Modified immune effector cells with improved resistance to natural killer cell-mediated allorejection

Abstract

Chimeric C-type lectin domain family 2, member D (chCLEC2d) polypeptides, immune effector cells expressing the chCLEC2d polypeptides, and methods for use of the immune effector cells in treating a disease or condition in a subject. Allogeneic immune effector cells expressing the chCLEC2d polypeptides have improved resistance to natural killer (NK) cell-mediated allorejection by the immune system of a subject to which the immune effector cells are administered.

Description

[0001] MODIFIED IMMUNE EFFECTOR CELLS WITH IMPROVED RESISTANCE TO NATURAL KILLER CELL-MEDIATED ALLOREJECTION

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims priority to U.S. Provisional Applications No. 63 / 564,681, filed March 13, 2024, 63 / 597,888, filed November 10, 2023, and 63 / 516,398, filed July 28, 2023, the entire contents of each of which are hereby incorporated by reference in its entirety.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The Sequence Listing XML file, created on July 22, 2024, is named 180802-049804PCT_SL.xml and is 3,378,536 bytes in size.

[0006] BACKGROUND

[0007] Autologous and allogeneic immunotherapies are approaches for treating a disease or disorder (e.g., a neoplasia or an autoimmune disease) in which immune cells (e.g., T cells) expressing chimeric antigen receptors are administered to a subject. According to some methods, to generate an immune cell that expresses a chimeric antigen receptor (CAR), the immune cell is first collected from the subject (autologous) or a donor separate from the subject receiving treatment (allogeneic) and genetically modified to express the chimeric antigen receptor. The resulting cell expresses the chimeric antigen receptor on its cell surface (e.g., CAR-T cell), and upon administration to the subject, the chimeric antigen receptor binds to the antigen expressed by a cell associated with the disease or disorder. This interaction with the antigen activates the CAR-expressing immune cell (e.g., a CAR-T cell), which then kills, inactivates, or neutralizes cells or molecules associated with the disease or disorder. But for autologous or allogeneic cell therapy to be effective and efficient, significant conditions and cellular responses, such as allorej ection mediated by T cells and / or natural killer (NK) cells must be addressed. Autologous cell therapies have a number of disadvantages, including long manufacturing times and the requirement that the patient cells are suitable despite previous therapies or disease state. Challenges are also associated with allogeneic cell therapy, including graft-versus-host disease (GVHD), and host rejection of CAR-T cells. Currently, CAR-expressing immune effector cells are susceptible to allorej ection by the immune cells of a subject to which they are administered. Thus, there is a significant need for techniques for improving immune effector cell resistance to allorej ection.

[0008] SUMMARY

[0009] As described herein, the present disclosure features chimeric C-type lectin domain family 2, member D (chCLEC2d) polypeptides, immune effector cells expressing the chCLEC2d polypeptides, and methods for use of the immune effector cells in treating a disease or condition in a subject. Allogeneic immune effector cells expressing the chCLEC2d polypeptides have improved resistance to natural killer (NK) cell-mediated allorej ection by the immune system of a subject to which the immune effector cells are administered.

[0010] In one aspect, the disclosure features a chimeric C-type lectin domain family 2, member D (CLEC2d) polypeptide containing in order from N-terminus to C-terminus a CLEC2d extracellular domain and a transmembrane domain.

[0011] In another aspect, the disclosure features a polynucleotide encoding the polypeptide of any aspect of the disclosure, or embodiments thereof.

[0012] In another aspect, the disclosure features a vector containing the polynucleotide of any aspect of the disclosure, or embodiments thereof.

[0013] In another aspect, the disclosure features a cell containing the polynucleotide, vector, or polypeptide of any aspect of the disclosure, or embodiments thereof.

[0014] In another aspect, the disclosure features a method for modifying a T cell to increase resistance of the T cell to natural killer cell killing. The method involves expressing in the T cell the polypeptide of any aspect of the disclosure, or embodiments thereof. Optionally, the T cell expresses reduced levels of cluster of differentiation 54 (CD54), cluster of differentiation 58 (CD58), human leukocyte antigen A, human leukocyte antigen B, and / or human leukocyte antigen C relative to a wild-type T cell.

[0015] In another aspect, the disclosure features a cell prepared by the method of any aspect of the disclosure, or embodiments thereof.

[0016] In another aspect, the disclosure features a pharmaceutical composition containing the cell of any aspect of the disclosure, or embodiments thereof, and a pharmaceutically acceptable excipient.

[0017] In another aspect, the disclosure features a method for treating a neoplasia in a subject in need thereof. The method involves administering to the subject the pharmaceutical composition of any aspect of the disclosure, or embodiments thereof, where the cell expresses a chimeric antigen receptor targeting a polypeptide associated with the neoplasia. In another aspect, the disclosure features a polypeptide containing in order from N- terminus to C-terminus: a CD8 signal peptide, a C-type lectin domain family 2, member D (CLEC2d) extracellular domain, a CD8 hinge domain, a CD8 transmembrane domain, and a cytoplasmic domain. The polypeptide is capable of being expressed on the surface of a T cell and of binding a CD161 polypeptide.

[0018] In another aspect, the disclosure features a T cell expressing the polypeptide of any aspect of the disclosure, or embodiments thereof. The T cell expresses a chimeric antigen receptor (CAR) and expresses reduced levels of beta-2-microglobulin, CD161, and / or CIITA relative to a wild-type T cell.

[0019] In another aspect, the disclosure features a T cell expressing the polypeptide of any aspect of the disclosure, or embodiments thereof. The T cell expresses a chimeric antigen receptor (CAR) and expresses reduced levels of beta-2-microglobulin, CD54, and / or CD58 relative to a wild-type cell. In another aspect, the disclosure features a method for preparing a modified T cell. The method involves A) contacting the T cell with a base editor system containing an ABE8.20 base editor, or a polynucleotide encoding the base editor, and two or more guide polynucleotides. The guide polynucleotides each target the base editor to alter a nucleotide within a polynucleotide encoding a polypeptide selected from one or more of a CD54, CD58, CD161 polypeptide, a beta-2-microglobulin (B2M) polypeptide, and a Class II Major Histocompatibility Complex Transactivator (CIITA) polypeptide, thereby reducing or eliminating expression of the CD54, the CD58, the CD161 polypeptide, the B2M polypeptide, and / or the CIITA polypeptide in the T cell. The method also involves B) expressing in the T cell a polypeptide containing an amino acid sequence with at least 85% sequence identity to the following amino acid sequence: MALPVTALLLPLALLLHAARPRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRF CDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYL NDKGASSARHYTERKWICSKSDIHVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSA (SEQ ID NO: 440; BTx_CM496 / mbCLEC2d), thereby increasing resistance of the T cell to being killed by a natural killer cell in a mixed leukocyte reaction.

[0020] In another aspect, the disclosure features a method for preparing a modified T cell. The method involves A) contacting the T cell with a base editor system containing an ABE8.20 base editor, or a polynucleotide encoding the base editor, and a guide polynucleotide. The guide polynucleotide targets the base editor to alter a nucleotide within a polynucleotide encoding a beta-2-microglobulin (B2M) polypeptide, thereby reducing or eliminating expression of the B2M polypeptide in the T cell. The method also involves B) expressing in the T cell a polypeptide containing an amino acid sequence with at least 85% sequence identity to the following amino acid sequence:

[0021] MALPVTALLLPLALLLHAARPRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRF CDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYL NDKGASSARHYTERKWICSKSDIHVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSA (SEQ ID NO: 440;

[0022] BTx_CM496 / mbCLEC2d), thereby increasing resistance of the T cell to being killed by a natural killer cell in a mixed leukocyte reaction.

[0023] In another aspect, the disclosure provides a kit suitable for use in any aspect of the disclosure, or embodiments thereof. The kit contains the polypeptide, polynucleotide, cell, and / or pharmaceutical composition of any aspect of the disclosure, or embodiments thereof.

[0024] In another aspect, the disclosure provides a chimeric C-type lectin domain family 2, member D (chCLEC2d) polypeptide containing a CLEC2d extracellular domain and a signal peptide.

[0025] In another aspect, the disclosure provides a method for preparing a modified T cell. The method involves A) contacting the T cell with a base editor system containing an ABE8.20 base editor, or a polynucleotide encoding the base editor, and a guide polynucleotide. The guide polynucleotide targets the base editor to alter a nucleotide within a polynucleotide encoding a beta-2-microglobulin (B2M) polypeptide, thereby reducing or eliminating expression of the B2M polypeptide in the T cell. The method also involves B) expressing in the T cell a polypeptide containing an amino acid sequence with at least 85% sequence identity to an amino acid sequence selected from one or more of: MALPVTALLLPLALLLHAARPRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRF CDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYL NDKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3325; BTx_LC001);

[0026] MYRMQLLSCIALSLALVTNSRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRFC DSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYLN DKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3326; BTx_LC002); and MKYTSYILAFQLCIVLGSLGCYCRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQ RFCDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECA YLNDKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3327; BTx_LC003 / sCLEC2d). The method increases resistance of the T cell to being killed by a natural killer cell in a mixed leukocyte reaction. In another aspect, the disclosure provides a method for preparing a modified T cell. The method involves A) contacting the T cell with a base editor system containing an ABE8.20 base editor, or a polynucleotide encoding the base editor, and two guide polynucleotides. One guide polynucleotide targets the base editor to alter a nucleotide within a polynucleotide encoding a beta-2-microglobulin (B2M) polypeptide, thereby reducing or eliminating expression of the B2M polypeptide in the T cell. Another guide polynucleotide targets the base editor to alter a nucleotide within a polynucleotide encoding a Major Histocompatibility Complex Transactivator (CIITA) polypeptide, thereby reducing or eliminating expression of the CIITA polypeptide in the T cell. The method also involves B) expressing in the T cell a polypeptide having an amino acid sequence with at least 85% sequence identity to an amino acid sequence selected from one or more of: MALPVTALLLPLALLLHAARPRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRF CDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYL NDKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3325; BTx_LC001); MYRMQLLSCIALSLALVTNSRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRFC DSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYLN DKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3326; BTx_LC002); and MKYTSYILAFQLCIVLGSLGCYCRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQ RFCDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECA YLNDKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3327; BTx_LC003 / sCLEC2d). The method increases resistance of the T cell to being killed by a natural killer cell in a mixed leukocyte reaction.

[0027] In another aspect, the disclosure features a method for preparing a modified T cell. The method involves A) contacting the T cell with a base editor system containing an ABE8.20 base editor, or a polynucleotide encoding the base editor, and three guide polynucleotides. One guide polynucleotide targets the base editor to alter a nucleotide within a polynucleotide encoding a beta-2-microglobulin (B2M) polypeptide, thereby reducing or eliminating expression of the B2M polypeptide in the T cell. Another guide polynucleotide targets the base editor to alter a nucleotide within a polynucleotide encoding a CD54 polypeptide, thereby reducing or eliminating expression of the CD54 polypeptide in the T cell. Another guide polynucleotide targets the base editor to alter a nucleotide within a polynucleotide encoding a CD58 polypeptide, thereby reducing or eliminating expression of the CD58 polypeptide in the T cell. The method further involves B) expressing in the T cell a polypeptide containing an amino acid sequence with at least 85% sequence identity to an amino acid sequence selected from one or more of:

[0028] MALPVTALLLPLALLLHAARPRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRF CDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYL NDKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3325; BTx_LC001);

[0029] MYRMQLLSCIALSLALVTNSRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRFC DSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYLN DKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3326; BTx_LC002); and MKYTSYILAFQLCIVLGSLGCYCRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQ RFCDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECA YLNDKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3327; BTx_LC003 / sCLEC2d). The method is associated with an increase in resistance of the T cell to being killed by a natural killer cell in a mixed leukocyte reaction.

[0030] In another aspect, the disclosure features a method for preparing a modified T cell. The method involves A) contacting the T cell with a base editor system containing an ABE8.20 base editor, or a polynucleotide encoding the base editor, and three guide polynucleotides. The guide polynucleotides target the base editor to alter a nucleotide within a polynucleotide encoding a beta-2-microglobulin (B2M) polypeptide, a polynucleotide encoding a cluster of differentiation 24 (CD54) polypeptide, and a polynucleotide encoding a cluster of differentiation 58 (CD58) polypeptide, thereby reducing or eliminating expression of the B2M polypeptide, the CD54 polypeptide, and the CD58 polypeptide in the T cell. The method further involves B) expressing in the T cell a polypeptide containing an amino acid sequence with at least 85% sequence identity to the following amino acid sequence: MALPVTALLLPLALLLHAARPRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRF CDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYL NDKGASSARHYTERKWICSKSDIHVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSA (SEQ ID NO: 440; BTx_CM496 / mbCLEC2d). The method is associated with an increase in resistance of the T cell to being killed by a natural killer cell in a mixed leukocyte reaction.

[0031] In another aspect, the disclosure features a method for preparing a modified T cell. The method involves A) contacting the T cell with a base editor system containing an ABE8.20 base editor, or a polynucleotide encoding the base editor, and at least two guide polynucleotides. One guide polynucleotide targets the base editor to alter a nucleotide within a polynucleotide encoding a beta-2-microglobulin (B2M) polypeptide, thereby reducing or eliminating expression of the B2M polypeptide in the T cell. Another guide polynucleotide targets the base editor to i) alter a nucleotide within a polynucleotide encoding a CD54 polypeptide, thereby reducing or eliminating expression of the CD54 polypeptide in the T cell, or ii) alter a nucleotide within a polynucleotide encoding a CD58 polypeptide, thereby reducing or eliminating expression of the CD58 polypeptide in the T cell. The method further involves B) expressing in the T cell a polypeptide containing an amino acid sequence with at least 85% sequence identity to an amino acid sequence selected from one or more of: MALPVTALLLPLALLLHAARPRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRF CDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYL NDKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3325; BTx_LC001);

[0032] MYRMQLLSCIALSLALVTNSRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRFC DSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYLN DKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3326; BTx_LC002); and MKYTSYILAFQLCIVLGSLGCYCRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQ RFCDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECA YLNDKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3327; BTx_LC003 / sCLEC2d). The method results in increasing resistance of the T cell to being killed by a natural killer cell in a mixed leukocyte reaction.

[0033] In another aspect, the disclosure features a method for preparing a modified T cell. The method involves A) contacting the T cell with a base editor system containing an ABE8.20 base editor, or a polynucleotide encoding the base editor, and at least two guide polynucleotides. One guide polynucleotide targets the base editor to alter a nucleotide within a polynucleotide encoding a beta-2-microglobulin (B2M) polypeptide, thereby reducing or eliminating expression of the B2M polypeptide. Another guide polynucleotide targets the base editor to i) alter a polynucleotide encoding a cluster of differentiation 58 (CD58) polypeptide, thereby reducing or eliminating expression of the CD58 polypeptide, or ii) alter a polynucleotide encoding a cluster of differentiation 54 (CD54) polypeptide, thereby reducing or eliminating expression of the CD54 polypeptide. The method further involves B) expressing in the T cell a polypeptide containing an amino acid sequence with at least 85% sequence identity to the following amino acid sequence: MALPVTALLLPLALLLHAARPRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRF CDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYL NDKGASSARHYTERKWICSKSDIHVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSA (SEQ ID NO: 440; BTx_CM496 / mbCLEC2d). The method results in increasing resistance of the T cell to being killed by a natural killer cell in a mixed leukocyte reaction.

[0034] In any aspect of the disclosure, or embodiments thereof, the CLEC2d extracellular domain contains an amino acid sequence with at least about 85% sequence identity to the following sequence and is capable of binding a CD161 polypeptide: RANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRFCDSQDADLAQVESFQELNFLL RYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYLNDKGASSARHYTERKWICSKS DIHV (SEQ ID NO: 461).

[0035] In any aspect of the disclosure, or embodiments thereof, the transmembrane domain contains a sequence with at least about 85% sequence identity to a sequence selected from one or more of: FFLIMFLTIIVCGMVAALSAI (SEQ ID NO: 463; CLEC2d transmembrane domain); MALIVLGGVAGLLLFIGLGIFF (SEQ ID NO: 470; CD4 transmembrane domain); IYIWAPLAGTCGVLLLSLVITLY (SEQ ID NO: 492; CD8a transmembrane domain); PFFFCCFIAVAMGIRFIIMVT (SEQ ID NO: 466; NKG2D transmembrane domain); and PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO: 474; decay accelerating factor transmembrane domain).

[0036] In any aspect of the disclosure, or embodiments thereof, the polypeptide further contains a hinge domain disposed between the CLEC2d extracellular domain and the transmembrane domain. In some embodiments, the hinge domain is between about 10 and 75 amino acids in length. In some embodiments, the hinge domain is between about 35 and 55 amino acids in length. In some embodiments, the hinge domain contains an amino acid sequence containing from about 20% to about 30% proline and / or cytosine amino acid residues. In some embodiments, the hinge domain contains an amino acid sequence with at least about 85% amino acid sequence identity to the following amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 468; CD8a hinge domain).

[0037] In any aspect of the disclosure, or embodiments thereof, the polypeptide shows increased levels of surface expression in a T cell relative to a wild-type CLEC2d polypeptide. In some embodiments, the increase is at least about 1.1-fold. In some embodiments, the increase is at least about 1.5-fold.

[0038] In any aspect of the disclosure, or embodiments thereof, the polypeptide further contains at the C-terminus a truncated cluster of differentiation zeta (CD3z) cytoplasmic domain. In some embodiments, the CD3z cytoplasmic domain contains the amino acid sequence RVKFSRSA (SEQ ID NO: 464). In any aspect of the disclosure, or embodiments thereof, the polypeptide further contains a signal peptide at the N-terminus. In some embodiments, the signal peptide contains an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 469; CD8a signal peptide).

[0039] In any aspect of the disclosure, or embodiments thereof, the polypeptide further contains a protein tag. In some embodiments, the tag is disposed between the CLEC2d extracellular domain and the transmembrane domain. In some embodiments, the protein tag contains an amino acid sequence with at least 85% amino acid sequence identity to the amino acid sequence ELPTQGTFSNVSTNVS (SEQ ID NO: 472; CD34 Qbend / 10 epitope).

[0040] In any aspect of the disclosure, or embodiments thereof, the cell is a T cell. In some embodiments, the T cell expresses a chimeric antigen receptor (CAR) polypeptide. In some embodiments, the T cell has been modified to reduce or eliminate expression of beta-2- microglobulin. In some embodiments, the T cell has been modified to reduce or eliminate expression of CD161. In some embodiments, the T cell has been modified to reduce or eliminate expression of cluster of differentiation CD54. In some embodiments, the T cell has been modified to reduce or eliminate expression of CD58. In some embodiments, the T cell has been modified using a base editor system to reduce or eliminate expression of beta-2-microglobulin, CD54, CD58, CD161, and / or Class II Major Histocompatibility Complex Transactivator (CIITA). In some embodiments, the T cell has been modified using a base editor system to reduce or eliminate expression of CD54 and / or CD58.

[0041] In any aspect of the disclosure, or embodiments thereof, the method involves contacting the T cell with a base editor system containing a base editor containing a nucleic acid programmable DNA binding protein (napDNAbp) domain and an adenosine deaminase domain, or one or more polynucleotides encoding the base editor, and one or more guide polynucleotides, or one or more polynucleotide encoding the guide polynucleotides, where the one or more guide polynucleotides target the base editor to alter a nucleotide within a polynucleotide encoding a CD161 polypeptide and / or to alter a nucleotide within a polynucleotide encoding a beta-2 - microglobulin (B2M) polypeptide, to alter a nucleotide within a polynucleotide encoding a Class II Major Histocompatibility Complex Transactivator (CIITA) polypeptide, to alter a polynucleotide encoding a cluster of differentiation 54 (CD54) polypeptide, and / or to alter a polynucleotide encoding a cluster of differentiation 58 (CD58) polypeptide.

[0042] In any aspect of the disclosure, or embodiments thereof, the deaminase domain contains a TadA*8 adenosine deaminase domain. In any aspect of the disclosure, or embodiments thereof, the deaminase domain contains a TadA*8.20 adenosine deaminase domain. In any aspect of the disclosure, or embodiments thereof, the base editor is an ABE8.20 base editor.

[0043] In any aspect of the disclosure, or embodiments thereof, the napDNAbp domain is a Cas9 domain.

[0044] In any aspect of the disclosure, or embodiments thereof, the one or more guide polynucleotides contain a spacer containing a nucleotide sequence containing at least 10 contiguous nucleotides of a nucleotide sequence selected from one or more of: UUACCCCGAGGAAGAGAUGA (SEQ ID NO: 426; IMM_169), UAACUUUUCAGAUGUCUGUC (SEQ ID NO: 427; IMM_170), AACUUUUCAGAUGUCUGUCA (SEQ ID NO: 428; IMM_171), UUUUUUACUUUAGAGAGACC (SEQ ID NO: 429: IMM_172), CUCCACAGCUUAGAAAUUAG (SEQ ID NO: 430; IMM_173), and CCUCACCAAAGGUCUGGAGC (SEQ ID NO: 3338;

[0045] IMM 163).

[0046] In any aspect of the disclosure, or embodiments thereof, the modified T cell has reduced levels of B2M, CD54, CD58, CD161, and / or CIITA expression relative to a wild-type T cell. In any aspect of the disclosure, or embodiments thereof, the T cell shows a reduction in specific lysis by NK cells of at least 10% relative to T cells with reduced levels of human leukocyte antigen A, human leukocyte antigen B, and / or human leukocyte antigen C relative to a wild-type T cell that do not express the polypeptide of any aspect of the disclosure, or embodiments thereof.

[0047] In any aspect of the disclosure, or embodiments thereof, the method further involves expressing a chimeric antigen receptor (CAR) polypeptide in the T cell.

[0048] In any aspect of the disclosure, or embodiments thereof, the T cells surface-express increased levels of the CLEC2d polypeptide relative to a T cell expressing a wild-type CLEC2d polypeptide. In any aspect of the disclosure, or embodiments thereof, the T cells surface-express increased levels of the CLEC2d polypeptide at least 5 days after first expressing the polypeptide of any aspect of the disclosure, or embodiments thereof.

[0049] In any aspect of the disclosure, or embodiments thereof, the polypeptide contains an amino acid sequence with at least 85% sequence identity to the following amino acid sequence: MALPVTALLLPLALLLHAARPRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRF CDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYL NDKGASSARHYTERKWICSKSDIHVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSA (SEQ ID NO: 440;

[0050] BTx_CM496 / mbCLEC2d). In any aspect of the disclosure, or embodiments thereof, the signal peptide is fused to the N-terminus of the CLEC2d extracellular domain.

[0051] In any aspect of the disclosure, or embodiments thereof, the chCLEC2d polypeptide does not contain a transmembrane domain.

[0052] In any aspect of the disclosure, or embodiments thereof, the CLEC2d extracellular domain contains an amino acid sequence with at least about 85% sequence identity to the following sequence and is capable of binding a CD161 polypeptide: RANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRFCDSQDADLAQVESFQELNFLL RYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYLNDKGASSARHYTERKWICSKS DIHV (SEQ ID NO: 461).

[0053] In any aspect of the disclosure, or embodiments thereof, the signal peptide contains an amino acid sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from one or more of MALPVTALLLPLALLLHAARP (SEQ ID NO: 469; CD8a signal peptide), MYRMQLLSCIALSLALVTNS (SEQ ID NO: 3331; IL-2 signal peptide), and MKYTSYILAFQLCIVLGSLGCYC (SEQ ID NO: 3332; IFN-gamma signal peptide).

[0054] In any aspect of the disclosure, or embodiments thereof, the polynucleotide contains a nucleotide sequence with at least 90% identity to a sequence selected from one or more of those listed in Table 7C and / or selected from one or more of the following: ATGGCCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGGCCCC GGGCCAATTGTCATCAGGAACCAAGTGTGTGCCTTCAGGCCGCGTGTCCTGAATCATGGATAGG CTTTCAACGGAAGTGTTTTTATTTTTCCGACGACACTAAAAACTGGACTAGTAGTCAACGGTTT TGCGATTCTCAAGATGCGGATCTTGCCCAAGTGGAGTCCTTCCAAGAGTTGAATTTTCTTCTTC GATATAAAGGTCCTTCAGACCATTGGATAGGGTTGTCCAGGGAACAGGGACAACCGTGGAAGTG GATAAACGGCACAGAATGGACCCGGCAGTTCCCTATATTGGGCGCGGGCGAGTGCGCTTACCTC AATGACAAAGGCGCTTCATCTGCCAGACATTACACGGAACGAAAGTGGATCTGTAGTAAGTCCG ATATTCACGTC (SEQ ID NO: 3328; BTx_LC001); ATGTACCGGATGCAGCTCCTGTCTTGTATAGCACTTTCTCTTGCGTTGGTAACTAATAGCCGGG CCAATTGTCATCAGGAACCAAGTGTGTGCCTTCAGGCCGCGTGTCCTGAATCATGGATAGGCTT TCAACGGAAGTGTTTTTATTTTTCCGACGACACTAAAAACTGGACTAGTAGTCAACGGTTTTGC GATTCTCAAGATGCGGATCTTGCCCAAGTGGAGTCCTTCCAAGAGTTGAATTTTCTTCTTCGAT ATAAAGGTCCTTCAGACCATTGGATAGGGTTGTCCAGGGAACAGGGACAACCGTGGAAGTGGAT AAACGGCACAGAATGGACCCGGCAGTTCCCTATATTGGGCGCGGGCGAGTGCGCTTACCTCAAT GACAAAGGCGCTTCATCTGCCAGACATTACACGGAACGAAAGTGGATCTGTAGTAAGTCCGATA TTCACGTC (SEQ ID NO: 3329; BTx_LC002); and ATGAAATACACGTCCTACATCCTTGCGTTTCAACTTTGTATTGTACTGGGTTCACTGGGCTGCT ACTGTCGGGCCAATTGTCATCAGGAACCAAGTGTGTGCCTTCAGGCCGCGTGTCCTGAATCATG GATAGGCTTTCAACGGAAGTGTTTTTATTTTTCCGACGACACTAAAAACTGGACTAGTAGTCAA CGGTTTTGCGATTCTCAAGATGCGGATCTTGCCCAAGTGGAGTCCTTCCAAGAGTTGAATTTTC TTCTTCGATATAAAGGTCCTTCAGACCATTGGATAGGGTTGTCCAGGGAACAGGGACAACCGTG GAAGTGGATAAACGGCACAGAATGGACCCGGCAGTTCCCTATATTGGGCGCGGGCGAGTGCGCT TACCTCAATGACAAAGGCGCTTCATCTGCCAGACATTACACGGAACGAAAGTGGATCTGTAGTA AGTCCGATATTCACGTC (SEQ ID NO: 3330; BTx_LC003).

[0055] In any aspect provided herein, or embodiments thereof, the method is not a process for modifying the germline genetic identity of human beings.

[0056] In any aspect provided herein, or embodiments thereof, the chCLEC2d polypeptide is secreted by a cell expressing the polypeptide.

[0057] In any aspect provided herein, or embodiments thereof, the chCLEC2d polypeptide contains or contains only an amino acid sequence having at least 85% sequence identity to a sequence selected from the group consisting of: BTx_LC003 (sCLEC2d) MKYTSYILAFQLCIVLGSLGCYCRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQ RFCDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECA YLNDKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3327);

[0058] BTx LCOOl :

[0059] MALPVTALLLPLALLLHAARPRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRF CDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYL NDKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3325); and BTx_LC002 MYRMQLLSCIALSLALVTNSRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRFC DSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYLN DKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3326).

[0060] In any aspect provided herein, or embodiments thereof, the T cell has been modified to reduce or eliminate expression of a functional T cell receptor complex. In any aspect provided herein, or embodiments thereof, the T cell has been modified to reduce or eliminate expression of CD3E. In any aspect provided herein, or embodiments thereof, the T cell has been modified using a base editor system to reduce or eliminate expression of beta-2-microglobulin, CIITA, a functional T cell receptor complex, and CD58. In any aspect provided herein, or embodiments thereof, the T cell has been modified using a base editor system to reduce or eliminate expression of beta-2-microglobulin and to reduce expression of one or more of CIITA, CD3E, CD54, CD58, and CD161.

[0061] In any aspect provided herein, or embodiments thereof, the T cell has increased persistence compared to a T cell that does not contain the polynucleotide or the vector of any aspect of the disclosure, or embodiments thereof, that does not express the sCLEC2d polypeptide of claim 25a, and that has been modified to reduce or eliminate expression of beta-2- microglobulin. In any aspect provided herein, or embodiments thereof, the T cell shows a reduction in specific lysis by NK cells relative to a reference T cell that does not contain the polynucleotide or vector of any aspect of the disclosure, or embodiments thereof, and that does not express the chCLEC2d polypeptide of claim 25a.

[0062] Definitions

[0063] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton etal., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The

[0064] Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0065] By “adenine” or “ 9J / -Purin-6-amine” is meant a purine nucleobase with the molecular formula C5H5N5, having the structure , and corresponding to CAS No. 73-

[0066] 24-5.

[0067] By “adenosine” or “ 4-Amino-l-[(2A,3A,45,5A)-3,4-dihydroxy-5-

[0068] (hydroxymethyl)oxolan-2-yl]pyrimidin-2(U7)-one” is meant an adenine molecule attached to a ribose sugar via a glycosidic bond, having the structure , and corresponding to CAS No. 65-46-3. Its molecular formula is C10H13N5O4.

[0069] By “adenosine deaminase” or “adenine deaminase” is meant a polypeptide or fragment thereof capable of catalyzing the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase catalyzing the hydrolytic deamination of adenosine to inosine or deoxy adenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases (e.g., engineered adenosine deaminases, evolved adenosine deaminases) provided herein may be from any organism (e.g., eukaryotic, prokaryotic), including but not limited to algae, bacteria, fungi, plants, invertebrates (e.g., insects), and vertebrates (e.g., amphibians, mammals). In some embodiments, the adenosine deaminase is an adenosine deaminase variant with one or more alterations and is capable of deaminating both adenine and cytosine in a target polynucleotide e.g., DNA, RNA) and may be referred to as a “dual deaminase”. Non-limiting examples of dual deaminases include those described in PCT / US22 / 22050. In some embodiments, the target polynucleotide is single or double stranded. In some embodiments, the adenosine deaminase variant is capable of deaminating both adenine and cytosine in DNA. In some embodiments, the adenosine deaminase variant is capable of deaminating both adenine and cytosine in singlestranded DNA. In some embodiments, the adenosine deaminase variant is capable of deaminating both adenine and cytosine in RNA. In embodiments, the adenosine deaminase variant is selected from those described in PCT / US2020 / 018192, PCT / US2020 / 049975, PCT / US2017 / 045381, PCT / US2021 / 016827, PCT / US2022 / 073781, PCT / US24 / 34189, or PCT / US2020 / 028568, the full contents of which are each incorporated herein by reference in their entireties for all purposes. Further non-limiting examples of adenosine deaminases include those disclosed or referenced in Rufflow, et al., “Design of highly functional genome editors by modeling of the universe of CRISPR-Cas Sequences,” bioRxiv, posted April 22, 2024, doi: 10.1101 / 2024.04.22.590591, the disclosure of which is incorporated herein by reference in its entirety for all purposes, which were designed using artificial intelligence. Further exemplary adenosine deaminase amino acid sequenes include: TadA-8e (SEQ ID NO: 3351), Tadl (SEQ ID NO: 471), Tad2 (SEQ ID NO: 3352), Tad3 (SEQ ID NO: 3353), Tad4 (SEQ ID NO: 3354), Tad6 (SEQ ID NO: 3356), Tad6-SR (SEQ ID NO: 3357), TadA9 (SEQ ID NO: 3358), TadA20 (SEQ ID NO: 3359), Staphylococcus aureus TadA (SEQ ID NO: 3360), Bacillus subtilis TadA (SEQ ID NO: 3361), Salmonella typhimurium TadA (SEQ ID NO: 3362), Shewanella putrefaciens (SEQ ID NO: 3363), Haemophilus influenzae F3031 TadA (SEQ ID NO: 3364), Caulobacter crescentus TadA (SEQ ID NO: 3365), Geobacter sulfurreducens TadA (SEQ ID NO: 3366), Streptococcus pyogenes TadA (SEQ ID NO: 3367), Aquifex aeolicus TadA (SEQ ID NO: 3368), and A. coli TadA deaminase (ecTadA) (SEQ ID NO: 3369).

[0070] By “adenosine deaminase activity” is meant catalyzing the deamination of adenine or adenosine to guanine in a polynucleotide.

[0071] By “Adenosine Base Editor (ABE)” is meant a base editor comprising an adenosine deaminase.

[0072] By “Adenosine Base Editor (ABE) polynucleotide” is meant a polynucleotide encoding an ABE.

[0073] By “Adenosine Base Editor 8 (ABE8) polypeptide” or “ABE8” is meant a base editor as defined herein comprising an adenosine deaminase or adenosine deaminase variant comprising one or more of the alterations listed in Table 5B, one of the combinations of alterations listed in Table 5B, or an alteration at one or more of the amino acid positions listed in Table 5B, where such alterations are relative to the following reference sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1), or a corresponding position in another adenosine deaminase. In embodiments, ABE8 comprises alterations at amino acids 82 and / or 166 of SEQ ID NO: 1. In some embodiments, ABE8 comprises further alterations, as described herein, relative to the reference sequence.

[0074] By “Adenosine Base Editor 8 (ABE8) polynucleotide” is meant a polynucleotide encoding an ABE8 polypeptide.

[0075] “Administering” is referred to herein as providing one or more compositions described herein to a patient or a subject. By way of example and without limitation, composition administration (e.g., injection) can be performed by intravenous (i.v.) injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more such routes can be employed. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. In some embodiments, parenteral administration includes infusing or injecting intravascularly, intravenously, intramuscularly, intraarterially, intrathecally, intratumorally, intradermally, intraperitoneally, transtracheally, subcutaneously, subcuticularly, intraarticularly, subcapsularly, subarachnoidly and intrasternally. Alternatively, or concurrently, administration can be by the oral route.

[0076] By “agent” is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

[0077] “Allogeneic,” as used herein, refers to cells that are genetically dissimilar and immunologically incompatible. In embodiments, allogeneic cells are administered to a genetically dissimilar and immunologically incompatible subject. In some embodiments, the allogeneic cells comprise modifications improving their persistence in the subject allogeneic to the cells.

[0078] By “alteration” is meant a change in the level, structure, or activity of an analyte, gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a change (e.g., increase or reduction) in expression levels. In embodiments, the increase or reduction in expression levels is by 10%, 25%, 40%, 50% or greater. In some embodiments, an alteration includes an insertion, deletion, or substitution of a nucleobase or amino acid (by, e.g., genetic engineering).

[0079] By “ameliorate” is meant reduce, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

[0080] By “analog” is meant a molecule that is not identical but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog’s function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog’s protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

[0081] As used herein, the term “antibody” refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen, and includes polyclonal, monoclonal, genetically engineered, and otherwise modified forms of antibodies, including but not limited to chimeric antibodies, humanized antibodies, heteroconjugate antibodies e.g., bi- tri- and quad-specific antibodies, diabodies, triabodies, and tetrabodies), and antigen binding fragments of antibodies, including, for example, Fab’, F(ab’)2, Fab, Fv, rlgG, and scFv fragments. Further non-limiting examples of antibodies include VHH domains. Unless otherwise indicated, the term “monoclonal antibody” (mAb) is meant to include both intact molecules, as well as antibody fragments (including, for example, Fab and F(ab’)2 fragments) that are capable of specifically binding to a target protein. As used herein, the Fab and F(ab’)2 fragments refer to antibody fragments that lack the Fc fragment of an intact antibody.

[0082] Antibodies (immunoglobulins) comprise two heavy chains linked together by disulfide bonds, and two light chains, with each light chain being linked to a respective heavy chain by disulfide bonds in a " Y" shaped configuration. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains (CH). Each light chain has a variable domain (VL) at one end and a constant domain (CL) at its other end. The variable domain of the light chain (VL) is aligned with the variable domain of the heavy chain (VL), and the light chain constant domain (CL) is aligned with the first constant domain of the heavy chain (CHI). The variable domains of each pair of light and heavy chains form the antigen binding site. The isotype of the heavy chain (gamma, alpha, delta, epsilon or mu) determines the immunoglobulin class (IgG, IgA, IgD, IgE or IgM, respectively). The light chain is either of two isotypes (kappa (K) or lambda (X)) found in all antibody classes. The terms "antibody" or "antibodies" include intact antibodies, such as polyclonal antibodies or monoclonal antibodies (mAbs), as well as proteolytic portions or fragments thereof, such as the Fab or F(ab')2 fragments, that are capable of specifically binding to a target protein. Antibodies may include chimeric antibodies; recombinant and engineered antibodies, and antigen binding fragments thereof. Exemplary functional antibody fragments comprising whole or essentially whole variable regions of both the light and heavy chains are defined as follows: (i) Fv, defined as a genetically engineered fragment consisting of the variable region of the light chain and the variable region of the heavy chain expressed as two chains; (ii) single-chain Fv (“scFv”), a genetically engineered singlechain molecule including the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker; (iii) Fab, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule, obtained by treating an intact antibody with the enzyme papain to yield the intact light chain and the Fd fragment of the heavy chain, which consists of the variable and CHI domains thereof; (iv) Fab', a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule, obtained by treating an intact antibody with the enzyme pepsin, followed by reduction (two Fab' fragments are generated per antibody molecule); and (v) F(ab')2, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule, obtained by treating an intact antibody with the enzyme pepsin (i.e., a dimer of Fab' fragments held together by two disulfide bonds). By “base editor (BE),” or “nucleobase editor polypeptide (NBE)” is meant an agent that binds a polynucleotide and has nucleobase modifying activity. In various embodiments, the base editor comprises a nucleobase modifying polypeptide (e.g., a deaminase) and a polynucleotide programmable nucleotide binding domain (e.g., Cas9 or Cpfl). Representative nucleic acid and protein sequences of base editors include those sequences having about or at least about 85% sequence identity to any base editor sequence provided in the sequence listing, such as those corresponding to SEQ ID NOs: 2-11.

[0083] By “beta-2 microglobulin (P2M; B2M) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to UniProt Accession No. P61769, or a fragment thereof having immunomodulatory activity.

[0084] By “beta-2-microglobulin (P2M; B2M) polynucleotide” is meant a nucleic acid molecule encoding an P2M polypeptide, as well as the introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. The beta-2-microglobulin gene encodes a serum protein associated with the major histocompatibility complex. P2M is involved in non-self-recognition by host CD8+ T cells. An exemplary P2M polynucleotide sequence is provided at GenBank Accession No. DQ217933.1.

[0085] By “BE4 cytidine deaminase (BE4) polypeptide,” is meant a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain, a cytidine deaminase domain, and two uracil glycosylase inhibitor domains (UGIs). In embodiments, the napDNAbp is a Cas9n (DIO A) polypeptide. Non-limiting examples of cytidine deaminase domains include rAPOBEC, ppAPOBEC, RrA3F, AmAPOBECl, and SsAPOBEC3B.

[0086] By “BE4 cytidine deaminase (BE4) polynucleotide,” is meant a polynucleotide encoding a BE4 polypeptide.

[0087] By “base editing activity” is meant acting to chemically alter a base within a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is cytidine deaminase activity, e.g., converting target OG to T»A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, e.g., converting A»T to G»C.

[0088] By “base editing efficiency” is meant the total percent of one or more target bases in a sample that have been modified using a base editor. In some cases, the base editing efficiency is calculated as the total percent of target polynucleotides in a sample containing a modified target base. In some instances, the base editing efficiency is calculated as the total percent of target polynucleotides in a sample containing a modification to one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) of 2, 3, 4, 5, 6, 7, 8, 9, or 10 target bases. Methods for measuring base editing efficiency for a base editor are known in the art (see, e.g., Gaudelli, et al. Nature 551 :464-471 (2017), the disclosure of which is incorporated herein in its entirety for all purposes). In some cases a base editing efficiency is a median base editing efficiency calculated across 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more target sites.

[0089] By “base editing window” for a base editor is meant bases within a target polynucleotide sequence that can be modified using the base editor. In some embodiments, the position of the nucleobases in the target polynucleotide sequence are numbered relative to a protospacer adjacent motif (PAM) for which a nucleic acid programmable DNA binding protein (napDNAbp) domain of the base editor has specificity, where base 1 corresponds to the base immediately adjacent to the PAM. In some embodiments, the position of the nucleobases in the target polynucleotide sequence are numbered relative to the 5' or 3' end of a spacer of a guide polynucleotide used to guide a nucleic acid programmable DNA binding protein (napDNAbp) domain of the base editor to a target site, where base 1 corresponds to the 5' or 3' terminal base of the spacer.

[0090] The term “base editor system” refers to an intermolecular complex for editing a nucleobase of a target nucleotide sequence. In various embodiments, the base editor (BE) system comprises (1) a polynucleotide programmable nucleotide binding domain, a deaminase domain (e.g., cytidine deaminase or adenosine deaminase) for deaminating nucleobases in the target nucleotide sequence; and (2) one or more guide polynucleotides (e.g., guide RNA) in conjunction with the polynucleotide programmable nucleotide binding domain. In various embodiments, the base editor (BE) system comprises a nucleobase editor domain selected from an adenosine deaminase or a cytidine deaminase, and a domain having nucleic acid sequence specific binding activity. In some embodiments, the base editor system comprises (1) a base editor (BE) comprising a polynucleotide programmable DNA binding domain and a deaminase domain for deaminating one or more nucleobases in a target nucleotide sequence; and (2) one or more guide RNAs in conjunction with the polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE) or a cytidine or cytosine base editor (CBE). In some embodiments, the base editor system (e.g., a base editor system comprising a cytidine deaminase) comprises a uracil glycosylase inhibitor or other agent or peptide (e.g., a uracil stabilizing protein such as provided in W02022015969, the disclosure of which is incorporated herein by reference in its entirety for all purposes) that inhibits the inosine base excision repair system.

[0091] The term “Cas9” or “Cas9 domain” refers to an RNA guided nuclease comprising a Cas9 protein, or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9, and / or the gRNA binding domain of Cas9). A Cas9 nuclease is also referred to sometimes as a casnl nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat) associated nuclease.

[0092] By “Intercellular Adhesion Molecule 1 (ICAM1)” or “Cluster of Differentiation 54 (CD54) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to GenBank Accession No. AAA52709.1, or a fragment thereof that functions in the immune system.

[0093] By “Cluster of Differentiation 54 (CD54) polynucleotide” is meant a nucleic acid molecule encoding an CD54 polypeptide, as well as the introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. An exemplary CD54 polynucleotide is provided at GenBank Accession No. J03132.1. The CD54 gene corresponds to Ensembl: ENSG00000090339.

[0094] By “Cluster of Differentiation 58 (CD58) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence Accession No. NP 001770.1, or a fragment thereof that functions in the immune system. CD58 and the immunobiology thereof is described in Zhang, et al. "CD58 Immunobiology at a Glance," Frontiers in Immunology, vol. 12, article 705260 (2021), the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0095] By “Cluster of Differentiation 58 (CD58) polynucleotide” is meant a nucleic acid molecule encoding an CD58 polypeptide, as well as the introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. An exemplary CD58 polynucleotide is provided at NCBI Accession No. NM_001779.3.The CD58 gene corresponds to Ensembl: ENSG00000116815.

[0096] By “class II, major histocompatibility complex, transactivator (CIITA) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Accession No. NP_001273331.1, or a fragment thereof having DNA binding activity.

[0097] By “class II, major histocompatibility complex, transactiv tor (CIITA) polynucleotide” is meant a nucleic acid molecule encoding an CIITA polypeptide, as well as the introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. An exemplary CIITA polynucleotide is provided at NCBI Accession No. NM_001286402.1. The CIITA gene corresponds to Ensembl: ENSG00000179583.

[0098] By “C-type lectin domain family 2, member D (CLEC2d) polypeptide” or “lectin-like transcript 1 (LLT1) polypeptide” is meant a protein with at least about 85% amino acid sequence identity to NCBI Ref. Seq. Accession No. NP_037401.1, which is provided below, or a fragment thereof. In embodiments, the fragment has immunomodulatory activity. In various embodiments, the CLEC2d polypeptide is capable of binding to a CD161 polypeptide. In some embodiments, a cell that expresses a CLEC2d polypeptide on its surface has increased resistance to NK-mediated lysis. In the below sequence, an exemplary CLEC2d transmembrane domain is shown in bold text, and an exemplary CLEC2d extracellular domain is shown in underlined text (see also Table 8) >NP_037401.1 C-type lectin domain family 2 member D isoform 1 [Homo sapiens] (f!CLEC2d) MHDSNNVEKDITPSELPANPGCLHSKEHSIKATLIWRLFFLIMFLTIIVCGMVAALSAIRANCH QEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRFCDSQDADLAQVESFQELNFLLRYKGP SDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYLNDKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 475). In various embodiments, a CLEC2d extracellular domain comprises an amino acid sequence with at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the following amino acid sequence: RANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRFCDSQDADLJAQVESFQELJNFLJLJ RYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYLNDKGASSARHYTERKWICSKS DIHV (SEQ ID NO: 461).

[0099] By “C-type lectin domain family 2, member D (CLEC2d) polynucleotide” or “lectin-like transcript 1 (LLT1) polynucleotide” is meant a nucleic acid molecule encoding an CLEC2d polypeptide, as well as the introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a CLEC2d polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and / or required for CLEC2d expression. An exemplary CLEC2d nucleotide sequence from Homo Sapiens is provided below (NCBI Ref. Seq. Accession No. NM_013269.6:23-598). An exemplary CLEC2d gene sequence is provided at Ensembl Accession No. ENSG00000069493. >NM_013269.6:23-598 Homo sapiens C-type lectin domain family 2 member D (CLEC2D), transcript variant 1, mRNA ATGCATGACAGTAACAATGTGGAGAAAGACATTACACCATCTGAATTGCCTGCAAACCCAGGTT GTCTGCATTCAAAAGAGCATTCTATTAAAGCTACCTTAATTTGGCGCTTATTTTTCTTAATCAT GTTTCTGACAATCATAGTGTGTGGAATGGTTGCTGCTTTAAGCGCAATAAGAGCTAACTGCCAT CAAGAGCCATCAGTATGTCTTCAAGCTGCATGCCCAGAAAGCTGGATTGGTTTTCAAAGAAAGT GTTTCTATTTTTCTGATGACACCAAGAACTGGACATCAAGTCAGAGGTTTTGTGACTCACAAGA TGCTGATCTTGCTCAGGTTGAAAGCTTCCAGGAACTGAATTTCCTGTTGAGATATAAAGGCCCA TCTGATCACTGGATTGGGCTGAGCAGAGAACAAGGCCAACCATGGAAATGGATAAATGGTACTG AATGGACAAGACAGTTTCCTATCCTGGGAGCAGGAGAGTGTGCCTATTTGAATGACAAAGGTGC CAGTAGTGCCAGGCACTACACAGAGAGGAAGTGGATTTGTTCCAAATCAGATATACATGTCTAG (SEQ ID NO: 476)

[0100] By “chimeric polypeptide” is meant a polypeptide comprising fragments that are heterologous to one another or whose domain architecture differs from the domain architecture of a wild-type polypeptide. For example, in some embodiments a chimeric polypeptide is a chimeric CLEC2d (chCLEC2d) polypeptide comprising from N- to C-terminus a CLEC2d extracellular domain and a CLEC2d transmembrane domain. See, for example, FIG. 14. For example, in some embodiments a chimeric polypeptide is a chimeric CLEC2d (chCLEC2d) polypeptide containing a CLEC2d extracellular domain and a signal peptide, hinge domain, transmembrane domain, and / or intracellular domain derived from a non-CLEC2d polypeptide (e.g., CD4, CD8, and / or NKG2D).

[0101] By “chimeric antigen receptor” or “CAR” is meant a synthetic or engineered receptor comprising an extracellular antigen binding domain operationally joined to one or more intracellular signaling domains where the CAR confers specificity for an antigen bound by the extracellular antigen binding domain onto an immune effector cell. In some cases, the intracellular signaling domain is a T cell signaling domain. In embodiments, the immune effector cell is a T cell, an NK cell, or a macrophage. In embodiments, the CAR is a SUPRA CAR, an anti-tag CAR, a TCR-CAR, or a TCR-like CAR (see, e.g., Guedan, etal. “Engineering and Design of Chimeric Antigen Receptors,” Methods and Clinical Development, 12: 145-156 (2019); Poorebrahim, etal., “TCR-like CARs and TCR-CARs targeting neoepitopes: an emerging potential,” Cancer Gene Therapy, 28:581-589 (2021); and Minutolo, et al. “The Emergence of Universal Immune Receptor T Cell Therapy for Cancer,” Front Oncol., 9:176 (2019), the disclosures of which are incorporated herein by reference in their entireties for all purposes). In some embodiments, the antigen binding domain binds one or more of the following proteins: CD123, CLL-1, CD33, CD34, CD70, FLT3, CD38, Siglec-6, c-Kit / CD117, ILT3, NKG2DL, WT1, CD2, CD4, CD5, CD7, BCL: CD19, CD20, CD22, CD79b, CD79a, Igk, Igl, Her2, ROR1, CD70, Claudinl8.2, mesothelin, and EGFR. In some instances, the antigen is associated with acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), B-cell leukemia (BCL), and / or a solid tumor.

[0102] By “chimeric antigen receptor (CAR) T cell” or “CAR-T cell” is meant a T cell expressing a CAR that has antigen specificity determined by the antibody-derived targeting domain of the CAR. As used herein, “CAR-T cells” includes T cells, regulatory T cells (TREG), macrophages, or NK cells. As used herein, the term “CAR-T cells” includes cells engineered to express a CAR or a T cell receptor (TCR, sometimes referred to as TCR-CARs or TCR-like CARs). Methods of making CARs (e.g., for treatment of cancer) are publicly available see, e.g., Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368: 1509-1518, 2013; Han et al., J. Hematol Oncol. 6:47, 2013; Haso et al, (2013) Blood, 121, 1165-1174; Mohseni, et al., (2020) Front. Immunol., 11, art. 1608, doi: 10.3389 / fimmu.2020.01608; Eggenhuizen, et al. Int. J. Mol. Sci. (2020), 21 :7015, doi: 10.3390 / ijms21197015; Poorebrahim, et al., Cancer Gene Ther 28, 581-589 (2021), doi.org / 10.1038 / s41417-021-00307-7, PCT Pubs. WO20 12 / 079000, WO2013 / 059593; and U.S. Pub. 2012 / 0213783, the disclosure of each of which is incorporated herein by reference herein in its entirety).

[0103] The term “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and Schirmer, R. H., Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids can be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and Schirmer, R. H., supra). Nonlimiting examples of conservative mutations include amino acid substitutions of amino acids, for example, lysine for arginine and vice versa such that a positive charge can be maintained; glutamic acid for aspartic acid and vice versa such that a negative charge can be maintained; serine for threonine such that a free -OH can be maintained; and glutamine for asparagine such that a free -NH2 can be maintained.

[0104] Amino acids generally can be grouped into classes according to the following common side- chain properties:

[0105] (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;

[0106] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0107] (3) acidic: Asp, Glu;

[0108] (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro;

[0109] (6) aromatic: Trp, Tyr, Phe.

[0110] In some embodiments, conservative substitutions can involve the exchange of a member of one of these classes for another member of the same class. In some embodiments, nonconservative amino acid substitutions can involve exchanging a member of one of these classes for another class.

[0111] By “cluster of differentiation 3 antigen, zeta subunit (CD3z; CD3 zeta; CD3Q polypeptide” or “cluster of differentiation 247 (CD247) polypeptide” is meant a protein with at least about 85% amino acid sequence identity to GenBank Accession No. AAA60394.1, which is provided below, or a fragment thereof having immunomodulatory activity. In the below sequence, an exemplary CD3 zeta truncated intracellular domain (CD3A(^; CD3z(trunc)) is shown in bold text (see also Table 8).

[0112] >AAA60394.1 T-cell receptor zeta chain [Homo sapiens] MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSAEPPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 477)

[0113] By “cluster of differentiation 3 antigen, zeta subunit (CD3z; CD3 zeta; CD3Q polynucleotide” or “cluster of differentiation 247 (CD247) polynucleotide” is meant a nucleic acid molecule encoding a CD3z polypeptide, as well as the introns, exons, 3 ' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a CD3z polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and / or required for CD3z expression. An exemplary CD3z nucleotide sequence from Homo Sapiens is provided below (GenBank Accession No. 104132.1 :75-566). An exemplary CD3z gene sequence is provided at Ensembl Accession No. ENSG00000198821.

[0114] >104132.1 :75-566 Human T cell receptor zeta-chain mRNA, complete cds ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCAC

[0115] AGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGG TGTCATTCTCACTGCCTTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGAGCCCCCCGCGTAC CAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTT TGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGA AGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAA GGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGG ACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (SEQ ID NO: 478) By “cluster of differentiation 4 (CD4) polypeptide” is meant a protein with at least about 85% amino acid sequence identity to GenBank Accession No. AAA35572.1, which is provided below, or a fragment thereof having immunomodulatory activity. In the below sequence, an exemplary CD4 transmembrane domain is shown in bold text, and an exemplary CD4 truncated intracellular domain is shown in underlined text (see also Table 8).

[0116] >AAA35572.1 T4 surface glycoprotein precursor [Homo sapiens] MNRGVPFRHLLLVLQLALLPAATQGKKWLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGN QGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFGLTAN SDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKK VEFKIDIWLAFQKASSIVYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKSWITFDL KNKEVSVKRVTQDPKLQMGKKLPLHLTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLWMRAT QLQKNLTCEVWGPTSPKLMLSLKLENKEAKVSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNI KVLPTWSTPVQPMALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQAERMSQIKRLLSEKKTCQCP HRFQKTCSPI (SEQ ID NO: 479)

[0117] By “cluster of differentiation 4 (CD4) polynucleotide” is meant a nucleic acid molecule encoding an CD4 polypeptide, as well as the introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a CD4 polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and / or required for CD4 expression. An exemplary CD4 nucleotide sequence from Homo Sapiens is provided below (GenBank Accession No. M12807.1 :76-1452). An exemplary CD4 gene sequence is provided at Ensembl Accession No. ENSG00000010610. >M12807.1 :76-1452 Human T-cell surface glycoprotein T4 mRNA, complete cds ATGAACCGGGGAGTCCCTTTTAGGCACTTGCTTCTGGTGCTGCAACTGGCGCTCCTCCCAGCAG CCACTCAGGGAAAGAAAGTGGTGCTGGGCAAAAAAGGGGATACAGTGGAACTGACCTGTACAGC TTCCCAGAAGAAGAGCATACAATTCCACTGGAAAAACTCCAACCAGATAAAGATTCTGGGAAAT CAGGGCTCCTTCTTAACTAAAGGTCCATCCAAGCTGAATGATCGCGCTGACTCAAGAAGAAGCC TTTGGGACCAAGGAAACTTCCCCCTGATCATCAAGAATCTTAAGATAGAAGACTCAGATACTTA CATCTGTGAAGTGGAGGACCAGAAGGAGGAGGTGCAATTGCTAGTGTTCGGATTGACTGCCAAC TCTGACACCCACCTGCTTCAGGGGCAGAGCCTGACCCTGACCTTGGAGAGCCCCCCTGGTAGTA GCCCCTCAGTGCAATGTAGGAGTCCAAGGGGTAAAAACATACAGGGGGGGAAGACCCTCTCCGT GTCTCAGCTGGAGCTCCAGGATAGTGGCACCTGGACATGCACTGTCTTGCAGAACCAGAAGAAG GTGGAGTTCAAAATAGACATCGTGGTGCTAGCTTTCCAGAAGGCCTCCAGCATAGTCTATAAGA AAGAGGGGGAACAGGTGGAGTTCTCCTTCCCACTCGCCTTTACAGTTGAAAAGCTGACGGGCAG TGGCGAGCTGTGGTGGCAGGCGGAGAGGGCTTCCTCCTCCAAGTCTTGGATCACCTTTGACCTG AAGAACAAGGAAGTGTCTGTAAAACGGGTTACCCAGGACCCTAAGCTCCAGATGGGCAAGAAGC TCCCGCTCCACCTCACCCTGCCCCAGGCCTTGCCTCAGTATGCTGGCTCTGGAAACCTCACCCT GGCCCTTGAAGCGAAAACAGGAAAGTTGCATCAGGAAGTGAACCTGGTGGTGATGAGAGCCACT CAGCTCCAGAAAAATTTGACCTGTGAGGTGTGGGGACCCACCTCCCCTAAGCTGATGCTGAGCT TGAAACTGGAGAACAAGGAGGCAAAGGTCTCGAAGCGGGAGAAGGCGGTGTGGGTGCTGAACCC TGAGGCGGGGATGTGGCAGTGTCTGCTGAGTGACTCGGGACAGGTCCTGCTGGAATCCAACATC AAGGTTCTGCCCACATGGTCCACCCCGGTGCAGCCAATGGCCCTGATTGTGCTGGGGGGCGTCG CCGGCCTCCTGCTTTTCATTGGGCTAGGCATCTTCTTCTGTGTCAGGTGCCGGCACCGAAGGCG CCAAGCAGAGCGGATGTCTCAGATCAAGAGACTCCTCAGTGAGAAGAAGACCTGCCAGTGCCCT CACCGGTTTCAGAAGACATGTAGCCCCATTTGA (SEQ ID NO: 480)

[0118] By “cluster of differentiation 8 alpha (CD8a; CD8a) polypeptide” is meant a protein with at least about 85% amino acid sequence identity to GenBank Accession No. AAB04637.1, which is provided below, or a fragment thereof having immunomodulatory activity. In the below sequence, an exemplary CD8a transmembrane domain is shown in bold text, an exemplary CD8a hinge domain is shown in underlined text, and an exemplary CD8a signal peptide is shown in italicized text (see also Table 8).

[0119] >AAB04637.1 cell surface glycoprotein T8 precursor [Homo sapiens] MALPVTALLLPLALLLJL4ARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAA ASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHF VPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLJDFACDIYIWAPLAGT CGVLLLSLVITLYCNHRNRRRVCKCPRPWKSGDKPSLSARYV (SEQ ID NO: 481)

[0120] By “cluster of differentiation 8 alpha (CD8a; CD8a) polynucleotide” is meant a nucleic acid molecule encoding an CD8a polypeptide, as well as the introns, exons, 3 ' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a CD8a polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and / or required for CD8a expression. An exemplary CD8a nucleotide sequence from Homo Sapiens is provided below (GenBank Accession No. M12828.1 :66-773). An exemplary CD8a gene sequence is provided at Ensembl Accession No. ENSG00000153563.

[0121] >M12828.1 :66-773 Homo sapiens T-cell surface protein T8 mRNA ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGA

[0122] GCCAGTTCCGGGTGTCGCCGCTGGATCGGACCTGGAACCTGGGCGAGACAGTGGAGCTGAAGTG CCAGGTGCTGCTGTCCAACCCGACGTCGGGCTGCTCGTGGCTCTTCCAGCCGCGCGGCGCCGCC GCCAGTCCCACCTTCCTCCTATACCTCTCCCAAAACAAGCCCAAGGCGGCCGAGGGGCTGGACA CCCAGCGGTTCTCGGGCAAGAGGTTGGGGGACACCTTCGTCCTCACCCTGAGCGACTTCCGCCG AGAGAACGAGGGCTACTATTTCTGCTCGGCCCTGAGCAACTCCATCATGTACTTCAGCCACTTC GTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGC CCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGC AGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACT TGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACCGAAGACGTG TTTGCAAATGTCCCCGGCCTGTGGTCAAATCGGGAGACAAGCCCAGCCTTTCGGCGAGATACGT CTAA (SEQ ID NO: 482)

[0123] By “cluster of differentiation 107a (CD 107a) polypeptide” is meant a protein with at least about 85% amino acid sequence identity to GenBank Accession No. AAH93044.1, or a fragment thereof having immunomodulatory activity.

[0124] By “cluster of differentiation 107a (CD 107a) polynucleotide” is meant a nucleic acid molecule encoding an CD107a polypeptide, as well as the introns, exons, 3 ' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a CD 107a polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and / or required for CD 107a expression. An exemplary CD 107a nucleotide sequence from Homo Sapiens is provided at GenBank Accession No. BC093044.1 : 150-1403. An exemplary CD107a gene sequence is provided at Ensembl Accession No. ENSG00000185896.

[0125] By “cluster of differentiation 161 (CD161) polypeptide” or “killer cell lectin-like receptor, subfamily B, member 1 (KLRB1) polypeptide” is meant a protein with at least about 85% amino acid sequence identity to NCBI Ref. Seq. Accession No. NP_002249.1, which is provided below, or a fragment thereof having immunomodulatory activity. In various embodiments, the CD161 polypeptide is capable of binding to a CLEC2d polypeptide. >NP_002249.1 killer cell lectin-like receptor subfamily B member 1 [Homo sapiens] MDQQAIYAELNLPTDSGPESSSPSSLPRDVCQGSPWHQFALKLSCAGIILLVLWTGLSVSVTS LIQKSSIEKCSVDIQQSRNKTTERPGLLNCPIYWQQLREKCLLFSHTVNPWNNSLADCSTKESS LLLIRDKDELIHTQNLIRDKAILFWIGLNFSLSEKNWKWINGSFLNSNDLEIRGDAKENSCISI SQTSVYSEYCSTEIRWICQKELTPVRNKVYPDS (SEQ ID NO: 483)

[0126] By “cluster of differentiation 161 (CD161) polynucleotide” or “killer cell lectin-like receptor, subfamily B, member 1 (KLRB1) polynucleotide” is meant a nucleic acid molecule encoding an CD161 polypeptide, as well as the introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a CD161 polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and / or required for CD161 expression. An exemplary CD161 nucleotide sequence from Homo Sapiens is provided below (NCBI Ref. Seq. Accession No.

[0127] NM_013269.6:23-598). An exemplary CD161 gene sequence is provided at Ensembl Accession No. ENSG00000111796.

[0128] >NM_002258.3:78-755 Homo sapiens killer cell lectin like receptor Bl (KLRB1), mRNA ATGGACCAACAAGCAATATATGCTGAGTTAAACTTACCCACAGACTCAGGCCCAGAAAGTTCTT CACCTTCATCTCTTCCTCGGGATGTCTGTCAGGGTTCACCTTGGCATCAATTTGCCCTGAAACT TAGCTGTGCTGGGATTATTCTCCTTGTCTTGGTTGTTACTGGGTTGAGTGTTTCAGTGACATCC TTAATACAGAAATCATCAATAGAAAAATGCAGTGTGGACATTCAACAGAGCAGGAATAAAACAA CAGAGAGACCGGGTCTCTTAAACTGCCCAATATATTGGCAGCAACTCCGAGAGAAATGCTTGTT ATTTTCTCACACTGTCAACCCTTGGAATAACAGTCTAGCTGATTGTTCCACCAAAGAATCCAGC CTGCTGCTTATTCGAGATAAGGATGAATTGATACACACACAGAACCTGATACGTGACAAAGCAA TTCTGTTTTGGATTGGATTAAATTTTTCATTATCAGAAAAGAACTGGAAGTGGATAAACGGCTC TTTTTTAAATTCTAATGACTTAGAAATTAGAGGTGATGCTAAAGAAAACAGCTGTATTTCCATC TCACAGACATCTGTGTATTCTGAGTACTGTAGTACAGAAATCAGATGGATCTGCCAAAAAGAAC TAACACCTGTGAGAAATAAAGTGTATCCTGACTCTTGA (SEQ ID NO: 484)

[0129] The term “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and Schirmer, R. H., Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids can be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and Schirmer, R. H., supra). Nonlimiting examples of conservative mutations include amino acid substitutions of amino acids, for example, lysine for arginine and vice versa such that a positive charge can be maintained; glutamic acid for aspartic acid and vice versa such that a negative charge can be maintained; serine for threonine such that a free -OH can be maintained; and glutamine for asparagine such that a free -NH2 can be maintained.

[0130] The term “coding sequence” or “protein coding sequence” as used interchangeably herein refers to a segment of a polynucleotide that codes for a protein. Coding sequences can also be referred to as open reading frames. The region or sequence is bounded nearer the 5' end by a start codon and nearer the 3' end with a stop codon. Stop codons useful with the base editors described herein include the following: TAG, TAA, and TGA. By “complex” is meant a combination of two or more molecules whose interaction relies on inter-molecular forces. Non-limiting examples of inter-molecular forces include covalent and non-covalent interactions. Non-limiting examples of non-covalent interactions include hydrogen bonding, ionic bonding, halogen bonding, hydrophobic bonding, van der Waals interactions (e.g., dipole-dipole interactions, dipole-induced dipole interactions, and London dispersion forces), and 7t-effects. In an embodiment, a complex comprises polypeptides, polynucleotides, or a combination of one or more polypeptides and one or more polynucleotides. In one embodiment, a complex comprises one or more polypeptides that associate to form a base editor (e.g., base editor comprising a nucleic acid programmable DNA binding protein, such as Cas9, and a deaminase) and a polynucleotide (e.g., a guide RNA). In an embodiment, the complex is held together by hydrogen bonds. It should be appreciated that one or more components of a base editor (e.g., a deaminase, or a nucleic acid programmable DNA binding protein) may associate covalently or non-covalently. As one example, a base editor may include a deaminase covalently linked to a nucleic acid programmable DNA binding protein (e.g., by a peptide bond).

[0131] Alternatively, a base editor may include a deaminase and a nucleic acid programmable DNA binding protein that associate noncovalently (e.g., where one or more components of the base editor are supplied in trans and associate directly or via another molecule such as a protein or nucleic acid). In an embodiment, one or more components of the complex are held together by hydrogen bonds.

[0132] By “cytosine” or “4-Aminopyrimidin-2 e nucleobase with the molecular formula C4H5N3O, having the struct corresponding to CAS

[0133] No. 71-30-7.

[0134] By “cytidine” is meant a cytosine molecule attached to a ribose sugar via a glycosidic bond, having the structure , and corresponding to CAS No. 65-46-3. Its molecular formula is C9H13N3O5. By “Cytidine Base Editor (CBE)” is meant a base editor comprising a cytidine deaminase.

[0135] By “Cytidine Base Editor (CBE) polynucleotide” is meant a polynucleotide encoding a CBE.

[0136] By “cytidine deaminase” or “cytosine deaminase” is meant a polypeptide or fragment thereof capable of deaminating cytidine or cytosine. In embodiments, the cytidine or cytosine is present in a polynucleotide. In one embodiment, the cytidine deaminase converts cytosine to uracil or 5 -methylcytosine to thymine. The terms “cytidine deaminase” and “cytosine deaminase” are used interchangeably throughout the application. Petromyzon marinus cytosine deaminase 1 (PmCDAl) (SEQ ID NO: 13-14), Activation-induced cytidine deaminase (AICDA) (SEQ ID NOs: 15-21), and APOBEC (SEQ ID NOs: 12-61) are exemplary cytidine deaminases. Further exemplary cytidine deaminase (CD A) sequences are provided in the Sequence Listing as SEQ ID NOs: 62-66 and SEQ ID NOs: 67-189. Non-limiting examples of cytidine deaminases include those described in PCT / US20 / 16288, PCT / US2018 / 021878, 180802-021804 / PCT, PCT / US2018 / 048969, and PCT / US2016 / 058344.

[0137] By “cytosine deaminase activity” is meant catalyzing the deamination of cytosine or cytidine. In one embodiment, a polypeptide having cytosine deaminase activity converts an amino group to a carbonyl group. In an embodiment, a cytosine deaminase converts cytosine to uracil (ie., C to U) or 5-methylcytosine to thymine (z.e., 5mC to T). In some embodiments, a cytosine deaminase as provided herein has increased cytosine deaminase activity (e.g., at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more) relative to a reference cytosine deaminase.

[0138] The term “deaminase” or “deaminase domain,” as used herein, refers to a protein or fragment thereof that catalyzes a deamination reaction.

[0139] The term “detect” refers to identifying the presence, absence or amount of the analyte to be detected. In one embodiment, a sequence alteration in a polynucleotide or polypeptide is detected. In another embodiment, the presence of indels is detected.

[0140] By “detectable label” is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an enzyme linked immunosorbent assay (ELISA)), biotin, digoxigenin, or haptens. By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. In some embodiments, the disease is a cancer (e.g., a hematological cancer or a solid tumor). In some instances, the disease is a disease that can be treated using the modified allogeneic T cells of the disclosure. In one embodiment, the disease is a neoplasia or cancer. In some instances, the disease is a malignancy. In some cases, the disease is a dysplasia, or a non-malignant or benign neoplasia. In some embodiments, the disease is a hematological cancer. By “hematological cancer” is meant a malignancy of immune system cells. In some embodiments, the hematological cancer is leukemia, myeloma, and / or lymphoma. Lymphomas and Leukemias are examples of “liquid cancers” or cancers present in the blood and are derived from the transformation of either a hematopoietic precursor in the bone marrow or a mature hematopoietic cell in the blood. Leukemias can be lymphoid or myeloid, and acute or chronic. In the case of myelomas, the transformed cell is a fully differentiated plasma cell that may be present as a dispersed collection of malignant cells or as a solid mass in the bone marrow. In the case of lymphomas, a transformed lymphocyte in a secondary lymphoid tissue generates a solid mass. Lymphomas are classified either Hodgkin lymphoma (HL) or nonHodgkin lymphoma (NHL). In some embodiments, the disease is an autoimmune disease.

[0141] By “dual editing activity” or “dual deaminase activity” is meant having adenosine deaminase and cytidine deaminase activity. In one embodiment, a base editor having dual editing activity has both A~>G and C~>T activity, wherein the two activities are approximately equal or are within about 10% or 20% of each other. In another embodiment, a dual editor has A->G activity that no more than about 10% or 20% greater than C~>T activity. In another embodiment, a dual editor has A->G activity that is no more than about 10% or 20% less than C~>T activity. In some embodiments, the adenosine deaminase variant has predominantly cytosine deaminase activity, and little, if any, adenosine deaminase activity. In some embodiments, the adenosine deaminase variant has cytosine deaminase activity, and no significant or no detectable adenosine deaminase activity. Non-limiting examples of proteins having dual deaminase activity include those described in International Patent Application Publications No. WO 2024 / 040083 and WO 2022 / 204574, the disclosures of which are hereby incorporated by reference in their entirities for all purposes.

[0142] By “effective amount” is meant the amount of an agent (e.g., a base editor, cell) as described herein, that is required to ameliorate the symptoms of a disease relative to an untreated patient or an individual without disease, i.e., a healthy individual, or is the amount of the agent sufficient to elicit a desired biological response. The effective amount of active compound(s) used to practice embodiments of the present disclosure for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount. In one embodiment, an effective amount is the amount of a base editor of the disclosure sufficient to introduce an alteration in a gene of interest in a cell (e.g., a cell in vitro or in vivo). In one embodiment, an effective amount is the amount of a base editor required to achieve a therapeutic effect. Such therapeutic effect need not be sufficient to alter a pathogenic gene in all cells of a subject, tissue or organ, but only to alter the pathogenic gene in about 1%, 5%, 10%, 25%, 50%, 75% or more of the cells present in a subject, tissue or organ. In one embodiment, an effective amount is sufficient to ameliorate one or more symptoms of a disease.

[0143] By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids. In some embodiments, the fragment is a functional fragment.

[0144] The term “graft versus host disease” (GVHD) refers to a pathological condition where transplanted cells of a donor generate an immune response against cells of the host.

[0145] By “guide polynucleotide” is meant a polynucleotide or polynucleotide complex which is specific for a target sequence and can form a complex with a polynucleotide programmable nucleotide binding domain protein (e.g., Cas9 or Cpfl). In an embodiment, the guide polynucleotide is a guide RNA (gRNA). gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule.

[0146] By “heterologous,” or “exogenous” is meant a polynucleotide or polypeptide that 1) has been experimentally incorporated into a polynucleotide or polypeptide sequence to which the polynucleotide or polypeptide is not normally found in nature; and / or 2) has been experimentally placed into a cell that does not normally comprise the polynucleotide or polypeptide. In some embodiments, “heterologous” means that a polynucleotide or polypeptide has been experimentally placed into a non-native context. In some embodiments, a heterologous polynucleotide or polypeptide is derived from a first species or host organism and is incorporated into a polynucleotide or polypeptide derived from a second species or host organism. In some embodiments, the first species or host organism is different from the second species or host organism. In some embodiments the heterologous polynucleotide is DNA. In some embodiments the heterologous polynucleotide is RNA. By “hinge region” or “hinge domain” is meant a flexible peptide linking two portions of a polypeptide to one another. In various embodiments, a hinge region is derived from a flexible stretch of amino acids that links two domains of a polypeptide to one another, such as an extracellular domain to a transmembrane domain (e.g., the hinge region of a CD8 polypeptide). In embodiments a hinge region is about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length. In embodiments a hinge region is no more than about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length. In some cases, about, or at least about, 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the amino acids of a hinge region are proline and / or cysteine amino acids. A non-limiting example of a hinge domain is a CD8a hinge domain.

[0147] By “major histocompatibility complex, class I, A (HLA-A) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to GenBank. Accession No. BAA07530.1, or a fragment thereof having immunomodulatory activity.

[0148] By “major histocompatibility complex, class I, A (HLA-A) polynucleotide” is meant a nucleic acid molecule encoding an HLA-A polypeptide, as well as the introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, an HLA-A polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and / or required for HLA-A expression. An exemplary HLA-A polynucleotide sequence is provided at GenBank Accession No. D38525.1. The HLA-A gene corresponds to Ensemble ENSG00000206503.

[0149] By “major histocompatibility complex, class I, B (HLA-B) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to GenBank Accession No. CAD30340.1, or a fragment thereof having immunomodulatory activity.

[0150] By “major histocompatibility complex, class I, B (HLA-B) polynucleotide” is meant a nucleic acid molecule encoding an HLA-B polypeptide, as well as the introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, an HLA-B polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and / or required for HLA-B expression. An exemplary HLA-B polynucleotide sequence is provided at GenBank Accession No. AJ458992.1. The HLA-B gene corresponds to Ensembl: ENSG00000234745.

[0151] By “major histocompatibility complex, class I, C (HLA-C) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to GenBank. Accession No. BBO94058.1, or a fragment thereof having immunomodulatory activity. By “major histocompatibility complex, class I, C (HLA-C) polynucleotide” is meant a nucleic acid molecule encoding an HLA-C polypeptide, as well as the introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, an HLA-C polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and / or required for HLA-C expression. An exemplary HLA-C polynucleotide sequence is provided at GenBank Accession No. LC508210.1. The HLA-C gene corresponds to Ensembl: ENSG00000204525.

[0152] “Host versus graft disease” (HVGD) or “host-versus-graft rejection” refers to a pathological condition where the immune system of a host generates an immune response against transplanted cells of an allogeneic donor.

[0153] “Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

[0154] By “immune cell” is meant a cell of the immune system capable of generating an immune response. Exemplary immune cells include, but are not limited to, T cells, NK cells, B cells, macrophages, hematopoietic stem cells, or precursors thereof. In embodiments, an immune cell is allogeneic to a subject to whom the cell is to be administered. In embodiments, an immune cell is from a donor and is allogeneic to a subject to which the immune cell will be administered after being modified according to the methods provided herein. The invention of the disclosure features methods for preparing modified allogeneic immune cells with improved characteristics (e.g., increased resistance to natural killer (NK) cell-mediated rejection in a subject) as well as the cells produced by these methods. In various embodiments, an immune cell expresses a chCLEC2d polypeptide of the disclosure.

[0155] By “immune effector cell” is meant a lymphocyte, once activated, capable of effecting an immune response upon a target cell. In some embodiments, immune effector cells are effector T cells. In some embodiments, the effector T cell is a naive CD8+T cell, a cytotoxic T cell, a natural killer T (NKT) cell, a natural killer (NK) cell, or a regulatory T (Treg) cell. In some embodiments, immune effector cells are effector NK cells. In some embodiments, the effector T cells are thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. In some embodiments the immune effector cell is a CD4+CD8+T cell or a CD4" CD8" T cell. In some embodiments the immune effector cell is a T helper cell. In some embodiments the T helper cell is a T helper 1 (Thl), a T helper 2 (Th2) cell, or a helper T cell expressing CD4 (CD4+ T cell). By “immunomodulatory activity” is meant increasing, decreasing, or sustaining an immune response.

[0156] By “increases” is meant a positive alteration of at least 10%, 25%, 50%, 75%, or 100%, or about 1.5 fold, about 2 fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7- fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, or about 100-fold.

[0157] The terms “inhibitor of base repair”, “base repair inhibitor”, “IBR” or their grammatical equivalents refer to a protein that is capable in inhibiting the activity of a nucleic acid repair enzyme, for example a base excision repair enzyme.

[0158] An “intein” is a fragment of a protein that is able to excise itself and join the remaining fragments (the exteins) with a peptide bond in a process known as protein splicing.

[0159] The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this disclosure is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0160] By “killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail, 1 (KIR2DL1) polypeptide” is meant a protein with at least about 85% amino acid sequence identity to GenBank Accession No. AAA69868.1, or a fragment thereof having immunomodulatory activity.

[0161] By “killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail, 1 (KIR2DL1) polynucleotide” is meant a nucleic acid molecule encoding an KIR2DL1 polypeptide, as well as the introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a KIR2DL1 polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and / or required for KIR2DL1 expression. An exemplary KIR2DL1 nucleotide sequence from Homo Sapiens is provided at GenBank Accession No. L41267.1 :33-1079. An exemplary KIR2DL1 gene sequence is provided at Ensembl Accession No. ENSG00000125498.

[0162] By “killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail, 2 (KIR2DL2) polypeptide” is meant a protein with at least about 85% amino acid sequence identity to GenBank Accession No. AAC50334.1, or a fragment thereof having immunomodulatory activity.

[0163] By “killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail, 2 (KIR2DL2) polynucleotide” is meant a nucleic acid molecule encoding an KIR2DL2 polypeptide, as well as the introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a KIR2DL2 polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and / or required for KIR2DL2 expression. An exemplary KIR2DL2 nucleotide sequence from Homo Sapiens is provided at GenBank Accession No. U24075.1. An exemplary KIR2DL2 gene sequence is provided at Ensembl Accession No. ENSG00000275914.

[0164] By “killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail, 3 (KIR2DL3) polypeptide” is meant a protein with at least about 85% amino acid sequence identity to GenBank Accession No. AAA69869.1, or a fragment thereof having immunomodulatory activity.

[0165] By “killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail, 3 (KIR2DL3) polynucleotide” is meant a nucleic acid molecule encoding an KIR2DL3 polypeptide, as well as the introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a KIR2DL3 polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and / or required for KIR2DL3 expression. An exemplary KIR2DL3 nucleotide sequence from Homo Sapiens is provided at GenBank Accession No. L41268.1 :38-1063. An exemplary KIR2DL3 gene sequence is provided at Ensembl Accession No. ENSG00000243772.

[0166] By “killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 1 (KIR3DL1) polypeptide” is meant a protein with at least about 85% amino acid sequence identity to GenBank Accession No. AAA69870.1, or a fragment thereof having immunomodulatory activity.

[0167] By “killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 1 (KIR3DL1) polynucleotide” is meant a nucleic acid molecule encoding an KIR3DL1 polypeptide, as well as the introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a KIR3DL1 polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and / or required for KIR3DL1 expression. An exemplary KIR3DL1 nucleotide sequence from Homo Sapiens is provided at GenBank Accession No. L41269.1 :20-1354. An exemplary KIR3DL1 gene sequence is provided at Ensembl Accession No. ENSG00000167633.

[0168] The term “linker”, as used herein, refers to a molecule that links two moieties. In one embodiment, the term “linker” refers to a covalent linker (e.g., covalent bond) or a non-covalent linker.

[0169] By “marker” is meant any protein or polynucleotide having an alteration in expression, level, structure, or activity that is associated with a disease or disorder. In some instances, the disease or disorder is a neoplasia.

[0170] The term “mutation,” as used herein, refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)).

[0171] By “natural killer group 2 D (NKG2D) polypeptide” is meant a protein with at least about 85% amino acid sequence identity to GenBank Accession No. AAF86973.1, which is provided below, or a fragment thereof having immunomodulatory activity. In the below sequence, an exemplary NKG2D transmembrane domain is shown in bold text (see also Table 8). >AAF86973.1 NK cell receptor D [Homo sapiens] MGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPWKSKCRENASPFFFCCFIAVAMG IRFIIMVTIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCM SQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALY ASSFKGYIENCSTPNTYICMQRTV (SEQ ID NO: 485)

[0172] By “natural killer group 2 D (NKG2D) polynucleotide” is meant a nucleic acid molecule encoding an NKG2D polypeptide, as well as the introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, an NKG2D polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and / or required for NKG2D expression. An exemplary NKG2D nucleotide sequence from Homo Sapiens is provided below (GenBank Accession No. AF260135.1). An exemplary NKG2D gene sequence is provided at Ensembl Accession No. ENSG00000213809. >AF260135.1 Homo sapiens NK cell receptor D (NKG2-D) mRNA, NKG2-D*02 allele, complete cds ATGGGGTGGATTCGTGGTCGGAGGTCTCGACACAGCTGGGAGATGAGTGAATTTCATAATTATA

[0173] ACTTGGATCTGAAGAAGAGTGATTTTTCAACACGATGGCAAAAGCAAAGATGTCCAGTAGTCAA AAGCAAATGTAGAGAAAATGCATCTCCATTTTTTTTCTGCTGCTTCATCGCTGTAGCCATGGGA ATCCGTTTCATTATTATGGTAACAATATGGAGTGCTGTATTCCTAAACTCATTATTCAACCAAG AAGTTCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACAA AAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGGTATGAGAGCCAGGCTTCTTGTATG TCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAAGAGGACCAGGATTTACTTAAACTGGTGA AGTCATATCATTGGATGGGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGG CTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTAT GCCTCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACGTACATCTGCATGCAAA GGACTGTGTAA (SEQ ID NO: 486)

[0174] The term "neoplasia" refers to cells or tissues exhibiting abnormal growth or proliferation. The term neoplasia encompasses cancer, liquid, and solid tumors. In some embodiments, the neoplasia is a solid tumor. In some embodiments, the neoplasia is a malignant T- or NK-cell neoplasia. In other embodiments, the neoplasia is a liquid tumor. In some embodiments, the neoplasia is a hematological cancer. In some embodiments, the hematological cancer is leukemia, myeloma, and / or lymphoma. In some embodiments, the hematological cancer is a B cell cancer. In some embodiments, the B cell cancer is a lymphoma or a leukemia. In some cases, the leukemia comprises a pre-leukemia. In some cases, the leukemia is an acute leukemia. Acute leukemias include, for example, an acute myeloid leukemia (AML). In some cases, the neoplasia is mycosis fungoides (MF), Sezary syndrome (SS), Peripheral T / NK-cell lymphoma, Anaplastic large cell lymphoma ALKA, Primary cutaneous T-cell lymphoma, T-cell large granular lymphocytic leukemia, Angioimmunoblastic T / NK-cell lymphoma, Hepatosplenic T-cell lymphoma, Primary cutaneous CD30 + lymphoproliferative disorders, Extranodal NK / T- cell lymphoma, Adult T-cell leukemia / lymphoma, T-cell prolymphocytic leukemia, Subcutaneous panniculitis-like T-cell lymphoma, Primary cutaneous gamma-delta T-cell lymphoma, Aggressive NK-cell leukemia, or Enteropathy-associated T-cell lymphoma. Acute leukemias also include, for example, an acute lymphoid leukemia or an acute lymphocytic leukemia (ALL); ALL includes B-lineage ALL; T-lineage ALL; and T-cell acute lymphocytic leukemia (T-ALL). Further non-limiting examples of neoplasias include renal cell carcinoma (RCC), lung cancer (e.g. non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma), ovarian cancer (e.g. endometrial, mucinous, serous) and breast cancer (e.g. triple negative breast cancer (TNBC)).

[0175] The terms “nucleic acid” and “nucleic acid molecule,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single and / or doublestranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA / RNA hybrid, or including non-naturally occurring nucleotides or nucleosides. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and / or similar terms include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5- propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2 -thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-7V-phosphoramidite linkages).

[0176] The term “nuclear localization sequence,” “nuclear localization signal,” or “NLS” refers to an amino acid sequence that promotes import of a protein into the cell nucleus. Nuclear localization sequences are known in the art and described, for example, in Plank et al., International PCT application, PCT / EP2000 / 011690, filed November 23, 2000, published as WO / 2001 / 038547 on May 31, 2001, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In other embodiments, the NLS is an optimized NLS described, for example, by Koblan et al. , Nature Biotech. 2018 doi: 10.1038 / nbt.4172. In some embodiments, an NLS comprises the amino acid sequence KRTADGSE FES PKKKRKV (SEQ ID NO: 190), KRPAATKKAGQAKKKK (SEQ ID NO: 191), KKTELQTTNAENKTKKL (SEQ ID NO: 192), KRGINDRNFWRGENGRKTR (SEQ ID NO: 193), RKSGKIAAIWKRPRK (SEQ ID NO: 194), PKKKRKV (SEQ ID NO: 195), MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 196), PKKKRKVEGADKRTADGSE FES PKKKRKV (SEQ ID NO: 328), or RKSGKIAAIWKRPRKPKKKRKV (SEQ ID NO: 329).

[0177] The term “nucleobase,” “nitrogenous base,” or “base,” used interchangeably herein, refers to a nitrogen-containing biological compound that forms a nucleoside, which in turn is a component of a nucleotide. The ability of nucleobases to form base pairs and to stack one upon another leads directly to long-chain helical structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Five nucleobases - adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) - are called primary or canonical. Adenine and guanine are derived from purine, and cytosine, uracil, and thymine are derived from pyrimidine. DNA and RNA can also contain other (non-primary) bases that are modified. Non-limiting exemplary modified nucleobases can include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5- methylcytosine (m5C), and 5-hydromethylcytosine. Hypoxanthine and xanthine can be created through mutagen presence, both of them through deamination (replacement of the amine group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can result from deamination of cytosine. A “nucleoside” consists of a nucleobase and a five carbon sugar (either ribose or deoxyribose). Examples of a nucleoside include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Examples of a nucleoside with a modified nucleobase includes inosine (I), xanthosine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), and pseudouridine ( ). A “nucleotide” consists of a nucleobase, a five carbon sugar (either ribose or deoxyribose), and at least one phosphate group. Non-limiting examples of modified nucleobases and / or chemical modifications that a modified nucleobase may include are the following: pseudo-uridine, 5-Methyl-cytosine, 2'-( - methyl-3'-phosphonoacetate, 2'-(9-methyl thioPACE (MSP), 2'-(9-methyl-PACE (MP), 2'-fluoro RNA (2'-F-RNA), constrained ethyl (S-cEt), 2'-(9-methyl (‘M’), 2'-O-methyl-3'- phosphorothioate (‘MS’), 2'-(9-methyl-3'-thiophosphonoacetate (‘MSP’), 5-methoxyuridine, phosphorothioate, and N1 -Methylpseudouridine.

[0178] The term “nucleic acid programmable DNA binding protein” or “napDNAbp” may be used interchangeably with “polynucleotide programmable nucleotide binding domain” to refer to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid or guide polynucleotide (e.g., gRNA), that guides the napDNAbp to a specific nucleic acid sequence. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable RNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 protein. A Cas9 protein can associate with a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas9 domain, for example a nuclease active Cas9, a Cas9 nickase (nCas9), or a nuclease inactive Cas9 (dCas9). Non-limiting examples of nucleic acid programmable DNA binding proteins include, Cas9 (e.g., dCas9 and nCas9), Casl2a / Cpfl, Casl2b / C2cl, Casl2c / C2c3, Casl2d / CasY, Casl2e / CasX, Casl2g, Casl2h, Casl2i, and Casl2j / Cas (Casl2j / Casphi). Non-limiting examples of Cas enzymes include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csnl or Csxl2), CaslO, CaslOd, Casl2a / Cpfl, Casl2b / C2cl, Casl2c / C2c3, Casl2d / CasY, Casl2e / CasX, Casl2g, Casl2h, Casl2i, Casl2j / Cas, Cpfl, Csyl , Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, Cse5e, Cscl, Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csxl l, Csfl, Csf2, CsO, Csf4, Csdl, Csd2, Cstl, Cst2, Cshl, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, Type II Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARF, DinG, homologues thereof, or modified or engineered versions thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, although they may not be specifically listed in this disclosure. See, e.g., Makarova et al. “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPR J. 2018 Oct; 1:325-336. doi: 10.1089 / crispr.2018.0033; Yan et a / ., “Functionally diverse type V CRISPR-Cas systems” Science. 2019 Jan 4;363(6422):88-91. doi: 10.1126 / science.aav7271, the entire contents of each are hereby incorporated by reference. Exemplary nucleic acid programmable DNA binding proteins and nucleic acid sequences encoding nucleic acid programmable DNA binding proteins are provided in the Sequence Listing as SEQ ID NOs: 197-231, 232-245, 254-257, 260, and 378. In some embodiments, the napDNAbp is a (CRISPR-associated system) Cas9 endonuclease, for example, Cas9 (Csnl) from Streptococcus pyogenes (e.g., SEQ ID NO: 197), Cas9 from Neisseria meningitidis (NmeCas9; SEQ ID NO: 208), Nme2Cas9 (SEQ ID NO: 209), Streptococcus constellatus (ScoCas9), or derivatives thereof (e.g., a sequence with at least about 85% sequence identity to a Cas9, such as Nme2Cas9 or spCas9). Further non-limiting examples of nucleic acid programmable DNA binding proteins include those disclosed or referenced in Rufflow, et al., “Design of highly functional genome editors by modeling of the universe of CRISPR-Cas Sequences,” bioRxiv, posted April 22, 2024, doi: 10.1101 / 2024.04.22.590591, the disclosure of which is incorporated herein by reference in its entirety for all purposes, which were designed using artificial intelligence. In some embodiments, the napDNAbp is OpenCRISPR-1, or a variant thereof (e.g., a variant comprising a D10A amino acid alteration and / or lacking an N-terminal methionine). Further non-limiting examples of nucleic acid programmable DNA binding proteins include those disclosed in International Patent Application No. PCT / US2019 / 047996.

[0179] The terms “nucleobase editing domain” or “nucleobase editing protein,” as used herein, refers to a protein or enzyme that can catalyze a nucleobase modification in RNA or DNA, such as cytosine (or cytidine) to uracil (or uridine) or thymine (or thymidine), and adenine (or adenosine) to hypoxanthine (or inosine) deaminations, as well as non-templated nucleotide additions and insertions. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., an adenine deaminase or an adenosine deaminase; or a cytidine deaminase or a cytosine deaminase).

[0180] As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.

[0181] By “OpenCRISPR-1 polypeptide” is meant a protein with an amino acid sequence having at least about 85% amino acid sequence identity to SEQ ID NO: 3349, or a fragment thereof that associates with a nucleic acid, such as a guide nucleic acid or guide polynucleotide, that guides the napDNAbp to a specific nucleic acid sequence. Further details relating to the OpenCRISPR-1 polypeptide are disclosed in Rufflow, et al., “Design of highly functional genome editors by modeling of the universe of CRISPR-Cas Sequences,” bioRxiv, posted April 22, 2024, doi: 10.1101 / 2024.04.22.590591, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0182] By “OpenCRISPR-1 polynucleotide” is meant a nucleic acid molecule encoding an OpenCRISPR-1 polypeptide, as well as the introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, an OpenCRISPR-1 polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and / or required for OpenCRISPR-1 expression. An exemplary OpenCRISPR-1 nucleotide sequence is provided at SEQ ID NO: 3350.

[0183] In various embodiments, a guide RNA suitable for use in combination with an OpenCRISPR-1 polypeptide contains a scaffold having at least 85% sequence identity to a nucleotide sequence selected from the following, or fragments thereof capable of binding to an OpenCRISPR-1 polypeptide: GUUUUAGAGCUGUGUUGAAAAACACAGCAAGUUAAAAUAAGGCUUUGUCCGUAUCCAACUUGAA AAAGUGAGCACCGAUUCGGUGC (SEQ ID NO: 3346); GUUUUAGAGCUGGAAACAGCAAGUUAAAAUAAGGCUUUGUCCGUAUCCAACUUGAAAAAGUGAG CACCGAUUCGGUGC (SEQ ID NO: 3347); and GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGC (SEQ ID NO: 3348).

[0184] By “persistence” in the context of an allogeneic transplant is meant the continued survival of a donor cell in a host organism. In some embodiments, allogeneic cell(s) comprising one or more of the edits described herein (e.g., a base edit in a CD5, CD3e, CD 3g, B2M, CD58, CD48, CD54, CD58, CD155, MICA, MICB, and / or CIITA gene, or regulatory element(s) thereof; or knockdown of a CD5, TCRaP, B2M, and / or CIITA polypeptide) persist in a subject allogeneic to the cell(s) at higher levels over time post-infusion than corresponding unedited allogeneic control cell(s). In some embodiments, allogeneic cell(s) comprising an edit resulting in reduced or eliminated expression of a CD54 and / or CD58 polypeptide in the cell(s) persist in a subject allogenic to the cell(s) at higher levels over time post-infusion than corresponding unedited allogeneic control cell(s). In embodiments, the percentage of edited cells (e.g., T cells, NK cells, or lymphocytes) persisting in a subject at a given time point (e.g., 7 days, 14 days, 1 month, 3 months, 6 months, 9 months, or greater than 1, 2, or 3 years is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% greater than the level of unedited control cells at the same time point. A cell(s) modified by methods of the present disclosure are more persistent than a reference unmodified cell(s). In some embodiments, cells prepared according to the methods of the disclosure show increased persistence relative to a reference cell. It can be advantageous for the increase in persistence to be 1.25-fold, 1.5-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, or greater relative to the reference cell.

[0185] By “subject” or “patient” is meant a mammal, including, but not limited to, a human or non-human mammal. In embodiments, the mammal is a bovine, equine, canine, ovine, rabbit, rodent, nonhuman primate, or feline. In an embodiment, “patient” refers to a mammalian subject with a higher than average likelihood of developing a disease or a disorder. Exemplary patients can be humans, non-human primates, cats, dogs, pigs, cattle, cats, horses, camels, llamas, goats, sheep, rodents (e.g., mice, rabbits, rats, or guinea pigs) and other mammalians that can benefit from the therapies disclosed herein. Exemplary human patients can be male and / or female.

[0186] “Patient in need thereof’ or “subject in need thereof’ is referred to herein as a patient diagnosed with, at risk or having, predetermined to have, or suspected of having a disease or disorder.

[0187] The terms “protein”, “peptide”, “polypeptide”, and their grammatical equivalents are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. A protein, peptide, or polypeptide can be naturally occurring, recombinant, or synthetic, or any combination thereof.

[0188] The term “fusion protein” as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins.

[0189] The term “recombinant” as used herein in the context of proteins or nucleic acids refers to proteins or nucleic acids that do not occur in nature but are the product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations as compared to any naturally occurring sequence.

[0190] By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

[0191] By “reference” is meant a standard or control condition. In one embodiment, the reference is a wild-type or healthy cell. In other embodiments and without limitation, a reference is an untreated cell that is not subjected to a test condition, or is subjected to placebo or normal saline, medium, buffer, and / or a control vector that does not harbor a polynucleotide of interest. In some cases, the reference is an unedited, untransduced, or wild-type cell (e.g., a T cell). In some cases, a reference is a healthy subject, such as a subject not having a neoplasia. In some embodiments, the reference is a cell lacking a nucleobase alteration and / or having an additional nucleobase alteration. In some cases, a reference is a cell that does not express a polypeptide of the disclosure (e.g., a chCLEC2d polypeptide) or a polypeptide of interest. The reference can be a cell that does not express one or more of the polypeptides described herein. The reference can be a subject before administration of a composition provided herein or treated according to a method provided herein and / or the subject before a change in a treatment (e.g., an alteration in dose or agent administered to the subject). The reference may be a wild-type CLEC2d polypeptide. In some embodiments, the reference is an HLA class I (HLA-I) mismatch or HLA-I deficient cell. In various instances the reference cell is a T cell that does not express a chimeric C-type lectin domain family 2, member D (CLEC2d) polypeptide of the present disclosure and / or that lacks one or more modifications (e.g., reduced expression of one or more polypeptides) of the disclosure.

[0192] A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween. In some embodiments, a reference sequence is a wild-type sequence of a protein of interest. In other embodiments, a reference sequence is a polynucleotide sequence encoding a wild-type protein.

[0193] The term “RNA-programmable nuclease,” and “RNA-guided nuclease” refer to a nuclease that forms a complex with (e.g., binds or associates with) one or more RNA(s) that is not a target for cleavage. In some embodiments, an RNA-programmable nuclease, when in a complex with an RNA, may be referred to as a nuclease-RNA complex. Typically, the bound RNA(s) is referred to as a guide RNA (gRNA).

[0194] As used herein, the term “scFv” refers to a single chain Fv antibody in which the variable domains of the heavy chain and the light chain from an antibody have been joined to form one chain. scFv fragments contain a single polypeptide chain that includes the variable region of an antibody light chain (VL) (e.g., CDR-L1 , CDR- L2, and / or CDR-L3) and the variable region of an antibody heavy chain (VH) (e.g., CDR-H1 , CDR-H2, and / or CDR-H3) separated by a linker. The linker that joins the VL and VH regions of a scFv fragment can be a peptide linker composed of proteinogenic amino acids. Alternative linkers can be used to so as to increase the resistance of the scFv fragment to proteolytic degradation (for example, linkers containing D- amino acids), in order to enhance the solubility of the scFv fragment (for example, hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine and serine residues), to improve the biophysical stability of the molecule (for example, a linker containing cysteine residues that form intramolecular or intermolecular disulfide bonds), or to attenuate the immunogenicity of the scFv fragment (for example, linkers containing glycosylation sites). It will also be understood by one of ordinary skill in the art that the variable regions of the scFv molecules described herein can be modified such that they vary in amino acid sequence from the antibody molecule from which they were derived. For example, nucleotide or amino acid substitutions leading to conservative substitutions or changes at amino acid residues can be made (e.g., in CDR and / or framework residues) so as to preserve or enhance the ability of the scFv to bind to the antigen recognized by the corresponding antibody.

[0195] By “specifically binds” is meant a nucleic acid molecule, polypeptide, polypeptide / polynucleotide complex, compound, or molecule that recognizes and binds a polypeptide and / or nucleic acid molecule of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample.

[0196] In some embodiments, conservative substitutions can involve the exchange of a member of one of these classes for another member of the same class. In some embodiments, nonconservative amino acid substitutions can involve exchanging a member of one of these classes for another class.

[0197] By “specifically binds” is meant a nucleic acid molecule, polypeptide, polypeptide / polynucleotide complex, compound, or molecule that recognizes and binds a polypeptide and / or nucleic acid molecule of the disclosure, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample.

[0198] By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence. In one embodiment, a reference sequence is a wild-type amino acid or nucleic acid sequence. In another embodiment, a reference sequence is any one of the amino acid or nucleic acid sequences described herein. In one embodiment, such a sequence is at least about 60%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or even 99.99%, identical at the amino acid level or nucleic acid level to the sequence used for comparison.

[0199] Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0200] Nucleic acid molecules useful in the methods of the disclosure include any nucleic acid molecule that encodes a polypeptide of the disclosure or a functional fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a doublestranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the disclosure include any nucleic acid molecule that encodes a polypeptide of the disclosure or a functional fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

[0201] By “split” is meant divided into two or more fragments.

[0202] A “split polypeptide” or “split protein” refers to a protein that is provided as an N- terminal fragment and a C-terminal fragment translated as two separate polypeptides from a nucleotide sequence(s). The polypeptides corresponding to the N-terminal portion and the C- terminal portion of the split protein may be spliced in some embodiments to form a “reconstituted” protein. In embodiments, the split polypeptide is a nucleic acid programmable DNA binding protein (e.g. a Cas9) or a base editor.

[0203] The term “target site” refers to a nucleotide sequence or nucleobase of interest within a nucleic acid molecule that is modified. In embodiments, the modification is deamination of a base. The deaminase can be a cytidine or an adenine deaminase. The fusion protein or base editing complex comprising a deaminase may comprise a dCas9-adenosine deaminase fusion protein, a Casl2b-adenosine deaminase fusion, or a base editor disclosed herein.

[0204] As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and / or symptoms associated therewith or obtaining a desired pharmacologic and / or physiologic effect. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated. In some embodiments, the effect is therapeutic, ie., without limitation, the effect partially or completely reduces, diminishes, abrogates, abates, alleviates, or reduces the intensity of, or cures a disease and / or adverse symptom attributable to the disease. In some embodiments, the effect is preventative, z.e., the effect protects or prevents an occurrence or reoccurrence of a disease or condition. To this end, the presently disclosed methods comprise administering a therapeutically effective amount of a composition as described herein.

[0205] By “uracil glycosylase inhibitor” or “UGI” is meant an agent that inhibits the uracil- excision repair system. Base editors comprising a cytidine deaminase convert cytosine to uracil, which is then converted to thymine through DNA replication or repair. In various embodiments, a uracil DNA glycosylase (UGI) prevent base excision repair which changes the U back to a C. In some instances, contacting a cell and / or polynucleotide with a UGI and a base editor prevents base excision repair which changes the U back to a C. An exemplary UGI comprises an amino acid sequence as follows: >splP14739IUNGI_BPPB2 Uracil-DNA glycosylase inhibitor MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPE YKPWALVIQDSNGENKIKML (SEQ ID NO: 231).

[0206] In some embodiments, the agent inhibiting the uracil-excision repair system is a uracil stabilizing protein (USP). See, e.g., WO 2022015969 Al, incorporated herein by reference.

[0207] As used herein, the term "vector" refers to a means of introducing a nucleic acid molecule into a cell, resulting in a transformed cell. Vectors include plasmids, transposons, phages, viruses, liposomes, lipid nanoparticles, and episomes.

[0208] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0209] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0210] All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains

[0211] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and / or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.

[0212] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended. This wording indicates that specified elements, features, components, and / or method steps are present, but does not exclude the presence of other elements, features, components, and / or method steps. Any embodiments specified as “comprising” a particular component(s) or element(s) are also contemplated as “consisting of’ or “consisting essentially of’ the particular component(s) or element(s) in some embodiments. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0213] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, z.e., the limitations of the measurement system.

[0214] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

[0215] BRIEF DESCRIPTION OF THE DRAWINGS

[0216] FIGs. 1A-1C provide flow cytometry scatter plots showing surface expression of CLEC2d in the B cell lymphoma (BCL) immortalized cell lines Jeko-1 (FIG. 1A), NALM-6 (FIG. IB), and Raji (FIG. 1C). For each of FIGs. 1A-1C, surface expression was measured for biological replicates. The numbers within each scatter plot indicate the percent of total cells counted that were designated as surface-expressing CLEC2d. In FIGs. 1A-1C, the term “APC” indicates the fluorophore allophycocyanin. FIGs. 2A and 2B provide schematic diagrams showing how immune cells of the disclosure inhibit natural killer (NK) cell activation. FIG. 2A provides a schematic diagram showing that beta-2-microglobulin (B2M) knock-out cells, which are deficient for human leukocyte antigen I (HLA-I) surface expression, are susceptible to NK cell killing via perforin and granzyme B release. FIG. 2B provides a schematic diagram showing that resistance of B2M knock-out CAR T-cells to killing by natural killer cells can be increased through surface expression of chimeric CLEC2d (chCLEC2d) in the CAR-T cells. The surface-expressed chimeric CLEC2d polypeptide interacts with CD161 expressed in natural killer (NK) cells to inhibit activation of the NK cells.

[0217] FIGs. 3A-3C provide a bar graph and flow cytometry scatter plots showing that CD161, the receptor for CLEC2d, was highly expressed on natural killer (NK) cells across a variety of natural killer (NK) cell subsets and donors. The human donors from which the NK cells were collected were referred to as DI 19 (Donor 1), D388 (Donor 2), D841 (Donor 3), and D949 (Donor 4). FIG. 3A provides a bar graph showing CD161 expression in natural killer (NK) cells collected from each of the donors. FIG. 3B provides flow cytometry scatter plots showing that CD161 was largely co-expressed with the HLA-E inhibitory receptor, NKG2A, in NK cells collected from each of the donors. FIG. 3C provides flow cytometry scatter plots showing that CD161 was largely co-expressed with the killer cell immunoglobulin-like receptors (KIRs), which are NK cell inhibitors, KIR2DL2 / 3, KIR3DL1, and KIR2DL1. In FIGs. 3B and 3C, the term “CD161 FMO” refers to a fluorescence minus one control where cells were stained with all fluorophores except for the fluorophore used to detect CD161. In FIGs. 3B and 3C, the numbers in each quadrant of each flow cytometry scatter plot indicate the percent of total counted cells that fell within the indicated quadrant.

[0218] FIGs. 4A and 4B provide a bar graph and flow cytometry scatter plots showing CD161 and CLEC2d expression in CD4+ and CD8+ T cells from human donors DI 19, D949, and D270. In FIG. 4B, the number above the square indicates the percent of total counted cells that were designated as surface-expressing CD161 or CLEC2d.

[0219] FIG. 5 provides a bar graphs showing percent A to G base editing in T cells at a target adenosine (A) carried out using base editor systems containing a TadA*8.20 adenosine deaminase and a guide polynucleotides containing one of the following spacers (see Table 1A for spacer sequences and target adenosines): IMM_169 (169), IMM_170 (170), IMM_171 (171), IMM 172 (172), or IMM_173 (173).

[0220] FIGs. 6A and 6B provide flow cytometry histograms showing that wild-type CLEC2d localized primarily intracellularly in T cells. FIG. 6A provides a flow cytometry histograms showing levels of surface expression of CLEC2d in T cells transduced with lentiviruses containing polynucleotides encoding CLEC2d (see BTx_CM449 of Tables 7A-7C and 8) and dihydrofolate reductase (mDHFR). The dihydrofolate reductase allowed for enrichment of cells successfully transduced with the lentiviruses using methotrexate (MTX). FIG. 6B provides a flow cytometry histograms showing levels of intracellular expression of CLEC2d in T cells transduced with lentiviruses containing polynucleotides encoding wild-type CLEC2d (see BTx_CM449 of Tables 7A-7C and 8) and dihydrofolate reductase (mDHFR). The dihydrofolate reductase allowed for enrichment of cells successfully transduced with the lentiviruses using methotrexate (MTX). In FIGs. 6A and 6B, the term “UTD” indicates untransduced cells that were not transduced using the lentiviruses. The numbers within the plots of FIGs. 6A and 6B indicate the percent of total cells counted that were designated as expressing CLEC2d on the cell surface (FIG. 6A) or intracellularly (FIG. 6B). In FIGs. 6A and 6B, the term “APC” indicates the fluorophore allophycocyanin, “-MTX” indicates cells that were not cultured in media containing methotrexate, and “+MTX” indicates cells that were cultured in media containing methotrexate.

[0221] FIGs. 7A-7C provide flow cytometry scatter plots demonstrating that the Q epitope (i.e., a peptide with the amino acid sequence ELPTQGTFSNVSTNVS (SEQ ID NO: 472)) inserted into a chCLEC2d polypeptide was expressed on the surface of a T cell. FIG. 7A provides a flow cytometry scatter plot where the numbers within the plot indicate the percent of total cells counted that were designated as surface-expressing the indicated CLEC2d polypeptide. FIG. 7B provides a flow cytometry scatter plot where the numbers within the plot indicate the percent of total cells counted that were designated as surface-expressing the Q epitope (“CD34”). FIG. 7C provides a flow cytometry scatter plot where the numbers within each quadrant indicate the percent of total cells counted that fell within the indicated quadrant. Cells that were determined to surface-express a CLEC2d polypeptide containing the Q epitope fell within the upper-right quadrant of each plot. In FIGs. 7A-7C, “529” indicates “CM529,” “530” indicates “CM529,” and “496” indicates “CM496.”

[0222] FIG. 8 provides a schematic diagram showing chimeric CLEC2d (chCLEC2d) polypeptides generated in an effort to disrupt intracellular retention of the chCLEC2d polypeptides. In FIG. 8, “TM” indicates a transmembrane domain, “ECD” indicates an extracellular domain, “CP” indicates a cytoplasmic domain, “M” indicates an N-terminal methionine, “CD3A(^” or “CD3z(trunc)” indicates an 8 amino acid peptide corresponding to a truncated CD3A(^ domain, “ATG” indicates a codon encoding methionine, and “CD3z(rev:trunc)” indicates a peptide containing from N-terminus to C-terminus the contiguous sequence of amino acids corresponding to CD3z(trunc) in order from C-terminus to N-terminus (i.e., the reverse sequence, or “rev”). At the bottom of FIG. 8 is provided a description of components in order from 5 '-end to 3 '-end of the polynucleotides “BTx_CM492” and “BTx_CM494” used for cell transduction (see Tables 7A-7C and 8 for sequences). Each of BTx_CM492 and BTx_CM494 contained an “ATG” codon followed by sequences encoding the indicated polypeptides. In FIG. 8, the “X” indicates a domain of CLEC2d to be replaced with a heterologous domain.

[0223] FIGs. 9A and 9B provide flow cytometry scatter plots showing levels of surface expression of CLEC2D observed in T cells transduced with BTx_CM492 or BTx_CM494 described in FIG. 8 at 3 days (FIG. 9A) and 5 days (FIG. 9B) post-transduction. In FIGs. 9A and 9B, “UTD” indicates untransduced (i.e., wild-type) T cells. In FIGs. 9A and 9B, all of the T cells were altered to knock out expression of beta-2-microglobulin and CD161 using base editor systems of the disclosure. In FIGs. 9A and 9B, the terms “D55” and “D41” indicate T cells from the human donors D55 and D41, respectively, the numbers within each plot indicate the percent of total counted cells that were determined to surface-express CLEC2d, and the term “APC” indicates the fluorophore allophycocyanin.

[0224] FIG. 10 provides a schematic diagram showing chimeric CLEC2d (chCLEC2d) polypeptides generated in an effort to disrupt intracellular retention of the chCLEC2d polypeptides. Each of the chCLEC2d polypeptides contained an NKG2D transmembrane domain. In FIG. 10, “TM” indicates a transmembrane domain, “ECD” indicates an extracellular domain, “CP” indicates a cytoplasmic domain, “M” indicates an N-terminal methionine, “CD3A ” or “CD3z(trunc)” indicates an 8 amino acid peptide corresponding to a truncated CD3A(^ domain, “ATG” indicates a codon encoding methionine, and “CD3z(rev:trunc)” indicates a peptide containing from N-terminus to C-terminus the contiguous sequence of amino acids corresponding to CD3z(trunc) in order from C-terminus to N-terminus (i.e., the reverse sequence, or “rev”). At the bottom of FIG. 10 is provided a description in order from 5 '-end to 3 '-end of components of the polynucleotides “BTx_CM488” and “BTx_CM490” used for cell transduction (see Tables 7A-7C and 8 for sequences). Each of BTx_CM488 and BTx_CM490 contained an “ATG” codon followed by sequences encoding the indicated polypeptides. In FIG. 10, the “X” terms indicate domains of CLEC2d to be replaced with a heterologous domain. Term “Type II integral membrane protein family” indicates membrane proteins containing in order from N-terminus to C-terminus a cytoplasmic domain, a transmembrane domain, and an extracellular domain. FIGs. 11A and 11B provide flow cytometry scatter plots showing levels of surface expression of CLEC2d observed in T cells transduced with BTx_CM488 or BTx_CM490 described in FIG. 10 at 3 days (FIG. 11 A) and 5 days (FIG. 11B) post-transduction. In FIGs. 11A and 11B, “UTD” indicates untransduced (i.e., wild-type) T cells. In FIGs. 11A and 11B, all of the T cells were altered to knock out expression of beta-2-microglobulin and CD161 using base editor systems of the disclosure. In FIGs. 11A and 11B, the terms “D55” and “D41” indicate T cells from the human donors D55 and D41, respectively, the numbers within each plot indicate the percent of total counted cells that were determined to surface-express CLEC2d, and the term “APC” indicates the fluorophore allophycocyanin.

[0225] FIGs. 12A and 12B provide schematic diagrams relating to the preparation of Type I membrane protein chCLEC2d polypeptides. FIG. 12A provides schematic diagrams defining “Type I” and “Type II” membrane proteins. FIG. 12B provides a schematic diagram describing the preparation of Type I membrane protein chCLEC2d polypeptides. In the top portion of FIG. 12B, the arrows represent the incorporation of the extracellular domain of CLEC2d in the reverse orientation into the indicated polypeptide containing a signal peptide (SP) from CD8 and a transmembrane domain (TM) from CD8 (CD8TM) or CD4 (CD4TM). By “reverse orientation” is meant a sequence containing from N-terminus to C-terminus the contiguous sequence of amino acids corresponding to the wild-type polypeptide in order from C-terminus to N-terminus (i.e., the reverse sequence). In some cases, the Type I membrane protein chCLEC2d polypeptide contained a hinge domain from CD8 together with a transmembrane domain from CD8 (i.e., CD8H-TM). In FIG. 12B, the term “T2A” indicates the self-cleaving peptide T2A, and the term “mutDHFR” indicates a dihydrofolate reductase variant (i.e., “mutant”). At the bottom of FIG. 12B is provided a description in order from 5 '-end to 3 '-end of components of the polynucleotides “BTx_CM454,” “BTx_CM455,” and “BTx_CM460” used for cell transduction (see Tables 7A-7C and 8 for sequences).

[0226] FIG. 13 provides flow cytometry histograms showing levels of surface expression of CLEC2d at three days post-transduction of the Type I membrane protein chCLEC2d polypeptides of FIG. 12B in T cells from human donor D949 or D41. The T cells were transduced with BTx_CM454, BTxCM455, or BTx_CM460, as indicated in the figure. In FIG. 13, the term “APC” indicates the fluorophore allophycocyanin. All of the T cells were altered to knock out expression of beta-2-microglobulin (B2M KO) and CD161 using base editor systems of the disclosure. In FIG. 13, the term “UTD” indicates cells that were not transduced with a polynucleotide encoding a Type I membrane protein chCLEC2d polypeptide. FIG. 14 provides a schematic diagram describing the preparation of Type I membrane protein CLEC2d (chCLEC2d) polypeptides. In FIG. 14, “SP” indicates a signal peptide, “TM” indicates a transmembrane domain, “H” indicates a hinge domain, and “ECD” indicates an extracellular domain. In FIG. 14, the arrows represent the incorporation of the extracellular domain of CLEC2d into a polypeptide containing a signal peptide (SP) from CD8 and a transmembrane domain (TM) from CD8 (8TM) or CD4 (4TM). In some cases, the Type I membrane protein chCLEC2d polypeptide contained a hinge domain from CD8 together with a transmembrane domain from CD8 (i.e., 8H / TM). In FIG. 14, the term “T2A” indicates the selfcleaving peptide T2A, and the term “mDHFR” indicates a dihydrofolate reductase variant (i.e., “mutant”). At the bottom of FIG. 14 is provided a description of components in order from 5'- end to 3 '-end of the polynucleotides “BTx_CM495,” “BTx_CM496,” and “BTx_CM497” used for cell transduction (see Tables 7A-7C and 8 for sequences).

[0227] FIGs. 15A and 15B provide flow cytometry scatter plots showing levels of surface expression of CLEC2d observed in T cells transduced with BTxCM495, BTxCM496, or BTxCM497 described in FIG. 14 at 3 days (FIG. 15A) and 5 days (FIG. 15B) posttransduction. In FIGs. 15A and 15B, “UTD” indicates untransduced (i.e., wild-type) T cells. In FIGs. 15A and 15B, all of the T cells were altered to knock out expression of beta-2- microglobulin and CD161 using base editor systems of the disclosure. In FIGs. 15A and 15B, the terms “D55” and “D41” indicate T cells from the human donors D55 and D41, respectively, the numbers within each plot indicate the percent of total counted cells that were determined to surface-express CLEC2d, and the term “APC” indicates the fluorophore allophycocyanin.

[0228] FIG. 16 provides flow cytometry scatter plots showing intracellular levels of CLEC2d expression observed in T cells transduced with BTxCM495, BTxCM496, or BTxCM497 described in FIG. 14 at 5 days post-transduction, as determined using intracellular staining (ICS). In FIG. 16, “UTD” indicates untransduced (i.e., wild-type) T cells. In FIG. 16, all of the T cells were altered to knock out expression of beta-2-microglobulin and CD161 using base editor systems of the disclosure. In FIG. 16, the terms “D55” and “D41” indicate T cells from the human donors D55 and D41, respectively, the numbers within each plot indicate the percent of total counted cells that were determined to intracellularly express CLEC2d, and the term “APC” indicates the fluorophore allophycocyanin.

[0229] FIGs. 17A and 17B provide plots showing results from a natural killer (NK) cell mixed leukocyte reaction (MLR) demonstrating a reduction in specific lysis of T cells expressing a chimeric CLEC2d polypeptide containing hinge and transmembrane domains from CD8. In each of FIGs. 17A and 17B, the x-axis indicates the effector to target ratio (E:T) at which the cells were cultured, where the effector cells were natural killer (NK) cells and the target cells were T cells altered using base editor systems of the disclosure to knock out expression of beta-2 - microglobulin that were (CM496) or were not (B2M KO) transduced with the polynucleotide BTx_CM496 described in FIG. 14. The NK cells were from donor DI 19 and the T cells were from either donor D41 (FIG. 17A) or donor D55 (FIG. 17B). Specific lysis was calculated as: %specific lysis = 100-[(TE / TC)*100] where TE=% of live on-target cells (HLA-ABC-) in test tubes (target+effector) and TC=%(average of replicates) of live on-target cells (HLA-ABC-) in control tubes (target only).

[0230] FIGs. 18A-18C provide plots showing results from natural killer (NK) cell mixed leukocyte reactions (MLR) demonstrating a reduction in specific lysis by NK cells from three different human donors (DI 19, D841, and D168) of T cells expressing a chimeric CLEC2d polypeptide containing hinge and transmembrane domains from CD8. The T cells expressing the chimeric CLEC2d polypeptide were prepared from human donor 55. In each of FIGs. 18A-18C, the x-axis indicates the effector to target ratio (E:T) at which the cells were cultured, where the effector cells were natural killer (NK) cells and the target cells were T cells altered using base editor systems of the disclosure to knock out expression of beta-2-microglobulin that were (CM496) or were not (B2M KO) transduced with the polynucleotide BTx_CM496 described in FIG. 14. The NK cells were from donor DI 19 (FIG. 18A), D841 (FIG. 18B), or D168 (FIG. 18C) and the T cells were from a different donor. Specific lysis was calculated as: %specific lysis = 100-[(TE / TC)*100] where TE=% of live on-target cells (HLA-ABC-) in test tubes (target+effector) and TC=%(average of replicates) of live on-target cells (HLA-ABC-) in control tubes (target only).

[0231] FIG. 19 provides flow cytometry scatter plots demonstrating that CLEC2d was expressed on the surface of a T cell as a GPI-anchored protein. GPI-anchored CLEC2d was detectable on the cell surface, and addition of a CD8 hinge domain further stabilized surface expression. In FIGs. 19, “531” indicates “CM531,” “532” indicates “CM532,” and “496” indicates “CM496.” FIG. 20 provides a schematic diagram and chart describing the preparation of secreted variants of CLEC2d. In FIG. 20, “SP” indicates a signal peptide, “TM” indicates a transmembrane domain, “H” indicates a hinge domain, “CLEC2d ECD” indicates an extracellular domain of CLEC2d, “T2A” indicates a self-cleaving peptide, “CD8SP” indicates a signal peptide from CD8, “IL-2SP” indicates an IL-2 signal peptide, “IFNgSP” indicates a signal peptide from interferon gamma, and the term “mDHFR” indicates a dihydrofolate reductase variant (i.e., “mutant”). The constructs in the chart of FIG. 20 were designed as variants of BTx_CM496 of FIG. 14. FIGs. 21A and 21B provide plots showing results from a natural killer (NK) cell mixed leukocyte reaction (MLR) demonstrating a reduction in specific lysis of T cells expressing a secreted CLEC2d variant of FIG. 20 by NK cells from donor 168 (FIG. 21A) and donor 655 (FIG. 21B). The T cells were deficient for expression of B2M. As a control, untransduced cells (i.e., cells not expressing a secreted variant of CLEC2d) were evaluated (B2M KO cells). The x- axis of FIGs. 21 A and 21B represents the ratio of effector (NK cells) to T cells (target cells) in the MLRs.

[0232] FIGs. 22A-22E provide bar graphs, heatmaps, histograms and FACS plots demonstrating that the CD161 inhibitory receptor was broadly expressed by alloreactive NK cells. FIG. 22A provides a bar graph showing the frequency of mature (CD56dimCD16+) and immature (CD56hlghCD16‘) subsets within total NK cells (CD56+CD3 ) from human PBMC or purified CD56+NK cell samples. Symbols represent independent donors. FIG. 22B provides a heatmap showing mean frequency of CD56+CD3‘, CD56dimCD16+and CD56hlghCD16 NK cells expressing the inhibitory receptors: CD161, CD94 / NKG2A, KIR2DL1, KIR2DL2 / L3, KIR3DL1, LIR-1, PD-1 and SIRPalpha. Data represents 7-9 independent donors. FIG. 22C provides a histogram showing HLA class I expression in b2MKO and unedited T cells. FIGs. 22D and 22E provides FACS plots (FIG. 22D) and summarized data in the form of a bar graph (FIG. 22E) showing the frequency of degranulating CD107a+NK cells that expressed the indicated inhibitory receptor after stimulation with b2MKO T cells from an unrelated donor. Symbols represent independent NK cell donors. For all data, bars represent mean and error bars show ±s.e.m.

[0233] FIGs. 23A-23F provide schematics, histograms and bar graphs demonstrating that secretion of CLEC2d protected HLA class Ldeficient T cells from NK cell-driven lysis in vitro. FIG. 23A provides a schematic demonstrating that secreted (s) CLEC2d by HLA class I- deficient (b2MKO) T cells inhibited CD161+NK cells. FIG. 23B provides a schematic of a lentivirus construct encoding methotrexate (MTX)-resistant dihydrofolate reductase (mDHFR) and sCLEC2d separated by an intervening T2A peptide. sCLEC2d contained the IFNgamma signal peptide (SP) fused to the extracellular domain (ECD) of CLEC2d replacing the intracellular (IC) and transmembrane (TM) regions. FIGs. 23C and 23D provide a histogram (FIG. 23C) and summarized data in the form of a bar graph (FIG. 23D) quantifying CLEC2d in the cell culture supernatant from non-transduced (NTD) T cells, or b2MKO T cells engineered to express sCLEC2d in the presence or absence of MTX. Data represents geometric mean fluorescence intensity (GMFI) of bead-bound CLEC2d. Symbols represent 3 independent donors. FIG. 23E provides histograms showing cell-surface and intracellular detection of CLEC2d by b2MK0 T cells that were NTD or expressed sCLEC2d. FIG. 23F provides a bar graph showing the results from an NK cell cytotoxicity assay. The bar graph of FIG. 23F demonstrates specific lysis of b2MK0 T cells that were NTD or expressed sCLEC2d at 48-hours post-culture with NK cells at the indicated E / T ratios. Symbols represent 4 independent NK cell donors in duplicate. Wilcoxon matched-pairs signed rank test was used to calculate significance. For all data, bars represent mean and error bars show ±s.e.m.

[0234] FIGs. 24A-24H provide schematics, bar graphs and histograms demonstrating that HLA class I-deficient T cells expressing membrane-bound CLEC2d resisted elimination by NK cells in vitro. FIG. 24A provides a schematic showing expression of membrane-bound (mb) CLEC2d on HLA class I-deficient (b2MK0) T cells inhibits CD161+NK cells. FIG. 24B provides a schematic of lentivirus constructs encoding mDHFR and full-length (fl) CLEC2d or a chimeric mbCLEC2d where the CD8alpha SP, and CD8alpha TM and IC domains flanked the N- and C- termini of the CLEC2d ECD respectively. FIG. 24C provides a histogram showing cell-surface and intracellular detection of CLEC2d by b2MK0 T cells that were NTD or engineered to express f!CLEC2d. FIG. 24D provides a histogram showing cell-surface expression of CD161 by b2MK0 T cells relative to NK cells. FIG. 24E provides a bar graph showing frequency of on-target A>G nucleotide conversion in T cells that were unedited or base-edited using a KERB 1 -specific sgRNA administered in combination with ABE8.20m mRNA. FIG. 24F provides histograms showing cell-surface and intracellular detection of CLEC2d by KLRB1K0 b2MK0 T cells that were NTD or expressed flCLEC2d. FIG. 24G provides a histogram showing surface expression of mbCLEC2d by b2MK0 T cells that were NTD or expressed chimeric mbCLEC2d. FIG. 24H provides a bar graph showing the results from an NK cell cytotoxicity assay. The bar graph of FIG. 24H demonstrates specific lysis of b2MK0 T cells that were NTD or expressed mbCLEC2d at 48-hours post-culture with NK cells at the indicated E / T ratios. Symbols represent 7 independent NK cell donors in duplicate. Wilcoxon matched- pairs signed rank test was used to calculate significance. For all data, bars represent mean.

[0235] FIGs. 25A-25E provide schematics, bar graphs, histograms and FACS plots demonstrating that genetic disruption of adhesion ligands CD54 and CD58 abrogated alloreactive NK cell responses. FIG. 25A provides a schematic showing that genetic disruption of CD54 and CD58 prevented T cells from binding to LFA-1 and CD2 on alloreactive NK cells, respectively. FIG. 25B provides a bargraph showing the frequency of on-target A>G nucleotide conversion in T cells that were base-edited using CD54- and CD58-specific sgRNAs administered in combination with ABE8.20m mRNA. FIG. 25C provides histograms showing cell-surface expression of CD54 and CD58 in unedited, CD54KO, and CD58KO T cells. FIGs. 25D and 25E provide results from an NK cell cytotoxicity assay. FIG. 25D provides FACS plots indicating the frequency of on-target T cell populations: b2M KO, CD54 KO b2M KO, and CD58 KO b2MK0 T cells relative to off-target HLA class I+T cells at 48-hours post-culture with NK cells at the indicated E / T ratios. FIG. 25E provides a bargraph showing specific lysis of on-target T cell populations at 48-hours post-culture with NK cells at the indicated E / T ratios. Symbols represent 5 independent NK cell donors in duplicate. Wilcoxon matched-pairs signed rank test was used to calculate significance. For all data, bars represent mean and error bars show ±s.e.m.

[0236] FIGs. 26A-26F provides flow cytometry (FACS) plots, bargraphs, a pie chart, and schematics demonstrating that mbCLEC2d expression and CD58KO enhanced resistance of b2MK0 T cells to NK cell-dependent lysis in vitro. FIGs. 26A and 26B provide a FACS plot (FIG. 26A) and summarized data in the form of a bargraph (FIG. 26B) showing frequency of CD56 CD3', CD56dimCD16+, CD56highCD16’ NK cell subsets expressing CD2 (n=8 donors) and CD161 (n=5 donors). Symbols represent independent donors. FIG. 26C provides a pie chart showing mean frequency (n=3 donors) of CD56+CD3‘ NK cells expressing the indicated combinations of CD2 and CD 161. FIG. 26D provides a schematic indicating that mbCLEC2d expression and CD58KO in b2MK0 T cells mitigated NK cell alloreactivity by, without intending to be bound by theory or any mechanism of action, engaging CD161 and abrogating CD2 ligation, respectively. FIGs. 26E and 26F provides results from an NK cell cytotoxicity assay. FIG. 26E provides FACS plots showing the frequency of on-target HLA class I" T cell populations: b2MK0 T cells, mbCLEC2d+b2MK0 T cells, CD58KO b2MK0 T cells, and mbCLEC2d+CD58KO b2MK0 T cells relative to off-target HLA class I+T cells at 48-hours post-culture with NK cells at the indicated E / T ratios. FIG. 26F provides a bargraph showing specific lysis of on-target T cell populations at 48-hours post-culture with NK cells at the indicated E / T ratios. Symbols represent 3 independent NK cell donors in duplicate. Wilcoxon matched-pairs signed rank test was used to calculate significance. For all data, bars represent mean and error bars show ±s.e.m.

[0237] FIGs 27A and 27B provide bar graphs demonstrating that CD58KO combined with HLA-E single-chain trimer or mbCLEC2d attenuated alloreactive NK cell responses to the same extent. FIGs. 27A-27B present results from an NK cell cytotoxicity assay. FIG. 27A provides a bar graph showing specific lysis of b2MK0 T cells, CD58KO b2MK0 T cells or CD58KO b2MK0 T cells expressing HLA-E single-chain (sc) at 48-hours post-culture with NK cells at the indicated E / T ratios. Symbols represent two independent NK cell donors in duplicate and bars indicate mean. FIG. 27B provides a bar graph showing specific lysis of NTD b2MK0 T cells and CD58KO b2MK0 T cells expressing mbCLEC2d or scHLA-E at 48-hours post-culture with NK cells at the indicated E / T ratios. Symbols represent average of 4 independent NK cell donors and error bars indicate ±s.e.m.

[0238] FIGs. 28A-28J provide bar graphs, histograms, and FACS plots demonstrating that combined mbCLEC2d expression and CD58KO averted in vivo NK cell-dependent allorej ection. FIG. 28A provides a bargraph presenting results from an experiement where NSG (n=5) and NSG-IL15tg (n=5) mice were infused with 5xl06primary human CD56+cells. The graph of FIG. 28A shows the peripheral concentration of CD56+CD3‘ NK cells at 1-, 2- and 3-weeks post-infusion. FIG. 28B provides a bargraph showing the frequency of on-target A>G nucleotide conversion in T cells that were base-edited using b2M- and CD3E-specific sgRNAs administered in combination with with ABE8.20m mRNA. FIG. 28C provides a histogram showing cellsurface expression of CD3 in unedited and CD3EK0 T cells. FIGs. 28D and 28E provide a FACS plot and a bargraph presenting data from NSG-IL15tg mice that were infused with 5xl06primary human CD56+cells (n=5, huNK mice) or remained unreconstituled (n=5, NSG-IL15tg) followed two-weeks later with 5xl06CD3EK0 T cells (HLA-I+) and 5xl06CD3EK0 b2MK0 T cells (HLA-F) from an allogeneic donor. In FIGs. 28D and 28E, FACS plots indicate frequency of (FIG. 28D) and summarized data in the form of a bargraph showing cells total for (FIG. 28E) allogeneic HLA-I+and HLA-F T cells in spleen 4-weeks post-T cell infusion into huNK mice and NSG-IL15tg mice. FIGs. 28F-28J present results from huNK mice (n=9 per group) that were infused with allogeneic T cell products comprising 5xl06CD3EK0 T cells (HLA-I+) and 5xl06CD3EK0 b2MK0 T cells (HLA-F), or 5xl06CD3EK0 T cells (HLA-I+) and 5xl06CD3EK0 CD58KO b2MK0 T cells expressing mbCLEC2d (HLA-F). Peripheral blood and spleen tissue were analyzed 5-days post-T cell infusion to quantify allogeneic cell persistence. FIG. 28F provides a FACS plot showing the frequency of peripheral T cell populations. FIG. 28G provides a bargraph showing the ratio of peripheral HLA CD3' to HLA+CD3‘ T cells from within individual mice. FIG. 28H provides a bargraph showing the peripheral concentrration of HLA CD3' T cells. FIG. 281 provides a bargraph showing the ratio of splenic HLA CD3' to HLA+CD3‘ T cells from within individual mice. FIG. 28J provides a bargraph showing the total number of HLA CD3' T cells in spleen tissue. For all data, symbols indicate independent animals or donors, bars represent mean and error bars show ±s.e.m. For FIG. 28E and FIGs. 28G-28J, Wilcoxon matched-pairs signed rank test was used to calculate significance.

[0239] FIG. 29 provides a bar graph showing percent A>G editing at a target adenosine within a cluster of differentiation 54 (CD54) polynucleotide encoded by T cells from two different donors (DI 19 or D949) using the adenosine deaminase base editor (ABE) ABE8.20 in combination with a guide RNA containing one of the spacers indicated along the x-axis (spacer sequences are provided in Table IB). The target A>G edit resulted in reduced expression of a CD54 polypeptide encoded by the CD54 polynucleotide. A>G editing was measured using nextgeneration sequencing (NGS). In FIG. 29, the terms “DI 19” and “D949” refer to two donors from which the T cells were originally collected.

[0240] FIG. 30 provides a plot showing results from a natural killer (NK) cell mixed leukocyte reaction (MLR) demonstrating a reduction in specific lysis of T cells base edited to knock out expression of both a cluster of differentiation 58 (CD58) polypeptide and a cluster of differentiation 54 (CD54) polypeptide and modified to express a chimeric CLEC2d (chCLEC2d) polypeptide containing a CD8 hinge domain and a CD8 transmembrane domain (CLEC2d- CD8H-TM). The T cells were originally obtained from donor DI 19. In FIG. 30, the term “B2M” indicates T cells base edited according to the methods provided herein to knock-out expression of beta-2-microglobulin, the term “CD58” indicates T cells base edited according to the methods provided herein to knock-out expression of CD58, the term “DKO” indicates T cells base edited according to the methods provided herein to knock out expression of both CD54 and CD58, and the term “KO” indicates “knock-out.” In the plot of FIG. 30, the lines containing circles, squares, or triangles filled-in with dark grey correspond to T cells that did not express the chCLEC2d polypeptide and the lines containing circles, squares, or triangles that not filled in with any color represent T cells that expressed the chCLEC2d polypeptide.

[0241] DETAILED DESCRIPTION

[0242] The disclosure features chimeric C-type lectin domain family 2, member D (chCLEC2d) polypeptides, immune effector cells expressing the chCLEC2d polypeptides, and methods for use of the immune effector cells in treating a disease or condition in a subject. Allogeneic immune effector cells expressing the chCLEC2d polypeptides have improved resistance to natural killer (NK) cell-mediated allorej ection by the immune system of a subject to which the immune effector cells are administered.

[0243] The aspects of the disclosure and embodiments thereof are based, at least in part, on the design of new chimeric CLEC2d polypeptides capable of being expressed on the surface of T cells. T cells expressing the chimeric CLEC2d polypeptides were found to have improved resistance to natural killer (NK) cell-mediated allorej ection thereof. The aspects of the disclosure are also based, at least in part, on the discovery that combined mbCLEC2d expression and CD58KO improved the in vivo persistence of HLA class I-deficient T cells by averting NK cell- driven rejection. NATURAL KILLER CELL-MEDIATED ALLOREJECTION OF T CELLS

[0244] When allogeneic T cells are administered to a subject, the cells are susceptible to killing (i.e., “allorej ection”) by immune cells of the subject, such as natural killer cells or T cells. To prevent allorej ection of the administered T cells by T cells of the subject’s immune system, the administered T cells can be modified, for example, to be deficient for expression of human leukocyte antigen class I (HLA-I) polypeptides. For instance, T cells can be rendered deficient for expression of HLA-I polypeptides by modifying the T cells according to the methods provided herein to knockout expression of beta-2-microglobulin. However, although modifying the administered T cells to be deficient for expression of HLA-I polypeptides increases resistance of the T cells to allorej ection by T cells of a subject to which they are administered, deficiency for expression of HLA-I polypeptides also renders the T cells susceptible to NK cell- mediated allorej ection in the subject (see FIG. 2A). Accordingly, as detailed in the Examples provided herein, and as shown in FIG. 2B, the T cells deficient for expression of HLA-I polypeptides can be modified to improve their resistance to NK cell-mediated allorej ection in the subject by being modified to surface-express CLEC2d polypeptides, such as the chimeric CLEC2d polypeptides of the disclosure. Not intending to be bound by any theory of operation, CLEC2d polypeptides expressed on the surface of the T cells inhibit activation of NK cells by interacting with the inhibitory CLEC2d receptor CD161 expressed on the surface of most NK cells. By binding to CD161, the CLEC2d polypeptide prevents activation of the NK cells, where activation of the NK cells is associated with increases in CD 107a surface expression, degranulation, and / or perforin and / or granzyme B release.

[0245] Accordingly, the present disclosure provides T cells, such as chimeric antigen receptorexpressing T cells (i.e., CAR T-cells) with increased resistance to NK cell-mediated allorejection and that surface-express chimeric CLEC2d receptors of the disclosure.

[0246] CHIMERIC C-TYPE LECTIN DOMAIN FAMILY 2, MEMBER D (CHCLEC2D) POLYPEPTIDES

[0247] In various aspects, the present disclosure features chimeric C-type lectin domain family 2, member D (chCLEC2d) polypeptides. The chCLEC2d polypeptides contain a CLEC2d extracellular domain and one or more of a signal peptide, a hinge domain, a transmembrane domain, and / or an intracellular domain that are heterologous to the CLEC2d extracellular domain. In embodiments, the heterologous peptide or domain is derived from CD8a, CD4, interleukin 2 (IL-2), interferon gamma (IFNg), a human leukocyte antigen (HLA), and / or NKG2D.

[0248] The chCLEC2d polypeptides may be Type I or Type II transmembrane proteins (see, FIG. 12A). In some embodiments, the chCLEC2d polypeptide is a Type I transmembrane protein containing, in order from N-terminus to C-terminus, an extracellular domain and a transmembrane domain. The chCLEC2d polypeptides may be secreted proteins. For example, the chCLEC2d polypeptide may contain a signal peptide (SP) from CD8, IL-2, or IFNg fused to a terminus of the extracellular domain of chCLEC2d (i.e., the N-terminus or the C-terminus). In some embodiments, a secreted chCLEC2d polypeptide contains no transmembrane domain and / or no hinge domain.

[0249] In various embodiments, it may be advantageous to integrate a polynucleotide encoding a chCLEC2d polypeptide of the disclosure into the genome of a cell. In some embodiments, the polynucleotide is incorporated into the genome of the cell within a gene in the cell. In various embodiments, the gene is a B2M or CIITA gene, or any gene associated with NK or T cell- mediated rejection of an allogeneic cell administered to a subject. In some cases, incorporation of the polynucleotide into the genome of the cell results in reduced or eliminated expression of a B2M polypeptide, CIITA polypeptide, and / or any other polypeptide in the cell associated with NK or T cell-mediated allorej ection of the cell in a subject.

[0250] In some cases, the chCLEC2d polypeptides contain in order from N-terminus to C- terminus, a CLEC2d extracellular domain, a hinge domain, and a transmembrane domain. In some cases, the hinge domain is derived from CD8a. In some embodiments, the transmembrane domain is derived from CD8a, decay accelerating factor (DAF), NKG2D, or CD4.

[0251] Transmembrane domains for use in the disclosed chCLEC2d polypeptides can include at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some embodiments, the transmembrane domain is derived from CD4, CD8a, CD28, or CD3

[0252] It can be advantageous for a chCLEC2d polypeptide of the disclosure to contain a peptide that may be used to select for T cells expressing the chCLEC2d polypeptide (e.g., in flow cytometry). In some cases, the peptide is a CD34 selection peptide, such as a peptide containing the amino acid sequence ELPTQGTFSNVSTNVS (SEQ ID NO: 472), which can also be referred to as a “Q” peptide, “Q tag,” or “Q epitope.” In embodiments, the Q peptide is used for the selection of cells surface-expressing the chCLEC2d polypeptide. In some cases, the chCLEC2d polypeptide contains a molecular switch, such as the kill switches described herein. The chCLEC2d polypeptides have increased levels of surface expression in T cells relative to a wild-type CLEC2d polypeptide. In embodiments, levels of surface expression of a chCLEC2d polypeptide of the disclosure in a T cell are about or at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold or greater than levels of surface expression for a wild-type CLEC2d polypeptide.

[0253] In various embodiments, an allogeneic T cell expressing a chCLEC2d polypeptide of the disclosure has increased resistance to NK cell-mediated allorej ection relative to T cells expressing a wild-type CLEC2d polypeptide or no CLEC2d polypeptide. In some embodiments, the T cells expressing chCLEC2d polypeptide show a reduction in specific lysis by NK cells of about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to T cells expressing a wild-type CLEC2d polypeptide or T cells expressing no CLEC2d polypeptide. NK cell-mediated allorejection of T cells may be measured using a mixed leukocyte reaction (MLR) (see, e.g., FIGs. 17A and 17B). In some cases, the chCLEC2d polypeptide binds a CD161 polypeptide expressed on the surface of an NK cell.

[0254] In various embodiments, a T cell expressing a CLEC2d polypeptide of the disclosure is modified according to methods provided herein to knockout expression of beta-2-microglobulin and / or CD161.

[0255] EDITING OF TARGET GENES

[0256] To produce the gene edits of the present disclosure, immune cells, such as immune effector cells (e.g., T cells), are contacted with one or more guide RNAs and a nucleobase editor polypeptide comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a cytidine deaminase or adenosine deaminase or comprising one or more deaminases with cytidine deaminase and / or adenosine deaminase activity (e.g., a “dual deaminase” which has cytidine and adenosine deaminase activity). In some embodiments, the immune effector cells are collected from a subject and the contacting is in vitro. In some embodiments, cells to be edited are contacted with at least one nucleic acid, wherein the at least one nucleic acid encodes one or more guide RNAs and a nucleobase editor polypeptide comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a cytidine deaminase. In some embodiments, the gRNA comprises nucleotide analogs. In some instances, the gRNA is added directly to a cell. These nucleotide analogs can inhibit degradation of the gRNA from cellular processes.

[0257] In various instances, it is advantageous for a spacer sequence to include a 5' and / or a 3' “G” nucleotide. In some cases, for example, any spacer sequence or guide polynucleotide provided herein comprises or further comprises a 5' “G”, where, in some embodiments, the 5' “G” is or is not complementary to a target sequence. In some embodiments, the 5' “G” is added to a spacer sequence that does not already contain a 5' “G ” For example, it can be advantageous for a guide RNA to include a 5' terminal “G” when the guide RNA is expressed under the control of a U6 promoter or the like because the U6 promoter prefers a “G” at the transcription start site (see Cong, L. el al. “Multiplex genome engineering using CRISPR / Cas systems. Science 339:819-823 (2013) doi: 10.1126 / science.l231143). In some cases, a 5' terminal “G” is added to a guide polynucleotide that is to be expressed under the control of a promoter but is optionally not added to the guide polynucleotide if or when the guide polynucleotide is not expressed under the control of a promoter.

[0258] Variants of the spacer sequences provided in the present disclosure comprising 1, 2, 3, 4, or 5 nucleobase alterations are contemplated. For example, variation of a target polynucleotide sequence within a population (e.g., single nucleotide polymorphisms) may require said alterations to a spacer sequence to allow the spacer to better bind a variant of a target sequence in a subject.

[0259] Variants of the spacer sequences listed in the following tables comprising 1, 2, 3, 4, or 5 nucleobase alterations are contemplated. For example, variation of a target polynucleotide sequence within a population (e.g., single nucleotide polymorphisms) may require said alterations to a spacer sequence to allow the spacer to better bind a variant of a target sequence in a subject.

[0260] Exemplary guide RNA sequences are provided in the following Tables 1A, IB, 2A, and

[0261] 2B

[0262] Table 1A. Guide RNA sequences

[0263] Table IB. Guide RNA sequences Table 2A. Exemplary guide RNAs suitable for use with one or more of the following editors: CBE, ABE, Cas9, and Casllb

[0264] Table 2B. Exemplary Spacer Sequences. In the following table, the right portion of the table is a continuation of the bottom of the left portion. NUCLEOBASE EDITORS

[0265] Useful in the methods and compositions described herein are nucleobase editors that edit, modify or alter a target nucleotide sequence of a polynucleotide. Nucleobase editors described herein typically include a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain (e.g, adenosine deaminase, cytidine deaminase, or a dual deaminase). A polynucleotide programmable nucleotide binding domain, when in conjunction with a bound guide polynucleotide (e.g., gRNA), can specifically bind to a target polynucleotide sequence and thereby localize the base editor to the target nucleic acid sequence desired to be edited.

[0266] Polynucleotide Programmable Nucleotide Binding Domain

[0267] Polynucleotide programmable nucleotide binding domains bind polynucleotides (e.g., RNA, DNA). A polynucleotide programmable nucleotide binding domain of a base editor can itself comprise one or more domains (e.g., one or more nuclease domains). In some embodiments, the nuclease domain of a polynucleotide programmable nucleotide binding domain comprises an endonuclease or an exonuclease.

[0268] Disclosed herein are base editors comprising a polynucleotide programmable nucleotide binding domain comprising all or a portion (e.g., a functional portion) of a CRISPR protein (i.e., a base editor comprising as a domain all or a portion (e.g., a functional portion) of a CRISPR protein (e.g., a Cas protein), also referred to as a “CRISPR protein-derived domain” of the base editor). A CRISPR protein-derived domain incorporated into a base editor can be modified compared to a wild-type or natural version of the CRISPR protein. A CRISPR protein-derived domain can comprise one or more mutations, insertions, deletions, rearrangements and / or recombinations relative to a wild-type or natural version of the CRISPR protein.

[0269] Cas proteins that can be used herein include class 1 and class 2. Non-limiting examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csnl or Csxl2), CaslO, Csyl , Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, Cse5e, Cscl, Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, CsO, Csf4, Csdl, Csd2, Cstl, Cst2, Cshl, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, Casl2a / Cpfl, Casl2b / C2cl (e.g, SEQ ID NO: 232), Casl2c / C2c3, Casl2d / CasY, Casl2e / CasX, Casl2g, Casl2h, Casl2i, and Casl2j / Cas, CARF, DinG, Turbo Cas9 (i.e., an SpCas9 with the amino acid alterations Q844R, V842L, F846Y, L847M, and I852F), homologues thereof, or modified versions thereof. A CRISPR enzyme can direct cleavage of one or both strands at a target sequence, such as within a target sequence and / or within a complement of a target sequence. For example, a CRISPR enzyme can direct cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.

[0270] A vector that encodes a CRISPR enzyme that is mutated to with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence can be used. A Cas protein (e.g., Cas9, Cast 2) or a Cas domain (e.g., Cas9, Cast 2) can refer to a polypeptide or domain with at least or at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology to a wild-type exemplary Cas polypeptide or Cas domain. Cas (e.g., Cas9, Cas 12) can refer to the wild-type or a modified form of the Cas protein that can comprise an amino acid change such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof. In some embodiments, a CRISPR protein-derived domain of a base editor can include all or a portion (e.g., a functional portion) of Cas9 from Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquis (NCBI Ref: NC 018721.1); Streptococcus thermophilus (NCBI Ref: YP 820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); Neisseria meningitidis (NCBI Ref: YP_002342100.1), Streptococcus pyogenes, or Staphylococcus aureus.

[0271] Some aspects of the disclosure provide high fidelity Cas9 domains. High fidelity Cas9 domains are known in the art and described, for example, in KI einstiver, B.P., et al. “High- fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.” Nature 529, 490-495 (2016); and Slaymaker, I.M., et al “Rationally engineered Cas9 nucleases with improved specificity.” Science 351, 84-88 (2015); the entire contents of each of which are incorporated herein by reference. An Exemplary high fidelity Cas9 domain is provided in the Sequence Listing as SEQ ID NO: 233.

[0272] In some embodiments, any of the Cas9 fusion proteins or complexes provided herein comprise one or more of a D10A, N497X, a R661X, a Q695X, and / or a Q926X mutation, or a corresponding mutation in any of the amino acid sequences provided herein, wherein X is any amino acid. . Typically, Cas9 proteins, such as Cas9 from S. pyogenes (spCas9), require a “protospacer adjacent motif (PAM)” or PAM-like motif, which is a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. The presence of an NGG PAM sequence is required to bind a particular nucleic acid region, where the “N” in “NGG” is adenosine (A), thymidine (T), or cytosine (C), and the G is guanosine. In some embodiments, any of the fusion proteins or complexes provided herein may contain a Cas9 domain that is capable of binding a nucleotide sequence that does not contain a canonical (e.g., NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequences have been described in the art and would be apparent to the skilled artisan. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., et cd.. “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, B. P., et al., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); the entire contents of each are hereby incorporated by reference.

[0273] In some embodiments, the napDNAbp is a circular permutant (e.g., SEQ ID NO: 238).

[0274] In some embodiments, the polynucleotide programmable nucleotide binding domain comprises a nickase domain. Herein the term “nickase” refers to a polynucleotide programmable nucleotide binding domain comprising a nuclease domain that is capable of cleaving only one strand of the two strands in a duplexed nucleic acid molecule (e.g., DNA). For example, where a polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9, the Cas9-derived nickase domain can include a D10A mutation and a histidine at position 840. In another example, a Cas9-derived nickase domain comprises an H840A mutation, while the amino acid residue at position 10 remains a D.

[0275] In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase, referred to as an “nCas9” protein (for “nickase” Cas9; SEQ ID NO: 201). The Cas9 nickase may be a Cas9 protein that is capable of cleaving only one strand of a duplexed nucleic acid molecule (e.g., a duplexed DNA molecule). In some embodiments the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the Cas9 nickases provided herein. Additional suitable Cas9 nickases will be apparent to those of skill in the art based on this disclosure and knowledge in the field and are within the scope of this disclosure. Also provided herein are base editors comprising a polynucleotide programmable nucleotide binding domain which is catalytically dead (z.e., incapable of cleaving a target polynucleotide sequence). For example, in the case of a base editor comprising a Cas9 domain, the Cas9 can comprise both a D10A mutation and an H840A mutation. In further embodiments, a catalytically dead polynucleotide programmable nucleotide binding domain comprises a point mutation (e.g., D10A or H840A) as well as a deletion of all or a portion (e.g., a functional portion) of a nuclease domain. dCas9 domains are known in the art and described, for example, in Qi etal., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.” Cell. 2013; 152(5): 1173-83, the entire contents of which are incorporated herein by reference.

[0276] The term “protospacer adjacent motif (PAM)” or PAM-like motif refers to a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by a nucleic acid programmable DNA binding protein. In some embodiments, the PAM can be a 5' PAM (z.e., located upstream of the 5' end of the protospacer). In other embodiments, the PAM can be a 3' PAM (z.e., located downstream of the 5' end of the protospacer). The PAM sequence can be any PAM sequence known in the art. Suitable PAM sequences include, but are not limited to, NGG, NGA, NGC, NGN, NGT, NGTT, NGCG, NGAG, NGAN, NGNG, NGCN, NGCG, NGTN, NNGRRT, NNNRRT, NNGRR(N), TTTV, TYCV, TYCV, TATV, NNNNGATT, NNAGAAW, or NAAAAC. Y is a pyrimidine; N is any nucleotide base; W is A or T.

[0277] By “protein tag,” is meant a peptide that is fused to a protein to facilitate purification or detection of that protein and / or selection or enrichment of a cell expressing that protein. In embodiments, the peptide is fused to a recombinant protein, a binding polypeptide or an antibody. Such tags may be fused to either or both the N (amino)-terminus, the C (carboxy)- terminus, or within a polypeptide.

[0278] A base editor provided herein can comprise a CRISPR protein-derived domain that is capable of binding a nucleotide sequence that contains a canonical or non-canonical protospacer adjacent motif (PAM) sequence.

[0279] In some embodiments, the PAM is an “NRN” PAM where the “N” in “NRN” is adenine (A), thymine (T), guanine (G), or cytosine (C), and the R is adenine (A) or guanine (G); or the PAM is an “NYN” PAM, wherein the “N” in NYN is adenine (A), thymine (T), guanine (G), or cytosine (C), and the Y is cytidine (C) or thymine (T), for example, as described in R.T. Walton et al., 2020, Science, 10.1126 / science.aba8853 (2020), the entire contents of which are incorporated herein by reference.

[0280] Several PAM variants are described in Table 3 below. Table 3. Cas9 proteins and corresponding PAM sequences. N is A, C, T, or G; and V is A, C, or G.

[0281] In some embodiments, the PAM is NGC. In some embodiments, the NGC PAM is recognized by a Cas9 variant. In some embodiments, the Cas9 variant contains one or more amino acid substitutions selected from DI 135V, G1218R, R1335Q, and T1337R (collectively termed VRQR) of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9. In some embodiments, the Cas9 variant contains one or more amino acid substitutions selected from DI 135V, G1218R, R1335E, and T1337R (collectively termed VRER) of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9. In some embodiments, the Cas9 variant contains one or more amino acid substitutions selected from E782K, N968K, and R1015H (collectively termed KHH) of saCas9 (SEQ ID NO: 218).

[0282] In some cases, a Cas9 variant has specificity for the PAM 5 " -NGC-3 " . In some embodiments, a Cas9 variant includes one or more amino acid substitutions selected from D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337K of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9. In some embodiments, a Cas9 variant includes one or more amino acid substitutions selected from DI 135M, SI 136Y, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337K of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9. In some embodiments, the a Cas9 variant includes one or more amino acid substitutions selected from DI 135L, SI 136Y, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9. In some embodiments, a Cas9 variant includes one or more amino acid substitutions selected from DI 135M, S1136Y, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337K of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9. In some embodiments, a Cas9 variant includes one or more amino acid substitutions selected from D1135L, S1136Y, G1218K, E1219F, A1283D, A1322R, D1332A, R1335E, and T1337K of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9. In some embodiments, a Cas9 variant includes one or more amino acid substitutions selected from DI 135L, SI 136Q, G1218K, E1219F, E1250K, A1283D, A1322R, D1332A, R1335E, and T1337K of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9. In some embodiments, a Cas9 variant includes one or more amino acid substitutions selected from DI 135M, SI 136Y, G1218K, E1219F, E1250K, A1283D, A1322R, D1332A, R1335E, and T1337R of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9. In some embodiments, a Cas9 variant includes one or more amino acid substitutions selected from R765A, Q768A, DI 135L, SI 136Y, G1218K, A1283D, E1219F, A1322R, D1332A, R1335E, and T1337K of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9. In some embodiments, any of the Cas9 proteins provided herein, including an SpCas9 comprises any one, two, three, four, five, six, seven, eight, nine, or ten of the following amino acid substitutions in a corresponding residue: R765A, Q768A, W1126R, R1359W, E1250K, A1239T, A1239V, A1283D, R1335D, D1135L, D1135M, D1135R, D1135W, S1136H, S1136Q, S1136Y, G1218D, G1218K, G1218R, G1218E, G1218L, E1219F, E1219K, E1219N, A1322A, A1322R, A1322K, D1332A, R1335V, T1337K, T1337T, D1332A, DI 135V and T1337R.

[0283] In some embodiments, a CRISPR protein-derived domain of a base editor comprises all or a portion (e.g., a functional portion) of a Cas9 protein with a canonical PAM sequence (NGG). In other embodiments, a Cas9-derived domain of a base editor can employ a non- canonical PAM sequence. Such sequences have been described in the art and would be apparent to the skilled artisan. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., etal., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, B. P., et cd., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); R.T. Walton et al. “Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants” Science 10.1126 / science.aba8853 (2020); Hu et al. “Evolved Cas9 variants with broad PAM compatibility and high DNA specificity,” Nature, 2018 Apr. 5, 556(7699), 57-63; Miller et al., “Continuous evolution of SpCas9 variants compatible with non-G PAMs” Nat. Biotechnol., 2020 Apr;38(4):471-481; the entire contents of each are hereby incorporated by reference.

[0284] Fusion Proteins or Complexes Comprising a NapDNAbp and a Cytidine Deaminase and / or

[0285] Adenosine Deaminase

[0286] Some aspects of the disclosure provide fusion proteins or complexes comprising a Cas9 domain or other nucleic acid programmable DNA binding protein (e.g., Cast 2) and one or more cytidine deaminase, adenosine deaminase, or cytidine adenosine deaminase domains. It should be appreciated that the Cas9 domain may be any of the Cas9 domains or Cas9 proteins (e.g., dCas9 or nCas9) provided herein. In some embodiments, any of the Cas9 domains or Cas9 proteins (e.g., dCas9 or nCas9) provided herein may be fused with any of the cytidine deaminases and / or adenosine deaminases provided herein. The domains of the base editors disclosed herein can be arranged in any order.

[0287] In some embodiments, the fusion proteins or complexes comprising a cytidine deaminase or adenosine deaminase and a napDNAbp (e.g., Cas9 or Casl2 domain) do not include a linker sequence. In some embodiments, a linker is present between the cytidine or adenosine deaminase and the napDNAbp. In some embodiments, cytidine or adenosine deaminase and the napDNAbp are fused via any of the linkers provided herein. For example, in some embodiments the cytidine or adenosine deaminase and the napDNAbp are fused via any of the linkers provided herein.

[0288] It should be appreciated that the fusion proteins or complexes of the present disclosure may comprise one or more additional features. For example, in some embodiments, the fusion protein or complex may comprise inhibitors, cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the fusion proteins or complexes. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags, also referred to as histidine tags or His-tags, maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1, Softag 3), strep-tags , biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable sequences will be apparent to those of skill in the art. In some embodiments, the fusion protein or complex comprises one or more His tags. Exemplary, yet nonlimiting, fusion proteins are described in International PCT Application Nos. PCT / US2017 / 045381, PCT / US2019 / 044935, and PCT / US2020 / 016288, each of which is incorporated herein by reference for its entirety.

[0289] Fusion Proteins or Complexes with Internal Insertions

[0290] Provided herein are fusion proteins or complexes comprising a heterologous polypeptide fused to a nucleic acid programmable nucleic acid binding protein, for example, a napDNAbp. The heterologous polypeptide can be fused to the napDNAbp at a C-terminal end of the napDNAbp, an N-terminal end of the napDNAbp, or inserted at an internal location of the napDNAbp. In some embodiments, the heterologous polypeptide is a deaminase (e.g., cytidine or adenosine deaminase) or a functional fragment thereof. For example, a fusion protein can comprise a deaminase flanked by an N- terminal fragment and a C-terminal fragment of a Cas9 or Casl2 (e.g., Casl2b / C2cl), polypeptide.

[0291] The deaminase can be a circular permutant deaminase. In some embodiments, the deaminase is a circular permutant TadA, circularly permutated at amino acid residue 116, 136, or 65 as numbered in a TadA reference sequence.

[0292] The fusion protein or complexes can comprise more than one deaminase. The fusion protein or complex can comprise, for example, 1, 2, 3, 4, 5 or more deaminases. The deaminases in a fusion protein or complex can be adenosine deaminases, cytidine deaminases, or a combination thereof.

[0293] In some embodiments, the napDNAbp in the fusion protein or complex contains a Cas9 polypeptide or a fragment thereof. The Cas9 polypeptide can be a variant Cas9 polypeptide. The Cas9 polypeptide can be a circularly permuted Cas9 protein.

[0294] The heterologous polypeptide (e.g., deaminase) can be inserted in the napDNAbp (e.g., Cas9 or Casl2 (e.g., Casl2b / C2cl)) at a suitable location, for example, such that the napDNAbp retains its ability to bind the target polynucleotide and a guide nucleic acid. A deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase (dual deaminase)) can be inserted into a napDNAbp without compromising function of the deaminase (e.g., base editing activity) or the napDNAbp (e.g., ability to bind to target nucleic acid and guide nucleic acid).

[0295] In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted in regions of the Cas9 polypeptide comprising higher than average B-factors (e.g., higher B factors compared to the total protein or the protein domain comprising the disordered region). Cas9 polypeptide positions comprising a higher than average B-factor can include, for example, residues 768, 792, 1052, 1015, 1022, 1026, 1029, 1067, 1040, 1054, 1068, 1246, 1247, and 1248 as numbered in SEQ ID NO: 197. Cas9 polypeptide regions comprising a higher than average B-factor can include, for example, residues 792-872, 792-906, and 2-791 as numbered in SEQ ID NO: 197.

[0296] In some embodiments, a heterologous polypeptide (e.g., deaminase) is inserted in a flexible loop of a Cas9 polypeptide. The flexible loop portions can be selected from the group consisting of 530-537, 569-570, 686-691, 943-947, 1002-1025, 1052-1077, 1232-1247, or 1298- 1300 as numbered in SEQ ID NO: 197, or a corresponding amino acid residue in another Cas9 polypeptide. The flexible loop portions can be selected from the group consisting of: 1-529, 538- 568, 580-685, 692-942, 948-1001, 1026-1051, 1078-1231, or 1248-1297 as numbered in SEQ ID NO: 197, or a corresponding amino acid residue in another Cas9 polypeptide.

[0297] A heterologous polypeptide (e.g., adenine deaminase) can be inserted into a Cas9 polypeptide region corresponding to amino acid residues: 1017-1069, 1242-1247, 1052-1056, 1060-1077, 1002 - 1003, 943-947, 530-537, 568-579, 686-691, 1242-1247, 1298 - 1300, 1066- 1077, 1052-1056, or 1060-1077 as numbered in SEQ ID NO: 197, or a corresponding amino acid residue in another Cas9 polypeptide.

[0298] A heterologous polypeptide (e.g., adenine deaminase) can be inserted in place of a deleted region of a Cas9 polypeptide. The deleted region can correspond to an N-terminal or C- terminal portion of the Cas9 polypeptide. Exemplary internal fusions base editors are provided in Table 4A below:

[0299] Table 4A: Insertion loci in Cas9 proteins

[0300] A heterologous polypeptide (e.g., deaminase) can be inserted within a structural or functional domain of a Cas9 polypeptide. A heterologous polypeptide (e.g., deaminase) can be inserted between two structural or functional domains of a Cas9 polypeptide. A heterologous polypeptide (e.g., deaminase) can be inserted in place of a structural or functional domain of a Cas9 polypeptide, for example, after deleting the domain from the Cas9 polypeptide. The structural or functional domains of a Cas9 polypeptide can include, for example, RuvC I, RuvC II, RuvC III, Reel, Rec2, PI, or HNH.

[0301] A fusion protein can comprise a linker between the deaminase and the napDNAbp polypeptide. The linker can be a peptide or a non-peptide linker. For example, the linker can be an XTEN, (GGGS)n (SEQ ID NO: 246), SGGSSGGS (SEQ ID NO: 330), (GGGGS)n (SEQ ID NO: 247), (G)n, (EAAAK)n (SEQ ID NO: 248), (GGS)n, SGSETPGTSESATPES (SEQ ID NO: 249). In some embodiments, the fusion protein comprises a linker between the N-terminal Cas9 fragment and the deaminase. In some embodiments, the fusion protein comprises a linker between the C-terminal Cas9 fragment and the deaminase. In some embodiments, the N-terminal and C-terminal fragments of napDNAbp are connected to the deaminase with a linker. In some embodiments, the N-terminal and C-terminal fragments are joined to the deaminase domain without a linker. In some embodiments, the fusion protein comprises a linker between the N- terminal Cas9 fragment and the deaminase but does not comprise a linker between the C- terminal Cas9 fragment and the deaminase. In some embodiments, the fusion protein comprises a linker between the C-terminal Cas9 fragment and the deaminase but does not comprise a linker between the N-terminal Cas9 fragment and the deaminase.

[0302] In some embodiments, the napDNAbp in the fusion protein or complex is a Casl2 polypeptide, e.g., Casl2b / C2cl, or a functional fragment thereof capable of associating with a nucleic acid (e.g., a gRNA) that guides the Casl2 to a specific nucleic acid sequence. The Casl2 polypeptide can be a variant Cast 2 polypeptide. In other embodiments, the N- or C-terminal fragments of the Casl2 polypeptide comprise a nucleic acid programmable DNA binding domain or a RuvC domain. In other embodiments, the fusion protein contains a linker between the Cast 2 polypeptide and the catalytic domain. In other embodiments, the amino acid sequence of the linker is GGSGGS (SEQ ID NO: 250) or GSSGSETPGTSESATPESSG (SEQ ID NO: 251). In other embodiments, the linker is a rigid linker. In other embodiments of the above aspects, the linker is encoded by GGAGGCTCTGGAGGAAGC (SEQ ID NO: 252) or GGCTCTTCTGGATCTGAAACACCTGGCACAAGCGAGAGCGCCACCCCTGAGAGCTCTGGC (SEQ ID NO: 253).

[0303] In other embodiments, the fusion protein or complex contains a nuclear localization signal (e.g., a bipartite nuclear localization signal). In other embodiments, the amino acid sequence of the nuclear localization signal is MAPKKKRKVGIHGVPAA (SEQ ID NO: 261). In other embodiments of the above aspects, the nuclear localization signal is encoded by the following sequence: ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCC (SEQ ID NO: 262). In other embodiments, the Cast 2b polypeptide contains a mutation that silences the catalytic activity of a RuvC domain. In other embodiments, the Casl2b polypeptide contains D574A, D829A and / or D952A mutations.

[0304] In some embodiments, the fusion protein or complex comprises a napDNAbp domain (e.g., Casl2-derived domain) with an internally fused nucleobase editing domain e.g., all or a portion (e.g., a functional portion) of a deaminase domain, e.g., an adenosine deaminase domain). In some embodiments, the napDNAbp is a Casl2b. In some embodiments, the base editor comprises a BhCasl2b domain with an internally fused TadA*8 domain inserted at the loci provided in Table 4B below.

[0305] Table 4B: Insertion loci in Casllb proteins In some embodiments, the base editing system described herein is an ABE with TadA inserted into a Cas9. Polypeptide sequences of relevant ABEs with TadA inserted into a Cas9 are provided in the attached Sequence Listing as SEQ ID NOs: 263-308.

[0306] Exemplary, yet nonlimiting, fusion proteins are described in International PCT Application Nos. PCT / US2020 / 016285 and U.S. Provisional Application Nos. 62 / 852,228 and 62 / 852,224, the contents of which are incorporated by reference herein in their entireties.

[0307] A to G Editing

[0308] In some embodiments, a base editor described herein comprises an adenosine deaminase domain. Such an adenosine deaminase domain of a base editor can facilitate the editing of an adenine (A) nucleobase to a guanine (G) nucleobase by deaminating the A to form inosine (I), which exhibits base pairing properties of G. In some embodiments, an A-to-G base editor further comprises an inhibitor of inosine base excision repair, for example, a uracil glycosylase inhibitor (UGI) domain or a catalytically inactive inosine specific nuclease. Without wishing to be bound by any particular theory, the UGI domain or catalytically inactive inosine specific nuclease can inhibit or prevent base excision repair of a deaminated adenosine residue (e.g., inosine), which can improve the activity or efficiency of the base editor.

[0309] A base editor comprising an adenosine deaminase can act on any polynucleotide, including DNA, RNA and DNA-RNA hybrids. In an embodiment an adenosine deaminase domain of a base editor comprises all or a portion (e.g., a functional portion) of an AD AT comprising one or more mutations which permit the AD AT to deaminate a target A in DNA. For example, the base editor can comprise all or a portion (e.g., a functional portion) of an ADAT from Escherichia coli (EcTadA) comprising one or more of the following mutations: D108N, A106V, D147Y, E155V, L84F, H123Y, I156F, or a corresponding mutation in another adenosine deaminase. Exemplary ADAT homolog polypeptide sequences are provided in the Sequence Listing as SEQ ID NOs: 1 and 309-315.

[0310] The adenosine deaminase can be derived from any suitable organism (e.g., E. coif). In some embodiments, the adenosine deaminase is from Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus, or Bacillus subtilis. In some embodiments, the adenine deaminase is a naturally-occurring adenosine deaminase that includes one or more mutations corresponding to any of the mutations provided herein (e.g., mutations in ecTadA). The corresponding residue in any homologous protein can be identified by e.g., sequence alignment and determination of homologous residues. The mutations in any naturally-occurring adenosine deaminase (e.g., having homology to ecTadA) that correspond to any of the mutations described herein (e.g., any of the mutations identified in ecTadA) can be generated accordingly.

[0311] In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences set forth in any of the adenosine deaminases provided herein. It should be appreciated that adenosine deaminases provided herein may include one or more mutations (e.g., any of the mutations provided herein). The disclosure provides any deaminase domains with a certain percent identify plus any of the mutations or combinations thereof described herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to a reference sequence, or any of the adenosine deaminases provided herein.

[0312] It should be appreciated that any of the mutations provided herein (e.g., based on a TadA reference sequence, such as TadA*7.10 (SEQ ID NO: 1)) can be introduced into other adenosine deaminases, such as E. coli TadA (ecTadA), S. aureus TadA (saTadA), or other adenosine deaminases (e.g., bacterial adenosine deaminases). In some embodiments, the TadA reference sequence is TadA*7.10 (SEQ ID NO: 1). It would be apparent to the skilled artisan that additional deaminases may similarly be aligned to identify homologous amino acid residues that can be mutated as provided herein. Thus, any of the mutations identified in a TadA reference sequence can be made in other adenosine deaminases (e.g., ecTada) that have homologous amino acid residues. It should also be appreciated that any of the mutations provided herein can be made individually or in any combination in a TadA reference sequence or another adenosine deaminase.

[0313] In some embodiments, the adenosine deaminase comprises an alteration or set of alterations selected from those listed in Tables 5A-5E below:

[0314] Table 5A. Adenosine Deaminase Variants. Residue positions in the E. coli TadA variant (TadA*) are indicated.

[0315] Table 5B. TadA*8 Adenosine Deaminase Variants. Residue positions in the E. coli TadA variant (TadA*) are indicated. Alterations are referenced to TadA*7.10 (first row). Table 5C. TadA*9 Adenosine Deaminase Variants. Alterations are referenced to TadA*7.10. Additional details of TadA*9 adenosine deaminases are described in International PCT Application No. PCT / US2020 / 049975, which is incorporated herein by reference in its entirety for all purposes.

[0316] In some embodiments, the adenosine deaminase comprises a TadA*8.20 adenosine deaminase variant further comprising an F149Y amino acid alteration. In some embodiments, the adenosine deaminase comprises a TadA*8.20 adenosine deaminase variant further comprising the amino acid alterations R147D, F149Y, T166I, and D167N (TadA*8.10+). In some embodiments, the adenosine deaminase comprises a TadA*8.20 adenosine deaminase variant further comprising the amino acid alterations S82T and F149Y (TadA*9vl). In some embodiments, the adenosine deaminase comprises a TadA*8.20 adenosine deaminase variant further comprising the amino acid alterations Y147D, F149Y, T166I, D167N and S82T (TadA*9v2).

[0317] In some embodiments, the adenosine deaminase comprises one or more of Mil, MIS, S2A, S2E, S2H, S2R, S2L, E3L, V4D, V4E, V4M, V4K, V4S, V4T, V4A, E5K, F6S, F6G, F6H, F6Y, F6I, F6E, S7K, H8E, H8Y, H8H, H8Q, H8E, H8G, H8S, E9Y, E9K, E9V, E9E, Y10F, Y10W, Y10Y, M12S, M12L, M12R, M12W, R13H, R13I, R13Y, R13R, R13G, R13S, H14N, A15D, A15V, A15L, A15H, T17T, T17A, T17W, T17L, T17F, T17R, T17S, L18A, L18E, L18N, L18L, L18S, A19N, A19H, A19K, A19A, A19D, A19G, A19M, R21N, K20K, K20A, K20R, K20E, K20G, K20C, K20Q R21A, R21R, R21N, R21Y, R21C G22P, A22W, A22R, W23D, R23H, W23G, W23Q, W23L, W23R, W23H W23D W23M, W23W, W23I, D24E, D24G, D24W, D24D, D24R, E25F, E25M, E25D, E25A, E25G, E25R, E25E, E25H E25V, E25S, E25Y, R26D, R26E, R26G, R26N, R26Q, R26C, R26L, R26K, R26W, R26C, R26P, R26R, R26A, R26H, E27E, E27Q, E27H, E27C, E27G, E27K, E27S, E27P, E27R, E27L, E27V, E27D, V28V, V28A, V28C, V28G, V28P, V28S, V28T, P29V, P29P, P29A, P29G, P29K, P29L, V30V, V30I, V30L, V30F, V30G, V30A, V30M, L34S, L34V, L34L, L34M, L34W, L34G, H36E, H36V, L36H, H36L, H36N, N37N, N37H, N37R, N37T, N37S, N38G, N38R, N38N, N38E, V40I, W45A, W45W, W45R, W45L, W45N, N46N, N46M, N46P, N46G, N46L, N46R, N46V, R46W, R46F, R46Q, R46M, R47A, R47Q, R47F, R47K, R47P, R47W, R47M, R47R, R47G, R47S, R47V, R47H, P48T, P48L, P48A, P48I, P48S, P48R, P48K, P48D, P48E, P48H, P48G, P48P, P48N, I49G, I49H, I49V, I49F, I49H, 1491, 149M, I49N, I49K, I49Q, I49T, G50L, G50S, G50R, G50G, R51H, R51L, R51N, L51W, R51Y, R51G, R51V, R51R, H52D, H52Y, H52I, H52H, D53D, D53E, D53G, D53P, P54C, P54T, P54P, P54E, A55H, T55A, T55I, T55V, T55G, T55T, A56A, A56H, A56W, A56E, A56S, H57P, H57A, H57H, H57N, A58G, A58E, A58A, A58R, E59A, E59G, E59I, E59Q, E59W, E59E, E59T, E59H, E59P, M61A, M61I, M61L, M61V, M61P, M61G, M61I, L63S, L63V, L63T, L63R, L63H, L63A, R64A, R64Q, R64R, R64D, Q65V, Q65H, Q65G, Q65P, Q65F, Q65Q, Q65R, G66V, G66E, G66T, G66G, G66C, G67G, G67W, G67I, G67A, G67D, G67L, G67V, L68Q, L68M, L68V, L68H, L68L, L68G,V69A, V69M, V69V, M70V, M70L, E70A, M70A, M70M, M70E, M70T, M70v, Q71M, Q71N, Q71L, Q71R, Q71Q, Q71I, N72A, N72K, N72S, N72D, N72Y, N72N, N72H, N72G, N72M, Y73G, Y73I, Y73K, Y73R, Y73S, Y73Y, Y73H, Y73A, R74A, R74Q, R74G, R74K, R74L, R74N, R74G, R74K, R74R, I76H, I76R, I76W, I76Y, I76V, I76Q, I76L, I76D, I76F, 1761, 176N, I76T, I76Y, D77G, D77D, D77A, D77Q, A78Y, A78T, A78G, A78A, A78I, T79M, T79R, T79L, T79T, L80M, L80Y, L80I, L80V, L80L, Y81D, Y81V, Y81Y, Y81M, V82A, V82S, V82G, V82T, V82V, V82Q, V82Y, T83L, T83F, T83T, T83N, L84E, L84F, L84Y, L84I, L84L, L84M, L84A, L84T, L84S, E85K, E85G, E85P, E85S, E85E, E85F, E85V, E85R, P86T, P86C, P86P, P86L, P86N, P86K, P86H, C87M, C87I, C87S, C87N, C87P, S87C, S87L, S87V, V88A, V88M, V88V, V88T, V88E, V88D, V88S, C90S, C90P, C90A, C90T, C90M, A91A, A91G, A91S, A91V, A91T, A91C, A91L, G92T, G92M, G92A, G92Y, G92G, A93I, A93C, A93M, A93V, A93A, M94M, M94T, M94A, M94V, M94L, M94I, M94H, 195 S, I95G, I95L, I95H, 195 V, H96A, H96L, H96R, H96S, H96H, H96N, H96E, S97C, S97G, S97I, S97M, S97R, S97S, S97P, R98K, R98I, R98N, R98Q, R98G, R98H, R98C, R98L, R98R, G100R, G100V, G100K, G100A, G1OOS, G100M, G1OOI, R101V, R101R, R1O1S, R101C, V102A, V102F, V102I, V102V, D103A, V103A, V103G, V1O3F, V103V, F104G, D104N, Fl 04V, Fl 041, F104L, F104A, F104F, F104R, G105V, G105W, G105G, G105M, G105A, A106T, V106Q, V106F, V106W, V106M, A106A, A106Q, A106F, A106G, A106W, A106M, Al 06V, A106R, A106L, A106S, A106B, Al 061, R107C, R107G, R107P, R107K, R107A, R107N, R107W, R107H, R107S, R107R, R107F, D108N, D108F, D108G, DI 08V, D108A, D108Y, D108H, D108I, D108K, D108L, D108M, D108Q, N108Q, N108F, N108W, N108M, N108K, D108K, D108F, D108M, D108Q, D108R, D108W, D108S, D108E, D108T, D108R, D108D, A109H, A109K, A109R, A109S, A109T, A109V, A109A, A109D, K110G, KI 10H, KI 101, KI 10R, KI 10T, KI 10K, KI 10A, KI 101, T111 A, T111G, Ti l 1H, T111R, T11 IT, T11 IK, G112A, G112G, G112H, G112T, G112R, Al 13N, Al 14G, Al 14H, Al 14V, A114C, A114S, A114A, G115S, G115G, G115M, G115L, G115A, G115F, L117M, L117L, L117W, L117A, L117S, L117N, LI 17V, M118D, M118G, M118K, M118N, Ml 18V, M118M, Ml 18L, Ml 18R, DI 19L, DI 19N, DI 19S, DI 19V, DI 19D, V120H, V120L, V120V, V120T, V120A, V120E, V120G, V120D, L121D, L121M, L121N, L121K, L121L, H122H, H122N, H122P, H122R, H122S, H122Y, H122G, H122T, H122L, H123C, H123G, H123P, H123V, H123Y, Y123H, H123Y, H123H, P124P, P124H, P124A, P124Y, P124D, P124G, P124I, P124L, P124W, G125H, G125I, G125A, G125M, G125K, G125G, G125P, M126D, M126H, M126K, M126I, M126N, M126O, M126S, M126Y, M126M, M126G, N127H, N127S, N127D, N127K, N127R, N127N, N127I, N127P, N127M, H128R, H128N, H128L, H128H, R129H, R129Q, R129V, R129I, R129E, R129V, R129R, R129M, R129P, V130R, V130V, V130E, V130D, E131E, E131I, E131V, E131K, I132I, I132F, I132T, I132L, I132V, I132E, T133V, T133E, T133G, T133K, T133T, T133A, T133H, T133F, T133I, E134A, E134E, E134G, E134I, E134H, E134K, E134T, G135G, G135V, G135I, G135P, G135E, I136G, I136L, I136T, I136I , 1137A, 1137D, 1137E, L137M, 1137S, L137L, L137I , A138D, A138E, A138G, S138A, A138N, A138S, A138T, A138V, A138Y, A138A, A138M, A138L, D139E, D139I, D139C, D139L, D139M, D139D, D139G, D139H, D139A, E140A, E140C, E140L, E140R, E140K, E140E, E140D, C141S, C141A, C141C, C141V, C141E, A142N, A142D, A142G, A142A, A142L, A142S, A142T, A142N, A142S, A142V, A142E, A142C, A143D, A143E, A143G, , A143D, A143G, A143E, A143L, A143W, A143M, A143S, A143Q, A143R, A143A, A143I, L144S, L144L, L144T, L144A, L145A, L145F, L145G, L145D, L145L, L145C, L145E, L145s, C146R, S146A, S146C, S146D, S146F, S146R, S146T, S146D, S146G, S146S, S146L, D147D, D147L, D147F, D147G, D147Y, Y147T, Y147R, Y147D, D147R, D147Y, D147A, D147T, D147H, D147F, D147U, DI 47V, DI 471, D147C, F148L, F148F, F148R, F148Y, Fl 48 A, F148T, F149C, F149M, F149R, F149Y, F149N, F149F, F149A, F149T, F149V R150R, R150M, R150D, R150F, M151F, M151P, M151R, M151V, M151M, M151E, R152C, R152F, R152H, R152P, R152R, R152P, R152Q, R152M, R152O, R153C, R153Q, R153R, R153V, R153E, R153A, R153P, Q154E, Q154H, Q154M, Q154R, Q154L, Q154S, QI 54V, Q154Q, Q154F, Q154I, Q154A, Q154K, E155F, E155G, E155I, E155K, E155P, E155V, E155D, E155E, E155L, E155Q, I156V, I156A, I156I, I156L, I156F, I156D, I156K, I156N, I156R, I156Y, E157A, E157F, El 571, E157P, E157T, El 57V, N157K, K157N, KI 57V, K157P, K157I, K157F, K157F, K157T, K157A, K157S, K157R, A158Q, A158K, A158V, A158A, A158D, A158S, A158T, A158N, Q159S, Q159Q, Q159A, Q159F, Q159K, Q159L, Q159N, K160A, K160S, K160E, K160K, K160N, K160F, K160Q, K161T, K161K, K161R, K161I, K161A, K161N, K161Q, K161S, K161T, A162D, A162Q, R162H, R162P, A162S, A162A, A162N, A162M, A162K, Q163G, Q163S, Q163Q, Q163A, Q163H, Q163N, Q163R, S164F, S164S, S164Q, S164I, S164R, S164Y, S165S, S165P, S165Q, S165A, S165D, S165I, S165T, S165Y, T166T, T166Q, T166E, T166S, T166D, T166K, T166I, T166N, T166P, T166R, D167S D167D, D167I, D167G, D167T, D167A and / or D167N mutation in a TadA reference sequence (e.g., TadA*7.10,ecTadA, or TadA8e), and any alternative mutation at the corresponding positioner one or more corresponding mutations in another adenosine deaminase. Additional mutations are described in U.S. Patent Application Publication No. 2022 / 0307003 Al U.S. Patent No. 11,155,803, and International Patent Application Publications No. WO 2023 / 288304 A2, PCT / CN2022 / 143408, WO 2018 / 027078 Al, WO 2021 / 158921 Al and WO 2023 / 034959 A2, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0318] In various embodiments, an adenosine deaminase of the disclosure lacks an N-terminal methionine.

[0319] In some embodiments, the disclosure provides TadA variants comprising an alteration at an amino acid selected from one or more of L36, 176, V82, Y147, Q154, and N157 comapred to TadA*7.10. In some embodiments, the disclosure provides TadA variants comprising one or more of the following alterations relative to TadA*7.10: L36H, I76Y, V82T, Y147T, Q154S, and N157K. In some embodiments, the disclosure provides TadA variants comprising the following alterations relative to TadA*7.10: L36H, I76Y, V82T, Y147T, Q154S, and N157K. In some embodiments, the disclosure provides TadA variants comprising the following alterations relative to TadA*7.10: F84Y, A109L, A109V, A109I, A109F, A109S, A109T, A109N, V155S, V155T, V155N, F156Y, F156W, F156R, F156N, and F156Q. In some embodiments, the disclosure provides TadA variants comprising the following alterations relative to TadA*7.10: E3N, E3K, E3G, F6A, HMD, L18A, W23I, W23R, P29T, P29Y, P29Q, V35Q, L36S, N38D, G42M, N46Y, P48A, G50A, H52L, A62V, L63R, L63F, Q65R, G67N, L68V, M70I, N72Y, T79H, Y81V, V82S, M94R, G100V, V102E, V102S, R107A, A114C, G115E, M118L, D119L, H122T, P124H, P124K, P124Q, H128R, V130F, I132K, I132T, E140L, A142N, A142S, L144Q, L145R, L145N, Y147A, F149A, R152P, F156N, and K160E.

[0320] In some embodiments, the disclosure provides TadA variants comprising a V82T, Y147T, and / or a Q154S mutation. In some embodiments, the disclosure provides TadA variants comprising a V82T, Y147T, and / or a Q154S mutation. In some embodiments, the disclosure provides TadA*8.8 further comprising a V82T mutation. In some embodiments, the disclosure provides TadA*8.8 further comprising a V82T, a Y147T, and a Q154S mutation. In some embodiments, the disclosure provides TadA*8.17 further comprising a V82T mutation. In some embodiments, the disclosure provides TadA*8.17 further comprising a V82T, a Y147T, and a Q154S mutation. In some embodiments, the disclosure provides TadA*8.20 further comprising a V82T mutation. In some embodiments, the disclosure provides TadA*8.20 further comprising a V82T, a Y147T, and a Q154S mutation.

[0321] In embodiments, a variant of TadA*7.10 comprises one or more alterations selected from any of those alterations provided herein.

[0322] In particular embodiments, an adenosine deaminase heterodimer comprises a TadA*8 domain and an adenosine deaminase domain selected from Staphylococcus aureus (S. aureus) Tad A, Bacillus subtilis (B. subtilis) Tad A, Salmonella typhimurium (S. typhimurium) Tad A, Shewanella putrefaciens (S. putrefaciens) TadA, Haemophilus influenzae F3031 (H. influenzae) TadA, Caulobacter crescentus (C. crescentus) TadA, Geobacter sulfurreducens (G. sulfurreducens) TadA, or TadA*7.10.

[0323] In some embodiments, the TadA*8 is a variant as shown in Table 5D. Table 5D shows certain amino acid position numbers in the TadA amino acid sequence and the amino acids present in those positions in the TadA-7.10 adenosine deaminase. Table 5D also shows amino acid changes in TadA variants relative to TadA-7.10 following phage-assisted non-continuous evolution (PANCE) and phage-assisted continuous evolution (PACE), as described in M. Richter et al.. 2020, Nature Biotechnology, doi.org / 10.1038 / s41587-020-0453-z, the entire contents of which are incorporated by reference herein. In some embodiments, the TadA*8 is TadA*8a, TadA*8b, TadA*8c, TadA*8d, or TadA*8e. In some embodiments, the TadA*8 is TadA*8e. In one embodiment, an adenosine deaminase is a TadA*8 that comprises or consists essentially of SEQ ID NO: 316 or a fragment thereof having adenosine deaminase activity.

[0324] Table 5D. Select TadA*8 Variants

[0325] In some embodiments, the TadA variant is a variant as shown in Table 5E. Table 5E shows certain amino acid position numbers in the TadA amino acid sequence and the amino acids present in those positions in the TadA*7.10 adenosine deaminase. In some embodiments, the TadA variant is MSP605, MSP680, MSP823, MSP824, MSP825, MSP827, MSP828, or MSP829. In some embodiments, the TadA variant is MSP828. In some embodiments, the TadA variant is MSP829.

[0326] Table 5E. TadA Variants

[0327] In particular embodiments, the fusion proteins or complexes comprise a single e.g., provided as a monomer) TadA* (e.g., TadA*8 or TadA*9). Throughout the present disclosure, an adenosine deaminase base editor that comprises a single TadA* domain is indicates using the terminology ABEm or ABE#m, where “#” is an identifying number (e.g., ABE8.20m), where “m” indicates “monomer.” In some embodiments, the TadA* is linked to a Cas9 nickase. In some embodiments, the fusion proteins or complexes of the disclosure comprise as a heterodimer of a wild-type TadA (TadA(wt)) linked to a TadA*. Throughout the present disclosure, an adenosine deaminase base editor that comprises a single TadA* domain and a TadA(wt) domain is indicates using the terminology ABEd or ABE#d, where “#” is an identifying number (e.g., ABE8.20d), where “d” indicates “dimer.” In other embodiments, the fusion proteins or complexes of the disclosure comprise as a heterodimer of a TadA*7.10 linked to a TadA*. In some embodiments, the base editor is ABE8 comprising a TadA* variant monomer. In some embodiments, the base editor is ABE comprising a heterodimer of a TadA* and a TadA(wt). In some embodiments, the base editor is ABE comprising a heterodimer of a TadA* and TadA*7.10. In some embodiments, the base editor is ABE comprising a heterodimer of a TadA*. In some embodiments, the TadA* is selected from Tables 5A-5E.

[0328] In some embodiments, the adenosine deaminase is expressed as a monomer. In other embodiments, the adenosine deaminase is expressed as a heterodimer. In some embodiments, the deaminase or other polypeptide sequence lacks a methionine, for example when included as a component of a fusion protein. This can alter the numbering of positions. However, the skilled person will understand that such corresponding mutations refer to the same mutation.

[0329] Any of the mutations provided herein and any additional mutations (e.g., based on the ecTadA amino acid sequence) can be introduced into any other adenosine deaminases. Any of the mutations provided herein can be made individually or in any combination in a TadA reference sequence or another adenosine deaminase (e.g., ecTadA).

[0330] Details of A to G nucleobase editing proteins are described in International PCT Application No. PCT / US2017 / 045381 (WO2018 / 027078) and Gaudelli, N.M., el al., “Programmable base editing of A»T to G»C in genomic DNA without DNA cleavage” Nature, 551, 464-471 (2017), the entire contents of which are hereby incorporated by reference.

[0331] Guide Polynucleotides

[0332] A polynucleotide programmable nucleotide binding domain, when in conjunction with a bound guide polynucleotide (e.g., gRNA), can specifically bind to a target polynucleotide sequence (i.e., via complementary base pairing between bases of the bound guide nucleic acid and bases of the target polynucleotide sequence) and thereby localize the base editor to the target nucleic acid sequence desired to be edited. In some embodiments, the target polynucleotide sequence comprises single-stranded DNA or double-stranded DNA. In some embodiments, the target polynucleotide sequence comprises RNA. In some embodiments, the target polynucleotide sequence comprises a DNA-RNA hybrid.

[0333] In an embodiment, a guide polynucleotide described herein can be RNA or DNA. In one embodiment, the guide polynucleotide is a gRNA.

[0334] In some embodiments, the guide polynucleotide is at least one single guide RNA (“sgRNA” or “gRNA”). In some embodiments, a guide polynucleotide comprises two or more individual polynucleotides, which can interact with one another via for example complementary base pairing (e.g., a dual guide polynucleotide, dual gRNA). For example, a guide polynucleotide can comprise a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA) or can comprise one or more trans-activating CRISPR RNA (tracrRNA).

[0335] A guide polynucleotide may include natural or non-natural (or unnatural) nucleotides (e.g., peptide nucleic acid or nucleotide analogs). In some cases, the targeting region of a guide nucleic acid sequence (e.g., a spacer) can be at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0336] In some embodiments, the methods described herein can utilize an engineered Cas protein. A guide RNA (gRNA) is a short synthetic RNA composed of a scaffold sequence necessary for Cas-binding and a user-defined ~20 nucleotide spacer that defines the genomic target to be modified. Exemplary gRNA scaffold sequences are provided in the sequence listing as SEQ ID NOs: 317-327 and 425. Thus, a skilled artisan can change the genomic target of the Cas protein specificity is partially determined by how specific the gRNA targeting sequence is for the genomic target compared to the rest of the genome. In embodiments, the spacer is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more nucleotides in length. The spacer of a gRNA can be or can be about 19, 20, or 21 nucleotides in length.

[0337] A gRNA or a guide polynucleotide can target any exon or intron of a gene target. In some embodiments, a composition comprises multiple gRNAs that all target the same exon or multiple gRNAs that target different exons. An exon and / or an intron of a gene can be targeted. A gRNA or a guide polynucleotide can target a nucleic acid sequence of about 20 nucleotides or less than about 20 nucleotides (e.g., at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 nucleotides), or anywhere between about 1-100 nucleotides (e.g, 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100). A target nucleic acid sequence can be or can be about 20 bases immediately 5' of the first nucleotide of the PAM. A gRNA can target a nucleic acid sequence. A target nucleic acid can be at least or at least about 1-10, 1-20, 1-30, 1- 40, 1-50, 1-60, 1-70, 1-80, 1-90, or 1-100 nucleotides.

[0338] The guide polynucleotides can comprise standard ribonucleotides, modified ribonucleotides (e.g, pseudouridine), ribonucleotide isomers, and / or ribonucleotide analogs.

[0339] In some embodiments, a base editor system may comprise multiple guide polynucleotides, e.g., gRNAs. For example, the gRNAs may target to one or more target loci (e.g., at least 1 gRNA, at least 2 gRNA, at least 5 gRNA, at least 10 gRNA, at least 20 gRNA, at least 30 g RNA, at least 50 gRNA) comprised in a base editor system. The multiple gRNA sequences can be tandemly arranged and may be separated by a direct repeat.

[0340] Modified Polynucleotides

[0341] To enhance expression, stability, and / or genomic / base editing efficiency, and / or reduce possible toxicity, the base editor-coding sequence (e.g., mRNA) and / or the guide polynucleotide (e.g., gRNA) can be modified to include one or more modified nucleotides and / or chemical modifications, e.g. using pseudo-uridine, 5-Methyl-cytosine, 2'-O-methyl-3'-phosphonoacetate, 2'-(9-methyl thioPACE (MSP), 2'-(9-methyl-PACE (MP), 2'-fluoro RNA (2'-F-RNA), =constrained ethyl (S-cEt), 2'-O-methyl (‘M’), 2'-O-methyl-3'-phosphorothioate (‘MS’), 2'-O- methyl-3'-thiophosphonoacetate (‘MSP’), 5-methoxyuridine, phosphorothioate, and Nl- Methylpseudouridine. Chemically protected gRNAs can enhance stability and editing efficiency in vivo and ex vivo. Methods for using chemically modified mRNAs and guide RNAs are known in the art and described, for example, by Jiang et al., Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope. Nat Commun 11, 1979 (2020). doi.org / 10.1038 / s41467-020-15892-8, Callum et al., Al -Methylpseudouridine substitution enhances the performance of synthetic mRNA switches in cells, Nucleic Acids Research, Volume 48, Issue 6, 06 April 2020, Page e35, and Andries et al., Journal of Controlled Release, Volume 217, 10 November 2015, Pages 337-344, each of which is incorporated herein by reference in its entirety.

[0342] In some embodiments, the guide polynucleotide comprises one or more modified nucleotides at the 5' end and / or the 3' end of the guide. In some embodiments, the guide polynucleotide comprises two, three, four or more modified nucleosides at the 5' end and / or the 3' end of the guide. In some embodiments, the guide polynucleotide comprises two, three, four or more modified nucleosides at the 5' end and / or the 3' end of the guide.

[0343] In some embodiments, the guide comprises at least about 50%-75% modified nucleotides. In some embodiments, the guide comprises at least about 85% or more modified nucleotides. In some embodiments, at least about 1-5 nucleotides at the 5' end of the gRNA are modified and at least about 1-5 nucleotides at the 3' end of the gRNA are modified. In some embodiments, at least about 3-5 contiguous nucleotides at each of the 5' and 3' termini of the gRNA are modified. In some embodiments, at least about 20% of the nucleotides present in a direct repeat or anti-direct repeat are modified. In some embodiments, at least about 50% of the nucleotides present in a direct repeat or anti-direct repeat are modified. In some embodiments, at least about 50-75% of the nucleotides present in a direct repeat or anti-direct repeat are modified. In some embodiments, at least about 100 of the nucleotides present in a direct repeat or antidirect repeat are modified. In some embodiments, at least about 20% or more of the nucleotides present in a hairpin present in the gRNA scaffold are modified. In some embodiments, at least about 50% or more of the nucleotides present in a hairpin present in the gRNA scaffold are modified. In some embodiments, the guide comprises a variable length spacer. In some embodiments, the guide comprises a 20-40 nucleotide spacer. In some embodiments, the guide comprises a spacer comprising at least about 20-25 nucleotides or at least about 30-35 nucleotides. In some embodiments, the spacer comprises modified nucleotides. In some embodiments, the guide comprises two or more of the following: at least about 1-5 nucleotides at the 5' end of the gRNA are modified and at least about 1-5 nucleotides at the 3' end of the gRNA are modified; at least about 20% of the nucleotides present in a direct repeat or anti-direct repeat are modified; at least about 50-75% of the nucleotides present in a direct repeat or anti-direct repeat are modified; at least about 20% or more of the nucleotides present in a hairpin present in the gRNA scaffold are modified; a variable length spacer; and a spacer comprising modified nucleotides.

[0344] In embodiments, the gRNA contains numerous modified nucleotides and / or chemical modifications. Such modifications can increase base editing ~2 fold in vivo or in vitro. In embodiments, the gRNA comprises 2'-O-methyl or phosphorothioate modifications. In an embodiment, the gRNA comprises 2'-O-methyl and phosphorothioate modifications. In an embodiment, the modifications increase base editing by at least about 2 fold.

[0345] A guide polynucleotide can comprise one or more modifications to provide a nucleic acid with a new or enhanced feature. A guide polynucleotide can comprise a nucleic acid affinity tag. A guide polynucleotide can comprise synthetic nucleotide, synthetic nucleotide analog, nucleotide derivatives, and / or modified nucleotides.

[0346] A gRNA or a guide polynucleotide can also be modified by 5' adenylate, 5' guanosinetriphosphate cap, 5' N7-Methylguanosine-triphosphate cap, 5' triphosphate cap, 3' phosphate, 3' thiophosphate, 5' phosphate, 5' thiophosphate, Cis-Syn thymidine dimer, trimers, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer 9, 3 '-3' modifications, -O- methyl thioPACE (MSP), 2'-O-methyl-PACE (MP), and constrained ethyl (S-cEt), 5 '-5' modifications, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-Biotin, dual biotin, PC biotin, psoralen C2, psoralen C6, TINA, 3' DABCYL, black hole quencher 1, black hole quencher 2, DABCYL SE, dT -DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker, thiol linkers, 2'- deoxyribonucleoside analog purine, 2'-deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2'-O-methyl ribonucleoside analog, sugar modified analogs, wobble / universal bases, fluorescent dye label, 2'-fluoro RNA, 2'-O-methyl RNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5 '-triphosphate, 5 '-methylcytidine-5 '-triphosphate, or any combination thereof.

[0347] In some cases, a phosphorothioate enhanced RNA gRNA can inhibit RNase A, RNase Tl, calf serum nucleases, or any combinations thereof. These properties can allow the use of PS- RNA gRNAs to be used in applications where exposure to nucleases is of high probability in vivo or in vitro. For example, phosphorothioate (PS) bonds can be introduced between the last 3- 5 nucleotides at the 5'- or 3 '-end of a gRNA which can inhibit exonuclease degradation. In some cases, phosphorothioate bonds can be added throughout an entire gRNA to reduce attack by endonucleases.

[0348] Fusion Proteins or Complexes Comprising a Nuclear Localization Sequence (NLS)

[0349] In some embodiments, the fusion proteins or complexes provided herein further comprise one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, for example a nuclear localization sequence (NLS). In one embodiment, a bipartite NLS is used. In some embodiments, a NLS comprises an amino acid sequence that facilitates the importation of a protein, that comprises an NLS, into the cell nucleus (e.g., by nuclear transport). In some embodiments, the NLS is fused to the N-terminus or the C-terminus of the fusion protein. In some embodiments, the NLS is fused to the C-terminus or N-terminus of an nCas9 domain or a dCas9 domain. In some embodiments, the NLS is fused to the N-terminus or C-terminus of the Casl2 domain. In some embodiments, the NLS is fused to the N-terminus or C-terminus of the cytidine or adenosine deaminase. In some embodiments, the NLS is fused to the fusion protein via one or more linkers. In some embodiments, the NLS is fused to the fusion protein without a linker. In some embodiments, the NLS comprises an amino acid sequence of any one of the NLS sequences provided or referenced herein. Additional nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al., PCT / EP2000 / 011690, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences.

[0350] In some embodiments, the NLS is present in a linker or the NLS is flanked by linkers, for example described herein. A bipartite NLS comprises two basic amino acid clusters, which are separated by a relatively short spacer sequence (hence bipartite - 2 parts, while monopartite NLSs are not). The NLS of nucleoplasmin, KR [P YATKKAGQA] KKKK (SEQ ID NO: 191), is the prototype of the ubiquitous bipartite signal: two clusters of basic amino acids, separated by a spacer of about 10 amino acids. The sequence of an exemplary bipartite NLS follows: PKKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 328).

[0351] In some embodiments, any of the fusion proteins or complexes provided herein comprise an NLS comprising the amino acid sequence EGADKRTADGSEFESPKKKRKV (amino acids 8 to 29 of SEQ ID NO 328). In some embodiments, any of the adenosine base editors provided herein comprise an NLS comprising the amino acid sequence EGADKRTADGSEFESPKKKRKV (amino acids 8 to 29 of SEQ ID NO: 328). In some embodiments, the NLS is at a C-terminal portion of the adenosine base editor. In some embodiemtns, the NLS is at the C-terminus of the adenosine base editor. Additional Domains

[0352] A base editor described herein can include any domain which helps to facilitate the nucleobase editing, modification or altering of a nucleobase of a polynucleotide. In some embodiments, a base editor comprises a polynucleotide programmable nucleotide binding domain (e.g., Cas9), a nucleobase editing domain (e.g., deaminase domain), and one or more additional domains. In some embodiments, the additional domain can facilitate enzymatic or catalytic functions of the base editor, binding functions of the base editor, or be inhibitors of cellular machinery (e.g., enzymes) that could interfere with the desired base editing result. In some embodiments, a base editor comprises a nuclease, a nickase, a recombinase, a deaminase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcriptional activator, or a transcriptional repressor domain.

[0353] In some embodiments, a base editor comprises an uracil glycosylase inhibitor (UGI) domain. In some cases, a base editor is expressed in a cell in trans with a UGI polypeptide. In some embodiments, cellular DNA repair response to the presence of U: G heteroduplex DNA can be responsible for a reduction in nucleobase editing efficiency in cells. In such embodiments, uracil DNA glycosylase (UDG) can catalyze removal of U from DNA in cells, which can initiate base excision repair (BER), mostly resulting in reversion of the U:G pair to a C:G pair. In such embodiments, BER can be inhibited in base editors comprising one or more domains that bind the single strand, block the edited base, inhibit UGI, inhibit BER, protect the edited base, and / or promote repairing of the non-edited strand. Thus, this disclosure contemplates a base editor fusion protein or complex comprising a UGI domain and / or a uracil stabilizing protein (USP) domain.

[0354] BASE EDITOR SYSTEM

[0355] Provided herein are systems, compositions, and methods for editing a nucleobase using a base editor system. In some embodiments, the base editor system comprises (1) a base editor (BE) comprising a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain (e.g., a deaminase domain) for editing the nucleobase; and (2) a guide polynucleotide (e.g., guide RNA) in conjunction with the polynucleotide programmable nucleotide binding domain. In some embodiments, the base editor system is a cytidine base editor (CBE) or an adenosine base editor (ABE). In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA or RNA binding domain. In some embodiments, the nucleobase editing domain is a deaminase domain. In some embodiments, a deaminase domain can be a cytidine deaminase or an cytosine deaminase. In some embodiments, a deaminase domain can be an adenine deaminase or an adenosine deaminase. In some embodiments, the adenosine base editor can deaminate adenine in DNA. In some embodiments, the base editor is capable of deaminating a cytidine in DNA.

[0356] Use of the base editor system provided herein comprises the steps of: (a) contacting a target nucleotide sequence of a polynucleotide (e.g., double- or single stranded DNA or RNA) of a subject with a base editor system comprising a nucleobase editor (e.g., an adenosine base editor or a cytidine base editor) and a guide polynucleotide (e.g., gRNA), wherein the target nucleotide sequence comprises a targeted nucleobase pair; (b) inducing strand separation of said target region; (c) converting a first nucleobase of said target nucleobase pair in a single strand of the target region to a second nucleobase; and (d) cutting no more than one strand of said target region, where a third nucleobase complementary to the first nucleobase base is replaced by a fourth nucleobase complementary to the second nucleobase. It should be appreciated that in some embodiments, step (b) is omitted. In some embodiments, said targeted nucleobase pair is a plurality of nucleobase pairs in one or more genes. In some embodiments, the base editor system provided herein is capable of multiplex editing of a plurality of nucleobase pairs in one or more genes. In some embodiments, the plurality of nucleobase pairs is located in the same gene. In some embodiments, the plurality of nucleobase pairs is located in one or more genes, wherein at least one gene is located in a different locus.

[0357] The components of a base editor system (e.g., a deaminase domain, a guide RNA, and / or a polynucleotide programmable nucleotide binding domain) may be associated with each other covalently or non-covalently. For example, in some embodiments, the deaminase domain can be targeted to a target nucleotide sequence by a polynucleotide programmable nucleotide binding domain, optionally where the polynucleotide programmable nucleotide binding domain is complexed with a polynucleotide (e.g., a guide RNA). In some embodiments, a polynucleotide programmable nucleotide binding domain can be fused or linked to a deaminase domain. In some embodiments, a polynucleotide programmable nucleotide binding domain can target a deaminase domain to a target nucleotide sequence by non-covalently interacting with or associating with the deaminase domain. For example, in some embodiments, the nucleobase editing component (e.g., the deaminase component) comprises an additional heterologous portion or domain that is capable of interacting with, associating with, or capable of forming a complex with a corresponding heterologous portion, antigen, or domain that is part of a polynucleotide programmable nucleotide binding domain and / or a guide polynucleotide (e.g., a guide RNA) complexed therewith. In some embodiments, the polynucleotide programmable nucleotide binding domain, and / or a guide polynucleotide (e.g., a guide RNA) complexed therewith, comprises an additional heterologous portion or domain that is capable of interacting with, associating with, or capable of forming a complex with a corresponding heterologous portion, antigen, or domain that is part of a nucleobase editing domain (e.g., the deaminase component). In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion is capable of binding to a polynucleotide linker. An additional heterologous portion may be a protein domain. In some embodiments, an additional heterologous portion comprises a polypeptide, such as a 22 amino acid RNA-binding domain of the lambda bacteriophage antiterminator protein N (N22p), a 2G12 IgG homodimer domain, an AB I, an antibody (e.g. an antibody that binds a component of the base editor system or a heterologous portion thereof) or fragment thereof (e.g. heavy chain domain 2 (CH2) of IgM (MHD2) or IgE (EHD2), an immunoglobulin Fc region, a heavy chain domain 3 (CH3) of IgG or IgA, a heavy chain domain 4 (CH4) of IgM or IgE, an Fab, an Fab2, miniantibodies, and / or ZIP antibodies), a bamase-barstar dimer domain, a Bcl-xL domain, a Calcineurin A (CAN) domain, a Cardiac phospholamban transmembrane pentamer domain, a collagen domain, a Com RNA binding protein domain (e.g. SfMu Com coat protein domain, and SfMu Com binding protein domain), a Cyclophilin-Fas fusion protein (CyP-Fas) domain, a Fab domain, an Fe domain, a fibritin foldon domain, an FK506 binding protein (FKBP) domain, an FKBP binding domain (FRB) domain of mTOR, a foldon domain, a fragment X domain, a GAI domain, a GID1 domain, a Glycophorin A transmembrane domain, a GyrB domain, a Halo tag, an HIV Gp41 trimerisation domain, an HPV45 oncoprotein E7 C-terminal dimer domain, a hydrophobic polypeptide, a K Homology (KH) domain, a Ku protein domain (e.g., a Ku heterodimer), a leucine zipper, a LOV domain, a mitochondrial antiviral-signaling protein CARD filament domain, an MS2 coat protein domain (MCP), a non-natural RNA aptamer ligand that binds a corresponding RNA motif / aptamer, a parathyroid hormone dimerization domain, a PP7 coat protein (PCP) domain, a PSD95-Dlgl-zo-l (PDZ) domain, a PYL domain, a SNAP tag, a SpyCatcher moiety, a SpyTag moiety, a streptavidin domain, a streptavidin-binding protein domain, a streptavidin binding protein (SBP) domain, a telomerase Sm7 protein domain (e.g. Sm7 homoheptamer or a monomeric Sm-like protein), and / or fragments thereof. In embodiments, an additional heterologous portion comprises a polynucleotide (e.g., an RNA motif), such as an MS2 phage operator stem-loop (e.g., an MS2, an MS2 C-5 mutant, or an MS2 F-5 mutant), a non-natural RNA motif, a PP7 operator stem-loop, an SfMu phate Com stemloop, a steril alpha motif, a telomerase Ku binding motif, a telomerase Sm7 binding motif,, and / or fragments thereof . Non-limiting examples of additional heterologous portions include polypeptides with at least about 85% sequence identity to any one or more of SEQ ID NOs: 380, 382, 384, 386-388, or fragments thereof. Non-limiting examples of additional heterologous portions include polynucleotides with at least about 85% sequence identity to any one or more of SEQ ID NOs: 379, 381, 383, 385, or fragments thereof.

[0358] In some instances, components of the base editing system are associated with one another through the interaction of leucine zipper domains (e.g., SEQ ID NOs: 387 and 388). In some cases, components of the base editing system are associated with one another through polypeptide domains (e.g., FokI domains) that associate to form protein complexes containing about, at least about, or no more than about 1, 2 (i.e., dimerize), 3, 4, 5, 6, 7, 8, 9, 10 polypeptide domain units, optionally the polypeptide domains may include alterations that reduce or eliminate an activity thereof.

[0359] In some instances, components of the base editing system are associated with one another through the interaction of multimeric antibodies or fragments thereof (e.g., IgG, IgD, IgA, IgM, IgE, a heavy chain domain 2 (CH2) of IgM (MHD2) or IgE (EHD2), an immunoglobulin Fc region, a heavy chain domain 3 (CH3) of IgG or IgA, a heavy chain domain 4 (CH4) of IgM or IgE, an Fab, and an Fab2). In some instances, the antibodies are dimeric, trimeric, or tetrameric. In embodiments, the dimeric antibodies bind a polypeptide or polynucleotide component of the base editing system.

[0360] In some cases, components of the base editing system are associated with one another through the interaction of a polynucleotide-binding protein domain(s) with a polynucleotide(s). In some instances, components of the base editing system are associated with one another through the interaction of one or more polynucleotide-binding protein domains with polynucleotides that are self-complementary and / or complementary to one another so that complementary binding of the polynucleotides to one another brings into association their respective bound polynucleotide-binding protein domain(s).

[0361] In some instances, components of the base editing system are associated with one another through the interaction of a polypeptide domain(s) with a small molecule(s) (e.g., chemical inducers of dimerization (CIDs), also known as “dimerizers”). Non-limiting examples of CIDs include those disclosed in Amara, et al., “A versatile synthetic dimerizer for the regulation of protein-protein interactions,” PNAS, 94:10618-10623 (1997); and VoB, et al. “Chemically induced dimerization: reversible and spatiotemporal control of protein function in cells,” Current Opinion in Chemical Biology, 28: 194-201 (2015), the disclosures of each of which are incorporated herein by reference in their entireties for all purposes. In some embodiments, the base editor inhibits base excision repair (BER) of the edited strand. In some embodiments, the base editor protects or binds the non-edited strand. In some embodiments, the base editor comprises UGI activity or USP activity. In some embodiments, the base editor comprises a catalytically inactive inosine-specific nuclease.

[0362] The base editors of the present disclosure can comprise any domain, feature or amino acid sequence which facilitates the editing of a target polynucleotide sequence. For example, in some embodiments, the base editor comprises a nuclear localization sequence (NLS). In some embodiments, an NLS of the base editor is localized between a deaminase domain and a polynucleotide programmable nucleotide binding domain. In some embodiments, an NLS of the base editor is localized C-terminal to a polynucleotide programmable nucleotide binding domain.

[0363] Protein domains included in the fusion protein can be a heterologous functional domain. Non-limiting examples of protein domains which can be included in the fusion protein include a deaminase domain (e.g., cytidine deaminase and / or adenosine deaminase), a uracil glycosylase inhibitor (UGI) domain, epitope tags, and reporter gene sequences.

[0364] In some embodiments, the adenosine base editor (ABE) can deaminate adenine in DNA. In some embodiments, ABE is generated by replacing APOBEC1 component of BE3 with natural or engineered A. coli TadA, human ADAR2, mouse ADA, or human ADAT2. In some embodiments, ABE comprises an evolved TadA variant. In some embodiments, the base editor is ABE8.1, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity: SEQ ID NO: 331. Other ABE8 sequences are provided in the attached sequence listing (SEQ ID NOs: 332-354).

[0365] In some embodiments, the base editor includes an adenosine deaminase variant comprising an amino acid sequence, which contains alterations relative to an ABE 7*10 reference sequence, as described herein. The term “monomer” as used in Table 6 refers to a monomeric form of TadA*7.10 comprising the alterations described. The term “heterodimer” as used in Table 6 refers to the specified wild-type E. coli TadA adenosine deaminase fused to a TadA*7.10 comprising the alterations as described. Table 6. Adenosine Deaminase Base Editor Variants

[0366] In some embodiments, the base editor comprises a domain comprising all or a portion (e.g., a functional portion) of a uracil glycosylase inhibitor (UGI) or a uracil stabilizing protein (USP) domain.

[0367] Linkers

[0368] In certain embodiments, linkers may be used to link any of the peptides or peptide domains of the disclosure. The linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length. In certain embodiments, the linker is a polypeptide or based on amino acids. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbonheteroatom bond, etc. . In some embodiments, any of the fusion proteins provided herein, comprise a cytidine or adenosine deaminase and a Cas9 domain that are fused to each other via a linker. Various linker lengths and flexibilities between the cytidine or adenosine deaminase and the Cas9 domain can be employed (e.g., ranging from very flexible linkers of the form (GGGS )n(SEQ ID NO: 246), (GGGGS)n (SEQ ID NO: 247), and (G)n to more rigid linkers of the form (EAAAK)n(SEQ ID NO: 248), (SGGS)n (SEQ ID NO: 355), SGSETPGTSESATPES (SEQ ID NO: 249) (see, e.g., Guilinger JP, et al. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014; 32(6): 577-82; the entire contents are incorporated herein by reference) and (XP)n) in order to achieve the optimal length for activity for the cytidine or adenosine deaminase nucleobase editor. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a (GGS)n motif, wherein n is 1, 3, or 7. In some embodiments, cytidine deaminase or adenosine deaminase and the Cas9 domain of any of the fusion proteins provided herein are fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 249), which can also be referred to as the XTEN linker.

[0369] In some embodiments, the domains of the base editor are fused via a linker that comprises the amino acid sequence of: SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 356), SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 357), GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG SAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 358), EGGSEEEEESGS (SEQ ID NO: 3344), or KGPKPKKEESEK (SEQ ID NO: 3345).

[0370] In some embodiments, domains of the base editor are fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 249), which may also be referred to as the XTEN linker. In some embodiments, a linker comprises the amino acid sequence SGGS (SEQ ID NO: 355). In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 359). In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence: SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 360). In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence: SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 361). In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence: PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS TEPSEGSAPGTSESATPESGPGSEPATS (SEQ ID NO: 362).

[0371] In some embodiments, a linker comprises a plurality of proline residues and is 5-21, 5-14, 5-9, 5- 7 amino acids in length, e.g., PAPAP (SEQ ID NO: 363), PAPAPA (SEQ ID NO: 364), PAPAPAP (SEQ ID NO: 365), PAPAPAPA (SEQ ID NO: 366), P(AP)4 (SEQ ID NO: 367), P(AP)7 (SEQ ID NO: 368), P(AP)10 (SEQ ID NO: 369) (see, e.g., Tan J, Zhang F, Karcher D, Bock R. Engineering of high-precision base editors for site-specific single nucleotide replacement. Nat Commun. 2019 Jan 25;10(l):439; the entire contents are incorporated herein by reference). Such proline-rich linkers are also termed “rigid” linkers.

[0372] Nucleic Acid Programmable DNA Binding Proteins with Guide RNAs

[0373] Provided herein are compositions and methods for base editing in cells. Further provided herein are compositions comprising a guide polynucleotide sequence, e.g., a guide RNA sequence, or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more guide RNAs as provided herein. In some embodiments, a composition for base editing as provided herein further comprises a polynucleotide that encodes a base editor, e.g., a C-base editor or an A-base editor. For example, a composition for base editing may comprise a mRNA sequence encoding a BE, a BE4, an ABE, and a combination of one or more guide RNAs as provided. A composition for base editing may comprise a base editor polypeptide and a combination of one or more of any guide RNAs provided herein. Such a composition may be used to effect base editing in a cell through different delivery approaches, for example, electroporation, nucleofection, viral transduction or transfection. In some embodiments, the composition for base editing comprises an mRNA sequence that encodes a base editor and a combination of one or more guide RNA sequences provided herein for electroporation.

[0374] Some aspects of this disclosure provide systems comprising any of the fusion proteins or complexes provided herein, and a guide RNA bound to a nucleic acid programmable DNA binding protein (napDNAbp) domain (e.g., a Cas9 (e.g., a dCas9, a nuclease active Cas9, or a Cas9 nickase) or Casl2) of the fusion protein or complex. These complexes are also termed ribonucleoproteins (RNPs). In some embodiments, the guide nucleic acid (e.g., guide RNA) is from 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the target sequence is a DNA sequence. In some embodiments, the target sequence is an RNA sequence. In some embodiments, the target sequence is a sequence in the genome of a bacteria, yeast, fungi, insect, plant, or animal. In some embodiments, the target sequence is a sequence in the genome of a human. In some embodiments, the 3' end of the target sequence is immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3' end of the target sequence is immediately adjacent to a non-canonical PAM sequence (e.g., a sequence listed in Table 3 or 5'- NAA-3'). In some embodiments, the guide nucleic acid (e.g., guide RNA) is complementary to a sequence in a gene of interest (e.g., a gene associated with a disease or disorder).

[0375] Some aspects of this disclosure provide methods of using the fusion proteins, or complexes provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA molecule with any of the fusion proteins or complexes provided herein, and with at least one guide RNA, wherein the guide RNA is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence.

[0376] The domains of the base editor disclosed herein can be arranged in any order.

[0377] A defined target region can be a deamination window. A deamination window can be the defined region in which a base editor acts upon and deaminates a target nucleotide. In some embodiments, the deamination window is within a 2, 3, 4, 5, 6, 7, 8, 9, or 10 base regions. In some embodiments, the deamination window is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases upstream of the PAM.

[0378] The base editors of the present disclosure can comprise any domain, feature or amino acid sequence which facilitates the editing of a target polynucleotide sequence.

[0379] Methods of Using Fusion Proteins or Complexes Comprising a Cytidine or Adenosine Deaminase and a Cas9 Domain

[0380] Some aspects of this disclosure provide methods of using the fusion proteins, or complexes provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA molecule with any of the fusion proteins or complexes provided herein, and with at least one guide RNA described herein.

[0381] In some embodiments, a fusion protein or complex of the disclosure is used for editing a target gene of interest. In particular, a cytidine deaminase or adenosine deaminase nucleobase editor described herein is capable of making multiple mutations within a target sequence. These mutations may affect the function of the target. For example, when a cytidine deaminase or adenosine deaminase nucleobase editor is used to target a regulatory region the function of the regulatory region is altered and the expression of the downstream protein is reduced or eliminated. Base Editor Efficiency

[0382] In some embodiments, the purpose of the methods provided herein is to alter a gene and / or gene product via gene editing. The nucleobase editing proteins provided herein can be used for gene editing-based human therapeutics in vitro or in vivo. It will be understood by the skilled artisan that the nucleobase editing proteins provided herein, e.g., the fusion proteins or complexes comprising a polynucleotide programmable nucleotide binding domain (e.g., Cas9) and a nucleobase editing domain (e.g., an adenosine deaminase domain or a cytidine deaminase domain) can be used to edit a nucleotide from A to G or C to T.

[0383] Advantageously, base editing systems as provided herein provide genome editing without generating double-strand DNA breaks, without requiring a donor DNA template, and without inducing an excess of stochastic insertions and deletions as CRISPR may do. In some embodiments, the present disclosure provides base editors that efficiently generate an intended mutation, such as a STOP codon, in a nucleic acid (e.g., a nucleic acid within a genome of a subject) without generating a significant number of unintended mutations, such as unintended point mutations.

[0384] The base editors of the disclosure advantageously modify a specific nucleotide base encoding a protein without generating a significant proportion of indels (i.e., insertions or deletions). Such indels can lead to frame shift mutations within a coding region of a gene.

[0385] In some embodiments, the base editors provided herein are capable of generating a ratio of intended mutations to indels (i.e., intended point mutations:unintended point mutations) that is greater than 1 : 1. In some embodiments, the base editors provided herein are capable of generating a ratio of intended mutations to indels that is at least 1.5:1, at least 2:1, at least 2.5: 1, at least 3: 1, at least 3.5: 1, at least 4: 1, at least 4.5: 1, at least 5: 1, at least 5.5: 1, at least 6: 1, at least 6.5: 1, at least 7:1, at least 7.5: 1, at least 8: 1, at least 10: 1, at least 12: 1, at least 15: 1, at least 20: 1, at least 25: 1, at least 30: 1, at least 40: 1, at least 50: 1, at least 100: 1, at least 200: 1, at least 300: 1, at least 400: 1, at least 500: 1, at least 600: 1, at least 700: 1, at least 800: 1, at least 900: 1, or at least 1000:1, or more. The number of intended mutations and indels may be determined using any suitable method.

[0386] In some embodiments, the base editors provided herein can limit formation of indels in a region of a nucleic acid. In some embodiments, the region is at a nucleotide targeted by a base editor or a region within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nucleotide targeted by a base editor. In some embodiments, any of the base editors provided herein can limit the formation of indels at a region of a nucleic acid to less than 1%, less than 1.5%, less than 2%, less than 2.5%, less than 3%, less than 3.5%, less than 4%, less than 4.5%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 12%, less than 15%, or less than 20%.

[0387] Base editing is often referred to as a “modification”, such as, a genetic modification, a gene modification and modification of the nucleic acid sequence and is clearly understandable based on the context that the modification is a base editing modification. A base editing modification is therefore a modification at the nucleotide base level, for example as a result of the deaminase activity discussed throughout the disclosure, which then results in a change in the gene sequence and may affect the gene product.

[0388] In some embodiments, the modification, e.g., single base edit results in about or at least about a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,

[0389] 85%, 90%, 95%, 99%, or 100% reduction, or reduction to an undetectable level, of the gene targeted expression.

[0390] The disclosure provides adenosine deaminase variants (e.g., ABE8 variants) that have increased efficiency and specificity. In particular, the adenosine deaminase variants described herein are more likely to edit a desired base within a polynucleotide and are less likely to edit bases that are not intended to be altered (e.g., “bystanders”).

[0391] In some embodiments, any of the base editing system comprising one of the ABE8 base editor variants described herein has reduced bystander editing or mutations by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,

[0392] 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to a base editor system comprising an ABE7 base editor, e.g., ABE7.10.

[0393] In some embodiments, any of the ABE8 base editor variants described herein has higher base editing efficiency compared to the ABE7 base editors. In some embodiments, any of the

[0394] ABE8 base editor variants described herein have at least 1%, 2%, 3%, 4%, 5%, 10%, 15%,

[0395] 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,

[0396] 95%, 99%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%,

[0397] 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 450%, or 500% higher base editing efficiency compared to an ABE7 base editor, e.g., ABE7.10.

[0398] The ABE8 base editor variants described herein may be delivered to a host cell via a plasmid, a vector, a LNP complex, or an mRNA. In some embodiments, any of the ABE8 base editor variants described herein is delivered to a host cell as an mRNA. In some embodiments, the method described herein, for example, the base editing methods has minimum to no off-target effects. In some embodiments, the method described herein, for example, the base editing methods, has minimal to no chromosomal translocations.

[0399] In some embodiments, the base editing method described herein results in about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of a cell population that have been successfully edited.

[0400] In some embodiments, the percent of viable cells in a cell population following a base editing intervention is greater than at least 60%, 70%, 80%, or 90% of the starting cell population at the time of the base editing event. In some embodiments, the percent of viable cells in a cell population following editing is about 70%. In some embodiments, the percent of viable cells in a cell population following editing is about 75%. In some embodiments, the percent of viable cells in a cell population following editing is about 80%. In some embodiments, the percent of viable cells in a cell population as described above is about 85%. In some embodiments, the percent of viable cells in a cell population as described above is about 90%, or about 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% of the cells in the population at the time of the base editing event.

[0401] In embodiments, the cell population is a population of cells contacted with a base editor, complex, or base editor system of the present disclosure.

[0402] The number of intended mutations and indels can be determined using any suitable method, for example, as described in International PCT Application Nos. PCT / US2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632); Komor, A.C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N.M., etal., “Programmable base editing of A»T to G*C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); and Komor, A.C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017); the entire contents of which are hereby incorporated by reference.

[0403] In some embodiments, to calculate indel frequencies, sequencing reads are scanned for exact matches to two 10-bp sequences that flank both sides of a window in which indels can occur. If no exact matches are located, the read is excluded from analysis. If the length of this indel window exactly matches the reference sequence the read is classified as not containing an indel. If the indel window is two or more bases longer or shorter than the reference sequence, then the sequencing read is classified as an insertion or deletion, respectively. In some embodiments, the base editors provided herein can limit formation of indels in a region of a nucleic acid. In some embodiments, the region is at a nucleotide targeted by a base editor or a region within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nucleotide targeted by a base editor.

[0404] Multiplex Editing

[0405] In some embodiments, the base editor system provided herein is capable of multiplex editing of a plurality of nucleobase pairs in one or more genes or polynucleotide sequences. In some embodiments, the plurality of nucleobase pairs is located in the same gene or in one or more genes, wherein at least one gene is located in a different locus. In some embodiments, the multiplex editing comprises one or more guide polynucleotides. In some embodiments, the multiplex editing comprises one or more base editor systems. In some embodiments, the multiplex editing comprises one or more base editor systems with a single guide polynucleotide or a plurality of guide polynucleotides. In some embodiments, the multiplex editing comprises one or more guide polynucleotides with a single base editor system. It should be appreciated that the characteristics of the multiplex editing using any of the base editors as described herein can be applied to any combination of methods using any base editor provided herein. It should also be appreciated that the multiplex editing using any of the base editors as described herein can comprise a sequential editing of a plurality of nucleobase pairs.

[0406] In some embodiments, the base editor system capable of multiplex editing of a plurality of nucleobase pairs in one or more genes comprises one of ABE7, ABE8, and / or ABE9 base editors.

[0407] Expression of Fusion Proteins or Complexes in a Host Cell

[0408] Fusion proteins or complexes of the disclosure comprising a deaminase may be expressed in virtually any host cell of interest, including but not limited to bacteria, yeast, fungi, insects, plants, and animal cells using routine methods known to the skilled artisan. For example, a DNA encoding an adenosine deaminase of the disclosure can be cloned by designing suitable primers for the upstream and downstream of CDS based on the cDNA sequence. The cloned DNA may be directly, or after digestion with a restriction enzyme when desired, or after addition of a suitable linker and / or a nuclear localization signal, ligated with a DNA encoding one or more additional components of a base editing system. The base editing system is translated in a host cell to form a complex.

[0409] A polynucleotide encoding a polypeptide described herein can be obtained by chemically synthesizing the polynucleotide, or by connecting synthesized partly overlapping oligo short chains by utilizing the PCR method and the Gibson Assembly method to construct a polynucleotide (e.g., DNA) encoding the full length thereof. The advantage of constructing a full-length polynucleotide by chemical synthesis or a combination of PCR method or Gibson Assembly method is that the codons to be used can be selected in according to the host into which the polynucleotide is to be introduced. In the expression from a heterologous DNA molecule, the protein expression level is expected to increase by converting the DNA sequence thereof to a codon highly frequently used in the host organism. Codon use data for a host cell (e.g., codon use data available at kazusa.or.jp / codon / index.html) can be used to guide codon optimization for a polynucleotide sequence encoding a polypeptide. Codons having low use frequency in the host may be converted to a codon coding the same amino acid and having high use frequency.

[0410] An expression vector containing a polynucleotide encoding a nucleic acid sequencerecognizing module and / or a nucleic acid base converting enzyme can be produced, for example, by linking the DNA to the downstream of a promoter in a suitable expression vector.

[0411] As the expression vector, Escherichia coli- v\ plasmids (e.g., pBR322, pBR325, pUC12, pUC13); Bacillus subtilis-denved plasmids (e.g., pUBUO, pTP5, pC194); yeast-derived plasmids (e.g., pSH19, pSH15); insect cell expression plasmids (e.g., pFast-Bac); animal cell expression plasmids (e.g., pAl-11, pXTl, pRc / CMV, pRc / RSV, pcDNAI / Neo); bacteriophages such as .lambda phage and the like; insect virus vectors such as baculovirus and the like (e.g., BmNPV, AcNPV); animal virus vectors such as retrovirus, vaccinia virus, adenovirus and the like, and the like are used.

[0412] Regarding the promoter to be used, any promoter appropriate for a host to be used for gene expression can be used. In a conventional method using double-stranded breaks, since the survival rate of the host cell sometimes reduces markedly due to the toxicity, it is desirable to increase the number of cells by the start of the induction by using an inductive promoter. However, since sufficient cell proliferation can also be afforded by expressing the nucleic acidmodifying enzyme complex of the present disclosure, a constitutive promoter can be used without limitation.

[0413] For example, when the host is an animal cell, an SR.alpha. promoter, SV40 promoter, LTR promoter, cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, Moloney mouse leukemia virus (MoMuLV), LTR, herpes simplex virus thymidine kinase (HSV- TK) promoter, and the like can be used. Of these, CMV promoter, SR.alpha. promoter and the like may be used.

[0414] When the host is Escherichia coli, a trp promoter, lac promoter, recA promoter, .lamda.P.sub.L promoter, Ipp promoter, T7 promoter, and the like can be used. When the host is in the genus Bacillus, the SPO1 promoter, SPO2 promoter, penP promoter, and the like can be used.

[0415] When the host is a yeast, the Gal 1 / 10 promoter, PHO 5 promoter, PGK promoter, GAP promoter, ADH promoter, and the like can be used.

[0416] When the host is an insect cell, the polyhedrin promoter, PIO promoter, and the like can be used.

[0417] When the host is a plant cell, the CaMV35S promoter, CaMV19S promoter, NOS promoter, and the like can be used.

[0418] Expression vectors for use in the present disclosure, besides those mentioned above, can comprise an enhancer, a splicing signal, a terminator, a polyA addition signal, a selection marker such as drug resistance gene, an auxotrophic complementary gene and the like, a replication origin, and the like can be used.

[0419] An RNA encoding a protein domain described herein can be prepared by, for example, in vitro transcription of a nucleic acid sequence encoding any of the fusion proteins or complexes disclosed herein.

[0420] A fusion protein or complex of the disclosure can be intracellularly expressed by introducing into the cell an expression vector comprising a nucleic acid sequence encoding the fusion protein or complex.

[0421] Host cells of interest, include but are not limited to bacteria, yeast, fungi, insects, plants, and animal cells. For example, a host cell may comprise bacteria from the genus Escherichia, such as Escherichia coli K12.cndot.DHl [Proc. Natl. Acad. Sci. USA, 60, 160 (1968)], Escherichia coli JM103 [Nucleic Acids Research, 9, 309 (1981)], Escherichia coli JA221 [Journal of Molecular Biology, 120, 517 (1978)], Escherichia coli HB 101 [Journal of Molecular Biology, 41, 459 (1969)], Escherichia coli C600 [Genetics, 39, 440 (1954)] and the like.

[0422] A host cell may comprise bacteria from the genus Bacillus, for example Bacillus subtilis Ml 114 [Gene, 24, 255 (1983)], Bacillus subtilis 207 -21 [Journal of Biochemistry, 95, 87 (1984)] and the like.

[0423] A host cell may be a yeast cell. Examples of yeast cells include Saccharomyces cerevisiae AH22, AH22R.sup.-, NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris KM71 and the like.

[0424] When the viral delivery methods utilize the virus AcNPV, cells from a cabbage armyworm larva-derived established line (Spodoptera frugiperda cell; Sf cell), MG1 cells derived from the mid-intestine of Trichoplusia ni, High Five™ cells derived from an ovary of Trichoplusia ni, Mamestra brassicae- v\ cells, Estigmena acrea- v\ cells and the like can be used. When the virus is BmNPV, cells of Bombyx mori- m established line (Bombyx mori N cell; BmN cell) and the like are used. As the Sf cell, for example, Sf9 cell (ATCC CRL1711), Sf21 cell [all above, In Vivo, 13, 213-217 (1977)] and the like are used.

[0425] An insect can be any insect, for example, larva of Bombyx mori, Drosophila, cricket, and the like [Nature, 315, 592 (1985)].

[0426] Animal cells contemplated in the present disclosure include, but are not limited to, cell lines such as monkey COS-7 cells, monkey Vero cells, Chinese hamster ovary (CHO) cells, dhfr gene-deficient CHO cells, mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, human FL cells and the like, pluripotent stem cells such as iPS cells, ES cells derived humans and other mammals, and primary cultured cells prepared from various tissues. Furthermore, zebrafish embryo, Xenopus oocyte, and the like can also be used.

[0427] Plant cells are also contemplated in the present disclosure. Plant cells include, but are not limited to, suspended cultured cells, callus, protoplast, leaf segment, root segment and the like prepared from various plants (e.g., grain such as rice, wheat, corn, and the like; product crops such as tomato, cucumber, eggplant and the like; garden plants such as carnations, Eustoma russellianum, and the like; and other plants such as tobacco, Arabidopsis thaliana and the like) are used.

[0428] All the above-mentioned host cells may be haploid (monoploid), or polyploid (e.g., diploid, triploid, tetrapioid, etc.). Using conventional methods, mutations, in principle, introduced into only one homologous chromosome produce a heterogenous cell. Therefore, the desired phenotype is not expressed unless the mutation is dominant. For recessive mutations, acquiring a homozygous cell can be inconvenient due to labor and time requirements. In contrast, according to the present disclosure, since a mutation can be introduced into any allele on the homologous chromosome in the genome, the desired phenotype can be expressed in a single generation even in the case of recessive mutation, thereby solving the problem associated with conventional mutagenesis methods.

[0429] An expression vector can be introduced by a known method (e.g., the lysozyme method, the competent method, the PEG method, the CaCh coprecipitation method, electroporation, microinjection, particle gun method, lipofection, Hgrotocterzwm-mediated delivery, etc.) according to the kind of the host.

[0430] Escherichia coli can be transformed according to the methods described in, for example, Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982).

[0431] The genus Bacillus can be introduced into a vector according to the methods described in, for example, Molecular & General Genetics, 168, 111 (1979). A yeast can be introduced into a vector according to the methods described in, for example, Methods in Enzymology, 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).

[0432] An insect cell and an insect can be introduced into a vector according to the methods described in, for example, Bio / Technology, 6, 47-55 (1988).

[0433] A vector can be introduced into an animal cell according to the methods described in, for example, Cell Engineering additional volume 8, New Cell Engineering Experiment Protocol, 263-267 (1995) (published by Shujunsha), and Virology, 52, 456 (1973).

[0434] A cell comprising a vector can be cultured according to a known method according to the kind of the host. For example, when Escherichia coli or genus Bacillus is cultured, a liquid medium may be used as a medium to be used for the culture. The medium may contain a carbon source, nitrogen source, inorganic substance and the like necessary for the growth of the transformant. Examples of the carbon source include glucose, dextrin, soluble starch, sucrose and the like; examples of the nitrogen source include inorganic or organic substances such as ammonium salts, nitrate salts, corn steep liquor, peptone, casein, meat extract, soybean cake, potato extract and the like; and examples of the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. The medium may contain yeast extract, vitamins, growth promoting factor and the like. The pH of the medium is between about 5 about 8 in embodiments.

[0435] As a medium for culturing Escherichia coli, for example, M9 medium containing glucose, casamino acid [Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972] can be used. Where necessary, for example, agents such as 3P-indolylacrylic acid may be added to the medium to ensure an efficient function of a promoter. Escherichia coli is cultured at generally about 15 to about 43°C. Where necessary, aeration and stirring may be performed.

[0436] The genus Bacillus is cultured at generally about 30 to about 40°C. Where necessary, aeration and stirring may be performed.

[0437] Examples of the medium for culturing yeast include Burkholder minimum medium [Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)], SD medium containing 0.5% casamino acid [Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)] and the like. The pH of the medium may be between about 5 to about 8. The culture is performed at generally about 20°C to about 35°C. Where necessary, aeration and stirring may be performed.

[0438] As a medium for culturing an insect cell or insect, for example, Grace’s Insect Medium [Nature, 195, 788 (1962)] containing an additive such as inactivated 10% bovine serum and the like as appropriate and the like are used. The pH of the medium is may be between about 6.2 to about 6.4. The culture is performed at generally about 27°C. Where necessary, aeration and stirring may be performed.

[0439] As a medium for culturing an animal cell, for example, minimum essential medium (MEM) containing about 5 to about 20% of fetal bovine serum [Science, 122, 501 (1952)], Dulbecco’s modified Eagle medium (DMEM) [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)] and the like are used. The pH of the medium may be between about 6 to about 8. The culture is performed at generally about 30°C.to about 40°C. Where necessary, aeration and stirring may be performed.

[0440] As a medium for culturing a plant cell, for example, MS medium, LS medium, B5 medium and the like are used. The pH of the medium may be between about 5- about 8. The culture is performed at generally about 20°C to about 30°C. Where necessary, aeration and stirring may be performed.

[0441] When a higher eukaryotic cell, such as animal cell, insect cell, plant cell and the like is used as a host cell, a polynucleotide encoding a base editing system of the present disclosure (e.g., comprising an adenosine deaminase variant) is introduced into a host cell under the regulation of an inducible promoter (e.g., metallothionein promoter (induced by heavy metal ion), heat shock protein promoter (induced by heat shock), Tet-ON / Tet-OFF system promoter (induced by addition or removal of tetracycline or a derivative thereof), steroid-responsive promoter (induced by steroid hormone or a derivative thereof) etc.), the induction substance is added to the medium (or removed from the medium) at an appropriate stage to induce expression of the nucleic acid-modifying enzyme complex, culture is performed for a given period to carry out a base editing and, introduction of a mutation into a target gene, transient expression of the base editing system can be realized.

[0442] Prokaryotic cells such as Escherichia coli and the like can utilize an inducible promoter. Examples of the inducible promoter include, but are not limited to, lac promoter (induced by IPTG), cspA promoter (induced by cold shock), araBAD promoter (induced by arabinose) and the like.

[0443] Alternatively, the above-mentioned inductive promoter can also be utilized as a vector removal mechanism when higher eukaryotic cells, such as animal cell, insect cell, plant cell and the like are used as a host cell. That is, a vector is mounted with a replication origin that functions in a host cell, and a nucleic acid encoding a protein necessary for replication (e.g., SV40 on and large T antigen, oriP and EBNA-1 etc. for animal cells), of the expression of the nucleic acid encoding the protein is regulated by the above-mentioned inducible promoter. As a result, while the vector is autonomously replicable in the presence of an induction substance, when the induction substance is removed, autonomous replication is not available, and the vector naturally falls off along with cell division (autonomous replication is not possible by the addition of tetracycline and doxycycline in Tet-OFF system vector).

[0444] CAR-T CELL THERAPIES

[0445] The present disclosure provides immune cells (e.g., T-cells) modified using nucleobase editors and / or nucleases described herein. The modified immune cells may express chimeric antigen receptors (CARs) (e.g., CAR-T cells). Modification of immune cells to express a chimeric antigen receptor can enhance an immune cell’s immunoreactive activity, wherein the chimeric antigen receptor has an affinity for an epitope on an antigen, wherein the antigen is associated with an altered fitness of an organism. For example, the chimeric antigen receptor can have an affinity for an epitope on a protein expressed in a diseased cell. In embodiments, the immune cells express a chCLEC2d polypeptide of the disclosure.

[0446] In embodiments, the immune cells contain a kill switch (e.g., RQR8 or an antibody-drug conjugate target). The kill switch can be contained within a chimeric antigen receptor and / or chCLEC2d polypeptide of the disclosure. In some cases, a chimeric antigen receptor expressed by the cell contains the kill switch.

[0447] Some aspects of the present disclosure provide for immune cells comprising a chimeric antigen receptor (CAR) and an altered endogenous gene that provides increased persistence, resistance to fratricide, enhances immune cell function, resistance to immunosuppression or inhibition, or a combination thereof. In some embodiments, the altered endogenous gene may be created by base editing. In some embodiments, the base editing may reduce or attenuate the gene expression. In some embodiments, the base editing may reduce or attenuate the gene activation. In some embodiments, the base editing may reduce or attenuate the functionality of the gene product. In some other embodiments, the base editing may activate or enhance the gene expression. In some embodiments, the base editing may increase the functionality of the gene product. In some embodiments, the altered endogenous gene may be modified or edited in an exon, an intron, an exon-intron injunction, or a regulatory element thereof. The modification may be edit to a single nucleobase in a gene or a regulatory element thereof. The modification may be in a exon, more than one exons, an intron, or more than one introns, or a combination thereof. The modification may be in an open reading frame of a gene. The modification may be in an untranslated region of the gene, for example, a 3'-UTR or a 5'-UTR. In some embodiments, the modification is in a regulatory element of an endogenous gene. In some embodiments, the modification is in a promoter, an enhancer, an operator, a silencer, an insulator, a terminator, a transcription initiation sequence, a translation initiation sequence (e.g., a Kozak sequence), or any combination thereof.

[0448] In some embodiments, each edited gene may comprise a single base edit. In some embodiments, each edited gene may comprise multiple base edits at different regions of the gene. In some embodiments, a single modification event (such as electroporation), may introduce one or more gene edits. In some embodiments at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more edits may be introduced in one or more genes simultaneously. In some embodiments, an immune cell, including but not limited to any immune cell comprising an edited gene selected from any of the aforementioned gene edits, can be edited to generate mutations in other genes that enhance the CAR-T’s function or reduce immunosuppression or inhibition of the cell.

[0449] In some embodiments, an immune cell of the disclosure comprises a chimeric antigen receptor and one or more edited genes. In some embodiments, an immune cell of the disclosure comprises and / or expresses a chimeric c-type lectin domain family 2, member D (chCLEC2d) polypeptide of the disclosure. The one or more gene edits may reduce or eliminate expression of the one or more edited genes. The one or more genes may be selected from beta-2 microglobulin, CD54, CD58, CD161, TAPI, TAP2, Tapasin, CIITA, CITA (NLRC5), ERP57, HLA-A, HLA-B, HLA-C, components of the T cell receptor complex (e.g., CD3E), and various combinations thereof. The one or more genes may be selected from, as non-limiting examples, beta-2- microglobulin, FKBP1A, and CD58. An edited gene may be an immune response regulation gene, an immunogenic gene, a checkpoint inhibitor gene, a gene involved in immune responses, a cell surface marker, e.g., a T cell surface marker, or any combination thereof. In some embodiments, an immune cell comprises a chimeric antigen receptor and an edited gene that is associated with activated T cell proliferation, alpha-beta T cell activation, gamma-delta T cell activation, positive regulation of T cell proliferation, negative regulation of T-helper cell proliferation or differentiation, or their regulatory elements thereof, or combinations thereof.

[0450] In embodiments, one or more genes are modified in an immune effector cell so that the modified immune cell has a reduced level of, lacks, or have virtually undetectable levels of beta- 2 -microglobulin and / or one or more of the following polypeptides relative to an unmodified immune cell: B cell leukemia / lymphoma 1 lb (Bell lb); B cell leukemia / lymphoma 2 related protein Aid (Bcl2ald); B cell leukemia / lymphoma 6 (Bcl6); butyrophilin-like 6 (Btnl6); CD151 antigen (Cdl51); chemokine (C-C motif) receptor 7 (Ccr7); discs large MAGUK scaffold protein 5 (Dlg5); erythropoietin( Epo); G protein-coupled receptor 18 (Gprl8); interferon alpha 15 (Ifnal5); interleukin 6 signal transducer (I16st); interleukin 7 receptor (I17r); Janus kinase 3 (Jak3); membrane associated ring-CH-type finger 7 (Marchf7); NCK associated protein 1 like (Nckapll); phospholipase A2, group IIF (Pla2g2f); runt related transcription factor 3 (Runx3); Signal-regulatory protein beta IB (Sirpblb); transforming growth factor, beta 1 (Tgfbl); tumor necrosis factor (ligand) superfamily, member 14 (Tnfsfl4); tumor necrosis factor (ligand) superfamily, member 18 (Tnfsfl8); tumor necrosis factor (ligand) superfamily, member 8 (Tnfsf8); zinc finger CCCH type containing 8 (Zc3h8); (Rac family small GTPase 2); (Slc4al); 5-azacytidine induced gene 2 (Azi2); a disintegrin and metalloprotease domain 17 (Adam 17); a disintegrin and metalloprotease domain 8 (Adam8); Acetyl-CoA Acetyltransferase 1 (ACAT1); ACLY; adapter related protein complex 3 beta 1 sububit (Ap3bl); adapter related protein complex 3 delta 1 sububit (Ap3dl); adenosine A2a receptor (Adora2a); adenosine deaminase (Ada); adenosine kinase (Adk); adenosine regulating molecule 1 (Adrml); advanced glycosylation end product-specific receptor (Ager) allograft inflammatory factor 1 (Aifl); AKT1; AKT2; amyloid beta (A4) precursor protein-binding family B member 1 interacting protein (Apbblip); ankyrin repeat and LEM domain (Anklel); annecin Al (Anxal); arginase liver (Arg 1); arginase type II (Arg 2); AtPase Cu++ transporting, alpha polypeptide (Atp7a); autoimmune regulator (Aire); autophagy related 5 (Atg5); AXL; B and T Lymphocyte Associated (BTLA); B and T lymphocyte associated (Btla); B cell leukemia / lymphoma 10 (BcllO); B cell leukemia / lymphoma I la (Bell la); B cell leukemia / lymphoma 2 (Bcl2); B cell leukemia / lymphoma 3 (Bcl3); basic leucine zipper transcription factor, ATF-like (Batf); BCL2- associated X protein (Bax); BCL2L11; beta 2 microglobulin (B2m); BL2-associated agonist of cell dealth (Bad); BLIMP 1; Bloom syndrome, RecQ like helicase (Blm); Bmil poly comb ring finger oncogene (Bmil); Bone morphogenic protein 4 (Bmp4); Braf transforming gene (Braf); butyrophilin, subfamily 2, member Al (Btn2al); butyrophilin, subfamily 2, member A2 (Btn2a2); butyrophilin-like 1 (Btnll); butyrophilin-like 2 (Btnl2); c-abl oncogene 1 (Abll); c-abl oncogene 2 (Abl2); cadherin-like 26(Cdh26); calcium channel, voltage dependent, beta 4 subunit (Cacnb4); CAMK2D; capping protein regulator and myosin 1 linker 2 (Carmil2); carcinoembryonic antigen-related cell adhesion molecule (Ceacaml); Casitas B-lineage lymphoma b (Cblb); CASP8; Caspase 3 (Casp3); caspase recruitment domain family member 11 (Cardl l); catenin (cadherin associated protein), beta 1 (Ctnnbl); caveolin 1 (Cavl); CBL-B;

[0451] CCAAT / enhancer binding protein (CZEBP), beta (Cebpb); CCR10; CCR4; CCR5; CCR6; CCR9; CD103; CDl la; CD122; CD123; CD127; CD130; CD132; CD160 antigen (Cdl60); CD161; CD 19; CDldl antigen (Cdldl); CDld2 antigen (CDld2); CD2 antigen (CD2); CD209e antigen (Cd209e); CD23; CD244 molecule A (Cd244a); CD24a antigen (Cd24a); CD27 antigen (CD27); CD274 antigen (Cd274); CD276 antigen (Cd276); CD28 antigen (Cd28); CD3 delta; CD3 epsilon; CD3 gamma; CD30; CD300A molecule (Cd300a); CD33; CD38; CD4 antigen (Cd4); CD40 ligand (Cd401g); CD44 antigen (Cd44); CD46 antigen, complement regulatory protein (Cd46); CD47 antigen (Rh-related antigen, integrin-associated signal transducer) (Cd47); CD48 antigen (Cd48); CD5 antigen (Cd5); CD52; CD58; CD59b antigen (Cd59b); CD6 antigen (Cd6); CD69; CD7; CD70; CD74 antigen (Cd74); CD8; CD8 antigen (Cd8); CD80 antigen (Cd80); CD81 antigen (Cd81); CD82; CD83 antigen (Cd83); CD86; CD86 antigen (Cd86); CD8A; CD96; CD99; CDK4; CDK8; CDKN1B; chemokine (C motif) ligand 1 (Xcll); chemokine (C-C motif) ligand 19 (Cell 9); chemokine (C-C motif) ligand 2 (Ccl2); chemokine (C-C motif) ligand 20 (Ccl20); chemokine (C-C motif) ligand 5 (Ccl5); chemokine (C-C motif) receptor 2 (Ccr2); chemokine (C-C motif) receptor 6 (Ccr6); chemokine (C-C motif) receptor 9 (Ccr9); chemokine (C-X-C motif) ligand 12 (Cxcll2); chemokine (C-X-C motif) receptor (Cxcr4); Chitinase 3 Like

[0452] 1 (Chi311); cholinergic receptor, nicotinic, alpha polypeptide 7 (Chma7); chromodomain helicase DNA binding protein 7 (Chd7); CLA; Class II Major Histocompatibility Complex Transactivator (CIITA); cleft lip and palate associated transmembrane protein 1 (Clptml); Cluster of Differentiation 123 (CD123); Cluster of Differentiation 3 (CD3); Cluster of Differentiation 33 (CD33); Cluster of Differentiation 52 (CD52); Cluster of Differentiation 7 (CD7); Cluster of Differentiation 96 (CD96); coagulation factor II (thrombin) receptor-like 1 (F2rll); coil-coil domain containing 88B (Ccdc88b); core-binding factor beta (Cbfb); coronin, actin binding protein 1A (Corola); coxsackie virus and adenovirus receptor (Cxadr); CS-1; CSF2CSK; c-src tyrosine kinase (Csk); C-type lectin domain family 2, member i (Clec2i); C- type lectin domain family 4, member a2 (Clec4a2); C-type lectin domain family 4, member d (Clec4d); C-type lectin domain family 4, member e (Clec4e); C-type lectin domain family 4, member f (Clec4f); C-type lectin domain family 4, member g (Clec4g); CUL3; CXCR3; cyclic GMP-AMP synthase (Cgas); cyclin D3 (Ccnd3); cyclin dependent kinase inhibitor 2A (Cdkn2a); cyclin-dependent kinase (Cdk6); CYLD lysine 63 deubiquitinase (Cyld); cysteine-rich protein 3 (Crip3); cytidine 5'-triphosphate synthase (Ctps); Cytochrome P450 Family 11 Subfamily A Member 1 (Cypl lal); cytochrome P450, family 26, subfamily b, polypeptide (Cyp26bl); Cytokine Inducible SH2 Containing Protein (CISH); cytotoxic T lymphocyte-associated protein

[0453] 2 alpha (Ctla2a); Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4); DCK; dedicator of cytokinesis 2 (Dock2); dedicator of cytokinesis 8 (Dock8); delta like canonical Notch ligand 4 (D114); deltex 1, E3 ubiquitin ligase (Dtxl); deoxyhypusine synthase (Dhps); DGKA; DGKZ; DHX37; dicer 1, ribonuclease type III (Dicerl); dipeptidylpeptidase 4 (Dpp4); discs large MAGUK scaffold protein 1 (Dlgl); DnaJ heat shock protein family (Hsp40) member A3 (Dnaja3); dolichyl-di-phosphooligosaccharide-protein glycotransferase (Ddost); double homeobox B-like 1 (Duxbll); drosha, ribonuclease type III (Drosha); dual specificity phosphatase 10 (DusplO); dual specificity phosphatase 22 (Dusp22); dual specificity phosphatase 3 (Dusp3); E74-like factor 4 (Elf4); early growth response l(Egrl); early growth response 3 (Egr3); ELOB (TCEB2); ENTPD1 (CD39); eomesodermin (Eomes); Eph receptor B4 (Ephb4); Eph receptor B6 (Ephb6); ephrin Bl (Efnbl); ephrin B2 (Efnb2); ephrin B3 (Efnb3); Epstein-Barr virus induced gene 3 (Ebi3); erb-b2 receptor tyrosine kinase (Erbb2); eukaryotic translation initiation factor 2 alpha kinase 4 (Eif2ak4); FADD; family with sequence similarity 49, member B (Fam49b); Fanconi anemia, complementation group A (Fanca); Fanconi anemia, complementation group D2 (Fancd2); Fas (TNF receptor superfamily member 6) (Fas); Fas (TNFRSF6)-associated via death domain (Fadd); Fas Cell Surface Death Receptor (FAS); Fc receptor, IgE, high affinity I, gamma polypeptide (Fcerlg); fibrinogen-like protein 1 (Fgl 1); fibrinogen-like protein 2 (Fgl2); FK506 binding protein la (Fkbpla); FK506 binding protein lb ((Fkbplb); flotillin 2 (Flot2); FMS-like tyrosine kinase (Flt3); forkhead box JI (Foxj l); forkhead box N1 (Foxnl); forkhead box Pl (Foxpl); forkhead box P3 (Foxp3); frizzled class receptor 5 (Fzd5); frizzled class receptor 7 (Fzd7); frizzled class receptor 8 (Fzd8); fucosyltransferase 7 (Fut7); Fyn proto-oncogene (Fyn); gap junction protein, alpha 1 (Gjal); GATA binding protein 3 (GATA3); GCN2 kinase (IDO pathway); gelsolin (Gsn); GLI-Kruppel family member GLI3 (Gli3); glycerol-3 -phosphate acyltransferase, mitochondrial (Gpam); growth arrest and DNA- damage-inducible 45 gamma (Gadd45g); GTPase, IMAP family member 1 (Gimapl); H1TET2; H2.0-like homeobox (Hix); haematopoietic l(heml); HCLS1 binding protein 3 (Hslbp3); heat shock 105kDa / l lOkDa protein l(Hsphl); heat shock protein 1 (chaperonin) (Hspdl); heat shock protein 90, alpha (cytosolic), class A member 1 (Hsp90aal); hematopoietic SH2 domain containing (Hsh2d); hepatitis A virus cellular receptor 2 (Havcr2); hes family bHLH transcription factor 1 (Hesl); histocompatibility 2, class II antigen A, alpha (H2-Aa); histocompatibility 2, class II antigen A, beta 1 (H2-Abl); histocompatibility 2, class II, locus DMa (H2-DMa); histocompatibility 2, M region locus 3(H3-M3); histocompatibility 2, O region alpha locus (H2-Oa); histocompatibility 2, T region locus 23 (H2-T23); HLA-DR; homeostatic iron regulator (Hfe); icos ligand (Icosl); IKAROS family zinc finger 1 (Ikzfl); IL10; IL10RA; IL2 inducible T cell kinase (Itk); IL6R; Indian hedgehog (Ihh); indoleamine 2,3-dioxygenase 1 (Idol); inducible T cell co-stimulator (Icos); inositol 1,4, 5 -trisphosphate 3-kinase B (Itpkb); insulin II (Ins2); insulin-like growth factor 1 (Igf 1 ); insulin-like growth factor 2 (Igf2); insulin- like growth factor binding protein 2 (Igfbp2); integrin alpha L (Itgal); integrin alpha M (Itgam); integrin alpha V (Itgav); integrin alpha X (Itgax); integrin beta 2 (Itgb2); integrin, alpha D (Itgad); intercellular adhesion molecule 1 (Icaml); interferon (alpha and beta) receptor l(Ifnarl); interferon alpha 1 (Ifinal); interferon alpha 11 (Ifnal l); interferon alpha 12 (Ifnal2); interferon alpha 13 (Ifnal3); interferon alpha 14 (Ifnal4); interferon alpha 16 (Ifnal6); interferon alpha 2 (Ifna2); interferon alpha 4 (Ifna4); interferon alpha 5 (Ifna5); interferon alpha 6 (Ifna6); interferon alpha 7 (Ifna7); interferon alpha 9 (Ifna9); interferon alpha B (Ifnab); interferon beta 1 (Ifnbl); interferon gamma (IFNg); interferon kappa (Ifnk); interferon regulatory factor 1 (Irfl); interferon regulatory factor 4 (Irf4); interferon zeta (Ifnz); interleukin 1 beta (Il lb; interleukin 1 family, member 8 (Il lf8); interleukin 1 receptor-like 2 (Il lrl2); interleukin 12 receptor, betal (I112rbl); interleukin 12a (1112a); interleukin 12b (1112b); interleukin 15 (1115); interleukin 18 (1118); interleukin 18 receptor 1 (Il 18rl); interleukin 2 (112); interleukin 2 receptor, alpha chain (I12ra); interleukin 2 receptor, gamma chain (I12rg); interleukin 20 receptor beta (I120rb); interleukin 21 (1121); interleukin 23, alpha subunit pl9 (1123a); interleukin 27 (1127); interleukin 4 (114); interleukin 4 receptor, alpha (I14ra); interleukin 6 (116); interleukin 7 (117); IRF8; itchy, E3 ubiquitin protein ligase (Itch); jagged 2 (Jag2); jumonji domain containing 6 (Jmjd6); JUNB; junction adhesion molecule like 9 (Jam9); K(lysine) acetyltransferase 2A (Kat2a); KDEL (Lys- Asp-Glu-Leu) endoplasmic reticulum protein retention receptor 1 (Kdelrl); KIT proto-oncogene receptor tyrosine kinase (Kit); LAG-3; LAIR-1 (CD305); LDHA; lectin, galactose binding, soluble 1 (Lgalsl); lectin, galactose binding, soluble 3 (Lgals3); lectin, galactose binding, soluble 8 (Lgals8); lectin, galactose binding, soluble 9 (Lgals9); leptin (Lep); leptin receptor (Lepr); leucine rich repeat containing 32 (Lrrc32); leukocyte immunoglobulin-like receptor, subfamily B, member 4A (Lilrb4a); LFNG O-fucosylpeptide 3-beta-N- acetylglucosaminyltransf erase (Lfing); LIF; ligase IV, DNA, ATP-dependent (Lig4); LIM domain only 1 (Lmol); limb region 1 like (Lmbrl); linker for activation of T cells (Lat); lymphocyte antigen 9 (Ly9); lymphocyte cytosolic protein 1 (Lcpl); lymphocyte protein tyrosine kinase (Lek); lymphocyte transmembrane adaptor 1 (Laxl); lymphocyte-activation gene 3 (Lag3); lymphoid enhancer binding factor 1 (Left); LYN; lysyl oxidase-like 3 (Loxl3); MAD1 mitotic arrest deficient 1 -like 1 (Madill); MA...

Claims

CLAIMSWhat is claimed:

1. A chimeric C-type lectin domain family 2, member D (CLEC2d) polypeptide comprising in order from N-terminus to C-terminus a CLEC2d extracellular domain and a transmembrane domain.

2. The polypeptide of claim 1, wherein the CLEC2d extracellular domain comprises an amino acid sequence with at least about 85% sequence identity to the following sequence and is capable of binding a CD161 polypeptide:RANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRFCDSQDADLAQVESFQELNFLL RYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYLNDKGASSARHYTERKWICSKS DIHV (SEQ ID NO: 461).

3. The polypeptide of claim 1, wherein the transmembrane domain comprises a sequence with at least about 85% sequence identity to a sequence selected from the group consisting of: FFLIMFLTI IVCGMVAALSAI (SEQ ID NO: 463; CLEC2d transmembrane domain);MALIVLGGVAGLLLFIGLGI FF (SEQ ID NO: 470; CD4 transmembrane domain);IYIWAPLAGTCGVLLLSLVITLY (SEQ ID NO: 492; CD8a transmembrane domain);PFFFCCFIAVAMGIRFI IMVT (SEQ ID NO: 466; NKG2D transmembrane domain); and PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO: 474; decay accelerating factor transmembrane domain).

4. The polypeptide of claim 1, further comprising a hinge domain disposed between the CLEC2d extracellular domain and the transmembrane domain.

5. The polypeptide of claim 4, wherein the hinge domain is between about 10 and 75 amino acids in length.

6. The polypeptide of claim 5, wherein the hinge domain is between about 35 and 55 amino acids in length.

7. The polypeptide of claim 5 or claim 6, wherein the hinge domain comprises an amino acid sequence comprising from about 20% to about 30% proline and / or cytosine amino acid residues.

8. The polypeptide of claim 7, wherein the hinge domain comprises an amino acid sequence with at least about 85% amino acid sequence identity to the following amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 468; CD8a hinge domain).

9. The polypeptide of claim 1, wherein the polypeptide shows increased levels of surface expression in a T cell relative to a wild-type CLEC2d polypeptide.

10. The polypeptide of claim 9, wherein the increase is at least about 1.1-fold.

11. The polypeptide of claim 10, wherein the increase is at least about 1.5-fold.

12. The polypeptide of claim 1 further comprising at the C-terminus a truncated cluster of differentiation zeta (CD3z) cytoplasmic domain.

13. The polypeptide of claim 12, wherein the CD3z cytoplasmic domain comprises the amino acid sequence RVKFSRSA (SEQ ID NO: 464).

14. The polypeptide of claim 1, further comprising a signal peptide at the N-terminus.

15. The polypeptide of claim 14, wherein the signal peptide comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence MALPVTALLLJPLJALJLJLJHAARP (SEQ ID NO: 469; CD8a signal peptide).

16. The polypeptide of claim 1, further comprising a protein tag.

17. The polypeptide of claim 16, wherein the tag is disposed between the CLEC2d extracellular domain and the transmembrane domain.

18. The polypeptide of claim 16, wherein the protein tag comprises an amino acid sequence with at least 85% amino acid sequence identity to the amino acid sequence ELPTQGTFSNVSTNVS (SEQ ID NO: 472; CD34 Qbend / 10 epitope).

19. A chimeric C-type lectin domain family 2, member D (chCLEC2d) polypeptide comprising a CLEC2d extracellular domain and a signal peptide.

20. The chCLEC2d polypeptide of claim 19, wherein the signal peptide is fused to the N- terminus of the CLEC2d extracellular domain.

21. The chCLEC2d polypeptide of claim 19 or claim 20, wherein the chCLEC2d polypeptide does not comprise a transmembrane domain.

22. The chCLEC2d polypeptide of claim 19, wherein the chCLEC2d polypeptide is secreted by a cell expressing the polypeptide.

23. The polypeptide of claim 19, wherein the CLEC2d extracellular domain comprises an amino acid sequence with at least about 85% sequence identity to the following sequence and is capable of binding a CD161 polypeptide:RANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRFCDSQDADLAQVESFQELNFLL RYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYLNDKGASSARHYTERKWICSKS DIHV (SEQ ID NO: 461).

24. The polypeptide of claim 19, wherein the signal peptide comprises an amino acid sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of MALPVTALLLPLALLLHAARP (SEQ ID NO: 469; CD8a signal peptide), MYRMQLLSCIALSLALVTNS (SEQ ID NO: 3331; IL-2 signal peptide), and MKYTSYILAFQLCIVLGSLGCYC (SEQ ID NO: 3332; IFN-gamma signal peptide).

25. A polynucleotide encoding the polypeptide of any one of claims 1-24.

26. The polynucleotide of claim 25, wherein the polynucleotide comprises a nucleotide sequence with at least 90% identity to a sequence selected from the group consisting of:ATGGCCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGGCCCC GGGCCAATTGTCATCAGGAACCAAGTGTGTGCCTTCAGGCCGCGTGTCCTGAATCATGGATAGG CTTTCAACGGAAGTGTTTTTATTTTTCCGACGACACTAAAAACTGGACTAGTAGTCAACGGTTT TGCGATTCTCAAGATGCGGATCTTGCCCAAGTGGAGTCCTTCCAAGAGTTGAATTTTCTTCTTC GATATAAAGGTCCTTCAGACCATTGGATAGGGTTGTCCAGGGAACAGGGACAACCGTGGAAGTG GATAAACGGCACAGAATGGACCCGGCAGTTCCCTATATTGGGCGCGGGCGAGTGCGCTTACCTC AATGACAAAGGCGCTTCATCTGCCAGACATTACACGGAACGAAAGTGGATCTGTAGTAAGTCCG ATATTCACGTC (SEQ ID NO: 3328; BTx_LC001);ATGTACCGGATGCAGCTCCTGTCTTGTATAGCACTTTCTCTTGCGTTGGTAACTAATAGCCGGG CCAATTGTCATCAGGAACCAAGTGTGTGCCTTCAGGCCGCGTGTCCTGAATCATGGATAGGCTT TCAACGGAAGTGTTTTTATTTTTCCGACGACACTAAAAACTGGACTAGTAGTCAACGGTTTTGC GATTCTCAAGATGCGGATCTTGCCCAAGTGGAGTCCTTCCAAGAGTTGAATTTTCTTCTTCGAT ATAAAGGTCCTTCAGACCATTGGATAGGGTTGTCCAGGGAACAGGGACAACCGTGGAAGTGGAT AAACGGCACAGAATGGACCCGGCAGTTCCCTATATTGGGCGCGGGCGAGTGCGCTTACCTCAAT GACAAAGGCGCTTCATCTGCCAGACATTACACGGAACGAAAGTGGATCTGTAGTAAGTCCGATA TTCACGTC (SEQ ID NO: 3329; BTx_LC002); andATGAAATACACGTCCTACATCCTTGCGTTTCAACTTTGTATTGTACTGGGTTCACTGGGCTGCT ACTGTCGGGCCAATTGTCATCAGGAACCAAGTGTGTGCCTTCAGGCCGCGTGTCCTGAATCATG GATAGGCTTTCAACGGAAGTGTTTTTATTTTTCCGACGACACTAAAAACTGGACTAGTAGTCAA CGGTTTTGCGATTCTCAAGATGCGGATCTTGCCCAAGTGGAGTCCTTCCAAGAGTTGAATTTTC TTCTTCGATATAAAGGTCCTTCAGACCATTGGATAGGGTTGTCCAGGGAACAGGGACAACCGTG GAAGTGGATAAACGGCACAGAATGGACCCGGCAGTTCCCTATATTGGGCGCGGGCGAGTGCGCT TACCTCAATGACAAAGGCGCTTCATCTGCCAGACATTACACGGAACGAAAGTGGATCTGTAGTA AGTCCGATATTCACGTC (SEQ ID NO: 3330; BTx_LC003).

27. A vector comprising the polynucleotide of claim 25.

28. The chCLEC2d polypeptide of claim 19, wherein the polypeptide comprises an amino acid sequence having at least 85% sequence identity to a sequence selected from the group consisting of:BTx_LC003 (sCLEC2d)MKYTSYILAFQLCIVLGSLGCYCRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQ RFCDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECA YLNDKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3327);BTx LCOOl:MALPVTALLLPLALLLHAARPRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRFCDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYLNDKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3325); andBTx_LC002MYRMQLLSCIALSLALVTNSRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRFCDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYLN DKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3326).

29. A cell comprising the polynucleotide of claim 25 or claim 26, or the vector of claim 27, or expressing the chCLEC2d polypeptide of claim 28.

30. The cell of claim 29, wherein the cell is a T cell.

31. The cell of claim 30, wherein the T cell expresses a chimeric antigen receptor (CAR) polypeptide.

32. The cell of claim 30 or claim 31, wherein the T cell has been modified to reduce or eliminate expression of beta-2-microglobulin.

33. The cell of claim 30 or claim 31, wherein the T cell has been modified to reduce or eliminate expression of CD161.

34. The cell of claim 30 or claim 31, wherein the T cell has been modified to reduce or eliminate expression of cluster of differentiation 54 (CD54).

35. The cell of claim 30 or claim 31, wherein the T cell has been modified to reduce or eliminate expression of cluster of differentiation 58 (CD58).

36. The cell of claim 30 or claim 31, wherein the T cell has been modified to reduce or eliminate expression of a functional T cell receptor complex.

37. The cell of claim 36, wherein the T cell has been modified to reduce or eliminate expression of CD3E.

38. The cell of claim 30 or claim 31, wherein the T cell has been modified using a base editor system to reduce or eliminate expression of beta-2-microglobulin, CD54, CD58, CD161, and / or Class II Major Histocompatibility Complex Transactivator (CIITA).

39. The cell of claim 30 or claim 31, wherein the T cell has been modified using a base editor system to reduce or eliminate expression of beta-2-microglobulin, CIITA, a functional T cell receptor complex, and CD58.

40. The cell of claim 39, wherein the T cell has been modified to reduce or eliminate expression of CD3E.

41. The cell of claim 30 or claim 31, wherein the T cell has been modified using a base editor system to reduce or eliminate expression of beta-2-microglobulin and to reduce expression of one or more of CIITA, CD3E, CD54, CD58, and CD161.

42. The cell of claim 30 or claim 31, wherein the T cell has been modified using a base editor system to reduce or eliminate expression of CD54 and / or CD58.

43. The cell of claim 30 or claim 31, wherein the T cell has increased persistence compared to a T cell that does not comprise the polynucleotide of claim 25 or claim 26, or the vector of claim 27, that does not express the chCLEC2d polypeptide of claim 28, and that has been modified to reduce or eliminate expression of beta-2-microglobulin.

44. The cell of claim 30 or claim 31, wherein the T cell shows a reduction in specific lysis by NK cells relative to a reference T cell that does not comprise the polynucleotide of claim 25 or claim 26, or the vector of claim 27, and that does not express the chCLEC2d polypeptide of claim 28.

45. A method for modifying a T cell to increase resistance of the T cell to natural killer cell killing, the method comprising expressing in the T cell the polypeptide of any one of claims 1- 34, wherein the T cell expresses reduced levels of CD54, CD58, human leukocyte antigen A, human leukocyte antigen B, and / or human leukocyte antigen C relative to a wild-type T cell.

46. The method of claim 45, wherein the method comprises contacting the T cell with a base editor system comprising a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and an adenosine deaminase domain, or one or more polynucleotides encoding the base editor, and one or more guide polynucleotides, or one or more polynucleotide encoding the guide polynucleotides, wherein the one or more guide polynucleotides target the base editor to alter a nucleotide within a polynucleotide encoding a CD161 polypeptide, to alter a nucleotide within a polynucleotide encoding a beta-2- microglobulin (B2M) polypeptide, to alter a nucleotide within a polynucleotide encoding a Class II Major Histocompatibility Complex Transactivator (CIITA) polypeptide, to alter a polynucleotide encoding a cluster of differentiation 54 (CD54) polypeptide, and / or to alter a polynucleotide encoding a cluster of differentiation 58 (CD58) polypeptide.

47. The method of claim 46, wherein the deaminase domain comprises a TadA*8 adenosine deaminase domain.

48. The method of claim 47, wherein the deaminase domain comprises a TadA*8.20 adenosine deaminase domain.

49. The method of claim 48, wherein the base editor is an ABE8.20 base editor.

50. The method of claim 46, wherein the napDNAbp domain is a Cas9 domain.

51. The method of claim 46, wherein the one or more guide polynucleotides comprise a spacer comprising a nucleotide sequence comprising at least 10 contiguous nucleotides of a nucleotide sequence selected from the group consisting of: UUACCCCGAGGAAGAGAUGA (SEQ ID NO: 426; IMM_169), UAACUUUUCAGAUGUCUGUC (SEQ ID NO: 427; IMM_170), AACUUUUCAGAUGUCUGUCA (SEQ ID NO: 428; IMM_171), UUUUUUACUUUAG G G CC (SEQ ID NO: 429: IMM_172), CUCCACAGCUUAGAAAUUAG (SEQ ID NO: 430; IMM_173), and CCUCACCAAAGGUCUGGAGC (SEQ ID NO: 3338; IMM_163).

52. The method of claim 46, wherein the modified T cell has reduced levels of B2M, CD54, CD58, CD161, and / or CIITA expression relative to a wild-type T cell.

53. The method of claim 46, wherein the T cell shows a reduction in specific lysis by NK cells of at least 10% relative to T cells with reduced levels of human leukocyte antigen A, human leukocyte antigen B, and / or human leukocyte antigen C relative to a wild-type T cell that do not express the polypeptide of any one of claims 1-18.

54. The method of claim 45 further comprising expressing a chimeric antigen receptor (CAR) polypeptide in the T cell.

55. The method of claim 45, wherein the T cells surface-express increased levels of the CLEC2d polypeptide relative to a T cell expressing a wild-type CLEC2d polypeptide.

56. The method of claim 55, wherein the T cells surface-express increased levels of the CLEC2d polypeptide at least 5 days after first expressing the polypeptide of any one of claims 1- 18.

57. A cell prepared by the method of any one of claims 45-56.

58. A pharmaceutical composition comprising the cell of any one of claims 29-44 or claim 57 and a pharmaceutically acceptable excipient.

59. A method for treating a neoplasia in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of claim 58, wherein the cell expresses a chimeric antigen receptor targeting a polypeptide associated with the neoplasia.

60. A polypeptide comprising in order from N-terminus to C-terminus: a CD8 signal peptide, a C-type lectin domain family 2, member D (CLEC2d) extracellular domain, a CD8 hinge domain, a CD8 transmembrane domain, and a cytoplasmic domain, wherein the polypeptide is capable of being expressed on the surface of a T cell and of binding a CD161 polypeptide.

61. The polypeptide of claim 60, wherein the polypeptide comprises an amino acid sequence with at least 85% sequence identity to the following amino acid sequence: MALPVTALLLPLALLLHAARPRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRF CDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYLNDKGASSARHYTERKWICSKSDIHVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSA (SEQ ID NO: 440;BTx_CM496 / mbCLEC2d).

62. A T cell expressing the polypeptide of claim 60, wherein the T cell expresses a chimeric antigen receptor (CAR) and expresses reduced levels of beta-2-microglobulin, CD161, and / or CIITA relative to a wild-type T cell.

63. A T cell expressing the polypeptide of claim 60, wherein the T cell expresses a chimeric antigen receptor (CAR) and expresses reduced levels of beta-2-microglobulin, CD54, and / or CD58 relative to a wild-type T cell.

64. A method for preparing a modified T cell, the method comprising:A) contacting the T cell with a base editor system comprising an ABE8.20 base editor, or a polynucleotide encoding the base editor, and two or more guide polynucleotides, wherein the guide polynucleotides each target the base editor to alter a nucleotide within a polynucleotide encoding a polypeptide selected from the group consisting of a CD54, CD58, CD161 polypeptide, a beta-2-microglobulin (B2M) polypeptide, and a Class II Major Histocompatibility Complex Transactivator (CIITA) polypeptide, thereby reducing or eliminating expression of the CD54, the CD58, the CD161 polypeptide, the B2M polypeptide, and / or the CIITA polypeptide in the T cell; andB) expressing in the T cell a polypeptide comprising an amino acid sequence with at least 85% sequence identity to the following amino acid sequence: MALPVTALLLPLALLLHAARPRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRF CDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYL NDKGASSARHYTERKWICSKSDIHVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSA (SEQ ID NO: 440; BTx_CM496 / mbCLEC2d), thereby increasing resistance of the T cell to being killed by a natural killer cell in a mixed leukocyte reaction.

65. A method for preparing a modified T cell, the method comprising:A) contacting the T cell with a base editor system comprising an ABE8.20 base editor, or a polynucleotide encoding the base editor, and a guide polynucleotide, wherein the guide polynucleotide targets the base editor to alter a nucleotide within a polynucleotide encoding abeta-2-microglobulin (B2M) polypeptide, thereby reducing or eliminating expression of the B2M polypeptide in the T cell; andB) expressing in the T cell a polypeptide comprising an amino acid sequence with at least 85% sequence identity to the following amino acid sequence: MALPVTALLLPLALLLHAARPRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRF CDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYL NDKGASSARHYTERKWICSKSDIHVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSA (SEQ ID NO: 440;BTx_CM496 / mbCLEC2d), thereby increasing resistance of the T cell to being killed by a natural killer cell in a mixed leukocyte reaction.

66. A method for preparing a modified T cell, the method comprising:A) contacting the T cell with a base editor system comprising an ABE8.20 base editor, or a polynucleotide encoding the base editor, and a guide polynucleotide, wherein the guide polynucleotide targets the base editor to alter a nucleotide within a polynucleotide encoding a beta-2-microglobulin (B2M) polypeptide, thereby reducing or eliminating expression of the B2M polypeptide in the T cell; andB) expressing in the T cell a polypeptide comprising an amino acid sequence with at least 85% sequence identity to an amino acid sequence selected from the group consisting of: MALPVTALLLPLALLLHAARPRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRF CDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYL NDKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3325; BTx_LC001);MYRMQLLSCIALSLALVTNSRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRFC DSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYLN DKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3326; BTx_LC002); and MKYTSYILAFQLCIVLGSLGCYCRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQ RFCDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECA YLNDKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3327; BTx_LC003); thereby increasing resistance of the T cell to being killed by a natural killer cell in a mixed leukocyte reaction.

67. A method for preparing a modified T cell, the method comprising:A) contacting the T cell with a base editor system comprising an ABE8.20 base editor, or a polynucleotide encoding the base editor, and two guide polynucleotides, wherein one guidepolynucleotide targets the base editor to alter a nucleotide within a polynucleotide encoding a beta-2-microglobulin (B2M) polypeptide, thereby reducing or eliminating expression of the B2M polypeptide in the T cell, and wherein another guide polynucleotide targets the base editor to alter a nucleotide within a polynucleotide encoding a Major Histocompatibility Complex Transactivator (CIITA) polypeptide, thereby reducing or eliminating expression of the CIITA polypeptide in the T cell; andB) expressing in the T cell a polypeptide comprising an amino acid sequence with at least 85% sequence identity to an amino acid sequence selected from the group consisting of: MALPVTALLLPLALLLHAARPRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRF CDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYL NDKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3325; BTx_LC001); MYRMQLLSCIALSLALVTNSRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRFC DSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYLN DKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3326; BTx_LC002); and MKYTSYILAFQLCIVLGSLGCYCRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQ RFCDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECA YLNDKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3327; BTx_LC003 / sCLEC2d); thereby increasing resistance of the T cell to being killed by a natural killer cell in a mixed leukocyte reaction.

68. A method for preparing a modified T cell, the method comprising:A) contacting the T cell with a base editor system comprising an ABE8.20 base editor, or a polynucleotide encoding the base editor, and at least two guide polynucleotides, wherein one guide polynucleotide targets the base editor to alter a nucleotide within a polynucleotide encoding a beta-2-microglobulin (B2M) polypeptide, thereby reducing or eliminating expression of the B2M polypeptide in the T cell, and wherein another guide polynucleotide targets the base editor to i) alter a nucleotide within a polynucleotide encoding a CD54 polypeptide, thereby reducing or eliminating expression of the CD54 polypeptide in the T cell, or ii) alter a nucleotide within a polynucleotide encoding a CD58 polypeptide, thereby reducing or eliminating expression of the CD58 polypeptide in the T cell; andB) expressing in the T cell a polypeptide comprising an amino acid sequence with at least 85% sequence identity to an amino acid sequence selected from the group consisting of:MALPVTALLLPLALLLHAARPRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRF CDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYL NDKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3325; BTx_LC001);MYRMQLLSCIALSLALVTNSRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRFC DSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYLN DKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3326; BTx_LC002); and MKYTSYILAFQLCIVLGSLGCYCRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQ RFCDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECA YLNDKGASSARHYTERKWICSKSDIHV (SEQ ID NO: 3327; BTx_LC003 / sCLEC2d); thereby increasing resistance of the T cell to being killed by a natural killer cell in a mixed leukocyte reaction.

69. A method for preparing a modified T cell, the method comprising:A) contacting the T cell with a base editor system comprising an ABE8.20 base editor, or a polynucleotide encoding the base editor, and at least two guide polynucleotides, wherein one guide polynucleotide targets the base editor to alter a nucleotide within a polynucleotide encoding a beta-2-microglobulin (B2M) polypeptide, thereby reducing or eliminating expression of the B2M polypeptide, and wherein another guide polynucleotide targets the base editor to i) alter a polynucleotide encoding a cluster of differentiation 58 (CD58) polypeptide, thereby reducing or eliminating expression of the CD58 polypeptide, or ii) alter a polynucleotide encoding a cluster of differentiation 54 (CD54) polypeptide, thereby reducing or eliminating expression of the CD54 polypeptide; andB) expressing in the T cell a polypeptide comprising an amino acid sequence with at least 85% sequence identity to the following amino acid sequence: MALPVTALLLPLALLLHAARPRANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRF CDSQDADLAQVESFQELNFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYL NDKGASSARHYTERKWICSKSDIHVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSA (SEQ ID NO: 440; BTx_CM496 / mbCLEC2d), thereby increasing resistance of the T cell to being killed by a natural killer cell in a mixed leukocyte reaction.

70. A kit suitable for use in any of the above claims, the kit comprising the polypeptide, polynucleotide, cell, and / or pharmaceutical composition of any of the above claims.

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