of a chimeric receptor, and one or more multicistronic elements are located upstream of the sequence of nucleotides encoding a portion of the chimeric receptor; and / or are located between the sequence of nucleotides encoding a portion of the chimeric receptor and a sequence of nucleotides encoding at least one additional protein; and / or The chimeric receptor is a CAR that is a multi-chain CAR, and one or more multicistronic elements are located between the sequence of nucleotides encoding one chain of the multi-chain CAR and the sequence of nucleotides encoding another chain of the multi-chain CAR. The genetically engineered T cells of the present invention. [The present invention 1028] 1028. The genetically engineered T cell of claim 1026 or 1027, wherein one or more multicistronic elements is or comprises a ribosomal skipping sequence, and optionally the ribosomal skipping sequence is a T2A, P2A, E2A, or F2A element. [The present invention 1029] The genetically engineered T cell of any of claims 1001 to 1028, wherein the modified CD247 locus comprises a promoter and / or control or regulatory elements of the endogenous CD247 locus operably linked to regulate expression of the nucleic acid sequence encoding the chimeric receptor; or wherein the modified CD247 locus comprises one or more heterologous control or regulatory elements operably linked to regulate expression of the chimeric receptor or a portion thereof. [The present invention 1030] 1029. The genetically engineered T cell of any of claims 1001 to 1029, wherein said T cell is a primary T cell derived from a subject, and optionally, said subject is a human. [The present invention 1031] The genetically engineered T cell of any of claims 1001 to 1030, wherein the T cell is a CD8+ T cell or a subtype thereof, or a CD4+ T cell or a subtype thereof. [The present invention 1032] (a) a nucleic acid sequence encoding a chimeric receptor or a portion thereof; (b) one or more homology arms linked to the nucleic acid sequence, the one or more homology arms comprising a sequence homologous to one or more regions of an open reading frame of the CD247 locus or a subsequence thereof; A polynucleotide comprising: [The present invention 1033] A polynucleotide of the present invention 1032, wherein the nucleic acid sequence (a) encodes a portion of a chimeric receptor, and the portion of the chimeric receptor encoded by the nucleic acid sequence comprises an extracellular region including a binding domain and a transmembrane domain, but does not include the entire CD3 zeta (CD3ζ) signaling domain of the intracellular region. [The present invention 1034] (a) a nucleic acid sequence encoding a portion of a chimeric receptor, the chimeric receptor including an intracellular region including a CD3 zeta (CD3ζ) signaling domain, the portion of the chimeric receptor encoded by the nucleic acid sequence including an extracellular region including a binding domain and a transmembrane domain, but not including the entire CD3 zeta (CD3ζ) signaling domain of the intracellular region; (b) one or more homology arms linked to the nucleic acid sequence, the one or more homology arms comprising a sequence homologous to one or more regions of an open reading frame of the CD247 locus or a subsequence thereof; A polynucleotide comprising: [This invention 1035] The polynucleotide of any of claims 1032 to 1034, wherein the nucleic acid sequence of (a) is a sequence that is foreign or heterologous to the open reading frame of the endogenous genomic CD247 locus of a T cell, optionally a human T cell. [The present invention 1036] 1036. The polynucleotide of any of claims 1032 to 1035, wherein the open reading frame or a subsequence thereof comprises at least one intron and at least one exon and / or 3'UTR of the endogenous CD247 locus of a T cell, optionally a human T cell. [This invention 1037] Any of the polynucleotides of 1032 to 1036 of the present invention, wherein when the chimeric receptor is expressed from a cell into which the polynucleotide has been introduced, at least a fragment of the CD3ζ signaling domain, and optionally the entire CD3ζ signaling domain, is encoded by the open reading frame of the endogenous CD247 locus or a partial sequence thereof. [The present invention 1038] Any of the polynucleotides of 1032 to 1037 of the present invention, wherein the nucleic acid sequence of (a) encodes a fragment of the CD3ζ signaling domain. [This invention 1039] Any of the polynucleotides of 1032 to 1037 of the present invention, wherein the nucleic acid sequence of (a) does not encode the CD3ζ signaling domain. [The present invention 1040] The polynucleotide of any one of 1032 to 1039, wherein the nucleic acid sequence of (a) does not contain a sequence encoding a 3'UTR and / or does not contain an intron. [This invention 1041] The polynucleotide of any of claims 1032 to 1040, wherein the nucleic acid sequence of (a) comprises a sequence of nucleotides that is in frame with one or more exons of the open reading frame of the CD247 locus or a subsequence thereof contained in one or more homology arms; optionally, one or more regions of the open reading frame are: a sequence upstream of exon 8 of the open reading frame of the CD247 locus; a sequence upstream of exon 3 of the open reading frame of the CD247 locus, optionally including exon 3 of the open reading frame of the CD247 locus; and / or a sequence comprising at least a portion of exon 2 of the open reading frame of the CD247 locus. [The present invention 1042] The polynucleotide of any of claims 1032 to 1041, wherein one or more homology arms do not include exon 1 of the open reading frame of the endogenous CD247 locus, do not include the entire length of exon 1, and / or do not include the entire length of exon 2. [This invention 1043] The polynucleotide of any of claims 1032 to 1042, wherein the chimeric receptor is capable of signaling via the CD3ζ signaling domain when expressed by a cell into which the polynucleotide has been introduced. [This invention 1044] A polynucleotide of any of claims 1033 to 1043 of the present invention, wherein the entire CD3ζ signaling domain comprises a sequence selected from any one of SEQ ID NOs: 13 to 15, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 13 to 15, or a fragment thereof. [This invention 1045] The polynucleotide of any of claims 1032 to 1045, wherein the one or more homology arms comprise a 5' homology arm and a 3' homology arm, and the polynucleotide comprises the structure [5' homology arm]-[nucleic acid sequence of (a)]-[3' homology arm]. [The present invention 1046] 1046. The polynucleotide of any of claims 1032 to 1045, wherein the 5' homology arm and the 3' homology arm are independently 200, 300, 400, 500, 600, 700, or 800, or about 200, 300, 400, 500, 600, 700, or 800 nucleotides in length, or any value between any of these, or 300 or more than 300 nucleotides in length, optionally 400, 500, or 600, or about 400, 500, or 600 nucleotides in length, or any value between any of these. [This invention 1047] Any of the polynucleotides 1032 to 1046 of the present invention, wherein the 5' homology arm comprises the sequence set forth in SEQ ID NO:80, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:80, or a subsequence thereof; and / or the 3' homology arm comprises the sequence set forth in SEQ ID NO:81, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:81, or a subsequence thereof. [This invention 1048] The polynucleotide of any of claims 1032 to 1047, wherein the chimeric receptor is a chimeric antigen receptor (CAR). [This invention 1049] The polynucleotide of any of claims 1033 to 1048, wherein the binding domain is or comprises an antibody or an antigen-binding fragment thereof. [The present invention 1050] The polynucleotide of any of claims 1033 to 1049, wherein the binding domain is capable of binding to a target antigen associated with, specific for, and / or expressed on cells or tissues of a disease, disorder, or condition, and optionally the target antigen is a tumor antigen. [This invention 1051] Target antigens include 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 mutant (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;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), kappa light chain, L1 cell adhesion molecule (L1-CAM), and the 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, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligand, melan-A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, melanoma-preferentially expressed antigen Gen (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein G (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;The polynucleotide of the present invention 1050 is selected from among dopachrome tautomerase, dopachrome delta-isomerase, or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms' tumor 1 (WT-1), a pathogen-specific antigen or pathogen-expressed antigen, or an antigen associated with a universal tag, and / or a biotinylated molecule, and / or a molecule expressed by HIV, HCV, HBV, or other pathogens; [This invention 1052] The polynucleotide of any one of 1033 to 1051, wherein the extracellular region comprises a spacer, and optionally, the spacer is operably linked between the binding domain and the transmembrane domain. [This invention 1053] The spacer may be an immunoglobulin hinge region and / or a C H 2 area and C H The polynucleotide of the present invention 1052, comprising three regions. [This invention 1054] The polynucleotide of any of claims 1033 to 1053, wherein the intracellular region comprises one or more costimulatory signaling domains. [This invention 1055] The polynucleotide of claim 1054, wherein the one or more costimulatory signaling domains comprise the intracellular signaling domain of CD28, 4-1BB, or ICOS, or a signaling portion thereof. [This invention 1056] the nucleic acid sequence of (a) optionally comprising, in order, a binding domain, optionally a single-chain Fv fragment (scFv); and a sequence derived from a human immunoglobulin hinge, optionally derived from IgG1, IgG2, or IgG4, or a modified version thereof; H 2 area and / or C H Any of the polynucleotides of 1032 to 1055 of the present invention, comprising a sequence of nucleotides encoding a spacer, optionally further comprising three regions; a transmembrane domain, optionally derived from human CD28; and an intracellular region, optionally derived from human 4-1BB, including a costimulatory signaling domain. [This invention 1057] The polynucleotide of any of claims 1032 to 1056, wherein the nucleic acid sequence of (a) comprises a sequence of nucleotides encoding at least one additional protein. [This invention 1058] The polynucleotide of the present invention 1057, wherein at least one further protein is a surrogate marker, optionally said surrogate marker is a truncated receptor, optionally said truncated receptor lacking an intracellular signaling domain and / or incapable of mediating intracellular signaling when bound by its ligand. [This invention 1059] The polynucleotide of any one of 1032 to 1058, wherein the nucleic acid sequence of (a) comprises one or more multicistronic elements. [The present invention 1060] the nucleic acid of (a) comprises a sequence of nucleotides encoding a portion of a chimeric receptor, and one or more multicistronic elements are located upstream of the sequence of nucleotides encoding the portion of the chimeric receptor; and / or are located between the sequence of nucleotides encoding the portion of the chimeric receptor and a sequence of nucleotides encoding at least one additional protein; and / or The chimeric receptor is a CAR that is a multi-chain CAR, and one or more multicistronic elements are located between the sequence of nucleotides encoding one chain of the multi-chain CAR and the sequence of nucleotides encoding another chain of the multi-chain CAR. The polynucleotide of the present invention 1059. [This invention 1061] 1060. The polynucleotide of claim 1059, wherein one or more multicistronic elements is or comprises a ribosomal skipping sequence, and optionally the ribosomal skipping sequence is a T2A, P2A, E2A, or F2A element. [This invention 1062] The polynucleotide of any of claims 1032 to 1061, wherein the nucleic acid sequence of (a) comprises one or more heterologous control or regulatory elements operably linked to regulate expression of the chimeric receptor or a portion thereof. [This invention 1063] A polynucleotide of any one of 1032 to 1062 of the present invention, which is contained in a viral vector. [This invention 1064] The polynucleotide of the present invention 1063, wherein the viral vector is an AAV vector, and optionally, the AAV vector is an AAV2 vector or an AAV6 vector. [This invention 1065] The polynucleotide of the present invention 1063, wherein the viral vector is a retroviral vector, optionally a lentiviral vector. [The present invention 1066] 1064. The polynucleotide of any one of claims 1032 to 1063, which is a linear polynucleotide, optionally a double-stranded polynucleotide or a single-stranded polynucleotide. [This invention 1067] The polynucleotide of any of claims 1032 to 1066, which is from 2500 or about 2500 to 5000 or about 5000 nucleotides, from 3500 or about 3500 to 4500 or about 4500 nucleotides, or from 3750 or about 3750 nucleotides to 4250 or about 4250 nucleotides in length. [The present invention 1068] A method for producing genetically engineered T cells, comprising the step of introducing any of the polynucleotides of the present inventions 1032 to 1067 into T cells containing a gene disruption in the CD247 locus. [This invention 1069] (a) introducing into a T cell one or more agents capable of inducing gene disruption at a target site within the endogenous CD247 locus of the T cell; and (b) introducing any one of the polynucleotides 1032 to 1067 of the present invention into T cells containing a gene disruption in the CD247 locus. 1. A method for producing genetically engineered T cells, comprising: [The present invention 1070] 1069. The method of claim 1068 or 1069, wherein the nucleic acid sequence encoding the chimeric receptor or a portion thereof is integrated into the endogenous CD247 locus via homology directed repair (HDR). [This invention 1071] 1. A method of making a genetically engineered T cell comprising introducing into a T cell a polynucleotide comprising a nucleic acid sequence encoding a chimeric receptor or a portion thereof, wherein the T cell has a gene disruption within the CD247 locus of the T cell, and wherein the nucleic acid sequence encoding the chimeric receptor or a portion thereof is integrated into the endogenous CD247 locus via homology directed repair (HDR). [This invention 1072] Any of the methods of inventions 1068, 1070, and 1071, wherein gene disruption is carried out by introducing into the T cell one or more agents capable of inducing gene disruption at a target site within the endogenous CD247 locus of the T cell. [This invention 1073] 1073. Any of the methods of claims 1068 to 1072, wherein the method creates a modified CD247 locus, the modified CD247 locus comprising a nucleic acid sequence encoding a chimeric receptor comprising an intracellular region comprising a CD3ζ signaling domain, and at least a fragment of the CD3ζ signaling domain is encoded by an open reading frame of the endogenous CD247 locus. [This invention 1074] Any of the methods of claims 1071 to 1073, wherein the polynucleotide comprises one or more homology arms linked to the nucleic acid sequence, and the one or more homology arms comprise a sequence homologous to one or more regions of an open reading frame of the CD247 locus. [This invention 1075] 1075. The method of any of claims 1071 to 1074, wherein the nucleic acid sequence encoding the chimeric receptor or a portion thereof does not include a sequence encoding a 3'UTR and / or does not include an intron. [This invention 1076] The method of any of claims 1073 to 1075, wherein the chimeric receptor, when expressed by a cell into which the polynucleotide has been introduced, is capable of signaling through the CD3ζ signaling domain. [This invention 1077] Any of the methods of claims 1073 to 1076, wherein the encoded CD3 zeta signaling domain comprises a sequence selected from any one of SEQ ID NOs:13 to 15, or 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 any one of SEQ ID NOs:13 to 15, or a fragment thereof. [This invention 1078] Any of the methods of claims 1074 to 1077, wherein the one or more homology arms comprise a 5' homology arm and a 3' homology arm, and the polynucleotide comprises the structure [5' homology arm]-[nucleic acid sequence encoding the chimeric receptor or a portion thereof]-[3' homology arm]. [This invention 1079] one or more agents capable of inducing gene disruption, DNA-binding proteins or DNA-binding nucleic acids that specifically bind or hybridize to a target site, fusion proteins comprising a DNA-targeting protein and a nuclease, or RNA-guided nucleases and optionally, the one or more agents include 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. Any of the methods of the present inventions 1069 and 1072 to 1078, comprising: [The present invention 1080] 1079. The method of any of claims 1069 and 1072-1079, wherein each of said one or more agents comprises a guide RNA (gRNA) having a targeting domain complementary to at least one target site. [This invention 1081] The method of claim 1080, wherein said one or more agents are introduced as a ribonucleoprotein (RNP) complex comprising a gRNA and a Cas9 protein, and optionally the RNP is introduced via electroporation, a particle gun, calcium phosphate transfection, compaction or squeezing of the cells, optionally via electroporation. [This invention 1082] 1081. The method of claim 1081, wherein the concentration of RNP is 1, 2, 2.5, 5, 10, 20, 25, 30, 40, or 50, or about 1, 2, 2.5, 5, 10, 20, 25, 30, 40, or 50 μM, or a range defined by any two of these values, and optionally, the concentration of RNP is 25 or about 25 μM. [This invention 1083] 1083. The method of any of claims 1080 to 1082, wherein the molar ratio of gRNA to Cas9 molecules in the RNP is 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5, or about 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5, or a range defined by any two of these values, and optionally, the molar ratio of gRNA to Cas9 molecules in the RNP is 2.6:1 or about 2.6:1. [This invention 1084] gRNA, TIFF0007754722000004.tif17140 The method of any one of claims 1080 to 1083, wherein the targeting domain sequence is selected from the group consisting of: [This invention 1085] gRNA, TIFF0007754722000005.tif4128 The method of any one of claims 1080 to 1084, wherein the targeting domain sequence is [The present invention 1086] gRNA, TIFF0007754722000006.tif4128 The method of any one of claims 1080 to 1084, wherein the targeting domain sequence is [This invention 1087] The method of any of claims 1068 to 1086, wherein said T cells are primary T cells derived from a subject, and optionally, said subject is a human. [This invention 1088] The method of any one of claims 1068 to 1087, wherein the T cells are CD8+ T cells or a subtype thereof, or CD4+ T cells or a subtype thereof. [This invention 1089] The method of any one of claims 1068 to 1088, wherein the polynucleotide is contained in a viral vector. [The present invention 1090] 1089. The method of claim 1089, wherein the viral vector is an AAV vector, and optionally, the AAV vector is an AAV2 vector or an AAV6 vector. [This invention 1091] 1089. The method of claim 1089, wherein the viral vector is a retroviral vector, optionally a lentiviral vector. [This invention 1092] The method of any of claims 1068 to 1088, wherein said polynucleotide is a linear polynucleotide, optionally a double-stranded polynucleotide or a single-stranded polynucleotide. [This invention 1093] The method of any of claims 1069 and 1072 to 1092, wherein said polynucleotide is introduced after introduction of said one or more agents. [This invention 1094] 1093. The method of claim 1093, wherein the polynucleotide is introduced immediately after introduction of the agent or within about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 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 thereafter. [This invention 1095] 10. The method of any of claims 1069 and 1072 to 1094, wherein prior to the introduction of said one or more agents, said method comprises incubating cells in vitro with one or more stimulatory agents under conditions to stimulate or activate one or more immune cells, optionally wherein said one or more stimulatory agents comprise anti-CD3 / anti-CD28 antibodies, optionally anti-CD3 / anti-CD28 beads, and optionally wherein the ratio of beads to cells is 1:1 or about 1:1. [This invention 1096] 1069 and 1072 to 1095, wherein 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 agents and / or the introduction of the polynucleotide, wherein optionally the one or more recombinant cytokines are selected from the group consisting of IL-2, IL-7, and IL-15, and optionally the one or more recombinant cytokines are added at a concentration selected from: IL-2 at a concentration of from 10 or about 10 U / mL to 200 or about 200 U / mL, optionally from 50 or about 50 IU / mL to 100 or about 100 U / ml; IL-7 at a concentration of from 0.5 ng / mL to 50 ng / mL, optionally from 5 or about 5 ng / mL to 10 or about 10 ng / mL; and / or IL-15 at a concentration of from 0.1 ng / mL to 20 ng / mL, optionally from 0.5 or about 0.5 ng / mL to 5 or about 5 ng / ml. [This invention 1097] 1097. The method of claim 1095 or 1096, wherein incubation is carried out for up to or about 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 introduction of said one or more agents and introduction of said polynucleotide, optionally for up to or about 7 days. [This invention 1098] at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the engineered cells produced by the method, or greater than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells, comprise a gene disruption of at least one target site within the CD247 locus; and / or or any of the methods of claims 1068 to 1097, wherein at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the engineered cells produced by said method, or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in said cells, express the chimeric receptor. [This invention 1099] A genetically engineered T cell or a plurality of genetically engineered T cells produced using the method of any of claims 1068 to 1098. [The present invention 1100] A composition comprising a genetically engineered T cell of any of claims 1001 to 1031 and 1099 or a plurality of genetically engineered T cells of any of claims 1001 to 1031 and 1099. [The present invention 1101] The composition of the present invention 1100, comprising CD4+ T cells and / or CD8+ T cells. [The present invention 1102] 1101. The composition of claim 1101, wherein 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, optionally 1:1. [The present invention 1103] Any of the compositions of claims 1100-1102, wherein cells expressing the chimeric receptor represent at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of all cells in the composition, or of all CD4+ T cells or CD8+ T cells in the composition. [The present invention 1104] A method of treatment comprising administering to a subject having a disease or disorder any one of the genetically engineered T cells, multiple genetically engineered T cells, or compositions of the present inventions 1001 to 1031 and 1100 to 1103. [This invention 1105] Use of a genetically engineered T cell, a plurality of genetically engineered T cells, or a composition of any of inventions 1001 to 1031 and 1100 to 1103 for the treatment of a disease or disorder. [The present invention 1106] Use of a genetically engineered T cell, a plurality of genetically engineered T cells, or a composition of any of inventions 1001-1031 and 1100-1103 in the manufacture of a medicament for treating a disease or disorder. [This invention 1107] The genetically engineered T cell, a plurality of genetically engineered T cells, or a composition of any of claims 1001 to 1031 and 1100 to 1103 for use in treating a disease or disorder. [This invention 1108] 8. The method, use, or genetically engineered T cell, a plurality of genetically engineered T cells, or composition for use of any of claims 1104 to 1107, wherein the disease or disorder is cancer or tumor. [This invention 1109] 1108. The method, use, or genetically engineered T cell, plurality of genetically engineered T cells, or composition for use of invention 1108, wherein said cancer or tumor is a hematological malignancy, optionally a lymphoma, leukemia, or plasma cell malignancy. [The present invention 1110] 1108. The method, use, or genetically engineered T cell, plurality of genetically engineered T cells, or composition for use of invention 1108, wherein said cancer or tumor is a solid tumor, and optionally said solid tumor is non-small cell lung cancer (NSCLC) or head and neck squamous cell carcinoma (HNSCC). [The present invention 1111] one or more agents capable of inducing gene disruption at a target site within the CD247 locus; Any one of the polynucleotides 1032 to 1067 of the present invention Includes a kit. [The present invention 1112] one or more agents capable of inducing gene disruption at a target site within the CD247 locus; a polynucleotide comprising a nucleic acid sequence encoding a chimeric receptor or a portion thereof, wherein the nucleic acid sequence encoding the chimeric receptor or a portion thereof is targeted for integration at or near a target site via homology directed repair (HDR); Instructions for carrying out any one of the methods of the present invention 1068 to 1098 and Includes a kit. [Brief explanation of the drawings]

[0107] [Figure 1] FIG. 1 shows CD3 and TCR surface expression assessed by flow cytometry in T cells electroporated with ribonucleoprotein (RNP) complexes containing one of four CD247-targeting gRNAs (gRNA1, 2, 3, 4) to introduce a gene disruption at the endogenous CD247 locus by CRISPR / Cas9-mediated gene editing, or mock-electroporated T cells containing no gRNA as a control (mock). [Figure 2A] Figure 1 shows surface expression of CD3 (detected using an anti-CD3ε antibody) and anti-BCMA chimeric antigen receptor (CAR) (detected using BCMA-Fc; soluble human BCMA fused at the C-terminus to the Fc region of IgG), assessed by flow cytometry, in T cells electroporated with RNP complexes containing gRNA3 targeting CD247 and incubated with an adeno-associated virus (AAV) construct containing one of four polynucleotides (polynucleotides A, B, C, D; listed in Table E1) containing a transgene sequence and regulatory elements and / or multicistronic elements encoding an anti-BCMA CAR or a portion thereof; or mock-electroporated and transduced T cells (mock) as a control. [Figure 2B]FIG. 1 shows the coefficient of variation (CV) (standard deviation of the signal within a cell population divided by the mean value of the signal in the respective population) and geometric mean fluorescence (gMFI) of expression of an exemplary anti-BCMA CAR engineered as described in Example 2.B. [Figure 3-1] Figure 3A shows the overall lysis rate from a cytolytic activity assay after co-culture of CAR-expressing T cells engineered with an AAV construct containing one of four polynucleotides (polynucleotides A, B, C, D; listed in Table E1) containing transgene sequences and regulatory and / or multicistronic elements encoding an anti-BCMA CAR or a portion thereof with RPMI8226 multiple myeloma cells (ATCC® CCL-155™; expressing low levels of BCMA) at an E:T ratio of 2:1, 1:1, or 1:2. The loss of NucLight Red (NLR)-labeled viable target cells, as determined by red fluorescent signal (using an IncuCyte® Live Cell Analysis System (Essen Bioscience)), was measured over 49 hours. Mock-electroporated and transduced cells (mock) and target cells cultured without CAR+ cells (target only) were assessed as controls. Lysis rates were determined and normalized to the CAR+ population. Figure 3B shows the overall lysis rate from a cytolytic activity assay after co-culture of engineered CAR-expressing T cells with K562 chronic myeloid leukemia (CML) cells (ATCC® CCL-243™; K562-BCMA, expressing high levels of BCMA) at E:T ratios of 2:1, 1:1, or 1:2. Figures 3C-3E show the lysis of RPMI8226 cells over time at E:T ratios of 2:1 (Figure 3C), 1:1 (Figure 3D), and 1:2 (Figure 3E), as determined by red fluorescence signal. Figures 3F-3H show the lysis of K562 cells over time at E:T ratios of 2:1 (Figure 3F), 1:1 (Figure 3G), and 1:2 (Figure 3H). [Figure 3-2] See description of Figure 3-1. [Figure 3-3] See description of Figure 3-1. [Figure 3-4]See description of Figure 3-1. [Figure 4A] Figures 4A-4C show levels of interferon gamma (IFNγ; Figure 4A), interleukin-2 (IL-2; Figure 4B), and tumor necrosis factor alpha (TNFα; Figure 4C) using a multiplex cytokine immunoassay after incubation of RPMI8226 or K562 target cells with CAR-expressing T cells engineered with AAV constructs containing one of four polynucleotides (polynucleotides A, B, C, D; listed in Table E1) at E:T ratios of 2:1, 1:1, or 1:2 E:T, as described in Example 3. Mock-electroporated and transduced cells (mock) and target cells cultured without CAR+ cells (target only) were assessed as controls. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 5] FIG. 1 shows surface expression of CD3 assessed by flow cytometry in T cells electroporated with ribonucleoprotein (RNP) complexes containing gRNA1 or gRNA3 targeting CD247, each with an Alt-R modification (IDT Technologies; Coralville, IA), at a gRNA-to-Cas9 protein ratio of approximately 2.6:1 and a concentration of 25 μM. [Figure 6A]Figures 6A-6B show surface expression of CD3 (detected using an anti-CD3ε antibody) and anti-BCMA chimeric antigen receptor (CAR) (detected using BCMA-Fc; soluble human BCMA fused at the C-terminus to the Fc region of IgG) assessed by flow cytometry in T cells from a representative donor (donor 1) electroporated with RNP complexes containing gRNA3 targeting CD247 and incubated with an adeno-associated virus (AAV) construct containing one of four polynucleotides (polynucleotides A, B, C, and D; listed in Table E1); or engineered to express an anti-BCMA CAR by lentiviral delivery (lentivirus; see Figure 6B); and, as controls, mock-electroporated and transduced T cells (mock) or mock-transduced and electroporated only with CD247-targeting RNP (KO only). Figure 6C shows a histogram of anti-BCMA CAR expression in each group. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 7] Figures 7A-7B show the overall lysis rate from a cytolytic activity assay after co-culture of CAR-expressing T cells engineered using an AAV construct containing one of four polynucleotides containing a transgene sequence encoding an anti-BCMA CAR (polynucleotides A, B, C, D; listed in Table E1, see Figure 7A) with MM.1S (ATCC® CRL-2974™) human B-lymphoblastoid target cells at an E:T ratio of 2:1 or 1:2. T cells engineered to express an anti-BCMA CAR by lentiviral delivery (lentivirus; see Figure 7B), as well as T cells that were mock-transduced and electroporated with CD247-targeting RNP (KO only), were also assessed as controls. Percent lysis values ​​were averaged from triplicate samples and normalized across the three donors. [Figure 8]8A-8C show levels of interferon-gamma (IFNγ; FIG. 8A), interleukin 2 (IL-2; FIG. 8B), and tumor necrosis factor alpha (TNFα; FIG. 8C) following incubation of CAR-expressing T cells engineered using an AAV construct containing one of four polynucleotides (polynucleotides A, B, C, D; listed in Table E1) with MM.1S target cells at E:T ratios of 2:1 and 1:2, as described in Example 4. T cells engineered to express an anti-BCMA CAR by lentiviral delivery (LV), T cells that were mock transduced and electroporated with CD247-targeting RNP only (KO), and mock-electroporated and transduced cells (Mock) were also assessed as controls. DETAILED DESCRIPTION OF THE INVENTION

[0108] Detailed Description Provided herein are genetically engineered cells, e.g., T cells, having a modified CD247 locus that includes one or more transgene sequences (hereinafter interchangeably referred to as "donor" sequences, e.g., sequences that are foreign or heterologous to the T cell) encoding a chimeric or recombinant receptor, e.g., a chimeric antigen receptor (CAR), or a portion thereof. In some aspects, the cells are engineered to express a chimeric receptor containing a CD3 zeta (CD3ζ) chain or a fragment thereof, typically present at the C-terminus of the chimeric receptor. In some embodiments, at least a portion of the CD3ζ chain or fragment is encoded by the genomic sequence of the endogenous CD247 locus (the genomic locus encoding CD3ζ) of the engineered cell, e.g., T cell, or a subsequence thereof. In some aspects, integration of the transgene sequence into the endogenous CD247 locus, e.g., by homology-directed repair (HDR), is performed such that the nucleic acid sequence encoding a portion of the chimeric receptor is fused, e.g., fused in-frame, with the open reading frame of the endogenous CD247 locus or a subsequence thereof, e.g., an exon of the open reading frame.

[0109] Also provided are methods for producing genetically engineered cells containing modified CD247 loci that express chimeric or recombinant receptors or portions thereof. Provided embodiments include specifically targeting a transgene sequence encoding a chimeric receptor (e.g., CAR) or a portion thereof to the endogenous CD247 locus. In some circumstances, provided embodiments include, for example, using gene editing methods to induce target-specific gene disruption, e.g., the generation of DNA breaks, and inducing HDR for target-specific integration of the transgene sequence encoding the chimeric receptor into the endogenous CD247 locus. Related cell compositions, nucleic acids, and kits for generating the engineered cells provided herein and / or for use in the methods provided herein are also provided.

[0110] In some embodiments, the transgene sequence encoding a portion of a chimeric or recombinant receptor, e.g., a CAR, contains a sequence of nucleotides encoding one or more domains or regions of the chimeric receptor, e.g., the extracellular region, the transmembrane domain, and the intracellular region. In some aspects, the extracellular region contains a binding domain (e.g., an antigen-binding domain or a ligand-binding domain) that provides specificity for a desired antigen (e.g., a tumor antigen) or ligand, and / or a spacer for linking the extracellular binding domain to the transmembrane domain and the intracellular region. In some aspects, the intracellular region encoded by the transgene sequence includes one or more costimulatory domains and / or other domains. In some embodiments, the intracellular region encoded by the transgene sequence (i.e., a sequence introduced exogenously to a cell) includes less than the entire length of the CD3 zeta chain or does not include the sequence encoding the CD3 zeta chain. The modified CD247 locus obtained by integrating the transgene sequence into the endogenous CD247 locus encodes a chimeric receptor encoded by a fusion of the HDR-targeted transgene sequence with the open reading frame of the endogenous CD247 locus or a subsequence thereof. The encoded chimeric receptor contains an intracellular region containing a functional CD3ζ chain or a fragment thereof, for example, a functional CD3ζ chain or a fragment thereof that can mediate, activate, or stimulate primary cytoplasmic or primary intracellular signals in T cells. The resulting genetically engineered cells or cell compositions can be used in adoptive cell therapy.

[0111] T cell-based therapies, e.g., adoptive T cell therapies (e.g., those involving the administration of engineered cells expressing recombinant, engineered, or chimeric receptors, e.g., chimeric antigen receptors (CARs), or other recombinant, engineered, or chimeric receptors, specific for a disease or disorder of interest) can be effective in treating cancer and other diseases and disorders. In certain situations, other approaches to generating engineered cells for adoptive cell therapy are not always entirely sufficient. In some situations, optimal efficacy may depend on the ability of the administered cells to express the chimeric receptor, e.g., uniform, homogeneous, and / or consistent receptor expression in cells, e.g., a population of immune cells and / or cells in a therapeutic cell composition, and the ability of the chimeric receptor to recognize and bind to a target, e.g., a target antigen, in the subject, tumor, and their environment.

[0112] In some cases, available methods for introducing chimeric receptors (e.g., CARs) into cells include the random integration of sequences encoding chimeric receptors, for example, by viral transduction.In certain respects, such methods are not entirely satisfactory.In some aspects, random integration may result in insertion mutagenesis and / or gene disruption at one or more random loci in cells, for example, loci that may be important for the function and activity of cells.In some aspects, the efficiency of chimeric receptor expression is limited in certain cells or certain cell populations engineered using currently available methods.In some cases, chimeric receptors are only expressed in certain cells within a cell population, and the expression level of chimeric receptors may vary widely between cells within the population.In certain aspects, the expression level of chimeric receptors may be difficult to predict, regulate, and / or control.In some cases, semi-random or random integration of receptor-encoding transgenes into the genome of cells may cause harmful and / or unwanted effects due to the integration of nucleic acid sequences into undesired locations within the genome, such as essential genes or genes important in controlling cellular activity.

[0113] In some cases, random integration can result in variable integration of sequences encoding recombinant or chimeric receptors, which can lead to inconsistent expression, variable copy numbers of nucleic acids, and / or variable receptor expression in cells of a cell composition, e.g., a therapeutic cell composition. In some cases, random integration of nucleic acid sequences encoding receptors can result in diverse, heterogeneous, non-uniform, and / or suboptimal expression or antigen binding, oncogenic transformation, and transcriptional silencing of the nucleic acid sequence, depending on the site of integration and / or the copy number of the nucleic acid sequence. In some aspects, heterogeneous and non-uniform expression in a cell population can result in inconsistent or unstable expression and / or antigen binding by recombinant or chimeric receptors, unpredictable or reduced function of the engineered cells, and / or heterogeneous drug products, thereby reducing the efficacy of the engineered cells. In some aspects, the use of certain random integration vectors, such as certain lentiviral vectors, requires confirming that the engineered cells do not contain replication-competent viruses, for example, by performing a replication-competent lentivirus (RCL) assay. Improved strategies are needed to achieve consistent expression levels and function of recombinant or chimeric receptors while minimizing random integration of nucleic acids and / or heterogeneous expression within a population.

[0114] In some aspects, the size of the payload (e.g., the transgene sequence or heterologous sequence to be inserted) within a particular polynucleotide or vector used to deliver a nucleic acid sequence encoding a chimeric receptor may be limiting. In some cases, the limited size may affect expression in cells and / or the efficiency of transduction and expression.

[0115] The provided embodiments relate to engineering cells, e.g., T cells, to integrate a nucleic acid encoding a chimeric receptor into their endogenous CD247 locus by homology-directed repair (HDR). In some aspects, HDR can mediate site-specific integration of a transgene sequence (e.g., a transgene sequence encoding a recombinant or chimeric receptor, or a portion, chain, or fragment thereof) at or near a target site of gene disruption, such as the endogenous CD247 locus. In some embodiments, the presence of a gene disruption (e.g., at a target site in the endogenous CD247 locus) and a polynucleotide, e.g., a template polynucleotide, containing one or more homology arms (e.g., containing a nucleic acid sequence homologous to the sequence surrounding the gene disruption) can induce or direct HDR, with the homologous sequence serving as a template for DNA repair. Based on the homology between the endogenous gene sequence surrounding the gene disruption and the homology arms contained in the polynucleotide, e.g., template polynucleotide, the cellular DNA repair machinery uses the polynucleotide, e.g., template polynucleotide, to repair the DNA break and resynthesize genetic information at the target site of the gene disruption, thereby efficiently inserting or integrating the sequence between the homology arms (e.g., a transgene sequence encoding a chimeric receptor or a portion thereof) into or near the target site of the gene disruption. Provided embodiments can generate cells containing a modified CD247 locus encoding a chimeric receptor or a portion thereof, in which a transgene sequence encoding a chimeric receptor or a portion thereof has been integrated into the endogenous CD247 locus by HDR.

[0116] In some aspects, the provided embodiments provide the advantage of creating engineered cells by improved and / or more efficient targeting of nucleic acids encoding chimeric or recombinant receptors into cells. In some cases, the methods minimize possible semi-random or random integration and / or heterogeneous or variable expression and / or unwanted expression from unintegrated nucleic acid sequences, resulting in improved, uniform, homogeneous, consistent, predictable, or stable expression of chimeric or recombinant receptors with reduced, low, or no possibility of insertional mutagenesis. In some aspects, compared to other methods of creating engineered immune cells expressing chimeric or recombinant receptors, such as CARs, the provided embodiments enable more stable, more physiological, more regulatable, or more uniform, consistent, or homogeneous expression of chimeric or recombinant receptors. In some cases, the methods result in the production of more consistent and more predictable drug products, such as cell compositions containing engineered cells, which can result in safer therapy for treated patients. In some aspects, the provided embodiments also enable predictable and consistent integration into a single locus or multiple loci of interest. In some embodiments, the provided embodiments can also result in the production of cell populations in which the number of copies of the nucleic acid integrated into the cells of the population is consistent (typically 1 or 2), which in some aspects provides consistent expression of the chimeric or recombinant receptor and the expression of the endogenous receptor gene within the cell population. In some cases, the provided embodiments do not involve the use of viral vectors for integration, thus reducing the need to ensure that the engineered cells do not contain replication-competent viruses, thereby improving the safety of the cell composition.

[0117] In the engineered cells provided herein, the chimeric receptor encoded by the modified CD247 locus can be encoded under the control of endogenous or exogenous control elements. In some aspects, the provided embodiments allow the chimeric receptor to be expressed under the control of endogenous CD247 control elements, which can provide a more physiological expression level in some cases. In some aspects, the provided embodiments allow the nucleic acid encoding the chimeric receptor to be expressed under the control of endogenous control or regulatory elements, such as cis-regulatory elements, such as the promoter or 5' and / or 3' untranslated region (UTR) of the endogenous CD247 locus. Thus, in some aspects, the provided embodiments allow the chimeric receptor, for example, CAR, or a portion thereof, to be expressed and / or its expression controlled at a level similar to that of the endogenous CD3ζ chain.

[0118] In some aspects, the provided embodiments can reduce or minimize antigen-independent signaling or activity (also known as "tonic signaling") through the chimeric receptor. In some cases, antigen-independent signaling can result from overexpression or unregulated activity of the expressed chimeric receptor, leading to unwanted effects such as increased differentiation and / or attrition of T cells expressing the chimeric receptor. In some embodiments, the provided engineered cells and cell compositions can reduce the effects of antigen-independent signaling that can result from overexpression or unregulated activity of the expressed chimeric receptor. Thus, the provided embodiments can facilitate the generation of engineered cells that exhibit improved expression, function, and uniformity of expression, and / or other desirable traits or characteristics, ultimately exhibiting greater efficacy. In some embodiments, the provided polynucleotides, transgenes, and / or vectors, when delivered to immune cells, result in the expression of chimeric receptors, e.g., CARs, that can modulate T cell activity and, in some cases, T cell differentiation and homeostasis.

[0119] In some aspects, provided embodiments allow the chimeric receptor to be expressed under the control of exogenous or heterologous control or regulatory elements, which in some aspects provides more tunable expression levels. In some aspects, provided embodiments allow target-specific, regulated expression of the chimeric receptor in a variety of cell types, including cells in which the endogenous promoter of the endogenous CD247 locus may not be active, e.g., cells that do not typically express the CD3ζ chain, e.g., non-T cells, e.g., NK cells, B cells, or certain induced totipotent stem cell (iPSC)-derived cells.

[0120] In some aspects, provided embodiments can prevent unregulated expression or expression from randomly integrated or non-integrated polynucleotides. In some embodiments, the introduced polynucleotide, e.g., the template polynucleotide, does not contain a nucleic acid sequence encoding the entire length of a functional receptor. In some cases, a portion of the CD3 zeta chain is not encoded by the introduced polynucleotide. In some aspects, transcription from a randomly integrated or non-integrated polynucleotide does not produce a functional receptor. In some aspects, only integration into a target locus, e.g., the endogenous CD247 locus, can generate a functional receptor containing all of the required signaling regions. In some aspects, provided embodiments can result in improved safety of the cell composition by preventing unregulated expression, e.g., from randomly integrated or non-integrated polynucleotides, e.g., non-integrated viral vector sequences.

[0121] In some aspects, provided embodiments may also result in the reduction and / or elimination (e.g., knockout) of expression of the extracellular portion of CD3ζ to reduce the immunogenicity of the administered cells, e.g., for applications in allogeneic adoptive cell therapy.

[0122] The provided embodiments can also reduce the length of the transgene sequence required to deliver a recombinant CAR to a cell, for example, by allowing sufficient space for packaging additional elements and / or transgenes into the same vector, e.g., a viral vector. In some aspects, the provided embodiments also enable the use of smaller nucleic acid sequence fragments for engineering compared to existing methods by utilizing part or all of the open reading frame sequence of an endogenous gene encoding the CD3ζ chain to encode all or a portion of the CD3ζ chain of the CAR. In some aspects, the methods utilize part or all of the open reading frame sequence of the endogenous gene encoding CD3ζ, CD247, to encode the chimeric receptor CD3ζ or a portion thereof, so the provided embodiments provide greater flexibility for engineering cells to express a CAR compared to existing methods. In some cases, the reduced length requirement for the nucleic acid sequence encoding the chimeric receptor or a portion thereof reduces the payload space for the sequence encoding the chimeric receptor or a portion thereof, leaving space for sequences encoding other components, such as other transgene sequences, homology arms, and regulatory elements. In some aspects, the length requirement of the nucleic acid sequence encoding a portion of a chimeric receptor, such as a CAR, is reduced, so the provided embodiments can accommodate larger homology arms and / or can accommodate nucleic acid sequences encoding additional molecules, compared to conventional embodiments that require the full length of the chimeric receptor, such as a CAR, in the polynucleotide to be introduced.In some aspects, the provided embodiments can be used to facilitate or improve the generation, delivery, and / or targeting efficiency of nucleic acid sequences, such as transgene sequences, by homology-directed repair (HDR).In other aspects, the provided embodiments can accommodate nucleic acid sequences encoding additional molecules for expression on or in cells.

[0123] Methods for manipulating, preparing, and generating engineered cells, as well as kits and devices for generating or producing engineered cells, are also provided. Cells and cell compositions produced by the methods are also provided. Polynucleotides, e.g., viral vectors, containing nucleic acid sequences encoding portions of chimeric receptors, and methods for introducing such polynucleotides into cells, e.g., by transduction or physical delivery, e.g., electroporation, are also provided. Compositions containing engineered cells, as well as methods, kits, and devices for administering the cells and compositions to a subject, e.g., for adoptive cell therapy, are also provided. In some aspects, cells are isolated from a subject, manipulated, and administered to the same subject. In other aspects, they are isolated from one subject, manipulated, and administered to another subject. The resulting genetically engineered cells or cell compositions can be used in adoptive cell therapy.

[0124] All publications, e.g., patent documents, scientific articles, and databases, mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. To the extent that a definition set forth herein contradicts or otherwise disagrees with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein takes precedence over the definition incorporated herein by reference.

[0125] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0126] I. Methods for generating cells expressing chimeric receptors by homology-directed repair Provided herein is a method for generating or producing genetically engineered cells that contain a modified CD247 locus, wherein the modified CD247 locus comprises a nucleic acid sequence encoding a chimeric receptor or recombinant receptor, such as a chimeric antigen receptor (CAR). In some aspects, the modified CD247 locus in the genetically engineered cell comprises a transgene sequence encoding a chimeric receptor or a portion of a chimeric receptor, integrated into the endogenous CD247 locus that normally encodes the CD3 zeta (CD3ζ) chain. In some embodiments, the method comprises using a polynucleotide (also referred to as "template polynucleotide") containing a transgene encoding a chimeric receptor or recombinant receptor or a portion of a chimeric receptor to induce targeted gene disruption and homology-dependent repair (HDR), thereby targeting the integration of the transgene into the CD247 locus. Also provided are cells and cell compositions produced by the method. In some embodiments, compositions containing populations of cells engineered to express a chimeric receptor, e.g., a CAR, such that they exhibit improved, uniform, homogeneous, and / or stable expression and / or antigen binding by the chimeric receptor, e.g., genetically engineered immune cells produced by any of the provided methods, as well as polynucleotides, e.g., template polynucleotides, and kits for use in the methods, are also provided.

[0127] In some aspects, the expressed chimeric receptor comprises a CD3 zeta (CD3ζ) chain or a fragment thereof, e.g., an intracellular region containing the signaling region or signaling domain of CD3ζ. In some embodiments, the encoded CD3ζ chain or fragment thereof is a functional CD3ζ chain or fragment thereof, e.g., a cytoplasmic signaling domain or cytoplasmic signaling region. In some embodiments, the CD3ζ chain or fragment thereof is at the C-terminus of the receptor. In some aspects, after integration of a transgene sequence encoding a portion of the chimeric receptor into the CD247 locus, at least a portion of the CD3ζ chain is encoded by an open reading frame or a subsequence thereof of the CD247 locus in the genome. In some aspects, the chimeric receptor is encoded by an exogenous nucleic acid sequence fused to an open reading frame or a subsequence thereof of the endogenous CD247 locus.

[0128] In some embodiments, the method utilizes HDR for target-specific integration of transgene sequence into CD247 locus.In some cases, the method comprises the combination of one or more target-specific gene disruption, for example, DNA breakage, in endogenous CD247 locus by gene editing technology, and the target-specific integration of the transgene sequence encoding chimeric receptor or a part of chimeric receptor by HDR.In some embodiments, the HDR process is accompanied by DNA breakage or damage, for example, double-strand breakage, at target genome position.In some embodiments, DNA breakage is induced by gene editing method, for example, by using target-specific nuclease.

[0129] In some aspects, the provided method comprises introducing into T cells one or more agents capable of inducing gene disruption at a target site within the CD247 locus; and introducing into T cells a polynucleotide, such as a template polynucleotide, comprising a transgene and one or more homology arms. In some aspects, the transgene contains a nucleotide sequence encoding a chimeric receptor or a portion thereof. In some embodiments, the nucleic acid sequence, such as a transgene, is targeted for integration into the CD247 locus via homology-directed repair (HDR). In some aspects, the provided method comprises introducing into T cells having a gene disruption within the CD247 locus a polynucleotide comprising a transgene sequence encoding a chimeric receptor or a portion thereof, wherein the gene disruption is induced by one or more agents capable of inducing gene disruption at one or more target sites within the CD247 locus, and the nucleic acid sequence, such as a transgene, is targeted for integration into the CD247 locus via HDR.

[0130] In some aspects, embodiments include target-specific genome disruption using gene editing methods and / or target-specific nucleases, e.g., generating target-specific DNA breaks, followed by HDR based on one or more polynucleotides, e.g., template polynucleotides, containing homologous sequences homologous to the sequence of the endogenous CD247 locus, linked to a transgene sequence encoding a portion of a chimeric receptor, and in some embodiments, also linked to nucleic acid sequences encoding other molecules, to specifically target or integrate the transgene sequence into or near the DNA break. Thus, in some aspects, the method includes inducing a target-specific gene disruption (e.g., via gene editing), and introducing a polynucleotide, e.g., template polynucleotide, containing the transgene sequence into a cell (e.g., via HDR).

[0131] In some embodiments, target-specific gene disruption and target-specific integration of the transgene sequence by HDR occur at one or more target sites of the endogenous CD247 locus that encodes CD3 zeta (CD3ζ) chain.In some aspects, target-specific integration occurs within the open reading frame sequence of the endogenous CD247 locus.In some aspects, the target-specific integration of the transgene sequence results in the in-frame fusion of the coding portion of the transgene with one or more exons of the open reading frame of the endogenous CD247 locus, for example, with the adjacent exon of the integration site.

[0132] In some embodiments, polynucleotide (for example, template polynucleotide) is introduced into engineered cell before, at the same time, or after the introduction of one or more agents that can induce one or more target gene disruption.In the presence of one or more target gene disruption (for example, DNA break), polynucleotide can be used as DNA repair template to effectively replicate and / or integrate transgene at or near the site of target gene disruption by HDR based on the homology between the endogenous gene sequence around gene disruption and one or more homology arms, such as the 5' and / or 3' homology arms contained in template polynucleotide.

[0133] In some aspects, two steps can be carried out sequentially.In some embodiments, gene editing and HDR step are carried out simultaneously and / or in one experimental reaction.In some embodiments, gene editing and HDR step are carried out continuously or sequentially in one or consecutive experimental reaction.In some embodiments, gene editing and HDR step are carried out simultaneously or at different times in separate experimental reaction.

[0134] Immune cells can include a population of cells containing T cells. Such cells can be obtained from a subject, for example, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product. In some embodiments, immune cells such as T cells are primary cells, such as primary T cells. In some embodiments, T cells can be separated or selected to enrich the T cells in the population using positive or negative selection and enrichment methods. In some embodiments, the population contains CD4+, CD8+, or CD4+ and CD8+ T cells. In some embodiments, the steps of introducing a polynucleotide (e.g., a template polynucleotide) and introducing an agent (e.g., Cas9 / gRNA RNP) can be performed simultaneously or sequentially, in any order. In some embodiments, the polynucleotide is introduced simultaneously with the introduction of one or more agents (e.g., Cas9 / gRNA RNP) capable of inducing gene disruption. In certain embodiments, the polynucleotide template is introduced into the immune cells after inducing gene disruption by introducing an agent (e.g., Cas9 / gRNA RNP). In some embodiments, the cells are cultured or incubated under conditions that stimulate cell expansion and / or proliferation before, during, and / or after the introduction of the polynucleotide template and one or more agents (e.g., Cas9 / gRNA RNP).

[0135] In certain embodiments of the provided method, the introduction of template polynucleotide is carried out after the introduction of one or more agents that can induce gene disruption.Depending on the specific agent used to induce gene disruption, any method for introducing one or more agents can be used as described.In some aspects, the disruption is carried out by gene editing, for example, using an RNA-guided nuclease, such as a clustered regularly interspaced short palindromic nucleic acid (CRISPR)-Cas system, specific to the CD247 locus to be disrupted, for example, a CRISPR-Cas9 system.In some aspects, the disruption is carried out using a CRISPR-Cas9 system specific to the CD247 locus.In some embodiments, an agent containing Cas9 and a guide RNA (gRNA) containing a targeting domain that targets a region of the CD247 locus is introduced into cells. In some embodiments, the agent is or includes a ribonucleoprotein (RNP) complex of Cas9 and a gRNA containing a targeting domain that targets CD247 (Cas9 / gRNA RNP). In some embodiments, the introduction includes contacting the agent, or a portion thereof, with the cell in vitro, which can include culturing or incubating the cell and agent for up to 24, 36, or 48 hours, or 3, 4, 5, 6, 7, or 8 days. In some embodiments, the introduction can further include delivering the agent into the cell. In various embodiments, methods, compositions, and cells according to the present disclosure utilize direct delivery of a ribonucleoprotein (RNP) complex of Cas9 and gRNA into the cell, for example, by electroporation. In some embodiments, the RNP complex includes a gRNA that has been modified to include a 3' polyA tail and a 5' Anti-Reverse Cap Analog (ARCA) cap.Optionally, electroporating the cells to be modified includes cold-shocking the cells, for example at 32° C., after electroporation of the cells and before plating.

[0136] In such aspects of the provided methods, the polynucleotide, e.g., template polynucleotide, is introduced into the cell after introduction of one or more agents, e.g., Cas9 / gRNA RNP, introduced, e.g., via electroporation. In some embodiments, the polynucleotide, e.g., template polynucleotide, is introduced immediately after introduction of one or more agents capable of inducing gene disruption. In some embodiments, a polynucleotide, e.g., a template polynucleotide, is introduced into a cell within at or about 30 seconds, within at or about 1 minute, within at or about 2 minutes, within at or about 3 minutes, within at or about 4 minutes, within at or about 5 minutes, within at or about 6 minutes, within at or about 6 minutes, within at or about 6 minutes, within at or about 8 minutes, within at or about 9 minutes, within at or about 10 minutes, within at or about 15 minutes, within at or about 20 minutes, within at or about 30 minutes, within at or about 40 minutes, within at or about 50 minutes, within at or about 60 minutes, within at or about 60 minutes, within at or about 90 minutes, within at or about 2 hours, within at or about 3 hours, or within at or about 4 hours after introduction of one or more agents capable of inducing a gene disruption.In some embodiments, the polynucleotide, e.g., template polynucleotide, is incubated for 15 minutes or about 15 minutes to 4 hours or about 4 hours, e.g., 15 minutes or about 15 minutes to 3 hours or about 3 hours, 15 minutes or about 15 minutes to 2 hours or about 2 hours, 15 minutes or about 15 minutes to 1 hour or about 1 hour, 15 minutes or about 15 minutes to 30 minutes or about 30 minutes, 30 minutes or about 30 minutes to 4 hours or about 4 hours, 30 minutes or about 30 minutes to 3 hours or about The polynucleotide, e.g., template polynucleotide, is introduced into the cell for a time period of 3 hours, between at or about 30 minutes and 2 or about 2 hours, between at or about 30 minutes and 1 or about 1 hour, between at or about 1 hour and 4 or about 4 hours, between at or about 1 hour and 3 or about 3 hours, between at or about 1 hour and 2 or about 2 hours, between 2 or about 2 hours and 4 or about 4 hours, between 2 or about 2 hours and 3 or about 3 hours, or between 3 or about 3 hours and 4 or about 4 hours. In some embodiments, the polynucleotide, e.g., template polynucleotide, is introduced into the cell 2 hours or about 2 hours after introduction of one or more agents, e.g., Cas9 / gRNA RNP, introduced, e.g., via electroporation.

[0137] Depending on the specific method used to deliver polynucleotides, such as template polynucleotides, to cells, any method for introducing polynucleotides, such as template polynucleotides, can be used as described.Exemplary methods include those for transferring receptor-encoding nucleic acids, including those via viruses, such as retroviruses or lentiviruses, transduction, transposons, and electroporation.In certain embodiments, viral transduction methods are used.In some embodiments, polynucleotides can be transferred or introduced into cells using recombinant infectious viral particles, such as vectors derived from Simian Virus 40 (SV40), adenovirus, or adeno-associated virus (AAV). In some embodiments, the recombinant nucleic acid is transferred into T cells using a recombinant lentiviral or retroviral vector, e.g., a gamma-retroviral vector (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 certain embodiments, the viral vector is an AAV, such as AAV2 or AAV6.

[0138] In some embodiments, prior to, during, or following contacting the cells with an agent, and / or prior to, during, or following delivery (e.g., electroporation), the provided methods comprise incubating the cells in the presence of a cytokine, a stimulatory agent, and / or an agent capable of inducing proliferation, stimulation, or activation of immune cells (e.g., T cells). In some embodiments, at least a portion of the incubation is in the presence of a stimulatory agent that is or includes an antibody specific for CD3, an antibody specific for CD28, and / or a cytokine, e.g., anti-CD3 / anti-CD28 beads. In some embodiments, at least a portion of the incubation is in 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 for up to 8 days, e.g., up to 24 hours, 36 hours, or 48 hours, or 3, 4, 5, 6, 7, or 8 days, e.g., before or after introduction of one or more agents, e.g., Cas9 / gRNA RNP, and polynucleotide, e.g., template polynucleotide, via electroporation.

[0139] In some embodiments, the methods include activating or stimulating the cells with a stimulatory agent (e.g., an anti-CD3 / anti-CD28 antibody) prior to introducing an agent, e.g., the Cas9 / gRNA RNP, and the polynucleotide template. In some embodiments, incubation in the presence of the stimulatory agent (e.g., anti-CD3 / anti-CD28) is for 6 to 96 hours, e.g., 24 to 48 hours, or 24 to 36 hours, prior to introduction of one or more agents, e.g., the Cas9 / gRNA RNP, e.g., via electroporation. In some embodiments, incubation with the stimulatory agent can 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 a recombinant cytokine, such as 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 stimulatory agent (e.g., anti-CD3 / anti-CD28 antibody) is washed or removed from the cells prior to introducing or delivering into the cells an agent Cas9 / gRNA RNP and / or polynucleotide template capable of inducing gene disruption. In some embodiments, the cells are rested prior to introducing the agent, e.g., by removing any stimulating or activating agents, hi some embodiments, stimulating or activating agents and / or cytokines are not removed prior to introducing the agent.

[0140] In some embodiments, following introduction of an agent, e.g., Cas9 / gRNA, and / or polynucleotide template, the cells are incubated, cultivated, or cultured in the presence of one or more recombinant cytokines, e.g., recombinant IL-2, recombinant IL-7, and / or recombinant IL-15. In some embodiments, the incubation is performed in the presence of a recombinant cytokine, 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). The cells can be incubated or cultured under conditions to induce cell proliferation or expansion, hi some embodiments, the cells can be incubated or cultured until a threshold cell number for harvest, e.g., a therapeutically effective amount, is achieved.

[0141] In some embodiments, incubation during any part of the process or the entire process can be at a temperature of 30°C ± 2°C to 39°C ± 2°C, e.g., 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 a portion of the incubation is at 30°C ± 2°C and at least a portion of the incubation is at 37°C ± 2°C.

[0142] In some embodiments, the nucleic acid sequence present at the modified CD247 locus through target-specific integration comprises a fusion of a transgene targeted by HDR (e.g., a portion of a chimeric receptor, e.g., a CAR, described herein) with an open reading frame of the endogenous CD247 locus, or a subsequence thereof. In some aspects, the nucleic acid sequence present at the modified CD247 locus comprises a portion of a transgene, e.g., a chimeric receptor, e.g., a CAR, described herein, integrated into the endogenous CD247 locus, including an open reading frame encoding the CD3ζ chain. In some aspects, through target-specific integration or fusion, e.g., in-frame fusion, a portion of the exogenous sequence of the transgene and a portion of the open reading frame of the endogenous CD247 locus together encode a chimeric receptor, e.g., a CAR, containing a CD3ζ signaling domain or a fragment thereof. Thus, provided embodiments utilize a portion or all of the open reading frame sequence of the endogenous CD247 locus to encode the CD3ζ signaling domain or a portion thereof of the chimeric receptor. In some embodiments, by target-specific in-frame integration of a transgene sequence, the modified CD247 locus contains a sequence encoding the entire, complete, or full-length chimeric receptor, e.g., a CAR, containing a CD3ζ signaling domain.

[0143] Exemplary methods for performing gene disruption at the endogenous CD247 locus and / or performing HDR for targeted integration of a transgene sequence, e.g., a portion of a chimeric receptor, e.g., a portion of a CAR, into the CD247 locus, are described in the following subsections.

[0144] A. Gene disruption In some embodiments, one or more target-specific gene disruptions are induced in the endogenous CD247 locus. In some embodiments, one or more target-specific gene disruptions are induced at one or more target sites at or near the endogenous CD247 locus. In some embodiments, the target-specific gene disruptions are induced in introns of the endogenous CD247 locus. In some embodiments, the target-specific gene disruptions are induced in exons of the endogenous CD247 locus. In some aspects, the presence of one or more target-specific gene disruptions and a polynucleotide, e.g., a template polynucleotide, containing a transgene sequence encoding a chimeric receptor or a portion thereof can result in the targeted integration of the transgene sequence into or near one or more gene disruptions (e.g., target sites) at the endogenous CD247 locus.

[0145] In some embodiments, gene disruption causes DNA breakage, such as double-strand break (DSB) or cleavage, or nick, such as single-strand break (SSB), at one or more target sites in the genome.In some embodiments, at the site of gene disruption, such as DNA breakage or nick, the action of cellular DNA repair mechanisms can cause knockout mutation, insertion mutation, missense mutation, or frameshift mutation, such as biallelic frameshift mutation, deletion of all or part of the gene; or, in the presence of a repair template, such as a template polynucleotide, the DNA sequence can be modified based on the repair template, for example, the nucleic acid sequence, such as a transgene, encoding all or part of the recombinant receptor contained in the template can be integrated or inserted.In some embodiments, gene disruption can be targeted to one or more exons of a gene or part thereof.In some embodiments, gene disruption can be targeted near the desired site of target-specific integration of an exogenous sequence, such as a transgene sequence encoding a chimeric receptor.

[0146] In some embodiments, a DNA binding protein or DNA binding nucleic acid that specifically binds or hybridizes with the sequence of a region near one of at least one target site is used for target-specific disruption.In some embodiments, a template polynucleotide, for example, a nucleic acid sequence encoding a portion of a chimeric receptor, for example, a template polynucleotide comprising a transgene and a homologous sequence, can be introduced for target-specific integration of the sequence encoding the chimeric receptor by HDR at or near the site of gene disruption, as described herein, for example, in section IB.

[0147] In some embodiments, gene disruption is carried out by introducing one or more agents that can induce gene disruption.In some embodiments, such agents include DNA-binding proteins or DNA-binding nucleic acids that specifically bind or hybridize with genes.In some embodiments, the agents include various components, such as fusion proteins that include DNA-targeting proteins and nucleases, or RNA-guided nucleases.In some embodiments, the agents can target one or more target sites or target positions.In some aspects, a pair of single-strand breaks (e.g., nicks) can be generated on each side of the target site.

[0148] In the provided embodiments, the term "introduction" encompasses a variety of methods for introducing nucleic acids and / or proteins, e.g., DNA, into cells, either in vitro or in vivo, 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, e.g., adenoviral vectors or adeno-associated vectors. Methods such as electroporation can also be used to introduce or deliver proteins, or ribonucleoproteins (RNPs), for example, containing a complex of Cas9 protein and targeting gRNA, into cells of interest.

[0149] In some embodiments, gene disruption occurs at a target site (also known as a "target location," "target DNA sequence," or "target position"), for example, at the endogenous CD247 locus. In some embodiments, the target site includes a site on target DNA (e.g., genomic DNA) that is modified by one or more agents capable of inducing gene disruption, for example, a Cas9 molecule complexed with a gRNA that specifies the target site. For example, the target site may include a position within the DNA of the endogenous CD247 locus where cleavage or DNA breakage occurs. In some aspects, integration of a nucleic acid sequence, such as a transgene encoding a recombinant receptor or a portion thereof, by HDR can occur at or near the target site or target sequence. In some embodiments, the target site can be a site between two nucleotides, e.g., adjacent nucleotides, on DNA where one or more nucleotides are added. The target site can include one or more nucleotides that are modified by the template polynucleotide. In some embodiments, the target site is within the target sequence (e.g., the sequence to which the gRNA binds). In some embodiments, the target site is upstream or downstream of the target sequence.

[0150] 1. Target site in the endogenous CD247 locus In some embodiments, gene disruption and / or integration of a transgene encoding a portion of a chimeric receptor via homology-directed repair (HDR) is targeted to the endogenous or genomic locus encoding the T cell surface glycoprotein CD3 zeta chain (also known as CD3 zeta; CD3ζ; T cell receptor T3 zeta chain; CD3Z; T3Z; TCRZ; cluster of differentiation 247; CD247; IMD25). In humans, CD3ζ is encoded by the cluster of differentiation 247 (CD247) gene. In some embodiments, gene disruption and integration of a transgene encoding a portion of a chimeric receptor via homology-directed repair (HDR) is targeted to the human CD247 locus. In some aspects, gene disruption is targeted to a target site within the CD247 locus containing the open reading frame encoding CD3ζ, so that target-specific integration, fusion, or insertion of the transgene sequence occurs at or near the site of gene disruption in the CD247 locus. In some aspects, the gene disruption is targeted to an exon or near an exon of the open reading frame encoding CD3ζ. In some aspects, the gene disruption is targeted to an intron or near an intron of the open reading frame encoding CD3ζ.

[0151] CD3ζ is part of the TCR-CD3 complex present on the surface of T cells involved in adoptive immune responses. CD3ζ forms the TCR-CD3 complex with the T cell receptor (TCR) alpha / beta (TCRαβ) or TCR gamma / delta (TCRγδ) heterodimer, CD3-gamma (CD3γ), CD3-delta (CD3δ), and CD3-epsilon (CD3ε). CD3ζ contains immunoreceptor tyrosine-based activation motifs (ITAMs) in its intracellular or cytoplasmic domain. The CD3ζ chain can couple antigen recognition to intracellular signal transduction pathways, for example, by stimulating or activating primary cytoplasmic or intracellular signaling via the ITAMs. Upon engagement of the TCR with a ligand (e.g., a peptide associated with an MHC molecule; an MHC-peptide complex), the ITAM motifs are phosphorylated by kinases such as the Src family protein tyrosine kinases LCK and FYN, leading to stimulation of downstream signaling pathways. In some aspects, phosphorylation of CD3ζ ITAM creates a docking site for the protein kinase ZAP70, leading to the phosphorylation and activation of ZAP70.

[0152] Exemplary human CD3 zeta precursor polypeptide sequences are set forth in SEQ ID NO:73 (isoform 1; mature polypeptide comprises residues 22-164 of SEQ ID NO:73; Uniprot Accession No. P20963; NCBI Reference Sequence: NP_932170.1; mRNA sequence set forth in SEQ ID NO:74, see NCBI Reference Sequence: NM_198053.2) or SEQ ID NO:75 (isoform 2; mature polypeptide comprises residues 22-163 of SEQ ID NO:75; NCBI Reference Sequence: NP_000725.1; mRNA sequence set forth in SEQ ID NO:76, see NCBI Reference Sequence: NM_000734.3). An exemplary mature CD3 zeta chain contains an extracellular region (comprising amino acid residues 22-30 of the human CD3 zeta chain precursor sequence shown in SEQ ID NO:73 or 75), a transmembrane region (comprising amino acid residues 31-51 of the human CD3 zeta chain precursor sequence shown in SEQ ID NO:73 or 75), and an intracellular region (comprising amino acid residues 52-164 of the human CD3 zeta chain precursor sequence shown in SEQ ID NO:73 or amino acid residues 52-163 of the human CD3 zeta chain precursor sequence shown in SEQ ID NO:75). The CD3 zeta chain contains three immunoreceptor tyrosine-based activation motifs (ITAMs) at amino acid residues 61-89, 100-128, or 131-159 of the human CD3 zeta chain precursor sequence shown in SEQ ID NO:73, or at amino acid residues 61-89, 100-127, or 130-158 of the human CD3 zeta chain precursor sequence shown in SEQ ID NO:75.

[0153] In humans, an exemplary genomic locus for CD247 contains an open reading frame containing eight exons and seven introns. An exemplary mRNA transcript for CD247 can span the sequence corresponding to chromosome 1:167,430,640 to 167,518,610 on the opposite strand, relative to human genome version GRCh38 (UCSC Genome Browser on Human Dec. 2013 (GRCh38 / hg38) Assembly). Table 1 shows the coordinates of the exons and introns and untranslated regions of the open reading frame of an exemplary human CD247 locus transcript.

[0154] Table 1. Exon and intron coordinates of an exemplary human CD247 locus (GRCh38, chromosome 1, opposite strand). TIFF0007754722000007.tif85165

[0155] In some aspects, the transgene (e.g., exogenous nucleic acid sequence) in the template polynucleotide can be used to guide the location of the target site and / or homology arm. In some aspects, the target site of gene disruption can be used as a guide to design the template polynucleotide and / or homology arm used for HDR. In some embodiments, the gene disruption can be targeted near the desired site of target-specific integration of the transgene sequence (e.g., encoding a chimeric receptor or a portion thereof). In some aspects, the gene disruption is targeted based on the amount of the CD3 zeta chain encoding sequence contained in the transgene sequence for integration. In some aspects, the target site is within an exon of the open reading frame of the endogenous CD247 locus. In some aspects, the target site is within an intron of the open reading frame of the CD247 locus.

[0156] In some embodiments, the target site for gene disruption is selected to contain a functional CD3 zeta chain or a fragment thereof, such that after integration of the transgene sequence, the chimeric receptor encoded by the modified CD247 locus can signal via the CD3 zeta chain or a fragment thereof. In some embodiments, one or more homology arm sequences of the template polynucleotide are designed to surround the site of gene disruption. In some aspects, the target site is located within or near an exon of the endogenous CD247 locus, such that the transgene encoding a portion of the chimeric receptor can be integrated in frame with the coding sequence of the CD247 locus.

[0157] In some embodiments, the target site is selected so that target-specific integration of the transgene generates a genetic fusion between the transgene and an endogenous sequence at the CD247 locus, which together encode a functional CD3 zeta chain. The endogenous sequence can, in some aspects, encode a functional CD3 zeta chain that is a portion of the CD3 zeta chain that can mediate, activate, or stimulate a primary cytoplasmic or intracellular signal, e.g., a portion of the CD3 zeta chain that includes the cytoplasmic domain of the CD3 zeta chain, e.g., an immunoreceptor tyrosine-based activation motif (ITAM). In some aspects, the target site is located in the intracellular region of the CD3 zeta chain, e.g., at or near the beginning of the endogenous open reading frame sequence encoding amino acid residues 52-164 of the human CD3 zeta chain precursor sequence set forth in SEQ ID NO:73, or amino acid residues 52-163 of the human CD3 zeta chain precursor sequence set forth in SEQ ID NO:75; or at or near exon 2 or exon 3 (e.g., at or near nucleotides 167,440,767-167,440,664 or nucleotides 167,439,400-167,439,344 of GrCh38 as set forth in Table 1 herein). In some aspects, the target site is located before or upstream of an endogenous open reading frame sequence encoding the ITAM domain of the CD3 zeta chain, e.g., amino acid residues 61-89, 100-128, or 131-159 of the human CD3 zeta chain precursor sequence shown in SEQ ID NO:73, or amino acid residues 61-89, 100-127, or 130-158 of the human CD3 zeta chain precursor sequence shown in SEQ ID NO:75.

[0158] In some aspects, the target site is within an exon of the endogenous CD247 locus. In some aspects, the target site is within an intron of the endogenous CD247 locus. In some aspects, the target site is within a control or regulatory element of the CD247 locus, such as a promoter, a 5' untranslated region (UTR), or a 3' UTR. In some embodiments, the target site is within any exon or intron of the CD247 genomic region sequence listed in Table 1 herein, or the CD247 genomic region sequence contained therein.

[0159] In some aspects, the target site is within an exon, e.g., an exon corresponding to the early coding region. In some embodiments, the target site is within or near an exon corresponding to the early coding region of the open reading frame of the endogenous CD247 locus (as described in Table 1 herein), e.g., exon 1, 2, or 3, e.g., the sequence immediately following the transcription start site, within exon 1, 2, or 3, or within 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 bp of exon 1, 2, or 3. In some aspects, the target site is within or near exon 1 of the endogenous CD247 locus, e.g., within 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 bp of exon 1. In some embodiments, the target site is in or near exon 2 of the endogenous CD247 locus, or within 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 bp of exon 2. In some aspects, the target site is in or near exon 3 of the endogenous CD247 locus, e.g., within 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 bp of exon 3. In some aspects, the target site is within a control or regulatory element, e.g., a promoter, of the CD247 locus.

[0160] In certain embodiments, the gene disruption is targeted to, near, or within the CD247 locus. In a specific embodiment, the gene disruption is targeted to, near, or within the open reading frame of the CD247 locus (as described in Table 1 herein). In certain embodiments, the gene disruption is targeted to, near, or within the open reading frame encoding the CD3 zeta chain. In some embodiments, the gene disruption is targeted to, near, or within the CD247 locus (as described in Table 1 herein), or all or a portion of the CD247 locus (as described in Table 1 herein), for example, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, or 4,000, or at least 500, 1,000, 1,500, 2,000, 2,500, Targeted to, near, or within a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity, or at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity for 3,000, 3,500, or 4,000 contiguous nucleotides.

[0161] In some embodiments, the gene disruption, e.g., DNA breakage, is targeted within an exon of the CD247 locus or its open reading frame. In certain embodiments, the gene disruption is within the first, second, third, or fourth exon of the CD247 locus or its open reading frame. In specific embodiments, the gene disruption is within the first exon of the CD247 locus or its open reading frame. In some embodiments, the gene disruption is within 500 base pairs (bp) downstream from the 5' end of the first exon of the CD247 locus or its open reading frame. In specific embodiments, the gene disruption is between the 5' nucleotide of exon 1 and the 3' nucleotide of exon 1. In certain embodiments, the gene disruption is 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 of the CD247 locus or its open reading frame. In specific embodiments, the gene disruption is 1 bp to 400 bp, 50 to 300 bp, 100 bp to 200 bp, or 100 bp to 150 bp (inclusive) downstream from the 5' end of the first exon of the CD247 locus or its open reading frame. In certain embodiments, the gene disruption is 100 bp to 150 bp (inclusive) downstream from the 5' end of the first exon of the CD247 locus or its open reading frame.

[0162] 2. Gene disruption methods In some aspects, the method for generating genetically engineered cells comprises introducing gene disruption into one or more target sites, for example, one or more target sites in the CD247 gene locus encoding CD3 zeta (CD3ζ).The method for generating gene disruption, including those described herein, can comprise the use of one or more agents capable of inducing gene disruption, such as the use of an engineered system that induces gene disruption, cleavage and / or double-strand break (DSB) or nick (e.g., single-strand break (SSB)) at a target site or target location in endogenous or genomic DNA, so that repair of the break by an error-generating process such as non-homologous end joining (NHEJ) or repair by HDR using a repair template can result in the insertion of a sequence of interest (e.g., an exogenous nucleic acid sequence encoding a portion of a chimeric receptor or a transgene) at or near the target site or location.Also provided herein are one or more agents capable of inducing gene disruption for use in the methods provided herein. In some aspects, one or more agents can be used in combination with the template nucleotides provided herein for homology-directed repair (HDR)-mediated targeted integration of transgene sequences.

[0163] In some embodiments, one or more agents that can induce gene disruption comprise specific site or position in genome, for example, DNA binding protein or DNA binding nucleic acid that specifically binds or hybridizes to target site or target location.In some aspects, targeted gene disruption, for example, DNA cutting or cleavage, at endogenous CD247 locus is achieved by using protein or nucleic acid, and said protein or nucleic acid is combined or complexed with gene editing nuclease, for example, in chimeric or fusion protein.In some embodiments, one or more agents that can induce gene disruption comprise RNA-guided nuclease, or the fusion protein that comprises DNA targeting protein and nuclease.

[0164] In some embodiments, the agent comprises various components, for example, an RNA-guided nuclease, or a fusion protein comprising a DNA targeting protein and a nuclease. In some embodiments, targeted gene disruption is carried out using a DNA targeting molecule comprising a DNA binding protein, for example, one or more zinc finger proteins (ZFPs) or transcription activator-like effectors (TALEs), fused to a nuclease, for example, an endonuclease. In some embodiments, targeted gene disruption is carried out using an RNA-guided nuclease, for example, 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 carried out using an agent that can induce gene disruption, such as sequence-specific or targeted nuclease, including DNA-binding targeted nuclease and gene editing nuclease, such as zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN), and RNA-guided nuclease, such as CRISPR-associated nuclease (Cas) system, which is specifically designed to target at least one target site, gene sequence, or a part thereof.Exemplary ZFN, TALE, and TALEN are described, for example, in Lloyd et al., Frontiers in Immunology, 4(221): 1-7 (2013).

[0165] The binding domains of zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs), and CRISPR systems can be "engineered" to bind to predetermined nucleotide sequences, for example, by manipulating the recognition helix region of naturally occurring ZFPs or TALEs (changing one or more amino acids). Engineered DNA binding proteins (ZFPs or TALEs) are proteins that do not occur in nature. Rational design criteria include the application of substitution rules and computerized algorithms to process information in databases that store information on the design and binding data of existing ZFPs and / or TALEs. For example, see U.S. Patent Nos. 6,140,081; 6,453,242; and 6,534,261. See also WO98 / 53058; WO98 / 53059; WO98 / 53060; WO02 / 016536, and WO03 / 016496 and U.S. Patent Application Publication No. 20110301073.

[0166] In some embodiments, one or more agents specifically target at least one target site at or near the CD247 locus. In some embodiments, the agents comprise ZFNs, TALENs, or CRISPR / Cas9 combinations that specifically bind to, recognize, or hybridize to the target site. In some embodiments, the CRISPR / Cas9 system comprises an engineered crRNA / tracr RNA ("single guide RNA") to guide specific cleavage. In some embodiments, the agent comprises a nuclease based on the Argonaute system (e.g., derived from T. thermophilus and known as "TtAgo" (Swarts et al. (2014) Nature 507(7491): 258-261). Targeted cleavage using any of the nuclease systems described herein can be exploited to insert a nucleic acid sequence, such as the sequence of a transgene encoding a portion of a chimeric receptor, into a specific target location in the endogenous CD247 locus using either HDR or NHEJ-mediated processes.

[0167] In some embodiments, a "zinc finger DNA-binding protein" (or binding domain) is a protein or domain within a larger protein that binds to DNA in a sequence-specific manner via one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through 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 in length, that target specific DNA sequences and are generated by the assembly of individual fingers. ZFPs include those with two, three, four, five, or six fingers, where a single finger domain is approximately 30 amino acids in length and contains an alpha helix containing two invariant histidine residues coordinated via zinc to two cysteines in a single beta turn. Generally, the sequence specificity of ZFPs can be altered by making amino acid substitutions at four helical positions (-1, 2, 3, and 6) on the zinc finger recognition helix. Thus, for example, the ZFP or ZFP-containing molecule is not naturally occurring but has been engineered, eg, to bind to a selected target site.

[0168] In some cases, the DNA targeting molecule is or comprises a zinc finger DNA binding domain that is fused with a DNA cleavage domain to form a zinc finger nuclease (ZFN).For example, the fusion protein comprises a cleavage domain (or cleavage half-domain) derived from at least one type IIS restriction enzyme, and one or more zinc finger binding domains, which may or may not be engineered.In some cases, the cleavage domain is derived from type IIS restriction endonuclease FokI, which generally catalyzes double-stranded cleavage of DNA at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand. For example, see U.S. Patent No. 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. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem. 269:978-982.Some gene-specific engineered zinc fingers are commercially available.For example, a platform called CompoZr for zinc finger construction is available, which provides zinc fingers that are 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 custom designed.

[0169] In some embodiments, for example, one or more target sites within the CD247 locus can be targeted for gene disruption by engineered ZFNs. Exemplary ZFNs that target the endogenous CD247 locus include, for example, those described in Rudemiller et al., (2014) Hypertension. 63(3):559-64, the entire disclosure of which is incorporated by reference.

[0170] Transcription activator-like effectors (TALEs) are proteins derived from the bacterial species Xanthomonas that contain multiple repeats, each containing two residues (RVDs) at positions 12 and 13 that are specific for each nucleotide base of a nucleic acid target sequence. Binding domains with similar modular base-per-base nucleic acid binding properties (MBBBDs) can also be derived from different bacterial species. The new modular proteins have the advantage of exhibiting more sequence diversity than TAL repeats. In some embodiments, the RVDs involved in recognizing different nucleotides are: HD for recognizing C, NG for recognizing T, NI for recognizing A, NN for recognizing G or A, NS for recognizing A, C, G, or T, HG for recognizing T, IG for recognizing T, NK for recognizing G, HA for recognizing C, ND for recognizing C, HI for recognizing C, HN for recognizing G, NA for recognizing G, SN for recognizing G or A, and YG for recognizing T, TL for recognizing A, VT for recognizing A or G, and SW for recognizing A. In some embodiments, the critical amino acids 12 and 13 can be mutated to other amino acid residues to modulate their specificity for the nucleotides A, T, C, and G, and in particular to enhance this specificity.

[0171] In some embodiments, a "TALE DNA binding domain" or "TALE" is a polypeptide comprising one or more TALE repeat domains / units. The repeat domains, each containing a repeat variable dipeptide (RVD), are responsible for binding of the TALE to its cognate target DNA sequence. A single "repeat unit" (also referred to as a "repeat") is typically 33-35 amino acids in length and exhibits at least some sequence homology to other TALE repeat sequences within naturally occurring TALE proteins. TALE proteins can be engineered to bind to target sites using canonical or non-canonical RVDs within the repeat unit. See, e.g., U.S. Patent Nos. 8,586,526 and 9,458,205.

[0172] In some embodiments, a "TALE nuclease" (TALEN) is a fusion protein comprising a nucleic acid binding domain, typically derived from a transcription activator-like effector (TALE), and a nuclease catalytic domain that cleaves a nucleic acid target sequence. Catalytic domains include nuclease domains or domains with endonuclease activity, such as I-TevI, ColE7, NucA, and Fok-I. In certain embodiments, the TALE domain can be fused to a meganuclease, such as I-CreI and I-OnuI, or functional variants thereof. 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, such as fusion of the engineered TAL repeat with the catalytic domain of I-TevI, as described in WO2012138927. TALEN has 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 the CD247 locus can be targeted for gene disruption by engineered TALEN.

[0173] In some embodiments, "TtAgo" refers to a prokaryotic Argonaute protein believed to be involved in gene silencing. TtAgo is derived from the bacterium Thermus thermophilus. See, e.g., 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 components required, including guide DNA, for cleavage by the TtAgo enzyme.

[0174] In some embodiments, the engineered zinc finger proteins, TALE proteins, or CRISPR / Cas systems are not found in nature, and their production primarily results from experimental processes such as phage display, interaction traps, or hybrid selection. See, e.g., U.S. Patent Nos. 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.

[0175] Zinc finger and TALE DNA binding domains can be engineered to bind to a predetermined nucleotide sequence, for example, through manipulation of the recognition helix region of naturally occurring zinc finger proteins (changing one or more amino acids) or by manipulating amino acids involved in DNA binding (the repeat variable dipeptide or RVD region). Thus, engineered zinc finger proteins or TALE proteins are non-naturally occurring proteins. A non-limiting example of a method for engineering zinc finger proteins and TALEs is design and selection. Designed proteins are non-naturally occurring proteins whose design / composition results from rational criteria. Rational criteria for design include substitution rules and the application of computerized algorithms to process information in databases that store information on existing ZFP or TALE designs (canonical and non-canonical RVDs) and binding data. See, for example, U.S. Patent Nos. 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.

[0176] Various methods and compositions for target cleavage of genomic DNA have been described.Such target cleavage events can be used to induce targeted mutagenesis, induce targeted deletion of cellular DNA sequence, and promote targeted recombination at predetermined chromosomal locus.For example, see U.S. Patent Nos. 9,255,250; 9,200,266; 9,045,763; 9,005,973; 9,150,847; 8,956,828; 8,945,868; 8,703,489; 8,586,526, the disclosure of which is incorporated by reference in its entirety. 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 Nos. 20030232410; 20050208489; 20050026157; 20050064474; 20060063231; 20080159996; 201000218264; 20120017290; 20110265198 See Nos. 20130137104; 20130122591; 20130177983; 20130196373; 20140120622; 20150056705; 20150335708; 20160030477, and 20160024474.

[0177] a. CRISPR / Cas9 In some embodiments, targeted gene disruption, e.g., DNA cleavage, of the endogenous gene encoding CD3 zeta (CD3ζ), such as CD247, in humans is performed using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. See Sander and Joung (2014) Nature Biotechnology, 32(4): 347-355.

[0178] Generally, a "CRISPR system" refers collectively to transcripts and other elements involved in directing the expression or activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracr RNA or active partial tracr RNA), tracr mate sequences (which encompass "direct repeats" and processed partial direct repeats of tracr RNA in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), and / or other sequences and transcripts from the CRISPR locus.

[0179] In some aspects, a CRISPR / Cas nuclease or CRISPR / Cas nuclease system comprises a non-coding guide RNA (gRNA) that binds to DNA in a sequence-specific manner and a Cas protein (e.g., Cas9) that has nuclease functionality.

[0180] Also provided are one or more agents capable of introducing a gene disruption. Also provided are polynucleotides (e.g., nucleic acid molecules) encoding one or more components of the one or more agents capable of inducing a gene disruption.

[0181] (i) Guide RNA (gRNA) In some embodiments, the one or more agents capable of inducing gene disruption comprise at least one of a guide RNA (gRNA) or at least one nucleic acid encoding a gRNA having a targeting domain complementary to a target site in the CD247 locus.

[0182] In some aspects, a "gRNA molecule" is a nucleic acid that facilitates specific targeting or homing of a gRNA molecule / Cas9 molecule complex to a target nucleic acid, such as a locus on the genomic DNA of a cell. A gRNA molecule can be unimolecular (having a single RNA molecule) or modular (comprising 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 that has sufficient complementarity with a target polynucleotide sequence, e.g., the CD247 locus in humans, to hybridize with the target sequence at the target site and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, in the context of CRISPR complex formation, a "target sequence" is a sequence to which the guide sequence is designed to have complementarity, such that hybridization between the target sequence and a domain, e.g., the targeting domain, of the guide RNA facilitates CRISPR complex formation. Complete complementarity is not necessarily required, provided that there is sufficient complementarity to cause hybridization and promote the formation of CRISPR complex.Generally, guide sequence is selected so as to reduce the degree of secondary structure in guide sequence.Secondary structure can be determined by any suitable polynucleotide folding algorithm.

[0183] In some embodiments, a guide RNA (gRNA) specific to a target locus of interest (e.g., the CD247 locus in humans) is used with an RNA-guided nuclease, such as Cas, to induce DNA cleavage at a target site or target position. Methods for designing gRNAs and exemplary targeting domains include those described in, for example, International PCT Publication Nos. WO2015 / 161276, WO2017 / 193107, and WO2017 / 093969.

[0184] Several exemplary gRNA structures with domains depicted thereon are described in WO2015 / 161276, e.g., in Figures 1A-1G therein. Without wishing to be bound by theory, regions of high complementarity with respect to the three-dimensional shape of the active form of the gRNA, or intra- or inter-strand interactions, are sometimes depicted as duplexes in WO2015 / 161276, e.g., in Figures 1A-1G therein, and in other depictions provided herein.

[0185] In some cases, the gRNA is a unimolecular or chimeric gRNA that includes, from 5' to 3', a targeting domain that is complementary to a target nucleic acid, such as a sequence from the CD247 gene (encoding the sequence set forth in SEQ ID NO:74); a first complementarity domain; a linking domain; a second complementarity domain (complementary to the first complementarity domain); a proximal domain; and, optionally, a tail domain.

[0186] In other cases, the gRNA is a modular gRNA comprising a first and second strand. In these cases, the first strand preferably comprises, from 5' to 3', a targeting domain (complementary to the target nucleic acid, such as the sequence from the CD247 gene encoding the sequence shown in SEQ ID NO: 74 or 76) and a first complementary domain. The second strand generally comprises, from 5' to 3', optionally a 5' extension domain; a second complementary domain; a proximal domain; and optionally a tail domain.

[0187] (a) Targeting Domain The targeting domain comprises 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, fully complementary. The strand of the target nucleic acid that comprises the target sequence is referred to herein as the "complementary strand" of the target nucleic acid. Guidance on the selection of the targeting domain can be found, for example, in Fu Y et al., Nat Biotechnol 2014 (doi: 10.1038 / nbt.2808) and Sternberg SH et al., Nature 2014 (doi: 10.1038 / nature13011). Examples of the arrangement of the targeting domain include those described in WO2015 / 161276, for example, in Figures 1A-1G therein.

[0188] The targeting domain is a portion of an RNA molecule and therefore contains the base uracil (U), whereas any DNA encoding a gRNA molecule contains the base thymine (T). Without wishing to be bound by theory, in some embodiments, the complementarity between the targeting domain and the target sequence is believed to contribute to the specificity of the interaction between the gRNA molecule / Cas9 molecule complex and the target nucleic acid. It is understood that in a pairing of a targeting domain and a target sequence, the uracil base in the targeting domain pairs with the adenine base in the target sequence. In some embodiments, the targeting domain itself comprises, in the 5' to 3' direction, any secondary domains and a core domain. In some embodiments, the core domain is fully complementary to the target sequence. In some embodiments, the targeting domain is 5 to 50 nucleotides in length. The strand of the target nucleic acid to which the targeting domain is complementary is referred to herein as the complementary strand. Some or all of the nucleotides of the domain may have modifications, e.g., to make them less susceptible to degradation or to improve biocompatibility. As a non-limiting example, the backbone of the targeting domain can be modified with phosphorothioates or other modifications. In some cases, the nucleotides of the targeting domain can include 2' modifications, e.g., 2-acetylations, e.g., 2' methylations, or other modifications.

[0189] In various embodiments, the targeting domain is 16 to 26 nucleotides in length (i.e., it is 16 nucleotides in length, or 17 nucleotides in length, or 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length).

[0190] (b) Exemplary Targeting Domains In some embodiments, the gRNA sequence is designed or specified, and the gRNA sequence is or comprises the targeting domain sequence that targets the target site in specific gene, such as CD247 locus.Genome-wide gRNA database for CRISPR genome editing is publicly available, and it contains the exemplary single guide RNA (sgRNA) sequence that targets the constitutional exon of gene in human genome 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, the gRNA sequence is or comprises the sequence that has minimal off-target binding to non-target site or position.

[0191] In some embodiments, the target sequence (target domain) is located at or near the CD247 locus, such as any portion of the CD247 coding sequence set forth in SEQ ID NO: 74 or 76. In some embodiments, the target nucleic acid complementary to the targeting domain is located in the early coding region of a gene of interest, such as CD247. Targeting the early coding region can be used for gene disruption (i.e., eliminating the expression) of a gene of interest. In some embodiments, the early coding region of a gene of interest includes a sequence immediately following the start codon (e.g., ATG) or within 500 bp of the start codon (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100, 50 bp, 40 bp, 30 bp, 20 bp, or 10 bp). In certain examples, the target nucleic acid is within 200bp, 150bp, 100bp, 50bp, 40bp, 30bp, 20bp, or 10bp 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 the CD247 locus, e.g., at least 80, 85, 90, 95, 98, or 99% complementary, e.g., fully complementary.

[0192] In some embodiments, the gRNA can target a site in the CD247 locus that is close to the desired site of target-specific integration of, for example, a transgene sequence encoding a chimeric receptor. In some aspects, the gRNA can target a site based on the amount of CD3 zeta chain encoding sequence contained in the transgene sequence for integration. In some aspects, the gRNA can target a site within an exon of the open reading frame of the endogenous CD247 locus. In some aspects, the gRNA can target a site within an intron of the open reading frame of the CD247 locus. In some aspects, the gRNA can target a site within a control or regulatory element of the CD247 locus, such as a promoter. In some aspects, the target site in the CD247 locus that can be targeted by the gRNA can be any of the target sites described herein, for example, in Section IA1. In some embodiments, the gRNA can target a site within or near an exon that corresponds to the early coding region of the open reading frame of the endogenous CD247 locus, e.g., exon 1, 2, or 3, e.g., a sequence immediately after the transcription start site, within exon 1, 2, or 3, or within 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 bp of exon 1, 2, or 3. In some embodiments, the gRNA can target a site at or near exon 2 of the endogenous CD247 locus, or within 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 bp of exon 2.

[0193] Exemplary target site sequences for disruption of the human CD247 locus using Cas9 can include any of those set forth in SEQ ID NOs:59-62 and 67-72. In some aspects, exemplary target site sequences containing an NGG PAM can include any of those set forth in SEQ ID NOs:63-66. Exemplary gRNAs can include a ribonucleic acid sequence capable of binding to or targeting a target site sequence set forth in any of SEQ ID NOs:59-62 and 67-72. Exemplary gRNA targeting domain sequences include: TIFF0007754722000008.tif37164 is included. Exemplary gRNA sequences for generating gene disruptions at the endogenous CD247 locus (encoding CD3 zeta) are described, for example, in International PCT Publication No. WO2017093969. Exemplary methods for gene editing at the endogenous CD247 locus (encoding CD3 zeta) include, for example, those described in WO2017093969. Any of the known methods that can be used to target the endogenous CD247 locus and generate gene disruptions thereat can be used in the embodiments provided herein.

[0194] In some embodiments, the targeting domain includes one for introducing a gene disruption into the CD247 gene using S. pyogenes Cas9 or N. meningitidis Cas9.

[0195] In some embodiments, targeting domains include those for introducing a gene disruption in the CD247 gene using S. pyogenes Cas9. Any of the targeting domains can be used with S. pyogenes Cas9 molecules that generate double-stranded breaks (Cas9 nuclease) or single-stranded breaks (Cas9 nickase).

[0196] In some embodiments, dual targeting is used to create two nicks in opposite DNA strands by using a Streptococcus pyogenes Cas9 nickase with two targeting domains complementary to opposite DNA strands; for example, any gRNA containing a minus-strand targeting domain can pair with any gRNA containing a plus-strand targeting domain. In some embodiments, the two gRNAs are oriented on the DNA so that the PAMs face outward and the distance between the 5' ends of the gRNAs is 0-50 bp. In some embodiments, two gRNAs are used to target two Cas9 nucleases or two Cas9 nickases, for example, 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 opposite strands of the target domain. In some embodiments, the two Cas9 nickases can comprise a molecule with HNH activity, e.g., a Cas9 molecule with abolished RuvC activity, e.g., a Cas9 molecule with a mutation at D10, e.g., a D10A mutation, a molecule with RuvC activity, e.g., a Cas9 molecule with abolished HNH activity, e.g., a Cas9 molecule with a mutation at H840, e.g., H840A, or a molecule with RuvC activity, e.g., a Cas9 molecule with abolished HNH activity, e.g., a Cas9 molecule with a mutation at N863, e.g., N863A. In some embodiments, each of the two gRNAs is complexed with a D10A Cas9 nickase.

[0197] (c) the first complementarity domain The first complementarity domain is complementary to the second complementarity domain described herein and generally has sufficient complementarity to form a double-stranded region under at least some physiological conditions. The first complementarity domain is typically 5-30 nucleotides in length, and may be 5-25 nucleotides in length, 7-25 nucleotides in length, 7-22 nucleotides in length, 7-18 nucleotides in length, or 7-15 nucleotides in length. In various embodiments, the first complementarity domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. Examples of first complementarity domains include those described in WO2015 / 161276, e.g., Figures 1A-1G therein.

[0198] Typically, the first complementary domain does not have exact complementarity with the second complementary domain target. In some embodiments, the first complementary domain may have 1, 2, 3, 4, or 5 nucleotides that are not complementary to the corresponding nucleotides of the second complementary domain. In some embodiments, a segment of 1, 2, 3, 4, 5, or 6 (e.g., 3) nucleotides of the first complementary domain may not be paired in the duplex and may form a non-duplex or looped-out region. In some cases, a non-paired or looped-out region, such as a 3-nucleotide loop-out, exists in the second complementary domain. This non-paired region optionally begins 1, 2, 3, 4, 5, or 6, for example, 4 nucleotides, from the 5' end of the second complementary domain.

[0199] The first complementarity domain can include three subdomains, which, from 5' to 3', are a 5' subdomain, a central subdomain, and a 3' subdomain. In some embodiments, the 5' subdomain is 4 to 9, e.g., 4, 5, 6, 7, 8, or 9 nucleotides in length. In some embodiments, the central subdomain is 1, 2, or 3, e.g., 1 nucleotide in length. In some embodiments, the 3' subdomain is 3 to 25, e.g., 4 to 22, 4 to 18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.

[0200] In some embodiments, the first and second complementary domains, when double stranded, can be represented by, for example, the gRNA sequence (one paired strand is underlined and one is in bold): Contains 11 paired nucleotides in TIFF0007754722000009.tif11163.

[0201] In some embodiments, the first and second complementary domains, when double stranded, can be represented by, for example, the gRNA sequence (one paired strand is underlined and one is in bold): Contains 15 paired nucleotides in TIFF0007754722000010.tif11163.

[0202] In some embodiments, the first and second complementary domains, when double stranded, can be represented by, for example, the gRNA sequence (one paired strand is underlined and one is in bold): Contains 16 paired nucleotides in TIFF0007754722000011.tif11163.

[0203] In some embodiments, the first and second complementary domains, when double stranded, can be represented by, for example, the gRNA sequence (one paired strand is underlined and one is in bold): Contains 21 paired nucleotides in TIFF0007754722000012.tif17163.

[0204] In some embodiments, for example, the gRNA sequence (the exchanged nucleotides are underlined): Nucleotides are exchanged to remove the poly-U tract in TIFF0007754722000013.tif44165.

[0205] The first complementarity domain may be homologous to or derived from a naturally occurring first complementarity domain, hi some embodiments, it has at least 50% homology to a first complementarity domain disclosed herein, e.g., a first complementarity domain of Streptococcus pyogenes, Staphylococcus aureus (S. aureus), Neisseria meningitidis, or S. thermophilus.

[0206] It should be noted that one or more, or even all, of the nucleotides of the first complementarity domain may have modifications along the lines described herein for the targeting domain.

[0207] (d) Linked Domain In a unimolecular or chimeric gRNA, the linking domain serves to link a first complementary domain to a second complementary domain of the unimolecular gRNA. The linking domain can link the first and second complementary domains covalently or non-covalently. In some embodiments, the linkage is a covalent bond. In some embodiments, the linking domain covalently joins the first and second complementary domains. See, e.g., WO2015 / 161276, e.g., Figures 1B-1E therein. In some embodiments, the linking domain is or includes a covalent bond inserted between the first complementary domain and the second complementary domain. Typically, the linking domain comprises one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, although in various embodiments, the linker may be 20, 30, 40, 50, or even 100 nucleotides in length. Examples of linking domains include those described in WO2015 / 161276, for example, in Figures 1A-1G therein.

[0208] In modular gRNA molecules, the two molecules are joined by hybridization of complementary domains and a linking domain may be absent. See, e.g., WO2015 / 161276, e.g., Figure 1A therein.

[0209] A wide variety of linking domains are suitable for use in a unimolecular gRNA molecule. Linking domains can consist of a covalent bond or can be as short as one or a few nucleotides, e.g., 1, 2, 3, 4, or 5 nucleotides in length. In some embodiments, linking domains are 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in length. In some embodiments, linking domains are 2-50, 2-40, 2-30, 2-20, 2-10, or 2-5 nucleotides in length. In some embodiments, linking domains are homologous to or derived from naturally occurring sequences, e.g., sequences of tracrRNAs that are 5' to the second complementarity domain. In some embodiments, linking domains have at least 50% homology to the linking domains disclosed herein.

[0210] As described herein for the first complementarity domain, some or all of the nucleotides of the linking domain may comprise modifications.

[0211] (e) 5' extension domain In some cases, the modular gRNA can include an additional sequence 5' to the second complementarity domain, referred to herein as a 5' extension domain. In some embodiments, the 5' extension domain is 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, or 2-4 nucleotides in length. In some embodiments, the 5' extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length. In some embodiments, examples of 5' extension domains include those described in WO2015 / 161276, e.g., in Figure 1A therein.

[0212] (f) a second complementarity domain The second complementarity domain is complementary to the first complementarity domain and generally has sufficient complementarity to form a double-stranded region under at least some physiological conditions. In some cases, the second complementarity domain can include a sequence that lacks complementarity with the first complementarity domain, e.g., a sequence that loops out from the double-stranded region, as shown, for example, in WO2015 / 161276, e.g., in Figures 1A-1B therein. Examples of second complementarity domains include those described in WO2015 / 161276, e.g., in Figures 1A-1G therein.

[0213] The second complementarity domain can be 5 to 27 nucleotides in length, and in some cases can be longer than the first complementarity region. In some embodiments, the second complementarity domain can be 7 to 27 nucleotides in length, 7 to 25 nucleotides in length, 7 to 20 nucleotides in length, or 7 to 17 nucleotides in length. More commonly, the complementarity domain can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length.

[0214] In some embodiments, the second complementarity domain comprises three subdomains, which, in the 5' to 3' direction, are a 5' subdomain, a central subdomain, and a 3' subdomain. In some embodiments, the 5' subdomain is 3 to 25, e.g., 4 to 22, 4 to 18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the central subdomain is 1, 2, 3, 4, or 5, e.g., 3 nucleotides in length. In some embodiments, the 3' subdomain is 4 to 9, e.g., 4, 5, 6, 7, 8, or 9 nucleotides in length.

[0215] In some embodiments, the 5' and 3' subdomains of a first complementarity domain are complementary, eg, fully complementary, to the 3' and 5' subdomains of a second complementarity domain, respectively.

[0216] The second complementarity domain may be homologous to or derived from a naturally occurring second complementarity domain, hi some embodiments, it has at least 50% homology to a second complementarity domain disclosed herein, e.g., a first complementarity domain of Streptococcus pyogenes, Staphylococcus aureus, Neisseria meningitidis, or S. thermophilus.

[0217] Some or all of the nucleotides of the second complementarity domain may have a modification, such as a modification described herein.

[0218] (g) Proximal domain Examples of proximal domains include those described in WO2015 / 161276, e.g., Figures 1A-1G therein. In some embodiments, the proximal domain is 5-20 nucleotides in length. In some embodiments, the proximal domain may be homologous to or derived from a naturally occurring proximal domain. In some embodiments, it has at least 50% homology to a proximal domain disclosed herein, e.g., a proximal domain of Streptococcus pyogenes, Staphylococcus aureus, Neisseria meningitidis, or S. thermophilus.

[0219] Some or all of the nucleotides of the proximal domain may have modifications along the lines described herein.

[0220] (h) Tail domain As can be seen from an examination of the tail domains in WO2015 / 161276, e.g., in Figures 1A and 1B-1F therein, a wide variety of tail domains are suitable for use in gRNA molecules. In various embodiments, the tail domain is 0 (none), 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In certain embodiments, the nucleotides of the tail domain are derived from or homologous to the 5'-terminal sequence of a naturally occurring tail domain. See, e.g., WO2015 / 161276, e.g., Figures 1D or 1E therein. The tail domain also optionally includes sequences that are complementary to each other and form a double-stranded region under at least some physiological conditions. Exemplary tail domains include those described in WO2015 / 161276, e.g., in Figures 1A-1G therein.

[0221] The tail domain may have homology to or may be derived from a naturally occurring proximal tail domain. As a non-limiting example, a given tail domain according to various aspects of the present disclosure may have at least 50% homology to a naturally occurring tail domain disclosed herein, e.g., a tail domain of Streptococcus pyogenes, Staphylococcus aureus, Neisseria meningitidis, or S. thermophilus.

[0222] In certain cases, the tail domain contains nucleotides at the 3' end that are relevant for in vitro or in vivo transcription methods. When a T7 promoter is used for in vitro transcription of gRNA, these nucleotides can be any nucleotides that precede the 3' end of the DNA template. When a U6 promoter is used for in vivo transcription, these nucleotides can be the sequence UUUUUU. When an alternative Pol-III promoter is used, these nucleotides can be a different number, or can be uracil bases, or can contain alternative bases.

[0223] As a non-limiting example, in various embodiments, the proximal and tail domains, taken together, comprise the following sequences: TIFF0007754722000014.tif31160.

[0224] In some embodiments, the tail domain comprises the 3' sequence UUUUUU, e.g., when a U6 promoter is used for transcription. In some embodiments, the tail domain comprises the 3' sequence UUUU, e.g., when an H1 promoter is used for transcription. In some embodiments, the tail domain comprises a variable number of 3' U's, depending, e.g., on the termination signal of the Pol-III promoter used. In some embodiments, the tail domain comprises a variable 3' sequence derived from the DNA template, e.g., when a T7 promoter is used. In some embodiments, the tail domain comprises a variable 3' sequence derived from the DNA template, e.g., when in vitro transcription is used to generate the RNA molecule. In some embodiments, the tail domain comprises a variable 3' sequence derived from the DNA template, e.g., when a Pol-II promoter is used to drive transcription.

[0225] In some embodiments, the gRNA has the following structure: 5' [targeting domain] - [first complementarity domain] - [linking domain] - [second complementarity domain] - [proximal domain] - [tail domain] - 3', where the targeting domain comprises a core domain and optionally a secondary domain and is 10-50 nucleotides in length; the first complementarity domain is 5-25 nucleotides in length and in some embodiments has at least 50, 60, 70, 80, 85, 90, 95, 98, or 99% homology with a reference first complementarity domain disclosed herein. the linking domain is 1-5 nucleotides in length; the proximal domain is 5-20 nucleotides in length and, in some embodiments, has at least 50, 60, 70, 80, 85, 90, 95, 98, or 99% homology to a reference proximal domain disclosed herein; the tail domain is absent or has a nucleotide sequence 1-50 nucleotides in length and, in some embodiments, has at least 50, 60, 70, 80, 85, 90, 95, 98, or 99% homology to a reference tail domain disclosed herein.

[0226] (i) Exemplary chimeric gRNAs In some embodiments, the unimolecular or chimeric gRNA preferably comprises, from 5' to 3', e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides: a targeting domain (which is complementary to the target nucleic acid); a first complementarity domain; a linking domain; a second complementarity domain (which is complementary to the first complementarity domain); a proximal domain; and a tail domain, wherein (a) the proximal and tail domains, taken together, are at least 15, 18, 20, 25, 30, 31, 35, or 40 nucleotides long. , 40, 45, 49, 50, or 53 nucleotides; (b) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain; or (c) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to the corresponding nucleotide of the first complementarity domain.

[0227] In some embodiments, the sequence from (a), (b), or (c) has at least 60, 75, 80, 85, 90, 95, or 99% homology to the corresponding sequence of a naturally occurring gRNA or to a gRNA described herein. In some embodiments, the proximal and tail domains collectively comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides. In some embodiments, at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides are present 3' to the last nucleotide of the second complementarity domain. In some embodiments, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to the corresponding nucleotide of the first complementarity domain. In some embodiments, the targeting domain comprises, has, or consists of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length.

[0228] In some embodiments, a unimolecular or chimeric gRNA molecule (comprising a targeting domain, a first complementarity domain, a linking domain, a second complementarity domain, a proximal domain, and optionally a tail domain) has the following sequence: TIFF0007754722000015.tif11162. In some embodiments, the monomolecular or chimeric gRNA molecule is a Streptococcus pyogenes gRNA molecule.

[0229] In some embodiments, a unimolecular or chimeric gRNA molecule (comprising a targeting domain, a first complementarity domain, a linking domain, a second complementarity domain, a proximal domain, and optionally a tail domain) has the following sequence: TIFF0007754722000016.tif11164. In some embodiments, the monomolecular or chimeric gRNA molecule is a Staphylococcus aureus gRNA molecule. The sequences and structures of exemplary chimeric gRNAs are also shown in WO2015 / 161276, e.g., Figures 10A-10B therein.

[0230] Any gRNA molecule described herein can be used with any Cas9 molecule to generate double-strand or single-strand breaks and change the sequence of target nucleic acid, for example, target position or target gene signature.In some examples, the target nucleic acid is located at or near the CD247 locus, such as any described.In some embodiments, a ribonucleic acid molecule, such as a gRNA molecule, and a protein, such as Cas9 protein or its variant, are introduced into any of the engineered cells provided herein.The gRNA molecules useful in these methods are described below.

[0231] In some embodiments, the gRNA, e.g., a chimeric gRNA, is configured to include one or more of the following properties: a) For example, when targeting a Cas9 molecule that creates a double-stranded break, it can position the double-stranded break (i) within 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nucleotides of the target location, or (ii) sufficiently close such that the target location is within the region of terminal resection; b) it has a targeting domain of at least 16 nucleotides, e.g., a targeting domain of (i) 16, (ii) 17, (iii) 18, (iv) 19, (v) 20, (vi) 21, (vii) 22, (viii) 23, (ix) 24, (x) 25, or (xi) 26 nucleotides; and c) (i) the proximal and tail domains, taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from the tail and proximal domains of naturally occurring Streptococcus pyogenes, S. thermophilus, Staphylococcus aureus, or Neisseria meningitidis, or a sequence that differs therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides; (ii) 3' to the last nucleotide of the second complementarity domain, there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis gRNA; (iii) 3' to the last nucleotide of the second complementarity domain that is complementary to the corresponding nucleotide of the first complementarity domain, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides, e.g., at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides from, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis gRNA; (iv) the tail domain is at least 10, 15, 20, 25, 30, 35, or 40 nucleotides in length, e.g., comprises at least 10, 15, 20, 25, 30, 35, or 40 nucleotides from, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, the tail domain of naturally occurring Streptococcus pyogenes, S. thermophilus, Staphylococcus aureus, or Neisseria meningitidis; or (v) the tail domain comprises 15, 20, 25, 30, 35, 40 nucleotides, or all of a corresponding portion of a naturally occurring tail domain, e.g., a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain.

[0232] In some embodiments, the gRNA is configured to include features a and b(i). In some embodiments, the gRNA is configured to include features a and b(ii). In some embodiments, the gRNA is configured to include features a and b(iii). In some embodiments, the gRNA is configured to include features a and b(iv). In some embodiments, the gRNA is configured to include features a and b(v). In some embodiments, the gRNA is configured to include features a and b(vi). In some embodiments, the gRNA is configured to include features a and b(vii). In some embodiments, the gRNA is configured to include features a and b(viii). In some embodiments, the gRNA is configured to include features a and b(ix). In some embodiments, the gRNA is configured to include features a and b(x). In some embodiments, the gRNA is configured to include features a and b(xi). In some embodiments, the gRNA is configured to include features a and c. In some embodiments, the gRNA is configured to include features a, b, and c. In some embodiments, the gRNA is configured to include characteristics a(i), b(i), and c(i). In some embodiments, the gRNA is configured to include characteristics a(i), b(i), and c(ii). In some embodiments, the gRNA is configured to include characteristics a(i), b(ii), and c(i). In some embodiments, the gRNA is configured to include characteristics a(i), b(ii), and c(ii). In some embodiments, the gRNA is configured to include characteristics a(i), b(iii), and c(i). In some embodiments, the gRNA is configured to include characteristics a(i), b(iii), and c(ii). In some embodiments, the gRNA is configured to include characteristics a(i), b(iv), and c(i). In some embodiments, the gRNA is configured to include characteristics a(i), b(iv), and c(ii).In some embodiments, the gRNA is configured to include features a(i), b(v), and c(i). In some embodiments, the gRNA is configured to include features a(i), b(v), and c(ii). In some embodiments, the gRNA is configured to include features a(i), b(vi), and c(i). In some embodiments, the gRNA is configured to include features a(i), b(vi), and c(ii). In some embodiments, the gRNA is configured to include features a(i), b(vii), and c(i). In some embodiments, the gRNA is configured to include features a(i), b(vii), and c(ii). In some embodiments, the gRNA is configured to include features a(i), b(viii), and c(i). In some embodiments, the gRNA is configured to include features a(i), b(viii), and c(ii). In some embodiments, the gRNA is configured to include features a(i), b(ix), and c(i). In some embodiments, the gRNA is configured to include features a(i), b(ix), and c(ii). In some embodiments, the gRNA is configured to include features a(i), b(x), and c(i). In some embodiments, the gRNA is configured to include features a(i), b(x), and c(ii). In some embodiments, the gRNA is configured to include features a(i), b(xi), and c(i). In some embodiments, the gRNA is configured to include features a(i), b(xi), and c(ii).

[0233] In some embodiments, the gRNA, e.g., a chimeric gRNA, is configured to include one or more of the following properties: a) For example, when targeting a Cas9 molecule that creates a single-strand break, one or both of the gRNAs can position the single-strand break (i) within 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nucleotides of the target location, or (ii) sufficiently close that the target location is within the region of the end resection; b) one or both have a targeting domain of at least 16 nucleotides, e.g., a targeting domain of (i) 16, (ii) 17, (iii) 18, (iv) 19, (v) 20, (vi) 21, (vii) 22, (viii) 23, (ix) 24, (x) 25, or (xi) 26 nucleotides; and c) (i) the proximal and tail domains, taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from the tail and proximal domains of naturally occurring Streptococcus pyogenes, S. thermophilus, Staphylococcus aureus, or Neisseria meningitidis, or a sequence that differs therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides; (ii) 3' to the last nucleotide of the second complementarity domain, there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis gRNA; (iii) 3' to the last nucleotide of the second complementarity domain that is complementary to the corresponding nucleotide of the first complementarity domain, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides, e.g., at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides from, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis gRNA; (iv) the tail domain is at least 10, 15, 20, 25, 30, 35, or 40 nucleotides in length, e.g., comprises at least 10, 15, 20, 25, 30, 35, or 40 nucleotides from, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, the tail domain of naturally occurring Streptococcus pyogenes, S. thermophilus, Staphylococcus aureus, or Neisseria meningitidis; or (v) the tail domain comprises 15, 20, 25, 30, 35, 40 nucleotides, or all of a corresponding portion of a naturally occurring tail domain, e.g., a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain.

[0234] In some embodiments, the gRNA is configured to include features a and b(i). In some embodiments, the gRNA is configured to include features a and b(ii). In some embodiments, the gRNA is configured to include features a and b(iii). In some embodiments, the gRNA is configured to include features a and b(iv). In some embodiments, the gRNA is configured to include features a and b(v). In some embodiments, the gRNA is configured to include features a and b(vi). In some embodiments, the gRNA is configured to include features a and b(vii). In some embodiments, the gRNA is configured to include features a and b(viii). In some embodiments, the gRNA is configured to include features a and b(ix). In some embodiments, the gRNA is configured to include features a and b(x). In some embodiments, the gRNA is configured to include features a and b(xi). In some embodiments, the gRNA is configured to include features a and c. In some embodiments, the gRNA is configured to include features a, b, and c. In some embodiments, the gRNA is configured to include characteristics a(i), b(i), and c(i). In some embodiments, the gRNA is configured to include characteristics a(i), b(i), and c(ii). In some embodiments, the gRNA is configured to include characteristics a(i), b(ii), and c(i). In some embodiments, the gRNA is configured to include characteristics a(i), b(ii), and c(ii). In some embodiments, the gRNA is configured to include characteristics a(i), b(iii), and c(i). In some embodiments, the gRNA is configured to include characteristics a(i), b(iii), and c(ii). In some embodiments, the gRNA is configured to include characteristics a(i), b(iv), and c(i). In some embodiments, the gRNA is configured to include characteristics a(i), b(iv), and c(ii).In some embodiments, the gRNA is configured to include features a(i), b(v), and c(i). In some embodiments, the gRNA is configured to include features a(i), b(v), and c(ii). In some embodiments, the gRNA is configured to include features a(i), b(vi), and c(i). In some embodiments, the gRNA is configured to include features a(i), b(vi), and c(ii). In some embodiments, the gRNA is configured to include features a(i), b(vii), and c(i). In some embodiments, the gRNA is configured to include features a(i), b(vii), and c(ii). In some embodiments, the gRNA is configured to include features a(i), b(viii), and c(i). In some embodiments, the gRNA is configured to include features a(i), b(viii), and c(ii). In some embodiments, the gRNA is configured to include features a(i), b(ix), and c(i). In some embodiments, the gRNA is configured to include features a(i), b(ix), and c(ii). In some embodiments, the gRNA is configured to include features a(i), b(x), and c(i). In some embodiments, the gRNA is configured to include features a(i), b(x), and c(ii). In some embodiments, the gRNA is configured to include features a(i), b(xi), and c(i). In some embodiments, the gRNA is configured to include features a(i), b(xi), and c(ii).

[0235] In some embodiments, the gRNA is used with a Cas9 nickase molecule with HNH activity, e.g., a Cas9 molecule with inactivated RuvC activity, e.g., a Cas9 molecule with a mutation at D10, e.g., a D10A mutation.

[0236] In some embodiments, the gRNA is used with a Cas9 nickase molecule that has RuvC activity, e.g., a Cas9 molecule that has inactivated HNH activity, e.g., a Cas9 molecule that has a mutation at H840, e.g., H840A.

[0237] In some embodiments, a gRNA pair, e.g., a chimeric gRNA pair, comprising a first and second gRNA is configured to include one or more of the following properties: a) For example, when targeting a Cas9 molecule that creates a single-strand break, one or more of the gRNAs can position the single-strand break (i) within 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nucleotides of the target location, or (ii) sufficiently close that the target location is within the region of the end resection; b) one or both have a targeting domain of at least 16 nucleotides, e.g., a targeting domain of (i) 16, (ii) 17, (iii) 18, (iv) 19, (v) 20, (vi) 21, (vii) 22, (viii) 23, (ix) 24, (x) 25, or (xi) 26 nucleotides; c) For one or both of the following: (i) the proximal and tail domains, taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from the tail and proximal domains of naturally occurring Streptococcus pyogenes, S. thermophilus, Staphylococcus aureus, or Neisseria meningitidis, or a sequence that differs therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides; (ii) 3' to the last nucleotide of the second complementarity domain, there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis gRNA; (iii) 3' to the last nucleotide of the second complementarity domain that is complementary to the corresponding nucleotide of the first complementarity domain, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides, e.g., at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides from, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis gRNA; (iv) the tail domain is at least 10, 15, 20, 25, 30, 35, or 40 nucleotides in length, e.g., comprises at least 10, 15, 20, 25, 30, 35, or 40 nucleotides from, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, the tail domain of naturally occurring Streptococcus pyogenes, S. thermophilus, Staphylococcus aureus, or Neisseria meningitidis; or (v) the tail domain comprises 15, 20, 25, 30, 35, 40 nucleotides, or all of a corresponding portion of a naturally occurring tail domain, e.g., a naturally occurring tail domain of S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis; d) the gRNAs are configured such that, when hybridized to the target nucleic acid, they are separated by 0-50, 0-100, 0-200, at least 10, at least 20, at least 30, or at least 50 nucleotides; e) the cleavage made by the first gRNA and the second gRNA is on a different strand; and f) PAM facing outwards.

[0238] In some embodiments, one or both of the gRNAs are configured to include characteristics a and b(i). In some embodiments, one or both of the gRNAs are configured to include characteristics a and b(ii). In some embodiments, one or both of the gRNAs are configured to include characteristics a and b(iii). In some embodiments, one or both of the gRNAs are configured to include characteristics a and b(iv). In some embodiments, one or both of the gRNAs are configured to include characteristics a and b(v). In some embodiments, one or both of the gRNAs are configured to include characteristics a and b(vi). In some embodiments, one or both of the gRNAs are configured to include characteristics a and b(vii). In some embodiments, one or both of the gRNAs are configured to include characteristics a and b(viii). In some embodiments, one or both of the gRNAs are configured to include characteristics a and b(ix). In some embodiments, one or both of the gRNAs are configured to include characteristics a and b(x). In some embodiments, one or both of the gRNAs are configured to include characteristics a and b(xi). In some embodiments, one or both of the gRNAs are configured to include properties a and c. In some embodiments, one or both of the gRNAs are configured to include properties a, b, and c. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(i), and c(i). In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(i), and c(ii). In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(i), c, and d. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(i), c, and e. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(i), c, d, and e. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(ii), and c(i).In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(ii), and c(ii). In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(ii), c, and d. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(ii), c, and e. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(ii), c, d, and e. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(iii), and c(i). In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(iii), and c(ii). In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(iii), c, and d. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(iii), c, and e. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(iii), c, d, and e. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(iv), and c(i). In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(iv), and c(ii). In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(iv), c, and d. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(iv), c, and e. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(iv), c, d, and e. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(v), and c(i). In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(v), and c(ii).In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(v), c, and d. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(v), c, and e. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(v), c, d, and e. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(vi), and c(i). In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(vi), and c(ii). In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(vi), c, and d. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(vi), c, and e. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(vi), c, d, and e. In some embodiments, one or both of the gRNAs are configured to include characteristics a(i), b(vii), and c(i). In some embodiments, one or both of the gRNAs are configured to include characteristics a(i), b(vii), and c(ii). In some embodiments, one or both of the gRNAs are configured to include characteristics a(i), b(vii), c, and d. In some embodiments, one or both of the gRNAs are configured to include characteristics a(i), b(vii), c, and e. In some embodiments, one or both of the gRNAs are configured to include characteristics a(i), b(vii), c, d, and e. In some embodiments, one or both of the gRNAs are configured to include characteristics a(i), b(viii), and c(i). In some embodiments, one or both of the gRNAs are configured to include characteristics a(i), b(viii), and c(ii). In some embodiments, one or both of the gRNAs are configured to include features a(i), b(viii), c, and d.In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(viii), c, and e. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(viii), c, d, and e. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(ix), and c(i). In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(ix), and c(ii). In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(ix), c, and d. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(ix), c, and e. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(ix), c, d, and e. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(x), and c(i). In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(x), and c(ii). In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(x), c, and d. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(x), c, and e. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(x), c, d, and e. In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(xi), and c(i). In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(xi), and c(ii). In some embodiments, one or both of the gRNAs are configured to include properties a(i), b(xi), c, and d. In some embodiments, one or both of the gRNAs are configured to include features a(i), b(xi), c, and e.In some embodiments, one or both of the gRNAs are configured to include features a(i), b(xi), c, d, and e.

[0239] In some embodiments, the gRNA is used with a Cas9 nickase molecule with HNH activity, e.g., a Cas9 molecule with inactivated RuvC activity, e.g., a Cas9 molecule with a mutation at D10, e.g., a D10A mutation.

[0240] In some embodiments, the gRNA is used with a Cas9 nickase molecule with RuvC activity, e.g., a Cas9 molecule with inactivated HNH activity, e.g., a Cas9 molecule with a mutation at H840, e.g., H840A. In some embodiments, the gRNA is used with a Cas9 nickase molecule with RuvC activity, e.g., a Cas9 molecule with inactivated HNH activity, e.g., a Cas9 molecule with a mutation at N863, e.g., N863A.

[0241] (j) Exemplary modular gRNAs In some embodiments, the modular gRNA comprises a first and a second strand: the first strand preferably comprises, from 5' to 3', a targeting domain comprising, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides; The second strand preferably comprises, from 5' to 3', an optional 5' extension domain; a second complementarity domain; a proximal domain; and a tail domain, wherein (a) the proximal and tail domains collectively comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides; (b) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain; or (c) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to the corresponding nucleotide of the first complementarity domain.

[0242] In some embodiments, the sequence from (a), (b), or (c) has at least 60, 75, 80, 85, 90, 95, or 99% homology to the corresponding sequence of a naturally occurring gRNA or to a gRNA described herein. In some embodiments, the proximal and tail domains collectively comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides. In some embodiments, at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides are present 3' to the last nucleotide of the second complementarity domain.

[0243] In some embodiments, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to the corresponding nucleotide of the first complementarity domain.

[0244] In some embodiments, the targeting domain has or consists of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides) that are complementary to the target domain, e.g., the targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length.

[0245] (k) Methods for designing gRNAs The method for designing gRNA is described herein, including the method for selecting, designing and verifying targeting domain.Exemplary targeting domain is also provided herein.The targeting domain discussed herein can be incorporated into the gRNA described herein.

[0246] Methods for target sequence selection and validation and 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., 2014 Nat Biotechnol, doi: 10.1038 / nbt.2808. PubMed PMID: 24463574; Heigwer et al., 2014 Nat Methods 11(2):122-3. doi: 10.1038 / nmeth.2812. PubMed PMID: 24481216; Bae et al., 2014 Bioinformatics PubMed PMID: 24463181; Xiao A et al., 2014 Bioinformatics PubMed Listed in PMID: 24389662.

[0247] In some embodiments, software tools can be used to optimize the selection of gRNAs within a user's target sequence, for example, to minimize total off-target activity across the genome. Off-target activity can be other than cleavage. For example, for each possible gRNA selection using Streptococcus pyogenes Cas9, the software tool can identify all potential off-target sequences (preceding either NAG or NGG PAM) across the genome that contain up to a certain number of mismatched base pairs (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). The cleavage efficiency at each off-target sequence can be predicted, for example, using an experimentally derived weighting scheme. Each possible gRNA can then be ranked according to its total predicted off-target cleavage; the top-ranked gRNAs represent those likely to have the greatest on-target and least off-target cleavage. Other functions may also be included in the tool, 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. Candidate gRNA molecules can be evaluated by methods known in the art or as described herein.

[0248] In some embodiments, gRNAs for use with Streptococcus pyogenes, Staphylococcus aureus, and Neisseria meningitidis Cas9 are identified using a DNA sequence search algorithm, e.g., using 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 calculates the genome-wide off-target propensity of the guide and then scores the guide. Typically, matches ranging from a perfect match to seven mismatches are considered for guides ranging in length from 17 to 24. In some aspects, once off-target sites are computationally determined, a total score is calculated for each guide and compiled in a tabular output using a web interface. In addition to identifying potential gRNA sites adjacent to the PAM sequence, the software can also identify all PAM-flanking sequences that differ from the selected gRNA site 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 repetitive elements using the publicly available RepeatMasker program. RepeatMasker searches the input DNA sequence for repetitive elements and low-complexity regions. The output is a detailed annotation of the repeats present in a given query sequence.

[0249] Once identified, gRNAs can be ranked into a hierarchy based on one or more of their distance to the target site, their orthogonality, and the presence of a 5'G (based on identifying exact matches in the human genome with 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 a minimal number of mismatches to the target sequence. "High level of orthogonality" or "good orthogonality" can refer, for example, to a 20-mer targeting domain that has no identical sequences in the human genome except for the intended target and no sequences containing one or two mismatches in the target sequence. Targeting domains with good orthogonality are selected to minimize off-target DNA cleavage. It is understood that this is a non-limiting example and that a variety of strategies can be utilized to identify gRNAs for use with Streptococcus pyogenes, Staphylococcus aureus, and Neisseria meningitidis or other Cas9 enzymes.

[0250] In some embodiments, gRNAs for use with S. pyogenes Cas9 can be identified using the publicly available web-based ZiFiT server (Fu et al., Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol. 2014 Jan 26. doi: 10.1038 / nbt.2808. PubMed PMID: 24463574. For original references, see Sander et al., 2007, NAR 35:W599-605; Sander et al., 2010, NAR 38: W462-8). In addition to identifying potential gRNA sites adjacent to the PAM sequence, the software also identifies all PAM-flanking sequences that differ from the selected gRNA site by one, two, three, or more nucleotides. In some aspects, the genomic DNA sequence for each gene can be obtained from the UCSC Genome browser, and the sequence can be screened for repetitive elements using the publicly available Repeat-Masker program. RepeatMasker searches the input DNA sequence for repetitive elements and low-complexity regions. The output is a detailed annotation of the repeats present in a given query sequence.

[0251] Once identified, gRNAs for use with S. pyogenes Cas9 can be ranked into a hierarchy, e.g., a tier of five. In some embodiments, targeting domains for first-tier gRNA molecules are selected based on their distance to the target site, their orthogonality, and the presence of a 5'G (based on ZiFiT identification of exact matches in the human genome containing an NGG PAM). In some embodiments, both 17-mer and 20-mer gRNAs are designed for the target. In some aspects, gRNAs are selected for both single gRNA nuclease cleavage and dual gRNA nickase strategies. The criteria for selecting gRNAs and the decision as to which gRNAs can be used for which strategies can be based on several considerations. In some embodiments, gRNAs are identified for both single gRNA nuclease cleavage and dual gRNA pairing "nickase" strategies. In some embodiments for selecting gRNAs, including determining which gRNAs can be used in a double gRNA pairing "nickase" strategy, the gRNA pair should be oriented on the DNA so that the PAM faces outward and cleavage by the D10A Cas9 nickase will result in a 5' overhang. In some aspects, it can be assumed that cleavage by a double nickase pair will result in the entire deletion of the intervening sequence with a reasonable frequency. However, cleavage by a double nickase pair can also frequently result in an indel mutation at only one site of the gRNA. Candidate pair members can be tested for how efficiently they remove the entire sequence compared to simply causing an indel mutation at one site of the gRNA.

[0252] In some embodiments, targeting domains for first-tier gRNA molecules can be selected based on (1) reasonable distance to the target location, e.g., within the first 500 bp of the coding sequence downstream of the start codon, (2) a high level of orthogonality, and (3) the presence of a 5'G. In some embodiments, for second-tier gRNA selection, the 5'G requirement can be eliminated, but distance restrictions are required and a high level of orthogonality is required. In some embodiments, third-tier selection uses the same distance restrictions and 5'G requirement, but eliminates the requirement for good orthogonality. In some embodiments, fourth-tier selection uses the same distance restrictions, but eliminates the requirement for good orthogonality and starting with a 5'G. In some embodiments, fifth-tier selection eliminates the requirement for good orthogonality and 5'G, and longer sequences (e.g., the remaining coding sequence, e.g., an additional 500 bp upstream or downstream of the transcription target site) are scanned. In certain cases, gRNAs are not identified based on specific tier criteria.

[0253] In some embodiments, gRNAs are specified for single gRNA nuclease cleavage and for dual gRNA pairing "nickase" strategies.

[0254] In some aspects, gRNAs for use with meningococcal and Staphylococcus aureus Cas9 can be manually identified by scanning genomic DNA sequences for the presence of PAM sequences. These gRNAs can be separated into two tiers. In some embodiments, for the first tier of gRNAs, targeting domains are selected within the first 500 bp of the coding sequence downstream of the start codon. In some embodiments, for the second tier of gRNAs, targeting domains are selected within the remaining coding sequence (downstream of the first 500 bp). In certain cases, gRNAs are not identified based on specific tier criteria.

[0255] In some embodiments, another strategy for identifying guide RNAs (gRNAs) for use with Streptococcus pyogenes, Staphylococcus aureus, and Neisseria meningitidis Cas9 can use a DNA sequence search algorithm. In some aspects, guide RNA design is performed using custom guide RNA design software based on the public tool cas-offinder (Bae et al. Bioinformatics. 2014;30(10):1473-1475). The custom guide RNA design software calculates the genome-wide off-target propensity of the guide and then scores the guide. Typically, matches ranging from a perfect match to seven mismatches are considered for guides ranging in length from 17 to 24. Once off-target sites are computationally determined, a total score is calculated for each guide and summarized in a tabular output using a web interface. In addition to identifying potential gRNA sites adjacent to the PAM sequence, the software also identifies all PAM-flanking sequences that differ from the selected gRNA site by one, two, three, or more nucleotides. In some embodiments, the genomic DNA sequence for each gene is obtained from the UCSC Genome browser, and the sequence is screened for repetitive elements using the publicly available RepeatMasker program. RepeatMasker searches the input DNA sequence for repetitive elements and low-complexity regions. The output is a detailed annotation of the repeats present in a given query sequence.

[0256] In some embodiments, once identified, gRNAs are ranked in a hierarchy based on their distance to the target site or their orthogonality (based on identifying exact matches in the human genome with 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). In some aspects, targeting domains with good orthogonality are selected to minimize off-target DNA cleavage.

[0257] As an example, 17-mer or 20-mer gRNAs can be designed for targets of Streptococcus pyogenes and Neisseria meningitidis. As another example, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, and 24-mer gRNAs can be designed for targets of Staphylococcus aureus.

[0258] In some embodiments, gRNAs are identified for both single gRNA nuclease cleavage and dual gRNA pairing "nickase" strategies. To select gRNAs, including determining which gRNAs can be used in the dual gRNA pairing "nickase" strategy, in some embodiments, the gRNA pair should be oriented on the DNA so that the PAM faces outward and cleavage by the D10A Cas9 nickase will result in a 5' overhang. In some aspects, it can be assumed that cleavage by a dual nickase pair will result in the entire deletion of the intervening sequence with a reasonable frequency. However, cleavage by a dual nickase pair can also frequently result in indel mutations at only one gRNA site. Candidate pair members can be tested for how efficiently they remove the entire sequence compared to simply creating an indel mutation at one gRNA site.

[0259] To design a strategy for gene disruption, in some embodiments, targeting domains for tier 1 gRNA molecules against S. pyogenes are selected based on their distance to the target site and their orthogonality (PAM is NGG). In some cases, targeting domains for tier 1 gRNA molecules are selected based on (1) a reasonable distance to the target location, e.g., within the first 500 bp of the coding sequence downstream of the start codon, and (2) a high level of orthogonality. In some aspects, a high level of orthogonality is not required for the selection of tier 2 gRNAs. In some cases, tier 3 gRNAs eliminate the requirement for good orthogonality, allowing longer sequences (e.g., the remaining coding sequence) to be scanned. In certain cases, gRNAs are not identified based on the criteria of a particular tier.

[0260] To design strategies for gene disruption, in some embodiments, targeting domains for tier 1 gRNA molecules against Neisseria meningitidis were selected within the first 500 bp of the coding sequence and had a high level of orthogonality. Targeting domains for tier 2 gRNA molecules against Neisseria meningitidis were selected within the first 500 bp of the coding sequence and did not require high orthogonality. Targeting domains for tier 3 gRNA molecules against Neisseria meningitidis were selected within the remainder of the coding sequence downstream of the 500 bp. Note that the tiers are non-inclusive (each gRNA is listed only once). In certain cases, gRNAs were not identified based on the criteria of a particular tier.

[0261] To design strategies for gene disruption, in some embodiments, targeting domains for tier 1 gRNA molecules against S. aureus are selected within the first 500 bp of the coding sequence, have a high level of orthogonality, and include an NNGRRT PAM. In some embodiments, targeting domains for tier 2 gRNA molecules against S. aureus are selected within the first 500 bp of the coding sequence, do not require a high level of orthogonality, and include an NNGRRT PAM. In some embodiments, targeting domains for tier 3 gRNA molecules against S. aureus are selected within the remainder of the downstream coding sequence and include an NNGRRT PAM. In some embodiments, targeting domains for tier 4 gRNA molecules against S. aureus are selected within the first 500 bp of the coding sequence and include an NNGRRV PAM. In some embodiments, targeting domains for tier 5 gRNA molecules against S. aureus are selected within the remainder of the downstream coding sequence and include an NNGRRV PAM. In certain cases, gRNAs are not identified based on criteria for a particular tier.

[0262] (ii) Cas9 In the methods and compositions described herein, the Cas9 molecules of various species can be used.Although the Cas9 molecules of Streptococcus pyogenes, Staphylococcus aureus, Neisseria meningitidis and S. thermophilus are the subject of much of the disclosure herein, the Cas9 molecules of other species of Cas9 proteins listed herein, the Cas9 molecules derived from said Cas9 proteins, or the Cas9 molecules based on said Cas9 proteins can also be used.In other words, although much of the description herein uses the Cas9 molecules of Streptococcus pyogenes, Staphylococcus aureus, Neisseria meningitidis and S. thermophilus, the Cas9 molecules from other species can be substituted therefor. Such species include: Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., Cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp. 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 ivanovii ivanovii, Listeria monocytogenes, 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 multocida 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.

[0263] As used herein, the term Cas9 molecule or Cas9 polypeptide refers to a molecule or polypeptide that can interact with a gRNA molecule and coordinate with the gRNA molecule to direct or localize to a site containing a target domain and a PAM sequence. As used herein, the term Cas9 molecule and Cas9 polypeptide refers to naturally occurring Cas9 molecules and engineered, altered, or modified Cas9 molecules or Cas9 polypeptides that differ from a reference sequence, such as the most similar naturally occurring Cas9 molecule, by at least one amino acid residue.

[0264] Crystal structures have been determined for two different naturally occurring bacterial Cas9 molecules (Jinek et al., Science, 343(6176):1247997, 2014) and for Streptococcus pyogenes Cas9 with guide RNAs (e.g., synthetic fusions of crRNA and tracrRNA) (Nishimasu et al., Cell, 156:935-949, 2014; and Anders et al., Nature, 2014, doi: 10.1038 / nature13579).

[0265] Naturally occurring Cas9 molecules contain two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe; each of these further contains the domains described herein. Exemplary schematic diagrams of the organization of key Cas9 domains in the primary structure are described in WO2015 / 161276, e.g., in Figures 8A-8B therein. The domain nomenclature and numbering of the amino acid residues contained in each domain used throughout this disclosure are as described in Nishimasu et al. The numbering of amino acid residues is with respect to Streptococcus pyogenes Cas9.

[0266] The REC lobe contains an arginine-rich bridge helix (BH), a REC1 domain, and a REC2 domain. The REC lobe has no structural similarity to other known proteins, indicating that it is a Cas9-specific functional domain. The BH domain is a long α-helical, arginine-rich region that includes amino acids 60-93 of the S. pyogenes Cas9 sequence. The REC1 domain is important for recognizing the repeat:anti-repeat duplex, for example, of gRNA or tracrRNA, and is therefore crucial for Cas9 activity by recognizing target sequences. The REC1 domain contains two REC1 motifs at amino acids 94-179 and 308-717 of the S. pyogenes Cas9 sequence. These two REC1 domains are separated by the REC2 domain in the linear primary structure but assemble to form the REC1 domain in the tertiary structure. The REC2 domain, or a portion thereof, may also play a role in recognizing the repeat:anti-repeat duplex. The REC2 domain contains amino acids 180 to 307 of the Streptococcus pyogenes Cas9 sequence.

[0267] The NUC lobe contains a RuvC domain (also referred to herein as a RuvC-like domain), an HNH domain (also referred to herein as an HNH-like domain), and a PAM-interacting (PI) domain. The RuvC domain has structural similarity to members of the retroviral integrase superfamily and cleaves single strands, e.g., non-complementary strands, of target nucleic acid molecules. The RuvC domain is assembled from three separate RuvC motifs (RuvC I, RuvC II, and RuvC III, commonly referred to as the RuvC I domain, or the N-terminal RuvC domain, RuvC II domain, and RuvC III domain) located at amino acids 1-59, 718-769, and 909-1098, respectively, of the Streptococcus pyogenes Cas9 sequence. Similar to the REC1 domain, the three RuvC motifs are linearly separated by other domains in the primary structure, but in the tertiary structure, the three RuvC motifs assemble to form the RuvC domain. The HNH domain has structural similarity to HNH endonucleases and cleaves a single strand, e.g., the complementary strand, of a target nucleic acid molecule. The HNH domain is located between the RuvC II and RuvC III motifs and contains amino acids 775-908 of the Streptococcus pyogenes Cas9 sequence. The PI domain interacts with the PAM of the target nucleic acid molecule and contains amino acids 1099-1368 of the Streptococcus pyogenes Cas9 sequence.

[0268] (a) RuvC-like domain and HNH-like domain In some embodiments, the Cas9 molecule or polypeptide comprises an HNH-like domain and a RuvC-like domain. In some embodiments, cleavage activity is dependent on the RuvC-like domain and the HNH-like domain. The Cas9 molecule or polypeptide, e.g., an eaCas9 molecule or polypeptide, can comprise one or more of the following domains: a RuvC-like domain and an HNH-like domain. In some embodiments, the Cas9 molecule or polypeptide is an eaCas9 molecule or polypeptide, and the eaCas9 molecule or polypeptide comprises a RuvC-like domain, e.g., a RuvC-like domain described herein, and / or an HNH-like domain, e.g., an HNH-like domain described herein.

[0269] (b) RuvC-like domain In some embodiments, the RuvC-like domain cleaves a single strand, e.g., a non-complementary strand, of a target nucleic acid molecule. A Cas9 molecule or polypeptide can contain more than one RuvC-like domain (e.g., one, two, three, or more RuvC-like domains). In some embodiments, the RuvC-like domain is at least 5, 6, 7, or 8 amino acids in length, but not more than 20, 19, 18, 17, 16, or 15 amino acids in length. In some embodiments, a Cas9 molecule or polypeptide contains an N-terminal RuvC-like domain about 10-20 amino acids in length, e.g., about 15 amino acids in length.

[0270] (c) N-terminal RuvC-like domain Some naturally occurring Cas9 molecules contain more than one RuvC-like domain and rely on an N-terminal RuvC-like domain for cleavage. Thus, a Cas9 molecule or Cas9 polypeptide can contain an N-terminal RuvC-like domain.

[0271] In embodiments, the N-terminal RuvC-like domain is cleavage competent.

[0272] In embodiments, the N-terminal RuvC-like domain is incompetent for cleavage.

[0273] In some embodiments, the N-terminal RuvC-like domain differs by as little as one, but not more than two, three, four, or five residues from the sequence of the N-terminal RuvC-like domain disclosed herein, e.g., in WO2015 / 161276, e.g., in Figures 3A-3B or 7A-7B therein. In some embodiments, one, two, or all three of the highly conserved residues identified in WO2015 / 161276, e.g., in Figures 3A-3B or 7A-7B therein, are present.

[0274] In some embodiments, the N-terminal RuvC-like domain differs in as little as one, but not more than two, three, four, or five residues from the sequence of the N-terminal RuvC-like domain disclosed herein, e.g., in WO2015 / 161276, e.g., in Figures 4A-4B or 7A-7B therein. In some embodiments, one, two, three, or all four of the highly conserved residues identified in WO2015 / 161276, e.g., in Figures 4A-4B or 7A-7B therein, are present.

[0275] (d) Additional RuvC-like domains In addition to the N-terminal RuvC-like domain, a Cas9 molecule or polypeptide, e.g., an eaCas9 molecule or polypeptide, can comprise one or more additional RuvC-like domains. In some embodiments, a Cas9 molecule or polypeptide can comprise two additional RuvC-like domains. Preferably, the additional RuvC-like domains are at least 5 amino acids in length and, e.g., less than 15 amino acids in length, e.g., 5-10 amino acids in length, e.g., 8 amino acids in length.

[0276] (e) HNH-like domain In some embodiments, the HNH-like domain cleaves a single-stranded complementary domain, e.g., the complementary strand of a double-stranded nucleic acid molecule. In some embodiments, the HNH-like domain is at least 15, 20, or 25 amino acids in length, but not more than 40, 35, or 30 amino acids in length, e.g., 20-35 amino acids in length, e.g., 25-30 amino acids in length. Exemplary HNH-like domains are described herein.

[0277] In some embodiments, the HNH-like domain is cleavable.

[0278] In some embodiments, the HNH-like domain is incapable of cleavage.

[0279] In some embodiments, the HNH-like domain differs by as little as one, but not more than two, three, four, or five residues, from the sequence of an HNH-like domain disclosed herein, e.g., in WO2015 / 161276, e.g., in Figures 5A-5C or 7A-7B therein. In some embodiments, one or both of the highly conserved residues identified in WO2015 / 161276, e.g., in Figures 5A-5C or 7A-7B therein, are present.

[0280] In some embodiments, the HNH-like domain differs in as little as one, but no more than two, three, four, or five residues from the sequence of an HNH-like domain disclosed herein, e.g., in WO2015 / 161276, e.g., in Figures 6A-6B or 7A-7B therein. In some embodiments, one, two, or all three of the highly conserved residues identified in WO2015 / 161276, e.g., in Figures 6A-6B or 7A-7B therein, are present.

[0281] (f) Nuclease and helicase activity In some embodiments, a Cas9 molecule or polypeptide can cleave a target nucleic acid molecule. Typically, wild-type Cas9 molecules cleave both strands of a target nucleic acid molecule. Cas9 molecules and polypeptides can be engineered to alter their nuclease cleavage (or other properties), e.g., to result in a Cas9 molecule or polypeptide that is a nickase or lacks the ability to cleave a target nucleic acid. A Cas9 molecule or polypeptide that can cleave a target nucleic acid molecule is referred to herein as an eaCas9 molecule or polypeptide.

[0282] In some embodiments, the eaCas9 molecule or eaCas9 polypeptide comprises one or more of the following activities: nickase activity, i.e., the ability to cleave a single strand of a nucleic acid molecule, e.g., a non-complementary or complementary strand; double-stranded nuclease activity, i.e., the ability to cleave both strands of a double-stranded nucleic acid to create a double-stranded break (which in some embodiments means there are two nickase activities); endonuclease activity; exonuclease activity; and helicase activity, i.e., the ability to unwind the helical structure of a double-stranded nucleic acid.

[0283] In some embodiments, an enzymatically active eaCas9 molecule or polypeptide cleaves both strands, resulting in a double-stranded break. In some embodiments, the eaCas9 molecule cleaves only one strand, e.g., the strand to which the gRNA hybridizes or the strand complementary to the strand to which the gRNA hybridizes. In some embodiments, the eaCas9 molecule or polypeptide comprises a cleavage activity associated with an HNH-like domain. In some embodiments, the eaCas9 molecule or polypeptide comprises a cleavage activity associated with an N-terminal RuvC-like domain. In some embodiments, the eaCas9 molecule or polypeptide comprises a cleavage activity associated with an HNH-like domain and a cleavage activity associated with an N-terminal RuvC-like domain. In some embodiments, the eaCas9 molecule or polypeptide comprises an active or cleavage-competent HNH-like domain and an inactive or cleavage-incompetent N-terminal RuvC-like domain. In some embodiments, the eaCas9 molecule or polypeptide comprises an inactive or cleavage-incompetent HNH-like domain and an active or cleavage-competent N-terminal RuvC-like domain.

[0284] Some Cas9 molecules or Cas9 polypeptides have the ability to interact with gRNA molecules and localize to the core target domain together with gRNA molecules, but cannot cleave target nucleic acids or cannot cleave at an efficient rate.Cas9 molecules that have no cleavage activity or no substantial cleavage activity are referred to herein as eiCas9 molecules or eiCas9 polypeptides.For example, when measured by the assays described herein, eiCas9 molecules or eiCas9 polypeptides may lack cleavage activity or have substantially less cleavage activity, for example, less than 20, 10, 5, 1, or 0.1% of the cleavage activity of reference Cas9 molecules or eiCas9 polypeptides.

[0285] (g) Targeting and PAM A Cas9 molecule or Cas9 polypeptide is a polypeptide that can interact with a guide RNA (gRNA) molecule and, in cooperation with the gRNA molecule, localizes to a site containing a target domain and a PAM sequence.

[0286] In some embodiments, the ability of an eaCas9 molecule or eaCas9 polypeptide to interact with and cleave a target nucleic acid is PAM sequence-dependent. The PAM sequence is a sequence within the target nucleic acid. In some embodiments, cleavage of the target nucleic acid occurs upstream from the PAM sequence. EaCas9 molecules from different bacterial species can recognize different sequence motifs (e.g., PAM sequences). In some embodiments, the eaCas9 molecule of Streptococcus pyogenes recognizes the sequence motifs NGG, NAG, and NGA, directing cleavage of the target nucleic acid sequence 1 to 10, e.g., 3 to 5 base pairs upstream from that sequence. See, e.g., Mali et al., Science 2013;339(6121):823-826. In some embodiments, S. thermophilus eaCas9 molecules recognize the sequence motifs NGGNG and / or NNAGAAW (W = A or T) and direct cleavage of the target nucleic acid sequence 1-10, e.g., 3-5 base pairs upstream from these sequences. See, e.g., Horvath et al., Science 2010;327(5962):167-170 and Deveau et al., J Bacteriol 2008;190(4):1390-1400. In some embodiments, S. mutans eaCas9 molecules recognize the sequence motifs NGG and / or NAAR (R = A or G) and direct cleavage of the core target nucleic acid sequence 1-10, e.g., 3-5 base pairs upstream from these sequences. See, e.g., Deveau et al., J Bacteriol 2008;190(4):1390-1400. In some embodiments, the S. aureus eaCas9 molecule recognizes the sequence motif NNGRR (R = A or G) and directs cleavage of the target nucleic acid sequence 1-10, e.g., 3-5 base pairs upstream from that sequence. In some embodiments, the S. aureus eaCas9 molecule recognizes the sequence motif NNGRRT (R = A or G) and directs cleavage of the target nucleic acid sequence 1-10, e.g., 3-5 base pairs upstream from that sequence. In some embodiments, the S. aureus eaCas9 molecule recognizes the sequence motif NNGRRV (R = A or G) and directs cleavage of the target nucleic acid sequence 1-10, e.g., 3-5 base pairs upstream from that sequence.In some embodiments, meningococcal eaCas9 molecules recognize the sequence motif NNNNGATT or NNNGCTT (R=A or G, V=A, G, or C) and direct cleavage of the target nucleic acid sequence 1-10, e.g., 3-5 base pairs upstream from that sequence. See, e.g., Hou et al., PNAS Early Edition 2013, 1-6. The ability of Cas9 molecules to recognize PAM sequences can be determined, for example, using the transformation assay described in Jinek et al., Science 2012 337:816. In the foregoing embodiments, N can be any nucleotide residue, e.g., A, G, C, or T.

[0287] As discussed herein, Cas9 molecules can be engineered to alter their PAM specificity.

[0288] Exemplary naturally occurring Cas9 molecules are described in Chylinski et al., RNA Biology 2013 10:5, 727-737. Such Cas9 molecules include Cas9 molecules from bacterial families of clusters 1-78.

[0289] Exemplary naturally occurring Cas9 molecules include those of the cluster 1 bacterial family. Examples include Cas9 molecules from Streptococcus pyogenes (e.g., strains SF370, MGAS10270, MGAS10750, MGAS2096, MGAS315, MGAS5005, MGAS6180, MGAS9429, NZ131, and SSI-1), S. thermophilus (e.g., strain LMD-9), S. pseudoporcinus (e.g., strain SPIN 20026), S. mutans (e.g., strains UA159, NN2025), S. macacae (e.g., strain NCTC11558), S. gallolyticus (e.g., strain UCN34, ATCC BAA-2069), S. equines (e.g., strain ATCC 9812, MGCS 124), S. dysdalactiae (e.g., strain GGS 124), S. bovis (e.g., strain ATCC 700338), S. anginosus (e.g., strain F0211), S. agalactiae (e.g., strains NEM316, A909), Listeria monocytogenes (e.g., strain F6854), Listeria innocua (e.g., strain Clip11262), Enterococcus italicus (e.g., strain DSM 15952), or Enterococcus faecium faecium) (e.g., strain 1,231,408). Another exemplary Cas9 molecule is the Cas9 molecule of Neisseria meningitidis (Hou et al., PNAS Early Edition 2013, 1-6).

[0290] In some embodiments, the Cas9 molecule or Cas9 polypeptide, e.g., the eaCas9 molecule or eaCas9 polypeptide, can be any Cas9 molecule sequence described herein, or a naturally occurring Cas9 molecule sequence, e.g., those listed herein (e.g., SEQ ID NOs: 112-115), or those described in Chylinski et al., RNA Biology 2013 10:5, 727-737; Hou et al., PNAS Early Edition 2013, 1-6; differs by no more than 2, 5, 10, 15, 20, 30, or 40% of amino acid residues compared to the Cas9 molecule sequence; differs from the Cas9 molecule sequence by at least 1, 2, 5, 10, or 20 amino acids, but no more than 100, 80, 70, 60, 50, 40, or 30 amino acids; or is identical to the Cas9 molecule sequence. In some embodiments, the Cas9 molecule or Cas9 polypeptide comprises one or more of the following activities: nickase activity; double-strand cleavage activity (e.g., endonuclease and / or exonuclease activity); helicase activity; or the ability to target nucleic acids together with a gRNA molecule.

[0291] In some embodiments, the Cas9 molecule or polypeptide comprises the amino acid sequence of the consensus sequence in WO2015 / 161276, e.g., in Figures 2A-2G therein, where "*" denotes any amino acid found at the corresponding position in the amino acid sequences of the Cas9 molecules of S. pyogenes, S. thermophilus, S. mutans, and L. innocua, and "-" denotes any amino acid. In some embodiments, the Cas9 molecule or polypeptide differs from the consensus sequence of SEQ ID NOs:112-117, or the sequence of the consensus sequence disclosed in WO2015 / 161276, e.g., in Figures 2A-2G therein, by at least one, but not more than 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acid residues. In some embodiments, the Cas9 molecule or polypeptide comprises the amino acid sequence of SEQ ID NO:117, or as set forth in WO2015 / 161276, e.g., Figures 7A-7B therein, where "*" denotes any amino acid found at the corresponding position in the amino acid sequence of a Cas9 molecule of Streptococcus pyogenes or Neisseria meningitidis, "-" denotes any amino acid, and "-" denotes any amino acid or absent. In some embodiments, the Cas9 molecule or polypeptide differs from the sequence of SEQ ID NO:116 or 117, or as set forth in WO2015 / 161276, e.g., Figures 7A-7B therein, by at least one, but not more than 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acid residues.

[0292] Comparison of the sequences of several Cas9 molecules shows that certain regions are conserved: these are identified as region 1 (residues 1-180, or 120-180 in the case of region 1'); region 2 (residues 360-480); region 3 (residues 660-720); region 4 (residues 817-900); and region 5 (residues 900-960).

[0293] In some embodiments, the Cas9 molecule or Cas9 polypeptide comprises regions 1-5, along with sufficient additional Cas9 molecule sequence to provide a biologically active molecule, e.g., a Cas9 molecule having at least one activity described herein. In some embodiments, each of regions 1-6 independently shares 50%, 60%, 70%, or 80% homology with the corresponding residues of a Cas9 molecule or Cas9 polypeptide described herein, e.g., as set forth in SEQ ID NOs:112-117, or the sequences disclosed in WO2015 / 161276, e.g., in Figures 2A-2G or 7A-7B therein.

[0294] In some embodiments, the Cas9 molecule or Cas9 polypeptide, e.g., the eaCas9 molecule or eaCas9 polypeptide, is selected from the group consisting of amino acids 1-180 of the amino acid sequence of Streptococcus pyogenes Cas9 (numbering according to the motif sequence in Figures 2A-2G of WO 2015 / 161276; WO 2015 / 161276 (52% of the residues in the four Cas9 sequences in Figures 2A-2G are conserved), and contains an amino acid sequence designated Region 1 that has 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity; differs by at least 1, 2, 5, 10, or 20 amino acids, but not more than 90, 80, 70, 60, 50, 40, or 30 amino acids, from amino acids 1-180 of the Cas9 sequence of S. pyogenes, S. thermophilus, S. mutans, or L. innocua; or is identical to amino acid sequences 1-180 of the Cas9 sequence of S. pyogenes, S. thermophilus, S. mutans, or L. innocua.

[0295] In some embodiments, the Cas9 molecule or Cas9 polypeptide, e.g., the eaCas9 molecule or eaCas9 polypeptide, comprises amino acids 120-180 of the amino acid sequence of Cas9 from Streptococcus pyogenes, S. thermophilus, S. mutans, or L. innocua (WO 2015 / 161276 (55% of the residues in the four Cas9 sequences in Figures 2A-2G are conserved), and contains an amino acid sequence designated Region 1' that has 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity; differs by at least 1, 2, or 5 amino acids, but not more than 35, 30, 25, 20, or 10 amino acids, from amino acids 120-180 of the Cas9 sequence of S. pyogenes, S. thermophilus, S. mutans, or L. innocua; or is identical to amino acid sequences 120-180 of the Cas9 sequence of S. pyogenes, S. thermophilus, S. mutans, or L. innocua.

[0296] In some embodiments, the Cas9 molecule or Cas9 polypeptide, e.g., the eaCas9 molecule or eaCas9 polypeptide, comprises amino acids 360-480 of the amino acid sequence of Cas9 from Streptococcus pyogenes, S. thermophilus, S. mutans, or L. innocua (WO 2015 / 161276 (52% of the residues in the four Cas9 sequences in Figures 2A-2G are conserved), and contains an amino acid sequence designated Region 2 that has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity; differs by at least 1, 2, or 5 amino acids, but not more than 35, 30, 25, 20, or 10 amino acids, from amino acids 360-480 of the Cas9 sequence of S. pyogenes, S. thermophilus, S. mutans, or L. innocua; or is identical to amino acid sequences 360-480 of the Cas9 sequence of S. pyogenes, S. thermophilus, S. mutans, or L. innocua.

[0297] In some embodiments, the Cas9 molecule or Cas9 polypeptide, e.g., the eaCas9 molecule or eaCas9 polypeptide, comprises amino acids 660-720 of the amino acid sequence of Cas9 from Streptococcus pyogenes, S. thermophilus, S. mutans, or L. innocua (WO 2A-2G of US Patent Application Publication No. 2015 / 161276, which contains an amino acid sequence designated region 3 that has 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of S. pyogenes, S. thermophilus, S. mutans, or L. innocua Cas9 (56% of the residues are conserved), or that differs by at least 1, 2, or 5 amino acids but not more than 35, 30, 25, 20, or 10 amino acids from amino acid sequence 660-720 of S. pyogenes, S. thermophilus, S. mutans, or L. innocua Cas9; or is identical to amino acid sequence 660-720 of S. pyogenes, S. thermophilus, S. mutans, or L. innocua Cas9.

[0298] In some embodiments, the Cas9 molecule or Cas9 polypeptide, e.g., the eaCas9 molecule or eaCas9 polypeptide, comprises amino acids 817-900 of the amino acid sequence of Cas9 from Streptococcus pyogenes, S. thermophilus, S. mutans, or L. innocua (WO 2015 / 161276 (55% of the residues in the four Cas9 sequences in Figures 2A-2G are conserved), contains an amino acid sequence designated Region 4 that has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity; differs by at least 1, 2, or 5 amino acids but not more than 35, 30, 25, 20, or 10 amino acids from amino acids 817-900 of the Cas9 sequence of S. pyogenes, S. thermophilus, S. mutans, or L. innocua; or is identical to amino acid sequences 817-900 of the Cas9 sequence of S. pyogenes, S. thermophilus, S. mutans, or L. innocua.

[0299] In some embodiments, the Cas9 molecule or Cas9 polypeptide, e.g., the eaCas9 molecule or eaCas9 polypeptide, comprises amino acids 900-960 of the amino acid sequence of Cas9 from Streptococcus pyogenes, S. thermophilus, S. mutans, or L. innocua (WO 2015 / 161276 (60% of the residues in the four Cas9 sequences in Figures 2A-2G are conserved), contains an amino acid sequence designated Region 5 that has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity; differs by at least 1, 2, or 5 amino acids but not more than 35, 30, 25, 20, or 10 amino acids from amino acids 900-960 of the Cas9 sequence of S. pyogenes, S. thermophilus, S. mutans, or L. innocua; or is identical to amino acid sequences 900-960 of the Cas9 sequence of S. pyogenes, S. thermophilus, S. mutans, or L. innocua.

[0300] (h) Engineered or modified Cas9 molecules and Cas9 polypeptides The Cas9 molecules and Cas9 polypeptides described herein, e.g., naturally occurring Cas9 molecules, can have any of several properties, including nickase activity, nuclease activity (e.g., endonuclease and / or exonuclease activity); helicase activity; the ability to functionally bind to a gRNA molecule; and the ability to target (or localize to) a site on a nucleic acid (e.g., PAM recognition and specificity). In some embodiments, a Cas9 molecule or Cas9 polypeptide can comprise all or a subset of these properties. In typical embodiments, a Cas9 molecule or Cas9 polypeptide has the ability to interact with a gRNA molecule and localize to a site in a nucleic acid in cooperation with the gRNA molecule. Other activities, e.g., PAM specificity, cleavage activity, or helicase activity, can vary more widely in Cas9 molecules and Cas9 polypeptides.

[0301] Cas9 molecules include engineered Cas9 molecules and engineered Cas9 polypeptides ("engineered" as used in this context simply means that the Cas9 molecule or Cas9 polypeptide differs from a reference sequence, without any limitations on process or origin). Engineered Cas9 molecules or Cas9 polypeptides can have altered enzymatic properties, such as altered nuclease activity (compared to naturally occurring or other reference Cas9 molecules) or altered helicase activity. As described herein, engineered Cas9 molecules or Cas9 polypeptides can have nickase activity (as opposed to double-stranded nuclease activity). In some embodiments, engineered Cas9 molecules or Cas9 polypeptides can have modifications that alter their size, such as deletion of one or more amino acid sequences that reduce their size without significantly affecting any Cas9 activity. In some embodiments, engineered Cas9 molecules or Cas9 polypeptides can have modifications that affect PAM recognition. For example, engineered Cas9 molecules can be altered to recognize PAM sequences other than those recognized by the endogenous wild-type PI domain. In some embodiments, a Cas9 molecule or Cas9 polypeptide can differ in sequence from a naturally occurring Cas9 molecule but have no significant alteration in one or more Cas9 activities.

[0302] Cas9 molecules or polypeptides with desirable properties can be generated in several ways, for example, by modifying a parent, e.g., naturally occurring, Cas9 molecule or polypeptide to result in an altered Cas9 molecule or polypeptide with desirable properties. For example, one or more mutations or differences can be introduced relative to a parent Cas9 molecule, e.g., a naturally occurring or engineered Cas9 molecule. Such mutations and differences include substitutions (e.g., conservative substitutions or substitutions of non-essential amino acids); insertions; or deletions. In some embodiments, a Cas9 molecule or polypeptide can contain one or more mutations or differences, e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50 mutations, but fewer than 200, 100, or 80 mutations, relative to a reference, e.g., parent Cas9 molecule.

[0303] In some embodiments, the mutation or mutations do not have a substantial effect on Cas9 activity, e.g., the Cas9 activity described herein. In some embodiments, the mutation or mutations have a substantial effect on Cas9 activity, e.g., the Cas9 activity described herein.

[0304] (i) Non-cleavable and modified cleavable Cas9 molecules and Cas9 polypeptides In some embodiments, the Cas9 molecule or Cas9 polypeptide comprises a cleavage property that differs from that of a naturally occurring Cas9 molecule, e.g., a Cas9 molecule that is most closely homologous. For example, the Cas9 molecule or Cas9 polypeptide can differ from a naturally occurring Cas9 molecule, e.g., a Streptococcus pyogenes Cas9 molecule, by, for example, its ability to modulate, e.g., increase or decrease, the cleavage of double-stranded nucleic acid (endonuclease and / or exonuclease activity) compared to a naturally occurring Cas9 molecule (e.g., a Streptococcus pyogenes Cas9 molecule); its ability to modulate, e.g., increase or decrease, the cleavage of a single strand of nucleic acid, e.g., a non-complementary strand of a nucleic acid molecule or a complementary strand of a nucleic acid molecule (nickase activity) compared to a naturally occurring Cas9 molecule (e.g., a Streptococcus pyogenes Cas9 molecule); or its ability to cleave nucleic acid molecules, e.g., double-stranded or single-stranded nucleic acid molecules, can be eliminated.

[0305] (j) Modified cleaved eaCas9 molecules and eaCas9 polypeptides In some embodiments, the eaCas9 molecule or eaCas9 polypeptide comprises one or more of the following activities: a cleavage activity associated with the N-terminal RuvC-like domain; a cleavage activity associated with the HNH-like domain; a cleavage activity associated with the HNH-like domain and a cleavage activity associated with the N-terminal RuvC-like domain.

[0306] In some embodiments, an eaCas9 molecule or polypeptide comprises an active or cleavage-competent HNH-like domain and an inactive or cleavage-incompetent N-terminal RuvC-like domain. Exemplary inactive or cleavage-incompetent N-terminal RuvC-like domains can have an aspartic acid mutation in the N-terminal RuvC-like domain, e.g., the aspartic acid at position 9 of the consensus sequences of SEQ ID NOs:112-117 or the consensus sequences disclosed in WO2015 / 161276, e.g., Figures 2A-2G therein, or the aspartic acid at position 10 of SEQ ID NO:117 can be substituted with, e.g., alanine. In some embodiments, the eaCas9 molecule or eaCas9 polypeptide differs from wild-type in its N-terminal RuvC-like domain and does not cleave a target nucleic acid or cleaves it with significantly less efficiency, e.g., less than 20, 10, 5, 1, or 0.1%, of the cleavage activity of a reference Cas9 molecule, e.g., as measured by an assay described herein. The reference Cas9 molecule may be a naturally occurring, unmodified Cas9 molecule, e.g., a naturally occurring Cas9 molecule, such as a Streptococcus pyogenes or S. thermophilus Cas9 molecule. In some embodiments, the reference Cas9 molecule is the naturally occurring Cas9 molecule with the closest sequence identity or homology.

[0307] In some embodiments, the eaCas9 molecule or polypeptide comprises an inactive or cleavage-incompetent HNH domain and an active or cleavage-competent N-terminal RuvC-like domain. Exemplary inactive or cleavage-incompetent HNH-like domains may have one or more of the following mutations: a histidine in the HNH-like domain, for example the histidine shown at position 856 of the consensus sequence of SEQ ID NOs: 112-117 or the consensus sequence disclosed in WO2015 / 161276, for example in Figures 2A-2G therein, may be substituted with, for example, an alanine; one or more asparagines in the HNH-like domain, for example the histidine shown at position 870 of the consensus sequence of SEQ ID NOs: 112-117 or the consensus sequence disclosed in WO2015 / 161276, for example in Figures 2A-2G therein, and / or the asparagine shown at position 879 of the consensus sequence of SEQ ID NOs: 112-117 or the consensus sequence disclosed in WO2015 / 161276, for example in Figures 2A-2G therein, may be substituted with, for example, an alanine. In some embodiments, eaCas9 has a different HNH-like domain from the wild-type, and does not cleave target nucleic acids or cleaves them with significantly lower efficiency, e.g., less than 20, 10, 5, 1, or 0.1% of the cleavage activity of a reference Cas9 molecule, as measured, for example, by an assay described herein. The reference Cas9 molecule may be a naturally occurring, unmodified Cas9 molecule, such as a naturally occurring Cas9 molecule from Streptococcus pyogenes or S. thermophilus. In some embodiments, the reference Cas9 molecule is the naturally occurring Cas9 molecule with the closest sequence identity or homology.

[0308] In some embodiments, the eaCas9 molecule or polypeptide comprises an inactive or cleavage-incompetent HNH domain and an active or cleavage-competent N-terminal RuvC-like domain. Exemplary inactive or cleavage-incompetent HNH-like domains may have one or more of the following mutations: a histidine in the HNH-like domain, for example the histidine shown at position 856 of the consensus sequence of SEQ ID NOs: 112-117 or the consensus sequence disclosed in WO2015 / 161276, for example in Figures 2A-2G therein, may be substituted with, for example, an alanine; one or more asparagines in the HNH-like domain, for example the histidine shown at position 870 of the consensus sequence of SEQ ID NOs: 112-117 or the consensus sequence disclosed in WO2015 / 161276, for example in Figures 2A-2G therein, and / or the asparagine shown at position 879 of the consensus sequence of SEQ ID NOs: 112-117 or the consensus sequence disclosed in WO2015 / 161276, for example in Figures 2A-2G therein, may be substituted with, for example, an alanine. In some embodiments, eaCas9 differs from wild-type in its HNH-like domain and does not cleave target nucleic acids or cleaves them with significantly lower efficiency, e.g., less than 20, 10, 5, 1, or 0.1% of the cleavage activity of a reference Cas9 molecule, as measured, for example, by an assay described herein. The reference Cas9 molecule may be a naturally occurring, unmodified Cas9 molecule, such as a Cas9 molecule from Streptococcus pyogenes or S. thermophilus. In some embodiments, the reference Cas9 molecule is the naturally occurring Cas9 molecule with the closest sequence identity or homology.

[0309] (k) Altering the ability to cleave one or both strands of a target nucleic acid In some embodiments, exemplary Cas9 activities include one or more of PAM specificity, cleavage activity, and helicase activity. The mutation can be present, for example, in one or more RuvC-like domains, e.g., the N-terminal RuvC-like domain; the HNH-like domain; or a region outside the RuvC-like and HNH-like domains. In some embodiments, the mutation is present in the RuvC-like domain, e.g., the N-terminal RuvC-like domain. In some embodiments, the mutation is present in the HNH-like domain. In some embodiments, the mutation is present in both the N-terminal RuvC-like domain and the HNH-like domain.

[0310] With respect to the S. pyogenes sequence, exemplary mutations that can be made in the RuvC or HNH domain include D10A, E762A, H840A, N854A, N863A, and / or D986A.

[0311] In some embodiments, the Cas9 molecule or Cas9 polypeptide is an eiCas9 molecule or eiCas9 polypeptide that includes one or more differences in the RuvC domain and / or the HNH domain compared to a reference Cas9 molecule, and the eiCas9 molecule or eiCas9 polypeptide does not cleave nucleic acids or cleaves them significantly less efficiently than a wild-type molecule, e.g., cleaves less than 50, 25, 10, or 1% of the nucleic acid of the reference Cas9 molecule as measured by an assay described herein, e.g., when compared to a wild-type molecule in a cleavage assay as described herein.

[0312] Whether a particular sequence, e.g., a substitution, is likely to affect one or more activities, e.g., targeting activity, cleavage activity, etc., can be assessed or predicted, e.g., by assessing whether the mutation is conservative. In some embodiments, a "non-essential" amino acid residue, when used in connection with a Cas9 molecule, is a residue that can be altered from the wild-type sequence of a Cas9 molecule, e.g., a naturally occurring Cas9 molecule, e.g., an eaCas9 molecule, without abolishing, or more preferably, substantially altering, a Cas9 activity (e.g., cleavage activity), whereas an alteration of an "essential" amino acid residue results in a substantial loss of activity (e.g., cleavage activity).

[0313] In some embodiments, the Cas9 molecule or Cas9 polypeptide comprises cleavage properties that differ from naturally occurring Cas9 molecules, e.g., that differ from the most closely homologous naturally occurring Cas9 molecule. For example, a Cas9 molecule or Cas9 polypeptide can differ from a naturally occurring Cas9 molecule, e.g., a Staphylococcus aureus, Streptococcus pyogenes, or C. jejuni Cas9 molecule, by, e.g., its ability to modulate, e.g., increase or decrease, cleavage of double-stranded breaks (endonuclease and / or exonuclease activity) compared to a naturally occurring Cas9 molecule (e.g., a Staphylococcus aureus, Streptococcus pyogenes, or C. jejuni Cas9 molecule); by, e.g., its ability to modulate, e.g., increase or decrease, cleavage of a single strand of nucleic acid, e.g., a non-complementary strand of a nucleic acid molecule or a complementary strand of a nucleic acid molecule (nickase activity) compared to a naturally occurring Cas9 molecule (e.g., a Staphylococcus aureus, Streptococcus pyogenes, or C. jejuni Cas9 molecule); or by its ability to cleave nucleic acid molecules, e.g., double-stranded or single-stranded nucleic acid molecules, being eliminated.

[0314] In some embodiments, the modified Cas9 molecule or Cas9 polypeptide is an eaCas9 molecule or eaCas9 polypeptide that comprises one or more of the following activities: a cleavage activity associated with the RuvC domain; a cleavage activity associated with the HNH domain; a cleavage activity associated with the HNH domain and a cleavage activity associated with the RuvC domain.

[0315] In some embodiments, the modified Cas9 molecule or polypeptide is an eiCas9 molecule or eaCas9 polypeptide that does not cleave nucleic acid molecules (either double-stranded or single-stranded nucleic acid molecules) or cleaves nucleic acid molecules with significantly less efficiency, e.g., less than 20, 10, 5, 1, or 0.1% of the cleavage activity of a reference Cas9 molecule, as measured, e.g., by the assays described herein. The reference Cas9 molecule may be a naturally occurring, unmodified Cas9 molecule, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule from Streptococcus pyogenes, S. thermophilus, Staphylococcus aureus, C. jejuni, or Neisseria meningitidis. In some embodiments, the reference Cas9 molecule is the naturally occurring Cas9 molecule with the closest sequence identity or homology. In some embodiments, the eiCas9 molecule or polypeptide lacks substantial cleavage activity associated with the RuvC domain and cleavage activity associated with the HNH domain.

[0316] In some embodiments, the modified Cas9 molecule or polypeptide is an eaCas9 molecule or polypeptide that includes the fixed amino acid residues of Streptococcus pyogenes set forth in the consensus sequence disclosed in WO2015 / 161276, e.g., in Figures 2A-2G therein, and has one or more amino acids that differ (e.g., have a substitution) from the amino acid sequence of Streptococcus pyogenes at one or more residues (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) of SEQ ID NO: 117 or at a residue represented by "-" in the consensus sequence disclosed in WO2015 / 161276, e.g., in Figures 2A-2G therein.

[0317] In some embodiments, the modified Cas9 molecule or polypeptide is one in which the sequence corresponding to a fixed sequence of the consensus sequence disclosed in Figures 2A-2G of WO2015 / 161276 differs by no more than 1, 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in Figures 2A-2G of WO2015 / 161276, or the sequence corresponding to a residue identified by an "*" in the consensus sequence disclosed in Figures 2A-2G of WO2015 / 161276 differs by no more than 1, 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in Figures 2A-2G of WO2015 / 161276. s9 molecule, e.g., a Streptococcus pyogenes Cas9 molecule, differs by no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the "*" residues; and sequences corresponding to residues identified by "-" in the consensus sequences disclosed in Figures 2A-2G of WO2015 / 161276 differ by no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the "-" residues from the corresponding sequence of a naturally occurring Cas9 molecule, e.g., a Streptococcus pyogenes Cas9 molecule.

[0318] In some embodiments, the modified Cas9 molecule or Cas9 polypeptide is an eaCas9 molecule or eaCas9 polypeptide that includes the fixed amino acid residues of S. thermophilus shown in the consensus sequence disclosed in Figures 2A-2G of WO2015 / 161276, and has one or more amino acids that differ from the amino acid sequence of S. thermophilus (e.g., has a substitution) at one or more residues represented by "-" in the consensus sequence disclosed in Figures 2A-2G of WO2015 / 161276 (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues).

[0319] In some embodiments, the modified Cas9 molecule or polypeptide is one in which the sequence corresponding to a fixed sequence of the consensus sequence disclosed in Figures 2A-2G of WO2015 / 161276 differs by no more than 1, 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in Figures 2A-2G of WO2015 / 161276, or a sequence corresponding to a residue identified by an "*" in the consensus sequence disclosed in Figures 2A-2G of WO2015 / 161276 differs by no more than 1, 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in Figures 2A-2G of WO2015 / 161276. and sequences corresponding to residues identified by a "-" in Figures 2A-2G of WO2015 / 161276 differ from the corresponding sequence of a naturally occurring Cas9 molecule, e.g., an S. thermophilus Cas9 molecule, by no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the "-" residues.

[0320] In some embodiments, the modified Cas9 molecule or Cas9 polypeptide is an...

Claims

1. A genetically engineered T cell comprising a modified CD247 locus, wherein the modified CD247 locus comprises a nucleic acid sequence encoding a chimeric antigen receptor comprising an intracellular region that includes a CD3 zeta (CD3ζ) signaling domain.

2. 2. The genetically engineered T cell of claim 1, wherein the modified CD247 locus is an endogenous CD247 locus, and the nucleic acid sequence comprises a transgene sequence encoding a portion of a chimeric antigen receptor, wherein the transgene sequence has been integrated into the endogenous CD247 locus of the T cell, optionally via homology directed repair (HDR).

3. The genetically engineered T cell of claim 2, wherein the entire CD3ζ signaling domain or a fragment of the CD3ζ signaling domain is encoded by an open reading frame of the endogenous CD247 locus or a subsequence thereof.

4. 4. The genetically engineered T cell of claim 2 or claim 3, wherein the nucleic acid sequence encoding the chimeric antigen receptor comprises an in-frame fusion of (i) a transgene sequence encoding a portion of the chimeric antigen receptor and (ii) an open reading frame of the endogenous CD247 locus or a subsequence thereof.

5. 5. The genetically engineered T cell of any one of claims 2-4, wherein the transgene sequence is in frame with one or more exons of the open reading frame of the endogenous CD247 locus or a subsequence thereof.

6. 6. The genetically engineered T cell of any one of claims 2 to 5, wherein the transgene sequence does not include a sequence encoding a 3'UTR and / or does not include an intron.

7. The genetically engineered T cell of any one of claims 2 to 6, wherein the transgene sequence encodes a fragment of the CD3ζ signaling domain or does not encode the CD3ζ signaling domain or a fragment thereof.

8. 8. The genetically engineered T cell of any one of claims 3-7, wherein the open reading frame or subsequence thereof comprises at least one intron and at least one exon of the endogenous CD247 locus and / or encodes the 3'UTR of the endogenous CD247 locus.

9. 9. The genetically engineered T cell of any one of claims 2-8, wherein the transgene sequence is downstream of exon 1 and upstream of exon 8 of the open reading frame of the endogenous CD247 locus; optionally downstream of exon 1 and upstream of exon 3 of the open reading frame of the endogenous CD247 locus.

10. 10. The genetically engineered T cell of any one of claims 2-9, wherein at least a fragment of the CD3 zeta signaling domain of the encoded chimeric antigen receptor, optionally the entire CD3 zeta signaling domain, is encoded by an open reading frame of an endogenous CD247 locus or a subsequence thereof, and optionally the CD3 zeta signaling domain is encoded by a sequence of nucleotides comprising at least a portion of exon 2 and exons 3-8 of the open reading frame of the endogenous CD247 locus; or a sequence of nucleotides that does not include exon 1, does not include the entirety of exon 1, and / or does not include the entirety of exon 2 of the open reading frame of the endogenous CD247 locus.

11. 11. The genetically engineered T cell of any one of claims 1 to 10, wherein the encoded chimeric antigen receptor is capable of signaling through the CD3ζ signaling domain.

12. 12. The genetically engineered T cell of any one of claims 1-11, wherein the encoded CD3 zeta signaling domain comprises a sequence selected from any one of SEQ ID NOs: 13-15, or a sequence exhibiting at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 13-15.

13. 13. The genetically engineered T cell of any one of claims 1-12, wherein the chimeric antigen receptor comprises: (i) an extracellular region comprising a binding domain; (ii) a transmembrane domain; and (iii) an intracellular region, and optionally, the binding domain is or comprises an antibody or an antigen-binding fragment thereof.

14. the binding domain is capable of binding to a target antigen associated with, specific for, and / or expressed on cells or tissues of a disease, disorder, or condition, and optionally the target antigen is a tumor antigen; and / or Target antigens include 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;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), kappa 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, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligand, melan-A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, preferentially expressed antigen in melanoma (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, 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;dopachrome tautomerase, dopachrome delta-isomerase, or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms' tumor 1 (WT-1), a pathogen-specific or pathogen-expressed antigen, or an antigen associated with a universal tag, and / or a biotinylated molecule, and / or a molecule expressed by HIV, HCV, HBV, or other pathogens; 14. The genetically engineered T cell of claim 13.

15. The extracellular region comprises a spacer, and optionally the spacer is operably linked between the binding domain and the transmembrane domain, and / or the spacer is linked to an immunoglobulin hinge region, and / or a C H 2 area and C H 15. The genetically engineered T cell of claim 13 or 14, comprising three regions.

16. 16. The genetically engineered T cell of any one of claims 1-15, wherein the intracellular region comprises one or more costimulatory signaling domains, optionally wherein the one or more costimulatory signaling domains comprise the intracellular signaling domain of CD28, 4-1BB, or ICOS, or a signaling portion thereof.

17. The transgene sequence optionally comprises, in order: (i) a binding domain, optionally a single chain Fv fragment (scFv); and (ii) a sequence derived from a human immunoglobulin hinge, optionally derived from IgG1, IgG2, IgG4, or modified versions thereof; H 2nd area and / or C H and (iii) a transmembrane domain, optionally from human CD28; and (iv) an intracellular region, optionally from human 4-1BB, comprising a costimulatory signaling domain; and / or The modified CD247 locus comprises, in order: (i) a binding domain, optionally an scFv; and (ii) a sequence derived from a human immunoglobulin hinge, optionally derived from IgG1, IgG2, IgG4, or a modified version thereof; H 2nd area and / or C H (iii) a transmembrane domain, optionally derived from human CD28; (iv) an intracellular region, optionally derived from human 4-1BB, comprising a sequence of nucleotides encoding a costimulatory signaling domain; and a CD3ζ signaling domain. The genetically engineered T cell of any one of claims 2 to 16.

18. 18. The genetically engineered T cell of any one of claims 2-17, wherein the transgene sequence comprises a sequence of nucleotides encoding at least one additional protein, optionally wherein the at least one additional protein is a surrogate marker, optionally wherein the surrogate marker is a truncated receptor, optionally wherein the truncated receptor lacks an intracellular signaling domain and / or is incapable of mediating intracellular signaling when bound by its ligand.

19. 19. The genetically engineered T cell of any one of claims 2-18, wherein the transgene sequence comprises one or more multicistronic elements, optionally wherein the one or more multicistronic elements is or comprises a ribosomal skipping sequence, and optionally wherein the ribosomal skipping sequence is a T2A, P2A, E2A, or F2A element.

20. the transgene sequence comprises a sequence of nucleotides encoding a portion of a chimeric antigen receptor, and one or more multicistronic elements are located upstream of the sequence of nucleotides encoding the portion of the chimeric antigen receptor; and / or are located between the sequence of nucleotides encoding the portion of the chimeric antigen receptor and a sequence of nucleotides encoding at least one additional protein; and / or The chimeric antigen receptor is a multi-chain CAR, and one or more multicistronic elements are located between the sequence of nucleotides encoding one chain of the multi-chain CAR and the sequence of nucleotides encoding another chain of the multi-chain CAR.

20. The genetically engineered T cell of claim 19.

21. 21. The genetically engineered T cell of any one of claims 2-20, wherein the modified CD247 locus comprises a promoter and / or control or regulatory elements of the endogenous CD247 locus operably linked to regulate expression of a nucleic acid sequence encoding a chimeric antigen receptor; or wherein the modified CD247 locus comprises one or more heterologous control or regulatory elements operably linked to regulate expression of the chimeric antigen receptor or a portion thereof.

22. 22. The genetically engineered T cell of any one of claims 1 to 21, wherein the T cell is a primary T cell derived from a subject, and optionally the subject is a human.

23. 23. The genetically engineered T cell of any one of claims 1 to 22, wherein the T cell is a CD8+ T cell or a subtype thereof or a CD4+ T cell or a subtype thereof.

24. (a) a nucleic acid sequence encoding a chimeric antigen receptor or a portion thereof; wherein the portion of the chimeric antigen receptor encoded by the nucleic acid sequence comprises (i) an extracellular region comprising a binding domain and (ii) a transmembrane domain; and (b) one or more homology arms linked 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 the CD247 locus or a subsequence thereof A polynucleotide comprising:

25. 25. The polynucleotide of claim 24, wherein the nucleic acid sequence of (a) encodes a portion of a chimeric antigen receptor, and the nucleic acid sequence of (a) does not encode the entire CD3 zeta (CD3ζ) signaling domain of the intracellular region.

26. 26. The polynucleotide of claim 24 or 25, wherein the nucleic acid sequence of (a) is a sequence that is foreign or heterologous to the open reading frame of the endogenous genomic CD247 locus of a T cell, optionally a human T cell.

27. 27. The polynucleotide of any one of claims 24-26, wherein said open reading frame or subsequence thereof comprises at least one intron and at least one exon and / or 3'UTR of an endogenous CD247 locus of a T cell, optionally a human T cell.

28. 28. The polynucleotide of any one of claims 24 to 27, wherein at least a fragment of the CD3 zeta signaling domain, and optionally the entire CD3 zeta signaling domain, is encoded by an open reading frame of the endogenous CD247 locus or a subsequence thereof when the chimeric antigen receptor is expressed from a cell into which the polynucleotide has been introduced.

29. 29. The polynucleotide of any one of claims 24 to 28, wherein the nucleic acid sequence of (a) encodes a fragment of the CD3 zeta signaling domain.

30. 29. The polynucleotide of any one of claims 24 to 28, wherein the nucleic acid sequence of (a) does not encode the CD3 zeta signaling domain or a fragment thereof.

31. 31. The polynucleotide of any one of claims 24 to 30, wherein the nucleic acid sequence of (a) does not include a sequence encoding a 3'UTR and / or does not include an intron.

32. 32. The polynucleotide of any one of claims 24 to 31, wherein the nucleic acid sequence of (a) comprises a sequence of nucleotides that is in frame with one or more exons of the open reading frame of the CD247 locus, or a subsequence thereof, comprised in one or more arms of homology; and optionally, one or more regions of the open reading frame is or comprises: a sequence upstream of exon 8 of the open reading frame of the CD247 locus; a sequence upstream of exon 3 of the open reading frame of the CD247 locus, optionally including exon 3 of the open reading frame of the CD247 locus; and / or a sequence comprising at least a portion of exon 2 of the open reading frame of the CD247 locus.

33. 33. The polynucleotide of any one of claims 24-32, wherein one or more of the homology arms does not include exon 1, does not include the entirety of exon 1, and / or does not include the entirety of exon 2 of the open reading frame of the endogenous CD247 locus.

34. 34. The polynucleotide of any one of claims 24-33, wherein the one or more homology arms comprise a 5' homology arm and a 3' homology arm, and wherein the polynucleotide comprises the structure [5' homology arm]-[nucleic acid sequence of (a)]-[3' homology arm], and optionally the 5' homology arm and the 3' homology arm are independently 200, 300, 400, 500, 600, 700, or 800, or about 200, 300, 400, 500, 600, 700, or 800 nucleotides in length, or any value between any of these, or 300 or more than 300 nucleotides in length, optionally 400, 500, or 600, or about 400, 500, or 600 nucleotides in length, or any value between any of these.

35. 35. The polynucleotide of any one of claims 24 to 34, wherein the 5' homology arm comprises the sequence shown in SEQ ID NO:80, or a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:80; and / or the 3' homology arm comprises the sequence shown in SEQ ID NO:81, or a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:

81.

36. 36. The polynucleotide of any one of claims 25 to 35, wherein the binding domain comprises an antibody or an antigen-binding fragment thereof.

37. The binding domain is capable of binding to a target antigen associated with, specific for, and / or expressed on cells or tissues of a disease, disorder, or condition, and optionally the target antigen is a tumor antigen and / or the target antigen is selected from the group consisting of 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; NY- also known as 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), epidermal growth factor receptor type III mutated (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;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), kappa 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, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligand, melan-A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, preferentially expressed antigen in melanoma (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, 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;37. The polynucleotide of any one of claims 25 to 36, wherein the polynucleotide is 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), a pathogen-specific antigen or pathogen-expressed antigen, or an antigen associated with a universal tag, and / or a biotinylated molecule, and / or a molecule expressed by HIV, HCV, HBV, or other pathogens;

38. 38. The polynucleotide of any one of claims 24-37, wherein the nucleic acid sequence of (a) comprises a sequence of nucleotides encoding at least one additional protein, optionally wherein the at least one additional protein is a surrogate marker, optionally wherein the surrogate marker is a truncated receptor, optionally wherein the truncated receptor lacks an intracellular signaling domain and / or is incapable of mediating intracellular signaling when bound by its ligand.

39. 39. The polynucleotide of any one of claims 24-38, wherein the nucleic acid sequence of (a) comprises one or more multicistronic elements, optionally wherein the one or more multicistronic elements are or comprise ribosomal skipping sequences, and optionally wherein the ribosomal skipping sequence is a T2A, P2A, E2A, or F2A element.

40. the nucleic acid of (a) comprises a sequence of nucleotides encoding a portion of a chimeric antigen receptor, and one or more multicistronic elements are located upstream of the sequence of nucleotides encoding the portion of the chimeric antigen receptor; and / or are located between the sequence of nucleotides encoding the portion of the chimeric antigen receptor and a sequence of nucleotides encoding at least one additional protein; and / or The chimeric antigen receptor is a multi-chain CAR, and one or more multicistronic elements are located between the sequence of nucleotides encoding one chain of the multi-chain CAR and the sequence of nucleotides encoding another chain of the multi-chain CAR.

40. The polynucleotide of claim 39.

41. 41. The polynucleotide of any one of claims 24-40, wherein the nucleic acid sequence of (a) comprises one or more heterologous control or regulatory elements operably linked to regulate expression of the chimeric antigen receptor or portion thereof.

42. 42. The polynucleotide of any one of claims 24 to 41, which is a linear polynucleotide, optionally a double-stranded or single-stranded polynucleotide, and optionally is from at or about 2500 to at or about 5000 nucleotides, from at or about 3500 to at or about 4500 nucleotides, or from at or about 3750 nucleotides to at or about 4250 nucleotides in length.

43. 43. A viral vector comprising the polynucleotide of any one of claims 24 to 42, wherein optionally the viral vector is an AAV vector, optionally the AAV vector is an AAV2 or AAV6 vector, or the viral vector is a retroviral vector, optionally a lentiviral vector.

44. 44. An in vitro method of generating a genetically engineered T cell, comprising introducing into a T cell the polynucleotide of any one of claims 24 to 42, or the viral vector of claim 43, under conditions to induce homology directed repair (HDR).

45. The method of claim 44, wherein the T cell comprises a gene disruption in the CD247 locus.

46. (a) introducing into a T cell one or more agents capable of inducing gene disruption at a target site within the endogenous CD247 locus of the T cell; and (b) introducing into said T cells the polynucleotide of any one of claims 24 to 42 or the viral vector of claim 43. Including, Steps (a) and (b) are simultaneous or sequential in any order; In vitro methods for generating genetically engineered T cells.

47. 47. The method of claim 46, wherein the polynucleotide is introduced after the introduction of one or more agents.

48. 48. The method of any one of claims 44-47, wherein the nucleic acid sequence encoding the chimeric antigen receptor or a portion thereof is integrated into the endogenous CD247 locus via homology directed repair (HDR).

49. 1. An in vitro method for producing a genetically engineered T cell, comprising the step of introducing into a T cell a polynucleotide comprising a nucleic acid sequence encoding a chimeric antigen receptor or a portion thereof, wherein the portion of the chimeric antigen receptor encoded by the nucleic acid sequence comprises (i) an extracellular region comprising a binding domain, and (ii) a transmembrane domain; The method, wherein the T cell has a gene disruption within the CD247 locus of the T cell, and a nucleic acid sequence encoding the chimeric antigen receptor or a portion thereof is integrated into the endogenous CD247 locus via homology directed repair (HDR).

50. 50. The method of any one of claims 44-49, wherein said method creates a modified CD247 locus, said modified CD247 locus comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising an intracellular region comprising a CD3 zeta signaling domain, wherein at least a fragment of the CD3 zeta signaling domain is encoded by an open reading frame of an endogenous CD247 locus.

51. 51. The method of any one of claims 49-50, wherein the nucleic acid sequence encoding the chimeric antigen receptor or a portion thereof does not include a sequence encoding a 3'UTR and / or does not include an intron.

52. 52. The method of any one of claims 50-51, wherein the chimeric antigen receptor, when expressed by a cell into which the polynucleotide has been introduced, is capable of signaling through the CD3 zeta signaling domain.

53. 53. The method of any one of claims 50-52, wherein the encoded CD3 zeta signaling domain comprises a sequence selected from any one of SEQ ID NOs:13-15, or a sequence exhibiting at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs:13-15.

54. the polynucleotide comprises one or more homology arms linked to the nucleic acid sequence, the one or more homology arms comprising a sequence homologous to one or more regions of an open reading frame; and The method of any one of claims 49-53, wherein the one or more homology arms comprise a 5' homology arm and a 3' homology arm, and wherein the polynucleotide comprises the structure [5' homology arm]-[nucleic acid sequence encoding a chimeric antigen receptor or a portion thereof]-[3' homology arm].

55. 55. The method of any one of claims 49-54, wherein said gene disruption is effected by introducing into the T cell one or more agents capable of inducing gene disruption at a target site within the CD247 locus of the T cell.

56. one or more agents capable of inducing gene disruption, DNA-binding proteins or DNA-binding nucleic acids that specifically bind or hybridize to a target site, fusion proteins comprising a DNA-targeting protein and a nuclease, or RNA-guided nucleases and optionally, the one or more agents include Combinations of zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), and / or CRISPR-Cas9 that specifically bind to, recognize, or hybridize with target sites and optionally, each of the one or more agents comprises a guide RNA (gRNA) having a targeting domain complementary to at least one target site.

56. The method of any one of claims 46-48 and 55.

57. 57. The method of claim 56, wherein the one or more agents are introduced as a ribonucleoprotein (RNP) complex comprising the gRNA and Cas9 protein, and optionally, the RNP is introduced via electroporation, particle gun, calcium phosphate transfection, compaction, or squeezing of cells.

58. gRNA, 58. The method of claim 56 or 57, wherein the targeting domain sequence is selected from the group consisting of:

59. gRNA, The method of any one of claims 56 to 58, wherein the targeting domain sequence is:

60. gRNA, The method of any one of claims 56 to 58, wherein the targeting domain sequence is:

61. 61. The method of any one of claims 44 to 60, wherein the T cells are primary T cells derived from a subject, and optionally, the subject is human.

62. The method of any one of claims 44 to 61, wherein the T cells are CD8+ T cells or subtypes thereof or CD4+ T cells or subtypes thereof.

63. 63. The method of any one of claims 44 to 62, wherein the polynucleotide is comprised in a viral vector, optionally wherein the viral vector is an AAV vector, optionally wherein the AAV vector is an AAV2 vector or an AAV6 vector, or wherein the viral vector is a retroviral vector, optionally a lentiviral vector.

64. 63. The method of any one of claims 44 to 62, wherein the polynucleotide is a linear polynucleotide, optionally a double-stranded or single-stranded polynucleotide.

65. 61. The method of any one of claims 46-48 and 55-60, wherein said polynucleotide is introduced after introduction of said one or more agents, optionally wherein said polynucleotide is introduced immediately after introduction of said agent or within about 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 thereafter.

66. 66. The method of any one of claims 46-48, 55-60, and 65, wherein prior to the introduction of said one or more agents, said method comprises incubating the cells in vitro with one or more stimulatory agents under conditions to stimulate or activate one or more immune cells, optionally wherein said one or more stimulatory agents comprise an anti-CD3 antibody and / or an anti-CD28 antibody, optionally anti-CD3 / anti-CD28 beads, optionally at a bead to cell ratio of 1:1 or about 1:

1.

67. at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the plurality of engineered cells produced by the method comprise a gene disruption of at least one target site within the CD247 locus; and / or 67. The method of any one of claims 44-66, wherein at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the plurality of engineered cells produced by said method, or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the cells, express a chimeric antigen receptor.

68. 24. A composition comprising the genetically engineered T cell of any one of claims 1 to 23, or a plurality of genetically engineered T cells of any one of claims 1 to 23.

69. 69. The composition of claim 68, wherein the composition comprises CD4+ T cells and / or CD8+ T cells, optionally CD4+ T cells and CD8+ T cells, wherein the ratio of CD4+ T cells to CD8+ T cells is at or about 1:3 to 3:1, optionally 1:

1.

70. 70. The composition of claim 68 or 69, wherein cells expressing a chimeric antigen receptor account for at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all cells in the composition, or of all CD4+ T cells or CD8+ T cells in the composition.

71. 71. The genetically engineered T cell, plurality of genetically engineered T cells, or composition of any one of claims 1 to 23 and 68 to 70 for use in the treatment of a disease or disorder.

72. 71. The engineered T cell, plurality of engineered T cells, or composition of any one of claims 1-23 and 68-70 for use in the manufacture of a medicament for treating a disease or disorder.

73. 73. The genetically engineered T cell, plurality of genetically engineered T cells, or composition for use of claim 71 or 72, wherein the disease or disorder is a cancer or tumor, optionally wherein the cancer or tumor is a hematological malignancy, optionally a lymphoma, leukemia, or plasma cell malignancy, or optionally wherein the cancer or tumor is a solid tumor, optionally wherein the solid tumor is non-small cell lung cancer (NSCLC) or head and neck squamous cell carcinoma (HNSCC).

74. one or more agents capable of inducing gene disruption at a target site within the CD247 locus; A polynucleotide according to any one of claims 24 to 42 or a viral vector according to claim 43. Includes a kit.

75. one or more agents capable of inducing gene disruption at a target site within the CD247 locus; a polynucleotide comprising a nucleic acid sequence encoding a chimeric antigen receptor or a portion thereof, wherein the nucleic acid sequence encoding the chimeric antigen receptor or a portion thereof is targeted for integration at or near the target site via homology-directed repair (HDR); A kit for carrying out the method of any one of claims 44 to 67.

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