Compositions and methods for treating liquid cancer

Abstract

The present invention is a composition and method for treating liquid cancer, as described below, the present invention proposes genetically modified immune cells having enhanced anti-tumorigenic activity, anti-immunosuppressive ability, and reduced risk of causing graft versus host response, or a composition thereof. The invention also proposes methods for producing and using these modified immune effector cells.

Description

[0001] This application is a continuation of PCT Patent Application No. PCT / US2021 / 053593, filed September 25, 2020, which claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 907,254, filed September 27, 2019, the entire contents of which are incorporated herein by reference in their entirety.

[0002] Cross Reference to Related Applications

[0003] This application is an international PCT application which claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 907,254, filed September 27, 2019, the entire contents of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0004] The present application relates to genetically modified immune cells having enhanced anti-tumorigenic activity, anti-immunosuppressive properties, reduced risk of causing graft versus host reactions, or combinations thereof. The present application also relates to methods of producing and using the genetically modified immune cells. BACKGROUND

[0005] Autologous and allogeneic immunotherapy is a method of tumor treatment in which immune cells expressing a chimeric antigen receptor are administered to a subject. To generate immune cells expressing a chimeric antigen receptor (CAR), immune cells are first collected from a subject (autologous) or a donor separate from the subject receiving treatment (allogeneic) and are genetically modified to express a chimeric antigen receptor. The resulting cells express a chimeric antigen receptor on their cell surface (e.g., CAR T cells), and when administered to a subject, the chimeric antigen receptor binds to a marker expressed by a tumor cell. This interaction with the tumor marker activates the CAR-T cells, which subsequently kill the tumor cells. But for autologous or allogeneic cell therapy to be effective or efficient, significant conditional and cellular responses such as T cell signaling inhibition must be reduced or avoided. For allogeneic cell therapy, graft versus host disease and host rejection of CAR-T cells can pose additional challenges. Editing genes involved in these processes can enhance CAR-T cell function and resistance to immune blockade or inhibition, and current methods for making such edits have the potential to induce large genomic rearrangements in CAR-T cells, thereby negatively impacting their efficacy. Thus, there is a significant need for technologies that more precisely modify immune cells, particularly CAR-T cells. The present application addresses this and other important needs. SUMMARY

[0006] As described below, the present application features genetically modified immune cells having enhanced anti-tumorigenic activity, resistance to immune suppression, and reduced risk of causing a graft versus host reaction or a host versus graft reaction, wherein host CD8+ T cells recognize the graft as non-self (e.g., wherein the graft recipient mounts an immune response against the transplanted organ), or a combination thereof. In one embodiment, CAR-T cells that lack or have reduced levels of functional TRAC are administered to a subject having or predisposed to developing graft versus host disease (GVHD). In one embodiment, CAR-T cells that lack or have reduced levels of beta 2 microglobulin (B2M) are administered to a subject having or predisposed to developing host versus graft disease (HVGD). The present application also features methods for producing and using these modified immune cells.

[0007] In one aspect, the application provides a composition comprising two or more immune cells, each immune cell comprising a) a different chimeric antigen receptor targeting an antigen selected from the group consisting of CD5, CD7, CD3, CD33, and CD123, wherein the immune cell comprises a mutation that reduces or eliminates expression of the targeted antigen; and b) one or more mutations that reduce or eliminate expression of an immunogenic polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In some embodiments, one of the immune cells comprises a chimeric antigen receptor targeting CD5, and the other immune cell comprises a chimeric antigen receptor targeting an antigen selected from the group consisting of CD7, CD3, CD33, and CD123. In some embodiments, the composition comprises at least three immune cells, each comprising a chimeric antigen receptor targeting a different antigen, wherein the targeted antigens are CD3, CD5, and CD7. In some embodiments, one of the immune cells expresses a chimeric antigen receptor targeting CD7, and the other immune cell expresses a chimeric antigen receptor targeting an antigen selected from the group consisting of CD3, CD33, and CD123. In some embodiments, one of the immune cells expresses a chimeric antigen receptor targeting CD3, and the other immune cell expresses a chimeric antigen receptor targeting an antigen selected from the group consisting of CD33 and CD123. In some embodiments, one of the immune cells expresses a chimeric antigen receptor targeting CD33, and the other immune cell expresses a chimeric antigen receptor targeting CD123. In some embodiments, one immune cell expresses two, three, four, or more different chimeric antigen receptors. In some embodiments, one of the chimeric antigen receptors targets CD5, and the other chimeric antigen receptor targets an antigen selected from the group consisting of CD7, CD3, CD33, and CD123. In some embodiments, one of the chimeric antigen receptors targets CD7, and the other chimeric antigen receptor targets an antigen selected from the group consisting of CD3, CD33, and CD123. In some embodiments, one of the chimeric antigen receptors targets CD3, and the other chimeric antigen receptor targets an antigen selected from the group consisting of CD33 and CD123. In some embodiments, one of the chimeric antigen receptors targets CD33, and the other chimeric antigen receptor targets CD123.

[0008] In another aspect, the application provides a composition comprising at least three immune cells, each comprising a different chimeric antigen receptor, wherein one chimeric antigen receptor targets CD3, one targets CD5, and a third targets CD7, wherein the cells further comprise a mutation that reduces or eliminates expression of the targeted antigen; and further comprise one or more mutations that reduce or eliminate expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0009] In another aspect, the application provides a composition comprising at least two immune cells, each comprising a different chimeric antigen receptor, wherein one chimeric antigen receptor targets CD3, and the other targets CD7, wherein the cells further comprise a mutation that reduces or eliminates expression of the targeted antigen; and further comprise one or more mutations that reduce or eliminate expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0010] In another aspect, the application provides a composition comprising at least two immune cells, each comprising a different chimeric antigen receptor, wherein one chimeric antigen receptor targets CD3, and the other targets CD7, wherein the cells further comprise a mutation that reduces or eliminates expression of the targeted antigen; and further comprise one or more mutations that reduce or eliminate expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0011] In another aspect, the application provides a composition comprising at least two immune cells, each comprising a different chimeric antigen receptor, wherein one chimeric antigen receptor targets CD3, and the other targets CD7, wherein the cells further comprise a mutation that reduces or eliminates expression of the targeted antigen; and further comprise one or more mutations that reduce or eliminate expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0012] In yet another aspect, the application provides a composition comprising at least two immune cells, each comprising a different chimeric antigen receptor, wherein one chimeric antigen receptor targets CD33 and the other targets CD123, wherein the cells further comprise a mutation that reduces or eliminates expression of the targeted antigen; and further comprise one or more mutations that reduce or eliminate expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0013] In some embodiments, the mutation is a C to T or A to G mutation that silences the gene or introduces a stop codon into the gene. In some embodiments, the mutation introduces a premature stop codon or alters a splice donor or acceptor site. In some embodiments, the mutation is generated by a base editor comprising a deaminase domain. In some embodiments, the deaminase is an adenosine deaminase. In some embodiments, the base editor is BE4. In some embodiments, the mutation reduces expression of a polypeptide by about 50% or more relative to a corresponding control cell lacking the mutation. In some embodiments, the composition comprises a population of immune cells. In some embodiments, at least 50% of the population comprises one or more mutations that reduce or eliminate expression of the targeted antigen and / or the immunogenic polypeptide. In some embodiments, the immune cells are anti- fratricidal. In some embodiments, the immune cells have increased anti-tumorigenic activity. In some embodiments, the immune cells do not comprise detectable translocations. In some embodiments, the immune cells comprise less than 1% indels. In some embodiments, the immune cells are mammalian cells. In some embodiments, the immune cells are human or rodent cells. In some embodiments, the immune cells are cytotoxic T cells, regulatory T cells, T helper cells, dendritic cells, B cells, or NK cells, or progenitors thereof. In some embodiments, the progenitors are hematopoietic stem cells.

[0014] In one aspect, the application provides a pharmaceutical composition comprising an effective amount of any of the compositions provided herein and a pharmaceutically acceptable excipient.

[0015] In another aspect, the application provides a base editor system comprising a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a deaminase domain, and at least two guide polynucleotides, each targeting a different antigen, wherein the antigen is selected from the group consisting of CD5, CD7, CD3, CD33, and CD123. In some embodiments, one guide polynucleotide targets CD5, and the other targets a member selected from the group consisting of CD7, CD3, CD33, and CD123. In some embodiments, the system comprises three guide polynucleotides, each targeting one of the antigens CD3, CD5, and CD7. In some embodiments, one guide polynucleotide targets CD7, and the other targets a member selected from the group consisting of CD3, CD33, and CD123. In some embodiments, one guide polynucleotide targets CD3, and the other targets a member selected from the group consisting of CD33 and CD123. In some embodiments, one guide polynucleotide targets CD33, and the other targets CD123.

[0016] In some embodiments, each of the guide polynucleotides comprises a nucleic acid sequence selected from Table 26. In some embodiments, the guide polynucleotides comprise a nucleic acid sequence selected from AGCGACUGCAGAAAGAAGAG or CAUACCAGCUGAGCCGUCCG. In some embodiments, the fusion protein further comprises one or more uracil glycosylase inhibitors (UGIs) and / or one or more nuclear localization sequences (NLSs). In some embodiments, the napDNAbp comprises a Cas9, Casl2a / Cpf l, Casl2b / C2cl, Casl2c / C2c3, Casl2d / CasY, Casl2e / CasX, Casl2g, Casl2h, Casl2i, or Casl2j / CasF polypeptide or a portion thereof. In some embodiments, the napDNAbp comprises a Casl2 polypeptide or a fragment thereof. In some embodiments, the napDNAbp comprises a Cas9 polypeptide or a fragment thereof. In some embodiments, the Cas9 is a dead Cas9 (dCas9) or a Cas9 nickase (nCas9). In some embodiments, the Cas9 is a modified Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (StlCas9), or a modified Streptococcus pyogenes Cas9 (SpCas9). In some embodiments, the Cas9 comprises an altered protospacer adjacent motif (PAM) specificity. In some embodiments, the altered PAM has specificity for the nucleic acid sequence 5'-NGC-3'.

[0017] In some embodiments, the deaminase domain is capable of deaminating cytidine or adenosine. In some embodiments, the deaminase domain is an adenosine deaminase domain. In some embodiments, the cytidine deaminase is an APOBEC deaminase. In some embodiments, the adenosine deaminase is a TadA variant. In some embodiments, the TadA variant is a TadA*8 variant. In some embodiments, the TadA*8 variant is TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, or TadA*8.24. In some embodiments, the TadA variant is a TadA*9 variant.

[0018] In one aspect, the present application provides a pharmaceutical composition comprising any of the base editor systems provided herein.

[0019] In another aspect, the present application provides a polynucleotide encoding any of the base editor systems and the guide polynucleotides provided herein.

[0020] In yet another aspect, the present application provides a vector comprising any of the polynucleotides provided herein. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector, an adenoviral vector, a lentiviral vector, a herpes viral vector, or an adeno-associated viral vector (AAV).

[0021] In one aspect, the present application provides a pharmaceutical composition comprising any of the polynucleotides or any of the vectors provided herein.

[0022] In one aspect, the application provides a method for producing a CAR-expressing immune cell having reduced immunogenicity. In some embodiments, the method comprises expressing in a CAR-expressing immune cell a base editor system comprising a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a deaminase domain and two guide polynucleotides each targeting a polynucleotide encoding a different antigen, wherein the antigen is selected from the group consisting of CD5, CD7, CD3, CD33, and CD123, thereby producing a CAR-expressing immune cell having reduced immunogenicity. In some embodiments, the immune cell expresses or is contacted with a guide polynucleotide targeting a polynucleotide encoding a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0023] In some embodiments, the method comprises (a) expressing in a CAR-expressing immune cell a base editor system comprising a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a deaminase domain and (b) contacting the CAR-expressing immune cell with at least two guide polynucleotides each targeting a polynucleotide encoding a different antigen, wherein the antigen is selected from the group consisting of CD5, CD7, CD3, CD33, and CD123, thereby producing a CAR-expressing immune cell having reduced immunogenicity.

[0024] In some embodiments, the method further comprises contacting the CAR-expressing immune cell with a guide polynucleotide targeting a polynucleotide encoding a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In some embodiments, the immune cell expresses a chimeric antigen receptor (CAR) targeting an antigen selected from the group consisting of CD5, CD7, CD3, CD33, and CD123. In some embodiments, the method further comprises introducing a mutation into the immune cell that reduces or eliminates expression of at least one polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1, thereby producing a population of CAR-expressing immune cells having reduced immunogenicity.

[0025] In one aspect, the present application provides a method for producing a population of CAR-expressing immune cells having reduced immunogenicity. In some embodiments, the method comprises a) introducing a mutation into an immune cell that reduces or eliminates expression of an antigen selected from the group consisting of CD5, CD7, CD3, CD33, and CD123 and introducing a different mutation into a second immune cell in one of the antigens; and b) introducing a mutation into the immune cell that reduces or eliminates expression of at least one polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1, thereby producing a population of CAR-expressing immune cells having reduced immunogenicity. In some embodiments, the chimeric antigen receptor expressed by the immune cell targets an antigen selected from the group consisting of CD3, CD5, CD7, CD33, and CD123. In some embodiments, the immune cell produced by the method expresses a chimeric antigen receptor that targets a CD3, CD5, and / or CD7 antigen, but fails to express or expresses a reduced level of the CD3, CD5, and / or CD7 antigen. In some embodiments, the immune cell produced by the method expresses a chimeric antigen receptor that targets a CD33 and CD123 antigen, but fails to express or expresses a reduced level of the CD33 and CD123 antigen. In some embodiments, the CAR is a CD5 chimeric antigen receptor (CAR). In some embodiments, the CD5 CAR is encoded by a CD5 CAR construct presented in Table 28.

[0026] In another aspect, the present application provides a method for producing an immune cell having reduced immunogenicity. In some embodiments, the method comprises a) introducing a mutation into an endogenous CD5 gene sequence or regulatory element that reduces or eliminates expression of CD5; and b) expressing a CD5 CAR construct presented in Table 28 in the cell. In some embodiments, the CD5 CAR construct encodes a CD5 CAR polypeptide comprising or consisting of an amino acid sequence selected from:

[0027] a)

[0028]

[0029]

[0030] b)

[0031]

[0032] c)

[0033]

[0034]

[0035] d)

[0036]

[0037] e)

[0038]

[0039]

[0040] In some embodiments, the immune cells produced by the method exhibit anti- fratricide and / or increased anti-tumorigenic activity compared to a corresponding control cell. In some embodiments, the method is performed in vivo or ex vivo. In some embodiments, the immune cells generated by the method do not comprise a detectable translocation. In some embodiments, the immune cells generated by the method comprise less than 1% indels. In some embodiments, the immune cells generated by the method comprise less than 5% non-target edits. In some embodiments, the immune cells generated by the method comprise less than 5% off-target edits. In some embodiments, the mutations are generated by nucleobase modification. In some embodiments, the mutations are in exons. In some embodiments, the mutations result in premature stop codons that reduce or eliminate protein expression. In some embodiments, the mutations are in splice donor sites or splice acceptor sites. In some embodiments, the one or more mutations are generated by contacting the target polynucleotide with a base editor system comprising a fusion protein having a nucleic acid programmable DNA binding protein (napDNAbp), a deaminase, and one or more guide polynucleotides.

[0041] In some embodiments, the deaminase is an adenosine or cytidine deaminase. In some embodiments, the cytidine deaminase is BE4. In some embodiments, the mutation reduces expression of the polypeptide by at least about 50% or more relative to a corresponding control cell lacking the mutation. In some embodiments, the guide polynucleotide comprises a sequence selected from those provided in Table 26. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence selected from AGCGACUGCAGAAAGAAGAG or CAUACCAGCUGAGCCGUCCG. In some embodiments, the one or more nucleic acid sequences each target the napDNAbp to a CD5, FAS, LAG-3, CD52, TRAC, B2M, CIITA, TRBC1, TRBC2, and / or PDC1 / PD-1 gene or regulatory element. In some embodiments, the base editor and one or more guide nucleic acid sequences are introduced into the immune cell by electroporation, nucleofection, cationic lipid-mediated methods, viral transduction, or a combination thereof. In some embodiments, the method further comprises expanding the immune cell in culture to generate a population of immune cells. In some embodiments, expression of the antigen or polypeptide is reduced in at least about 50% of the population of immune cells. In some embodiments, the method further comprises depleting TCRa / b+ cells from the population of modified immune cells.

[0042] In one aspect, the application provides an immune cell expressing a CAR having reduced immunogenicity produced by any of the methods provided herein.

[0043] In another aspect, the application provides a pharmaceutical composition comprising any of the immune cells provided herein. In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable excipient.

[0044] In yet another aspect, the application provides a method for killing a tumor cell. In some embodiments, the method comprises contacting a tumor cell expressing an antigen selected from the group consisting of CD5, CD7, CD3, CD33, and CD123 with two or more immune cells, each immune cell expressing a different chimeric antigen receptor targeting two of the antigens expressed by the cell, wherein the immune cells comprise one or more mutations that reduce or eliminate expression of the targeted antigen, and one or more mutations that reduce or eliminate expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In some embodiments, the method is performed in vitro or in vivo. In some embodiments, the tumor cell is derived from a neoplasia.

[0045] In one aspect, the present application provides a method for treating a neoplasia in a subject. In some embodiments, the method comprises administering to the subject two or more immune cells, each immune cell expressing a different chimeric antigen receptor targeting an antigen expressed by a tumor cell of the subject, the antigen selected from the group consisting of CD5, CD7, CD3, CD33, and CD123, wherein the immune cells comprise one or more mutations that reduce or eliminate expression of the antigen, and the immune cells each further comprise one or more mutations that reduce or eliminate expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0046] In some embodiments, the neoplasia is a hematological cancer. In some embodiments, the neoplasia is a liquid cancer. In some embodiments, the hematological cancer is a leukemia, myeloma, and / or lymphoma. In some embodiments, the hematological cancer is a B-cell cancer. In some embodiments, the hematological cancer is selected from at least one of T-cell acute lymphoblastic leukemia (T-ALL), mycosis fungoides (MF), Sézary syndrome (SS), peripheral T / NK cell lymphoma, anaplastic large cell lymphoma ALK + , primary cutaneous T-cell lymphoma, T-cell large granular lymphocyte-type 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 gd T-cell lymphoma, aggressive NK-cell leukemia, and enteropathy-associated T-cell lymphoma. In some embodiments, the hematological cancer is a T-cell acute lymphoblastic leukemia (T-ALL) cell. In some embodiments, the hematological cancer is a T-cell acute myeloid leukemia (AML).

[0047] In another aspect, the present application provides a method for treating a neoplasia in a selected subject. In some embodiments, the method comprises administering to the selected subject two or more immune cells, each expressing a different chimeric antigen receptor targeting an antigen expressed by a tumor cell of the subject, wherein the antigen is selected from the group consisting of CD5, CD7, CD3, CD33, and CD123, wherein the immune cells comprise one or more mutations that reduce or eliminate expression of the antigen, and the immune cells each further comprise one or more mutations that reduce or eliminate expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1, wherein the subject is selected as having a neoplasia expressing an antigen selected from the group consisting of CD5, CD7, CD3, CD33, and CD123. In some embodiments, the hematological cancer is a T-cell acute lymphoblastic leukemia (T-ALL) cell. In some embodiments, the hematological cancer is a T-cell acute myeloid leukemia (AML). In some embodiments, the two or more immune cells expressing different chimeric antigen receptors are administered sequentially. In some embodiments, the two or more immune cells expressing different chimeric antigen receptors are administered simultaneously.

[0048] In yet another aspect, the present application provides a method for antigen-dependent killing of a tumor cell in a subject. In some embodiments, the method comprises administering to a subject having a neoplasia expressing an antigen selected from the group consisting of CD5, CD7, CD3, CD33, and CD123, two or more immune cells, each expressing a different chimeric antigen receptor targeting one of the antigens, wherein the immune cells comprise one or more mutations that reduce or eliminate expression of the targeted antigen, and one or more mutations that reduce or eliminate expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0049] In one aspect, the present application provides a method for antigen-dependent killing of an acute myeloid leukemia (AML) cell in a subject. In some embodiments, the method comprises administering to a subject having an AML expressing CD33 and CD123 antigens, two or more immune cells, each expressing a different chimeric antigen receptor targeting one of the antigens, wherein the immune cells comprise one or more mutations that reduce or eliminate expression of the targeted antigen, and one or more mutations that reduce or eliminate expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0050] In one aspect, the application provides a method for antigen-dependent killing of T-cell acute lymphoblastic leukemia (T-ALL) cells in a subject. In some embodiments, the method comprises administering to a subject having T-ALL expressing CD3, CD5, and CD7 antigens at least three immune cells, each immune cell expressing a different chimeric antigen receptor targeting one of the antigens, wherein the immune cells comprise one or more mutations that reduce or eliminate expression of the targeted antigen, and one or more mutations that reduce or eliminate expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0051] In another aspect, the application provides a method for treating a cancer in a selected subject. In some embodiments, the method comprises administering to the subject at least two immune cells, each immune cell expressing a chimeric antigen receptor targeting a CD33 or CD123 antigen, wherein the immune cells comprise one or more mutations that reduce or eliminate expression of the targeted antigen, and one or more mutations that reduce or eliminate expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1, wherein the subject is selected by characterizing the cancer as expressing CD33 and CD123 antigens. In some embodiments, the cancer expressing CD33 and CD123 antigens is AML.

[0052] In yet another aspect, the application provides a method for treating a cancer in a selected subject. In some embodiments, the method comprises administering to the subject three or more immune cells, each immune cell expressing a different chimeric antigen receptor targeting CD3, CD5, and CD7 antigens, wherein the immune cells comprise one or more mutations that reduce or eliminate expression of the targeted antigen, and one or more mutations that reduce or eliminate expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1, wherein the subject is selected by characterizing the cancer as expressing CD3, CD5, and CD7 antigens. In some embodiments, the cancer expressing CD3, CD5, and CD7 antigens is T-ALL.

[0053] In some embodiments, the immune cell is a cytotoxic T cell, a regulatory T cell, a T helper cell, a dendritic cell, a B cell, or an NK cell. In some embodiments, the subject has been previously treated with lymphodepletion. In some embodiments, the lymphodepletion comprises administration of cyclophosphamide, fludarabine, and / or alemtuzumab (Cy / Flu / Campath). In some embodiments, the subject is refractory to chemotherapy or has a high tumor burden. In some embodiments, the subject is subsequently treated with allogeneic hematopoietic stem cell transplantation (allo-HSCT). In some embodiments, the immune cells are derived from a single human donor. In some embodiments, the immune cells are autologous to the subject. In some embodiments, the immune cells are allogeneic to the subject. In some embodiments, the subject is a mammalian subject. In some embodiments, the subject is a human or a rodent. In some embodiments, the subject is a human pediatric subject.

[0054] In one aspect, the present disclosure provides a kit comprising any of the compositions provided herein for use in treating cancer. In one aspect, the present disclosure provides a kit comprising any of the base editor systems provided herein for use in generating immune cells expressing a CAR with reduced immunogenicity. In some embodiments, any of the kits provided herein comprise written instructions for using the kit.

[0055] The specification and examples herein show embodiments of the present disclosure in detail. It is to be understood that the present disclosure is not limited to the specific embodiments described herein and is therefore variable. Those skilled in the art will recognize that there are numerous alterations and modifications that can be made to the present disclosure that come within the scope of the present disclosure.

[0056] Unless otherwise specified, the practice of some of the embodiments disclosed herein employs conventional techniques in immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genetics, and recombinant DNA, techniques that are available in the art. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th edition (2012); Current Protocols in Molecular Biology series (FMAusubel, et al. eds.); Methods in Enzymology series (Academic Press, Inc.); PCR 2: A Practical Approach (MJ MacPherson, B.D. Hames and G.T. Taylor eds. (1995)); Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual; and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th edition (RI Freshney, ed. (2010)).

[0057] While various features of this disclosure may be described in the context of a single embodiment, these features may also be provided individually or in any combination. Conversely, although the invention may be described in the context of a single embodiment for clarity, the invention may also be practiced in a single embodiment. Section headings used herein are for organizational purposes only and are not intended to be limiting of the subject matter described.

[0058] The features of this disclosure are described in detail in the appended claims. A better understanding of the features and advantages of this disclosure can be obtained by referring to the detailed description of exemplary embodiments (in which the principles of this disclosure are used) and the accompanying drawings.

[0059] definition

[0060] The following definitions are supplementary to those defined in this field and are specific to this application, and do not apply to any relevant or irrelevant cases, such as any duplicate patents or applications. While any methods and materials similar or equivalent to those described herein may be used to practice or test this disclosure, preferred materials and methods are as described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0061] 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 this application belongs. The following references provide one of skill with a general definition of many of the terms used in this application: Singleton et al., 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 Harper Collins Dictionary of Biology (1991).

[0062] 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, articles such as "a," "an," and "the" can mean one or more, unless otherwise specified. In this application, the use of "or" means "and / or" and is understood to be inclusive, unless otherwise specified. Furthermore, the use of terms like "including," as well as other forms such as "consisting of," "consisting essentially of," and variations thereof, is not limiting.

[0063] As used in the specification and claims, the terms "comprising," "having," "including," "containing," or "characterized by" and variations thereof are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in the 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.

[0064] 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, i.e., the limitations of the measurement system. For example, "about" can mean within one (1) or more than one (1) standard deviation, per the practice in the art. Alternatively, "about" can mean ranges deemed to be equivalent in the art to within a certain percentage, such as up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, especially with respect to biological systems or lengths, the term can mean within an order of magnitude, such as within 5-fold or within 2-fold. When a value is described in the application and claims as being "about" a particular value, unless other wise specified, the term is construed to mean that the particular value is an approximate value that can vary from a precise measured or true value up to a range of ± 10% from the particular value.

[0065] 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, or sub-range from a group consisting of 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.

[0066] "Some embodiments," "an embodiment," "one embodiment," or "other embodiments" in the specification mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least some embodiments, but not necessarily included in all embodiments of the disclosure.

[0067] "Adenosine deaminase" means a polypeptide or fragment thereof that is capable of catalyzing the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain catalyzes the hydrolytic deamination of adenosine to inosine or deoxyadenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases provided herein (e.g., engineered adenosine deaminases, evolved adenosine deaminases) can be from any organism such as a bacterium.

[0068] In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the adenosine deaminase or deaminase domain does not exist in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to a naturally occurring deaminase. In some embodiments, the adenosine deaminase is from a bacterium such as E. coli, S. Aureus, B. subtilis, S. typhi, S. putrefaciens, H. influenzae, C. crescentus, or G. sulfurreducens. In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is an E. coli TadA (ecTadA) deaminase or fragment thereof.

[0069] In some embodiments, the ecTadA cytidine deaminase is ecTadA. For example, a truncated ecTadA can lack one or more N-terminal amino acids relative to full-length ecTadA. In some embodiments, a truncated ecTadA8 lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relative to full-length ecTadA. In some embodiments, a truncated ecTadA8 lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues relative to full-length ecTadA. In some embodiments, the ecTadA deaminase does not comprise an N-terminal methionine. In some embodiments, the TadA deaminase is an N-terminally truncated TadA. In particular embodiments, the TadA is any one of the TadAs described in PCT / US2017 / 045381, which is incorporated by reference herein.

[0070] In some embodiments, the TadA deaminase is a TadA variant. In some embodiments, the TadA variant is TadA*7.10. In some embodiments, the TadA variant is TadA*8. In some embodiments, TadA*8 is TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, or TadA*8.24. In some embodiments, TadA*8 is TadA*8a, TadA*8b, TadA*8c, TadA*8d, or TadA*8e. In some embodiments, TadA*8 is TadA*8e. In some embodiments, the TadA variant is TadA*9.

[0071] “Adenosine deaminase base editor 8 (ABE8) polypeptide” or “ABE8” means a base editor as defined herein comprising an adenosine deaminase variant comprising an alteration at amino acid position 82 and / or 166 of the following reference sequence:

[0072] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

[0073] In some embodiments, the ABE8 comprises further alterations relative to the reference sequence as described herein.

[0074] “Adenosine deaminase base editor 8 (ABE8) polypeptide” means a polynucleotide encoding an ABE8.

[0075] “Adenosine deaminase base editor 9 (ABE9) polypeptide” or “ABE9” means a base editor comprising an adenosine deaminase variant comprising one or more of the following alterations: R21N, R23H, E25F, N38G, L51W, P54C, M70V, Q71M, N72K, Y73S, V82T, M94V, P124W, T133K, D139L, D139M, C146R, and A158K, in the reference sequence:

[0076]

[0077] The relevant bases altered in the reference sequence are shown in underlined and bold font. In some embodiments, ABE9 comprises further alterations relative to the reference sequence as described herein. Details of the ABE9 base editor are described in International PCT Application No. PCT / 2020 / 049975, which is incorporated by reference herein in its entirety.

[0078] “Adenosine deaminase base editor 9 (ABE9) polynucleotide” means a polynucleotide encoding ABE9.

[0079] “Administering” means providing one or more compositions described herein to a patient or subject. For example, but not by way of limitation, administration (e.g., injection) of a composition can be performed by intravenous (i.v.) injection, subcutaneous (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 by, for example, bolus injection or by gradual infusion over time. In some embodiments, parenteral administration includes intravascular, intravenous, intramuscular, intracranial, intrathecal, intratumoral, intradermal, intraperitoneal, transorgan, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, and intrasternal infusion or injection. Alternatively or concurrently, administration can be by oral routes.

[0080] “Agent” means any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof.

[0081] “Alteration” means a change (e.g., increase or decrease) in the structure, expression level, or activity of a gene or polypeptide as detected by standard known methods in the art, such as those described herein. As used herein, an alteration (e.g., increase or decrease) includes a change in the structure or expression level of a polynucleotide or polypeptide, such as a 10% change, a 25% change, a 40% change, or a 50% change or greater.

[0082] “Allogeneic” as used herein refers to a cell of the same species that is genetically different from the cell being compared.

[0083] “Alleviate” means to reduce, suppress, attenuate, eliminate, prevent the development or progression of, or stabilize the development or progression of a disease.

[0084] “Analog” means a molecule that is not identical but has similar functional or structural features. For example, a polynucleotide or polypeptide analog retains the biological activity of the corresponding naturally occurring polynucleotide or polypeptide, while having certain modifications that enhance the function of the analog relative to the naturally occurring polynucleotide or polypeptide. Such modifications can increase the affinity, efficiency, specificity, protease or protease resistance, membrane permeability, and / or half-life of the analog to DNA, without altering, for example, ligand binding. An analog can include non-natural nucleotides or amino acids.

[0085] “Anti-neoplasia activity” means to prevent or inhibit the mutation and / or proliferation of a tumor.

[0086] As used herein, “autologous” refers to cells from the same subject.

[0087] As used herein, the term “antibody” refers to an immunoglobulin molecule that specifically binds to a particular antigen or has immunological reactivity with a particular antigen, and includes polyclonal, monoclonal, genetically engineered, and other modified forms of antibodies, including but not limited to chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bispecific antibodies, trispecific antibodies, tetraspecific antibodies, diabodies, triabodies, and tetrabodies), and antigen binding fragments of antibodies, including, for example, Fab', F(ab')2, Fab, Fv, rlgG, and scFv fragments. Unless otherwise specified, the term “monoclonal antibody” (mAb) is intended 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, Fab and F(ab')2 fragments refer to antibody fragments that lack the Fc fragment of intact antibodies. These antibody fragments are exemplified herein.

[0088] “B-cell maturation antigen, or tumor necrosis factor receptor superfamily member 17 polypeptide, (BCMA)” means a protein expressed on mature B lymphocytes that has at least about 85% amino acid sequence identity to NCBI Accession No. NP_001183 or fragments thereof. An exemplary BCMA polypeptide sequence is provided below.

[0089] >NP_001183.2 tumor necrosis factor receptor superfamily member 17 [Homo sapiens]

[0090] MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAILWTCLGLSLIISLAVFVLMFLLRKINSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGLEYTVEECTCEDCIKSKPKVDSDHCFPLPAMEEGATILVTTKTNDYCKSLPAALSATEIEKSISAR

[0091] This antigen can be targeted in the therapy of relapsed or refractory multiple myeloma and other hematological tumors.

[0092] By "B cell maturation antigen, or tumor necrosis factor receptor superfamily member 17, (BCMA) polynucleotide" is meant a nucleic acid molecule encoding a BCMA polypeptide. The BCMA gene encodes a cell surface antigen that recognizes a B cell activating factor. An exemplary B2M polynucleotide sequence is provided below.

[0093] NM_001192.2 Homo sapiens (Human) TNF receptor superfamily member 17 (TNFRSF17), mRNA

[0094] AAGACTCAAACTTAGAAACTTGAATTAGATGTGGTATTCAAATCCTTAGCTGCCGCGAAGACACAGACAGCCCCCGTAAGAACCCACGAAGCAGGCGAAGTTCATTGTTCTCAACATTCTAGCTGCTCTTGCTGCATTTGCTCTGGAATTCTTGTAGAGATATTACTTGTCCTTCCAGGCTGTTCTTTCTGTAGCTCCCTTGTTTTCTTTTTGTGATCATGTTGCAGATGGCTGGGCAGTGCTCCCAAAATGAATATTTTGACAGTTTGTTGCATGCTTGCATACCTTGTCAACTTCGATGTTCTTCTAATACTCCTCCTCTAACATGTCAGCGTTATTGTAATGCAAGTGTGACCAATTCAGTGAAAGGAACGAATGCGATTCTCTGGACCTGTTTGGGACTGAGCTTAATAATTTCTTTGGCAGTTTTCGTGCTAATGTTTTTGCTAAGGAAGATAAACTCTGAACCATTAAAGGACGAGTTTAAAAACACAGGATCAGGTCTCCTGGGCATGGCTAACATTGACCTGGAAAAGAGCAGGACTGGTGATGAAATTATTCTTCCGAGAGGCCTCGAGTACACGGTGGAAGAATGCACCTGTGAAGACTGCATCAAGAGCAAACCGAAGGTCGACTCTGACCATTGCTTTCCACTCCCAGCTATGGAGGAAGGCGCAACCATTCTTGTCACCACGAAAACGAATGACTATTGCAAGAGCCTGCCAGCTGCTTTGAGTGCTACGGAGATAGAGAAATCAATTTCTGCTAGGTAATTAACCATTTCGACTCGAGCAGTGCCACTTTAAAAATCTTTTGTCAGAATAGATGATGTGTCAGATCTCTTTAGGATGACTGTATTTTTCAGTTGCCGATACAGCTTTTTGTCCTCTAACTGTGGAAACTCTTTATGTTAGATATATTTCTCTAGGTTACTGTTGGGAGCTTAATGGTAGAAACTTCCTTGGTTTCATGATTAAACTCTTTTTTTTCCTGA

[0095] A "base editor (BE)" or "nucleobase editor (NBE)" means an agent that binds to a polynucleotide and has nucleobase modification activity. In one embodiment, the agent binds to a polynucleotide at a specific sequence using a nucleic acid programmable DNA binding protein. In another embodiment, the base editor is an enzyme capable of modifying a cytosine base within a nucleic acid molecule (e.g., DNA). In some embodiments, the base editor is capable of deaminating a base within a nucleic acid molecule. In some embodiments, the base editor is capable of deaminating a base within a DNA molecule. In some embodiments, the base editor is capable of deaminating cytosine within DNA. In some embodiments, the base editor is a fusion protein comprising a cytosine deaminase or an adenosine deaminase. In some embodiments, the base editor is a Cas9 protein fused to a cytosine deaminase or an adenosine deaminase. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to a cytosine deaminase or an adenosine deaminase. In some embodiments, the base editor is fused to an inhibitor of base excision repair, e.g., a UGI domain. In some embodiments, the fusion protein comprises a Cas9 nickase fused to a deaminase and an inhibitor of base excision repair, such as a UGI domain.

[0096] In some embodiments, the cytosine deaminase or adenosine deaminase nucleobase editor is a polypeptide comprising the following structure A-B: NH2-[A-B]-COOH, wherein A comprises a cytosine deaminase domain, an adenosine deaminase domain, or an active fragment thereof, and wherein B comprises one or more domains having nucleic acid sequence specific binding activity. In one embodiment, the cytosine or adenosine deaminase nucleobase editor polypeptide of the previous aspect contains: NH2-[A n -B o ]-COOH, wherein A comprises a cytosine deaminase domain, an adenosine deaminase domain, or an active fragment thereof, wherein n is an integer: 1, 2, 3, 4, or 5; and wherein B comprises a domain having nucleic acid sequence specific binding activity; and wherein o is an integer: 1, 2, 3, 4, or 5. In one embodiment, the polypeptide contains one or more nuclear localization sequences. In one embodiment, the polypeptide contains at least one of said nuclear localization sequences located at the N-terminus or C-terminus. In one embodiment, the polypeptide contains a nuclear localization signal that is a bipartite nuclear localization signal. In one embodiment, the polypeptide contains one or more domains linked by a linker.

[0097] In some embodiments, the base editor is a cytosine base editor (CBE). In some embodiments, the base editor is an adenosine base editor (ABE). In some embodiments, the base editor is an adenosine base editor (ABE) and a cytosine base editor (CBE). In some embodiments, the base editor is a nuclease inactive Cas9 (dCas9) fused to an adenosine deaminase. In some embodiments, the Cas9 is a circular permutant Cas9 (e.g., spCas9 or saCas9). Circular permutant Cas9s are known in the art and described in, e.g., Oakes et al., Cell 176, 254-267, 2019. In some embodiments, the base editor is fused to an inhibitor of base excision repair, e.g., a UGI domain or a dISN domain. In some embodiments, the fusion protein comprises a Cas9 nickase fused to a deaminase and an inhibitor of base excision repair, such as a UGI or dISN domain. In other embodiments, the base editor is a baseless base editor.

[0098] In some embodiments, the adenosine deaminase is evolved from TadA. In some embodiments, the polynucleotide programmable DNA binding domain is a CRISPR-associated (e.g., Cas or Cpf1) enzyme. In some embodiments, the base editor is a catalytically dead Cas9 (dCas9) fused to a deaminase domain. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to a deaminase domain. In some embodiments, the base editor is fused to an inhibitor of base excision repair (BER). In some embodiments, the inhibitor of base excision repair is a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the inhibitor of base excision repair is a inosine base excision repair inhibitor.

[0099] In some embodiments, the base editor is generated by cloning an adenosine deaminase variant (e.g., TadA*7.10) into a scaffold comprising a circular permutant Cas9 (e.g., spCAS9) and a bipartite nuclear localization sequence. In some embodiments, the base editor (e.g., ABE8) is generated by cloning an adenosine deaminase variant (e.g., TadA*8) into a scaffold comprising a circular permutant Cas9 (e.g., spCAS9 or saCAS9) and a bipartite nuclear localization sequence. Circular permutant Cas9s are known in the art and described in, e.g., Oakes et al., Cell 176, 254-267, 2019.

[0100] In some embodiments, the polynucleotide programmable DNA binding domain is a CRISPR-associated (e.g., Cas or Cpf1) enzyme. In some embodiments, the base editor is a catalytically dead Cas9 (dCas9) fused to a deaminase domain. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to a deaminase domain. In some embodiments, the base editor is fused to an inhibitor of base excision repair (BER). In some embodiments, the inhibitor of base excision repair is a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the inhibitor of base excision repair is an inosine base excision repair inhibitor.

[0101] Details of base editors are described in International PCT Application Nos. PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), each of which is incorporated by reference herein in its entirety. See also 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., et al., “Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); 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: eaoo4774 (2017); and Rees, H.A., et al., “Base editing: precision chemistry on the genome and transcriptome of living cells.” Nat Rev Genet. 2018 Dec; 19(12): 770-788. doi: 10.1038 / s41576-018-0059-1, the entire contents of which are incorporated by reference herein.

[0102] For example, an adenosine base editor (ABE) for use in the base editing compositions, systems, and methods described herein has the nucleic acid sequence (8877 base pairs) as described below (Addgene, Watertown, MA.; Gaudelli NM, et al., Nature. 2017 Nov 23; 551(7681): 464-471. doi: 10.1038 / nature24644; Koblan LW, et al., Nat Biotechnol. 2018 Oct; 36(9): 843-846. doi: 10.1038 / nbt.4172.). Also encompassed are polynucleotide sequences having at least 95% or greater identity to the ABE nucleic acid sequence.

[0103]

[0104] In some embodiments, the base editor is an adenosine deaminase base editor 8 (ABE8). In some embodiments, the ABE8 is selected from the base editors from Tables 13, 14, or 16 below. In some embodiments, the ABE8 contains an adenosine deaminase evolved from TadA. In some embodiments, the adenosine deaminase variant of the ABE8 is a TadA*8 variant as described in Tables 11, 13, or 14 below. In some embodiments, the adenosine deaminase is a TadA*7.10 variant (e.g., TadA*8) comprising one or more of the following alterations selected from the group: Y147T, Q154S, Y123H, V82S, T166R, and / or Q154R. In various embodiments, the ABE8 comprises a TadA*7.10 variant (e.g., TadA*8) having a combination of alterations selected from the group: Y147T + Q154R; Y147T + Q154S; Y147R + Q154S; V82S + Q154S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; Y147R + Q154R + Y123H; Y147R + Q154R + I76Y; Y147R + Q154R + T166R; Y123H + Y147R + Q154R + I76Y; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R. In some embodiments, the ABE8 is a monomeric construct. In some embodiments, the ABE8 is a heterodimeric construct. In some embodiments, the ABE8 base editor comprises the sequence:

[0105] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD.

[0106] For example, a cytidine base editor (CBE) as used in the base editing compositions, systems, and methods described herein has the following nucleic acid sequence (8877 base pairs) provided below (Addgene, Watertown, MA.; Komor AC, et al., 2017, Sci Adv., 30; 3(8): eaao4774. doi: 10.1126 / sciadv.aao4774). Also encompassed are polynucleotide sequences having at least 95% or greater identity to the BE4 nucleic acid sequence.

[0107]

[0108]

[0109]

[0110] In some embodiments, the cytidine base editor is BE4 having a nucleic acid sequence selected from one of:

[0111] Original BE4 nucleic acid sequence:

[0112]

[0113] BE4 codon-optimized 1 nucleic acid sequence:

[0114]

[0115] BE4 codon-optimized 2 nucleic acid sequence:

[0116]

[0117] "Base editing activity" means to function 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 a cytidine deaminase activity, e.g., converting a target C G to T A. In another embodiment, the base editing activity is an adenosine or adenine deaminase activity, e.g., converting a target A T to G C. In another embodiment, the base editing activity is a cytidine deaminase activity, e.g., converting a target C G to T A, and an adenosine or adenine deaminase activity, e.g., converting a target A T to G C.

[0118] In some embodiments, base editing activity is assessed by editing efficiency. Base editing efficiency can be measured by any suitable means, e.g., by sequencing or next generation sequencing. In some embodiments, base editing efficiency is measured by the percentage of total sequencing reads having a nucleobase conversion effected by the base editor, e.g., the percentage of total sequencing reads having a target A T base pair converted to a G C base pair or a target C G base pair converted to a T A base pair. In some embodiments, base editing efficiency is measured by the percentage of total cells having a nucleobase conversion effected by the base editor when base editing is performed in a population of cells.

[0119] The term "base editor system" refers to a system for editing a nucleobase of a target nucleotide sequence. In various embodiments, a base editor system comprises (1) a polynucleotide programmable nucleotide binding domain (e.g., Cas9); (2) a deaminase domain (e.g., an adenosine deaminase or a cytidine deaminase) for deaminating the nucleobase; and (3) one or more guide polynucleotides (e.g., guide RNAs). 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 system is an adenosine deaminase base editor 8 (ABE8).

[0120] In some embodiments, ABE8 is a monomeric construct. In some embodiments, ABE8 is ABE8.1-m, ABE8.2-m, ABE8.3-m, ABE8.4-m, ABE8.5-m, ABE8.6-m, ABE8.7-m, ABE8.8-m, ABE8.9-m, ABE8.10-m, ABE8.11-m, ABE8.12-m, ABE8.13-m, ABE8.14-m, ABE8.15-m, ABE8.16-m, ABE8.17-m, ABE8.18-m, ABE8.19-m, ABE8.20-m, ABE8.21-m, ABE8.22-m, ABE8.23-m, ABE8.24-m. In some embodiments, ABE8 is a heteromeric construct. In some embodiments, ABE8 is ABE8.1-d, ABE8.2-d, ABE8.3-d, ABE8.4-d, ABE8.5-d, ABE8.6-d, ABE8.7-d, ABE8.8-d, ABE8.9-d, ABE8.10-d, ABE8.11-d, ABE8.12-d, ABE8.13-d, ABE8.14-d, ABE8.15-d, ABE8.16-d, ABE8.17-d, ABE8.18-d, ABE8.19-d, ABE8.20-d, ABE8.21-d, ABE8.22-d, ABE8.23-d, or ABE8.24-d.

[0121] In some embodiments, a base editor system can comprise more than one base editing component. For example, a base editor system can include more than one deaminase. In some embodiments, a base editor system can include one or more cytidine deaminases. In some embodiments, a base editor system can include one or more adenosine deaminases. In some embodiments, different deaminases can be targeted to a target nucleic acid sequence using a single guide polynucleotide. In some embodiments, different deaminases can be targeted to a target nucleic acid sequence using a pair of guide polynucleotides.

[0122] The nucleobase editing system nucleobase component and the polynucleotide programmable nucleotide binding component can be associated or interacted with each other covalently or non-covalently, or any combination thereof. For example, in some embodiments, the deaminase domain can be targeted to a target nucleotide sequence by the polynucleotide programmable nucleotide binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused or linked to the deaminase domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can target the deaminase domain to the target nucleotide sequence by non-covalently interacting or associating with the deaminase domain. For example, in some embodiments, the nucleobase editing component, e.g., the deaminase domain, can comprise an additional heterologous moiety or domain that is capable of interacting, associating or forming a complex with an additional heterologous moiety or domain that is part of the polynucleotide programmable nucleotide binding domain. In some embodiments, the additional heterologous moiety can be capable of binding, interacting, associating or forming a complex with a polypeptide. In some embodiments, the additional heterologous moiety can be capable of binding, interacting, associating or forming a complex with a polynucleotide. In some embodiments, the additional heterologous moiety can be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous moiety can be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous moiety can be capable of binding to a polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous moiety can be a K homology (KH) domain, a MS2 coat protein domain, a PP7 coat protein domain, a SfMuCom coat protein domain, an aseptic alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0123] The base editor system can further comprise a guide polynucleotide component. It will be appreciated that the components of the base editor system can be associated with one another via covalent bonds, non-covalent interactions, or any combination of their association and interaction. In some embodiments, the deamination domain can be targeted to a target nucleotide sequence by a guide polynucleotide. For example, in some embodiments, the nucleobase editing component of the base editor system, e.g., the deaminase domain, can comprise an additional heterologous moiety or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) that is capable of interacting with, associating with, or forming a complex with a portion or segment of the guide polynucleotide (e.g., a polynucleotide motif). In some embodiments, the additional heterologous moiety or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) can be fused or linked to the deaminase domain. In some embodiments, the additional heterologous moiety can be capable of binding to, interacting with, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous moiety can be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous moiety can be capable of binding to the guide polynucleotide. In some embodiments, the additional heterologous moiety can be capable of binding to the polypeptide linker. In some embodiments, the additional heterologous moiety can be capable of binding to the polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous moiety can be a K homology (KH) domain, a MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, an ariance alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0124] In some embodiments, the base editor system can further comprise an inhibitor of base excision repair (BER) component. It will be appreciated that the components of the base editor system can be associated with one another via covalent bonds, non-covalent interactions, or any combination of their association and interaction. The inhibitor of BER component can comprise a BER inhibitor. In some embodiments, the inhibitor of BER can be a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the inhibitor of BER can be an inosine BER inhibitor. In some embodiments, the inhibitor of BER can be targeted to a target nucleotide sequence by a polynucleotide programmable nucleotide binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused or linked to the inhibitor of BER. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused or linked to the deaminase domain and the inhibitor of BER. In some embodiments, the polynucleotide programmable nucleotide binding domain can target the inhibitor of BER to a target nucleotide sequence by non-covalently interacting or associating with the inhibitor of BER. For example, in some embodiments, the inhibitor of BER component of the base editor system can comprise an additional heterologous moiety or domain that is capable of interacting, associating, or forming a complex with an additional heterologous moiety or domain that is part of the polynucleotide programmable nucleotide binding domain.

[0125] In some embodiments, the inhibitor of BER can be targeted to a target nucleotide sequence by a guide polynucleotide. For example, in some embodiments, the inhibitor of BER component of the base editor system can comprise an additional heterologous moiety or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) that is capable of interacting, associating, or forming a complex with a portion or segment of the guide polynucleotide (e.g., a polynucleotide motif). In some embodiments, the additional heterologous moiety or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) of the guide polynucleotide can be fused or linked to the inhibitor of BER. In some embodiments, the additional heterologous moiety can be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous moiety can be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous moiety can be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous moiety can be capable of binding to a polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous moiety can be a K homology (KH) domain, a MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, an aseptic alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0126] A "beta-2 microglobulin (B2M) polypeptide" means a protein having at least about 85% amino acid sequence identity to UniProt accession number P61769 or a fragment thereof and having immunomodulatory activity. An exemplary B2M polypeptide sequence is provided below.

[0127] >sp|P61769|B2MG_HUMAN Beta-2-microglobulin OS=Homo sapiens OX=9606 GN=B2M PE=1 SV=1

[0128] MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM

[0129] A "beta-2 microglobulin (B2M) polynucleotide" means a nucleic acid molecule encoding a B2M polypeptide. The beta-2 microglobulin gene encodes a serum protein that associates with the major histocompatibility complex. B2M is implicated in non-self recognition by host CD8+ T cells. An exemplary B2M polynucleotide sequence is provided below.

[0130] >DQ217933.1 Homo sapiens Beta-2 microglobulin (B2M) gene, complete coding sequence

[0131]

[0132] The term“Cas9” or“Cas9 domain” refers to an RNA-guided nuclease or fragment thereof, which comprises a Cas9 protein (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9 and / or a gRNA binding domain of Cas9). Cas9 nucleases are sometimes referred to as Casnl nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)-associated nucleases. CRISPR is a system of adaptive immunity that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain spacer sequences, sequences complementary to protospacers of mobile elements, and target invader nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, proper processing of pre-crRNA requires a reverse-coded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and a Cas9 protein. The tracrRNA acts as a guide for ribonuclease 3-assisted processing of pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA endonucleolytically cleaves linear or circular dsDNA targets complementary to the spacer sequences. The target strand not complementary to the crRNA is first endonucleolytically cleaved, then 3’-5’ exonucleolytically trimmed. In nature, DNA binding and cleavage typically require the protein and two RNAs. However, a single guide RNA (“sgRNA” or simply“gRNA”) can be engineered to incorporate both the crRNA and the tracrRNA into a single RNA species. See, e.g., Jinek M. et al., Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes a short motif in the CRISPR repeat sequence (PAM or protospacer adjacent motif) to help distinguish self from non-self.Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., "Complete genome sequence of an Ml strain of Streptococcus pyogenes." Ferretti, J.J. et al., Natl. Acad. Sci. U.S.A. 98:4658-4663 (2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E. et al., Nature 471 :602-607 (201 1); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M. et al., Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in a variety of species, including S. pyogenes and S. thermophilus. Other suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on the present disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from organisms and loci as disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.

[0133] A Cas9 protein that is nuclease inactive can be interchangeably referred to as a "dCas9" protein (for "dead" Cas9 with respect to nuclease activity) or a Cas9 without catalytic activity. Cas9 proteins (or fragments thereof) for generating Cas9 proteins with inactive DNA cleavage domains are known (see, e.g., Jinek et al., Science. 337: 816-821 (2012); Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression" (2013) Cell. 28; 152(5): 1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains: an HNH nuclease subdomain and a RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations in these subdomains can silence the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337: 816-821 (2012); Qi et al., Cell. 28; 152(5): 1173-83 (2013)). In some embodiments, a dCas9 corresponds to or comprises, in part or in whole, a Cas9 amino acid sequence with one or more mutations that inactivate the Cas9 nuclease activity. In some embodiments, a dCas9 domain comprises D10A and H840A mutations or a corresponding mutation in another Cas9. In some embodiments, a Cas9 nuclease has an inactivated (e.g., inactivated) DNA cleavage domain, in other words, the Cas9 is a nickase, referred to as a "nCas9" protein (for "nickase" Cas9). It will be appreciated that other Cas9 proteins (e.g., nuclease dead Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease active Cas9), including variants and homologs thereof, are within the scope of the present disclosure. Exemplary Cas9 proteins include, but are not limited to, those provided herein. In some embodiments, a Cas9 protein is a nuclease dead Cas9 (dCas9). In some embodiments, a Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, a Cas9 protein is a nuclease active Cas9.

[0134] In some embodiments, a protein comprising a fragment of Cas9 is provided. For example, in some embodiments, the protein comprises one or two Cas9 domains: (1) a gRNA binding domain of Cas9; or (2) a DNA cleavage domain of Cas9. In some embodiments, the protein comprising Cas9 or a fragment thereof is referred to as a "Cas9 variant." A Cas9 variant shares homology with Cas9 or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas9. In some embodiments, a Cas9 variant can have 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 amino acid changes compared to wild-type Cas9. In some embodiments, a Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA cleavage domain) such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, 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% of the amino acid length of the corresponding wild-type Cas9.

[0135] In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.

[0136] In some embodiments, Cas9 refers to a 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), or Neisseria meningitidis (NCBI Ref: YP_002342100.1); or a Cas9 from any other organism.

[0137] In some embodiments, the Cas9 is from Neisseria meningitidis (Nme). In some embodiments, the Cas9 is Nme1, Nme2, or Nme3. In some embodiments, the PAM interaction domains for Nme1, Nme2, or Nme3 are N4GAT, N4CC, and N4CAAA, respectively (see, e.g., Edraki, A., et al., A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing, Molecular Cell (2018)).

[0138] In some embodiments, a Cas9 fusion protein as provided herein comprises the full-length amino acid sequence of a Cas9 protein (e.g., one of the Cas9 sequences provided herein). However, in other embodiments, a fusion protein provided herein does not comprise a full-length Cas9 sequence, but only one or more fragments thereof. For example, in some embodiments, a Cas9 fusion protein provided herein comprises a Cas9 fragment, wherein the fragment binds a crRNA and a tracrRNA or sgRNA, but does not comprise a functional nuclease domain, e.g., in the case that it only comprises a truncated version of a nuclease domain or does not comprise a nuclease domain at all.

[0139] Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and other suitable sequences of Cas9 domains and fragments will be apparent to those skilled in the art.

[0140] In some embodiments, Cas9 refers to Cas9 from archaea (e.g., nanarchae), which constitute a domain and kingdom of single-celled prokaryotic microorganisms. In some embodiments, Cas9 refers to CasX or CasY, which have been described, e.g., in Burstein et al., “New CRISPR-Cas systems from uncultivated microbes.” Cell Res. 2017 Feb 21. doi: 10.1038 / cr.2017.21, the entire contents of which are incorporated herein by reference. Using genomic-resolved metagenomics, a large number of CRISPR-Cas systems were identified, including a Cas9 first reported in the archaeal domain of life. This distinct Cas9 protein was discovered in the poorly studied nanarchaea as an active CRISPR-Cas system. Two previously unknown systems, CRISPR-CasX and CRISPR-CasY, were discovered in bacteria, one of the most compact systems ever discovered. In some embodiments, Cas9 refers to CasX or a variant of CasX. In some embodiments, Cas9 refers to CasY or a variant of CasY. It will be appreciated that other RNA-guided DNA binding proteins can be used as a nucleic acid programmable DNA binding protein (napDNAbp) and are within the scope of the present disclosure.

[0141] In particular embodiments, napDNAbps useful in the methods of the application include circular arrangements, which are known in the art and described by, e.g., Oakes et al., Cell 176, 254-267, 2019.

[0142] Non-limiting examples of polynucleotide programmable nucleotide binding domains that can be incorporated into a base editor include domains derived from CRISPR proteins, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs).

[0143] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) or any fusion protein provided herein can be a CasX or CasY protein. In some embodiments, the napDNAbp is a CasX protein. In some embodiments, the napDNAbp is a CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring CasX or CasY protein. In some embodiments, the napDNAbp is a naturally occurring CasX or CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a CasX or CasY protein described herein. It will be appreciated that CasX and CasY from other bacterial species can also be used in accordance with the present disclosure.

[0144] The term “Cas12b” or “Cas12b domain” refers to an RNA-guided nuclease or fragment thereof comprising a Cas12b, C2c1 protein (e.g., a protein comprising a DNA cleavage domain of Cas12b that is active, inactive, or partially active and / or a gRNA binding domain of Cas12b). The contents of each are incorporated herein by reference. Cas12b homologs have been described in various species, including but not limited to, Alicyclobacillus acidoterrestris, Alicyclobacillus acidophilus (Teng et al., Cell Discov. 2018 Nov 27;4:63), Bacillus hisashi, and Bacillus sp. V3-13. Other suitable Cas12b nuclease sequences will be apparent to those of skill in the art based on the present disclosure.

[0145] In some embodiments, a protein comprising Cas12b or a fragment thereof is referred to as a “Cas12b variant.” A Cas12b variant shares homology with Cas12b or a fragment thereof. For example, a Cas12b variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas12b. In some embodiments, a Cas12b variant can have 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 amino acid changes compared to wild-type Cas12b. In some embodiments, a Cas12b variant comprises a fragment of Cas12b (e.g., a gRNA binding domain or a DNA cleavage domain) such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas12b. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, 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% of the amino acid length of the corresponding wild-type Cas12b. Exemplary Cas12b polypeptides are set forth herein.

[0146] A “Cbl proto-oncogene B (CBLB) polypeptide” means a protein that shares at least about 85% amino acid sequence identity to GenBank Accession No. ABC86700.1 or a fragment thereof and is involved in the regulation of immune responses. An exemplary CBLB polypeptide sequence is provided below.

[0147] >ABC86700.1 CBL-B [Homo sapiens]

[0148] MANSMNGRNPGGRGGNPRKGRILGIIDAIQDAVGPPKQAAADRRTVEKTWKLMDKVVRLCQNPKLQLKNSPPYILDILPDTYQHLRLILSKYDDNQKLAQLSENEYFKIYIDSLMKKSKRAIRLFKEGKERMYEEQSQDRRNLTKLSLIFSHMLAEIKAIFPNGQFQGDNFRITKADAAEFWRKFFGDKTIVPWKVFRQCLHEVHQISSGLEAMALKSTIDLTCNDYISVFEFDIFTRLFQPWGSILRNWNFLAVTHPGYMAFLTYDEVKARLQKYSTKPGSYIFRLSCTRLGQWAIGYVTGDGNILQTIPHNKPLFQALIDGSREGFYLYPDGRSYNPDLTGLCEPTPHDHIKVTQEQYELYCEMGSTFQLCKICAENDKDVKIEPCGHLMCTSCLTAWQESDGQGCPFCRCEIKGTEPIIVDPFDPRDEGSRCCSIIDPFGMPMLDLDDDDDREESLMMNRLANVRKCTDRQNSPVTSPGSSPLAQRRKPQPDPLQIPHLSLPPVPPRLDLIQKGIVRSPCGSPTGSPKSSPCMVRKQDKPLPAPPPPLRDPPPPPPERPPPIPPDNRLSRHIHHVESVPSRDPPMPLEAWCPRDVFGTNQLVGCRLLGEGSPKPGITASSNVNGRHSRVGSDPVLMRKHRRHDLPLEGAKVFSNGHLGSEEYDVPPRLSPPPPVTTLLPSIKCTGPLANSLSEKTRDPVEEDDDEYKIPSSHPVSLNSQPSHCHNVKPPVRSCDNGHCMLNGTHGPSSEKKSNIPDLSIYLKGDVFDSASDPVPLPPARPPTRDNPKHGSSLNRTPSDYDLLIPPLGEDAFDALPPSLPPPPPPARHSLIEHSKPPGSSSRPSSGQDLFLLPSDPFVDLASGQVPLPPARRLPGENVKTNRTSQDYDQLPSCSDGSQAPARPPKPRPRRTAPEIHHRKPHGPEAALENVDAKIAKLMGEGYAFEEVKRALEIAQNNVEVARSILREFAFPPPVSPRLNL

[0149] "Cbl proto-oncogene B (CBLB) polynucleotide" means a nucleic acid molecule encoding a CBLB polypeptide. The CBLB gene encodes an E3 ubiquitin ligase. An exemplary CBLB nucleic acid sequence is provided below.

[0150] >DQ349203.1 Homo sapiens CBL-B mRNA, complete cds

[0151]

[0152] “Chimeric antigen receptor” or “CAR” means a synthetic or engineered receptor comprising an extracellular antigen binding domain that confers antigen specificity on an immune effector cell, joined to one or more intracellular signaling domains (e.g., T cell signaling domains). In some embodiments, a CAR includes a transmembrane domain.

[0153] “Chimeric antigen receptor T cell” or “CAR-T cell” means a T cell that expresses a CAR, having antigen specificity dictated by the targeting domain of the antibody-derived CAR. As used herein, “CAR-T cell” includes a T cell or an NK cell. As used herein, “CAR-T cell” includes a cell engineered to express a CAR or a T cell receptor (TCR). In some embodiments, a CAR-T cell can be a T helper CD4+and / or T effector CD8+cell, optionally present in defined proportions. Methods of making CARs (e.g., for treating 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; PCT publications WO2012 / 079000, WO2013 / 059593, and U.S. Patent Publication 2012 / 0213783, each of which is incorporated herein by reference in its entirety).

[0154] “Class II major histocompatibility complex transactivator (CIITA)” means a protein having at least about 85% amino acid sequence identity to NCBI Accession No. NP_001273331.1 or a fragment thereof and having immunomodulatory activity. An exemplary amino acid sequence is provided below.

[0155] >NP_001273331.1 Class II, MHC transactivator isoform 1 [Homo sapiens]

[0156]

[0157] By "class II major histocompatibility complex transactivator (CIITA)" is meant a nucleic acid encoding a CIITA polypeptide. An exemplary CIITA nucleic acid sequence is provided below.

[0158] > NM_001286402.1 Homo sapiens class II major histocompatibility complex transactivator (CIITA), transcript variant 1, mRNA

[0159]

[0160] In this disclosure, "comprise," "comprising," "include," "including," and "has" have the meaning given the term under 35 U.S.C. § 112, and can mean "including," "comprising," and the like; "consisting essentially of' and the like have the meaning as recited in the U.S. Patent Law, and the term is open-ended, allowing for elements not recited, so long as the essential nature of the subject matter is not changed by the addition of such elements.

[0161] "Cytotoxic T-Lymphocyte-Associated Protein 4 (CTLA-4) polypeptide" means a protein having at least about 85% sequence identity to NCBI Accession No. EAW70354.1 or a fragment thereof. An exemplary amino acid sequence is provided below:

[0162] > EAW70354.1 Cytotoxic T-Lymphocyte-Associated Protein 4 [Homo sapiens]

[0163] MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN

[0164] "Cytotoxic T-Lymphocyte-Associated Protein 4 (CTLA-4) polynucleotide" means a nucleic acid molecule encoding a CTLA-4 polypeptide. The CTLA-4 gene encodes an immunoglobulin superfamily and encodes a protein that transmits an inhibitory signal to T cells. An exemplary CTLA-4 nucleic acid sequence is provided below.

[0165] > BC074842.2 Homo sapiens Cytotoxic T-Lymphocyte-Associated Protein 4, mRNA (cDNA clone MGC: 104099 IMAGE: 30915552), complete cds

[0166] GACCTGAACACCGCTCCCATAAAGCCATGGCTTGCCTTGGATTTCAGCGGCACAAGGCTCAGCTGAACCTGGCTACCAGGACCTGGCCCTGCACTCTCCTGTTTTTTCTTCTCTTCATCCCTGTCTTCTGCAAAGCAATGCACGTGGCCCAGCCTGCTGTGGTACTGGCCAGCAGCCGAGGCATCGCCAGCTTTGTGTGTGAGTATGCATCTCCAGGCAAAGCCACTGAGGTCCGGGTGACAGTGCTTCGGCAGGCTGACAGCCAGGTGACTGAAGTCTGTGCGGCAACCTACATGATGGGGAATGAGTTGACCTTCCTAGATGATTCCATCTGCACGGGCACCTCCAGTGGAAATCAAGTGAACCTCACTATCCAAGGACTGAGGGCCATGGACACGGGACTCTACATCTGCAAGGTGGAGCTCATGTACCCACCGCCATACTACCTGGGCATAGGCAACGGAACCCAGATTTATGTAATTGATCCAGAACCGTGCCCAGATTCTGACTTCCTCCTCTGGATCCTTGCAGCAGTTAGTTCGGGGTTGTTTTTTTATAGCTTTCTCCTCACAGCTGTTTCTTTGAGCAAAATGCTAAAGAAAAGAAGCCCTCTTACAACAGGGGTCTATGTGAAAATGCCCCCAACAGAGCCAGAATGTGAAAAGCAATTTCAGCCTTATTTTATTCCCATCAATTGAGAAACCATTATGAAGAAGAGAGTCCATATTTCAATTTCCAAGAGCTGAGG

[0167] "Differentiation cluster 2 (CD2) polypeptide" means a protein that has at least about 85% amino acid sequence identity to NCBI Accession No. NP_001758.2 or a fragment thereof and has immunomodulatory activity. An exemplary amino acid sequence is provided below.

[0168] >NP_001758.2 T-cell surface antigen CD2, isoform 2 precursor [Homo sapiens]

[0169]

[0170] The CD2 cytoplasmic domain (amino acid residues 235 to 351) is shown in bold. The architecture of an exemplary CD2 polypeptide from Homo sapiens is shown in Figure 4

[0171] A “cluster of differentiation 2 (CD2) polynucleotide” means a nucleic acid that encodes a CD2 polypeptide. An exemplary CD2 nucleic acid sequence is provided below.

[0172]

[0173]

[0174] A “cluster of differentiation 5 (CD5) polypeptide” means a protein that has at least about 85% amino acid sequence identity to NCBI Accession No. NP_001333385.1 or a fragment thereof and has immunomodulatory activity. An exemplary amino acid sequence is provided below.

[0175] >NP_001333385.1 T cell surface glycoprotein CD5 subtype 2 [Homo sapiens]

[0176] MVCSQSWGRSSKQWEDPSQASKVCQRLNCGVPLSLGPFLVTYTPQSSIICYGQLGSFSNCSHSRNDMCHSLGLTCLEPQKTTPPTTRPPPTTTPEPTAPPRLQLVAQSGGQHCAGVVEFYSGSLGGTISYEAQDKTQDLENFLCNNLQCGSFLKHLPETEAGRAQDPGEPREHQPLPIQWKIQNSSCTSLEHCFRKIKPQKSGRVLALLCSGFQPKVQSRLVGGSSICEGTVEVRQGAQWAALCDSSSARSSLRWEEVCREQQCGSVNSYRVLDAGDPTSRGLFCPHQKLSQCHELWERNSYCKKVFVTCQDPNPAGLAAGTVASIILALVLLVVLLVVCGPLAYKKLVKKFRQKKQRQWIGPTGMNQNMSFHRNHTATVRSHAENPTASHVDNEYSQPPRNSHLSAYPALEGALHRSSMQPDNSSDSDYDLHGAQRL

[0177] A “cluster of differentiation 5 (CD5) polynucleotide” means a nucleic acid that encodes a CD5 polypeptide. An exemplary CD5 nucleic acid sequence is provided below.​

[0178] NM_001346456.1 Homo sapiens CD5 molecule (CD5), transcript variant 2, mRNA

[0179]

[0180]

[0181] "Differentiation cluster 7 (CD7) polypeptide" means a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence NP_006128.1 or a fragment thereof and having immunomodulatory activity. An exemplary amino acid sequence is provided below.

[0182] >NP_006128.1 T-cell antigen precursor [Homo sapiens]

[0183]

[0184] "Differentiation cluster 7 (CD7) polynucleotide" means a nucleic acid molecule encoding a CD7 polypeptide. An exemplary CD7 nucleic acid sequence is provided below.

[0185] >NM_006137.7 Homo sapiens CD7 molecule (CD7), mRNA

[0186]

[0187]

[0188] "Differentiation cluster 33 (CD33) polypeptide" means a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence NP_001763.3 or a fragment thereof. CD33 is also known as Siglec-3. An exemplary amino acid sequence is provided below.

[0189] >NP_001763.3 Myeloid cell surface antigen CD33 subtype 1 precursor [Homo sapiens]

[0190]

[0191] "Differentiation cluster 33 (CD33) polynucleotide" means a nucleic acid molecule encoding a CD33 polypeptide. An exemplary CD33 nucleic acid sequence is provided below.

[0192] >NM_001772.4 Homo sapiens CD33 molecule (CD33), transcript variant 1, mRNA

[0193]

[0194] “Cluster of differentiation 52 (CD52) polypeptide” means a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence NP_001794.2, or a fragment thereof. CD52 is also known as CAMPATH-1. An exemplary amino acid sequence is provided below.

[0195] >NP_001794.2 CAMPATH-1 antigen precursor [Homo sapiens]

[0196] 1 MKRFLFLLLT ISLLVMVQIQ TGLSGQNDTS QTSSPSASSN ISGGIFLFFV ANAIIHLFCF

[0197] 61 S

[0198] “Cluster of differentiation 52 (CD52) polynucleotide” means a nucleic acid molecule encoding a CD52 polypeptide. An exemplary CD52 nucleic acid sequence is provided below.

[0199] >NM_001803.3 Homo sapiens CD52 molecule (CD52), mRNA

[0200]

[0201] “Cluster of differentiation 123 (CD123) polypeptide” means a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence NP_002174.1, or a fragment thereof. CD123 is also known as Interleukin-3 receptor. An exemplary amino acid sequence is provided below.

[0202] >NP_002174.1 Interleukin-3 receptor subunit alpha, isoform 1 precursor [Homo sapiens]

[0203]

[0204] “Cluster of differentiation 123 (CD123) polynucleotide” means a nucleic acid molecule encoding a CD123 polypeptide. An exemplary CD123 nucleic acid sequence is provided below.

[0205] >NM_002183.4 Homo sapiens Interleukin 3 receptor subunit alpha (IL3RA), transcript variant 1, mRNA

[0206]

[0207]

[0208] “Cluster of Differentiation 137 (CD137) polypeptide” means a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence NP_001552.2 or a fragment thereof. Cd137 is also known as 4-1BB. An exemplary amino acid sequence is provided below.

[0209] >NP_001552.2 Tumor necrosis factor receptor superfamily member 9 precursor [Homo sapiens]

[0210]

[0211] “Cluster of Differentiation 137 (CD137) polynucleotide” means a nucleic acid molecule encoding a CD137 polypeptide. An exemplary CD137 nucleic acid sequence is provided below.

[0212] >NM_001561.6 Homo sapiens TNF receptor superfamily member 9 (TNFRSF9), mRNA

[0213]

[0214]

[0215]

[0216] “Cluster of Differentiation 247 (CD247) polypeptide” means a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence NP_932170.1 or a fragment thereof. CD137 is also known as CD3 zeta. An exemplary amino acid sequence is provided below.

[0217] >NP_932170.1 T cell surface glycoprotein CD3 zeta chain isoform 1 precursor [Homo sapiens]

[0218] 1 mkwkalftaa ilqaqlpite aqsfglldpk lcylldgilf iygviltalf lrvkfsrsad

[0219] 61 apayqqgqnq lynelnlgrr eeydvldkrr grdpemggkp qrrknpqegl ynelqkdkma

[0220] 121 eayseigmkg errrgkghdg lyqglstatk dtydalhmqa lppr

[0221] A "cluster of differentiation 247 (CD247) polynucleotide" means a nucleic acid molecule that encodes a CD247 polypeptide. Exemplary CD247 nucleic acid sequences are provided below.

[0222] > NM_ NM_198053.3 Homo sapiens CD247 molecule (CD247), transcript variant 1, mRNA

[0223]

[0224] "Co-administration" or "co-administering" refers to the administration of two or more therapeutic agents or pharmaceutical compositions during the course of therapy. The co-administration can be concurrent or sequential administration. Sequential administration of a later administered therapeutic agent or pharmaceutical composition can occur at any time during the course of administration of the first pharmaceutical composition or therapeutic agent.

[0225] The term "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid by another amino acid with shared properties. A functional way to define shared properties among individual amino acids is to analyze the normalized frequency 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)). From such an analysis, groups of amino acids can be defined in which amino acids within a group preferentially exchange with each other and thus are most similar to each other in their impact on the overall protein structure (Schulz, G. E. and Schirmer, R. H., supra). Non-limiting examples of conservative mutations include amino acid substitutions of amino acids, e.g., 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.

[0226] The term "coding sequence" or "protein coding sequence," used interchangeably herein, refers to a segment of a polynucleotide that encodes a protein. The region or sequence is bound at the 5' end by a start codon and at the 3' end by a stop codon. A coding sequence can also be referred to as an open reading frame.

[0227] "Codon optimization" means the process of modifying a nucleic acid sequence for enhanced expression in a host cell of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with a codon that is more frequently or most frequently used in genes of the host cell while maintaining the native amino acid sequence. Various species exhibit certain biases for particular amino acids for certain codons. Codon bias (differences in codon usage between organisms) is often reflected in the dominance of certain transfer RNA (tRNA) molecules available in a cell. The predominance of a selected tRNA in a cell often reflects the most common codons used in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, e.g., at the "Codon Usage Database" available at www.kazusa.orjp / codon / (accessed July 9, 2002), and these tables can be adapted in a variety of ways. See, Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000). Computer algorithms are also available for codon optimizing a particular sequence for expression in a particular host cell, such as Gene Forge (Aptagen; Jacobus, Pa.). In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding an engineered nuclease correspond to the most frequently used codons for a particular amino acid.

[0228] "Cytidine deaminase" means a polypeptide or fragment thereof capable of catalyzing a deamination reaction that converts an amino group to a carbonyl group. In one embodiment, a cytidine deaminase converts cytosine to uracil or 5-methylcytosine to thymine. PmCDA1 (Petromyzon marinus cytosine deaminase 1, ("PmCDA1")), derived from sea lamprey (Petromyzon marinus), or AID (activation-induced cytidine deaminase; ("AICDA")) and APOBEC, derived from mammals (e.g., humans, pigs, cows, horses, monkeys, etc.), are exemplary cytidine deaminases.

[0229] The base and amino acid sequences of PmCDA1 and the base and amino acid sequences of human AID are shown below.

[0230] >tr|A5H718|A5H718_PETMA Cytidine deaminase OS=Petromyzon marinus OX=7757 PE=2 SV=1

[0231] MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQS

[0232] GTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHT

[0233] LKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWL

[0234] EKTLKRAEKRRSELSIMIQVKILHTTKSPAV

[0235] >EF094822.1 Petromyzon marinus isolate PmCDA.21 cytidine deaminase mRNA, complete cds

[0236] TGACACGACACAGCCGTGTATATGAGGAAGGGTAGCTGGATGGGGGGGGGGGGAATACGTTCAGAGAGGACATTAGCGAGCGTCTTGTTGGTGGCCTTGAGTCTAGACACCTGCAGACATGACCGACGCTGAGTACGTGAGAATCCATGAGAAGTTGGACATCTACACGTTTAAGAAACAGTTTTTCAACAACAAAAAATCCGTGTCGCATAGATGCTACGTTCTCTTTGAATTAAAACGACGGGGTGAACGTAGAGCGTGTTTTTGGGGCTATGCTGTGAATAAACCACAGAGCGGGACAGAACGTGGAATTCACGCCGAAATCTTTAGCATTAGAAAAGTCGAAGAATACCTGCGCGACAACCCCGGACAATTCACGATAAATTGGTACTCATCCTGGAGTCCTTGTGCAGATTGCGCTGAAAAGATCTTAGAATGGTATAACCAGGAGCTGCGGGGGAACGGCCACACTTTGAAAATCTGGGCTTGCAAACTCTATTACGAGAAAAATGCGAGGAATCAAATTGGGCTGTGGAACCTCAGAGATAACGGGGTTGGGTTGAATGTAATGGTAAGTGAACACTACCAATGTTGCAGGAAAATATTCATCCAATCGTCGCACAATCAATTGAATGAGAATAGATGGCTTGAGAAGACTTTGAAGCGAGCTGAAAAACGACGGAGCGAGTTGTCCATTATGATTCAGGTAAAAATACTCCACACCACTAAGAGTCCTGCTGTTTAAGAGGCTATGCGGATGGTTTTC

[0237] >tr|Q6QJ80|Q6QJ80_Homo sapiens activation-induced cytidine deaminase OS=Homo sapiens OX=9606 GN=AICDA PE=2 SV=1

[0238] MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKAPV

[0239] >NG_011588.1:5001-15681 Homo sapiens Activating Inducible Cytidine Deaminase (AICDA), RefSeqGene (LRG_17) on chromosome 12

[0240]

[0241] Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) is an evolutionarily conserved family of cytidine deaminases. Members of this family are C to U editing enzymes. The N-terminal domain of APOBEC-like proteins is the catalytic domain, while the C-terminal domain is a pseudocatalytic domain. More specifically, the catalytic domain is a zinc-dependent cytidine deaminase domain and is important for cytidine deamination. APOBEC family members include APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D (now called “APOBEC3E”), APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and activation-induced (cytidine) deaminase. Many of the modified cytidine deaminases are commercially available, including but not limited to, SaBE3, SaKKH-BE3, VQR-BE3, EQR-BE3, VRER-BE3, YE1-BE3, EE-BE3, YE2-BE3, and YEE-BE3, which are available from Addgene (plasmids 85169, 85170, 85171, 85172, 85173, 85174, 85175, 85176, 85177).

[0242] Other exemplary deaminases that can be fused to Cas9 in accordance with aspects of the present disclosure are provided herein. It should be appreciated that in some embodiments, active domains of the respective sequences can be used, e.g., domains that do not have localization signals (nuclear localization sequence, no nuclear export signal, cytoplasmic localization signal).

[0243] As used herein, the term "deaminase" or "deaminase domain" refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase or deaminase domain is a cytidine deaminase that catalyzes the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the deaminase or deaminase domain is a cytosine deaminase that catalyzes the hydrolytic deamination of cytosine to uracil. In some embodiments, the deaminase is an adenosine deaminase that catalyzes the hydrolytic deamination of adenine to hypoxanthine. In some embodiments, the deaminase is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine or adenine (A) to inosine (I). In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases provided herein (e.g., engineered adenosine deaminases, evolved adenosine deaminases) can be from any organism such as a bacterium. In some embodiments, the adenosine deaminase is from a bacterium such as Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Shewanella putrefaciens, Haemophilus influenzae, or Caulobacter crescentus.

[0244] In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the adenosine deaminase or deaminase domain does not exist in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to a naturally occurring deaminase.

[0245] For example, base editors are described in International PCT Application Nos. PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), each of which is incorporated by reference herein in its entirety. See also 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., et al., “Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); 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: eaoo4774 (2017); and Rees, H.A., et al., “Base editing: precision chemistry on the genome and transcriptome of living cells.” Nat Rev Genet. 2018 Dec; 19(12): 770-788. doi: 10.1038 / s41576-018-0059-1, the entire contents of which are incorporated by reference herein.

[0246] “Detecting” means identifying the presence, absence, or amount of an analyte to be detected.

[0247] “Detectable label” means a composition that is linked to a molecule of interest such that the latter can be detected via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radiolabels, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in enzyme-linked immunoabsorbent assay (ELISA)), biotin, digoxigenin, or haptens.

[0248] "Disease" means any condition or pathology that impairs or interferes with the normal functioning of a cell, tissue, or organ. In an embodiment, the disease is a neoplasia or a cancer. In some embodiments, the disease is a hematological cancer. "Hematological cancer" means a malignant pathology of cells of the immune system. In some embodiments, the hematological cancer is a leukemia, myeloma, and / or lymphoma. Lymphomas and leukemias are examples of "liquid cancers" or cancers that exist in the blood and arise from the transformation of hematopoietic precursors in the bone marrow or mature hematopoietic cells in the blood. Leukemias can be lymphoid or myeloid and acute or chronic. In the case of myeloma, the transformed cells are fully differentiated plasma cells, which can exist as a diffuse collection of malignant cells or as a solid mass in the bone marrow. In the case of lymphoma, transformed lymphocytes in secondary lymphoid tissues give rise to solid masses. Lymphomas are classified as Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL).

[0249] 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 includes a pre-leukemia. In some cases, the leukemia is an acute leukemia. Acute leukemias include, for example, acute myeloid leukemia (AML). Acute leukemias also include, for example, acute lymphoid leukemia or acute lymphoblastic leukemia (ALL); ALL includes B-lineage ALL, T-lineage ALL, and T-cell acute lymphoblastic leukemia (T-ALL).

[0250] Non-limiting examples of diseases include T-cell acute lymphoblastic leukemia (T-ALL), mycosis fungoides (MF), Sezary syndrome (SS), peripheral T / NK cell lymphoma, anaplastic large cell lymphoma ALK+, primary cutaneous T-cell lymphoma, T-cell large granular lymphocyte type 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 gd T-cell lymphoma, aggressive NK-cell leukemia, and enteropathy-associated T-cell lymphoma. In some embodiments, the disease is a liquid tumor. In some embodiments, the disease is T-cell acute lymphoblastic leukemia (T-ALL). In some embodiments, the disease is T-cell acute myeloid leukemia (AML).

[0251] As used herein, the term "effective amount" refers to the amount of a biologically active agent sufficient to elicit a desired biological response. In some embodiments, an effective amount is the amount required to alleviate disease symptoms relative to untreated patients. Effective amounts of the active agents used to practice the present application for therapeutic treatment of a disease vary depending upon the manner of administration and 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 present application (e.g., a fusion protein comprising a programmable DNA binding protein, a nucleobase editor, and a gRNA) sufficient to introduce an alteration into a gene of interest within 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 (e.g., to reduce or control a disease or a symptom or condition thereof). Such a therapeutic effect need not be sufficient to alter the gene of interest in all cells of a subject, tissue, or organ, but can be sufficient to alter the gene of interest in only about 1%, 5%, 10%, 25%, 50%, 75%, or more of the cells present in a subject, tissue, or organ.

[0252] In some embodiments, an effective amount of a fusion protein provided herein, e.g., a nucleobase editor comprising a nCas9 domain and a deaminase domain (e.g., an adenosine deaminase domain or a cytidine deaminase domain), refers to an amount of the fusion protein sufficient to induce editing of a target site specifically bound and edited by the nucleobase editor described herein. As will be appreciated by those skilled in the art, the amount of an agent (e.g., a fusion protein, a nuclease, a hybrid protein, a protein dimer, a complex of a protein (or protein dimer) and a polynucleotide, or a polynucleotide) can vary depending on various factors, e.g., depending on the desired biological response, e.g., depending on the specific allele, genome, or target site to be edited, depending on the cell or tissue to be targeted, and / or depending on the agent used. In the context of CAR-T cells, an "effective amount" refers to the number of cells necessary to be administered to a patient to achieve a therapeutic response.

[0253] The term "epitope" as used herein means an antigenic determinant. An epitope is a portion of an antigenic molecule that is recognized by and binds to a specific antibody.

[0254] "Fas cell surface death receptor (FAS) polypeptide" means a protein having at least about 85% amino acid sequence identity to NCBI Accession No. NP_000034.1 or a fragment thereof. An exemplary amino acid sequence is provided below.

[0255] >NP_000034.1 Tumor necrosis factor receptor superfamily member 6 isoform 1 precursor [Homo sapiens]

[0256]

[0257] "Fas cell surface death receptor (FAS) polynucleotide" means a nucleic acid encoding a FAS polypeptide. Exemplary FAS nucleic acid sequences are provided below.

[0258] > NM_000043.6 Homo sapiens Fas cell surface death receptor (FAS), transcript variant 1, mRNA

[0259]

[0260]

[0261]

[0262]

[0263] "Fragment" means a portion of a polypeptide or nucleic acid molecule. The 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 can 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.

[0264] "Fratricide" means killing of an immune cell by other immune cells, including self-antigen driven killing of immune cells. In certain embodiments, immune cells of the present application are genetically modified to prevent or reduce expression of an antigen recognized by immune cells expressing a chimeric antigen receptor (CAR), thereby preventing or reducing fratricide. In various embodiments, fratricide can occur in vivo (e.g., in a subject) or ex vivo (e.g., in a preparation of immune cells).

[0265] "GVHD" or "graft versus host disease" refers to a pathological condition in which transplanted donor cells mount an immune response against host cells.

[0266] "Guide RNA" or "gRNA" means a polynucleotide that can have specificity 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). A gRNA can exist as a complex of two or more RNAs, or as a single RNA molecule. A gRNA that exists as a single RNA molecule can be referred to as a single guide RNA (sgRNA), but "gRNA" is used interchangeably to refer to a guide RNA that exists as a single molecule or as a complex of two or more molecules. Typically, a gRNA that exists as a single RNA species comprises two domains: (1) a domain that shares homology with a target nucleic acid (e.g., and directs binding of a Cas9 complex to the target); and (2) a domain that binds a Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as a tracrRNA and comprises a stem loop structure. For example, in some embodiments, domain (2) is identical or homologous to the tracrRNA provided in Jinek et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Other examples of gRNAs (e.g., those that include domain 2) can be found in US20160208288 entitled "Switchable Cas9 Nucleases and Uses Thereof" and US 9,737,604 entitled "Delivery System For Functional Nucleases," the entire contents of each patent are incorporated herein by reference in their entirety. In some embodiments, a gRNA comprises two or more domains (1) and (2) and can be a "spreading gRNA." A spreading gRNA will bind two or more Cas9 proteins and bind a target nucleic acid at two or more different regions, as described herein. A gRNA comprises a nucleotide sequence that is complementary to a target site, which mediates binding of a nuclease / RNA complex to the target site, providing sequence specificity of the nuclease:RNA complex. As will be appreciated by those skilled in the art, RNA polynucleotide sequences, e.g., gRNA sequences, include the nucleobase uracil (U), a pyrimidine derivative, rather than the nucleobase thymine (T), which is included in DNA polynucleotide sequences. In RNA, uracil base pairs with adenine and substitutes for thymine during DNA transcription.

[0267] By "heterodimer" is meant a fusion protein comprising two domains, such as a wild-type TadA domain and a variant of a TadA domain (e.g., TadA*8), or two variant TadA and dizziness (e.g., TadA*7.10 and TadA*8, or two TadA*8 domains).

[0268] By "host versus graft disease" (HVGD) is meant a pathological condition in which the immune system of a host mounts an immune response against transplanted donor cells.

[0269] By "hybridize" is meant hydrogen bonding between complementary nucleobases, which can be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonding. For example, adenine and thymine are complementary nucleobases that pair by forming hydrogen bonds.

[0270] By "immune cell" is meant a cell of the immune system that is capable of mounting an immune response.

[0271] By "immune effector cell" is meant a lymphocyte that, once activated, is capable of effecting an immune response against a target cell. In some embodiments, the immune effector cell is an effector T cell. In some embodiments, the effector T cell is a natural 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, the immune effector cell is an effector NK cell. In some embodiments, the effector T cell is a thymocyte, an immature T lymphocyte, a mature T lymphocyte, a resting T lymphocyte, or an activated T lymphocyte. 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 CD4-expressing helper T cell (CD4+ T cell).

[0272] By "immune response modulating gene" or "immune response modulating factor" is meant a gene that encodes a polypeptide involved in the modulation of an immune response. An immune response modulating gene can modulate an immune response in a variety of mechanisms or at different levels. For example, an immune response modulating gene can inhibit or promote the activation of an immune cell, such as a T cell. An immune response modulating gene can increase or decrease the activation threshold of an immune cell. In some embodiments, an immune response modulating gene positively modulates an immune cell signaling pathway. In some embodiments, an immune response modulating gene negatively modulates an immune cell signaling pathway. In some embodiments, an immune response modulating gene encodes an antigen, an antibody, a cytokine, or a neuroendocrine.

[0273] An "immunogenic gene" means a gene that encodes a polypeptide that elicits an immune response. For example, an immunogenic gene can encode an immunogen that elicits an immune response. In some embodiments, an immunogenic gene encodes a cell surface protein. In some embodiments, an immunogenic gene encodes a cell surface antigen or a cell surface marker. In some embodiments, a cell surface marker is a T cell marker or a B cell marker. In some embodiments, an immunogenic gene encodes a CD2, CD3e, CD3 delta, CD3 gamma, TRAC, TRBC1, TRBC2, CD4, CD5, CD7, CD8, CD19, CD23, CD27, CD28, CD30, CD33, CD52, CD70, CD127, CD122, CD130, CD132, CD38, CD69, CD11a, CD58, CD99, CD103, CCR4, CCR5, CCR6, CCR9, CCR10, CXCR3, CXCR4, CLA, CD161, B2M, or CIITA polypeptide.

[0274] The term "inhibitor of base repair" or "IBR" refers to a protein that is capable of inhibiting the activity of a nucleic acid repair enzyme (e.g., a base excision repair (BER) enzyme). In some embodiments, the IBR is an inhibitor of inosine base excision repair. Exemplary editing repair inhibitors include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGGl, hNEILl, T7 Endol, T4PDG, UDG, hSMUGl, and hAAG. In some embodiments, the IBR is an inhibitor of Endo V or hAAG. In some embodiments, the IBR is a catalytically inactive Endo V or a catalytically inactive hAAG. In some embodiments, the base repair inhibitor is an inhibitor of Endo V or hAAG. In some embodiments, the base repair inhibitor is a catalytically inactive Endo V or a catalytically inactive hAAG.

[0275] In some embodiments, the base repair inhibitor is a uracil glycosylase inhibitor (UGI). UGI refers to a protein that is capable of inhibiting the uracil-DNA glycosylase base excision repair enzyme. In some embodiments, the UGI domain comprises a wild-type UGI or a fragment of a wild-type UGI. In some embodiments, the UGI proteins provided herein include fragments of UGI and proteins homologous to UGI or UGI fragments. In some embodiments, the base repair inhibitor is an inhibitor of inosine base excision repair. In some embodiments, the base repair inhibitor is an "apoenzyme inosine-specific nuclease" or a "dead inosine-specific nuclease." Without wishing to be bound by a particular theory, an apoenzyme inosine glycosylase (e.g., alkyladenine glycosylase (AAG)) can bind to inosine, but is unable to create an abasic site or remove the inosine, thereby sterically blocking the newly formed inosine moiety from the DNA damage / repair machinery. In some embodiments, an apoenzyme inosine-specific nuclease can be able to bind to inosine in a nucleic acid but not cleave the nucleic acid. Non-limiting exemplary apoenzyme inosine-specific nucleases include an apoenzyme alkyladenine glycosylase (AAG nuclease), e.g., from humans; and an apoenzyme endonuclease V (EndoV nuclease), e.g., from E. coli. In some embodiments, the apoenzyme AAG nuclease comprises an E125Q mutation or a corresponding mutation in another AAG nuclease.

[0276] "increasing" means a positive change of at least 10%, 25%, 50%, 75%, or 100%.

[0277] An "intein" is a segment of a protein that is capable of excising itself and joining the remaining segment (extein) with a peptide bond in a process known as protein splicing. Inteins are also known as "protein introns." The process by which an intein excises itself and joins the remaining portion of a protein is referred to herein as "protein splicing" or "intein-mediated protein splicing." In some embodiments, the intein of a precursor protein (a protein containing an intein prior to intein-mediated protein splicing) is from two genes. Such an intein is referred to herein as a split intein (e.g., split intein-N and split intein-C). For example, in cyanobacteria, DnaE (i.e., the catalytic subunit of DNA polymerase III) is encoded by two separate genes (i.e., dnaE-n and dnaE-c). The intein encoded by the dnaE-n gene can be referred to herein as "intein-N." The intein encoded by the dnaE-c gene can be referred to herein as "intein-C."

[0278] Other intein systems can also be used. For example, synthetic inteins based on the dnaE intein, i.e., the Cfa-N (e.g., split intein-N) and Cfa-C (e.g., split intein-C) intein pair, have been described (e.g., in Stevens et al., J Am Chem Soc. 2016 Feb. 24; 138(7):2162-5, which is incorporated herein by reference). Non-limiting examples of intein pairs that can be used in accordance with the present disclosure include: Cfa DnaE intein, Ssp GyrB intein, Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein, and Cne Prp8 intein (e.g., as described in U.S. Patent No. 8,394,604, which is incorporated herein by reference).

[0279] Nucleotide and amino acid sequences of exemplary inteins are provided below.

[0280] DnaE Intein-N DNA:

[0281] TGCCTGTCATACGAAACCGAGATACTGACAGTAGAATATGGCCTTCTGCCAATCGGGAAGATTGTGGAGAAACGGATAGAATGCACAGTTTACTCTGTCGATAACAATGGTAACATTTATACTCAGCCAGTTGCCCAGTGGCACGACCGGGGAGAGCAGGAAGTATTCGAATACTGTCTGGAGGATGGAAGTCTCATTAGGGCCACTAAGGACCACAAATTTATGACAGTCGATGGCCAGATGCTGCCTATAGACGAAATCTTTGAGCGAGAGTTGGACCTCATGCGAGTTGACAACCTTCCTAAT

[0282] DnaE Intein-N Protein:

[0283] CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN

[0284] DnaE Intein-C DNA:

[0285] ATGATCAAGATAGCTACAAGGAAGTATCTTGGCAAACAAAACGTTTATGATATTGGAGTCGAAAGAGATCACAACTTTGCTCTGAAGAACGGATTCATAGCTTCTAAT

[0286] Intein-C: MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN

[0287] Cfa-N DNA:

[0288] TGCCTGTCTTATGATACCGAGATACTTACCGTTGAATATGGCTTCTTGCCTATTGGAAAGATTGTCGAAGAGAGAATTGAATGCACAGTATATACTGTAGACAAGAATGGTTTCGTTTACACACAGCCCATTGCTCAATGGCACAATCGCGGCGAACAAGAAGTATTTGAGTACTGTCTCGAGGATGGAAGCATCATACGAGCAACTAAAGATCATAAATTCATGACCACTGACGGGCAGATGTTGCCAATAGATGAGATATTCGAGCGGGGCTTGGATCTCAAACAAGTGGATGGATTGCCA

[0289] Cfa-N protein:

[0290] CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP

[0291] Cfa-C DNA:

[0292] ATGAAGAGGACTGCCGATGGATCAGAGTTTGAATCTCCCAAGAAGAAGAGGAAAGTAAAGATAATATCTCGAAAAAGTCTTGGTACCCAAAATGTCTATGATATTGGAGTGGAGAAAGATCACAACTTCCTTCTCAAGAACGGTCTCGTAGCCAGCAAC

[0293] Cfa-C protein: MKRTADGSEFESPKKKRKVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN

[0294] Intein-N and Intein-C can be fused to the N-terminal portion of split Cas9 and the C-terminal portion of split Cas9, respectively, for joining the N-terminal portion of split Cas9 to the C-terminal portion of split Cas9. For example, in some embodiments, Intein-N is fused to the C-terminus of the N-terminal portion of split Cas9, i.e., to form the structure N— [N-terminal portion of split Cas9]— [Intein-N]— C. In some embodiments, Intein-C is fused to the N-terminus of the C-terminal portion of split Cas9, i.e., to form the structure N— [Intein-C]— [C-terminal portion of split Cas9]— C. Intein-mediated protein splicing mechanisms for proteins to which Inteins are fused (e.g., split Cas9) are known in the art, e.g., as described in Shah et al., Chem Sci. 2014; 5(1): 446-461, which is incorporated herein by reference. Methods for designing and using inteins are known in the art and described by, e.g., WO2014004336, WO2017132580, US20150344549, and US20180127780, each of which is incorporated herein by reference in its entirety.

[0295] The terms "isolated," "purified," or "biologically pure," denote a material that is not in its natural environment (e.g., in its native state). "Isolation" denotes a degree of separation that is greater than that of the original source or surrounding material. "Purification" denotes a degree of separation that is greater than that of isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not affect the biological properties of the protein or cause other adverse consequences at the level of the material. In other words, when produced by recombinant DNA technology, a nucleic acid or peptide of the application is purified if it is substantially free of cellular material, viral material, or culture medium; or when chemically synthesized, it is purified if it is free of chemical precursors or other chemicals. Purity is typically determined using analytical chemistry techniques, e.g., polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can mean that a nucleic acid or protein produces substantially one band on an electrophoretic gel. For proteins that can be modified, e.g., phosphorylated or glycosylated, different modifications can produce different isolated proteins, which can be purified independently.

[0296] "Isolated polynucleotide" means a nucleic acid (e.g., DNA) that is free of the genes that, in the natural genome of the organism from which the nucleic acid molecule of the application is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction analysis) independent of other sequences. Moreover, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequences.

[0297] "Isolated polypeptide" means a polypeptide of the application that has been separated from components that naturally accompany it. Typically, the polypeptide is at least 60% by weight of the protein and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight of the polypeptide of the application. An isolated polypeptide of the application can be obtained, for example, by extraction from natural sources, by expression of recombinant nucleic acids encoding the polypeptide, or by chemical synthesis of the protein. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0298] "Leader peptide" means a short amino acid sequence (e.g., about 16 to 30 amino acids in length) that directs newly synthesized secreted or membrane proteins to and through the membrane (e.g., the endoplasmic reticulum membrane). The leader peptide is typically positioned at the N-terminus of the polypeptide, and can be removed by signal peptidase after the polypeptide has crossed the membrane. Leader peptide sequences typically contain three consensus structural features: an N-terminal polar basic region (n-region), a hydrophobic core, and a hydrophilic c-region. In some embodiments, the CAR of the application includes a leader peptide sequence (e.g., at the N-terminus of the antigen binding domain). An exemplary leader peptide amino acid sequence is: METDTLLLWVLLLWVPGSTG.

[0299] As used herein, the term "linker" refers to a bond (e.g., a covalent bond), chemical group, or molecule that links two molecules or two moieties, e.g., two components of a protein complex or a ribonucleic acid complex or two domains of a fusion protein, e.g., a polynucleotide programmable DNA binding domain (e.g., dCas9) and a deaminase domain (e.g., an adenosine deaminase, a cytidine deaminase). A linker can join different components or different portions of multiple components of a base editor system. For example, in some embodiments, a linker can join a guide polynucleotide binding domain of a polynucleotide programmable nucleotide binding domain to a catalytic domain of a deaminase. In some embodiments, a linker can join a CRISPR polypeptide to a deaminase. In some embodiments, a linker can join a Cas9 to a deaminase. In some embodiments, a linker can join a dCas9 to a deaminase. In some embodiments, a linker can join a nCas9 to a deaminase. In some embodiments, a linker can join a guide polynucleotide to a deaminase. In some embodiments, a linker can join a deaminase component of a base editor system to a polynucleotide programmable nucleotide binding component. In some embodiments, a linker can join an RNA binding portion of a deaminase component of a base editor system to a polynucleotide programmable nucleotide binding component. In some embodiments, a linker can join an RNA binding portion of a deaminase component of a base editor system to an RNA binding portion of a polynucleotide programmable nucleotide binding component. A linker can be located between or flanking two groups, molecules, or other moieties and coupled to one another via a covalent bond or a non-covalent interaction, thereby coupling the two. In some embodiments, a linker can be an organic molecule, group, polymer, or chemical moiety. In some embodiments, a linker can be a polynucleotide. In some embodiments, a linker can be a DNA linker. In some embodiments, a linker can be an RNA linker. In some embodiments, a linker can comprise an aptamer capable of binding to a ligand. In some embodiments, a ligand can be a carbohydrate, a peptide, a protein, or a nucleic acid. In some embodiments, a linker can comprise an aptamer that can be derived from a riboswitch. A riboswitch from which an aptamer is derived can be selected from theophylline riboswitch, pyridoxal 5'-phosphate (TPP) riboswitch, adenosylcobalamin (AdoCbl) riboswitch, S-adenosylmethionine (SAM) riboswitch, SAH riboswitch, flavin mononucleotide (FMN) riboswitch, tetrahydrofolate riboswitch, lysine riboswitch, glycine riboswitch, purine riboswitch, GlmS riboswitch, or Q riboswitch precursor 1 (PreQ1) riboswitch. In some embodiments, a linker can comprise an aptamer that binds to a polypeptide or protein domain, such as a polypeptide ligand.In some embodiments, the polypeptide ligand can be a K homology (KH) domain, a MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, an asexuality alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif. In some embodiments, the polypeptide ligand can be part of a base editor system component. For example, a nucleic acid base editing component can comprise a deaminase domain and an RNA recognition motif.

[0300] In some embodiments, the linker can be one amino acid or a plurality of amino acids (e.g., a peptide or a protein). In some embodiments, the linker can be about 5 to 100 amino acids in length, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, or 90 to 100 amino acids in length. In some embodiments, the linker can be about 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 400 to 450, or 450 to 500 amino acids in length. Longer or shorter linkers can also be contemplated.

[0301] In some embodiments, a linker joins the gRNA binding domain of an RNA programmable nuclease (including a Cas9 nuclease domain) to the catalytic domain of a nucleic acid editing protein (e.g., a cytidine or adenosine deaminase). In some embodiments, a linker joins dCas9 to a nucleic acid editing protein. For example, a linker is positioned between or flanked by two groups, molecules, or other moieties, and is linked to each other via a covalent bond, thereby linking the two. In some embodiments, a linker is one amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, a linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, a linker is 5 to 200 amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 45, 50, 55, 60, 60, 65, 70, 70, 75, 80, 85, 90, 90, 95, 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 175, 180, 190, or 200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, a linker comprises the amino acid sequence of SGSETPGTSESATPES, which can also be referred to as an XTEN linker. In some embodiments, a linker comprises the amino acid sequence SGGS. In some embodiments, a linker comprises the (SGGS)n, (GGGS)n, (GGGGS)n, (G)n, (EAAAK)n, (GGS)n, SGSETPGTSESATPES, or (XP)nmotif, or a combination of any of these, where n is independently an integer between 1 and 30, and where X is any amino acid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, a linker comprises a plurality of proline residues and is 5 to 21, 5 to 14, 5 to 9, 5 to 7 amino acids in length, e.g., PAPAP, PAPAPA, PAPAPAP, PAPAPAPA, P(AP)4, P(AP)7, P(AP)10. Such proline-rich linkers are also referred to as “stiff” linkers.

[0302] In some embodiments, the chimeric antigen receptor comprises at least one linker. The at least one linker joins or links a variable heavy (VH) region to a constant heavy (CH) region of an extracellular binding domain of the chimeric antigen receptor. The linker can also link a variable light (VL) region to a variable constant (VC) region of the extracellular binding domain.

[0303] In some embodiments, the domains of the base editor are fused via a linker comprising the amino acid sequence of SGSETPGTSESATPES, also referred to as an XTEN linker. In some embodiments, the linker comprises the amino acid sequence SGGS. In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES. In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS. In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGSSGGS. In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS.

[0304] SGGSSGSETPGTSESATPESSGGS,

[0305] SGGSSGGSSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS.

[0306] In some embodiments, the domains of the base editor are fused via a linker comprising the amino acid sequence of SGSETPGTSESATPES, also referred to as an XTEN linker. In some embodiments, the linker comprises the amino acid sequence SGGS. In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES. In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS. In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGSSGGS. In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS.

[0307] As used herein, the term "liquid cancer" refers to cancer cells that are present in body fluids, such as blood, lymph, and bone marrow. Liquid cancers include, but are not limited to, leukemias, myelomas, and liquid lymphomas. As used herein, liquid cancers do not include solid tumors such as sarcomas and carcinomas, or solid lymphomas that do not contain cysts or liquid areas. A "liquid cancer" can be recurrent, refractory, or metastatic. Liquid cancers to be treated using the methods described herein can be, for example, also lymphomas; liquid lymphomas include lymphomas that contain cysts or liquid areas.

[0308] "Lymphocyte activation gene 3 (LAG-3) polypeptide" means a protein having at least about 85% amino acid sequence identity to NCBI Accession No. NP_002277.4 or a fragment thereof and having immunomodulatory activity. An exemplary amino acid sequence is provided below.

[0309] >NP_002277.4 Lymphocyte activation gene 3 protein precursor [Homo sapiens]

[0310]

[0311] "Lymphocyte activation gene 3 (LAG-3) polynucleotide" means a nucleic acid encoding a LAG-3 polypeptide. An exemplary LAG-3 nucleic acid sequence is provided below.

[0312] >NM_002286.6 Homo sapiens lymphocyte activation 3 (LAG3), mRNA

[0313]

[0314]

[0315]

[0316] "Marker" means any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.

[0317] As used herein, the term "mutation" refers to a substitution of a residue within a sequence (e.g., a nucleic acid or amino acid sequence) for another residue, or a deletion or insertion of one or more residues within a sequence. Herein, mutations are generally described as: the identity of the original residue, then the position of that residue in the sequence, then the identity of the new substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art and are provided by, e.g., Molecular Cloning: A Laboratory Manual (4th ed., Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). In some embodiments, the base editors disclosed herein can be effective to generate a "desired mutation" such as a point mutation in a nucleic acid (e.g., a nucleic acid within a subject's genome) without generating a significant number of undesired mutations such as undesired point mutations. In some embodiments, the desired mutation is a mutation generated by a specific base editor (e.g., a cytidine base editor or an adenosine base editor) that is designed to generate the desired mutation in conjunction with a guide polynucleotide (e.g., a gRNA).

[0318] Generally, mutations made or identified in a sequence (e.g., an amino acid sequence described herein) are numbered relative to a reference (or wild-type) sequence (i.e., the sequence that does not contain the mutation). Those of skill in the art will readily understand how to determine the location of a mutation in an amino acid and nucleic acid sequence relative to a reference sequence.

[0319] A "neoplasia" refers to a cell or tissue that exhibits abnormal growth or proliferation. The term neoplasia encompasses cancer, liquid and solid tumors. In some embodiments, the neoplasia is a solid tumor. 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 a 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 leukemia. In some cases, the leukemia includes a pre-leukemia. In some cases, the leukemia is an acute leukemia. Acute leukemias include, for example, acute myeloid leukemia (AML). Acute leukemias also include, for example, acute lymphoid leukemia or acute lymphoblastic leukemia (ALL); ALL includes B-lineage ALL, T-lineage ALL, and T-cell acute lymphoblastic leukemia (T-ALL).

[0320] Non-limiting examples of neoplasia include T-cell acute lymphoblastic leukemia (T-ALL), mycosis fungoides (MF), Sezary Syndrome (SS), peripheral T / NK cell lymphoma, anaplastic large cell lymphoma ALK+, primary cutaneous T-cell lymphoma, T-cell large granular lymphocyte type 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 gd T-cell lymphoma, aggressive NK-cell leukemia, and enteropathy-associated T-cell lymphoma. In some embodiments, the neoplasia is T-cell acute lymphoblastic leukemia (T-ALL). In some embodiments, the neoplasia is T-cell acute myeloid leukemia (AML).

[0321] “Activated T-cell nuclear factor 1 (NFATcl) polypeptide” means a protein having at least about 85% sequence identity to NCBI Accession No. NM_172390.2 or a fragment thereof and is a component of the activated T-cell DNA-binding transcription complex. An exemplary amino acid sequence is provided below.

[0322] >NP_765978.1 Activated T-cell nuclear factor, cytoplasmic 1 isoform A [Homo sapiens]

[0323] MPSTSFPVPSKFPLGPAAAVFGRGETLGPAPRAGGTMKSAEEEHYGYASSNVSPALPLPTAHSTLPAPCHNLQTSTPGIIPPADHPSGYGAALDGGPAGYFLSSGHTRPDGAPALESPRIEITSCLGLYHNNNQFFHDVEVEDVLPSSKRSPSTATLSLPSLEAYRDPSCLSPASSLSSRSCNSEASSYESNYSYPYASPQTSPWQSPCVSPKTTDPEEGFPRGLGACTLLGSPRHSPSTSPRASVTEESWLGARSSRPASPCNKRKYSLNGRQPPYSPHHSPTPSPHGSPRVSVTDDSWLGNTTQYTSSAIVAAINALTTDSSLDLGDGVPVKSRKTTLEQPPSVALKVEPVGEDLGSPPPPADFAPEDYSSFQHIRKGGFCDQYLAVPQHPYQWAKPKPLSPTSYMSPTLPALDWQLPSHSGPYELRIEVQPKSHHRAHYETEGSRGAVKASAGGHPIVQLHGYLENEPLMLQLFIGTADDRLLRPHAFYQVHRITGKTVSTTSHEAILSNTKVLEIPLLPENSMRAVIDCAGILKLRNSDIELRKGETDIGRKNTRVRLVFRVHVPQPSGRTLSLQVASNPIECSQRSAQELPLVEKQSTDSYPVVGGKKMVLSGHNFLQDSKVIFVEKAPDGHHVWEMEAKTDRDLCKPNSLVVEIPPFRNQRITSPVHVSFYVCNGKRKRSQYQRFTYLPANGNAIFLTVSREHERVGCFF

[0324] "Activating T cell nuclear factor 1 (NFATcl) polynucleotide" means a nucleic acid that encodes a NFATcl polypeptide. The NFATcl gene encodes a protein that is involved in inducible expression of cytokine genes, particularly IL-2 and IL-4, in T cells. An exemplary nucleic acid sequence is provided below.

[0325] NM_172390.2 Homo sapiens Activating T cell nuclear factor 1 (NFATC1), transcript variant 1, mRNA

[0326] GGCGGGCGCTCGGCGACTCGTCCCCGGGGCCCCGCGCGGGCCCGGGCAGCAGGGGCGTGATGTCACGGCA

[0327] GGGAGGGGGCGCGGGAGCCGCCGGGCCGGCGGGGAGGCGGGGGAGGTGTTTTCCAGCTTTAAAAAGGCAG

[0328] GAGGCAGAGCGCGGCCCTGCGTCAGAGCGAGACTCAGAGGCTCCGAACTCGCCGGCGGAGTCGCCGCGCC

[0329] AGATCCCAGCAGCAGGGCGCGGGCACCGGGGCGCGGGCAGGGCTCGGAGCCACCGCGCAGGTCCTAGGGC

[0330] CGCGGCCGGGCCCCGCCACGCGCGCACACGCCCCTCGATGACTTTCCTCCGGGGCGCGCGGCGCTGAGCC

[0331] CGGGGCGAGGGCTGTCTTCCCGGAGACCCGACCCCGGCAGCGCGGGGCGGCCGCTTCTCCTGTGCCTCCG

[0332] CCCGCCGCTCCACTCCCCGCCGCCGCCGCGCGGATGCCAAGCACCAGCTTTCCAGTCCCTTCCAAGTTTC

[0333] CACTTGGCCCTGCGGCTGCGGTCTTCGGGAGAGGAGAAACTTTGGGGCCCGCGCCGCGCGCCGGCGGCAC

[0334] CATGAAGTCAGCGGAGGAAGAACACTATGGCTATGCATCCTCCAACGTCAGCCCCGCCCTGCCGCTCCCC

[0335] ACGGCGCACTCCACCCTGCCGGCCCCGTGCCACAACCTTCAGACCTCCACACCGGGCATCATCCCGCCGG

[0336] CGGATCACCCCTCGGGGTACGGAGCAGCTTTGGACGGTGGGCCCGCGGGCTACTTCCTCTCCTCCGGCCA

[0337] CACCAGGCCTGATGGGGCCCCTGCCCTGGAGAGTCCTCGCATCGAGATAACCTCGTGCTTGGGCCTGTAC

[0338] CACAACAATAACCAGTTTTTCCACGATGTGGAGGTGGAAGACGTCCTCCCTAGCTCCAAACGGTCCCCCT

[0339] CCACGGCCACGCTGAGTCTGCCCAGCCTGGAGGCCTACAGAGACCCCTCGTGCCTGAGCCCGGCCAGCAG

[0340] CCTGTCCTCCCGGAGCTGCAACTCAGAGGCCTCCTCCTACGAGTCCAACTACTCGTACCCGTACGCGTCC

[0341] CCCCAGACGTCGCCATGGCAGTCTCCCTGCGTGTCTCCCAAGACCACGGACCCCGAGGAGGGCTTTCCCC

[0342] GCGGGCTGGGGGCCTGCACACTGCTGGGTTCCCCGCGGCACTCCCCCTCCACCTCGCCCCGCGCCAGCGT

[0343] CACTGAGGAGAGCTGGCTGGGTGCCCGCTCCTCCAGACCCGCGTCCCCTTGCAACAAGAGGAAGTACAGC

[0344] CTCAACGGCCGGCAGCCGCCCTACTCACCCCACCACTCGCCCACGCCGTCCCCGCACGGCTCCCCGCGGG

[0345] TCAGCGTGACCGACGACTCGTGGTTGGGCAACACCACCCAGTACACCAGCTCGGCCATCGTGGCCGCCAT

[0346] CTGGAGCAGCCGCCCTCAGTGGCGCTCAAGGTGGAGCCCGTCGGGGAGGACCTGGGCAGCCCCCCGCCCC

[0347] CTGGAGCAGCCGCCCTCAGTGGCGCTCAAGGTGGAGCCCGTCGGGGAGGACCTGGGCAGCCCCCCGCCCC

[0348] CGGCCGACTTCGCGCCCGAAGACTACTCCTCTTTCCAGCACATCAGGAAGGGCGGCTTCTGCGACCAGTA

[0349] CCTGGCGGTGCCGCAGCACCCCTACCAGTGGGCGAAGCCCAAGCCCCTGTCCCCTACGTCCTACATGAGC

[0350] CCGACCCTGCCCGCCCTGGACTGGCAGCTGCCGTCCCACTCAGGCCCGTATGAGCTTCGGATTGAGGTGC

[0351] AGCCCAAGTCCCACCACCGAGCCCACTACGAGACGGAGGGCAGCCGGGGGGCCGTGAAGGCGTCGGCCGG

[0352] AGGACACCCCATCGTGCAGCTGCATGGCTACTTGGAGAATGAGCCGCTGATGCTGCAGCTTTTCATTGGG

[0353] ACGGCGGACGACCGCCTGCTGCGCCCGCACGCCTTCTACCAGGTGCACCGCATCACAGGGAAGACCGTGT

[0354] CCACCACCAGCCACGAGGCCATCCTCTCCAACACCAAAGTCCTGGAGATCCCACTCCTGCCGGAGAACAG

[0355] CATGCGAGCCGTCATTGACTGTGCCGGAATCCTGAAACTCAGAAACTCCGACATTGAACTTCGGAAAGGA

[0356] GAGACGGACATCGGGAGGAAGAACACACGGGTACGGCTGGTGTTCCGCGTTCACGTCCCGCAACCCAGCG

[0357] GCCGCACGCTGTCCCTGCAGGTGGCCTCCAACCCCATCGAATGCTCCCAGCGCTCAGCTCAGGAGCTGCC

[0358] TCTGGTGGAGAAGCAGAGCACGGACAGCTATCCGGTCGTGGGCGGGAAGAAGATGGTCCTGTCTGGCCAC

[0359] AACTTCCTGCAGGACTCCAAGGTCATTTTCGTGGAGAAAGCCCCAGATGGCCACCATGTCTGGGAGATGG

[0360] AAGCGAAAACTGACCGGGACCTGTGCAAGCCGAATTCTCTGGTGGTTGAGATCCCGCCATTTCGGAATCA

[0361] GAGGATAACCAGCCCCGTTCACGTCAGTTTCTACGTCTGCAACGGGAAGAGAAAGCGAAGCCAGTACCAG

[0362] CGTTTCACCTACCTTCCCGCCAACGGTAACGCCATCTTTCTAACCGTAAGCCGTGAACATGAGCGCGTGG

[0363] GGTGCTTTTTCTAAAGACGCAGAAACGACGTCGCCGTAAAGCAGCGTGGCGTGTTGCACATTTAACTGTG

[0364] TGATGTCCCGTTAGTGAGACCGAGCCATCGATGCCCTGAAAAGGAAAGGAAAAGGGAAGCTTCGGATGCA

[0365] TTTTCCTTGATCCCTGTTGGGGGTGGGGGGCGGGGGTTGCATACTCAGATAGTCACGGTTATTTTGCTTC

[0366] TTGCGAATGTATAACAGCCAAGGGGAAAACATGGCTCTTCTGCTCCAAAAAACTGAGGGGGTCCTGGTGT

[0367] GCATTTGCACCCTAAAGCTGCTTACGGTGAAAAGGCAAATAGGTATAGCTATTTTGCAG

[0368] GCACCTTTAGG AATAAACTTTGCTTTTAAGCCTGTAAAAAAAAAAAAAA

[0369] The term“non-conservative mutation” includes amino acid substitutions between different groups, e.g., lysine for tryptophan, or phenylalanine for serine, etc. In this case, preferably the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the wild-type protein.

[0370] The term“nuclear localization sequence,”“nuclear localization signal,” or“NLS” refers to an amino acid sequence that facilitates the entry of a protein into the nucleus. Nuclear localization sequences are known in the art and are described, for example, in International PCT Application PCT / EP2000 / 011690 to Plank et al., filed November 23, 2000, and WO / 2001 / 038547, published 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 by, e.g., Koblan et al., Nature Biotech. 2018 doi: 10.1038 / nbt.4172. In some embodiments, the NLS comprises the amino acid sequence PKKKRKVEGADKRTADGSEFESPKKKRKV, KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRK, PKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

[0371] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, a polymeric nucleic acid, e.g., a nucleic acid molecule comprising three or more nucleotides, is a linear molecule in which adjacent nucleotides are linked to one another via phosphodiester linkages. In some embodiments, "nucleic acid" refers to a single nucleic acid residue (e.g., a nucleotide and / or a nucleoside). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more single nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" are 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 double-stranded DNA. A nucleic acid can be naturally occurring, e.g., in the context of a transcript, mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, a cosmid, a chromosome, a chromatid, or other naturally occurring nucleic acid molecule genome. On the other hand, a nucleic acid molecule can be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, a garand chromosome, an engineered genome, or a fragment thereof, or a synthetic DNA, RNA, DNA / RNA hybrid, or that includes non-naturally occurring nucleotides or nucleosides. Moreover, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs such as, for example, those with analog backbones. A nucleic acid can be purified from a natural source, produced using a recombinant expression system and optionally purified, chemically synthesized, etc. If applicable, e.g., in the case of a chemically synthesized molecule, a nucleic acid can comprise nucleoside analogs such as those with chemically modified bases or sugars as well as backbone modifications. Unless otherwise indicated, a nucleic acid sequence is presented in the 5' to 3' direction. In some embodiments, a nucleic acid is or comprises a natural nucleoside (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromo uridine, C5-fluoro uridine, C5-iodo uridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); a chemically modified base; a biologically modified base (e.g., a methylated base); an intervening base; a modified sugar (2'-e.g., fluoro ribose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or a modified phosphate group (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).The term “nucleic acid programmable DNA binding protein” or “napDNAbp” can be used interchangeably with “polynucleotide programmable nucleotide binding domain” and refers 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), which directs 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. The Cas9 protein can associate with a guide RNA, which directs the Cas9 protein to a specific DNA sequence complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas9 domain, e.g., 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), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, and Cas12j / CasF.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 Csx 12), CaslO, CaslOd, Casl2a / Cpfl, Casl2b / C2cl, Casl2c / C2c3, Casl2d / CasY, Casl2e / CasX, Casl2g, Casl2h, Casl2i, Casl2j / CasF, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx 17, Csx 14, Csx 10, Csx 16, CsaX, Csx3, Csx1, Csx1S, Csx 11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Type II Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARF, DinG, homologs thereof, or modified or engineered versions thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of the present disclosure, although they can not be specifically listed in the present 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 al., “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 of which are incorporated herein by reference.

[0372] The terms "nucleobase," "nitrogenous base," or "base," used interchangeably herein, refer to a nitrogen-containing biological compound that forms a nucleoside, which in turn serves as a component of a nucleotide. The ability of nucleobases to form base pairs and stack on top of one another directly leads to long chain helical structures, such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The five nucleobases, adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), are referred to as the primary nucleobases or standard nucleobases. Adenine and guanine are derived from purines, while cytosine, uracil, and thymine are derived from pyrimidines. 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-hydroxymethylcytosine. Both hypoxanthine and xanthine can be produced by deamination (replacement of an amino group with a carbonyl group) in the presence of mutagens. Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can come from a deamination reaction of cytosine. A "nucleoside" consists of a nucleobase and a five-carbon sugar (ribose or deoxyribose). Examples of nucleosides include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Examples of nucleosides with modified nucleobases include inosine (I), xanthosine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), and pseudouridine (Ψ). A "nucleotide" consists of a nucleobase, a five-carbon sugar (ribose or deoxyribose), and at least one phosphate group.

[0373] As used herein, the term "nucleobase editing domain" or "nucleobase editing protein" refers to a protein or enzyme that catalyzes nucleobase modifications 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), deamination, as well as non-templated nucleotide addition and insertion. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., an adenine deaminase or adenosine deaminase; or a cytidine deaminase or cytosine deaminase). In some embodiments, the nucleobase editing domain is more than one deaminase domain (e.g., an adenine deaminase or adenosine deaminase and a cytidine deaminase or cytosine deaminase). In some embodiments, the nucleobase editing domain can be a naturally occurring nucleobase editing domain. In some embodiments, the nucleobase editing domain can be a nucleobase editing domain engineered or evolved from a naturally occurring nucleobase editing domain. The nucleobase editing domain can be from any organism, such as a bacterium, a human, a chimpanzee, a gorilla, a monkey, a cow, a dog, a rat, or a mouse.

[0374] As used herein, “obtaining” in “obtaining a reagent” includes synthesizing, purchasing, or other means of acquiring the reagent. As used herein, “patient” or “subject” refers to a mammalian subject or individual diagnosed with or susceptible to or predisposed to developing or at risk of developing a disease or condition. In some embodiments, the term “patient” refers to a mammalian subject having a greater than average likelihood of developing a disease or condition. Exemplary patients can be humans, non-human primates, cats, dogs, pigs, cows, cats, horses, camels, llamas, goats, sheep, rodents (e.g., mice, rabbits, rats, hamsters, or guinea pigs), and other mammals that can benefit from the therapies disclosed herein. Exemplary human patients can be male and / or female.

[0375] Herein, “a patient in need thereof’ or “a subject in need thereof’ refers to a patient diagnosed with, having, expected to have, or predisposed to a disease or condition (e.g., a T or NK cell malignancy) or at risk of the disease or condition.

[0376] The term “disease-causing mutation,” “disease-causing variant,” “disease-causing mutation,” “disease-causing variant,” “deleterious mutation,” or “predisposing mutation” refers to a genetic alteration or mutation that increases an individual’s susceptibility or predisposition to a certain disease or condition. In some embodiments, a disease-causing mutation comprises at least one substitution of a wild-type amino acid to at least one disease-causing amino acid in a protein encoded by a gene.

[0377] The term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium stearate, or stearic acid), or solvent or encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. A pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of a formulation and not injurious to the subject, e.g., physiologically compatible, sterile, physiologically pH, etc. The terms such as “excipient,” “carrier,” “pharmaceutically acceptable carrier,” “vehicle,” etc., are used interchangeably herein.

[0378] The term “pharmaceutical composition” means a composition formulated for pharmaceutical use. In some embodiments, a pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition comprises additional reagents (e.g., for specific delivery, to increase half-life, or other therapeutic compounds).

[0379] "Programmed cell death 1 (PDCD1 or PD-1) polypeptide" means a protein having at least about 85% amino acid sequence identity to NCBI Accession No. AJS10360.1 or fragments thereof. PD-1 protein is believed to be involved in the regulation of T cell function during immune responses and in the setting of tolerance. An exemplary B2M polypeptide sequence is provided below.

[0380] >AJS10360.1 Programmed cell death 1 protein [Homo sapiens]

[0381] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0382] "Programmed cell death 1 (PDCD1 or PD-1) polynucleotide" means a nucleic acid molecule encoding a PD-1 polypeptide. The PDCD1 gene encodes an inhibitory cell surface receptor that suppresses T cell effector function in an antigen-specific manner. An exemplary PDCD1 nucleic acid sequence is provided below.

[0383] >AY238517.1 Homo sapiens Programmed cell death 1 (PDCD1) mRNA, complete cds ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCTGCTCCCGGGCCGCACGAGGGACAATAGGAGCCAGGCGCACCGGCCAGCCCCTGAAGGAGGACCCCTCAGCCGTGCCTGTGTTCTCTGTGGACTATGGGGAGCTGGATTTCCAGTGGCGAGAGAAGACCCCGGAGCCCCCCGTGCCCTGTGTCCCTGAGCAGACGGAGTATGCCACCATTGTCTTTCCTAGCGGAATGGGCACCTCATCCCCCGCCCGCAGGGGCTCAGCTGACGGCCCTCGGAGTGCCCAGCCACTGAGGCCTGAGGATGGACACTGCTCTTGGCCCCTCTGA

[0384] 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. The term refers to proteins, peptides, or polypeptides of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids in length. A protein, peptide, or polypeptide can refer to a single protein or a collection of proteins. One or more amino acids in a protein, peptide, or polypeptide can be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnyl group, an isofarnyl group, a fatty acid group; a linker for conjugation; functionalization; or other modification. A protein, peptide, or polypeptide can also be a single molecule or can be a multi-molecular complex. A protein, peptide, or polypeptide can be only a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide can be naturally occurring, recombinant, or synthetic, or any combination thereof. As used herein, the term "fusion protein" refers to a hybrid polypeptide that comprises protein domains from at least two different proteins. A protein can be positioned at either the amino-terminal (N-terminal) portion or at the carboxy-terminal (C-terminal) protein of a fusion protein, thus forming an amino-terminal fusion protein or a carboxy-terminal fusion protein, respectively. A protein can comprise different domains, for example, a nucleic acid binding domain (e.g., a gRNA binding domain of Cas9 that directs binding of the protein to a target site) and a nucleic acid cleavage domain, or a catalytic domain of a nucleic acid editing protein. In some embodiments, a protein comprises a protein portion (e.g., an amino acid sequence that constructs a nucleic acid binding domain) and an organic compound (e.g., a compound that can act as a nucleic acid cleavage agent). In some embodiments, a protein is complexed or associated with a nucleic acid (e.g., RNA or DNA). Any of the proteins provided herein can be produced by any method known in the art. For example, the proteins provided herein can be produced via recombinant protein expression and purification, which is particularly suitable for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.

[0385] The polypeptides and proteins disclosed herein, including functional portions and functional variants thereof, can comprise synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexane carboxylic acid, norleucine, a-amino normal decanoic acid, homoserine, S-acetylamino methyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, b-phenylserine, b-hydroxyphenylalanine, phenylglycine, a-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl- lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, a-aminocyclopentane carboxylic acid, a-aminocyclohexane carboxylic acid, a-aminocycloheptane carboxylic acid, a-(2-amino-2-norleucine)-carboxylic acid, a,g-diaminobutyric acid, a,b-diaminopropionic acid, homoproline, and a-tert-butylglycine. The polypeptides and proteins can be associated with post-translational modifications of one or more amino acids of the polypeptide construct. Non-limiting examples of post-translational modifications include phosphorylation, acylation (including acetylation and formylation), glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation (including methylation and ethylation), ubiquitination, addition of pyrrolidone carboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation, prenylation, farnesylation, geranylation, glycosylphosphatidylinositolation, lipidation, and iodination.

[0386] A "promoter" means an array of nucleic acid control sequences that direct transcription of a nucleic acid. A promoter includes the necessary nucleic acid sequences near the transcription initiation site. A promoter also optionally includes a distal enhancer or suppressor sequence element. A "constitutive promoter" is a promoter that is continuously active and not modulated by external signals or molecules. In contrast, the activity of an "inducible promoter" is modulated by external signals or molecules (e.g., transcription factors). For example, the promoter can be a CMV promoter.

[0387] As used herein, the term "recombinant" in the context of a protein or nucleic acid refers to a protein or nucleic acid that does not exist in nature but is a 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 one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations compared to any naturally occurring sequence.

[0388] "Decrease" means at least a 10%, 25%, 50%, 75%, or 100% inverse change.

[0389] "Reference" means a standard or control condition. In one embodiment, the reference is a wild-type or healthy cell. In other embodiments and without limitation, the reference is a treated cell that does not experience the test condition or that experiences a placebo or a control vehicle that does not carry a polynucleotide of interest.

[0390] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset of or all 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 typically 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 typically be at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, and about 100 nucleotides or about 300 nucleotides or any integral number of nucleotides therein or thereabout. In some embodiments, the reference sequence is the wild-type sequence of a protein of interest. In other embodiments, the reference sequence is a polynucleotide sequence that encodes a wild-type protein.

[0391] The terms "RNA-programmable nuclease" and "RNA-guided nuclease" are used in reference to one or more RNAs that are not cleaved at a target site (e.g., bound or associated with). In some embodiments, when the RNA-programmable nuclease is complexed with RNA, it can be referred to as a nuclease:RNA complex. Typically, the bound RNA is referred to as a guide RNA (gRNA). The gRNA can exist as a complex of two or more RNAs, or as a single RNA molecule. A gRNA that exists as a single RNA molecule can be referred to as a single guide RNA (sgRNA), but "gRNA" is used interchangeably to refer to a guide RNA that exists as a single molecule or as a complex of two or more molecules. Typically, a gRNA that exists as a single RNA species comprises two domains: (1) a domain that shares homology with a target nucleic acid (e.g., and directs binding of a Cas9 complex to the target); and (2) a domain that binds a Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as tracrRNA and comprises a stem loop structure. For example, in some embodiments, domain (2) is identical or homologous to the tracrRNA provided in Jinek et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Other examples of gRNAs (e.g., those that include domain 2) can be found in U.S. Patent Application No. 61 / 874,682, filed September 6, 2013, entitled "Switchable Cas9 Nucleases and Uses Thereof," and U.S. Provisional Patent Application No. 61 / 874,746, filed September 6, 2013, entitled "Delivery System For Functional Nucleases," the entire contents of each of which are incorporated herein by reference in their entirety. In some embodiments, a gRNA comprises two or more domains (1) and (2) and can be a "spreading gRNA." For example, a spreading gRNA will bind two or more Cas9 proteins and bind a target nucleic acid at two or more different regions, as described herein. The gRNA comprises a nucleotide sequence that is complementary to a target site, which mediates binding of the nuclease / RNA complex to the target site, providing sequence specificity of the nuclease:RNA complex.

[0392] In some embodiments, the RNA programmable nuclease is a (CRISPR-associated system) Cas9 endonuclease, e.g., Cas9 (Csnl) from Streptococcus pyogenes (see, e.g., “Complete genome sequence of an Ml strain of Streptococcus pyogenes.” Ferretti J.J., et al., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663 (2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., et al., Nature 471:602-607 (2011)). Because RNA programmable nucleases (e.g., Cas9) use RNA:DNA hybridization to target the DNA cleavage site, these proteins can in principle be targeted to any sequence for which a guide RNA is specific.Methods for site-specific cleavage using RNA-programmable nucleobases such as Cas9 (e.g., to modify a genome) are known in the art (see, e.g., Cong, L. et al, Multiplex genome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013); Mali, P. et al, RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, W. Y. et al, Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et al, RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, J. E. et al, Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research (2013); Jiang, W. et al RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature biotechnology 31, 233-239 (2013); the entire contents of each of which are incorporated herein by reference).

[0393] By "signaling domain" is meant the intracellular portion of a protein expressed within a T cell that transduces a T cell effector function signal (e.g., an activation signal) and directs the T cell to perform a specialized function. T cell activation can be induced by a variety of factors, including the binding of an antigen to a T cell receptor on the surface of a T cell and the binding of a cognate ligand to a costimulatory molecule on the surface of a T cell. A T cell costimulatory molecule is a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules. In some embodiments, the costimulatory domain is a CD2 cytoplasmic domain. Activation of a T cell leads to an immune response, such as T cell proliferation and differentiation (see, e.g., Smith-Garvin et al., Annu. Rev. Immunol., 27:591-619, 2009). Exemplary T cell signaling domains are known in the art. Non-limiting examples include CD2, CD3 zeta, CD8, CD28, CD27, CD154, GITR (TNFRSF18), CD134 (OX40), and CD137 (4-1BB) signaling domains.

[0394] By "single chain antibody" or "scFv" is meant a genetically engineered molecule containing the VH and VL domains of one or more antibodies linked as a single chain molecule by a peptide linker verified to be stable in vivo (see, e.g., Bird et al., Science, 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879-5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi: 10.1155 / 2012 / 980250: Marbry, Idrugs, 13:543-549, 2010). In some embodiments, the intramolecular orientation of the VH and VL domains in the scFv is VH domain-linker domain-VL domain. In some embodiments, the intramolecular orientation of the VH and VL domains in the scFv is VL domain-linker domain-VH domain.

[0395] The term "single nucleotide polymorphism (SNP)" is a variation in a single nucleotide that occurs at a specific location in the genome, where each variation is present in a population to a suitable degree (e.g., >1%). For example, at a specific base position in the human genome, a C nucleotide can occur in most individuals, but in a few individuals, the position is occupied by an A. This means that there is a SNP at this specific position, and the two possible nucleotide variations, C or A, are called alleles of this position. SNPs are the basis of differences in susceptibility to disease. Severity of disease and our body's response to treatment are also manifestations of genetic variations. SNPs can fall within coding regions of genes, within non-coding regions of genes, or within intergenic regions (regions between genes). In one embodiment, SNPs within coding sequences do not necessarily change the amino acid sequence of the protein produced due to the degeneracy of the genetic code. SNPs within coding regions are of two types: synonymous and nonsynonymous SNPs. Synonymous SNPs do not affect protein sequence, while nonsynonymous SNPs change the amino acid sequence of the protein. Nonsynonymous SNPs are of two types: missense and nonsense. SNPs that are not within protein coding regions can still affect gene splicing, transcription factor binding, messenger RNA degradation, or sequence of non-coding RNA. Gene expression affected by this type of SNP is called eSNP (expression SNP) and can be upstream or downstream of the gene. Single nucleotide variants (SNVs) are variations in a single nucleotide without any significant frequency, and can occur in somatic cells. Somatic single nucleotide variations (e.g., associated with cancer) can also be referred to as single nucleotide alterations.

[0396] "Specifically binds" means that a nucleic acid molecule, polypeptide, or complex thereof (e.g., a nucleic acid programmable DNA binding protein, a guide nucleic acid, and a chimeric antigen receptor), a compound, or a molecule recognizes and binds to a polypeptide and / or nucleic acid molecule of the application, but does not substantially recognize and bind to other molecules in a sample (e.g., a biological sample). For example, a chimeric antigen receptor specifically binds to a particular marker expressed on the surface of a cell, but does not bind to other polypeptides, carbohydrates, lipids, or any other compounds on the surface of the cell.

[0397] Nucleic acid molecules useful in the methods of the application include any nucleic acid molecule that encodes a polypeptide of the application or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence typically will hybridize to at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the application include any nucleic acid molecule that encodes a polypeptide of the application or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence typically will hybridize to at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant to pair between complementary polynucleotide sequences (e.g., genes described herein) or fragments thereof under various conditions of stringency to form a double-stranded molecule. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

[0398] For example, stringent salt concentration will generally be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than 500 mM NaCl and 50 mM trisodium citrate, and more preferably about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridizations can be achieved in the absence of organic solvents, such as formamide, while high stringency hybridizations are achieved in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will generally include temperatures of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Various other parameters, such as hybridization time, concentration of surfactant, such as sodium dodecyl sulfate (SDS), and inclusion or exclusion of carrier DNA, are well known to those skilled in the art. By combining these conditions as desired, one achieves various levels of stringency. In one embodiment, hybridization will occur in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS at 30°C. In another embodiment, hybridization will occur in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA) at 37°C. In another embodiment, hybridization will occur in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA at 42°C. Useful variations on these conditions will be apparent to those skilled in the art.

[0399] For most applications, the washing step after hybridization will also change the stringency. Washing stringency conditions can be defined by salt concentration and by temperature. As above, washing stringency can be increased by decreasing salt concentration or increasing temperature. For example, a stringent salt concentration for a washing step will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably about 15 mM NaCl and 1.5 mM trisodium citrate. A stringent temperature condition for a washing step generally will include a temperature of at least about 25°C, more preferably at least about 42°C, even more preferably at least about 68°C. In one embodiment, the washing step will occur at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step will occur at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step will occur at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Other variations of these conditions will be apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described in, for example, Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York).

[0400] "fragment" means divided into two or more pieces.

[0401] A“split Cas9 protein” or“split Cas9” refers to a Cas9 protein that is provided as an N-terminal fragment and a C-terminal fragment encoded by two separate nucleotide sequences. The polypeptides corresponding to the N-terminal portion and the C-terminal portion of the Cas9 protein can be spliced to form a“reconstituted” Cas9 protein. In particular embodiments, the Cas9 protein is split into two fragments located within the disordered region of the protein, for example, as described in Nishimasu et al., Cell, Volume 156, Issue 5, pp. 935-949, 2014 or as described in Jiang et al. (2016) Science 351 :867-871. PDB file: 5F9R, each of which is incorporated herein by reference. In some embodiments, the protein is split into two fragments located at any C, T, A, or S within the region between amino acids A292 to G364, F445 to K483, or E565 to T637 of SpCas9 or at the corresponding positions in any other Cas9, Cas9 variant (e.g., nCas9, dCas9), or other napDNAbp. In some embodiments, the protein is split into two fragments located at SpCas9 T310, T313, A456, S469, or C574. In some embodiments, the process of splitting the protein into two fragments is referred to as“cleaving” the protein.

[0402] In other embodiments, the N-terminal portion of the Cas9 protein comprises amino acids 1 to 573 or 1 to 637 of S. pyogenes Cas9 wild-type (SpCas9) (NCBI Reference Sequence: NC_002737.2, Uniprot Reference Sequence: Q99ZW2), and the C-terminal portion of the Cas9 protein comprises the portion of SpCas9 wild-type from amino acids 574 to 1368 or 638 to 1368 or the corresponding positions thereof.

[0403] The C-terminal portion of split Cas9 can be joined with the N-terminal portion of split Cas9 to form a complete Cas9 protein. In some embodiments, the C-terminal portion of the Cas9 protein begins where the N-terminal portion of the Cas9 protein ends. Thus, in some embodiments, the C-terminal portion of split Cas9 comprises the portion of spCas9 from amino acids (551 to 651) to 1368. By "(551 to 651) to 1368" is meant beginning at an amino acid between and including amino acids 551 to 651 and ending at amino acid 1368.For example, the C-terminal portion of a split Cas9 can comprise any of amino acids 551 to 1368, 552 to 1368, 553 to 1368, 554 to 1368, 555 to 1368, 556 to 1368, 557 to 1368, 558 to 1368, 559 to 1368, 560 to 1368, 561 to 1368, 562 to 1368, 563 to 1368, 564 to 1368, 565 to 1368, 566 to 1368, 567 to 1368, 568 to 1368, 569 to 1368, 570 to 1368, 571 to 1368, 572 to 1368, 573 to 1368, 574 to 1368, 575 to 1368, 576 to 1368, 577 to 1368, 578 to 1368, 579 to 1368, 580 to 1368, 581 to 1368, 582 to 1368, 583 to 1368, 584 to 1368, 585 to 1368, 586 to 1368, 587 to 1368, 588 to 1368, 589 to 1368, 590 to 1368, 591 to 1368, 592 to 1368, 593 to 1368, 594 to 1368, 595 to 1368, 596 to 1368, 597 to 1368, 598 to 1368, 599 to 1368, 600 to 1368, 601 to 1368, 602 to 1368, 603 to 1368, 604 to 1368, 605 to 1368, 606 to 1368, 607 to 1368, 608 to 1368, 609 to 1368, 610 to 1368, 611 to 1368, 612 to 1368, 613 to 1368, 614 to 1368, 615 to 1368, 616 to 1368, 617 to 1368, 618 to 1368, 619 to 1368, 620 to 1368, 621 to 1368, 622 to 1368, 623 to 1368, 624 to 1368, 625 to 1368, 626 to 1368, 627 to 1368, 628 to 1368, 629 to 1368, 630 to 1368, 631 to 1368, 632 to 1368, 633 to 1368, 634 to 1368, 635 to 1368, 636 to 1368, 637 to 1368, 638 to 1368, 639 to 1368, 640 to 1368, 641 to 1368, 642 to 1368, 643 to 1368, 644 to 1368, 645 to 1368, 646 to 1368, 647 to 1368, 648 to 1368, 649 to 1368, 650 to 1368, or 651 to 1368 of spCas9. In some embodiments, the C-terminal portion of a split Cas9 protein comprises a portion of amino acids 574 to 1368 or 638 to 1368 of SpCas9.

[0404] "Subject" means a mammal, including, but not limited to, a human and a non-human mammal, such as a cow, horse, dog, sheep, or cat. Subjects include domesticated animals, animals used for labor and to provide commodities such as food, including, but not limited to, cows, goats, chickens, horses, pigs, rabbits, and sheep.

[0405] "Substantially identical" means that a polypeptide or nucleic acid molecule exhibits at least 50% identity to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid molecule (e.g., any of the nucleic acid sequences described herein). In one embodiment, the sequence is at least 60%, 80% or 85%, 90%, 95%, or even 99% identical at the amino acid level or nucleic acid level to the nucleic acid used for comparison.

[0406] Sequence identity is typically measured using sequence analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705), BLAST, BESTFIT, COBALT, EMBOSS Needle, 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 groups of amino acids that are replaced with other amino acids in the same group: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In one exemplary method of determining degrees of identity, the BLAST program can be used, with a probability score between e-3 and e-100 indicating closely related sequences.

[0407] COBALT is used, for example, with the following parameters:

[0408] a) Alignment parameters: gap penalty -11, -1, and end gap penalty -5, -1,

[0409] b) CDD parameters: Use RPS BLAST on; Blast E value 0.003; Find Conserved columns and Recompute on, and

[0410] c) Query cluster parameters: open with query clusters; word length 4; maximum inter-cluster distance 0.8; Alphabet Regular.

[0411] Using EMBOSS Needle, for example, using the following parameters:

[0412] a) Matrix: BLOSUM62;

[0413] b) GAP OPEN: 10;

[0414] c) GAP EXTEND: 0.5;

[0415] d) OUTPUT FORMAT; yes;

[0416] e) END GAP PENALTY: false;

[0417] f) END GAP OPEN: 10; and

[0418] g) END GAP EXTEND: 0.5.

[0419] The term“target site” refers to a sequence within a nucleic acid molecule that is modified by a nucleobase editor. In one embodiment, the target site is determined by a deaminase or a fusion protein comprising a deaminase (e.g., a cytidine or adenosine deaminase).

[0420] “T cell receptor alpha constant (TRAC) polypeptide” means a protein having at least about 85% amino acid sequence identity to NCBI Accession No. P01848.2 or a fragment thereof and having immunomodulatory activity. An exemplary amino acid sequence is provided below.

[0421] >sp|P01848.2|TRAC_HUMAN RecName: Full=T cell receptor alpha constant region

[0422] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0423] “T cell receptor alpha constant (TRAC) polynucleotide” means a nucleic acid encoding a TRAC polypeptide. An exemplary TRAC nucleic acid sequence is provided below.

[0424] UCSC Human Genome Database, Gene ENSG00000277734.8 Human T cell receptor alpha chain (TCR-alpha)

[0425]

[0426] The nucleotides represented by lower case letters in the above are untranslated regions or introns, and the nucleotides represented by upper case letters are exons.

[0427] > X02592.1 mRNA for human T cell alpha chain (TCR-alpha)

[0428]

[0429] “T cell receptor beta constant region 1 polypeptide (TRBC1)” means a protein having at least about 85% amino acid sequence identity to NCBI Accession No. P01850 or a fragment thereof and having immunomodulatory activity. An exemplary amino acid sequence is provided below.

[0430] >sp|P01850|TRBC1_HUMAN T cell receptor beta constant region 1 OS=Homo sapiens OX=9606 GN=TRBC1 PE=1 SV=4 DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF

[0431] “T cell receptor beta constant region 1 polynucleotide (TRBC1)” means a nucleic acid encoding a TRBC1 polypeptide. An exemplary TRBC1 nucleic acid sequence is provided below.

[0432] >gi|X00437.1|ref|NP_000545.1| T cell receptor beta chain constant region 1 OS=Homo sapiens OX=9606 GN=TRBC1 PE=1 SV=1

[0433]

[0434] “T cell receptor beta constant region 2 polypeptide (TRBC2)” means a protein that has at least about 85% amino acid sequence identity to NCBI Accession No. A0A5B9 or a fragment thereof and has immunomodulatory activity. An exemplary amino acid sequence is provided below.

[0435] >sp|A0A5B9|TRBC2_HUMAN T cell receptor beta constant region 1 OS=Homo sapiens OX=9606 GN=TRBC2 PE=2 SV=2

[0436] DLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0437] “T cell receptor beta constant region 2 polynucleotide (TRBC2)” means a nucleic acid that encodes a TRAC polypeptide. An exemplary TRBC2 nucleic acid sequence is provided below.

[0438] >NG_001333.2:655095-656583 T cell receptor beta genomic on chromosome 7 Homo sapiens

[0439]

[0440] By "tet methylcytosine dioxygenase 2 (TET2) polypeptide" is meant a protein having at least about 85% amino acid sequence identity to NCBI Accession No. FM992369.1 or a fragment thereof and having catalytic activity to convert methylcytosine to 5- hydroxymethylcytosine. Defects in this gene have been associated with myeloid proliferative disorders, and the enzyme's ability to methylate cytosine contributes to transcriptional regulation. An exemplary TET2 amino acid sequence is provided below.

[0441] > CAX30492.1 tet oncogene family member 2 [Homo sapiens]

[0442]

[0443] By "tet methylcytosine dioxygenase 2 (TET2) polynucleotide" is meant a nucleic acid molecule encoding a TET2 polypeptide. TET polypeptides encode methylcytosine dioxygenases and have transcriptional regulatory activity. An exemplary TET2 nucleic acid sequence is presented below.

[0444] > FM992369.1 Homo sapiens tet oncogene family member 2 (TET2 gene) mRNA

[0445]

[0446] “Transforming growth factor receptor 2 (TGFBRII) polypeptide” means a protein having at least about 85% amino acid sequence identity to NCBI Accession No. ABG65632.1 or a fragment thereof and having immunosuppressive activity. An exemplary amino acid sequence is provided below.

[0447] >ABG65632.1 Transforming growth factor beta receptor II [Homo sapiens] MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK

[0448] “Transforming growth factor receptor 2 (TGFBRII) polynucleotide” means a nucleic acid molecule encoding a TGFBRII polypeptide. The TGFBRII gene encodes a transmembrane protein with serine / threonine kinase activity. An exemplary TGFBRII nucleic acid sequence is provided below.

[0449]

[0450] “T cell immunoreceptor with Ig and ITIM domains (TIGIT) polypeptide” means a protein having at least about 85% amino acid sequence identity to NCBI Accession No. ACD74757.1 or a fragment thereof and having immunomodulatory activity. An exemplary TIGIT amino acid sequence is provided below.

[0451] >ACD74757.1 T cell immunoreceptor with Ig and ITIM domains [Homo sapiens]

[0452] MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPLLGAMAATLVVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSCVQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRSLGNCSFFTETG

[0453] “T cell immunoreceptor with Ig and ITIM domains (TIGIT) polynucleotide” means a nucleic acid encoding a TIGIT polypeptide. The TIGIT gene encodes an inhibitory immune receptor associated with neoplasia and T cell exhaustion. An exemplary nucleic acid sequence is provided below.

[0454] >EU675310.1 Homo sapiens T cell immunoreceptor with Ig and ITIM domains (TIGIT) mRNA, complete cds

[0455] CGTCCTATCTGCAGTCGGCTACTTTCAGTGGCAGAAGAGGCCACATCTGCTTCCTGTAGGCCCTCTGGGCAGAAGCATGCGCTGGTGTCTCCTCCTGATCTGGGCCCAGGGGCTGAGGCAGGCTCCCCTCGCCTCAGGAATGATGACAGGCACAATAGAAACAACGGGGAACATTTCTGCAGAGAAAGGTGGCTCTATCATCTTACAATGTCACCTCTCCTCCACCACGGCACAAGTGACCCAGGTCAACTGGGAGCAGCAGGACCAGCTTCTGGCCATTTGTAATGCTGACTTGGGGTGGCACATCTCCCCATCCTTCAAGGATCGAGTGGCCCCAGGTCCCGGCCTGGGCCTCACCCTCCAGTCGCTGACCGTGAACGATACAGGGGAGTACTTCTGCATCTATCACACCTACCCTGATGGGACGTACACTGGGAGAATCTTCCTGGAGGTCCTAGAAAGCTCAGTGGCTGAGCACGGTGCCAGGTTCCAGATTCCATTGCTTGGAGCCATGGCCGCGACGCTGGTGGTCATCTGCACAGCAGTCATCGTGGTGGTCGCGTTGACTAGAAAGAAGAAAGCCCTCAGAATCCATTCTGTGGAAGGTGACCTCAGGAGAAAATCAGCTGGACAGGAGGAATGGAGCCCCAGTGCTCCCTCACCCCCAGGAAGCTGTGTCCAGGCAGAAGCTGCACCTGCTGGGCTCTGTGGAGAGCAGCGGGGAGAGGACTGTGCCGAGCTGCATGACTACTTCAATGTCCTGAGTTACAGAAGCCTGGGTAACTGCAGCTTCTTCACAGAGACTGGTTAGCAACCAGAGGCATCTTCTGG

[0456] As used herein, “transduction” means the transfer of a gene or genetic material into a cell via a viral vector.

[0457] As used herein, “transformation” refers to the process of introducing a genetic alteration into a cell by the introduction of exogenous nucleic acid.

[0458] "Transfection" refers to the transfer of genetic material or genetic material into a cell via chemical or physical means.

[0459] "Translocation" means the rearrangement of nucleic acid segments between nonhomologous chromosomes.

[0460] "Transmembrane domain" means an amino acid sequence that inserts into a lipid bilayer, such as that of a cell or virus or virus-like particle. A transmembrane domain can be used to anchor a protein of interest (e.g., a CAR) to a membrane. A transmembrane domain can be derived from a natural source or from a synthetic source. If derived from a natural source, the domain can be derived from any membrane-bound or transmembrane protein. A transmembrane domain for use in the disclosed CARs can include at least the transmembrane region of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, the transmembrane domain is derived from CD4, CD8 alpha, CD28, and CD3 zeta. In some embodiments, the transmembrane domain is a CD8 alpha hinge and transmembrane domain.

[0461] As used herein, the terms "treat," "treatment," and the like, refer to reducing or alleviating a disease or condition, and / or symptoms associated therewith, or to obtaining a desired pharmacologic and / or physiologic effect. It will be understood, without being limited by theory, that treatment of a disorder or condition does not require complete elimination of the disorder, condition, or symptoms associated therewith. In some embodiments, the effect is therapeutic, i.e., the effect partially or completely alleviates, eliminates, abrogates, decreases, slows, or cures a disease and / or negative symptom attributable to the disease. In some embodiments, the effect is prophylactic, i.e., the effect protects against or prevents the occurrence or recurrence of a disease or condition. To this end, the methods of the present disclosure include administering a therapeutically effective amount of a composition described herein.

[0462] The term "uracil glycosylase inhibitor" or "UGI" means an agent that inhibits the uracil excision repair system. In one embodiment, the agent is a protein or fragment thereof that binds to host uracil-DNA glycosylase and prevents removal of uracil residues from DNA. In one embodiment, the UGI is a protein, fragment or domain thereof that is capable of inhibiting the uracil-DNA glycosylase base-excision repair enzyme. In some embodiments, the UGI domain comprises a wild-type UGI or a modified version thereof. In some embodiments, the UGI domain comprises a fragment of an exemplary amino acid sequence detailed below. In some embodiments, the UGI fragment comprises an amino acid sequence comprising 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 100% of an exemplary UGI sequence provided below. In some embodiments, the UGI comprises an amino acid sequence that is homologous to an exemplary UGI amino acid sequence detailed below or a fragment thereof. In one embodiment, the UGI or portion thereof is 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%, at least 99.5%, at least 99.9%, or 100% identical to a wild-type UGI or UGI sequence detailed below or a fragment thereof. An exemplary UGI comprises an amino acid sequence as follows:

[0463] >sp|P14739| UNGI_BPPB2 Uracil-DNA glycosylase inhibitor

[0464] MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVM LLTSDAPEYKPWALVIQDSNGENKIKML. The term "vector" refers to a means for introducing a nucleic acid sequence into a cell to result in a transformed cell. Vectors include plasmids, transposons, bacteriophage, viruses, liposomes, and cosmids. An "expression vector" is a nucleic acid sequence that comprises a nucleic acid sequence to be expressed in a recipient cell. An expression vector can include additional nucleic acid sequences to facilitate and / or promote expression of the introduced sequences, such as initiators, terminators, promoters, and secretion sequences.

[0465] A "T cell receptor zeta chain-associated protein kinase 70 (ZAP70) polypeptide" means a protein that has at least about 85% amino acid sequence identity to NCBI Accession No. AAH53878.1 and has kinase activity. An exemplary amino acid sequence is provided below.

[0466] >AAH53878.1 Zeta chain (TCR) associated protein kinase 70 kDa [Homo sapiens]

[0467] MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTLNSDGYTPEPARITSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAEAACA

[0468] By "Zeta Chain Associated Protein Kinase 70 (ZAP70)" is meant a nucleic acid encoding a ZAP70 polypeptide. The ZAP70 gene encodes a tyrosine kinase involved in T cell development and lymphocyte activation. Loss of functional ZAP10 can result in severe combined immunodeficiency characterized by a deficiency in CD8+ T cells. An exemplary ZAP70 nucleic acid sequence is provided below.

[0469] >BC053878.1 Homo sapiens Zeta chain (TCR) associated protein kinase 70 kDa, mRNA (cDNA clone MGC:61743 IMAGE:5757161), complete cds

[0470]

[0471] A recitation of a series of chemical groups in any variable definition herein includes the definition of that variable as any single group or combination of the groups listed. A recitation of embodiments of a variable or aspect herein includes that embodiment as any single embodiment or in combination with other embodiments or portions thereof.

[0472] Any composition or method provided herein can be used in combination with one or more of any other composition and method provided herein.

[0473] DNA editing has emerged as a viable means to alter disease states by correcting pathogenic mutations at the genetic level. Until recently, all DNA editing platforms functioned by inducing a DNA double-strand break (DSB) at a specific genomic site and relied on endogenous DNA repair pathways to determine product outcomes in a semi-random fashion, resulting in a complex population of genetic products. While precise, user-defined repair outcomes can be achieved through the homology directed repair (HDR) pathway, efficient repair using HDR in therapeutically relevant cell types faces many challenges. In practice, this pathway is inefficient compared to the competing, error-prone non-homologous end joining pathway. Furthermore, HDR is strictly limited to the G1 and S phases of the cell cycle, precluding precise repair of DSBs in post-mitotic cells. Thus, efficient alteration of genomic sequences in a user-defined, programmable fashion in these populations has proven difficult or impossible.

[0474] INCORPORATED BY REFERENCE

[0475] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF DRAWINGS

[0476] The novel features of the application are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present application will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the application are utilized, and the accompanying drawings of which:

[0477] Figures 1A-1B is an illustrative depiction of three proteins that affect T cell function. Figure 1A is an illustrative depiction of the TRAC protein, which is a key component of graft versus host disease. Figure 1B is an illustrative depiction of the B2M protein, which is a component of MHC class 1 antigen presenting complex present on nucleated cells that can be recognized by the host's CD8+ T cells. Figure 1Cis an exemplary illustration of T cell signaling that leads to expression of the PDCD1 gene, and the resulting PD-1 protein acts to inhibit T cell signaling.

[0478] Figure 2 is a graph of the percentage of cells with knockdown of target gene expression following base editing. "EP" indicates electroporation.

[0479] Figure 3 is a graph of the percentage of gene modification types observed in cells that were not transduced or were transduced with the BE4 base editing system or a Cas9 nuclease.

[0480] Figure 4 is a graph depicting the percentage of target nucleotide modification by percentage of cells that were negative for target protein expression as determined by flow cytometry (FC) in cells transduced with BE4 and sgRNAs that direct BE4 to splice acceptor (SA) or donor (SD) sites, or that generate a STOP codon. Control cells were subjected to virtual electroporation (EP).

[0481] Figure 5 is a schematic of the BE4 system interrupting a splice acceptor (SA), splice donor (SD), or generating a STOP codon.

[0482] Figure 6 is a graph summarizing off-target binding sites for sgRNAs used to interrupt target genes.

[0483] Figure 7 is a graph summarizing flow cytometry (FC) data for cells edited with BE4 or Cas9 that exhibit reduced protein expression. Cells were gated on B2M or CD3, the latter being an indicator of TRAC expression.

[0484] Figure 8A is a scatter plot of FACS data for unedited control cells. Figure 8B is a scatter plot of FACS data for cells that have been edited at the B2M, TRAC, and PD1 loci.

[0485] Figure 9 is a graph exemplifying the effectiveness of the base editing technology described herein in modifying specific genes that can have a negative impact on CAR-T immunotherapy.

[0486] Figure 10 is a schematic depicting a droplet digital PCR (ddPCR) protocol used to detect and quantify gene modification and translocation.

[0487] Figure 11Two figures are presented showing data generated from next generation sequencing (NGS) analysis or ddPCR of cells edited using the BE4 system or Cas9 system.

[0488] Figure 12 is a schematic illustrating the role Cbl-b plays in suppressing T cell activation.

[0489] Figure 13 is a graph depicting the efficiency of Cbl-b knockdown by interrupting splice sites. SA = splice acceptor; SD = splice donor; STOP - generates STOP codon; 2° Only = secondary antibody only; C373 refers to loss-of-function variant (C373R); RL1-A::APC-A = laser; ICS = intracellular staining.

[0490] Figure 14 is an illustration of Cas12b-mediated indel rates in the GRIN2B and DNMT1 genes in T cells. EP indicates electroporation.

[0491] Figure 15 is a graph summarizing fluorescence assisted cell sorting (FACS) data of cells transduced via electroporation (EP) with bvCas12b and guide RNAs specific for TRAC, GRIN2B, and DNMT1 and gated for CD3.

[0492] Figure 16 is a scatter plot of fluorescence assisted cell sorting data of cells transduced with CAR-P2A-mCherry lentivirus, indicating CAR expression.

[0493] Figure 17 is a scatter plot of fluorescence assisted sorting data, indicating CAR expression in cells transduced with poly(l,8-octanediol citrate) (POC) lentivirus vector.

[0494] Figure 18 is a graph showing that BE4 produces efficient, durable gene knockout with high product purity.

[0495] Figure 19A is a representative FACS analysis showing loss of protein expression due to gene knockout by BE4 or spCas9. Figure 19B is a graph showing loss of B2M surface expression due to gene knockout by BE4 or spCas9.

[0496] Figure 20 is a schematic depicting the positioning of B2M, TRAC, and PD-1 target sites. When B2M, TRAC, and PD-1 sequences recombine, translocation can be detected.

[0497] Figure 21 is a graph showing that multiple base editing does not significantly impair cell expansion.

[0498] Figure 22 is a graph showing that BE4 generates triple-edited T cells with similar on-target editing efficiency and cell phenotypes as spCas9.

[0499] Figure 23 Flow cytometry analysis is described showing generation of triple-edited CD3-, B2M-, PD1- T cells.

[0500] Figure 24 Flow cytometry analysis is described showing CAR expression in BE4 and Cas9 edited cells.

[0501] Figure 25 is a graph showing that CAR-T cells kill or antigen positive cells.

[0502] Figure 26 is a graph showing that Cas12b and BE4 can be paired for efficient multiple editing in T cells.

[0503] Figure 27 is a graph showing that Cas12b can direct insertion of a chimeric antigen receptor (CAR) into a gene locus by introducing a double-stranded DNA template encoding the CAR into a cell in the presence of a Cas12 nuclease and sgRNAs targeting the gene locus.

[0504] Figure 28A and Figure 28B is an illustration showing base editing pathways used to silence genes to engineer CAR-T cells. Figure 28A Targets for CAR-T editing are shown. Figure 28B Two strategies for silencing using base editors are shown: creating a stop codon with CBE and interrupting splicing with CBE.

[0505] Figure 29 is an illustrative cartoon representation that in some cases, multiple CAR-Ts are needed to address clonality in AML.

[0506] Figure 30 is an illustrative cartoon representation that in some cases, multiple edits are needed to eliminate fratricide of T-ALL CAR-T combinations.

[0507] Figure 31A and Figure 31B Results of experiments testing four simultaneous base edits for T-ALL are illustratively shown; CAR-Ts do not impact yield compared to nucleases. Figure 31AExemplary illustration of theoretical yields in a four-way T-ALL editing race for the following: no electroporation (no EP; dark circles), EP only (dark squares), CBE variant 1 (light, upright triangles), CBE variant 2 (light, inverted triangles), and Cas9 (light diamonds). Figure 31B Exemplary illustration of cell survival rates after electroporation. For each set of results at 24 hours (hr), 48 hr, 72 hr, 96 hr, and 168 hr, results are shown from right to left as no EP, EP only, CBE variant 1, CBE variant 2, and Cas9.

[0508] Figure 32 Exemplary illustration that over 90% of four-way knockouts were caused by BE4. Electroporations were performed at 5M cell scale, and four-way edits were performed in a single electroporation (EP) step. Greater than 90% editing efficiency was achieved for all 4 targets using rBE4. For each set of results, data is from left to right: PD1, CD7, TRAC, and CD52.

[0509] Figure 33 Exemplary illustration that base editing did not cause differences in cell yield. Data is: no P (circles), EP only (squares), rBE4 (triangles), ppBE4 (inverted triangles), and Cas9 (diamonds).

[0510] Figure 34 Exemplary illustration of edited CAR-T targeting CD3 and CD7 on model tumor cells. Data is: UTD 1:1 (circles), 7CAR8 1:1 (squares), and 3CAR2 1:1 (triangles).

[0511] Figure 35 is a schematic of an exemplary CD7 CAR-T cell for targeting T-AL tumor cells.

[0512] Figure 36 is a flowchart depicting a clinical protocol for treating a patient with CD7 CAR-T cells.

[0513] Figures 37A-37C Describes CD7 CAR-T cell production. Figure 37A is a flowchart depicting a protocol for producing TALL017 hetero-fused CD7 CAR-T cells. Figure 37B is a flowchart depicting a protocol for producing TALL083 CD7 CAR-T cells. Figure 37C is a scatter plot of fluorescence-assisted cell sorting data indicating that TALL017 CAR-T cells are highly activated after fusion.

[0514] Figure 38is a graph depicting total number of edits in TALL017 and TALL038 CD7 CAR-T cells measured by next generation sequencing (NGS).

[0515] Figure 39A and Figure 39B Depicts expression of TCRa / b, CD7, and CD52 in TALL017 and TALL038 CD7 CAR-T cells at 24 hours post-thaw. Figure 39A is a scatter plot of fluorescence assisted cell sorting data for TALL017 and TALL038 CD7 CAR-T cells. Figure 39B is a graph depicting residual protein expression measured via FACS.

[0516] Figure 40 Depicts gating strategy for identity groups.

[0517] Figure 41 is a graph depicting final cell product identity as a mixture of CD2+ / - and CD56+ / - cells.

[0518] Figure 42 is a graph depicting CD7 CAR-T cell expression at 24 and 48 hours post-thaw.

[0519] Figure 43 Comprises multiple graphs depicting lower CD25 expression in TALL038 CD7 CAR-T cells than in TALL017 CD7 CAR-T cells post-thaw.

[0520] Figure 44 is a flow chart depicting a CD7 CAR-T bead-based potency strategy for in vitro characterization.

[0521] Figure 45A and Figure 45B Depicts TALL038 CD7 CAR-T cell release of IFNy, TNFa, and IL-2 in response to CD7 antigen. Figure 45A is a graph depicting TALL038 CD7 CAR-T cell release of IFNy. Figure 45B is a graph depicting TALL038 CD7 CAR-T cell release of TNFa, IL-10, and IL-2.

[0522] Figure 46 is a schematic depicting a co-culture strategy for measuring CAR-directed T cell killing of tumor cells.

[0523] Figure 47A and Figure 47BDescribes the primary challenge. TALL038 CD7 CAR-T cells exhibit increased CCRF upon re-challenge compared to TALL017 CAR T. Figure 47A Is a graph describing the primary challenge. Figure 47B Is a graph describing the secondary challenge.

[0524] Figure 48 Is a graph describing bioluminescent radiation data (mean, SEM) at day 10 post CCRF transplant / day -1 post CD7 CAR-T treatment. Total flux is measured on a linear scale.

[0525] Figure 49A and Figure 49B Describes bioluminescent radiation data (mean, SEM) at day 19 post CCRF transplant / day 8 post CD7 CAR-T treatment. Figure 49A Is a graph describing the average tumor burden. Total flux is measured on a linear scale. Figure 49B Is a graph describing the average mouse weight.

[0526] Figure 50A and Figure 50B Describes bioluminescent radiation data (mean, SEM) at day 38 post CCRF transplant / day 27 post CD7 CAR-T treatment. Figure 50A Is a graph describing the average tumor burden. Total flux is measured on a linear scale. Figure 50B Is a graph describing the average tumor burden. Total flux is measured on a log scale.

[0527] Figure 51A and Figure 51B Describes bioluminescent radiation data (individual mice) at day 38 post CCRF transplant / day 27 post CD7 CAR-T treatment. Figure 51A Is a graph describing the average tumor burden. Total flux is measured on a linear scale. Figure 51B Is a graph describing the average tumor burden. Total flux is measured on a log scale.

[0528] Figure 52A and Figure 52B Describes bioluminescent radiation data (individual mice) at day 38 post CCRF transplant / day 27 post CD7 CAR-T treatment. Figure 52A Includes a graph describing the average tumor burden. Total flux is measured on a linear scale (top) and on a log scale (bottom). Figure 52B Includes a graph describing the average tumor burden. Total flux is measured on a linear scale (top) and on a log scale (bottom).

[0529] Figure 53 Is a graph describing the editing efficiency of CD5 gRNA candidates.

[0530] Figure 54 FIG. 34 is a graph depicting editing efficiency via NGS of CD5 gRNA candidates g103 and g104 in combination with BE4.

[0531] Figure 55 FIG. 34 is a graph depicting editing efficiency via NGS of CD5 gRNA candidates g103 and g104 in combination with BE4.

[0532] Figure 56 FIG. 34 is a graph depicting editing efficiency via NGS of CD5 gRNA candidates g103 and g104 in combination with BE4.

[0533] Figure 57 FIG. 34 is a graph depicting editing efficiency via NGS of CD5 gRNA candidates g103 and g104 in combination with BE4.

[0534] Figures 58A-58D FIG. 34 is a graph depicting editing efficiency via NGS of CD5 gRNA candidates g103 and g104 in combination with BE4.

[0535] Figure 59A FIG. 34 is a graph depicting editing efficiency via NGS of CD5 gRNA candidates g103 and g104 in combination with BE4. Figure 59B FIG. 34 is a graph depicting editing efficiency via NGS of CD5 gRNA candidates g103 and g104 in combination with BE4. Figure 59A (Left) is a graph depicting production of IFNy by CD5 CAR LVV (LV63-69) transduced T cells edited with sgRNA 103 alone or in combination with CCRF cells. Figure 59A (Right) is a graph depicting production of IFNy by CD5 CAR LVV (LV63-69) transduced T cells edited with sgRNA 104 alone or in combination with CCRF cells. Figure 59A (Bottom) is a graph depicting production of IFNy by CCRF cells or T cells transduced with unedited CD5 CAR LVV (LV63-69). Figure 59B (Top) is a graph depicting production of IFNy by T cells transduced with unedited or edited with sgRNA 103 or 104 CD5 CAR LVV (LV63-69) alone. Figure 59B (Bottom) is a graph depicting production of IFNy by CCRF cells transduced with unedited or edited with sgRNA 103 or 104 CD5 CAR LVV (LV63-69). DETAILED DESCRIPTION

[0536] The present invention provides genetically modified immune cells having enhanced anti-neoplasia activity, anti-immunosuppressive properties, and reduced risk of causing graft versus host reaction or host versus graft reaction, or compositions thereof. The present invention also provides methods for producing and using these modified immune effector cells (e.g., immune effector cells, such as T cells).

[0537] In one embodiment, CAR-T cells are administered to a subject having or predisposed to developing graft versus host disease (GVHD), the cells lacking or having reduced levels of functional TRAC. In one embodiment, CAR-T cells are administered to a subject having or predisposed to developing host versus graft disease (HVGD), the cells lacking or having reduced levels of beta 2 microglobulin (B2M).

[0538] Modifications to immune effector cells to express chimeric antigen receptors as well as knock out or knock down of specific genes to reduce the negative impact their expression can have on immune cell function are carried out using base editor systems comprising cytidine deaminases or adenosine deaminases as described herein.

[0539] Autologous, patient-derived chimeric antigen receptor-T cell (CAR-T) therapies have shown remarkable efficacy in treating some hematological cancers. While these products have led to significant clinical benefit for patients, the need for generating personalized therapies has created substantial manufacturing challenges and economic burden. Allogeneic gene CAR-T therapies are a potential solution developed to address these challenges, with similar clinical efficacy as autologous products while treating many patients with cells derived from a single healthy donor, substantially reducing the cost of goods and batch-to-batch variability.

[0540] Most first generation allogeneic gene CAR-Ts use nucleases to introduce two or more targeted genomic DNA double strand breaks (DSBs) into a target T cell population, relying on error-prone DNA repair to generate mutations that knock out target genes in a semi-random pattern. Such nuclease-based gene knockout strategies aid in reducing the risk of graft versus host disease and host rejection of CAR-Ts. However, the simultaneous introduction of multiple DSBs results in the final cell product containing a large number of genomic rearrangements such as balanced and unbalanced translocations, and a relatively high frequency of local rearrangements including inversions and large deletions. Furthermore, as the number of simultaneous genetic modifications made through the introduced DSBs increases, appreciable genotoxicity is observed in the treated cell population. This has the potential to significantly reduce the cell expansion potential in each production process, reducing the number of patients that can be treated per healthy donor.

[0541] A base editor (BE) is a class of emerging gene editing reagents that can efficiently, user- definably modify target genomic DNA without generating DSBs. Herein, a method of producing allogeneic CAR-T cells is presented by using base editing technology to reduce or eliminate detectable genomic rearrangements while also improving cell expansion. As shown herein, in contrast to editing strategies using nucleases alone, efficient gene knockout without detectable translocation events is generated by base editing to simultaneously modify one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more genes) gene loci.

[0542] In some embodiments, at least one or more genes or regulatory elements thereof within an immune cell are modified using the base editing compositions and methods provided herein. In some embodiments, the at least one or more genes or regulatory elements thereof are selected from ACAT1, ACLY, ADORA2A, AXL, B2M, BATF, BCL2L11, BTLA, CAMK2D, cAMP, CASP8, CBLB, CCR5, CD2, CD3D, CD3E, CD3G, CD4, CD5, CD7, CD8A, CD33, CD38, CD52, CD70, CD82, CD86, CD96, CD123, CD160, CD244, CD276, CDK8, CDKN1B, Chi3l1, CIITA, CISH, CSF2CSK, CTLA-4, CUL3, Cyp11a1, DCK, DGKA, DGKZ, DHX37, ELOB (TCEB2), ENTPD1 (CD39), FADD, FAS, GATA3, IL6, IL6R, IL10, IL10RA, IRF4, IRF8, JUNB, Lag3, LAIR-1 (CD305), LDHA, LIF, LYN, MAP4K4, MAPK14, MCJ, MEF2D, MGAT5, NR4A1, NR4A2, NR4A3, NT5E (CD73), ODC1, OTULINL (FAM105A), PAG1, PDCD1, PDIA3, PHD1 (EGLN2), PHD2 (EGLN1), PHD3 (EGLN3), PIK3CD, PIKFYVE, PPARa, PPARd, PRDMI1, PRKACA, PTEN, PTPN2, PTPN6, PTPN11, PVRIG (CD112R), RASA2, RFXANK, SELPG / PSGL1, SIGLEC15, SLA, SLAMF7, SOCS1, Spry1, Spry2, STK4, SUV39, H1TET2, TGFbRII, TIGIT, Tim-3, TMEM222, TNFAIP3, TNFRSF8 (CD30), TNFRSF10B, TOX, TOX2, TRAC, TRBC1, TRBC2, UBASH3A, VHL, VISTA, XBP1, YAP1, and ZC3H12A. In some embodiments, the at least one or more genes or regulatory elements thereof are selected from CD3, CD5, CD7, CD33, CD123, TRAC, LAG-3, FAS, CD52, TRBC1, TRBC2, B2M, and CIITA and PD-1.In some embodiments, the modified immune cell comprises a modification in CD5 and at least one or more genes or regulatory elements thereof selected from the group consisting of TRAC, LAG-3, FAS, CD52, TRBC1, TRBC2, B2M, and CIITA and PD-1. In some embodiments, the modified immune cell comprises a modification in CD7 and at least one or more genes or regulatory elements thereof selected from the group consisting of TRAC, LAG-3, FAS, CD52, TRBC1, TRBC2, B2M, and CIITA and PD-1. In some embodiments, the modified immune cell comprises a modification in CD33 and at least one or more genes or regulatory elements thereof selected from the group consisting of TRAC, LAG-3, FAS, CD52, TRBC1, TRBC2, B2M, and CIITA and PD-1. In some embodiments, the modified immune cell comprises a modification in CD3 and at least one or more genes or regulatory elements thereof selected from the group consisting of TRAC, LAG-3, FAS, CD52, TRBC1, TRBC2, B2M, and CIITA and PD-1. In some embodiments, the modified immune cell comprises a modification in CD123 and at least one or more genes or regulatory elements thereof selected from the group consisting of TRAC, LAG-3, FAS, CD52, TRBC1, TRBC2, B2M, and CIITA and PD-1. Multiplexed editing of genes can be used to create CAR-T cell therapies with improved therapeutic properties. This approach addresses known limitations of multiplexed edited T cell products and is a promising development towards next generation precision cell therapies.

[0543] In one aspect, the present disclosure provides a universal CAR-T cell. In some embodiments, the CAR-T cell described herein is an allogeneic cell. In some embodiments, the universal CAR-T cell is an allogeneic T cell that can be used to express a desired CAR and can be universally applicable regardless of the immunogenic compatibility of the donor and recipient. The allogeneic immune cell can be derived from one or more donors. In certain embodiments, the allogeneic immune cell is derived from a single human donor. For example, the allogeneic T cell can be derived from PBMCs of a single healthy human donor. In certain embodiments, the allogeneic immune cell is derived from multiple human donors. In some embodiments, the universal CAR-T cell can be generated by introducing simultaneous modifications into multiple loci, e.g., three, four, five, six, seven, eight, nine, ten or more loci, using genetic modifications, as described herein. The modification or simultaneous modification as described herein can be a gene edit generated by a base editor, such as a base editing. The base editor can be a C base editor or an A base editor. As discussed herein, the base editing can be used to achieve a gene disruption such that the gene is not expressed. The modification by base editing can be used to achieve a reduction in gene expression. In some embodiments, the base editor can be used to introduce a genetic modification such that the edited gene does not generate a protein product that is structurally or functionally viable. In some embodiments, the modification, such as the simultaneous modification described herein, can comprise a gene edit, such as a base editing, such that the expression or functionality of the gene product is altered in any way. For example, the expression of the gene product can be enhanced or upregulated compared to a baseline expression level. In some embodiments, the activity or functionality of the gene product can be upregulated as a result of the base editing or multiple base editing events that synergize.

[0544] In some embodiments, the generation of universal CAR-T cells can be advantageous over autologous T cells (CAR-T), which can be difficult to generate for immediate use. Allogeneic approaches are more amenable to many situations related to the uncertainty of engineered autologous T cells expressing CARs, and ultimately to obtain the cell product needed for transplantation in medical emergencies, as compared to autologous cell preparations. However, for allogeneic T cells or“off-the-shelf” T cells, it is important to carefully navigate the host’s reactivity to CAR-T cells (HVGD) and the potential hostility of allogeneic T cells to host cells (GVHD). In this context, base editing can be successfully used to generate multiple simultaneous gene editing events such that (a) expression of antigens can be reduced or downregulated to generate alloreactive immune cells; (b) a platform cell type can be generated that is completely devoid or expresses low levels of endogenous T cell receptors, e.g., TCRa chain (such as via base editing of TRAC), or TCRP chain (such as by base editing of TRBC1 / TRBC2); and / or (c) expression of antigens that can be incompatible with the host tissue system can be reduced or downregulated, and vice versa.

[0545] In some embodiments, the methods described herein can be used to generate autologous T cells expressing CAR-T. In some embodiments, multiple base editing events can be implemented in a single electroporation event, thereby reducing electroporation event related toxicity. Any known method for incorporating exogenous genetic material into a cell can be used in place of electroporation, and thus it is contemplated that such methods known in the art be used in any of the methods described herein.

[0546] In some aspects, an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of CD2 and expresses a CD2 chimeric antigen receptor of a CD2 costimulatory domain is administered to a subject having a neoplasia (e.g., a T or NK cell malignancy) or having a potential to develop a neoplasia. In some embodiments, the CD2 modified immune cells administered to the subject are further modified in one or more genes or regulatory elements thereof (e.g., CD52, TRAC, PD-1) with the base editing compositions and methods provided herein.

[0547] As shown herein, the combination of base editing with CAR insertion is a useful strategy for generating alloreactive allogeneic T cells with minimal genomic rearrangements. Multiplex editing of genes can also be used to create CAR-T cell therapies with improved therapeutic properties. This approach addresses known limitations of CAR-T therapies and is a promising development towards next generation precision cell therapies.

[0548] In one experiment, base editor BE4 demonstrated efficient multiplex base editing of three cell surface targets (TRAC, B2M, and PD-1) in T cells, knocking out 95%, 95%, and 88% of gene expression, respectively, in one electroporation to generate a cell population with a high percentage of cells with reduced B2M and CD3 protein expression. Editing each of these genes can be used to create CAR-T cell therapies with improved therapeutic properties. Each of these genes can be silenced by a single targeted base change (C to T) without generating a double-strand break. Thus, cells treated with BE4 also did not show any measurable translocations (large-scale genomic rearrangements), whereas cells with the same triple edit with nucleases did show detectable genomic rearrangements.

[0549] Thus, coupling nuclease-based TRAC gene knockout with simultaneous BE-mediated knockout of two additional genes produces an allogeneic T cell population with minimal genomic rearrangements, enabling targeted insertion of a CAR transgene at the TRAC locus. Taken together, this demonstrates that base editing alone or in combination with a single nuclease knockout and CAR insertion is a useful strategy compared to methods using nucleases alone, which can produce allogeneic T cells with minimal genomic rearrangements. This approach addresses known limitations of multiplex edited T cell products and is a promising development toward next-generation precision cell therapies.

[0550] Chimeric antigen receptors and CAR-T cells

[0551] The present disclosure provides immune cells modified using the nucleobase editors described herein that express a chimeric antigen receptor (CAR). Modifying an immune cell to express a chimeric antigen receptor can enhance the immune response activity of the immune cell, where the chimeric antigen receptor has an affinity for an epitope on an antigen, where the antigen is associated with an altered organism fitness. For example, the chimeric antigen receptor can have an affinity for an epitope on a protein expressed in a tumor cell. Because CAR-T cells can act independent of major histocompatibility complex (MHC), activated CAR-T cells can kill tumor cells that express the antigen. The direct action of CAR-T cells circumvents tumor cell defense mechanisms that evolved as a response to MHC presenting antigens to immune cells.

[0552] In some embodiments, the present disclosure provides immune effector cells expressing a chimeric antigen receptor that targets B cells involved in an autoimmune response (e.g., a B cell of a subject that expresses an antibody generated against a subject’s own tissue).

[0553] Some embodiments comprise an autologous immune cell immunotherapy, in which immune cells are obtained from a subject having a disease characterized by cancerous or otherwise altered cells expressing surface markers or an altered fitness. The obtained immune cells are genetically modified to express a chimeric antigen receptor and effectively re-directed against a specific antigen. Thus, in some embodiments, immune cells are obtained from a subject in need of CAR-T immunotherapy. In some embodiments, after these autologous immune cells are obtained from a subject, the cells are cultured and modified in a short time. In other embodiments, autologous cells are obtained and subsequently stored for further use. This approach can be suitable for individuals who can undergo a parallel therapy in the future that will reduce immune cell counts. In allogeneic immune cell immunotherapy, immune cells can be obtained from a donor other than the subject who will receive the therapy. In some embodiments, immune cells are obtained from a healthy subject or donor and genetically modified to express a chimeric antigen receptor and effectively re-directed against a specific antigen. The immune cells, after being modified to express a chimeric antigen receptor, are administered to a subject for the treatment of neoplasia (e.g., leukemia). In some embodiments, immune cells to be modified to express a chimeric antigen receptor can be obtained from existing stock immune cell cultures.

[0554] Immune cells and / or immune effector cells can be isolated or purified from a sample collected from a subject or donor using standard techniques known in the art. For example, immune effector cells can be isolated or purified from a whole blood sample by lysing red blood cells and removing peripheral mononuclear blood cells by centrifugation. Immune effector cells can be further isolated or purified using a selective purification method that isolates immune effector cells based on cell-specific markers such as CD25, CD3, CD4, CD8, CD28, CD45RA, or CD45RO. In one embodiment, CD25+is used as a marker to select regulatory T cells. In one embodiment, CD4+is used as a marker to select T cells. In one embodiment, CD8+is used as a marker to select T cells. In one embodiment, CD4+and CD8+are used as markers to select T cells. In one embodiment, CD4+and CD25+are used as markers to select T cells.

[0555] In other embodiments, the present invention provides T cells with a targeted gene knockout at the TCR constant region (TRAC) responsible for TCRαβ surface expression. TCRαβ-deficient CAR T cells are compatible with allogeneic immunotherapy (Qasim et al., Sci. Transl. Med. 9, eaaj 2013 (2017); Valton et al., Mol Ther. 2015 Sep; 23(9):1507–1518). If necessary, residual TCRαβ T cells are depleted using CliniMACS magnetic beads to minimize the risk of GVHD. In another embodiment, the present invention provides donor T cells that are indirectly selected in vivo to recognize minimal histocompatibility antigens expressed on recipient hematopoietic cells, thereby minimizing the risk of graft-versus-host disease (GVHD), a leading cause of post-transplant morbidity and mortality (Warren et al., Blood 2010; 115(19):3869–3878). Another technique for isolating or purifying immune effector cells is flow cytometry. In fluorescently activated cell sorting, immune effector cells in the sample are labeled with fluorescently labeled antibodies that have affinity for immune effector cell markers. Cells are isolated using a gating strategy suitable for cells expressing that marker. For example, T cells can be separated from other cells in the sample by using, for example, fluorescently labeled antibodies specific to immune effector cell markers (e.g., CD4, CD8, CD28, CD45) and corresponding gating strategies. In one embodiment, a CD45 gating strategy is used. In some embodiments, a gating strategy specific to immune effector cells is used instead of or in combination with the CD45 gating strategy. In one embodiment, a CD4 gating strategy is used. In one embodiment, a CD8 gating strategy is used. In one embodiment, a CD25 gating strategy is used. In one embodiment, a CD4 and CD8 gating strategy is used. In one embodiment, a CD4 and CD26 gating strategy is used. In some embodiments, a gating strategy specific to immune effector cells is used instead of or in combination with the CD4, CD25, and / or CD8 gating strategies. In some embodiments, a gating strategy specific to immune effector cells is used instead of or in combination with the CD4, CD25, and / or CD8 gating strategies. Figure 40 The gating strategy provided in the middle.

[0556] The immune effector cells contemplated by the present application are effector T cells. In some embodiments, the effector T cells are natural CD8+ T cells, cytotoxic T cells, natural killer T (NKT) cells, natural killer (NK) cells, or regulatory T (Treg) 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 cells are CD4+ CD8+ T cells or CD4- CD8- T cells. In some embodiments, the immune effector cells are T helper cells. In some embodiments, the T helper cells are T helper 1 (Thl), T helper 2 (Th2) cells, or CD4-expressing helper T cells (CD4+ T cells). In some embodiments, the immune effector cells are effector NK cells. In some embodiments, the immune effector cells are other T cell subsets. The modified immune effector cells can express exogenous cytokines, different chimeric receptors, or any other agents that would enhance immune effector cell signaling or function in addition to expressing the chimeric antigen receptor. For example, co-expression of a chimeric antigen receptor and a cytokine can enhance the ability of a CAR-T cell to lyse target cells.

[0557] The chimeric antigen receptors contemplated by the present application comprise an extracellular domain, a transmembrane domain, and an intracellular domain. Antigen binding to the extracellular binding domain can activate the CAR-T cell and generate an effector response that includes CAR-T cell proliferation, cytokine production, and other processes that result in the death of the antigen-expressing cell. In some embodiments of the present application, the chimeric antigen receptor further comprises a linker. In some embodiments, the linker is a (GGGGS)nlinker. In some embodiments, the linker is a (GGGGS)3linker. In some embodiments, the CAR of the present application comprises a leader peptide sequence (e.g., N-terminal to the antigen binding domain). An exemplary leader peptide amino acid sequence is: METDTLLLWVLLLWVPGSTG.

[0558] The extracellular binding domain of the chimeric antigen receptors contemplated herein comprises an amino acid sequence of an antibody or antigen-binding fragment thereof that has affinity for a specific antigen. In various embodiments, the CAR specifically binds 5T4. Exemplary anti-5T4 CARs include, but are not limited to, CART-5T4 (Oxford BioMedica plc) and UCART-5T4 (Cellectis SA).

[0559] In various embodiments, the CAR specifically binds alpha-fetoprotein. Exemplary anti-alpha-fetoprotein CARs include, but are not limited to, ET-1402 (Eureka Therapeutics Inc).

[0560] In various embodiments, the CAR specifically binds Axl. Exemplary anti-Axl CARs include, but are not limited to, CCT-301-38 (F1 Oncology Inc).

[0561] In various embodiments, the CAR specifically binds B7H6. Exemplary anti-B7H6 CARs include, but are not limited to, CYAD-04 (Celyad SA).

[0562] In various embodiments, the CAR specifically binds BCMA. Exemplary anti-BCMA CARs include, but are not limited to, ACTR-087 + SEA-BCMA (Seattle Genetics Inc), ALLO-715 (Cellectis SA), ARI-0002 (Institut d’Investigacions Biomediques August Pi I Sunyer), bb-2121 (bluebird bio Inc), bb-21217 (bluebird bio Inc), CART-BCMA (University of Pennsylvania), CT-053 (Carsgen Therapeutics Ltd), Descartes-08 (Cartesian Therapeutics), FCARH-143 (Juno Therapeutics Inc), ICTCAR-032 (Innovative Cellular Therapeutics Co Ltd), IM21 CART (Beijing Immunochina Medical Science & Technology Co Ltd), JCARH-125 (Memorial Sloan-Kettering Cancer Center), KITE-585 (Kite Pharma Inc), LCAR-B38M (Nanjing Legend Biotech Co Ltd), LCAR-B4822M (Nanjing Legend Biotech Co Ltd), MCARH-171 (Memorial Sloan-Kettering Cancer Center), P-BCMA-101 (Poseida Therapeutics Inc), P-BCMA-ALLO1 (Poseida Therapeutics Inc), spCART-269 (Shanghai Unicar-Therapy Bio-medicine Technology Co Ltd), and BCMA02 / bb2121 (bluebird bio Inc). The polypeptide sequence of the BCMA02 / bb2121 CAR is provided below:

[0563] MALPVTALLLPLALLLHAARPDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0564] In various embodiments, the CAR specifically binds CCK2R. Exemplary anti-CCK2R CARs include, but are not limited to, anti-CCK2R CAR-T linker molecule (CAM) + anti-FITC CAR T cell therapy (cancer), Endocyte / Purdue (Purdue University).

[0565] In various embodiments, the CAR specifically binds CD antigen. Exemplary anti-CD antigen CARs include, but are not limited to, VM-802 (ViroMed Co Ltd).

[0566] In various embodiments, the CAR specifically binds CD123. Exemplary anti-CD123 CARs include, but are not limited to, MB-102 (Fortress Biotech Inc), RNA CART123 (University of Pennsylvania), SFG-iMC-CD123ζ (Bellicum Pharmaceuticals Inc), and UCART-123 (Cellectis SA).

[0567] In various embodiments, the CAR specifically binds CD133. Exemplary anti-CD133 CARs include, but are not limited to, KD-030 (Nanjing Kaedi Biotech Inc).

[0568] In various embodiments, the CAR specifically binds CD138. Exemplary anti-CD138 CARs include, but are not limited to, ATLCAR.CD138 (UNC Lineberger Comprehensive Cancer Center) and CART-138 (Chinese PLA General Hospital).

[0569] In various embodiments, the CAR specifically binds CD171. Exemplary anti-CD171 CARs include, but are not limited to, JCAR-023 (Juno Therapeutics Inc).

[0570] In various embodiments, the CAR specifically binds CD19. Exemplary anti-CD19 CARs include, but are not limited to, 1928z-41BBL (Memorial Sloan-Kettering Cancer Center), 1928z-E27 (Memorial Sloan-Kettering Cancer Center), 19-28z-T2 (Guangzhou Biomedical Institute), 4G7-CARD (University College London), 4SCAR19 (Shenzhen ImmuneGene Therapy Research Institute), ALLO-501 (Pfizer Inc), ATA-190 (QIMR Berghofer Medical Research Institute), AUTO-1 (University College London), AVA-008 (Avacta Ltd), axicabtagene ciloleucel (Kite Pharma Inc), BG-T19 (Guangzhou Biogenetech Co Ltd), BinD-19 (Shenzhen BinDeBio Ltd.), BPX-401 (Bellicum Pharmaceuticals Inc), CAR19h28TM41BBz (Westmead Institute for Medical Research), C-CAR-011 (General Hospital of People's Liberation Army), CD19CART (Innovative Cellular Therapeutics Co Ltd), CIK-CAR.CD19 (Formula Pharmaceuticals Inc), CLIC-1901 (Ottawa Hospital Research Institute), CSG-CD19 (Carsgen Therapeutics Ltd), CTL-119 (University of Pennsylvania), CTX-101 (CRISPR Therapeutics AG), DSCAR-01 (Shanghai Hrain Biotechnology), ET-190 (Eureka Therapeutics Inc), FT-819 (Memorial Sloan-Kettering Cancer Center), ICAR-19 (Immune Cell Therapy Inc), IM19 CAR-T (Beijing Immunochina Medical Science & Technology Co Ltd), JCAR-014 (Juno Therapeutics Inc), JWCAR-029 (MingJu Therapeutics (Shanghai) Co., Ltd), KD-C-19 (Nanjing Kaedi Biotech Inc), LinCART19 (iCellGene Therapeutics), lisocabtagene maraleucel (Juno Therapeutics Inc), MatchCART (Shanghai Hrain Biotechnology), MB-CART19.1 (Shanghai Children's Medical Center), PBCAR-0191 (Precision BioSciences Inc), PCAR-019 (PersonGen Biomedicine (Suzhou) Co Ltd), pCAR-19B (Chongqing Precision Biotechnology Co Ltd), PZ-01 (Pinze Life Technology Co Ltd), RB-1916 (Refuge Biotechnologies Inc), SKLB-083019 (Chengdu Silverlink Pharmaceutical Co Ltd), spCART-19 (Shanghai Yookang Biomedicine Technology Co Ltd), TBI-1501 (Takara Bio Inc), TC-110 (TCR2 Therapeutics Inc), TI-1007 (Timmune Biotech Inc), tisagenlecleucel (Abramson Cancer Center of the University of Pennsylvania), U-CART (Shanghai Bioray Laboratory Inc), UCART-19 (Wugen Inc), UCART-19 (Cellectis SA), vadacabtagene leucel (Memorial Sloan-Kettering Cancer Center), XLCART-001 (Nanjing Medical University), and yinnuoka ti-19 (Shenzhen Yinnuo Immunotech Co Ltd).

[0571] In various embodiments, the CAR specifically binds CD2. Exemplary anti-CD2 CARs include, but are not limited to, UCART-2 (Wugen Inc).

[0572] In various embodiments, the CAR specifically binds CD20. Exemplary anti-CD20 CARs include, but are not limited to, ACTR-087 (National University of Singapore), ACTR-707 (Unum Therapeutics Inc), CBM-C20.1 (General Hospital of People's Liberation Army), MB-106 (Fred Hutchinson Cancer Research Center), and MB-CART20.1 (Miltenyi Biotec GmbH).

[0573] In various embodiments, the CAR specifically binds CD22. Exemplary anti-CD22 CARs include, but are not limited to, anti-CD22 CAR T cell therapy (B-cell acute lymphoblastic leukemia), University of Pennsylvania, CD22-CART (Shanghai Yookang Biomedicine Technology Co., Ltd.), JCAR-018 (Opus Bio Inc), MendCART (Shanghai Hrain Biotechnology), and UCART-22 (Cellectis SA).

[0574] In various embodiments, the CAR specifically binds CD30. Exemplary anti-CD30 CARs include, but are not limited to, ATLCAR.CD30 (UNC Lineberger Comprehensive Cancer Center), CBM-C30.1 (General Hospital of People’s Liberation Army), and Hu30-CD28zeta (National Cancer Institute).

[0575] In various embodiments, the CAR specifically binds CD33. Exemplary anti-CD33 CARs include, but are not limited to, anti-CD33 CAR gd T cell therapy (acute myeloid leukemia), TC BioPharm / University College London, CAR33 VH (Opus Bio Inc), CART-33 (General Hospital of People’s Liberation Army), CIK-CAR.CD33 (Formula Pharmaceuticals Inc), UCART-33 (Cellectis SA), and VOR-33 (Columbia University).

[0576] In various embodiments, the CAR specifically binds CD38. Exemplary anti-CD38 CARs include, but are not limited to, UCART-38 (Cellectis SA).

[0577] In various embodiments, the CAR specifically binds CD38 A2. Exemplary anti-CD38 A2 CARs include, but are not limited to, T-007 (TNK Therapeutics Inc).

[0578] In various embodiments, the CAR specifically binds CD4. Exemplary anti-CD4 CARs include, but are not limited to, CD4 CAR (iCell Gene Therapeutics).

[0579] In various embodiments, the CAR specifically binds CD44. Exemplary anti-CD44 CARs include, without limitation, CAR-CD44v6 (Istituto Scientifico H San Raffaele).

[0580] In various embodiments, the CAR specifically binds CD5. Exemplary anti-CD5 CARs include, without limitation, CD5 CAR (iCell Gene Therapeutics). Exemplary CD5 CAR nucleic acid sequences are provided below:

[0581]

[0582]

[0583]

[0584]

[0585]

[0586]

[0587]

[0588] In various embodiments, the CAR specifically binds CD7. Exemplary anti-CD7 CARs include, without limitation, CAR-pNK (PersonGen Biomedicine (Suzhou) Co Ltd) and CD7.CAR / 28z CAR T cells (Baylor College of Medicine), UCART7 (Washington University in St Louis). An exemplary CD7 CAR amino acid sequence is provided below:

[0589]

[0590]

[0591] In various embodiments, the CAR specifically binds CDH17. Exemplary anti-CDH17 CARs include, without limitation, ARB-001.T (Arbele Ltd).

[0592] In various embodiments, the CAR specifically binds CEA. Exemplary anti-CEA CARs include, without limitation, HORC-020 (HumOrigin Inc).

[0593] In various embodiments, the CAR specifically binds Claudin 18.2. Exemplary anti-Claudin 18.2 CARs include, but are not limited to, CAR-CLD18 T cells (Carsgen Therapeutics Ltd) and KD-022 (Nanjing Kaedi Biotech Inc).

[0594] In various embodiments, the CAR specifically binds Claudin 18.2. Exemplary anti-Claudin 18.2 CARs include, but are not limited to, CAR-CLD18 T cells (Carsgen Therapeutics Ltd) and KD-022 (Nanjing Kaedi Biotech Inc).

[0595] In various embodiments, the CAR specifically binds CLL1. Exemplary anti-CLL1 CARs include, but are not limited to, KITE-796 (Kite Pharma Inc).

[0596] In various embodiments, the CAR specifically binds DLL3. Exemplary anti-DLL3 CARs include, but are not limited to, AMG-119 (Amgen Inc).

[0597] In various embodiments, the CAR specifically binds dual BCMA / TACI (APRIL). Exemplary anti-dual BCMA / TACI (APRIL) CARs include, but are not limited to, AUTO-2 (Autolus Therapeutics Limited).

[0598] In various embodiments, the CAR specifically binds dual CD19 / CD22. Exemplary anti-dual CD19 / CD22 CARs include, but are not limited to, AUTO-3 (Autolus Therapeutics Limited) and LCAR-L10D (Nanjing Legend Biotech Co Ltd).

[0599] In various embodiments, the CAR specifically binds CD19.

[0600] In various embodiments, the CAR specifically binds dual CLL1 / CD33. Exemplary anti-dual CLL1 / CD33 CARs include, but are not limited to, ICG-136 (iCell Gene Therapeutics).

[0601] In various embodiments, the CAR specifically binds dual EpCAM / CD3. Exemplary anti-dual EpCAM / CD3 CARs include, but are not limited to, IKT-701 (Icell Kealex Therapeutics).

[0602] In various embodiments, the CAR specifically binds dual ErbB / 4ab. Exemplary anti-dual ErbB / 4ab CARs include, but are not limited to, LEU-001 (King's College London).

[0603] In various embodiments, the CAR specifically binds dual FAP / CD3. Exemplary anti-dual FAP / CD3 CARs include, but are not limited to, IKT-702 (IcellKealex Therapeutics).

[0604] In various embodiments, the CAR specifically binds EBV. Exemplary anti-EBV CARs include, but are not limited to, TT-18 (Tessa Therapeutics Pte Ltd).

[0605] In various embodiments, the CAR specifically binds EGFR. Exemplary anti-EGFR CARs include, but are not limited to, anti-EGFR CAR T cell therapy (CBLB MegaTAL, cancer), bluebird bio Inc; anti-EGFR CAR T cell therapy expressing CTLA-4 checkpoint inhibitor + PD-1 checkpoint inhibitor mAbs (EGFR-positive advanced solid tumors), Shanghai Cell Therapy Research Institute; CSG-EGFR (Carsgen Therapeutics Ltd); and EGFR-IL12-CART (Pregene (Shenzhen) Biotechnology Co Ltd).

[0606] In various embodiments, the CAR specifically binds EGFRvIII. Exemplary anti-EGFRvIII CARs include, but are not limited to, KD-035 (Nanjing Kaedi Biotech Inc) and UCART-EgfrVIII (Cellectis SA).

[0607] In various embodiments, the CAR specifically binds Flt3. Exemplary anti-Flt3 CARs include, but are not limited to, ALLO-819 (Pfizer Inc) and AMG-553 (Amgen Inc).

[0608] In various embodiments, the CAR specifically binds folate receptor. Exemplary anti-folate receptor CARs include, but are not limited to, EC17 / CAR T (Endocyte Inc).

[0609] In various embodiments, the CAR specifically binds G250. Exemplary anti-G250 CARs include, but are not limited to, Autologous T-Lymphocyte Therapy (G250-scFV transduced, renal cell carcinoma), Erasmus Medical Center (Daniel den Hoed Cancer Center).

[0610] In various embodiments, the CAR specifically binds GD2. Exemplary anti-GD2 CARs include, but are not limited to, 1RG-CART (University College London), 4SCAR-GD2 (Shenzhen Geno-Immune Medical Institute), C7R-GD2.CART cells (Baylor College of Medicine), CMD-501 (Baylor College of Medicine), CSG-GD2 (Carsgen Therapeutics Ltd), GD2-CART01 (Bambino Gesu Hospital and Research Institute), GINAKIT cells (Baylor College of Medicine), iC9-GD2-CAR-IL-15 T cells (UNC Lineberger Comprehensive Cancer Center), and IKT-703 (Icell Kealex Therapeutics).

[0611] In various embodiments, the CAR specifically binds GD2 and MUC1. Exemplary anti-GD2 / MUC1 CARs include, but are not limited to, PSMACAR-T (University of Pennsylvania).

[0612] In various embodiments, the CAR specifically binds GPC3. Exemplary anti-GPC3 CARs include, but are not limited to, ARB-002.T (Arbele Ltd), CSG-GPC3 (Carsgen Therapeutics Ltd), GLYCAR (Baylor College of Medicine), and TT-14 (Tessa Therapeutics Pte Ltd).

[0613] In various embodiments, the CAR specifically binds Her2. Exemplary anti-Her2 CARs include, but are not limited to, ACTR-087 + trastuzumab (Unum Therapeutics Inc), ACTR-707 + trastuzumab (Unum Therapeutics Inc), CIDeCAR (Bellicum Pharmaceuticals Inc), MB-103 (Mustang Bio Inc), RB-H21 (Refuge Biotechnologies Inc), and TT-16 (Baylor College of Medicine).

[0614] In various embodiments, the CAR specifically binds IL13R. Exemplary anti-IL13R CARs include, but are not limited to, MB-101 (City of Hope) and YYB-103 (YooYoung Pharmaceuticals Co Ltd).

[0615] In various embodiments, the CAR specifically binds Integrin β-7. Exemplary anti- Integrin β-7 CARs include, but are not limited to, MMG49 CAR T cell therapy (Osaka University).

[0616] In various embodiments, the CAR specifically binds LC antigen. Exemplary anti-LC antigen CARs include, but are not limited to, VM-803 (ViroMed Co Ltd) and VM-804 (ViroMed Co Ltd).

[0617] In various embodiments, the CAR specifically binds mesothelin. Exemplary anti-mesothelin CARs include, but are not limited to, CARMA-hMeso (Johns Hopkins University), CSG-MESO (Carsgen Therapeutics Ltd), iCasp9M28z (Memorial Sloan-Kettering Cancer Center), KD-021 (Nanjing Kaedi Biotech Inc), m-28z-T2 (Guangzhou Institutes of Biomedicine and Health), MesoCART (University of Pennsylvania), meso-CAR-T+PD-78 (MirImmune LLC), RB-M1 (Refuge Biotechnologies Inc), and TC-210 (TCR2 Therapeutics Inc).

[0618] In various embodiments, the CAR specifically binds MUC1. Exemplary anti-MUC1 CARs include, but are not limited to, anti-MUC1 CAR T-cell therapy+PD-1 knockout T cell therapy (esophageal cancer / NSCLC), Guangzhou Anjie Biomedical Technology / University of Technology Sydney (Guangzhou Anjie Biomedical Technology Co LTD), ICTCAR-043 (Innovative Cellular Therapeutics Co Ltd), ICTCAR-046 (Innovative Cellular Therapeutics Co Ltd), P-MUC1C-101 (Poseida Therapeutics Inc), and TAB-28z (OncoTab Inc).

[0619] In various embodiments, the CAR specifically binds MUC16. Exemplary anti-MUC16 CARs include, but are not limited to, 4H1128Z-E27 (Eureka Therapeutics Inc) and JCAR-020 (Memorial Sloan-Kettering Cancer Center).

[0620] In various embodiments, the CAR specifically binds nfP2X7. Exemplary anti- nfP2X7 CARs include, but are not limited to, BIL-022c (Biosceptre International Ltd).

[0621] In various embodiments, the CAR specifically binds PSCA. Exemplary anti-PSCA CARs include, but are not limited to, BPX-601 (Bellicum Pharmaceuticals Inc).

[0622] In various embodiments, the CAR specifically binds PSMA. CIK-CAR.PSMA (Formula Pharmaceuticals Inc) and P-PSMA-101 (Poseida Therapeutics Inc).

[0623] In various embodiments, the CAR specifically binds ROR1. Exemplary anti-ROR1 CARs include, but are not limited to, JCAR-024 (Fred Hutchinson Cancer Research Center).

[0624] In various embodiments, the CAR specifically binds ROR2. Exemplary anti-ROR2 CARs include, but are not limited to, CCT-301-59 (F1 Oncology Inc).

[0625] In various embodiments, the CAR specifically binds SLAMF7. Exemplary anti-SLAMF7 CARs include, but are not limited to, UCART-CS1 (Cellectis SA).

[0626] In various embodiments, the CAR specifically binds TRBC1. Exemplary anti-TRBC1 CARs include, but are not limited to, AUTO-4 (Autolus Therapeutics Limited).

[0627] In various embodiments, the CAR specifically binds TRBC2. Exemplary anti-TRBC2 CARs include, but are not limited to, AUTO-5 (Autolus Therapeutics Limited).

[0628] In various embodiments, the CAR specifically binds TSHR. Exemplary anti-TSHR CARs include, but are not limited to, ICTCAT-023 (Innovative Cellular Therapeutics Co Ltd).

[0629] In various embodiments, the CAR specifically binds to VEGFR-1. Exemplary anti-VEGFR-1 CARs include, but are not limited to, SKLB-083017 (Sichuan University).

[0630] In various embodiments, the CAR is AT-101 (AbClon Inc); AU-101, AU-105, and AU-180 (Aurora Biopharma Inc); CARMA-0508 (Carisma Therapeutics); CAR-T (Fate Therapeutics Inc); CAR-T (CellDesign Labs Inc); CM-CX1 (Celdara Medical LLC); CMD-502, CMD-503, and CMD-504 (Baylor College of Medicine); CSG-002 and CSG-005 (Carsgen Therapeutics Ltd); ET-1501, ET-1502, and ET-1504 (Eureka Therapeutics Inc); FT-61314 (Fate Therapeutics Inc); GB-7001 (Shanghai GeneChem Co Ltd); IMA-201 (Immatics Biotechnologies GmbH); IMM-005 and IMM-039 (Immunome Inc); ImmuniCAR (TC BioPharm Ltd); NT-0004 and NT-0009 (BioNTech Cell and Gene Therapies GmbH), OGD-203 (OGD2 Pharma SAS), PMC-005B (PharmAbcine), and TI-7007 (Timmune Biotech Inc).

[0631] Also provided herein are nucleic acids encoding the chimeric antigen receptors described herein. In some embodiments, the nucleic acids are isolated or purified. Indirect in vivo delivery of nucleic acids can be performed using methods known in the art. For example, immune cells obtained from a subject can be transformed with a nucleic acid vector encoding a chimeric antigen receptor. This vector can then be used to transform recipient immune cells, such that these cells will subsequently express the chimeric antigen receptor. Effective means of transforming immune cells include transfection and transduction. Such methods are well known in the art. For example, methods suitable for delivering nucleic acid molecules encoding chimeric antigen receptors (and nucleic acids encoding base editors) can be found in International Patent Application No. PCT / US2009 / 040040 and U.S. Patent Nos. 8,450,112, 9,132,153, and 9,669,058, each of which is incorporated herein in its entirety. Moreover, those methods and vectors described herein for delivering nucleic acids encoding base editors are suitable for delivering nucleic acids encoding chimeric antigen receptors.

[0632] Extracellular binding domain

[0633] The chimeric antigen receptors of the present invention include an extracellular binding domain. The extracellular binding domain of the chimeric antigen receptors contemplated herein comprises an amino acid sequence of an antibody or antigen-binding fragment thereof, which has affinity for a specific antigen. In some embodiments, the antigen is CD3. In some embodiments, the antigen is CD5. In some embodiments, the antigen is CD7. In some embodiments, the antigen is CD33. In some embodiments, the antigen is CD123.

[0634] In some embodiments, the chimeric antigen receptor comprises an amino acid sequence of an antibody. In some embodiments, the chimeric antigen receptor comprises an amino acid sequence of an antigen-binding fragment of an antibody. The antibody (or fragment thereof) portion of the extracellular binding domain recognizes and binds to an epitope of an antigen. In some embodiments, the antibody fragment portion of the chimeric antigen receptor is a single-chain variable fragment (scFv). scFvs comprise the light and variable fragments of a monoclonal antibody. In other embodiments, the antibody fragment portion of the chimeric antigen receptor is a multi-chain variable fragment, which can comprise more than one extracellular binding domain and thus bind to more than one antigen simultaneously. In multi-chain variable fragment embodiments, a hinge region can separate the different variable fragments, providing the necessary spatial arrangement and flexibility.

[0635] In other embodiments, the antibody portion of the chimeric antigen receptor comprises at least one heavy chain and at least one light chain. In some embodiments, the antibody portion of the chimeric antigen receptor comprises two heavy chains and two light chains that are joined by disulfide bridges, where the light chains are each joined to one of the heavy chains by a disulfide bridge. In some embodiments, the light chains comprise a constant region and a variable region. Complementarity determining regions located within the variable region of an antibody are responsible for the affinity of the antibody for a particular antigen. Thus, antibodies that recognize different antigens comprise different complementarity determining regions. The complementarity determining regions are located within the variable domains of the extracellular binding domain, and the variable domains (i.e., variable heavy and variable light) can be joined using a linker, or in some embodiments, a disulfide bridge. In some embodiments, the variable heavy chain and the variable light chain are joined by a (GGGGS)nlinker, where n is an integer from 1 to 10. In some embodiments, the linker is a (GGGGS)3linker.

[0636] In some embodiments, the antigen recognized and bound by the extracellular domain is a protein or peptide, a nucleic acid, a lipid, or a polysaccharide. The antigen can be heterologous, such as those expressed in pathogenic bacteria or viruses. The antigen can also be synthetic; for example, some individuals are extremely allergic to synthetic latex, and exposure to that antigen can result in an extreme immune response. In some embodiments, the antigen is autologous, and is expressed on a diseased or otherwise altered cell. For example, in some embodiments, the antigen is expressed in a tumor cell. In some embodiments, the tumor cell is a solid tumor cell. In other embodiments, the tumor cell is a liquid tumor cell. In other embodiments, the tumor cell is a hematological cancer, such as a B-cell cancer. In some embodiments, the B-cell cancer is a lymphoma or a leukemia.

[0637] The liquid cancer to be treated using the methods described herein can be, for example, a leukemia. In some cases, the leukemia includes a pre-leukemia. In some cases, the leukemia is an acute leukemia. Acute leukemias include, for example, acute myeloid leukemia (AML). Acute leukemias also include, for example, acute lymphoid leukemia or acute lymphoblastic leukemia (ALL); ALL includes B-lineage ALL, T-lineage ALL, and T-cell acute lymphoblastic leukemia (T-ALL).

[0638] Non-limiting examples of neoplasia include T-cell acute lymphoblastic leukemia (T-ALL), mycosis fungoides (MF), Sezary syndrome (SS), peripheral T / NK cell lymphoma, anaplastic large cell lymphoma ALK+, primary cutaneous T-cell lymphoma, T-cell large granular lymphocyte type 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 gd T-cell lymphoma, aggressive NK-cell leukemia, and enteropathy-associated T-cell lymphoma. In some embodiments, the neoplasia is T-cell acute lymphoblastic leukemia (T-ALL). In some embodiments, the neoplasia is T-cell acute myeloid leukemia (AML).

[0639] Antibody-antigen interactions are noncovalent interactions resulting from hydrogen bond bonding, electrostatic or hydrophobic interactions, or van der Waals forces. The affinity of the extracellular binding domain of a chimeric antigen receptor for an antigen can be calculated using the following equation:

[0640] KA = [Ab-Ag] / [Ab][Ag], where

[0641] [Ab] = molar concentration of unoccupied binding sites on the antibody;

[0642] [Ag] = molar concentration of unoccupied binding sites on the antigen; and

[0643] [Ab-Ag] = molar concentration of antibody-antigen complex.

[0644] Antibody-antigen interactions can also be characterized based on the dissociation of antigen from antibody. The dissociation constant (KD) is the ratio of the association rate to the dissociation rate and is inversely related to the affinity constant. Thus, KD = 1 / KA. Those skilled in the art will be familiar with these concepts and will know that traditional methods such as ELISA assays can be used to calculate these constants.

[0645] Transmembrane domain

[0646] The chimeric antigen receptors of the present application include a transmembrane domain. The transmembrane domain of the chimeric antigen receptors described herein spans the CAR-T cell lipid bilayer cell membrane and separates the extracellular binding domain from the intracellular signaling domain. In some embodiments, this domain is derived from other receptors with transmembrane domains, while in other embodiments, this domain is synthetic. In some embodiments, the transmembrane domain can be derived from a non-human transmembrane domain, and in some embodiments can be humanized. By "humanized" is meant a sequence having a nucleic acid encoding a transmembrane domain that is optimized so as to be more easily or efficiently expressed in a human subject. In some embodiments, the transmembrane domain is derived from another transmembrane domain expressed in a human immune effector cell. Examples of such proteins include, but are not limited to, the subunits of the T cell receptor (TCR) complex PD1, or any cluster of differentiation protein, or other proteins expressed in immune effector cells and proteins with transmembrane domains. In some embodiments, the transmembrane domain will be synthetic, and such sequences will comprise a number of hydrophobic residues.

[0647] The transmembrane domain for the disclosed CARs can include at least the transmembrane region 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, CD154. In some embodiments, the transmembrane domain is derived from CD4, CD8 alpha, CD28, or CD3 zeta. In some embodiments, the transmembrane domain is a CD28 transmembrane domain. In some embodiments, the transmembrane domain is a CD8 alpha transmembrane domain.

[0648] In some embodiments, the transmembrane domain is a CD8 alpha hinge and transmembrane domain. In some embodiments, the CD8 alpha hinge and transmembrane domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the exemplary amino acid sequence provided below:

[0649] SDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

[0650] In some embodiments, the chimeric antigen receptor is designed to include a spacer sequence between the transmembrane domain and the extracellular domain, the intracellular domain, or both. Such a spacer sequence can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the spacer sequence can be 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids in length. In yet other embodiments, the spacer sequence can be between 100 and 500 amino acids in length. The spacer sequence can be any polypeptide that links one domain to another and serves to position such linked domains to enhance or optimize chimeric antigen receptor function. In some embodiments, the hinge / spacer sequence is selected from CH3, CD8a, or CD28.

[0651] Intracellular signaling domain

[0652] The chimeric antigen receptors of the present application include an intracellular signaling domain. An intracellular signaling domain is the intracellular portion of a protein expressed within a T cell that transduces a T cell effector function signal (e.g., an activation signal) and directs the T cell to perform a specialized function. T cell activation can be induced by a variety of factors, including the binding of an antigen to a T cell receptor on the surface of a T cell and the binding of a cognate ligand to a costimulatory molecule on the surface of a T cell. A T cell costimulatory molecule is a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules. Activation of a T cell leads to an immune response, such as T cell proliferation and differentiation (see, e.g., Smith-Garvin et al., Annu. Rev. Immunol., 27:591-619, 2009). Exemplary T cell signaling domains are known in the art. Non-limiting examples include CD3 zeta, CD8, CD28, CD27, CD154, GITR (TNFRSF18), CD134 (OX40), and CD137 (4-1BB) signaling domains.

[0653] The intracellular signaling domain of the chimeric antigen receptors contemplated herein comprises a primary signaling domain. In some embodiments, the chimeric antigen receptor comprises a primary signaling domain and the term or a costimulatory signaling domain.

[0654] In some embodiments, the primary signaling domain comprises one or more immunoreceptor tyrosine-based activation motifs or ITAMs. In some embodiments, the primary signaling domain comprises more than one ITAM. ITAMs incorporated into a chimeric antigen receptor can be derived from ITAMs from other cellular receptors. In some embodiments, the primary signaling domain comprising an ITAM can be derived from a subunit of the TCR complex, such as CD3y, CD3s, CD3z, or CD3d (see Figure 1A ). In some embodiments, the primary signaling domain comprising an ITAM can be derived from FcRy, FcRP, CD5, CD22, CD79a, CD79b, or CD66d.

[0655] In some embodiments, the primary signaling domain is selected from the group consisting of CD8, CD28, CD134 (OX40), CD137 (4-1BB), and CD3z. In some embodiments, the primary signaling domain is a CD3z signaling domain. In some embodiments, the CD3z signaling domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the exemplary amino acid sequence provided below:

[0656] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0657] In some embodiments, the primary signaling domain is a CD134 (OX40) signaling domain. In some embodiments, the CD134 (OX40) signaling domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the exemplary amino acid sequence provided below:

[0658] rrdqrlppdahkppgggsfrtpiqeeqadahstlaki

[0659] In some embodiments, the secondary or costimulatory signaling domain is derived from 4-1BB, CD2, CD4, CD28, CDS, CD8a, CD83, CD134, CD137, ICOS, or CD154. In some embodiments, the secondary signaling domain is a CD28 signaling domain. In some embodiments, the CD28 signaling domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the exemplary amino acid sequence provided below:

[0660] SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0661] In some embodiments, the secondary signaling domain is a CD137 (4-1BB) signaling domain. In some embodiments, the CD137 (4-1BB) signaling domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the exemplary amino acid sequence provided below:

[0662] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0663] In some embodiments, the CD137 (4-1BB) signaling domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the exemplary amino acid sequence provided below:

[0664] RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0665] In some embodiments, the CAR comprises one or more signaling domains. In some embodiments, the CAR comprises a 4-1BB signaling domain and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a CD28 signaling domain and a CD3 zeta signaling domain.

[0666] Editing of target genes in immune cells

[0667] The present disclosure provides immune cells comprising a chimeric antigen receptor (CAR) and one or more edited genes, one or more regulatory elements thereof, or a combination thereof, wherein expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cells comprise a CAR and an altered endogenous gene that provides resistance to fratricide, enhances immune cell function, resistance to immune checkpoint or suppression, or a combination thereof. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are CAR-T cells. In some embodiments, the immune cells are NK cells. In some embodiments, each edited gene can comprise a single base edit. In some embodiments, each edited gene can comprise multiple base edits located at different regions of the gene.

[0668] In some embodiments, a single modification event, such as electroporation, can introduce one or more base 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 can be introduced into one or more genes simultaneously. In some embodiments, immune cells, including but not limited to any immune cell comprising an edited gene selected from any of the foregoing gene edits, can be edited to generate a mutation in other genes that enhances the function of the CAR-T or reduces immune suppression or suppression of the cell.

[0669] In some embodiments, the CAR-T cells have increased fratricide resistance compared to similar CAR-T cells that do not further have one or more edits described herein. In some embodiments, the CAR-T cells have reduced immunogenicity compared to similar CAR-T cells that do not further have one or more edits described herein. In some embodiments, the CAR-T cells have a lower activation threshold compared to similar CAR-T cells that do not further have one or more edits described herein. In some embodiments, the CAR-T cells have increased anti-tumor forming activity compared to similar CAR-T cells that do not further have one or more edits described herein.

[0670] In some embodiments, provided herein is an immune cell having at least one modification in an endogenous gene or regulatory element thereof. In some embodiments, the immune cell can comprise further modifications in at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more endogenous genes or regulatory elements thereof.

[0671] In some embodiments, the one or more genes, or one or more regulatory elements thereof, or combinations thereof can be selected from the group consisting of CD3 antigen (CD3); CD5 antigen (CD5); CD7 antigen (CD7); CD33 antigen (CD33); CD52 antigen (CD52); CD123 antigen (CD123); T cell receptor alpha constant region (TRAC); programmed cell death 1 (PDCD1 or PD-1); Fas cell surface death receptor (FAS); lymphocyte-activation gene 3 (LAG-3); class II major histocompatibility complex transactivator (CIITA); T cell receptor beta constant region 1 (TRBC1); T cell receptor beta constant region 2 (TRBC2); and beta-2 microglobulin (B2M). In some embodiments, CD3, CD5, CD7, CD33, or CD123 is edited. In some embodiments, the immune cell comprises an edited CD3 gene, and additionally at least one edited gene. In some embodiments, the immune cell comprises an edited CD5 gene, and additionally at least one edited gene. In some embodiments, the immune cell comprises an edited CD7 gene, and additionally at least one edited gene. In some embodiments, the immune cell comprises an edited CD33 gene, and additionally at least one edited gene. In some embodiments, the immune cell comprises an edited CD123 gene, and additionally at least one edited gene. The at least one edited gene can be selected from the list of genes mentioned in the preceding paragraph. In some embodiments, CD3, CD5, CD7, CD33, or CD123 is edited in combination with one or more of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0672] In various embodiments, the modified immune cell comprises a mutation in one or more or a combination of CD5, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In various embodiments, the modified immune cell comprises a mutation in two, three, four, five, six, seven, eight, nine, or ten of CD5, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In various embodiments, the modified immune cell comprises a mutation in CD5 and one or more or a combination of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In one embodiment, the modified immune cell comprises a mutation in CD5, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0673] In various embodiments, the modified immune cell comprises a mutation in one or more or a combination of CD7, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In various embodiments, the modified immune cell comprises a mutation in two, three, four, five, six, seven, eight, nine, or ten of CD7, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In various embodiments, the modified immune cell comprises a mutation in CD7 and one or more or a combination of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In one embodiment, the modified immune cell comprises a mutation in CD7, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0674] In various embodiments, the modified immune cell comprises a mutation in one or more or a combination of CD3, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In various embodiments, the modified immune cell comprises a mutation in two, three, four, five, six, seven, eight, nine, or ten of CD3, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In various embodiments, the modified immune cell comprises a mutation in CD3 and one or more or a combination of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In one embodiment, the modified immune cell comprises a mutation in CD3, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0675] In various embodiments, the modified immune cell comprises a mutation in one or more or a combination of CD33, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In various embodiments, the modified immune cell comprises a mutation in two, three, four, five, six, seven, eight, nine, or ten of CD33, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In various embodiments, the modified immune cell comprises a mutation in CD33 and one or more or a combination of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In one embodiment, the modified immune cell comprises a mutation in CD33, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0676] In various embodiments, the modified immune cell comprises a mutation in one or more of CD123, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1, or a combination thereof. In various embodiments, the modified immune cell comprises a mutation in two, three, four, five, six, seven, eight, nine, or ten of CD123, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In various embodiments, the modified immune cell comprises a mutation in CD123 and one or more of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1, or a combination thereof. In one embodiment, the modified immune cell comprises a mutation in CD123, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.

[0677] In some embodiments, at least one modification is a single nucleobase modification. In some embodiments, the altered endogenous gene can be created by base editing. In some embodiments, base editing can reduce or impair gene expression. In some embodiments, base editing can reduce or impair gene activation. In some embodiments, base editing can reduce or impair functionality of a gene product. In some other embodiments, base editing can activate or enhance gene expression. In some embodiments, base editing can increase functionality of a gene product.

[0678] Allogeneic immune cells expressing endogenous immune cell receptors as well as chimeric antigen receptors can recognize and adhere to host cells, a condition known as graft versus host disease (GVHD). The alpha component of the immune cell receptor complex is encoded by the TRAC gene, and in some embodiments, this gene is edited such that the alpha subunit of the TCR complex is nonfunctional or absent. Because this subunit is necessary for endogenous immune cell signaling, editing this gene can reduce the risk of graft versus host disease caused by allogeneic immune cells.

[0679] In some embodiments, gene editing to provide fratricide resistance, enhance immune cell function, or reduce immune checkpoint or suppression can occur in an immune cell prior to the cell being transformed to express a chimeric antigen receptor. In another aspect, gene editing to enhance immune cell function or reduce immune checkpoint or suppression can occur in a CAR-T cell, i.e., after the immune cell has been transformed to express a chimeric antigen receptor.

[0680] In some embodiments of the application, CD5 in a CAR-T cell is edited to knock out or knock down expression. The CAR-T is then transformed to express a chimeric antigen receptor with a CD5 scFv. By knocking out or knocking down expression of the CD5 gene, the modified CAR-T cell is less likely to perform fratricide.

[0681] In some embodiments of the application, CD7 in a CAR-T cell is edited to knock out or knock down expression. The CAR-T is then transformed to express a chimeric antigen receptor with a CD7 scFv. By knocking out or knocking down expression of the CD7 gene, the modified CAR-T cell is less likely to perform fratricide.

[0682] In some embodiments of the application, CD33 in a CAR-T cell is edited to knock out or knock down expression. The CAR-T is then transformed to express a chimeric antigen receptor with a CD33 scFv. By knocking out or knocking down expression of the CD33 gene, the modified CAR-T cell is less likely to perform fratricide.

[0683] In some embodiments of the application, CD3 in a CAR-T cell is edited to knock out or knock down expression. The CAR-T is then transformed to express a chimeric antigen receptor with a CD3 scFv. By knocking out or knocking down expression of the CD3 gene, the modified CAR-T cell is less likely to perform fratricide.

[0684] In some embodiments of the application, CD123 in a CAR-T cell is edited to knock out or knock down expression. The CAR-T is then transformed to express a chimeric antigen receptor with a CD123 scFv. By knocking out or knocking down expression of the CD123 gene, the modified CAR-T cell is less likely to perform fratricide.

[0685] Host immune cells can potentially recognize allogeneic CAR-T cells as non-self and mount an immune response to remove the non-self cells. B2M is expressed in nearly all nucleated cells and associates with MHC class I complexes Figure 1B ). Circulating host CD8+ T cells can recognize this B2M protein as non-self and kill the allogeneic cells. To overcome this graft rejection, in some embodiments, the B2M gene is edited to knock out or knock down expression. In some embodiments, provided herein is an immune cell having an edited B2M gene such that the immune cell does not express endogenous functional B2M. In some embodiments, provided herein is a CAR-T cell having an edited B2M gene such that the CAR-T cell exhibits reduced or negligible or no expression of B2M.

[0686] In some embodiments, the immune cell comprises a chimeric antigen receptor and one or more edited genes, regulatory elements thereof, or a combination thereof. The edited gene can be an immune response modulating gene, an immunogen gene, a checkpoint inhibitor gene, a gene implicated in an immune response, a cell surface marker such as a T cell surface marker, or any combination thereof. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited gene related to 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 regulatory elements thereof, or a combination thereof. In some embodiments, the edited gene can be a checkpoint inhibitor gene, such as a PD1 gene, a PDC1 gene, a member of a pathway related to or regulating the pathway. In some embodiments, the edited gene is a TRAC gene. In some embodiments, the edited gene is a CD5 gene. In some embodiments, the edited gene is a CD7 gene. In some embodiments, the edited gene is a CD33 gene. In some embodiments, the edited gene is a CD3 gene. In some embodiments, the edited gene is a CD123 gene. In some embodiments, the edited gene is a B2M gene. In some embodiments, the edited gene is a CIITA gene. In some embodiments, the edited gene is a TRBC1 / 2 gene. In some embodiments, the edited gene is a CD5 gene. In some embodiments, the edited gene is a CD7 gene. In some embodiments, the edited gene is a CD52 gene. In some embodiments, the at least one edited gene is selected from the group consisting of PD-1, CD2, CD3, CD5, CD7, CD52, B2M, TRBC1 / 2, CIITA, and TRAC, or a combination thereof. In some embodiments, the PD-1, CD2, CD52, and TRAC genes are edited. In some embodiments, the PD-1, CD2, CD52, B2M, TRBC1 / 2, CIITA, and TRAC genes are edited. In some embodiments, the PD-1, CD5, CD52, and TRAC genes are edited. In some embodiments, the PD-1, CD3, CD7, and CD52 genes are edited.

[0687] In some embodiments, the editing of the endogenous gene reduces expression of the gene. In some embodiments, the editing of the endogenous gene reduces expression of the gene by at least 50% as compared to a control cell without modification. In some embodiments, the editing of the endogenous gene reduces expression of the gene by at least 60% as compared to a control cell without modification. In some embodiments, the editing of the endogenous gene reduces expression of the gene by at least 70% as compared to a control cell without modification. In some embodiments, the editing of the endogenous gene reduces expression of the gene by at least 80% as compared to a control cell without modification. In some embodiments, the editing of the endogenous gene reduces expression of the gene by at least 90% as compared to a control cell without modification. In some embodiments, the editing of the endogenous gene reduces expression of the gene by at least 100% as compared to a control cell without modification. In some embodiments, the editing of the endogenous gene eliminates gene expression.

[0688] In some embodiments of the application, PDCD1 in a CAR-T cell is edited to knock out or knock down expression. The PDCD1 gene encodes the cell surface receptor PD-1, an immune system checkpoint expressed in immune cells, and is implicated in reducing immunity by promoting apoptosis of antigen-specific immune cells. By knocking out or knocking down expression of the PDCD1 gene, the modified CAR-T cell is less susceptible to apoptosis, more likely to proliferate, and can escape from the programmed cell death immune checkpoint.

[0689] CBLB encodes an E3 ubiquitin ligase that plays a significant role in inhibiting activation of immune effector cells. Referring to Figure 1C , the CBLB protein favors signaling pathways that lead to tolerance of immune effector cells and actively inhibits signaling pathways that lead to activation of immune effector cells. Because activation of immune effector cells is necessary for proliferation in vivo after transplantation of CAR-T cells, in some embodiments of the application, the CBLB gene is edited to knock out or knock down expression.

[0690] In some embodiments, the application provides an immune cell having an edited TRAC gene, wherein the TRAC gene can comprise one, two, three, four, five, six, seven, eight, nine, or ten or more base edits, such that the immune cell does not express an endogenous functional T cell receptor chain. In some embodiments, the immune cell is a T cell expressing a chimeric antigen receptor (CAR-T cell). In some embodiments, provided herein is a CAR-T cell having a base edit in the TRAC gene, such that the CAR-T cell has reduced or negligible or no expression of an endogenous T cell receptor alpha protein.

[0691] In some embodiments, provided herein is an immune cell having an edited CIITA gene such that the immune cell does not express endogenous functional class II major histocompatibility complex reverse activator. In some embodiments, provided herein is a CAR-T cell having an edited CIITA gene such that the CAR-T cell exhibits reduced or negligible or no expression of endogenous class II major histocompatibility complex reverse activator.

[0692] In some embodiments, provided herein is an immune cell having an edited TRBC1 or TRBC2 gene such that the immune cell does not express endogenous functional T cell receptor beta chain. In some embodiments, provided herein is a CAR-T cell having an edited TRBC1 / TRBC2 gene such that the CAR-T cell exhibits reduced or negligible or no expression of endogenous T cell receptor beta chain.

[0693] In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited TRAC, B2M, PDCD1, CBLB gene, or a combination thereof, wherein expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited TRAC gene, wherein expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited TRAC and B2M gene, wherein expression of the edited gene is either knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited TRAC and PDCD1 gene, wherein expression of the edited gene is either knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited TRAC and CBLB gene, wherein expression of the edited gene is either knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited TRAC, B2M, and PDCD1 gene, wherein expression of the edited gene is either knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited TRAC, B2M, and CBLB gene, wherein expression of the edited gene is either knocked out or knocked down. In some embodiments, the immune cell or immune effector cell comprises a chimeric antigen receptor and an edited TRAC, PDCD1, and CBLB gene, wherein expression of the edited gene is either knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited TRAC, B2M, PDCD1, and CBLB gene, wherein expression of the edited gene is either knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited B2M gene, wherein expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited B2M and PDCD1 gene, wherein expression of the edited gene is either knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited B2M and CBLB gene, wherein expression of the edited gene is either knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited B2M, PDCD1, and CBLB gene, wherein expression of the edited gene is either knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited PDCD gene, wherein expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited PDCD and CBLB gene, wherein expression of the edited gene is either knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited CBLB gene, wherein expression of the edited gene is knocked out or knocked down.

[0694] In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited CD5 gene, wherein expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited CD7 gene, wherein expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited CD33 gene, wherein expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited CD3 gene, wherein expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited CD123 gene, wherein expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited FAS gene, wherein expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited LAG-3 gene, wherein expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited CIITA gene, wherein expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited TRBC1 gene, wherein expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited TRBC2 gene, wherein expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and an edited CD52 gene, wherein expression of the edited gene is knocked out or knocked down. In some embodiments, the immune cell comprises a chimeric antigen receptor and one or more edited CD3, CD5, CD7, CD33, CD123, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and / or PD1 genes, wherein expression of the edited gene is knocked out or knocked down.

[0695] Base editing can be at any suitable location in a gene, or in a regulatory element of the gene. Thus, single base editing at the start codon, for example, can completely abrogate expression of the gene. In some embodiments, an altered endogenous gene can be modified or edited in an exon, an intron, an exon-intron splice junction, or a regulatory element thereof. The modification can be an edit of a single nucleobase in the gene or a regulatory element thereof. The modification can be in an exon, more than one exon, an intron, or more than one intron, or a combination thereof. The modification can be in the open reading frame of the gene. The modification can be within an untranslated region of the gene, e.g., in the 3'-UTR or 5'-UTR. In some embodiments, the modification is in a regulatory element of the endogenous gene. In some embodiments, the modification is in a promoter, an enhancer, an operator, a silencer, an insulator, a terminator, a transcriptional start sequence, a translational start sequence (e.g., a Kozak sequence), or a combination thereof. In some embodiments, base editing can introduce a premature STOP codon into an exon, resulting in a lack or truncation of translation products that can be misfolded and thus eliminated by degradation, or base editing can produce unstable mRNA that is readily degraded.

[0696] In some embodiments, base editing can be performed, for example, on exon 1, exon 2, or exon 3, or exon 4 or exon 5 of the human PDC1 / PD-1 gene. In some embodiments, base editing in the human PDC1 / PD-1 gene is performed at a site within exon 1. In some embodiments, base editing in the human PDC1 / PD-1 gene is performed at a site within exon 2. In some embodiments, base editing in the human PDC1 / PD-1 gene is performed at a site within exon 3. In some embodiments, base editing in the human PDC1 / PD-1 gene is performed at a site within exon 4. In some embodiments, base editing in the human PDC1 / PD-1 gene is performed at a site within exon 5. In some embodiments, one or more base editing acts can be performed on the human PDC1 / PD-1 gene at exon 1, exon 2, exon 3, exon 4, exon 5, or any combination thereof.

[0697] In some embodiments, base editing in the human PDC1 / PD-1A gene is performed by editing position 4, 6, 7, 8, or 9 of a guide RNA spacer sequence targeting exon 1. In some embodiments, base editing in the human PDC1 / PD-1A gene is performed by editing position 4, 6, 7, 8, or 9 of a guide RNA spacer sequence targeting exon 1. In some embodiments, base editing in the human PDC1 / PD-1A gene is performed by editing position 7, 8, or 9 of a guide RNA spacer sequence targeting exon 2. In some embodiments, base editing in the human PDC1 / PD-1A gene is performed by editing position 5, 7, or 8 of a guide RNA spacer sequence targeting exon 3. In some embodiments, base editing in the human PDC1 / PD-1A gene is performed by editing position 5 or 8 of a guide RNA spacer sequence targeting exon 5.

[0698] In some embodiments, base editing can be performed, for example, on exon 1, exon 2, or exon 3 of the human CD7 gene. In some embodiments, base editing in the human CD7 gene is performed at a site within exon 1. In some embodiments, base editing in the human CD7 gene is performed at a site within exon 2. In some embodiments, base editing in the human CD7 gene is performed at a site within exon 3. In some embodiments, one or more base editing acts can be performed on the human CD7 gene at exon 1, exon 2, exon 3, or any combination thereof. In some embodiments, base editing in the human CD7 gene is performed at position 4, 8, 9 within exon 1. In some embodiments, base editing in the human CD7 gene is performed by editing position 5, 6, 7, 8, or 9 of a guide RNA spacer sequence targeting exon 2. In some embodiments, base editing in the human CD7 gene is performed by editing position 4 or 9 of a guide RNA spacer sequence targeting exon 3.

[0699] In some embodiments, base editing can be performed, for example, on exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or exon 8 of the human LAG-3 gene. In some embodiments, base editing in the human LAG-3 gene is performed at a site within exon 1. In some embodiments, base editing in the human LAG-3 gene is performed at a site within exon 2. In some embodiments, base editing in the human LAG-3 gene is performed at a site within exon 3. In some embodiments, base editing in the human LAG-3 gene is performed at a site within exon 4. In some embodiments, base editing in the human LAG-3 gene is performed at a site within exon 5. In some embodiments, base editing in the human LAG-3 gene is performed at a site within exon 6. In some embodiments, base editing in the human LAG-3 gene is performed at a site within exon 7. In some embodiments, base editing in the human LAG-3 gene is performed at a site within exon 8. In some embodiments, one or more base editing acts can be performed on the human LAG-3 gene at exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, or any combination thereof. In some embodiments, base editing in the human LAG-3 gene is performed by editing position 4 or 8 of a guide RNA spacer sequence targeting exon 1. In some embodiments, base editing in the human LAG-3 gene is performed by editing position 4, 6, or 8 of a guide RNA spacer sequence targeting exon 2. In some embodiments, base editing in the human LAG-3 gene is performed by editing position 4, 5, 6, or 7 of a guide RNA spacer sequence targeting exon 3. In some embodiments, base editing in the human LAG-3 gene is performed by editing position 4, 8, or 9 of a guide RNA spacer sequence targeting exon 4. In some embodiments, base editing in the human LAG-3 gene is performed by editing position 8 or 9 of a guide RNA spacer sequence targeting exon 5. In some embodiments, base editing in the human LAG-3 gene is performed by editing position 4, 6, 7, or 8 of a guide RNA spacer sequence targeting exon 6. In some embodiments, base editing in the human LAG-3 gene is performed by editing position 4, 6, or 7 of a guide RNA spacer sequence targeting exon 7. In some embodiments, base editing in the human LAG-3 gene is performed by editing position 8 of a guide RNA spacer sequence targeting exon 8.

[0700] In some embodiments, base editing can be performed, for example, on exon 1, exon 2, exon 3, exon 4, exon 5, or exon 6 of the human CD33 gene. In some embodiments, base editing in the human CD33 gene is performed at a site within exon 1. In some embodiments, base editing in the human CD33 gene is performed at a site within exon 2. In some embodiments, base editing in the human CD33 gene is performed at a site within exon 3. In some embodiments, base editing in the human CD33 gene is performed at a site within exon 4. In some embodiments, base editing in the human CD33 gene is performed at a site within exon 5. In some embodiments, base editing in the human CD33 gene is performed at a site within exon 6. In some embodiments, one or more base editing actions can be performed on the human CD33 gene at exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, or any combination thereof. In some embodiments, base editing in the human CD33 gene is performed by editing position 7, 8, or 1 of a guide RNA spacer sequence targeting exon 9. In some embodiments, base editing in the human CD33 gene is performed by editing position 4, 5, 6, or 8 of a guide RNA spacer sequence targeting exon 2. In some embodiments, base editing in the human CD33 gene is performed by editing position 4, 5, 6, or 7 of a guide RNA spacer sequence targeting exon 3. In some embodiments, base editing in the human CD33 gene is performed by editing position 6 or 7 of a guide RNA spacer sequence targeting exon 4. In some embodiments, base editing in the human CD33 gene is performed by editing position 7 or 8 of a guide RNA spacer sequence targeting exon 5. In some embodiments, base editing in the human CD33 gene is performed by editing position 4, 5, or 6 of a guide RNA spacer sequence targeting exon 6.

[0701] In some embodiments, base editing can be performed, for example, on exon 1, exon 2, exon 3, exon 4, exon 5, exon 7, exon 8, exon 10, or exon 11 of the human CD123 gene. In some embodiments, base editing in the human CD123 gene is performed at a site within exon 1. In some embodiments, base editing in the human CD123 gene is performed at a site within exon 2. In some embodiments, base editing in the human CD123 gene is performed at a site within exon 3. In some embodiments, base editing in the human CD123 gene is performed at a site within exon 4. In some embodiments, base editing in the human CD123 gene is performed at a site within exon 5. In some embodiments, base editing in the human CD123 gene is performed at a site within exon 7. In some embodiments, base editing in the human CD123 gene is performed at a site within exon 8. In some embodiments, base editing in the human CD123 gene is performed at a site within exon 10. In some embodiments, base editing in the human CD123 gene is performed at a site within exon 11. In some embodiments, one or more base editing acts can be performed on the human CD123 gene at exon 1, exon 2, exon 3, exon 4, exon 5, exon 7, exon 8, exon 10, exon 11, or any combina...

Claims

1. An ex vivo method for producing a CAR-expressing immune cell or a population of CAR-expressing immune cells having reduced immunogenicity, the method comprising: a) contacting a target polynucleotide in an immune cell or a population of immune cells with: 1) a base editor system comprising a guide polynucleotide and a base editor comprising a fusion protein comprising a nucleic acid programmable DNA binding protein and a cytidine deaminase domain; or 2) a base editor system comprising a guide polynucleotide, a nucleic acid programmable DNA binding protein, and a cytidine deaminase domain; b) introducing a mutation that disrupts a splice site in a CD5 gene and / or a CD7 gene, thereby reducing or eliminating expression of an antigen selected from the group consisting of CD5 and CD7, wherein the CD5 gene encodes the amino acid sequence of SEQ ID NO: 27 and the CD7 gene encodes the amino acid sequence of SEQ ID NO: 29; c) introducing one or more mutations that disrupt a splice site in a CD52 gene, a PDl gene, and / or a TRAC gene, thereby reducing or eliminating expression of one or more polypeptides encoded by the genes, wherein the CD52 gene encodes the amino acid sequence of SEQ ID NO: 33, the PDl gene encodes the amino acid sequence of SEQ ID NO: 91, and the TRAC gene encodes the amino acid sequence of SEQ ID NO: 93; and d) expressing a chimeric antigen receptor that targets the CD5 or CD7 antigen having reduced or eliminated expression in the immune cell or population of immune cells, thereby producing a CAR-expressing immune cell or a population of CAR-expressing immune cells having reduced immunogenicity.

2. The method of claim 1, wherein the cytidine deaminase domain is an apolipoprotein B mRNA- editing complex selected from the group consisting of pp apolipoprotein B mRNA-editing complex and rat apolipoprotein B mRNA-editing complex.

3. The method of claim 1, wherein the cytidine deaminase domain comprises the amino acid sequence of SEQ ID NO: 1380 (ppAPOBEC-l).

4. The method of claim 1, wherein the cytidine deaminase domain comprises the amino acid sequence of SEQ ID NO: 1378 (rAPOBEC-l).

5. The method of claim 1, wherein the nucleic acid programmable DNA binding protein is spCas9.

6. The method of claim 1, wherein the base editor is a BE4 base editor.

7. The method of claim 1, wherein the base editor system further comprises two UGI domains.

8. The method of claim 1, wherein the CAR-expressing immune cell or population of CAR-expressing immune cells produced by the method does not comprise a detectable translocation.

9. The method of claim 1, wherein the CAR comprises, in order from N-terminus to C- terminus, an extracellular antigen binding domain targeting the CD5 or CD7 antigen, a CD8a hinge domain, a CD8 transmembrane domain, and a CD28z signaling domain.

10. The method of claim 1, wherein the method comprises reducing or eliminating expression of a CD7 antigen, and wherein the CAR targets the CD7 antigen and consists of the amino acid sequence set forth in SEQ ID NO:

113.

11. The method of claim 1, wherein the method comprises reducing or eliminating expression of a CD5 antigen, and wherein the CAR is a CD5 chimeric antigen receptor consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-7 and 112.

12. The method of claim 1, wherein the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of UUCGUAUCUGUAAAACCAAG (SEQ ID NO: 824), UUACCUGUACCAUAACCAGG (SEQ ID NO: 1156), CUCUUACCUGUACCAUAACC (SEQ ID NO: 1155), UGCACCUCUGGGGAGGACCU (SEQ ID NO: 1123), CCUACCUGUCACCAGGACCA (SEQ ID NO: 1128), and CACCUACCUAAGAACCAUCC (SEQ ID NO: 897).

13. The method of claim 1, wherein the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of: ususcsGUAUCUGUAAAACCAAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu (SEQ ID NO: 1452); ususasCCUGUACCAUAACCAGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu (SEQ ID NO: 1453); csuscsUUACCUGUACCAUAACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu (SEQ ID NO: 1454); ​ ​ ​ usgscsACCUGGGGAGGACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu (SEQ ID NO: 1455); cscsusACCUGUCACCAGGACCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu (SEQ ID NO: 1456); and csascsCUACCUAAGAACCAUCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu (SEQ ID NO: 1457); wherein a, c, g, or u represents a 2'-0-methyl analog, and s represents a 3' phosphorothioate internucleotide linkage.

14. The method of claim 1, wherein the CAR-expressing immune cell or population of CAR- expressing immune cells produced by the method exhibits anti-self reactivity and / or increased anti-tumorigenic activity as compared to a corresponding control cell.

15. The method of claim 1, wherein the base editor and one or more guide nucleic acid sequences are introduced into the immune cell or population of immune cells by electroporation, nucleofection, cationic lipid-mediated methods, viral transduction, or a combination thereof.

16. The method of claim 1, further comprising depleting TCRa / b+cells from the CAR- expressing immune cell or population of CAR-expressing immune cells.

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